Photooxidative Stress in Chlamydomonas reinhardtii
A Promoter Study of the Gluthatione peroxidase
homologous Gene and Response to UV Radiation
pBF31
pBF32
Gpxh-Ars
AACGTTGACGCCAGTTAGAGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCC
TGACGCCAGagAcgGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCC
AACGTTGACGCCAGTTAGAGaagccaagtttgctaatcgcgggatgatgacaccgcccgc
60
60
60
pBF31
pBF32
Gpxh-Ars
CCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTT
CCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTT
agttgagg caattccctgcagatgttgacgcgctggctattgaggagtctctgttaTaT
120
120
120
pBF31
pBF32
Gpxh-Ars
AAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAAC
AAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAAC
AAAccctttcactcacatgctgtctgcatacgcttgcggttcgcctttgcatctactgaa
180
180
180
pBF31
pBF32
Gpxh-Ars
ACCTAGATCACTACCACTTCTACACAGGCCACTCGAGGCCAACGCTGTCAAGGGATCGTT
ACCTAGATCACTACCACTTCTACACAGGCCACTCGAGGCCAACGCTGTCAAGGGATCGTT
ccagcgacgattgcaatcgatatcgaatt
AGGCCAACGCTGTCAAGGGATCGTT
240
240
240
Diploma Thesis
2006
Yvonne Schwarzenbach
Zoological Museum, University Zürich
performed at the Department of Ecotoxicology, Eawag Dübendorf
Supervision
Beat Fischer, Georg Ribi, Wolf Blanckenhorn, Rik Eggen, Paul Ward
Acknowledgement
I would like to thank Beat Fischer for his support during my diploma thesis. I appreciated
getting introduced to the field of molecular ecotoxicology and into the way of scientific
thinking, working and writing.
A special thank goes also to Régine and Manu of our little Chlamy-group. I would like to
thank them for helping me out a lot and also for all the funny moments in the lab but also
else where for example by harvesting grapes in the Valais.
Thanks a lot to Ksenia, who never got tiered in answering my questions, especially at
times where people are not usually working at the EAWAG. But it was nice to have here
company while getting some fresh air on the balcony. Also a big thank to thank Enrique
for helping me by the setup of the UV experiments and by the data analysis.
Thanks to all the people in the E-floor and the F-floor to contribute to the good
atmosphere and giving me a lot of advises for my work, especially Karin, Christoph,
Thommy, Hansueli, Mirjam, Jane, Bettina, Nathalie, Etienne and Jules.
I also really enjoyed playing unihockey on Wednesday with Christoph, Hansueli, Thomas,
Michi, Kim, Andreas, Tobias, Anatol… Thanks a lot to all this people for coming along.
I would also like to thank Adi for the big support and understanding. I would also like to
thank my friends and family.
Finally, I would like to thank Rik for his support during my diploma thesis at the
department of Ecotoxicology at the EAWAG. Also a big thank to Georg for the helpful
inputs and his interest in my work. I would also like to thank Wolf for helping me out
with statistics and Paul Ward for his agreement with my external diploma thesis.
Summary
Risk assessment for chemicals released to the environment is a major issue in
Ecotoxicology. In the past this was often based on extrapolation of data gained from
classical toxicity tests. Recent approaches have focused on genetic responses in the
organisms to a cellular stress caused by a toxic agent. Genes, which are specifically
induced by a toxic agent, can be used as molecular biosensors. Thus, the specific
response of such a biosensor can be used to indicate and identify the pollution in a
complex mixture of chemicals in an environmental sample. However, a biosensor should
only be used in risk assessment if its reaction is specific, highly sensitive and if the
molecular mechanism of the response is known.
Oxidative stress is a common mode of toxic action of many pollutants and is also a
consequence of high light intensities or UV radiation in photosynthetic organisms. It is
caused by an increased formation of reactive oxygen species, such as singlet oxygen,
which can seriously damage the cells. In the green alga Chlamydomonas reinhardtii,
which has achieved recognition as a photosynthetic model organism, the Gluthatione
peroxidase homologous gene (Gpxh) was found to be specifically induced by singlet
oxygen. The specific induction of this gene could therefore serve as an ideal endpoint for
a biosensor for photooxidative stress, caused by singlet oxygen formed by a pollutant in
the environment.
However, before the Gpxh gene can be used as a biosensor it is necessary to know
whether the response is specific to singlet oxygen and how the gene is activated. Since
ultraviolet radiation is known to increase the cellular level of reactive oxygen species like
singlet oxygen in cells, we were interested if our singlet-oxygen-specific gene was
induced by exposure to high UVA and high UVB. Therefore, we measured its expression
after a short-time exposure to UV radiation with real time RT-PCR, but we found no
induction of the Gpxh gene, which could indicate that singlet oxygen is not the major
ROS produced in C.reinhardtii upon exposure to UV radiation. Furthermore we
investigated the response mechanism of the Gpxh gene by testing several Gpxh promoterreporter gene constructs, in which different elements in the Gpxh promoter were mutated.
In this manner we searched for elements which are required for the induction of the Gpxh
gene and revealed two new regulatory elements involved in the induction of the gene.
The first one is a GC-box and the second one a CAAT-box on the antisense strand, both
located upstream of the TATA-box in the promoter of Gpxh. Since a third element
required for Gpxh induction by singlet oxygen was isolated earlier, we conclude that
these three regulatory elements involved in the induction of the Gpxh gene could be
responsible for the high specificity of the response of this gene to singlet oxygen. Still,
the signalling pathway and the exact mechanism of the Gpxh induction need to be further
investigated. Therefore, it is too early to use the Gpxh promoter-reporter gene construct
as a commercial biosensor to detect photooxidative stress caused by singlet oxygen in
environmental risk assessment.
Table of Contents
Abbreviations ......................................................................................................6
1 Introduction..................................................................................................7
1.1
Ecotoxicology .........................................................................................7
1.2
Photooxidative Stress in Chlamydomonas reinhardtii.............................8
1.3
Induction of Gluthatione peroxidase homologous gene (Gpxh) by Singlet
Oxygen .................................................................................................10
1.4
Ultraviolet Radiation as a Putative Source of Singlet Oxygen ..............15
2 Material and Methods ................................................................................18
2.1
Strains and Growth Condition...............................................................18
2.2
Molecular Methods ...............................................................................19
2.2.1
Digestion, Fill in of 5’ Overhanging Ends and Dephosphorylation ......... 19
2.2.2
Separation, Purification and Quantification of DNA Fragments.............. 20
2.2.3
Ligation ..................................................................................................... 21
2.2.4
Plasmid Transformation in Escherichia coli............................................. 21
2.2.5
Isolation of Plamids from E.coli ............................................................... 21
2.2.6
DNA-Sequencing and Alignments ........................................................... 22
2.2.7
DNA Amplification by Polymerase Chain Reaction (PCR)..................... 22
2.3
Plasmid Cloning....................................................................................24
2.4
Transformation of Plasmids into C. reinhardtii ......................................30
2.5
Stress Treatment with Neutral Red and Measuring Reporter Gene
Expression............................................................................................30
2.5.1
Exposure to Oxidative Stress Caused by NR............................................ 30
2.5.2
Arylsulfatase Assay and Calculation of Induction.................................... 31
2.6
Stress Treatment by UV Radiation .......................................................32
2.6.1
Optimizing the Exposure Condition by Varying the Intensity of UVA,
UVB and PAR Light with Different Lamps, Filters and the Distance
Between Lamp and Culture ...................................................................... 32
2.6.2
Exposure of Algal Cultures to High UV Radiation .................................. 34
2.7
Measuring Physiological Parameters of Algae Exposed to High UV
Radiation ..............................................................................................35
2.7.1
Growth ...................................................................................................... 35
2.7.2
Photosynthetic Parameters ........................................................................ 35
2.7.3
Pigment Content........................................................................................ 35
2.8
Measuring the Genetic Response in Algae Exposed to UV Radiation..36
2.8.1
Isolation and Quantification of mRNA..................................................... 36
2.8.2
Quantification of the Gene Expression by real time RT-PCR.................. 37
2.9
Data Analysis........................................................................................37
3 Results & Discussion ................................................................................38
3.1
The Molecular Mechanisms of the Gpxh Induction by Singlet Oxygen .38
3.1.1
Effect of the Position of the CRE-Element on the Gpxh Induction by
Singlet Oxygen.......................................................................................... 38
3.1.2
Effect of the Missing TATA Consensus Sequence in the β-Tubulin
Promoter of pBF31mod and pBF32mod on Induction by Singlet Oxyge 40
3.1.3
The Involvement of Three Putative Transcription Factor Binding Sites of
the Gpxh Promoter in the Response to Singlet Oxygen............................ 42
3.2
4
5
6
Molecular and Physiological Response of Chlamydomonas reinhardtii
Exposed to Ultraviolet Radiation...........................................................45
3.2.1
Establishing Experimental Conditions for UV Treatment........................ 45
3.2.2
Effect of Preadaptation and Exposure of Cultures to UVA and UVB
Conditions Compared to a Culture Kept under our Standard Conditions 50
3.2.3
Physiological Parameters of Cultures Exposed to the Selected Conditions
of the UVA and UVB Treatment .............................................................. 52
3.2.4
Genetic Response to UV Radiation: The Induction of Gpxh Gene by UVA
and UVB-Treatment.................................................................................. 57
3.2.4.1 Normalization of Varying mRNA Levels............................................. 58
3.2.4.2 Comparison of the Expression of Three Stress Response Genes in the
UV-Control Conditions and in the Multitron........................................ 58
3.2.4.3 Genetic Response of the Gpxh, GST and HSP Gene to High UVA and
UVB Radiation...................................................................................... 59
Conclusion .................................................................................................63
References .................................................................................................65
Appendix ....................................................................................................70
Abbreviations
Arg
Ars
bp
cDNA
CRE
Ct
DNA
F
FE
G
Gpxh
GST
H2O2
HSP
kb
LB
M
mRNA
Multitron
NR
OD
O2•1
O2
OH•
PAR
PCR
PSI/PSII
Q
Rbcs2
RNA
ROS
rpm
TAP
U
UV
UVA
UVA1-4
UVB
UVB1-4
X-Gal
18Sr
Arginine
Arylsulfatase
base pair
complementary DNA
cAMP responsive regulatory element on the Gpxh promoter
Threshold cycle number
Deoxyribonucleic acid
Overhead foil
Plexiglas filter
Glass filter
Glutatione peroxidase homologous gene
Gluthatione-S-transferase gene
Hydrogen peroxide
Heat shock gene
Kilo base pair
Luria Bertani Broth medium for bacteria
Lubriflon filter
Messenger RNA
Incubator for algae
Exogenous photsensitizer Neutral Red
Optical density
Superoxide radical anion
Singlet oxygen
Hydroxyl radical
Photosynthetically active radiation
Polymerase chain reaction
Photosystem I/II
Quartz glass
Rubisco gene
Ribonucleic acid
Reactive oxygen species
Rounds per minute
Tris-Acetat-Phosphate medium for algae
Unit
Ultraviolet radiation
Ultraviolet A radiation
UVA conditions with different intensities in UVA radiation
Ultraviolet B radiation
UVB conditions with different intensities in UVA radiation
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
gene for 18S ribosomal RNA
Introduction
1 Introduction
1.1 Ecotoxicology
In recent years the environmental pollution and its possible consequences for humans and
the wildlife have received increasing attention. Humans tend to live close to the places
with easy access to resources. Hence, human impact and the resulting pollution endanger
the most important resource locations since they are not spread equally throughout the
world. There is a wide range of environmental stressors to the environment including
toxic chemicals, increased UV radiation, hypoxia, pathogen-induced diseases and habitat
disturbance [1]. Therefore risk assessment of endangered environments became very
important in Ecotoxicology to ensure the possibility to act before it is too late. The aim is
to establish a ranking of the relative toxicities and monitor chemicals that have an impact
or are of potential danger for the natural environment.
In the past most risk assessments for chemicals released to the environment were based
on the classical toxicity tests. One example of such a toxicity test is the standard algal
growth inhibition test. It is based on negative effects of pollutants on the growth of green
unicellular algae such as various Scenedesmus or Chlorella strains. The growth rate of
cultures exposed to increasing concentrations of the tested chemical is measured over a
period of 72 hours. Then, the resulting dose-response relationships of the pollutant
concentration and the growth reduction are used to calculate the ‘no observed effect
concentrations’ (NOEC) and the ‘lethal dose 50’ (LD50). The LD50 is the dose of the
toxic agent at which 50% of the initial algal population is dead after an exposure of 72
hours. Data obtained in such well defined standard bioassays allows to calculate the
predicted ‘no-effect concentrations’ (PNEC), which are applied in environmental risk
assessment [2]. However, the application of such assays does not allow a detection of an
effect of the toxic agent before the algae are seriously stressed. Another disadvantage of
toxicity tests is that the mode of toxic action can not be investigated with these tests.
Recent approaches in Ecotoxicology are focusing on gene expression as a response to
cellular stress caused by a toxic agent. Defence mechanisms are activated when an
organism is exposed to a toxic agent, for example by increasing the expression of defence
genes. The resulting proteins might directly inactivate the toxic agent or help the cell to
combat the cellular stress or activate further defence pathways. The induction of the gene
expression can be a general stress response, induced by main stressors, or a specific
response to only one stressor. The specifically induced genes provide direct information
about the cellular stress and the mode of action of a treatment and therefore they are of
special interest in Ecotoxicology. In a molecular biosensor, the expression of a gene with
a specific mode of action can be used to monitor a certain effect in an environmental
sample. In comparison to classical toxicity tests, molecular biosensors are often much
more sensitive because the cells already express their defence at low levels of stress.
7
Introduction
Therefore, such biosensors usually allow detecting an effect at very low doses of the toxic
agent, whereas the same dose would not cause any effect on growth in a classical toxicity
test. Additionally, a biosensor often allows a prediction of the mode of toxic action
caused by a toxic agent. Instead of only detecting a general negative biological effect, the
specific response of a biosensor to a pollutant indicates and helps to identify the actual
pollution in a complex mixture of chemicals found in most environmental samples. On
the other hand, a biosensor can only be efficiently used in risk assessment if the reaction
to the stressor is highly sensitive, specific and if the molecular mechanism of the response
is known in order to ensure a specific response of the biosensor to the stressor.
1.2 Photooxidative Stress in Chlamydomonas reinhardtii
Oxidative stress caused by reactive oxygen species (ROS) is a common mode of toxic
action of many pollutants in photosynthetic organisms. Under natural conditions the main
source of ROS are electron transport chains, for example the one located in the
photosynthetic apparatus in the chloroplast of a photosynthetic organisms [3]. There, the
splitting of water, the transport of electrons between the two photosystems and the high
local concentration of oxygen increase the chance of an electron transfer to molecular
oxygen (3O2) leading to the formation of low levels of ROS, like superoxide radicals
(O2•-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH•) (type I reaction) [4].
Especially in photosynthetic organisms the presence of high levels of endogenous
photosensitizers, such as the light absorbing pigments chlorophyll a and b, increase the
probability of uncontrolled energy transfer from excited photosensitizers to molecular
oxygen, leading to the formation of singlet oxygen (1O2) (type II reaction). The level of
ROS produced by such photochemical reactions can be dramatically increased by high
light intensities, UV radiation, heat, herbicides and other environmental pollutants. These
factors lead to a blockage of the electron flow in the photosynthetic apparatus, called
photoinhibition, which leads to an increased production of ROS. When the level of these
ROS in a cell exceeds the cellular capacity to cope with them, the photosynthetic
organism suffers a photooxidative stress.
ROS are highly reactive molecules which can modify lipids, proteins and DNA. ROS,
such as hydroxyl radicals or singlet oxygen, were found to react with unsaturated lipids in
membranes and form lipid hydroperoxides. This leads to a disruption of the structure and
loss of function of membranes which results in cell injury [5]. Additionally, many
intracellular proteins are very sensitive to ROS [3]. One effect in photosynthetic
organisms is the singlet oxygen dependent degradation of the D1 protein, which is the
central chlorophyll binding protein in the photosystem II (PSII) [6]. A degradation of this
protein results in the disassembling of the PSII and photoinhibition, which leads to the
complete inactivation of the photosynthesis. However, this process can be quickly
repaired again and therefore disturbs the cell fitness only for a limited time .The
modification of the DNA, on the other hand, can occur via the production of hydroxyl
8
Introduction
radicals or singlet oxygen and results in DNA strand breaks and mutations which can be
lethal to the cell [7]. Thus, increased levels of ROS can damage essential components of
the cell, inhibiting their function and result in an oxidative stress and even cell death.
Photosynthetic organisms have evolved defence mechanisms against photooxidative
stress caused by ROS by preventing the production of ROS, removing them or repairing
damaged molecules. Preventive defence strategies include the excretion or inactivation of
ROS generating compounds or reducing the synthesis of light absorbing pigments, and
thus prevent the formation of light induced oxidative stress. ROS can be removed either
in a non-enzymatic way by scavengers like ascorbate, gluthatione and carotenoids, or by
enzymatic degradation with superoxide dismutases, catalases or different types of
peroxidases. Finally, damaged molecules can be directly repaired, degraded or excreted
by proteases, chaperones and gluthatione-S-transferases [3, 8]. With low expression of
these defence mechanisms the cell is able to cope with low levels of ROS and keep up the
cellular functions. Whereas if a high level of ROS is present the cell is photooxidatively
stressed and therefore many of these defence genes are strongly induced in their
expression [9, 10]. The upregulation of these genes is either regulated by ROS production
directly, by damaged cell components or by signals from the overexcited photosynthetic
machinery [10, 11]. Depending on the function and regulation mechanism of the gene,
the upregulation of the gene expression can be a general stress response, induced by
many stressors, or a specific response to oxidative stress or even a specific ROS.
The unicellular green alga Chlamydomonas reinhardtii has achieved recognition as a
model organism to study the physiological, biochemical and genetic responses of
environmental stressors in photosynthetic organisms and was used to investigate
photooxidative stress. A Gluthatione peroxidase homologous gene (Gpxh), which was
found to be specifically induced upon exposure to singlet oxygen, could be an ideal
endpoint as a biomarker of photooxidative stress, caused by singlet oxygen, in
environmental risk assessment [6, 12].
9
Introduction
1.3 Induction of Gluthatione peroxidase homologous gene
(Gpxh) by Singlet Oxygen
Before, the Gpxh gene can be used as biosensor for singlet oxygen in environmental risk
assessment it is necessary to know the exact mechanism of the transcriptional activation
of this gene and whether the response is really specific for 1O2. Thus, besides the
identification of a 1O2 sensor and its signalling pathway, the promoter region needs to be
investigated.
A promoter is the region upstream of the transcription start site of a gene. Specific
regulatory sequence elements in the promoter region are recognized by proteins, called
transcription factors, which, after binding to the DNA, can activate the transcription. The
most important sequence element in a promoter is the TATA-box. The TATA-box is
sufficient for the formation of the basal transcription apparatus composed of general
transcription initiation factors and the RNA polymerase. However, the transcriptional
activation of many genes in eukaryotic organisms is controlled by several regulatory
elements and is therefore very complex. Several transcription factors receive a signal and
transmit this signal by binding to a specific sequence element upstream of the TATA-box.
Only the entire complex of all transcription factors and the polymerase is sufficient to
stimulate the high transcription rate of induced genes [13].
The promoter region of the Gpxh gene of C. reinhardtii was already investigated by
analyzing a promoter reporter gene construct. Since Gpxh is specifically induced by 1O2
(Fig 1.1a), its promoter region was isolated and fused to the reporter gene arylsulfatase
(Fig1.1b) by using common cloning techniques. The resulting plasmid pASpro1 was then
transformed into a C.reinhardtii strain where the construct was integrated into the
genome (Fig 1.1c). When a culture of this strain was exposed to singlet oxygen, not only
the endogenous Gpxh gene but also the integrated Gpxh promoter-arylsulfatase construct
was induced (Fig 1.1c). Such report gene constructs allow an easy detection of the
induction. The expression of the reporter gene arylsulfatase is measured by the
arylsulfatase assay, which is based on a quantification of the enzyme by a colour reaction
of the added substrate catalyzed by the arylsulfatase enzyme (Fig 1.1d). Now, this
reporter gene approach allows to investigate the regulation of the Gpxh gene and to
determine promoter elements involved in the singlet oxygen-induced Gpxh activation by
deletion and mutation studies.
10
Introduction
(a)
(b)
Reporter gene
(c)
(d)
Fig 1.1 Schematic drawing of the construction and the application of the Gpxh promoter-arylsulfatase
reporter gene construct. (a) The Gpxh gene in C.reinhardtii was found to be significantly induced by singlet
oxygen (triangles). (b) Isolation of the Gpxh promoter and fusion to the reporter gene arylsulfatase in E.coli.
(c) Exposure to singlet oxygen (triangles) of a strain which integrated the reporter gene constructs into the
genome results in the higher production of the arylsulfatase enzyme. (d) The arylsulfatase assay, based on a
quantification of the colour reaction catalyzed by the arylsulfatase enzyme, is used to analyze the gene
expression.
The first step in the characterisation of the promoter of Gpxh was a deletion study
performed by Leisinger, in which several Gpxh promoter fragments of decreasing length
were cloned in front of the arylsulfatase reporter gene and tested for their induction by
singlet oxygen (Fig 1.2) [12]. A singlet-oxygen responsive promoter region was found to
be located in the region between –179 and –104 relative to the transcription start-site,
because the induction of a 104bp long fragment (pASpro5) was clearly reduced compared
to the whole promoter region (pASpro1) or to a 179bp long fragment (pASpro4).
Homology search with the sequence between –179 and –104 of the promoter region of
Gpxh to the sequence of other promoters identified a sequence that is homologous to the
mammalian cAMP response element (CRE) and the activator protein 1 (AP1) recognition
site [12].
11
Introduction
ARS
pASpro1
ARS
pASpro4
ARS
pASpro5
Fig 1.2 Gpxh promoter arylsulfatase reporter gene constructs varying in the length of the Gpxh promoter
region, which were tested by Leisinger and his group [12]. The Gpxh promoter fragments are coloured in
green, the arylsulfatase reporter gene in blue. The first bright green box indicates the CRE/AP1
homologous sequence and the second the TATA-box.
The importance of this CRE/AP1 (CRE) homologous sequence was further investigated
by Fischer [14]. A significantly lower induction was found in constructs in which the
CRE element was mutated (pBF19) (Fig 1.3). However, testing the induction of
constructs were the CRE element was fused to a β-tubulin promoter in front of the
arylsulfatase gene (pBF31 and pBF32) still exhibited a lower induction than in constructs
with the wild type promoter (pASpro2) (Fig 1.3) The CRE element is therefore required
but not sufficient for the induction of Gpxh by singlet oxygen.
CRE
TATA
pASpro2
pBF19
pBF31
pBF32
pBF33
Fig 1.3 Different Gpxh promoter-arylsulfatase reporter gene constructs, tested by Fischer [14]. The
plasmids pASpro2 and pBF19 consist of 562bp long Gpxh promoter fragment, but in pBF19 the CRE
element was mutated. In pBF31 and pBF32 the CRE element, with and without flanking sequences, were
fused to a β-tubulin promoter and the plasmid pBF33 without the CRE element was used as a negative
control. The Gpxh promoter fragments are coloured in green, the β-tubulin promoter in red and the
arylsulfatase reporter gene in blue. The most upstream bright green box indicates the CRE/AP1
homologous sequence, the second boxes in bright green or orange show the TATA-box of the Gpxh or βtubulin promoter, respectively.
12
Introduction
In this study we further investigated the Gpxh promoter and its activation by 1O2 and tried
to answer the question why the CRE element fused to a β-tubulin promoter in pBF31,
pBF32 did not result in the same induction by singlet oxygen than the positive control
construct pASpro2 (Fig 1.3).
To explain this, we had three hypotheses:
I. The low induction in pBF31 and pBF32 could be due to the position of the CREelement, because the CRE-element in pBF31 and pBF31 was found to be located
one nucleotide further upstream compared to the position of the TATA-box in the
Gpxh promoter (Fig 1.4a). This difference could affect the three dimensional
structure and the efficiency of the interaction between a CRE-binding
transcription factor and the TATA-box-binding transcriptional apparatus.
II. The low induction in these constructs could be due to the missing consensus
TATA-box of the introduced β-tubulin promoter in these constructs (Fig 1.3). The
Gpxh promoter, among many other eukaryotic promoters, consists of a TATAbox with a consensus sequence of 5’-TATAAA-3’. However, the TATA-box of
the β-tubulin promoter is not a consensus TATA-box and thus its sequence is not
identical to the one of the Gpxh promoter. Since the TATA-box is very important
for a efficient activation of the transcription, changes within this element could
strongly affect the induction.
III. Since there is a long part in the constructs pBF31and pBF32 between the CRE
element and the transcription start, which is different to the Gpxh promoter, other
elements in the Gpxh promoter might be involved in the induction but are not
present in pBF31 and pBF32. Leisinger also analyzed the Gpxh promoter
fragment from the CRE-element till the TATA-box and found three putative
regulatory elements known to be involved in the transcriptional regulation of
genes. The first, most upstream element in the Gpxh promoter is a putative GCbox (5’-CCGCCC-3’), the second a CAAT-box on the sense strand (5’-CAAT-3’)
and the third a CAAT-box on the antisense strand (5’-ATTG-3’) (Fig 1.5). These
putative regulatory elements could be involved in the transcriptional activation of
Gpxh, and since they are missing in pBF31 and pBF32 their absence could be the
reason for the low induction of these constructs.
13
Introduction
(a)
pBF31
AACGTTGACGCCAGTTAGAGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCC
pBF32
TGACGCCAGagAcgGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCC
Gpxh-Ars AACGTTGACGCCAGTTAGAGaagccaagtttgctaatcgcgggatgatgacaccgcccgc
60
60
60
pBF31
CCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTT 120
pBF32
CCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTT 120
Gpxh-Ars agttgagg caattccctgcagatgttgacgcgctggctattgaggagtctctgttaTaT 120
pBF31
AAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAAC 180
pBF32
AAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAAC 180
Gpxh-Ars AAAccctttcactcacatgctgtctgcatacgcttgcggttcgcctttgcatctactgaa 180
pBF31
ACCTAGATCACTACCACTTCTACACAGGCCACTCGAGGCCAACGCTGTCAAGGGATCGTT 240
pBF32
ACCTAGATCACTACCACTTCTACACAGGCCACTCGAGGCCAACGCTGTCAAGGGATCGTT 240
Gpxh-Ars ccagcgacgattgcaatcgatatcgaatt
AGGCCAACGCTGTCAAGGGATCGTT 240
(b)
CRE
TATA
1
2
3
Fig 1.4 (a) Sequence alignment of the promoter region of pBF31, pBF32 and pASpro1 (Gpxh-Ars). In bold
the CRE-element and the TATA box, in red the additional nucleotide in pBF31 and pBF32 and underlined
the transcription start site are shown. (b) Gpxh promoter region (green) fused to the arylsulfatase reporter
gene (blue). The bright green boxes indicate the position of the CRE-element, the TATA-box and the three
putative regulatory elements which are a putative GC-box (1), a putative CAAT-box (2), and a putative
CAAT-box on the antisense strand (3).
In this study we tested the three hypotheses mentioned above in order to get a better
understanding of the mechanism of induction by singlet oxygen in the Gpxh gene. This
will help to identify the 1O2-specific promoter elements in Gpxh and to construct a
minimal 1O2 responsive reporter construct. This knowledge is crucial for future
application of a Gpxh promoter-arylsulfatase reporter gene construct as biosensor in
environmental risk assessment.
14
Introduction
1.4 Ultraviolet Radiation as a Putative Source of Singlet
Oxygen
Ultraviolet radiation (UV) is part of the full sunlight spectrum that reaches the surface of
the earth. The UV spectrum is by convention divided into three sections: UVA (320nm400nm), UVB (280-320nm) and UVC (100-280nm). Of these, only UVA and shorter
wavelength UVB are of biological importance because the stratospheric ozone layer
absorbs very effectively UV radiation with a wavelength below 280nm. Since reduction
in the stratospheric ozone concentration leads to an increase in UVB radiation reaching
the Earth’s surface, most research has been focusing on the effects caused by high UVB
radiation. However, UVA and UVB have both been shown to have adverse effects on
terrestrial and aquatic organisms [15, 16].
Absorption of UVB photons can cause substantial damage to biomolecules, including
DNA and RNA, but also proteins and lipids [17-20]}. Furthermore, in photosynthetic
organisms different physiological parameters have been shown to be seriously affected by
UVA and UVB radiation such as growth, photosynthesis and pigment content. Jokiel’s
group showed that growth of different microalgae was highest in pure photosynthetically
active radiation (PAR), but was significantly decreased by addition of UVA and even
more by UVB [21, 22]. Additionally, UV was shown to affect the photosynthetic
efficiency since the fluorescence yield was decreased in different photosynthetic
organisms upon exposure to UVA and UVB radiation what indicates a blockage of the
electron flow in the photosynthetic apparatus leading to photoinhibition [23-26].
Moreover, the oxygen production was clearly reduced by UV [27, 28], and upon
exposure to UVB radiation pigment bleaching is a wide spread phenomena, caused by a
decline in chlorophyll and carotenoid content [18, 23, 25, 28, 29]. Even though some
studies show contrasting results [26], it is generally believed that the damaging efficiency
of UVA is smaller than of UVB and that the target sites in the photosynthetic apparatus
are similar for UVA and UVB. Both UVA and UVB are probably affecting the electron
donor site of the photosystem II, the oxygen evolution complex. However, the
impairment might be via non-interacting mechanism for example by an increase of
reactive oxygen species which play either a role in the signalling transduction pathway or
by causing oxidative stress [24, 27, 30-32].
The defence mechanisms of organisms include the synthesis of various enzymes,
accumulation of protecting pigments and different morphological and behavioural
changes. The list of enzymes with increased level upon exposure to UV depends very
much on the organism, but they include mainly oxidative stress response proteins such as
superoxide dismutases, catalases, gluthatione reductases and enzymes involved in the
flavonoid synthesis or the synthesis of other UV absorbing components [33-35]. In
UVA/blue light, photolyase is a key enzyme in the DNA protection which can repair
photoproduct by enzymatic reactivation [34]. A variety of protecting pigments has been
15
Introduction
found to accumulate in different plants exposed to UV radiation. Flavonoids and sinapate
esters, for example, were shown to protect Arapidopsis plants from harmful effects of
UVB [36, 37]. In cyanobacteria and different algae the content of mycrosporine-like
amino acids (MAAs), absorbing from 310nm to 360nm, has been shown to be increased
upon exposure to UV radiation [38-40]. In addition, morphological changes, such as
increased size of the cuticula and behavioural changes like a different vertical orientation
of Antarctic cyanobacteria in a water column were also detected as a response to UV
radiation [32]}Quesada, 1997 #1889}.
Analysis of UV effects on the genetic level in photosynthetic organisms revealed that
gene expression is clearly altered by UV radiation. Genes involved in photosynthesis
were found to be downregulated, whereas genes coding for general and oxidative stress
response proteins, transcription factors, signal transduction molecules and for cell cycle
regulating proteins were found to be upregulated in higher plants by exposure to UVB
[41-44].
However, the underlying mechanisms governing these integrated responses to UV
radiation are still unclear. It is generally believed that there are two main sensor pigments
involved in the transduction of UV signals in higher plants: cryptochrome for sensing
UVA or blue light and a still unknown photoreceptor for UVB (<350nm) [33, 34].
Besides that, other UV-signalling components are shared with other stress signalling
pathways such as oxidative stress caused by high light intensities, salt stress or pathogen
induced stress. This includes reactive oxygen species (ROS), Ca2+Ions, nitric oxide, or
various plant hormones shown to be involved in the UVA and the UVB signalling
pathways of different plant species [26, 44-47]. For example, singlet oxygen production
in plants was recently found to be induced by UVA [48]. Interestingly, also in mammals
1
O2 was found to be a major ROS produced upon exposure to UV radiation [49-51].
In this study, we were interested in the genetic response of C.reinhardtii to high UV
radiation. Genes involved in the response to oxidative stress are often upregulated by
several stress conditions because many stressors can increase the intracellular level of
ROS. Since there is evidence that the production of 1O2 is increased by UV treatment, we
were especially interested if the 1O2 specific response of the Gpxh gene in C.reinhardtii is
also induced upon exposure to either high UVA or high UVB radiation. Since some
studies on the effects of UVA and UVB are contradictory [26], often because of
contaminations of UV radiation of by other wavelengths [52], we exposed cultures to
different intensities of either high UVA or high UVB radiation (Fig 1.6). On the other
hand, the intensity of the UV radiation had to be carefully controlled in order to
investigate the genetic response in cells which are seriously stressed but still vital
(Fig1.6).
16
Introduction
UVA
light intensity
effec
t of U
VA
?
effect of
U VB
?
UVB
light intensity
Fig 1.6 Schematic drawing of the light intensities needed to investigate the genetic response to either UVA
radiation (dotted arrow) or UVB radiation (black arrow). The grey lines indicate the highest UV intensity
level at which the cells are still vital during the time of exposure.
17
Material and Methods
2 Material and Methods
2.1 Strains and Growth Condition
Escherichia coli strain DH5α was grown in a Luria Bertani Broth medium (LB) either in
liquid cultures or on plates containing 15g/l agar (Tab 2.1). When required 50µg/ml
ampicillin was added. For LB-X-gal plates 4µl/l Isopropyl β-D-1-thiogalactopyranoside
(IPTG) and 40µl/l of the sugar X-galactose (X-Gal) was added to the medium. Liquid
cultures were grown in 50ml Erlenmeyer flasks at 37°C and agitated on a shaker at
200rpm. After transformation of plasmids cells were kept on plates in the fridge at 4°C or
conserved in 15%(v/v) Glycerol at -80°C. The competent cells were stored at -80°C
(Tab5.1).
The Chlamydomonas reinhardtii strain cw15arg7mt- was used for transformation of
plasmids and concomitant induction experiments. This strain is mutated in a gene for cell
wall biosynthesis, allowing transformation of exogenous DNA, and has a deletion in the
argininosuccinate lyase gene for selection of transformed cells with plasmid pARG7.8
(see section 2.4). For experiments under UV radiation the clone pASpro1β (cw15arg7mtcontaining the plasmid pASpro1) was used. Cultures were grown either in liquid cultures
in Tris-Acetat-Phosphate (TAP) media or on 15g/l Agar plates (Tab 2.1). To avoid
bacterial infection the TAP media was supplemented with 50µg /ml ampicillin. The strain
cw15arg7mt- was additionally supplemented with 50µg/ml arginine. The liquid cultures
were kept in 50-200ml Erlenmeyer flasks at 25°C under constant illumination with white
light of 100-150µmolm-2s-1 photosynthetically active radiation (PAR) and agitated on a
rotary shaker at 150rpm. Cell density was determined by measuring the optical density at
750nm or the cell density using the Z2Coulter counter.
18
Material and Methods
Table 2.1 Contents of media used for bacterial (L-Broth) and algal (TAP) cultures. For plates 15g/l Agar
was added.
L-Broth (per l)
10g
tryptone
5g
yeast extract
5g
NaCl
TAP-media (per l)
2.42g
Tris(hydroxymethyl)aminomethane (Fluka)
25ml
TAP salts:
15g/l
NH4Cl
4g/l
MgSO4x7H2O
2g/l
CaCl2x2H2O
0.375ml
phosphate solution:
288g/l K2HPO4 (anhydrous)
144g/l KH2PO4
1ml
of each Hunter solution:
Hunter solution 1:
50g/l
Na2EDTA
~16g/l KOH (pH 7.0)
Hunter solution 2:
4.99g/l
FeSO4
1ml/l
concentrated H2SO4
Hunter solution 3:
11.4g/l
H3BO3
Hunter solution 4:
22g/l
ZnSO4x7H2O
5.06g/l
MnCl2x4H20
1.61g/l
CoCl2x6H2O
1.57g/l
CuSO4x5H2O
1.1g/l
(NH4)6Mo7O24x4 H20
~0.2ml
acetic acid to adjust to pH 7.0
2.2 Molecular Methods
2.2.1 Digestion, Fill in of 5’ Overhanging Ends and
Dephosphorylation
Digestions were performed in volumes of 10-50µl by mixing the appropriate amount of
DNA, always less than 100ng/µl with dH2O, 1-5µl 10xrestriction buffer (Fermentas) and
5-10U of restriction enzymes (Fermentas). The digestions were carried out using
restriction enzymes and the restriction buffer as suggested by the supplier. The digestion
mixture was usually incubated at 37°C for at least one hour. SmaI digestions were
performed at 30°C.
The 5’ overhanging ends were filled in with Klenow polymerase to generate blunt end
dsDNA at the 5’overhanging ends of DNA fragments previously treated with restriction
enzymes. For this 0.1-4µg linearized DNA, 3µl 10xKlenow buffer (10xfilling-in buffer,
19
Material and Methods
500mM Tris-HCl, pH 7.5, 100mM MgCl2, 10mM DTT, 500µg/ml BSA), 2µl 10mM
solution of each dNTP and 1µl Klenow DNA polymerase I (Applied Biosystems) were mixed in
an eppendorf tube and filled up with dH2O to an end volume of 30µl. Then the reaction mixture
was incubated at 30°C for 15min. The reaction was stopped by heating to 75°C for 10min.
Dephosphorylation using Shrimp Alkaline Phosphatase (SAP) was carried out by
pipetting 26µl of a digested DNA fragment from a vector digestion mixture with 3µl
10xSAP buffer and 1u SAP reaching an total volume of 30µl. The mixture was incubated
at 37°C for 1hour and afterwards the enzyme was inactivated by incubating at 65°C for
15min.
2.2.2 Separation, Purification and Quantification of DNA Fragments
After DNA digestion or PCR amplification the DNA fragments were separated by their
size using agarose gel electrophoresis on a 1% agarose gel with Tris-acetate-EDTA (TAE)
buffer according to Sambrook (Sambrook, Fritsch et al. 1989). To detect fragments
smaller than 300bp, a 2% agarose gel was used. The agarose-TAE buffer mixture was
carefully heated in the microwave to melt agarose, and supplemented with 0.5µg/l
ethidium bromide before pouring into a gel chamber. When the gel was solid it was put
into the electrophoresis chamber previously filled with 1xTAE buffer. The DNA samples
was mixed with 1-2µl loading buffer (0.25% bromophenolblue, 0.25% xylene cyanol,
30% glycerol), loaded into the slots of the gel and run at 0.6V/cm for the first 10min and
at 1.2V/cm for further 30-60min. To determine the size of the fragments 5µl of a DNA
marker (Fermentas, St.Leon-Rot, Germany) was run in parallel to the samples with a
known amount of DNA for each band of the marker.
For extraction of the DNA fragments from the agarose gels JETsorbGel Extraction Kit
300 was used according to the manufacturer’s protocol (Genomed, Chemie Brunschwig
AG, Basel, Switzerland). Mostly dH2O was used to elute the DNA instead of the TE
buffer (10mM Tris-HCl pH 8.0 and 1mM EDTA) recommended by the supplier. To
purify PCR products GenElutePCR Clean-Up Kit was used (Sigma, Missouri, USA).
Ethanol Precipitation was occasionally used for buffer exchange and cleaning up of DNA.
Therefore, 2.5vol. of 100% ethanol and 0.1vol 3M sodium acetate (pH 5.2) was added to
the DNA and incubated at -80°C for 30-60min. After centrifugation of 14000rpm at 4°C
for 15min, the probe was washed with 70% ethanol and centrifuged for another 5min.
The probe was dried in the speed vac and solved in dH2O.
20
Material and Methods
2.2.3 Ligation
For DNA-ligation a mixture containing 50ng of a linearized vector, 300ng of an insert
fragment, 2µl 10xT4 ligase buffer, 1µl T4 DNA ligase (0.5u/µl) and dH2O up to a total
volume of 20µl, was incubated either at 37°C for 1-2h or on 4°C overnight.
2.2.4 Plasmid Transformation in Escherichia coli
To produce competent cells used for transformations, one clone of the E.coli strain DH5α
was picked and incubated overnight in a glass tube filled with 5ml liquid LB at 37°C and
agitated at 200rpm. The next morning 1000ml of LB were inoculated with the whole preculture and left at 37°C till the culture reached an OD600 between 0.4 and 0.5. Then the
culture was left on ice for 20min. After transferring the cells into 150ml-centrifugation
vials they were centrifuged at 6000rpm at 4°C for 10min. The supernatant was removed
and each pellet was resuspended in 15ml of pre-chilled 100mM CaCl2 and left on ice for
30-45min. Then the cells were centrifuged at 6000rpm at 4°C for 10min, the supernatant
was discarded, the pellet resuspended in 3.5ml of a pre-chilled mixture of 100mMCaCl2
and 13%(v/v) Glycerin (86%) and finally the mixture was transferred into several
Eppendorf tubes. Aliquots of 100µl were shock-frozen in liquid nitrogen and stored at 80°C. This competent cells were used for general transformation of plasmids into E.coli.
For transformation of plasmids into E.coli competent cells were thawed on ice for 30min.
For each transformation 2-5µl of ligation mix or 50-100ng plasmid DNA was added to an
aliquot of 100µl cell suspension and placed on ice for 30 minutes. After a heat-shock at
42°C for 1 minute, the probe was put on ice for 2 minutes, supplemented with 900µl of
LB-medium and incubated at 37°C for 1 hour. Finally, 200µl of a 1:10 and 1:1 dilution of
the probe were plated on LB agar plates, supplemented with 50µg /ml ampicillin and
incubated overnight at 37°C. After using PCR-ScriptTM Amp Cloning Kit (Stratagene, La
Jolla, USA) or QuikChangeSite-Directed Mutagenesis Kit (Stratagene, La Jolla, USA)
the transformation was carried out according to the supplier's protocol using the
competent cells delivered with the kit.
2.2.5 Isolation of Plamids from E.coli
To control plasmids after cloning, plasmids were isolated by the Boiling Miniprep
method. A bacteria culture of each clone containing a plasmid of interest was inoculated
by picking a colony with a toothpick and transferring it into 3ml LB media. The
inoculates were then incubated at 37°C overnight. Of this culture 1.5ml was transferred
into an Eppendorf tube and centrifuged at 13000rpm for 1min. The supernatant was
removed and again 1.5ml of the remaining culture was transferred to the same tube and
centrifuged. To lyse the cells, the pellet was resuspended in 500µl STET solution (10mM
21
Material and Methods
Tris-HCl (pH 8.0), 50mM EDTA (pH 8.0), 8%(w/v) sucrose and 0.5%(w/v) Triton X100)) and 10µl lysozyme mix (10ng/µl lysozyme, 50µl /ml RNAse [2ng/ml]). The
suspension was incubated at 100°C for 1min and centrifuged at 14000rpm at room
temperature for 15min. The mucous pellet was removed with a sterile toothpick. The
supernatant was supplemented with 500µl of isopropanol, mixed by inverting and
incubated at room temperature for 5min. After centrifugation at 14000rpm for 15-60min,
the supernatant was removed, the pellet washed with 600µl 70% Ethanol and centrifuged
for 5min. Finally, the pellet was dried in the speed vac and dissolved in 50µl dH2O before
incubating the tube at 50°C for 5min and resuspending the probe with a pipette. The
DNA was stored at –20°C.
To isolate a high amount of plasmid-DNA cultures of the strains of interest were grown
in 50ml LB medium at 37°C overnight. Then, the JETstar Plasmid MIDI Kit 50 2.0
(Genomed, Chemie Brunschwig AG, Basel, Switzerland) was applied according to the
supplier’s protocol. At the end DNA was dissolved in 200-500µl volume of dH2O and
stored at –20°C.
2.2.6 DNA-Sequencing and Alignments
Sequencing was performed by Synergene Biotech GmbH (Synergene, Schlieren,
Switzerland). For all sequencing reactions the primer Mano-lo was used. The sequence
files were opened with the Chromas software (Version 1.45, Conor Mc Carthy, Southport,
Australia), exctracted as text file and aligned to the theoretical sequence using DNAMAN
Version 5.1. Because the Mano-lo primer is on the antisense strand, reverse complement
sequence had to be generated for alignments with the genomic Gpxh sequence.
2.2.7 DNA Amplification by Polymerase Chain Reaction (PCR)
A list of all primers used for PCR can be found in Table 2.2. They were designed to have
an annealing temperature (Tm) of 60-65°C calculated with the formula: Tm=
(G+C)*4+(T+A)*2. Primers for Site-directed Mutagenesis were designed using with the
program PrimerX (http://bioinformatics.org/primerex). The primers were synthesized by
Microsynth and delivered in a lyophilized state (Microsynth GmbH, Balgach,
Switzerland). They were then dissolved in the recommended volume of dH2O in order to
get a concentration of 100µM. For better dissolve the DNA, the tube was incubated at
60°C for 10min and mixed by pipetting. All primers were stored at -20°C.
22
Material and Methods
Table 2.2 List of primers used in this study for PCR, sequencing, reverse transcription or real time RT-PCR
primer
Mano-lo
Ars461rev
GpxhTATAssp
GpxhCREfor
TubdelTfor,
TubdelTrev
CGCC140TTAT+for
CGCC140TTAT+rev
CAAT155TGCGfor
CAAT155TGCGrev
ATTG185GCGTfor
ATTG185GCGTrev
18SRT1
18SQPCR1for
18SQPCR1rev
Rbcsfor
Rbcsrev
Gpxhfor
Gpxhrev
GST2for
GST2rev
BF864288for
BF864288rev
sequence
5’-ATCTTGATGGTTTCGTCCTGAGC-3’
5’-GGTGACGAAGTACTGAGACAGC-3’
5’-TCAATATTATATAAACCCTTTCACTCACATG-3’
5’-ACGTTGACGCCAGTTAGAGAAG-3’
5’-CTGCATGGGCGCTCCG.TGCCGCTCCAGG-3’
5’-CCTGGAGCGGCA.CGGAGCGCCCATGCAG-3’
5’-CGCGGGATGATGACACTTATCGCAGTTGAGG
CAATTCCCTGC-3’
5’-GCAGGGAATTGCCTCAACTGCGATAAGTGTC
ATCATCCCGCG-3’
5’-ATGACACCGCCCGCAGTTGAGGTGCGTCCC
TGCAGATGTTGA-3’
5’-TCAACATCTGCAGGGACGCACCTCAACTGCG
GGCGGTGTCAT-3’
5’-CAGATGTTGACGCGCTGGCTGCGTAGGAGTC
TCTGTTATATAAAC-3’
5’-GTTTATATAACAGAGACTCCTACGCAGCCAG
CGCGTCAACATCTG-3’
5’-GAGCTGGAATTACCGCGGCT-3’
5’-GATGGCTACCACATCCAAGGAA-3’
5’-AAGCGCCCGGTATTGTTATTTATT-3’
5’-GCAGCGTGTCTTGCCTGTACT-3’
5’-GCGGCAGCCGAAGATG-3’
5’-ACGTGTTTGACACGGTTATGAGA-3’
5’-GCAATTGGCATGATGGATAGTG-3’
5’-TACCCATCGTAATCTCATCGTACATTT-3’
5’-TGCGTAAGCGTTACGTCTATGAA-3’
5’-GGGTCACGCATTACGGTCTAA-3’
5’-TGGCTGGGTCCCACAGTTA-3’
For PCR the following ingredients were mixed in a PCR tube: 5µl of DNA template
[10ng/µl] , 5µl 10xReaction Buffer, 1µl of 25mM dNTP, 34µl dH2O, 2µl of each primers
(10µM) and 0.5-1µl of BioTherm DNA polymerase or 0.5µl-2µl of a proofreading Pfupolymerase (Pfu DNA Polymerase (Promega, Wallisellen, Switzerland), AccuTherm
(GeneCraft, Germany) PfuTurbo Polymerase (Stratagene, La Jolla, USA)) when high
accuracy was required. After a short spin down tubes were put in a PCR thermocycler,
where the PCR takes place. The PCR reaction started with a denaturation step at 95°C for
5min, followed by 30-34 cycles consisting of a denaturation step at 95°C for 30sec, an
annealing step at 52-61°C for 30sec and an extension step at 72°C for 60-90sec. The PCR
reaction was finished by a final elongation step at 72°C for 5min to polymerize
incomplete products. The annealing temperature varied depending on the optimal
temperature for primer binding and product specificity. The duration of the extension step
depended on the length of the PCR product and the polymerase, since Pfu polymerase
23
Material and Methods
needs a longer. A detailed description of all PCRs carried out can be found in the
Appendix (Tab5.2).
The QuikChange®Site-Directed Mutagenesis Kit (Stratagene, La Jolla, USA) was used
to exchange specific nucleotiedes in a plasmid by site-directed mutagenesis. It was used
to delete a single amino acid in the β-tubulin promoter sequence of pYS1mod, pYS2mod,
pYS3mod, pBF28mod, pBF29mod and pBF30mod with the unmodified plasmids as
templates. Furthermore, it was also applied to introduce several point mutations in the
putative regulatory elements of the Gpxh promoter of pASpro2 resulting in plasmids
pGC1, pCAAT2 and pATTG3. The site-directed Mutagenesis Kit involves a
PfuTurbo®DNA polymerase and two primers each complementary to opposite strands of
the vector at the position where the mutation should be introduced and containing the
desired mutation (Tab2.2). While performing the PCR, the PfuTurbo®DNA polymerase
replicates both plasmid strands with high fidelity in every cycle resulting in a plamid with
two nicked strands. After the PCR amplification, the probe is treated with the DpnI
restriction enzyme which only digested the methylated parental DNA template. After this
the mutated plasmids are transformed into XL1-Blue supercompetent cells, where the
nicked strands are repaired. Application of the Kit was carried out according to the
supplier's protocol. For pGC1, pCAAT2 and pATTG3 the cycling parameters were
modified because the paternal template pASpro2 (10485bp) exceeded the recommended
size. Therefore, a primary denaturation step at 95°C for 30sec was followed by 9 cycles
of a denaturation step at 95°C for 30sec, an annealing step at 55°C for 60sec and an
extension step at 68°C for 13min. The reaction was then stopped and again 1µl of
polymerase added before repeating the same PCR procedure for a second time. A detailed
description all site-directed mutagenesis carried out can be found in the Appendix
(Tab5.2).
2.3 Plasmid Cloning
Table 2.3 Overview of the plasmids used and constructed in this study
Name
pASpro1β
Original
Plasmid
pJD54
pASpro2
pJD54
pBF28
pUC28
Description
Size
(bp)
contains a 1304 bp fragment of 11401
the Gpxh promoter fused to Ars
reporter gene construct
contains a 562bp fragment of the 10485
Gpxh promoter fused to Ars
reporter gene construct
3992
contains CRE with flanking
nucleotides fused to a β-tubulin
promoter sequence and an Ars
reporter gene fragment
24
Origin
Leisinger
Leisinger
Fischer
Material and Methods
pBF29
pUC28
pBF30
pUC28
pBF28-30mod
pBF28
pBF31
pBF28
pBF32
pBF29
pBF33
pBF30
pBF31-33mod
pBF2830mod
pYS1
pBF28
pYS2
pBF29
pYS3
pBF30
pYS1-3mod
pYS1-3
pYS4
pPCRScript
Amp
SK(+)
pPCRScript
Amp
SK(+)
pASpro1β
pYS5
pCRETA1mod
contains CRE fused to a βtubulin promoter sequence and
an Ars reporter gene fragment
contains aβ-tubulin promoter
sequence fused to an Ars
reporter gene fragment
pBF28-30 with one deleted
nucleotide in the β-tubulin
promoter region
contains CRE with flanking
nucleotides fused to a β-tubulin
promoter sequence and an Ars
reporter gene construct
contains CRE fused to a βtubulin promoter sequence and
an Ars reporter gene construct
contains a β-tubulin promoter
sequence fused to an Ars
reporter gene construct
pBF31-33 with one deleted
nucleotide in the β-tubulin
promoter region
contains a 199bp long Gpxh
promoter fragment with a
deletion between CRE (flanked)
and the TATA box
contains a 199bp long Gpxh
promoter fragment with a
deletion between CRE and the
TATA box
the same as pYS2 but without
the CRE element
pYS1-3 with one deleted
nucleotide in the β-tubulin
promoter region
667bp PCR product from
pASpro1 using primers
GpxhCREfor and Ars461rev in
pPCR Script Amp vector
555 PCR product from pASpro1
with primersGpxhTATAssp and
ARS461rev in pPCR Script Amp
contains a 199bp long Gpxh
promoter fragment with a
25
3985
Fischer
3977
Fischer
3991
3984
3976
10221
this work
10214
Fischer
10206
Fischer
10222
10215
10207
3396
this work
3389
this work
3381
this work
3395
3388
3380
3629
this work
2782
this work
10222
this work
Fischer
this work
this work
Material and Methods
pCRETA2mod
pASpro1β
pTA3mod
pASpro1β
pCRETATO
pASpro1β
pGC1
pASpro2
pCAAT2
pASpro2
pATTG3
pASpro2
parg7.8
unknown
pPCR-Script
Amp SK(+)
pPCRScript
Amp
SK(+)
deletion between the
CRE(flanked) and the TATA box
fused to the Ars reporter gene
(one nucleotide was removed)
contains a 199bp long Gpxh
promoter fragment with a
deletion between the CRE and
the TATA box fused to the Ars
reporter gene (one nucleotide
was removed)
like pCRETA2mod but without
the CRE element
contains a 199bp fragment of the
Gpxh promoter fused to Ars
reporter gene construct
contains a 562bp fragment of the
Gpxh promoter fused to Ars
reporter gene construct, mutated
in the putative GC-box
contains a 562bp fragment of the
Gpxh promoter fused to Ars
reporter gene construct mutated
in the putative CAAT-box
contains a 562bp fragment of the
Gpxh promoter fused to Ars
reporter gene construct mutated
in the putative CAAT-box on the
antisense strand
plasmid carries gene necessary
for arginine synthesis
cloning vector for blunt PCR
products, blue- white colour
selection by adding IPTG/X-gal
10215
this work
10207
this work
10234
this work
10485
this work
10485
this work
10485
this work
10485
this work
3000
Stratagene
All primers and plasmids used for constructing the plasmids are summarized in table 2.2.
The molecular methods used for cloning the plasmids were described previously. For the
construction of plasmids pCRETA1mod, pCRETA2mod and pTA3mod, a 555bp
fragment of the Gpxh promoter region from pASpro1 was amplified by PCR using the
primers GpxhTATAsspI and Ars461rev (Tab2.2). The product was first subcloned into
the pPCR-Skript™AmpSK(+) vector (Stratagene, La Jolla, USA) resulting in plasmid
pYS5 (Fig 2.1a). Subsequently, pYS5 was digested with the restriction enzymes SspI and
SacI and the smaller fragment was ligated into the Eco 47III and SacI digested pBF28,
pBF29 or pBF30, resulting in pYS1, pYS2 or pYS3, respectively. The site-directed
mutagenesis Kit (Stratagene, La Jolla, USA) was applied according to the manufactureres
26
Material and Methods
protocoll to delete one thymidine within the introduced β-tubulin promoter of the
plasmids pYS1, pYS2 or pYS3, using the primers TubdelTfor and TubdelTrev (Tab2.2).
The resulting plasmids pYS1mod, pYS2mod and pYS3mod were further digested with
SalI and EcoRV and the smaller fragment was ligated into the plasmid pASpro1,
previously digested with the same restriction enzymes, resulting in pCRETA1mod,
pCRETA2mod and pTA3mod. For the construction of pBF28mod, pBF29mod and
pBF30mod in which the same thymidine has been deleted, the site-directed mutagenesis
Kit was applied in the same manner as for pYS1-3 but with pBF28; pBF29 and pBF30 as
templates. The resulting plasmids pBF28mod, pBF29mod and pBF30mod were further
digested with SalI and ClaI and the resulting smaller fragment was ligated in a SalI and
ClaI digested pASpro1, resulting in pBF31mod, pBF32mod and pBF33mod (Fig 2.1a).
For the construction of pCRETATO a 667bp fragment from the Gpxh promoter region in
pASpro1 was amplified by PCR using the primers GpxhCREfor and Ars461rev (Table
2.2). The PCR-product was first subcloned into the pPCR-Skript™AmpSK(+) vector
(Stratagene, La Jolla, USA) resulting in pYS4 (Fig 2.1b). The plasmid pASpro1 was
digested with SalI and EcoRV. The sticky end of the SalI restriction site of the 11kb
fragment was then filled up with the Klenow polymerase resulting in a fragment with two
blunt ends, purified by ethanol precipitaion and dephosphorylated, in order to prevent a
backligation of the vector. Subsequently, the smaller fragment of an EcoRV digested
pYS4 was cloned into the blunt ends of this pASpro1 vector resulting in plasmid
pCRETATO (Fig 2.1b). Normally, the constructs are controlled after a cloning step by
digestion with suitable restriction enzymes and checked on size and orientation. However,
blunt-end ligated heterogeneous sites as created in pCRETATO can not be tested by
restriction analysis and other suitable sites in the vector pASpro1 were not available. For
this reason a PCR-test with primers CREfor and Ars461rev (Tab2.2) was applied to check
for the clones of interest. If a 667bp-long PCR fragment was produced, it was expected to
be the desired plasmid pCRETATO. This was the case for 3 out of 47 different clones
tested. In the clones, for which no PCR product was found, the vector pASpro1 was
probably backligated and therefore the primer CREfor could not bind to the template.
27
PCR product (555bp):
primer
-GpxhTATAsspI
-Ars461rev
TATA
SalI (1158)
pUC28 sequence
1
Gpxh promotor sequence
pASpro1
11401bp
EcoRV (2559)
SspI (19)
1
sspI
pYS5
arylsulfatase sequence
part of the Gpxh part of the reporter
gene Arylsulfatase
promoter
SalI & EcoRV
SspI (733)
TATA
Gpxh promotor sequence
2782bp
PCR
Reporter gene arylsulfatase
pPCR-Script Amp SK(+) cloning vector
SalI & ClaI
SacI (1321)
SspI & SacI
SalI & ClaI
SalI (138)
EcoRV (360)
CRE+
tubulin promotor sequence
TATA (Gpxh)
SalI (435)
1
1
pCRETA1mod
pYS1mod
3395bp
SalI & EcoRV
10222bp
Ars
Eco47III (568)
tubulin promotor sequence
TATA (tubulin)
CRE+
CRE+
TATA
tub
EcoRV (656)
-1 nucleotide
1
pYS1
3396bp
Eco47III & SacI
pBF28
3992bp
pBF28mod
Arylsulfatase
-1 nucleotide
3991bp
pBF31mod
SalI&ClaI 10222bp
PCR Skript Amp+
Arylsulfatase
SalI (138) EcoRV (360)
CRE TATA (Gpxh)
tubulin promotor sequence
1
1
pCRETA2mod
SalI & EcoRV
10215bp
pYS2mod
3388bp
SalI (435)
CRE tub
TATA
EcoRV (649)
Ars
-1 nucleotide
pPCR Script Amp SK+
Arylsulfatase
SalI (138)
tubulin promotor sequence
1
pYS2
3389bp
Eco47III & SacI
1
1
pYS3mod
3380bp
TATA
tub
EcoRV (641)
Ars
-1 nucleotide
pPCR Script Amp SK+
SacI (1141)
Arylsulfatase
pBF29mod
-1 nucleotide
3984bp
pBF32mod
SalI&ClaI 10215bp
SacI (1744)
SalI (435)
pTA3mod
10207bp
Arylsulfatase
Eco47III (561)
CRE
TATA (tubulin)
tubulin promotor sequence
pBF29
3985bp
SacI (1149)
TATA (Gpxh)
EcoRV (345)
SalI & EcoRV
SacI (1751)
SacI (1156)
1
pYS3
3381bp
Eco47III & SacI
Eco47III (553)
TATA (tubulin)
tubulin promotor sequence
pBF30
3977bp
Arylsulfatase
pBF30mod
-1 nucleotide
3976bp
pBF33mod
10207bp
SacI (1736)
Fig 2.1a Schematic overview of the cloning pathway of pCRETA1mod, pCRETA2mod, pTA3mod, pBF31mod, pBF32mod and pBF33mod. Blue arrows
indicate PCR amplifications, red arrows indicated digestion/ligation steps and green arrows indicate applications of site-directed mutagenesis.
CRE/AP1
homologous
sequence
SalI (1158)
pUC28 sequence
1
Gpxh promotor sequence
pASpro1
11401bp
EcoRV (2559)
PCR product (667bp):
primers: GpxhCREfor &Ars461rev
EcoRV (697)
TATA
1
PCR
pYS4
3629bp
part of the Gpxh
promoter
Reporter gene arylsulfatase
part of the reporter
gene Arylsulfatase
CRE
TATA
Ars
EcoRV (927)
pPCR-Script Amp SK(+) cloning vector
SalI & EcoRV
EcoRV
Gpxh promotor sequence
CRE
TATA
EcoRV (1392)
1
pASpro1
fill in 4 nucleotides
to get a blunt end
(Klenov)
pCRETATO
10234bp
pASpro1
Arylsulfatase
Mutations in:
box1 (5’-CCGCCC-3’)
primers: CGCC140TTAT+for
CGCC140TTAT+rev
box2 (5’-CAAT-3’)
primers: CAAT155TGCGfor
CAAT155TGCGrev
KpnI (1024) Gpxh promotor sequence
CRE box1
box2
1
TATA box3
pASpro2
10485bp
CRE/AP1
homologous
sequence
TATA
box3 (5’-ATTG-3’)
primers: ATTG1855GCGTfor
ATTG185GCGTrev
pGC1
10485bp
pCAAT2
10485bp
pATTG3
10485bp
Site- directed
Arylsulfatasemutagenesis
part of the Gpxh
promotor
part of the reporter
gene Arylsulfatase
Fig 2.1b,c Schematic overview of the cloning pathway of pCRETATO(b) and pGC1, pCAAT2 and pATTG3 (c). Blue arrows indicate PCR amplifications, red
arrows indicated digestion/ligation steps, green arrows indicate application of site-directed mutagenesis and purple arrows indicate applications of Klenow method..
Material and Methods
Mutations in a GC-box (5’-CCGCCC-3’)), a CAAT-box (5’-CAAT-3’) and a CAAT-box
on the antisense strand (5’-ATTG-3’) in the Gpxh promoter region of pASpro2 were
introduced using Site-directed mutagenesis Kit (Stratagene, La Jolla, USA). The plasmid
pASpro2 was used as DNA template and the primers CGCC140TTAT+for and
CGCC140TTAT+rev for mutating the GC-box resulting in pGC1, the primers
CAAT155TGCGfor and CAAT155TGCGrev for mutating the CAAT-box resulting in
pCAAT2 and primers ATTG185GCGTfor and ATTG185GCGTrev for mutating the
CAAT-box on the antisense strand resulting in pATTG3 (Fig 2.1c).
2.4 Transformation of Plasmids into C. reinhardtii
A freshly inoculated culture of C.reinhardtii cw15arg7 was grown to a cell density of 12*106 cells per ml or a corresponding OD750 of 0.07-0.1. For each transformation 25-40ml
of algal culture were centrifuged at room temperature and 1500rpm for 10min. Almost all
the supernatant was removed leaving ~500µl in the tube. Then 100µl of 20%(w/v)
Polyethylenglycol M3350 was added and the pellet was resuspended with a pipette before
300µl aliquots were transferred into a sterile conical glass tube containing 300mg glass
beads. To each aliquot 2µg of the plasmid parg7.8 and 2µg of the plasmid of interest
were added and the tube was vortexed for 15sec. Finally, the tubes were incubated at
room temperature for 1-2hours before 150µl were plated on TAP plates supplemented
with ampicillin. A detailed description of all transformations carried out can be found in
the Appendix (Tab5.3). Growth of the clones on TAP agar plates without arginine was
only possible by an integration of the parg7.8 plasmid into the genome of the
argininosynthetase mutant strain (cw15arg7), because parg7.8 carries the gene responsible
for arginine synthesis. Since C.reinhardtii has a high frequency of co-transformation of
two plasmids, about 5% of the arginine synthesizing clones are expected to also carry the
second plasmid transformed. For screening of clones which additionally integrated the
second plasmid, the plates were sprayed with a sterile-filtered X-SO4 solution, containing
27mg 5-Bromo-4-chloro-3-indoxylsulfate potassium salt (Sigma, Saint Louis, USA)
dissolved in dH2O. The plates were left for 1-2 days at 25°C in the light and then the
colonies were screened for blue clones. The colour reaction is due to a conversion of the
substrate X-SO4 by the arylsulfatase gene on the plasmids of interest in a blue substrate.
2.5 Stress Treatment with Neutral Red and Measuring Reporter
Gene Expression
2.5.1 Exposure to Oxidative Stress Caused by NR
In this study the exogenous photosensitizer Neutral Red (NR) was added to the algae to
induce the reporter gene expression, because NR causes an oxidative stress by
30
Material and Methods
transferring excitation energy to molecular oxygen, resulting in the formation of singlet
oxygen, known to upregulate the Gpxh promoter-reporter gene constructs. For NR
treatment two 5ml aliquots of each algal culture with an OD750 of 0.25±0.015 were
transferred in a 6-well culture plate. One of the aliquots served as control while the other
aliquot was supplemented with 1µM NR. The plates were incubated at 25°C under
constant illumination with white light of 150µmolm-2s-1 and agitated on a rotary shaker at
150rpm for 4 hours. Two samples of 300µl were taken after 2, 3 and 4hours for the
arylsulfatase assay, used to examine the gene expression of the different promoterarylsulfatase constructs exposed to NR. Each clone was tested in 3 independent
experiments at 3 different days, but not later then one month after its transformation to
exclude effects caused by silencing. For each construct 4-19 clones were tested, except
for pASpro1 where only the standard-clone pASpro1ß was used to control the
experimental conditions of each experiment.
2.5.2 Arylsulfatase Assay and Calculation of Induction
The arylsulfatase assay was used for easy detection of the induction by NR of the
different Gpxh promoter-arylsulfatase constructs. To do so, two samples of 300µl were
transferred into a 96-well plate to measure the accumulation of the arylsufatase in the
medium. The samples were centrifuged at 2500rpm for 3min and 200µl of the
supernatant was transferred into a new slot and mixed with 20µl of 10X substrate solution
containing 2M glycine-NaOH (pH 9.0), 200mM imidazole and 90mM pNitrophenylsulfate (Fluka) (10xGin-solution). For the background substration (blind
value) 20µl of the Gin-solution was added to 200µl of TAP medium. The plate was
incubated at 30°C for 20min and absorbance at 410nm was measured. The induction was
calculated by dividing the background-corrected absorption factor at 410nm of the
exposed sample through the background-corrected absorption factor of the control (Fig
2.2).
31
Material and Methods
Toxic agent
Cell suspension
Colour reaction
blind
ARS
Control
Sampel
Control
Sampel
Induction =
Sample OD
410
– blind OD 410
Control OD410 – blind OD 410
Figure 2.2 Schematic drawing of the principle of the arylsulfatase assay to measure the induction of a
reporter gene construct. The algae is exposed to NR (triangle) which causes an activation of the
arylsulfatase reporter gene. The arylsulfatase protein produced can be quantified by addition of a substrate
(Gin solution) which causes a colour reaction. This reaction can be quantified measuring the absorption at
410nm, and induction calculated with the formula stated.
2.6 Stress Treatment by UV Radiation
2.6.1 Optimizing the Exposure Condition by Varying the Intensity of
UVA, UVB and PAR Light with Different Lamps, Filters and the
Distance Between Lamp and Culture
The UV-exposure of the cultures was performed in two different experimental setups
(Fig2.3) in order to get a high UVA (315nm-400nm) and a high UVB (280-315 nm)
exposure condition. The UV light was provided by using 1 UV lamp (Osram HTC400241) in both setups but could be varied by the distance to the sample and different filters.
Photosynthetically Active Radiation (PAR 400-700 nm) which is required for normal
growth of the algae was provided by PAR lamps with Philips growth bulbs (HPL
Comfort 400W). In the UVA setup the PAR intensity was already high without PAR
lamps because the UV lamp also emits in the PAR region. In order to get the same
amount of PAR in both setups we used one PAR lamp in the UVA and 2 PAR lamps in
the UVB setup.
32
Material and Methods
Table 2.4 The different filters used achieve different intensities of UVA, UVB and PAR conditions. The
relative reduction in UVA, UVB and PAR light intensity by the different filters are stated.
name
type
FE
F
G
M
Q
Plexiglas
Overhead foil
Glass
Lubriflon
Quartz glass
reduction
UVA
-66%
-16%
-10%
-16%
-6%
in reduction in
UVB
-94%
-91%
-47%
-36%
-6%
reduction in
PAR
-7%
-9%
-8%
-13%
-8%
Besides the distance from the UV-lamps to the sample different filters were used to vary
the UV radiation. The five different filters transmitted different levels of UVA, UVB and
PAR radiation (Tab 2.4). The reduction of UV-radiation was highest with the FE-filter, a
UV-filtering Plexiglas (type XT 20070, Röhm, Switzerland), and lowest with the Q-filter,
a quartz glass (Wisag, Zurich, Switzerland). The F-filter was a normal overhead foil used
in presentations, the G-filter was a glas and the M-filter was a white LubriflonPTFEfoil (freely provided by Angst+Pfister, Zurich, Switzerland).
(a)
PAR lamp
UV lamp
Filter
UVA setup
UVB setup
(b)
Figure 2.3 Schematic drawing (a) and picture (b) of the setups for UVA and UVB exposure. Light intensity
was varied with the number of UV and PAR lamps, the distance between lamp and sample and by different
filters.
33
Material and Methods
The light intensities we measured with a PMA2100 Photometer/Radiometer (Solar Light
Co., Oak Line, USA) equipped with a UVA and UVB sensor (UVA-2110WP, UVB2106WP). A Photosynthetic Active Radiation (PAR) quantum sensor connected to a LI1000 Data-logger (Li-Cor, USA) was used to measure PAR light.
2.6.2 Exposure of Algal Cultures to High UV Radiation
For UV-exposure experiments a culture of the C.reinhardtii strain cw15arg7mt- ,which
was cultured in a HT Multitron incubator (Infors, Bottimingen, Switzerland) with
continuous illumination of 120 µmolm-2s-1 (Philips Coolwhite TLD 15W fluorescent
lamps) at 25ºC and at 150 rpm, was adjusted to and optical density at 750nm (OD750) of
0.1. The culture was divided in two 200ml cultures and preadapted for 14 hours in either
the UVA or UVB control condition at 25ºC and steered at 100rpm with a magnetic stirrer
(Fig 2.4 a,d). After preadaptation, the OD750 of the two precultures were adjusted to 0.25
and the cultures were divided into several 30ml cultures in 60ml glass beakers (Huber,
Switzerland)(Fig 2.4 b,e). Now, the cultures were exposed to individual light conditions
as listed in figure 3.5b by using different combinations of filters. Thus, for experiments to
determine gene expression and physiological parameters in the different conditions in the
UVA or UVB setup four samples were exposed to different intensities of UVA reaching
from 1.04-4.59mW cm-2 UVA by using the following filters: G/2FE/3M for the control
UVA1, G/2F/3M for UVA2, G/F/3M for UVA3 and G/2F/2M for UVA4 containing the
highest UVA intensity. To get the different light intensities in the UVB setup, reaching
from 0.00-0.17 mW cm-2 UVB, the control UVB1 was covered with G/2FE/2M, UVB2
with G/4M, UVB3 with Q/5M and UVB4 with the highest intensity of UVB radiation
with 4M as filters. The cultures of the different conditions were exposed to the different
UV intensities and a PAR illumination of ∼270 µmolm-2s-1 in both setups. All samples
were kept at 25ºC and stirred with a magnetic stirrer (Fig 2.4 c,f).
(a)
(b)
(c)
(d)
(e)
(f)
Fig 2.4 The pictures show the preadaptation of C.reinhardtii cultures in big containers (a,d), the divided
30ml cultures in the small beakers (b,e) and the cultures during the exposure covered by different filters (c,f)
in the UVA and UVB setup, respectively.
34
Material and Methods
2.7 Measuring Physiological Parameters of Algae Exposed to
High UV Radiation
2.7.1 Growth
Growth was determined as an indicator for the fitness and survival of the algae when
exposed to UV. The growth of exposed and control cultures was monitored by measuring
the optical density at 750nm at several time points during the exposure.
2.7.2 Photosynthetic Parameters
In photosynthetic organisms like the green alga C. reinhardtii, an overexitation of the
reaction centre in the Photosystem II (PSII) of the photosynthetic apparatus results in the
emission of excess energy as fluorescent light. The fluorescence can be detected using a
ToxY-PAM fluorometer (Prototyp manufactured by Gademann Instruments, Würzburg,
Germany; series production by Heinz Walz, Effeltrich, Germany),.and be used as an
indicator for the physiological state of the cell. The effective quantum yield of energy
conversion at the PSII reaction centre, Y, was calculated with the following equation: Y=
((FM’-F)/FM’), where F is the baseline fluorescence measured by actinic light and FM’ the
maximum fluorescence induced by a 5µs saturation pulse at 470nm [53]. A decrease in
the fluorescence yield indicates that cells of a culture are stressed.
The oxygen production was measured as a direct indicator of effects on the
photosynthetic activity. It was measured with a Clark-type oxygen electrode (Hansatech,
Norfolk, UK) using 3ml of a C.reinhardtii culture at OD750 of 0.25 at 25°C and 200µmol
m-2 s-1 PAR light. Cells were dark adapted for 10min and constant oxygen production
was measured for 5-10min. In parallel, the cell number was determined with a Z2
Beckman Counter (Beckman Coulter GmbH, Krefeld, Germany) to calculate the oxygen
production of each cell.
The data was normalized by the cell number counting the cells with Z2 Beckman Counter
(Beckman Coulter GmbH, Krefeld, Germany) of molecules between 3.5-9.483µM size at
the corresponding time points were performed.
2.7.3 Pigment Content
The pigment content of Chlorophyll a, Chlorophyll b and Carotenoid, as the most
important light absorbing pigments in photosynthetic organisms, were investigated in
cultures exposed to UV by pipetting 200µl algal culture of each condition into an
eppendorf tube and adding 800µl Aceton. The mixture was kept on ice in the dark for
35
Material and Methods
10min and then centrifuged at maximal speed at 4°C for 5min. After this, the absorption
at 470, 646 and 663nm was analyzed. The absorption measured was corrected by the
absorption of a background consisting of 200µl dH2O and 800µl Aceton. For calculations
of Chlorophyll a, Chlorophyll b and Carotenoid content, the following equations were
used.
Chlorophyll a (µg/ml)= 12.21(A663)-2.81(A646)
Chlorophyll b (µg/ml)= 20.13 (A646)- 5.03 (A663)
Carotenoids (µg/ml)= (1000A470-3.27 [Chla]-104 [Chlb])/227
The data was normalized by the cell number counting the cells with Z2 Beckman Counter
(Beckman Coulter GmbH, Krefeld, Germany) of molecules between 3.5-9.483µM size at
the corresponding time points were performed.
2.8 Measuring the Genetic Response in Algae Exposed to UV
Radiation
2.8.1 Isolation and Quantification of mRNA
Gene expression was measured after 1h and 3h of exposure to UV light in conditions
UVA1, UVA4, UVB1, UVB2 and UVB3 by quantifying the mRNA levels of genes of
interest. For MRNA isolation, cells of 10ml of individual culture were harvested by
centrifugation at 4000rpm at 4°C for 4min. The pellet was resuspended in 1ml Trizol
(Gibco BRL, Life Technologies Ltd.) by repetitive pipetting and then transferred to an
eppendorf tube. After incubation at room temperature for 5min , 200 µl chloroform was
added, the samples were shaken by hand for 15sec and incubated at room temperature for
2-3min. Then samples were centrifuged at max. speed at 4°C for 15min. The upper phase
was transferred to a fresh tube using a pipette and mixed with 500µl isopropanol. The
tubes were kept at room temperature for 10min before they were centrifuged at max.
speed at 2-8°C for 10min. The supernatant was removed and 1ml 75%, ethanol (stored at
-20°C) was added to the pellet. Afterwards, the suspension was vortexed and centrifuged
at 8000rpm at 2-8°C for 5min. Again, the supernatant was decanted and the pellet dried at
room temperature for 5min. The pellet was dissolved in 50µl dH2O by pipetting a few
times and incubating at 55-60°C for 10min.
Total mRNA was quantified spectrophotometrically by measuring the optical density of
the probes at 260nm in quartz cuvette. The mRNA concentrations of the samples varied
in a range of 0.3-2.0µg/µl but were adjusted to the same concentration for easier handling.
36
Material and Methods
2.8.2 Quantification of the Gene Expression by real time RT-PCR
Reverse transcription of mRNA into cDNA was carried out with a TaqMan Reverse
Transcription Reagents (Applied Biosystems, Rotkreuz, Switzerland) with an oligo d(T)16
primer that binds to the poly-A tail of all nucleonic mRNAs and with the primer 18SRT1
in order to produce cDNA of the ribosomal 18S mRNA. The synthesis of cDNA was
carried out in a total volume of 10µl in 0.5ml PCR tubes. The reaction mixture contained
200ng mRNA, 1µl of 10x TaqMan RT buffer, 5.5mM MgCl2, 2.5mM dNTP each, 2.5µM
oligo d(T)16, 2,5µM 18SRT1 primer, 0.4 U/µl RNase inhibitor and 1.25U/µl
MultiScribe Reverse Transcriptase each. After mixing the samples were held at 25°C
for 10min and then the reverse transcription was performed in a PCR cycler at 48°C for
30min followed by an inactivation step at 95°C for 5min.
The Quantification of mRNA levels of the Gpxh, a Glutathione transferase, a heat shock,
a Rubisco and a18S ribosomal gene was performed by real time PCR with the ABI
Prism7000 or 7500 Sequence Detection System using gene specific primers. Probes were
pipetted into a MicroAmp optical 96-well reaction plate (Applied Biosystems).
Samples had a total volume of 25µl and contained 1µl of cDNA (~20ng), 12.5µl SYBR
Green PCR Master Mix (Applied Biosystems, Rotkreuz, Switzerland), 1.5µl primers of
each primer solution (5mM) and 8.5µl dH2O. The PCR product is detected by increasing
fluorescence caused by binding of SYBR Green to double-stranded DNA. After the
amplification, a dissociation step was performed to control the specificity of the product.
Threshold cycle (Ct) values were determined for all reactions in the logarithmic
amplification phase, and the average Ct value was calculated for each sample out of 2-3
replicates. The Ct value of the ribosomal 18S gene was used for normalization. Average
induction factors for the different conditions were calculated for each gene as an average
with standard error out of the independent experiments.
2.9 Data Analysis
Data analysis of the different constructs in 3.1 was performed using a nested design in
GLM analysis (SPSS 11.0). Differences in the physiological parameters and the genetic
response was tested using repeated-measures ANOVA and one-way ANOVA when
necessary, followed by Fisher LSD post-hoc test when significant differences (p>0.05)
were found. These statistical analyses were computed using Statistica 6.0 (Statsoft, Inc.,
USA). In all graphs the error bars are expressed as standard errors.
37
Results and Discussion
3 Results & Discussion
3.1 The Molecular Mechanisms of the Gpxh Induction by
Singlet Oxygen
The Gpxh gene in C.reinhardtii is known to be specifically induced by singlet oxygen
(1O2). By using different Gpxh promoter-arylsulfatase reporter gene constructs it was
shown that this induction by 1O2 is mediated via transcriptional activation requiring an
8bp CRE/AP1-homologous sequence element (CRE) in the Gpxh promoter region [12].
However, the presence of a CRE-element in a promoter is not sufficient for a full
induction by 1O2, as shown by the response of two reporter gene constructs in which the
CRE-element was fused to the β-tubulin promoter [14] (Fig 1.3). The low induction in
these constructs, pBF31 and pBF32, by singlet oxygen could be due to the wrong position
of the CRE-element relative to the transcription start site, it could be that the missing
consensus sequence of the TATA-box within the Tub2B promoter sequence reduced the
induction or that other regulatory elements within the promoter sequence are also needed
for the full induction of the Gpxh gene.
3.1.1 Effect of the Position of the CRE-Element on the Gpxh
Induction by Singlet Oxygen
In the plasmids pBF31 and pBF32 the distance between the CRE-element and the
transcription start site was found to be one nucleotide longer than in the Gpxh promoter
(Fig 1.4a). This could affect the three dimensional structure and the efficiency of the
interaction between a CRE-binding transcription factor and the TATA-box-binding
transcriptional apparatus. In order to find out whether the low induction of pBF31 and
pBF32 was due to this additional nucleotide, a thymidine was removed in these
constructs resulting in the modified plasmids pBF31mod and pBF32mod (Fig 3.1a). The
difference between pBF31mod and pBF32mod is that in pBF31mod the CRE-element is
flanked by 6 nucleotides of the original Gpxh promoter on both sides The plasmid
pBF33mod containing the same constructs but without the CRE-element, served as a
negative control. The plasmid pCRETATO, consisting of a 199bp Gpxh promoter
fragment from the CRE-element to the transcription-start site, served as a positive control
(Fig 3.1a). Several clones of each construct were tested for their response to 1O2 by
incubating cultures with 1µM of the 1O2-oxygen producing exogenous photosensitizer
Neutral Red (NR) for several hours and testing the arylsulfatase activity. The average
induction of pBF31mod and pBF32mod were 1.5±0.5 and 1.1± 0.5fold respectively,
which were not significantly higher than the response of the negative control pBF33mod
but significantly lower than the positive control pCRETATO which had an average
38
Results and Discussion
induction of 7.1± 2.2fold (Fig 3.1). The plasmids pBF31mod and pBF32mod showed
even a slightly lower induction than the published induction factors of the original
constructs pBF31 (2.4) and pBF32 (2.3) [14].
Interestingly, the variability of the induction factors in the clones with the positive control
construct pCRETATO was very high, ranging from 0.9-29.3. However, this high range of
the 13 tested clones is due to three clones which showed an extremely low (0.9-1.1) and
one clone with an extremely high induction (29.3) compared to the average induction
factor of 7.1±2.2. It is known that the induction of reporter gene constructs is influenced
by the position where the plasmid has been integrated into the genome of the alga. If the
construct is integrated in a low expression position of the genome, the induction might be
lowered compared to a high expression site. This leads to variability in induction factors
of clones containing the same plasmid. In this study we can not exclude such an effect
and therefore we tested different clones with the same construct. However, the extremely
high and low induction factors of the four clones with the pCRETATO plasmid are very
unlikely to be only due to their position in the genome of the algae. However, we do not
have a reasonable explanation for this observation so fare. It can be argued that such
induction factors should be treated as outliers and not be included in the analysis, but
because this would not change the results significantly, we kept these data points in the
analysis. Another similar example is the relatively high average induction of the negative
control pBF33 (2.2 fold) which is due to the induction factor of one out of five clones
tested, which showed a 6 fold induction. Still, even though we did include this data point
in the analysis, the induction levels of pBF31mod, pBF32mod and pBF33mod were
significantly lower than in the positive control pCRETATO.
In this study we could therefore reduce the chance that the position of the CRE element
caused the low induction in pBF31 and pBF32, because a deletion of one nucleotide in
these constructs did not increase the induction by 1O2 (Fig 3.1). Thus, the additional
nucleotide in pBF31 and pBF32 between the CRE element and the TATA-box increasing
the distance between the two elements compared to the corresponding sequence in the
Gpxh promoter (Fig 1.4a) was not affecting the induction. Still, relative to the
transcription start site, pBF31mod and pBF32mod have a longer distance than Gpxh
because the location of the TATA-box of the Tub2B promoter is not fully defined (Fig
1.4a). However, it is rather unlikely that this difference has such a strong effect and is the
only reason for the low response of pBF31 and pBF32.
39
Results and Discussion
(a)
fold induction ±SE
10
CRE
8
TATA
1
pBF31m od
4
2
pBF32m od
2
3
pBF33m od
0
4
pCRETATO
6
1
2
3
4
(b)
fold induction
range
pBF31mod
PBF32mod
PBF33mod
pCRETATO
1.5
1.1
2.2
7.1
0.7-2.3
1.0-1.1
1.0-6.0
0.9-29.3
4
3
5
13
<0.000
<0.000
<0.000
-
n (clones)
P value
Figure 3.1 (a) Induction and schematic drawing of three reporter gene constructs, pBF31mod, pBF32mod
and pBF33mod and the positive control pCRETATO in cultures exposed to 1µM Neutral Red. Fragments
of the constructs which are identical to the sequence in the Gpxh promoter are coloured in green, while
fragments of the β-tubulin promoter are in red. The deletion of a thymidine is indicated by a black line. (b)
Table of the detailed responses to Neutral Red of the four different constructs shown in figure 3.1a. The Pvalues are derived from the pairwise comparison to the positive pCRETATO control using a GLM analysis.
3.1.2 Effect of the Missing TATA Consensus Sequence in the βTubulin Promoter of pBF31mod and pBF32mod on Induction by
Singlet Oxygen
In eukaryotic cells, gene expression is induced by the binding of a transcriptional
apparatus to the TATA-box with a consensus sequence 5’-TATAAA-3’ in most
promoters including the promoter of Gpxh. The TATA-box of the β-tubulin promoter, on
the other hand, is not a consensus TATA-box and thus its sequence not identical to the
one of the Gpxh promoter (Fig 1.4a). To investigate whether this missing consensus
sequence of the Tub2B TATA-box in the plasmids pBF31 and pBF32 caused the low
induction of these constructs by 1O2, a part of the Tub2B promoter starting from the
TATA-box till the transcription-start site was replaced in pBF31mod and pBF32mod by a
corresponding fragment of the Gpxh promoter (Fig 3.2a). Thus, the resulting constructs
pCRETA1mod and pCRETA2mod consist again of a Gpxh promoter with a CRE element,
either with or without flanking sequences, but they have a long deletion between the CRE
element and the TATA-box (Fig 3.2a). The plasmid pTA3mod with the same promoter
40
Results and Discussion
but no CRE-element served as a negative control and the plasmid pCRETATO with a
Gpxh promoter fragment of the same size was included as a positive control. Upon
exposure to Neutral Red almost no response could be found in cultures containing
pCRETA1mod, pCRETA2mod or pTA3mod with average fold inductions of 1.3±0.2,
1.4±0.2 or 1.3±0.1, respectively (Fig 3.2). Additionally, these responses were not
significantly different from pBF31mod, pBF32mod and pBF33mod, but significantly
reduced compared to the positive control pCRETATO which had an average induction of
7.1±2.2fold (Fig 3.2).
(a)
fold induction ±SE
10
CRE
8
TATA
1
pCRETA1mod
2
pCRETA2mod
2
3
pTA3mod
0
4
pCRETATO
6
4
1
2
3
4
(b)
fold induction
range
n (clones)
P value
pCRETA1mod
pCRETA2mod
pTA3mod
pCRETATO
1.3
1.4
1.3
7.1
0.8-2.4
0.9-2.6
0.8-1.7
0.9-29.3
8
7
6
13
<0.000
<0.000
<0.000
-
Figure 3.2 (a) Induction and schematic drawing of the three reporter gene constructs, pCRETA1mod,
pCRETA2mod and pTA3mod and the positive control pCRETATO in cultures exposed to 1µM Neutral
Red. Fragments of the constructs which are identical to the sequence of the Gpxh promoter are coloured in
green, while fragments of the β-tubulin promoter are in red. The deletion of a thymidine is indicated by a
black line. (b) Table with detailed responses to Neutral Red of the four different constructs shown in figure
3.2a. The P-values are from the pairwise comparison to the positive control pCRETATO using a GLM
analysis.
This proves that the low responses of pBF31 and pBF32 or pBF31mod and pBF32mod to
1O2, are not due to the missing consensus sequence of the Tub2B TATA-box within
these constructs. Even by replacing the β-tubulin TATA-box with the original TATA-box
of Gpxh in pCRETA1mod, pCRETA2mod and pTA3mod, the constructs were not fully
induced by 1O2 (Fig 3.2). Now, also the problem with the not well defined position of the
Tub2B TATA-box in pBF31 and pBF32 is definitively solved, because in pCRETA1mod
41
Results and Discussion
and pCRETA2mod the locations of the elements are exactly the same as in the Gpxh
promoter. At this point, it became very likely, that other putative elements are involved in
the induction of Gpxh which are missing in the constructs pCRETA1mod and
pCRETA2mod. Since these two constructs have a long deletion between the CREelement and the TATA-box the chance that such additional elements are found in this
region is high. Therefore it is important to further investigate this region in the original
Gpxh promoter.
3.1.3 The Involvement of Three Putative Transcription Factor Binding
Sites of the Gpxh Promoter in the Response to Singlet Oxygen
After having excluded an effect of the position of the CRE-element and the missing
consensus sequence of the TATA-box on the response of pF31 and pBF32 to 1O2, we
investigated a role of the region between the CRE-element and the TATA-box on Gpxh
induction. Analyzing the sequence of this part of the Gpxh promoter by homology search,
Leisinger found three putative regulatory elements homologous to either a GC-box or a
CAAT-box [12]. These elements are known transcription factor binding sites and
involved in the transcriptional activation of many nuclear genes in different organisms
[54-56]. The most upstream of the putative transcription factor binding sites found in the
Gpxh promoter is a putative GC-box and is located between nucleotides –93 and –88
relative to the transcription-start site. The two other elements are putative CAAT-boxes,
located at position -77 to –74 on the sense or -47 to –44 on the antisense strand in relation
to the transcription start site [12] (Fig 1.4b).
To test a role of the three putative transcription factor binding sites in the response of
Gpxh to 1O2, three constructs were cloned based on a 562bp Gpxh promoter fragment, in
which each of these elements was deleted independently (Fig 3.3a). The resulting
constructs pGC1, mutated in the GC-box, pCAAT2 and pATTG3, with a CAAT-box
deletion on the sense or antisense strand respectively, were tested for induction by
Neutral Red and compared to the positive control pASpro1β containing the full 1,4kb
Gpxh promoter fragment. In cultures of strains containing pGC1 or pATTG3 the
induction was significantly reduced compared to the positive control resulting in an
average induction of 0.9±0.1fold for pGC1 and 0.9±0.1fold for pATTG3 (Fig 3.3). On
the contrary, the construct pCAAT2 was induced 5.8±0.2 fold in average, and did not
significantly differ from the response of pASpro1β which had also a 5.8 fold induction
(Fig 3.3).
One should mention that the use of pASpro1 as a control for pGC1, pCAAT2 and
pATTG3 is not the ideal construct. The construct consists of a 1304bp long promoter
fragment while pGC1, pCAAT2 and pATTG3 were cloned based on pASpro2 consisting
of a 562bp long promoter fragment of the Gpxh promoter. However, the difference in
length between the positive control and the samples does not influence the results, since
42
Results and Discussion
Leisinger found no significant difference between the induction factors of pASpro1 and
pASpro2. It should also be noted that only one clone with the pASpro1 construct was
tested as a positive control, namely the clone pASpro1β, but since the clone pASpro1β is
our standard clone and its induction was stable since it had been created it is a reliable
control. Furthermore, its induction was not significantly different to the control
pCRETATO.
(a)
fold induction ±SE
10
CRE
8
TATA
1
pGC1
2
pCAAT2
3
pATTG3
4
pASpro1
6
4
2
0
1
2
3
4
(b)
fold induction
range
n (clones)
P value
pGC1
pCAAT2
pATTG3
pASpro1β
0.9
5.8
0.9
5.8
0.7-1.0
4.6-7.2
0.6-1.1
-
5
14
5
1
<0.000
1.000
<0.000
-
Figure 3.3 (a) Induction and schematic drawing of the reporter gene constructs deleted in one of the three
putative transcription factor binding sites found by their homology to either a GC-box (pGC1) or a CAATbox (pCAAT2 and pATTG3), and the positive control pASpro1β in cultures exposed to 1µM Neutral Red.
The green parts of the constructs represent elements identical to the Gpxh promoter, whereas the red part
shows the position of the mutated boxes. (b) Table with the detailed responses of the four different
constructs shown in figure 3.3 to Neutral Red. The P-values are from the pairwise comparison to the
positive control pCRETATO using a GLM analysis.
The results showed a full induction of pCAAT2 by 1O2 which was not significantly
different to the positive control pASpro1β (Fig 3.3). This shows that the presence of the
putative CAAT element on the sense strand does not affect the induction by 1O2. Hence,
this putative CAAT-box can be excluded as a regulatory element involved in the
induction of Gpxh by 1O2. On the contrary, the significantly lower induction of pGC1
and pATTG3, in which a putative GC-box and a putative CAAT-box on the antisense
strand were deleted, proofed that these elements are essential for an induction by 1O2.
Thus, in this study we found two more regulatory elements beside the CRE-element
which are involved in the transcription activation of the Gpxh gene by 1O2.
43
Results and Discussion
Genes with a polymerase II promoter, such as the Gpxh, consist of conserved promoter
elements which are associated with the regulation of these genes. The gene is only
induced by binding of specific transcription factors to the regulatory elements. In this
study we found two further regulatory elements in the promoter region of Gpxh. The first
one is a GC-box. This element is similar to GC-rich elements found in the promoter
regions of other genes of C. reinhardtii. A 5’-CGCGCC-3’ box found in the CAH1 gene
by Kuchos group might be involved in CO2- or light response of this gene [56].
Furthermore, a 5’-CGCGGAC-3’-element was found in four light-induced genes carrying
out a homology search [57]. This indicates that the GC-box in the promoter region of
Gpxh could be involved in regulation by light, in agreement with the fact that
measurements of Gpxh induction by 1O2 were always carried out in the presence of light.
This would mean that the induction of Gpxh by 1O2 always needs the presence of light
which activates the GC binding transcription factor. Since in photosynthetic organisms
the production of 1O2 is connected to the presence of light, such a combination of 1O2and light regulation seems reasonable. The second element found in this study is a
putative CAAT box. Such elements are known to be involved in the induction of many
genes by binding of a transcription factor termed α-CP1 [54, 55, 58]. The CAAT box we
found to be involved in the induction of Gpxh by singlet oxygen lies on the antisense
strand. This means the box is inverted with a sequence of 5’-ATTG-3’ on the sense strand.
However, CAAT box are known to be orientation independent. For example, mutations in
an inverted CAAT box in the major late promoter of a human adenoviruses reduced the
transcription initiation [59]. However, with our findings we can not be sure if we found
all regulatory elements involved in the induction of Gpxh.
44
Results and Discussion
3.2 Molecular and Physiological Response of Chlamydomonas
reinhardtii Exposed to Ultraviolet Radiation
Exposure to high Ultraviolet radiation (UV) has been reported to cause severe effects in
an organism [17, 19, 60]. A primary effect caused upon exposure to UV radiation is the
production of reactive oxygen species and free radicals, which are produced quickly and
cause serious damage to many cellular components [32]. Singlet oxygen was found to be
one of the main ROS produced in mammalian cells exposed to high UV radiation and
1O2 is considered to cause a major part of UV-induced damages [49, 50]. Since in the
green alga C.reinhardtii the Gpxh gene is specifically induced by 1O2, we investigated
whether Gpxh is, besides induction by high light intensities and exogenous
photosensitizers, also upregulated upon exposure to UV radiation. In order to measure
gene expression it was crucial to first establish experimental conditions under which the
algae were stressed by UV radiation but not dying within the time of exposure. For this
purpose, growth, photosynthetic activity and pigment bleaching were analyzed in each
experiment as indicators for the cellular stress and the physiological state of the cell
during the treatment.
3.2.1 Establishing Experimental Conditions for UV Treatment
In order to test the response of the Gpxh gene in cultures of C.reinhardtii exposed to high
UV-radiation, two independent exposure conditions were planned with either high UVA
or high UVB radiation (Fig 1.6). Since only lamps with the full UV spectrum are
available, the two conditions of high UVA or UVB radiation, with minimal
contamination from wavelengths of the other UV regions, had to be achieved by varying
the distance from the UV lamps to the samples, by using different UV filters (Tab 2.4)
and by changing the number and distance of PAR lamps (Fig 3.4). PAR lamps were used
to supplement the algae with photosynthetic active radiation (PAR 400-700nm) for
photosynthesis. High UVA radiation, for example, was produced by a combination of a
short lamp-to-sample distance and the use of strong UVB filters. However, a short
distance of the UV-lamps to the samples also caused an increased PAR irradiation. Since
in our experimental setup the three parameters UVA, UVB and PAR are strongly
dependent on each other, we tested the effect of many different combinations of UVA,
UVB and PAR intensities on the growth and the photosynthetic efficiency of C.
reinhardtii to find the combination affecting the cell fitness most seriously (Tab3.1)
45
Results and Discussion
PAR
a
UV
b
Figure 3.4 Schematic drawing of the experimental setup used to expose cultures to high UV radiation. The
light intensity could be changed by using different filters (a), varying the distance from the UV lamp and
PAR lamp to the samples (b) or varying the number of PAR lamps.
Table 3.1 Effects of different UVA, UVB and PAR light intensity combinations on growth and the
fluorescence yield in C.reinhardtii. The different intensities of UVA, UVB and PAR, which were achieved
by using different filters and varying the distance and number of PAR and UV lamps as indicated, were
divided into six colour labelled categories from low to high intensities with the following categories: bright
yellow: UVA(0-2.99), UVB(0-0.15), PAR(100-199); Yellow: UVA(3-5.99), UVB(0.16-0.31), PAR(200299); dark yellow: UVA(6-8.99), UVB(0.32-0.47), PAR(300-399); bright orange: UVA(9-11.99),
UVB(0.48-0.63), PAR(400-499); dark orange: UVA(12-14.99), UVB(0.64-0.79), PAR(500-599); brown:
UVA(15-17.99), UVB(0.80-1.00), PAR (600-699). Measurements for UVA and UVB are given in mW cm2, whereas measurements for the PAR values are given in µmol m-2 s-1. To categorize effects on growth,
the OD750 after 180min of exposure was expressed as percentage of the OD750 at the start of exposure. To
categorize effects on the fluorescence yield, the fluorescence yield measured after 180min of exposure was
expressed as the percentage of the fluorescence after the preadaptation. The effect on growth and on
fluorescence was then divided into six colour labelled categories from a very harmful effect (one) to no
effect (six) to better correlate the effect of UV radiation with the light parameters. The following categories
were used: 1 dark blue: OD750 (<12%), fluorescence yield (0-14%); 2 blue: growth(12-100%),
fluorescence yield(15%-29%), 3 bright blue: OD750 (100%), fluorescence yield (30-44%); 4 dark grey:
OD750 (116%), fluorescence yield(45-59%); 5 grey: OD750 (120%), fluorescence yield (60-89%); 6 light
grey: OD750 (>120%), fluorescence yield (90-100%).
46
Results and Discussion
filter
Quarz
Quarz
Quarz
quarz
Quarz
1M
G
5M
F/G
Quarz/FE
G/FE
G/F
G/FF
4M
G/F
Quarz/F
G
Quarz/7M
G
G
G
G
Quarz/5M
FE/FE/G
G/F
Quarz/F
G/FE
G/FE
G/FE
G/FE/FE
G/FE/FE
G/FE
G/F/3M
G/FF/2M
G/FF/3M
FE/FE/G
G/FE/FE/2M
FE/G
G/FE/FE/3M
G/4M
lamps
2PAR/UV
2 PAR/UV
2 PAR/UV
3PAR/2UV
3PAR/2UV
2 PAR/UV
2PAR/UV
2 PAR/UV
1 PAR/UV
3PAR/2UV
2PAR/UV
1 PAR/UV
1 PAR/UV
2 PAR/UV
2PAR/UV
3PAR/2UV
2 PAR/UV
2 PAR/UV
2 PAR/UV
2PAR/UV
3PAR/2UV
3PAR/2UV
2 PAR/UV
1 PAR/UV
3PAR/2UV
2 PAR/UV
3PAR/2UV
2PAR/UV
3PAR/2UV
1 PAR/UV
2 PAR/UV
1 PAR/UV
1 PAR/UV
1 PAR/UV
1 PAR/UV
2 PAR/UV
2 PAR/UV
2PAR/UV
1 PAR/UV
2 PAR/UV
UVA
7.98
5.27
5.78
4.29
4.29
3.87
11.86
1.86
17.14
1.53
4.09
11.48
9.92
1.90
7.93
3.57
6.30
1.44
5.47
7.62
4.12
4.12
1.46
4.26
3.45
3.95
1.50
3.31
1.50
2.91
1.27
5.00
4.30
4.59
3.69
1.08
0.35
2.86
1.04
1.65
UVB
0.93
0.64
0.64
0.53
0.53
0.45
0.44
0.21
0.12
0.04
0.04
0.09
0.05
0.17
0.06
0.05
0.20
0.11
0.21
0.32
0.19
0.19
0.12
0.01
0.03
0.03
0.01
0.03
0.01
0.01
0.00
0.05
0.04
0.03
0.02
0.01
0.00
0.03
0.00
0.04
PAR
339
310
313
196
196
254
673
131
673
184
619
459
425
314
546
182
343
124
302
337
198
198
250
649
183
300
186
546
186
438
307
460
270
295
259
268
299
460
272
254
47
growth
1
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
6
6
6
fluorescence
1
1
1
1
1
1
1
1
1
1
2
1
1
2
2
2
3
3
4
2
2
2
2
2
3
3
3
4
4
4
5
2
3
4
4
6
6
3
5
6
Results and Discussion
The effects on growth and the fluorescence yield of cultures exposed to various
combinations of UVA, UVB and PAR light intensities for 180min are summarized in
table 3.1. A categorization of all light parameters (yellow) and all physiological
parameters (grey) was performed to make it easier to correlate the effects to the exposure
conditions. Therefore, the severity of the effects on physiological parameters were
divided into six categories, where the most serious impact on the algae was in category
one whereas no effect could be detected in category six. Growth, as an indicator for
viability and the fitness of the cells, was detected by the relative changes of the optical
density at 750nm over 180min of exposure. The chlorophyll fluorescence yield, as an
indicator of the photosynthetic efficiency, was measured after 180min of exposure and its
reduction during the treatment was also divided in six categories (Tab 3.1). After sorting
the table 3.1 according to the effect on growth we could see a correlation between high
UVB intensities and the effect on the physiological parameters. Thus cells exposed to
high intensities of UVB are dying early after exposure and encounter an inhibition of the
photosynthetic activity. High UVA intensities affected growth less seriously but strongly
inhibited the photosynthesis. Only in combination with high PAR level the high UVA
intensities were also very toxic. However, the intensity of PAR light alone had only little
effect on growth. Thus, high UVB was found to have the most hazardous impact on
growth and the photosynthetic activity. A threshold level of ~0.3mW cm–2 UVB
intensity could be identified, above which all cultures started to die during the exposure.
No such threshold level could be identified for UVA and PAR light alone since there was
a direct correlation between high UVA radiation and PAR for which a strong effect on
the physiological parameters was found.
Maximal response of a stress induced gene is expected at a high level of UV radiation.
On the other hand, cells should still be in a vital state in order to be able to efficiently
express the genetic response. Therefore, the cells should survive the exposure but, except
for the control, be seriously stressed by the UV radiation. According to this restriction,
we chose the light conditions for further experiments (Fig 3.5). In all conditions selected,
the cultures were growing (categories 4-6) and the photosynthetic efficiency was reduced
(categories 1-4), except for the control conditions (UVA1 and UVB1) where only a
residual level of UVA and UVB intensity was present. Unfortunately, it was not possible
to find conditions with absolutely no contamination of the other UV-part, but the levels
were very low and should not have disturbed the experiments. The selected conditions of
the UVA (UVA1-4) and the UVB (UVB1-4) treatment chosen for further experiments
and the exact light intensities of UVA, UVB and PAR radiation are summarized in figure
3.5b.
48
Results and Discussion
(a)
UVA
mW/cm2
~0.3 mW/cm2
6
5
4
3
2
1
0
0
0.1
0.2
0.3
UVB
mW/cm2
(b)
UVA treatment
UVA1
UVA2
UVA3
UVA4
1.04
3.69
4.30
4.59
UVB (mW cm )
0.00
0.02
0.04
0.03
PAR (µmolm-2s-1)
272
259
270
295
UVB1
UVB2
UVB3
UVB4
0.35
1.65
1.46
1.90
0.00
0.04
0.12
0.17
299
254
250
314
UVA (mW cm-2)
-2
UVB treatment
UVA (mW cm-2)
-2
UVB (mW cm )
-2
1
PAR (µmolm s- )
Figure 3.5 (a) The selected treatments to measure the response to UVA (dotted grey arrow) and UVB (grey
arrow). The squares are the conditions used for UVA treatment (UVA1-4) and the triangles are the
conditions used for the UVB treatment (UVB1-4). The control samples are UVA1 and UVB1, whereas
UVA4 and UVB4 conditions are performed under the highest intensities of UVA and UVB, respectively.
The dotted line shows the threshold level of 0.3 mW/cm2 UVB intensity above which the cells started to
die. (b) Shown are the light intensities in the different conditions in the UVA and UVB treatment.
The highest UVA intensity (UVA4) of the selected conditions was 1.6 fold higher than
the level measured on a sunny day in September in front of the EAWAG building,
whereas the highest UVB intensity (UVB4) was similar to the natural level. The PAR
values are 2.5 fold higher than in our standard condition in the Multitron, where we
normally incubate algal cultures. In order to provide the same level of PAR light in all
conditions of the UVA and the UVB treatment, it was necessary to perform the UVA and
UVB exposure in two different experimental setups. In the UVA setup, one UV lamp and
one PAR lamp was used, while for UVB exposure one UV lamp and two PAR lamps
49
Results and Discussion
were used. Like this, the average PAR intensity in the UVA treatments was 284µmolm2s-1 and in the UVB treatment 277µmolm-2s-1, which is practically the same. To
preadapt the cultures to this higher PAR levels, the specific light spectra and the low
residual UV radiation, a culture was incubated in each of the control conditions of the
UVA and UVB setup for 14 hours.
3.2.2 Effect of Preadaptation and Exposure of Cultures to UVA and
UVB Conditions Compared to a Culture Kept under our
Standard Conditions
Cultures under the control conditions UVA1 and UVB1 are exposed to a residual level of
UVA and UVB (Tab 3.2). Furthermore, the PAR light was higher than under our standard
condition in the Multitron (25°C, 100-150µmolm-2s-1). Since we used cultures from the
Multitron for the exposure to the UVA and UVB treatment, we preadapted the algae in
order to let them adjust their metabolic activity to the new environment. To investigate
the effect of the preadaptation, we compared the preadapted cultures of the UVA and
UVB setup to a culture at standard condition by analyzing growth, oxygen production
and the pigment content.
Table 3.2 Light parameters of the two controls for the UV treatment and of the standard condition in the
Multitron.
UVA1
UVB1
Multitron
-2
1.04
0.352
0.052
-2
0.003
0.003
0.007
-2 -1
272
299
110
UVA (mW cm )
UVB (mW cm )
PAR (µmolm s )
For measurements of growth we investigated the optical density at 750nm of preadapted
cultures under the control conditions UVA1 and UVB1 and in the Multitron at different
time points over 210min. No significant difference in the growth rate was detected
between controls UVA1, UVB1 and a standard culture in the Multitron during an
exposure of 210min (Fig 3.6a). This means that the preadaption of the control conditions
UVA1 and UVB1 did not have an effect on growth. However, the average growth rate
seemed to be slightly increased in the UV controls compared to the standard culture. This
could be due to the higher photosynthetically active radiation (PAR) in the UV conditions.
The effect of the preadaptation on photosynthesis was tested by analyzing the oxygen
production of the controls UVA1 and UVB1 after the 14 hours of preadaptation and
comparing it to a Multitron control culture. The oxygen production of all three controls
was not significantly different. Still, the average oxygen emission was highest in the
Multitron culture with 0.074±0.006 µmol oxygen h-1 per 106 cells, while the oxygen
50
Results and Discussion
emission in the UV control UVA1 was 0.049±0.014 and in UVB1 0.052±0.005 (Fig3.6b).
This indicates that the photosynthetic activity might be slightly but not significantly
reduced by the preadaptation of the controls.
The pigment content was analyzed since chlorophylls and carotenoid are the main light
absorbing pigments in the photosynthetic organisms and their level is influenced by the
amount of UV and PAR radiation [17, 33]. The pigments were measured after 14h of
preadaptation in the conditions UVA1 and UVB1 and a culture in the Multitron and
normalized by the cell number. The chlorophyll a and carotenoid content of preadapted
algae in the UVA and UVB setup was significantly lower than in a culture kept in the
Multitron (Tab 3.3). The amount of chlorophyll a in the UV controls was two-fold lower,
and the amount of carotenoid 1,5 fold lower than in the Multitron. The Chlorophyll a
content was 0.23±0.01 µg per 106 cells in the Multitron, 0.12±0.00µg per 106 cells in
UVA1 and 0.10±0.01µg per 106 cells UVB1, whereas the Carotenoid content was 0.08±
0.00µg per 106 cells in the Multitron, 0.06±0.00µg per 106 cells in UVA1 and
0.05±0.00µg per 106 cells UVB1 (Fig 3.6c). On the contrary, the Chlorophyll b content
was not significantly different in UVA1, UVB1 and the Multitron culture (Fig 3.6c). The
difference in pigment contents shows clearly an adjustment of the algae towards the new
environment. The decrease of chlorophyll a and carotenoid in the UV controls is probably
caused by the increase of PAR intensity in these conditions. The slight increase of UVA
might have an additional effect. By lowering the amount of light absorbing pigments, less
light energy is absorbed by the cell which can protect the cells from photoinhibition. This
is also in agreement with a lower photosynthetic activity and might be a preventive
response to light stress. Thus, the preadapted cultures might already be more resistant to
the UV-stress than cultures form the Multitron would be.
Table 3.3 P-values of a One-way ANOVA analysis of the (a) Chlorophyll a content and (b) Carotenoid
content after 14h of preadaptation in UV treatments and of a culture in the Multitron
(a)
UVA1
UVB1
Multitron
UVA1
-
0.109
<0.000
UVB1
0.109
-
<0.000
Multitron
<0.000
<0.000
-
UVA1
UVB1
Multitron
UVA1
-
0.131
<0.005
UVB1
0.131
-
<0.001
Multitron
<0.005
<0.001
-
(b)
51
growth rate h-1 ±SE
0.12
0.10
O2 evolution/106 cells (umol h-1)±SE
Results and Discussion
(a)
0.08
0.06
0.04
0.02
0.00
UVA1
6
pigment content/10 cells (ug)
0.25
UVB1
0.10
(b)
0.08
0.06
0.04
0.02
0.00
Multitron
UVA1
UVB1
Multitron
(c)
(c)
0.20
0.15
0.10
0.05
0.00
UVA1
UVB1
Multitron
Figure 3.6 Growth rate (a), oxygen production (b) and pigment content (c) of the preadapted controls in the
UV treatment UVA1and UVB1 and of a Multitron culture. In (c) the pigment content of Chlorophyll a is in
dark grey, Chlorophyll b grey and Carotenoid white.
3.2.3 Physiological Parameters of Cultures Exposed to the Selected
Conditions of the UVA and UVB Treatment
The physiological state of cultures grown in the four selected conditions of increasing
level of UVA (UVA1-4) or UVB intensity (UVB1-4)(Fig 3.5), was investigated by
measuring growth, effects on photosynthesis and pigments. By this we tested whether the
cultures are viable under these conditions and whether they might be stressed by the
increasing UV intensities in the conditions UVA2-4 and UVB2-4 (Fig 3.5).
Analyzing growth by measuring the optical density at 750nm (OD750), we found that the
OD750 after 210min of exposure was significantly higher than at the start of the
experiment in all conditions of the UVA treatment (Tab 3.4). In the UVB treated cultures,
on the other hand, the OD750 after 210min of exposure was only significantly increased
in UVB1 and UVB2 (Tab 3.4)(Fig 3.7).
A significant increase in the OD750 indicates that the cultures were growing under the
conditions stated. We found that all conditions in the UVA treatment and UVB1 and
UVB2 in the UVB treatment were growing over an exposure of 210min. However, the
52
Results and Discussion
increase in OD750 in the conditions UVB3 and UVB4 was not significant (Tab 3.4). This
indicates that growth in these conditions was seriously affected. On the other hand we can
exclude, that the cells in these conditions died during the exposure since OD750 was not
significantly lower than the OD750 at the start of exposure. In general, the OD750 was
lower in the UVB conditions compared to the UVA conditions. This is supported by
several findings which show that high UVB intensities can seriously affect organisms
[27]}.
1.50
1.50
UVA
UVB
1.40
rel growth ±SE
rel growth+SE
1.40
1.30
1.20
1.10
1.00
1.30
1.20
1.10
1.00
0.90
0.90
0
50
100
150
0
200
min of exposure
50
100
150
200
min of exposure
Figure 3.7 Growth over 210min of exposure, expressed as the change in the relative optical density at
750nm. Conditions of the UVA treatment are shown in the left graph, while conditions of the UVB
treatments are shown in the right graph: controls UVA1/UVB1 (diamonds), UVA2/UVB2 (triangles),
UVA3/UVB3 (circle) and UVA4/UVB4 (squares). Growth of a culture growing under standard condition
in the Multitron is indicated with the dotted line.
Table 3.4 P-values of repeated measures ANOVA of growth across time (0 and 210min) in the UVA and
the UVB treatment
(a)
(b)
P-values
P-values
UVA1
<0.000
UVB1
0.005
UVA2
<0.000
UVB2
0.001
UVA3
<0.000
UVB3
0.107
UVA4
<0.000
UVB4
0.460
The relative fluorescence yield was tested at different time points over an exposure time
of 210min. The fluorescence yield indicates effects on the photosynthesis: a negative
effect on the photosynthetic apparatus results in a decreased fluorescence yield. This was
the case in all cultures exposed to UVA radiation (UVA2-4) during 210min (Fig 3.8a).
The fluorescence yield was decreased in UVA2-4, but was stable in the control UVA1 on
the level after preadaptation. The decrease in the UVB exposed conditions UVB3 and
UVB4 was even more pronounced. After 210min of exposure the fluorescence yield in
UVB4 was zero (Fig 3.8a). However, in the control UVB1, but also in UVB2 the
fluorescence yield was stable on a high level. In contrast to the UVA control, the level of
53
Results and Discussion
the fluorescence yield in UVB1 and UVB2 was higher than after the preadaptation (Fig
3.8a).
The fluorescence yields in figure 3.8a are normalized to the fluorescence yield after
preadaptation and not to the level at the start of the experiment. This because a transient
increase of the fluorescence yield at the start of exposure was measured in the UVA and
in the UVB treatment (Fig 3.8b), which was reduced to the level after preadaptation after
30-100min of exposure in the UVA treatment (Fig 3.8a). We argue that this increase in
fluorescence yield is artificially caused by the adjustment of the optical density and the
division of the precultures into smaller samples which was not performed under UV
radiation. It is likely that the preadapted cultures were able to recover during this time,
because the recovery time of photoinhibited cells in low light conditions is known to be
short. This could be a reason why the fluorescence yield was higher just at the start of
exposure compared to the level after the preadaptation (Fig 3.8b). However, we can not
explain why the level of the fluorescence yield in the UVB1 control and the UVB2
control remained on a much higher level during the whole experiment. Since a decrease
in the fluorescence yield indicates an inhibition of the photosynthesis, we concluded that
the cells were stressed under these conditions. This is supported by several studies which
show that UVA and UVB have an adverse effect on the photosynthetic efficiency [24, 26].
(a)
2.00
2.00
UVA
1.80
rel fluorescence yield
rel fluorescence yield
1.60
1.40
1.20
1.00
0.80
0.60
0.40
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.20
0.00
0.00
0
50
100
150
200
250
0
(b)
0.50
0.40
0.30
0.20
0.10
0.00
after 14h of preadaptation
50
100
150
min of exposure
min of exposure
fluorescence yield ±SE
UVB
1.80
at the start of exposure
54
200
250
Results and Discussion
Figure 3.8 (a) The fluorescence yield of the different conditions in the UVA treatment (left) and the UVB
treatment (right), at different time points of exposure, relative to the value of the fluorescence yield after
preadaptation. Diamonds are used for UVA1and UVB1; triangles for UVA2 and UVB2; circles for UVA3
an UVB3 and squares for UVA4 and UVB4. The dotted line indicates the level of the fluorescence yield
after preadaptation. (b) The difference level of the fluorescence yield after 14 hours of preadaptation and at
the start of the experiments. In dark grey the cultures of the UVA setup, in grey the cultures of the UVB
setup are shown.
Effects on photosynthesis were also tested by measuring the oxygen production in the
control condition (UVA1, UVB1) and the condition with highest intensities in UVA and
UVB (UVA4, UVB4) during the exposure. The oxygen production was significantly
reduced upon exposure to high UVA and UVB (Fig 3.9), whereas in UVA1, oxygen
production was significantly increased during the exposure. In UVA4 only half of the
initial amount of oxygen (0.015±0.003 µmol h-1/106 cells) was produced after 210min of
exposure, whereas in UVB4 no oxygen was produced anymore after 180min of exposure.
In the UVB control and in a standard culture in the Multitron, on the other hand, oxygen
production remained constant over the time of exposure (Fig 3.9).
A decrease in the oxygen production as found in UVA4 and UVB4 indicate a serious
effect on the photosynthetic activity. These findings correlate with the findings of the
chlorophyll fluorescence measurements indicating that the photosynthesis damage is
more seriously than only an inhibition of the electron transport chain, which is able to
recover again. This effect is more pronounced for high UVB than for high UVA radiation.
The increase of oxygen production in UVA1 is unclear. It might be due to the adjustment
of the optical density at the start of exposure, which caused a change in the culture, or the
fresh media might have enhanced the oxygen production in this condition.
1.6
UVA
1.4
1.4
1.2
1.2
rel oxygen emission
rel oxygen emission
1.6
1.0
0.8
0.6
0.4
0.2
0.0
UVB
1.0
0.8
0.6
0.4
0.2
0.0
0
50
100
150
200
0
250
min of exposure
50
100
150
200
250
min of exposure
Figure 3.9 Oxygen production relative to the oxygen production after preadaptation. Conditions: controls
UVA1 and UVB1 (diamonds); exposed samples UVA4 and UVB4 (squares). The dotted line in the left
graph indicates the values of a Multitron culture.
The pigment content was measured after 120min of exposure and normalized to the cell
number. There was hardly any difference in the pigment content among all conditions of
the UVA and UVB treatments (Fig 3.10). The only significant difference was found in
55
Results and Discussion
the chlorophyll a content of condition UVB2, which was significantly lower after an
exposure of 120min than after preadaptation. In all other conditions of the UVA or UVB
treatment the content of chlorophyll a, chlorophyll b and carotenoid did not significantly
differ after 120min of exposure to the content after preadaption (Fig 3.10).
This is surprising since UVB has been shown to be involved in the reduction of
chlorophyll a and carotenoid and that 99% of incident UVB is suggested to be absorbed
by chlorophylls [17, 28, 61]. On the other hand, a decrease in the pigment content by UV
might take longer than only two hours. Döhler exposed marine chlorophycean Dunaliella
tertiolecta to UVA (1.02 mW/cm2) and high UVB (1.6mW/cm2). After 2hours of
exposure to UVA he found an increase in chlorophyll a, b and of carotenoid, whereas he
did not find a change in this pigments after a 2 hours of exposure to UVB. [29]. The
pigment content is therefore influenced by the intensity and wavelength of irradiation but
can also vary strongly among different species [62].
rel pigment content ±SE
1.60
1.40
UVA
1.20
1.00
0.80
0.60
0.40
0.20
0.00
rel pigment content ±SE
1.60
1.40
UVA1
UVA2
UVA3
UVA4
UVB1
UVB2
UVB3
UVB4
UVB
1.20
1.00
0.80
0.60
0.40
0.20
0.00
Figure 3.10 Pigment content ±SE after 120min of exposure to UVA (top) and UVB (bottom) relative to the
content after preadaptation. In dark grey Chlorophyll a, in grey Chlorophyll b and in white the Carotenoid
content is shown.
To conclude, none of the cultures of the investigated conditions of high UVA or UVB
radiation were dying during the exposure to UV. However, measurements of the
chlorophyll fluorescence and oxygen production show a severe effect on photosynthesis.
56
Results and Discussion
This indicates that the cells were stressed under conditions UVA2-4 and conditions
UVB3-4. On the other hand, hardly any change in the pigment content could be detected
during an exposure of 120min. Table 3.5 summarizes the effects of the different UV
treatments on the physiological parameters of the cultures (Tab 3.5).
Table 3.5 Summary of the effect of the different light intensities of UVA, UVB and PAR on the
physiological state of the selected conditions to investigate an effect of UVA radiation (UVA1-4) and an
effect in UVB (UVB1-4). Growth was measured as an indicator for survival, the fluorescence yield and the
oxygen emission as and indicator of the photosynthetic activity. Content of the pigments chlorophyll a,
chlorophyll b and carotenoid was measured since they are the most important light absorbing pigments of
the photosynthetic apparatus. Up pointing arrows () indicate an increase of the parameter, an equation
mark (=) that the parameter was stable, and a down pointing arrow indicates a decrease () in the parameter
during the time of exposure.
UVA1
UVA UVB PAR Growth Fluorescence Chl Chl Carotenoid O2
yield
a b
production
G/FE/FE/3M 1.04 0.00 272
=
= =
=
=
UVA2
G/FF/3M
3.69 0.02 259
UVA3
G/F/3M
4.30 0.04 270
UVA4
G/FF/2M
4.59 0.03 295
UVB1
G/FE/FE/2M 0.35 0.00 299
UVB2
G/4M
1.65 0.04 254
UVB3
Q/5M
1.46 0.12 250
UVB4
4M
1.90 0.17 314
Treatment
Filter
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
3.2.4 Genetic Response to UV Radiation: The Induction of Gpxh Gene
by UVA and UVB-Treatment
After having evaluated the effect of UV-exposure on the physiological state of the algae
we chose the UV-conditions which were thought to be suitable to test the genetic
response. For this, the cultures had to be still vital and except for the control, be seriously
stressed by UV radiation. To test the effect of UV on the induction of the Gpxh gene, and
the two general stress response genes coding for gluthatione-S-transferase (GST) and a
heat shock protein (HSP), UVA1 and UVB1 were selected as controls and UVA4 and
UVB3 as UV-treated conditions, respectively. Additionally, UVB2 was used as a
negative control for UVB to detect putative effects of the small UVA contamination
within the UVB3 treatment. We investigated the genetic response of the three genes to
UVA and UVB after 1h and 3h of exposure by quantifying the amount of mRNA of each
gene in the collected sample with real time RT-PCR.
57
Results and Discussion
3.2.4.1
Normalization of Varying mRNA Levels
In real time RT-PCR, the amount of individual mRNA of each gene is quantified by
determine the number of PCR cycles (Ct) to reach a fixed amount of PCR product. These
Ct values are determined for all reactions in the logarithmic amplification phase of the
PCR. A low Ct value means that there was a high amount of this mRNA present in the
sample whereas a high Ct value means that the initial amount of mRNA was low. To
compare the amount of mRNA in between different samples, the Ct values have to be
normalized to the Ct value of a gene which is known to be constitutively expressed under
the stress condition. Initially, we used the Rbcs2 gene, coding for Rubisco was used for
normalizing of real time RT-PCR data. However, we found a high variation in the Ct
value for RbcS2 gene in our experiment. This means that this gene is downregulated upon
exposure to high UVB (Fig 3.11). This is supported by several studies which showed, that
the gene coding for Rubisco is downregulated by UVB [18, 63]. In contrast, we found no
significant differences in the Ct values of mRNA of the ribosomal 18SrRNA gene (Fig
3.11). Therefore, this gene could be used to normalize varying mRNA level of the tested
genes in the UV experiment.
PCR Cycle Number (CT)±SE
20.0
18.4
18.0
16.1
16.6
16.1
16.0
15.5
14.0
12.0
10.0
8.0
6.0
4.0
3.7
4.1
4.1
3.7
3.8
UVA1
UVA4
UVB1
UVB2
UVB3
2.0
0.0
UVA1
18Sr
18S
UVA4
UVB1
UVB2
UVB3
Rbcs2
Figure 3.11 PCR Cycle Number of two genes frequently used for normalization of the varying mRNA
levels in real time RT-PCR experiments.
3.2.4.2
Comparison of the Expression of Three Stress Response Genes in the UVControl Conditions and in the Multitron
In order to investigate the basal gene activity of Gpxh and the two stress response genes
GST and HSP in the UV control conditions after preadaptation we compared the
normalized amount of mRNA in the UV conditions with the amount of mRNA in the
standard condition (Fig 3.12). The average mRNA level of Gpxh and GST was not
58
Results and Discussion
significantly different in the UV controls and in the Multitron culture.
level of HSP in UVA1 was significantly higher than in UVB1 and
culture (Fig 3.12). The 10fold higher level of HSP in UVA1 than
condition indicates that this culture is already stressed, even though
growth and oxygen evolution was seen (Fig 3.6).
Only the mRNA
in the Multitron
in the Multitron
no effect on the
100000
14718
14459
5594
mRNA amount ±SE
10000
2710
1766
1000
276
129
100
9
9
UVB1
3h
Multitron
3h
10
1
UVA1
3h
UVB1
3h
Gpxh
Multitron
3h
UVA1
3h
UVB1
3h
GST1
Multitron
3h
UVA1
3h
HSP1
Figure 3.12 The normalized amount of mRNA of the GST, Gpxh and HSP gene after a preadaptation of 14
hours and subsequent exposure of 3h under the control conditions UVA1, UVB1 and of a culture grown
under standard condition (Multitron).
3.2.4.3
Genetic Response of the Gpxh, GST and HSP Gene to High UVA and UVB
Radiation
Since it is known that Gpxh is specifically induced by singlet oxygen and that singlet
oxygen is produced in mammalian cells upon exposure to UV, [50] we investigated the
induction of Gpxh by high UVA and UVB radiation in C. reinhardtii . Additionally, we
measured the induction of the two stress response genes GST and HSP. To investigate the
genetic response to high UVA and UVB we exposed the cultures under the conditions
UVA1, UVA4, UVB1, UVB2 and UVB4 and subsequently measured the gene expression
by real-time RT-PCR. To directly see the effect of the increased UVA or UVB radiation,
the mRNA levels of the treated conditions (UVA4, UVB2, UVB3) were related to the
level of the corresponding control sample (UVA1, UVB1) by calculating induction
factors.
There was no induction of the Gpxh gene by the exposure to UVA or UVB radiation
since the average induction after 3h of treatment were only 1.11±0.14 fold in UVA4 ,
59
Results and Discussion
1.02±0.10 fold in UVB2 and 1.26±0.26 fold in UVB3 (Fig 3.13). None of cultures had a
Gpxh expression which was significantly different in the UV-conditions compared to the
controls UVA1 or UVB1 and the induction of Gpxh by UV radiation was found to be
negligible compared to the 20 to 30 fold induction by high light conditions [6]. The stress
response genes HSP and GST, on the other hand, were both upregulated by high UVB
(Fig 3.13). Thus, in the condition UVB3 the HSP and the GST gene were significantly
induced with a high average induction of 130.0±29.2 and 176.7±66.4 fold for HSP after
1h and 3h of exposure and a lower induction of 2.0±0.2 and 2.8±1.1 fold for GST after 1h
and 3h of exposure, respectively. This data support our earlier findings by measuring
physiological parameters which showed that the UVB3 sample encounter a strong stress
condition by high UVB radiation resulting in a reduced growth. Support for a good
indication of a cellular stress condition by the response of the GST and HSP gene is
coming from several studies which showed that many genes upregulated by UVB are also
upregulated by other stress conditions such as oxidative stress, salt stress, pathogen
induced stress or drought [41-43, 47]. However, in the conditions UVA4 and UVB2 the
gene expression of GST and HSP did not significantly differ from the controls at both
time points (Fig 3.13). Since in UVB2 we could neither detect an effect on growth, the
fluorescence yield (Fig 3.7/3.8) or on the genetic response (Fig 3.13), we believe that
cells exposed to such intensities of UVA and UVB are not stressed during this short-time
exposure to UV. On the other hand, we do not know why the stress response genes GST
and HSP were not induced by high UVA which clearly affected photosynthesis in the
treated cultures (Fig3.8/3.9). It might be that the UVA intensities tested were too low to
mediate a genetic response because the cells were still not stressed even though the
photosynthetic activity was reduced.
60
Results and Discussion
(a)
1000.0
log fold induction ±SE
130.0
100.0
10.0
1.7
1.0
1.0
2.0
1.0
0.8
1.4
0.7
1.0
UVA4 UVB2 UVB3 UVA4 UVB2 UVB3 UVA4 UVB2 UVB3
1h
1h
1h
1h
1h
1h
1h
1h
1h
0.1
Gpxh
GST1
HSP1
(b)
1000.0
log fold induction ±SE
176.7
100.0
10.0
2.8
1.1
1.0
1.3
1.4
0.9
0.5
0.7
1.0
0.1
UVA4 UVB2 UVB3 UVA4 UVB2 UVB3 UVA4 UVB2 UVB3
3h
3h
3h
3h
3h
3h
3h
3h
3h
Gpxh
GST1
HSP1
Figure 3.13 Induction of Gpxh and the two stress response gene GST and HSP after (a) 1h and (b) 3h of
exposure to UVA or UVB radiation measured by real-time RT-PCR.
Recently, Barta et al. showed the formation of high levels of singlet oxygen in spinach
leaves exposed to UVA [48]. However, the singlet-oxygen specific Gpxh gene in our
model organism C. reinhardtii was not significantly induced by UVA or UVB indicating
that no 1O2 was produced in cultures exposed to UV in our experiments. This is
supported by findings of Hideg who found a clear increase of singlet oxygen in spinach
61
Results and Discussion
leaves upon exposure to high light but not to UV radiation. They suggested that singlet
oxygen might be the major ROS produced by high PAR intensities whereas superoxide
radicals are the major ROS produced upon exposure to UV radiation [64]. Furthermore, it
has been reported that the target sites of UV and high PAR light in the PSII are different
[30]. Exposure to high light leads to the impairment of the electron transport chain at the
electron acceptors QA and QB in photosystem II, what results in the formation of an
excited state of the reactive centre and the subsequent formation of singlet oxygen by
energy transfer reactions [65]. In contrast, the major target site of UVA and UVB
radiation was found to be the donor site of the photosystem II, namely the oxygen
evolution complex, where under normal conditions liberated electrons from the water
oxidation are transferred to the reaction centre of photosystem II [27, 31]. An impairment
of the electron donor site of the photosystem II by UV might therefore not result in the
formation of singlet oxygen but of other ROS. This might explain why the Gpxh gene is
not induced by the high UVB radiation. However, we are not able to exclude a production
of singlet oxygen by high UVA because it is possible that our intensities of UVA were
too low to mediate the production of singlet oxygen and the Gpxh induction as indicated
by the absence of response of the two other stress response genes. Still, it seems that the
Gpxh gene in C. reinhardtii is not or only to a minor extent induced by UV radiation.
62
Conclusion
4 Conclusion
In this study we further investigated the transcriptional activation of the Gpxh gene by
1
O2 and tested its response upon exposure to ultraviolet radiation since UV is known to
increase the 1O2 level in mammalian cells. We found that the Gpxh gene is not strongly
induced upon exposure to either high UVA or UVB. This indicates that 1O2 is not the
major ROS produced in C.reinhardtii upon exposure to UV radiation. However, the two
stress response genes GST and HSP, which are significantly induced by UVB, were not
induced by UVA, indicating that the UVA intensities tested might have been too low to
mediate a genetic response. Still, since the UVA and UVB intensities tested were similar
or even higher than under natural conditions it seems that the Gpxh gene in C.reinhardtii
is not or only to a minor extent induced by UV radiation. This lack of response to the UV
treatment excludes this stress as a direct or indirect signal to stimulate Gpxh expression
and by this further supports the specificity of this gene to 1O2. This is an important factor
of a gene to be used as a biosensor.
One part of the regulatory mechanism which could be responsible for this specificity of
the Gpxh gene to 1O2 was found in this study by identifying two additional regulatory
elements, a putative GC-element and a CAAT-box like element, in the promoter region of
Gpxh which are needed for a full induction of the gene. All regulatory elements in the
promoter region of Gpxh, namely the CRE-element, the GC-box and the CAAT-box, are
homologous to well-known regulatory elements and found in promoter regions of many
other genes in different organisms. Having several regulatory elements in one promoter,
like in the Gpxh promoter, makes the induction mechanism of the gene more complex but
also allows a more specific regulation of the gene expression. Therefore, the presence of
three regulatory elements in the promoter of Gpxh could be the reason for the specific
induction of this gene since the chance to find the same combination of elements within a
promoter region of other genes in the genome of C.reinhardtii is lower than for a single
element. Additionally, the three DNA-binding proteins might be regulated by different
pathways. These pathways might be activated by various signals including 1O2, but only
1
O2 would be involved in the activation of all three pathways, resulting in the induction of
the Gpxh gene. Such a multiple activating mechanism would eliminate the requirement of
one specific 1O2 sensor in the cell, but involve several less specific 1O2 sensors which all
together would activate the specific response of Gpxh to 1O2.
As a final conclusion we state that it is still too early to use the Gpxh promoter-reporter
gene construct as a commercial biosensor to detect photooxidative stress caused by 1O2.
Even though the induction of Gpxh by 1O2 seems to be specific and not induced by UV
radiation and other oxidative stress conditions, the exact mechanism of the induction is
still not known and probably even more complicated than expected. The response
therefore needs to be further investigated, for example, it is still not known which
transcription factors are binding to the promoter and how they are regulated further
63
Conclusion
upstream in the signalling pathway. It might be possible that the transcription factors
binding to the three characterized promoter elements are activated by different signals
than 1O2 and that the combination of different signals induces the Gpxh expression under
a condition not involving 1O2 production. Thus, the results of this study could clarify an
important point in the response of the Gpxh gene to 1O2 but it also opened new questions
which have to be answered in order to understand the whole process of the 1O2-specific
Gpxh induction.
64
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69
Appendix
6 Appendix
Table 6.1 Glycerol stocks
Stock Nr.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Date
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
18.09.2005
Name
pYS1
pYS2
pYS3
pCRETATO
pCRETATO
pCRETA1
pCRETA1
pCRETA2
pCRETA2
pTA3
pTA3
pYS1mod
pYS2mod
pYS3mod
pBF28mod
pBF29mod
pBF30mod
pCRETA1mod
pCRETA1mod
pCRETA2mod
pCRETA2mod
pTA3mod
pTA3mod
pBF31mod
pBF31mod
pBF32mod
pBF32mod
pBF33mod
pBF33mod
70
Table 6.2a PCR protocol showing aim, paternal templates, cycling parameters, polymerase, primers and results of the performed PCR’s. Products of shaded
boxes were used for further cloning or transformed into C. reinhardtii and tested with arylsulfatase essay.
x ul of DNA
1.step 2.step 3.step
4.step 5.step Cycles polymerase Primer1
Date/Aim
Primer2 Remarks
[10ng/ul]
(min/° (sec/°C) (sec/Ta°C) (sec/°C) (min/°C) (2-4)
C)
13/06/05
none
30/95 45/52
60/72 5/72
34
Taq
GpxhTATAssp Mano-lo control not ok
5/95
get promoter 5 pT7bluepro5
30/95
45/52
60/72
5/72
34
Taq
GpxhTATAssp
Mano-lo ok
5/95
30/95 45/54
60/72 5/72
34
Taq
GpxhTATAssp Mano-lo ok
fragment and 5 pT7bluepro5
5/95
establish
5 pT7bluepro5
30/95 45/56
60/72 5/72
34
Taq
GpxhTATAssp Mano-lo ok
5/95
PCR method 5 pT7bluepro5
30/95
45/58
60/72
5/72
34
Taq
GpxhTATAssp Mano-lo recommended
5/95
none
30/95 45/52
60/72 5/72
34
Taq
GpxhCREfor Mano-lo control ok
5/95
5 pT7bluepro5
30/95 45/52
60/72 5/72
34
Taq
GpxhCREfor Mano-lo ok
5/95
5 pT7bluepro5
30/95 45/54
60/72 5/72
34
Taq
GpxhCREfor Mano-lo ok
5/95
5 pT7bluepro5
30/95 45/56
60/72 5/72
34
Taq
GpxhCREfor Mano-lo ok
5/95
5 pT7bluepro5
30/95 45/58
60/72 5/72
34
Taq
GpxhCREfor Mano-lo recommended
5/95
14/06/05
none
get promoter 5 pT7bluepro5
fragment and 5 pT7bluepro5
establish
none
PCR method 5 pT7bluepro5
5 pT7bluepro5
5/95
5/95
5/95
5/95
5/95
5/95
30/95
30/95
30/95
30/95
30/95
30/95
45/58
45/58
45/58
45/58
45/58
45/58
60/72
60/72
60/72
60/72
60/72
60/72
5/72
5/72
5/72
5/72
5/72
5/72
34
34
34
34
34
34
Taq
Taq
Pfu
Taq
Taq
Pfu
GpxhTATAssp
GpxhTATAssp
GpxhTATAssp
GpxhCREfor
GpxhCREfor
GpxhCREfor
Mano-lo
Mano-lo
Mano-lo
Mano-lo
Mano-lo
Mano-lo
control not ok
little product
recommended
control ok
no product
recommended
23/06/05
get promoter
fragment and
establish
PCR method
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
45/55
45/55
45/55
45/58
45/61
45/64
45/55
45/55
45/55
45/58
60/72
60/72
60/72
60/72
60/72
60/72
60/72
60/72
60/72
60/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
34
34
34
34
34
34
34
34
34
34
Taq
Taq
Taq
Taq
Taq
Taq
Taq
Taq
Taq
Taq
GpxhCREfor
GpxhCREfor
GpxhCREfor
GpxhCREfor
GpxhCREfor
GpxhCREfor
GpxhTATAssp
GpxhTATAssp
GpxhTATAssp
GpxhTATAssp
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
control ok
no product
no product
no product
no product
no product
control ok
recommended
no product
little product
none
1 pASpro1
5 pASpro1
5 pASpro1
5 pASpro1
5 pASpro1
none
1 pASpro1
5 pASpro1
5 pASpro1
5 pASpro1
5 pASpro1
5/95
5/95
30/95
30/95
45/61
45/64
60/72
60/72
5/72
5/72
34
34
Taq
Taq
GpxhTATAssp Ars461rev little product
GpxhCREfor Ars461rev very little product
27/06/05
none
get promoter 5 pASpro1
fragment and 5 pASpro1
establish
5 pASpro1
PCR method 5 pASpro1
5 pASpro1
5 pASpro1
none
5 pASpro1
5 pASpro1
5 pASpro1
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
45/54
45/54
45/54
45/52
45/53
45/55
45/56
45/55
45/55
45/55
45/58
60/72
60/72
60/72
60/72
60/72
60/72
60/72
60/72
60/72
60/72
60/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
34
34
34
34
34
34
34
34
34
34
34
Taq
Taq
Pfu
Taq
Taq
Taq
Taq
Taq
Taq
Pfu
Pfu
GpxhCREfor
GpxhCREfor
GpxhCREfor
GpxhCREfor
GpxhCREfor
GpxhCREfor
GpxhCREfor
GpxhTATAssp
GpxhTATAssp
GpxhTATAssp
GpxhTATAssp
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
control ok
ok
no product
ok
ok
recommended
ok
control ok
recommended
no product
no product
none
5 pASpro1
5 pASpro1
none
5 pASpro1
5 pASpro1
5/95
5/95
5/95
5/95
5/95
5/95
30/95
30/95
30/95
30/95
30/95
30/95
45/55
45/55
45/55
45/55
45/55
45/55
60/72
60/72
60/72
60/72
60/72
60/72
5/72
5/72
5/72
5/72
5/72
5/72
34
34
34
34
34
34
Taq
Taq
Pfu
Taq
Taq
Pfu
GpxhCREfor
GpxhCREfor
GpxhCREfor
GpxhTATAssp
GpxhTATAssp
GpxhTATAssp
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
control ok
ok
no product
5 pASpro1
29/06/05
get promoter 5 pASpro1
fragment and 5 pASpro1
testing
5 pASpro1
of different
5 pASpro1
Polymerase 5 pASpro1
5 pASpro1
5 pASpro1
5 pASpro1
5 pASpro1
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
45/55
45/55
45/55
45/55
45/55
45/55
45/55
45/55
45/55
45/55
90/72
90/72
90/72
90/72
90/72
90/72
90/72
90/72
90/72
90/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
34
34
34
34
34
34
34
34
34
34
Taq
GpxhCREfor
GpxhCREfor
Pfu
Cloned Pfu GpxhCREfor
Accu Therm GpxhCREfor
Pfu Turbo GpxhCREfor
Taq
GpxhTATAssp
GpxhTATAssp
Pfu
Cloned Pfu GpxhTATAssp
Accu Therm GpxhTATAssp
Pfu Turbo GpxhTATAssp
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
ok
recommended
no product
recommended
no product
ok
little product
no product
little product
no product
1 pASpro1
5/95
30/95
45/53
90/72
5/72
34
Taq
28/06/05
get promoter
fragment and
establish
PCR method
30/06/05
control ok
ok
no product
GpxhTATAssp Ars461rev little product
get promoter
fragment and
establish
PCR method
1 pASpro1
1 pASpro1
5 pASpro1
5 pASpro1
5 pASpro1
1 pASpro1
1 pASpro1
1 pASpro1
5 pASpro1
5 pASpro1
5 pASpro1
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
5/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
30/95
45/53
45/53
45/53
45/53
45/53
45/55
45/55
45/55
45/55
45/55
45/55
90/72
90/72
90/72
90/72
90/72
90/72
90/72
90/72
90/72
90/72
90/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
5/72
34
34
34
34
34
34
34
34
34
34
34
Accu Therm GpxhTATAssp
GpxhTATAssp
Pfu
Taq
GpxhTATAssp
Accu Therm GpxhTATAssp
GpxhTATAssp
Pfu
Taq
GpxhTATAssp
Accu Therm GpxhTATAssp
GpxhTATAssp
Pfu
Taq
GpxhTATAssp
Accu Therm GpxhTATAssp
GpxhTATAssp
Pfu
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
Ars461rev
5 pASpro1
05/07/06
get promoter 5 pASpro1
5 pASpro1
fragment
5 pASpro1
5 pASpro1
5 pASpro1
5 pASpro1
5/95
5/95
30/95
30/95
30/95
45/53
45/53
45/53
90/72
90/72
90/72
5/72
5/72
5/72
34
34
34
Taq
30/95
30/95
30/95
30/95
45/55
45/55
45/55
45/55
90/72
90/72
90/72
90/72
5/72
5/72
5/72
5/72
34
34
34
34
30/95
30/55
90/72
5/72
30
GpxhTATAssp
Accu Therm GpxhTATAssp
GpxhTATAssp
Pfu
Accu Therm GpxhTATAssp
Taq
GpxhCREfor
Accu Therm GpxhCREfor
GpxhCREfor
Pfu
Taq
GpxhCREfor
dito
30/95
dito
30/55
dito
90/72
dito
5/72
30
30
dito
Taq
Ars461rev ok
Ars461rev little product
Ars461rev for ligation
(25ng/ul)
Ars461rev little product
Ars461rev ok
Ars461rev little product
Ars461rev for ligation
(25ng/ul)
Ars461rev found 3 clones
but
clone
dito
29=pCRETATO
Ars461rev control ok
5/95
5/95
5/95
5/95
5/95
12/8/06
8 "pCRETATO"
5/95
testing
47 potential clones dito
"pCRETATO" 5 pASpro1
5/95
dito
GpxhCREfor
ok
no product
ok
recommended
recommended
little product
ok
no product
ok
recommended
no product
Tab 6.2b: Aim, paternal templates, cycling parameters, primers and results of performed QuikChange®Site-Directed Mutagenesis (Stratagene). Products of
shaded boxes were transformed into Chlamydomonas reinhardtii and tested with arylsulfatase essay.
Date/Aim
x ul of DNA
1.step
2.step
3.step
4.step
5.step
[5ng/ul]
(sec/°C) (sec/°C) (sec/Ta°C) (min/°C) (min/°C)
31/08/05
5 pYS1
30/95
30/95
60/55
4/68
18
Pfu Turbo
tubdelTfor
tubdelTrev
no product
removal of
5 pYS2
60/55
4/68
18
Pfu Turbo
tubdelTfor
tubdelTrev
ok
pYS2mod
-
cycles polymerase Primer1
Primer2
Result
(2-4)
mutated
plasmid
30/95
30/95
1 nucleotide 5 pYS3
in tub
5 pBF28
30/95
30/95
60/55
4/68
18
Pfu Turbo
tubdelTfor
tubdelTrev
ok
pYS3mod
30/95
30/95
60/55
4/68
18
Pfu Turbo
tubdelTfor
tubdelTrev
ok
pBF28mod
sequence
5 pBF29
30/95
30/95
60/55
4/68
18
Pfu Turbo
tubdelTfor
tubdelTrev
no product
5 pBF30
30/95
30/95
60/55
4/68
12/09/05
7 pYS1
30/95
30/95
60/55
4/68
littel product pBF30mod
ok
pYS1mod
repetition
7 pBF29
30/95
30/95
60/55
4/68
11/11/05
6 pASpro2
30/95
30/95
60/55
mutation of
6 pASpro2
30/95
30/95
60/55
box 1-3
6 pASpro2
30/95
30/95
60/55
06/12/05
6 pASpro2
30/95
30/95
60/55
30/95
30/95
60/55
11/68
30/95
30/95
60/55
11/68
30/95
30/95
60/55
11/68
30/95
30/95
60/55
13/68
mut. box 2+3 6 pASpro2
30/95
06/02/06
6pT7bluepro5 30/95
other option 6pT7bluepro5 30/95
30/95
60/55
13/68
30/95
60/55
4/68
30/95
60/55
4/68
mut. box 2+3 6 pASpro2
23/01/06
6 pASpro2
mut. box 2+3 6 pASpro2
30/01/06
6 pASpro2
18
Pfu Turbo
tubdelTfor
tubdelTrev
-
18
Pfu Turbo
tubdelTfor
tubdelTrev
18
Pfu Turbo
11/68
+ 1ul Pfu
2x9
Pfu Turbo
tubdelTfor
tubdelTrev
ok
pBF29mod
CGCC140TTAT+for CGCC140TTAT+rev littel product pGC1
11/68
and repeat 2x9
Pfu Turbo
CAAT155GCGfor
11/68
step 1-4
2x9
Pfu Turbo
ATTG185GCGTfor
ATTG185GCGTrev littel product
11/68
dito
2x9
Pfu Turbo
CAAT155GCGfor
CAAT155GCGrev
2x9
Pfu Turbo
ATTG185GCGTfor
ATTG185GCGTrev littel product
2x9
Pfu Turbo
CAAT155GCGfor
CAAT155GCGrev
2x9
Pfu Turbo
ATTG185GCGTfor
ATTG185GCGTrev littel product
dito
2x9
Pfu Turbo
CAAT155GCGfor
CAAT155GCGrev
2x9
Pfu Turbo
ATTG185GCGTfor
ATTG185GCGTrev littel product pATTG3
5/68
18
Pfu Turbo
CAAT155GCGfor
CAAT155GCGrev
18
Pfu Turbo
ATTG185GCGTfor
ATTG185GCGTrev littel product
dito
CAAT155GCGrev
littel product
littel product
littel product
littel product pCAAT2
littel product
Tab 6.3: Parameters of Co-Transformation of plasmids into C. reinhardtii: Date, OD750nm of algae for the transformation, volume used for one transformation,
Co-plasmid, plasmid, number of replicates per plasmid, number of clones grown after the transformation, clones of interest with both plasmids integrated (shaded
column) and remarks.
Date/ TNr
Algae
OD750
14/10/05
T1
18/10/05
T2
0.100
0.086
Co-Plasmid
(ml/transformation
(2ug DNA)
)
Plasmid
Replicate Output
(2ug DNA)
Remarks
(clones/plate) ( blue clones)
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
pCRETA1mod
pCRETA2mod
pTA3mod
pBF31mod
pBF32mod
pBF33mod
pCRETA1
pCRETA2
pTA3
pBF31
pBF32
pBF33
pCRETATO
pASpro1
pASpro4
pASpro2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
25ml
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
pCRETA1mod
pCRETA2mod
pTA3mod
pBF31mod
pBF32mod
pBF33mod
pCRETA1
pCRETA2
pTA3
1
1
1
1
1
1
1
1
1
3.5
13
4.5
0
0
0
0
0
0
1
-most of the
plasmids are
still solved
in TE buffer
could be
inhibiting
1
1
1
1
-most of the
plasmids are
still solved
in TE buffer
could be
inhibiting
31/10/05
T3
11/11/05
T4
mixture of
algea
with OD:
0.130
0.060
0.066
0.105
25ml
25ml
25ml
25ml
25ml
25ml
25ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
pBF31
pBF32
pBF33
pCRETATO
pASpro1
pASpro4
pASpro2
pCRETA1mod
pCRETA2mod
pTA3mod
pBF31mod
pBF32mod
pBF33mod
pCRETA1
pCRETA2
pTA3
pCRETATO
pBF31
pBF32
pBF33
pASpro1
pASpro2
pASpro4
pASpro4 +TE
1
1
1
1
1
1
1
2
2
3
1
1
1
1
1
1
3
1
1
1
1
1
1
1
0
0
0
0
0
25
0
0
127.5
42.5
20
37.5
10
2.5
95
95
60.6
175
35
17.5
41.5
1
1
0.5
40ml
40ml
40ml
40ml
40ml
40ml
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
pCRETA1mod
pCRETA2mod
pTA3mod
pBF31mod
pBF32mod
pBF33mod
?
?
?
?
?
?
?
?
?
?
?
?
2
2
-plasmids of
new Midiprep
solved in
dH2O
2
-new Midi of parg7.8
-fresh PEG
2
-after transformation
incubation
at room temp
for a few
hours
- but: bacterial
contamination!!!!
1
16/11/05
?
T5
18/11/05
T6
?
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
pCRETA1
pCRETA2
pTA3
pCRETATO
pBF31
pBF32
pBF33
pASpro1
pASpro2
pASpro4
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
pCRETA1mod
pCRETA2mod
pTA3mod
pBF31mod
pBF32mod
pBF33mod
pCRETA1
pCRETA2
pTA3
pCRETATO
pBF31
pBF32
pBF33
pASpro1
pASpro2
pASpro4
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
1
1
2
2
2
40ml
40ml
40ml
40ml
parg7.8
parg7.8
parg7.8
parg7.8
pCRETA1mod
pCRETA2mod
pTA3mod
pBF31mod
?
?
?
?
?
?
?
?
3
4
2
2
1
1
1
1
1
1
01/12/05
T7
09/12/05
T8
?
0.081
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
pBF32mod
pBF33mod
pCRETA1
pCRETA2
pTA3
pCRETATO
pBF31
pBF32
pBF33
pASpro1
pASpro2
pASpro4
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
8
2
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
40ml
50ml
50ml
50ml
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
pCRETA1mod
pCRETA2mod
pTA3mod
pBF31mod
pBF32mod
pBF33mod
pCRETA1
pCRETA2
pTA3
pCRETATO
pBF31
pBF32
pBF33
pASpro1
pASpro2
pASpro4
pGC1
pCRETA1mod
pCRETA2mod
4
4
4
3
4
4
1
1
1
3
1
1
1
3
3
3
2
4
4
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
3
1
1
4
4
2
5
6
8
1
-more replicates
for the important
14/12/05
T9
March/06
T10/11/12
R. Dayer
0.118
?
50ml
50ml
50ml
50ml
50ml
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
pTA3mod
pCRETATO
pASpro1
pASpro2
pASpro4
4
4
2
2
2
?
?
?
?
?
3
7
6
5
9
50ml
50ml
50ml
40ml
40ml
40ml
50ml
50ml
50ml
50ml
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
parg7.8
pCRETA1mod
pCRETA2mod
pTA3mod
pBF31mod
pBF32mod
pBF33mod
pCRETATO
pASpro1
pASpro2
pASpro4
5
5
5
3
3
3
5
2
2
2
?
?
?
?
?
?
?
?
?
?
1
2
?
?
?
parg7.8
parg7.8
parg7.8
pCAAT2
pATTG3
pATTG3
4
4
3
30
28.75
10
19
5
0
clones
2
5
2
clones blurred
clones blurred
Appendix
80