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University
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G e y e r , P am ela K ent
CONSTRUCTION OF A RECOMBINANT COMPLEMENTARY-DNA LIBRARY
AND ISOLATION OF A PLASMID CONTAINING THYMIDYLATE SYNTHETASE
COMPLEMENTARY-DNA SEQ UENCES
Ph.D.
The Ohio State University
University
Microfilms
International
300 N. Zeeb R oad, Ann Arbor, MI 48106
Copyright 1983
by
Geyer, Pamela Kent
All Rights Reserved
1983
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University
Microfilms
International
Construction of a Recombinant cDNA Library and Isolation of a
Plasmid Containing Thymidylate Synthetase cDNA Sequences
Dissertation
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Pamela Kent Geyer, B.Sc.
*****
The Ohio State University
1983
Reading Committee:
Approved By
Dr. Lee F. Johnson
Dr. George Marzluf
Dr. David Ives
fvwA'riA
Advisor
Department of Biochemistry
To John
ACKOWLEDGEMEOTS
I would like to thank Dr. Johnson for his constant help and
encouragement during my graduate training.
I would also like to
thank all the people in the laboratry and department whose
friendships have helped me through these years.
VITA
August 21,
1956.......
Born- Teaneck, N.J.
1978...................
B.Sc., Biochemistry,
McGill University, Montreal,
P .Q ., Canada
Research Assistant, The Ohio
1978-1983..............
State University,
Columbus, Ohio.
PUBLICATIONS
Regulation of Ribosomal Protein mRNA Content and Translation
in Serum Stimulated 3T6 Cells. Pamela K. Geyer and Lee F.
Johnson,
(1981), J.Cell.Biol 91:373a.
Regulation of Ribosomal Protein Messenger RNA Content and
Translation in Growth Stimulated Mouse Fibroblasts.
Pamela
K. Geyer, Oded Meyuhas, Robert Perry, and Lee ,F. Johnson,
(1982), Mol.Cell.Biol 2:
685-693.
Regulation of Thymidylate Synthetase Gene Expression in
Overproducing Cells.
Lakshmi Rao, Cindy Rossana, Pamela
Geyer, Lee F. Johnson,
(1982), J.Cell.Biol 95:455a.
FIELD OF STUDY
Major Field:
Molecular Biology
TABLE OF CONTENTS
DEDICATION....................................................
ii
ACKNOWLEDGEMENTS.............................................
iii
VITA..........................................................
iv
LIST OF TABLES................................................ vii
LIST OF FIGURES.....................
viii
ABBREVIATIONS.................. ...............................
x
INTRODUCTION..................................................
1
MATERIALS and METHODS
Cell culture.............................................. 19
Nucleic acid
isolation..................................
20
Construction
of recombinant cDNA plasmids..............
23
Bacterial procedures.....................................
28
Gel electrophoresis......................................
32
Preparation of probes....................................
40
Characterization of recombinant cDNA library............ 41
RESULTS
Construction
of a recombinant cDNA library.............
47
Differential
colony filter hybridization...............
65
Identification of a plasmid containing thymidylate
synthetase coding sequences.............................. 71
Restriction Analysis of.. p 4 .............................
80
Analysis of thymidylate synthetase messenger RNA size... 80
v
Structure of thymidylate synthetase gene................ 87
DISCUSSION ................................................... 91
APPENDIX A ....................................................
100
APPENDIX B ...................................................
103
BIBLIOGRAPHY.................................................
108
vi
LIST OF TABLES
Table
Page
1. Characteristics of the double stranded
cDNA preparation used to construct the
recombinant cDNAlibrary.................... .........
58
2. Effects of annealing buffer and carrier
tRNA on the transformation frequency of
closed circularpBR322................................
63
3. Comparisons of transformation frequencies obtained
from cDNA annealed with different amounts
of pBR322............................................
64
LIST OF FIGURES
Figure
Page
1.
The biochemistry of thymidylate synthetase............
2.
The strategy used to construct a recombinant
cDNA library...........................................
3.
4.
Comparison of two methods used for cDNA synthesis
5.
Effect of vanadyl ribonucloside complexes
on cDNA synthesis......................................
54
57
60
Estimation of background levels in colony
filter hybridization analysis.........................
9.
51
Method for inserting double stranded cDNA into
pBR322 by blunt end ligation..........................
8.
48
Sucrose gradient profile of cDNA used for the
construction of the recombinant library...............
7.
16
Comparison of in vivo and in vitro protein
synthesis from 3T6, M50L3 and and LU3-7 cells........
6.
4
66
Determination of sensitivity of colony
filter hybridization...................................
69
10. Differential filter colony hybridization analysis of
a filter containing recombinant cDNA colonies........
72
11. In vitro translation of messenger RNA selected
by hybridization to a mixture of recombinant plasmids.
75
12. In vitro translation and immunoprecipitation
of p4 selected messenger R N A ..........................
viii
78
13. In vitro translation and immunoprecipitation
14.
to confirm identity of p 4 ................. ............
81
Restriction map of p 4 .................................
83
15. Analysis of thymidylate synthetase
messenger RNA in LU3-7, M50L3 and 3T6 cells...........
16.
85
Southern blot analysis of genomic DNA from
the resistant and sensitive cell lines................
88
ABBREVIATIONS
MTX
methotrexate
5FdU
5 fluorodeoxyuridine
SDS
sodium dodecyl sulfate
TCA
trichloroacetic acid
LB
Luria broth
Tet
tetracycline
TEMED
tetramethylethylenediamine
PBS
phosphate buffered saline
x
INTRODUCTION
Mechanisms involved in the regulation of gene expression in
eukaryotic cells are poorly understood.
Advances in the area of
recombinant DNA technology have allowed for a mere detailed
examination of these control processes.
Purified DNA sequences
have been isolated and used as probes to quantitate levels of
RNA, in addition to determining the structure and organization of
particular genes.
These investigations have indicated that each
cell possesses a large number of ways by which it can regulate the
expression of a gene during the conversion of information from
DNA into protein.
Much of the current knowledge in this area has
come from work on of viral genes as well as genes which code for
highly abundant proteins. Transcriptional control of gene
expression has been the predominant mode of regulation
demonstrated (Darnell, 1982).
Following transcription,
heterogeneous nuclear RNA must be polyadenylated, processed and
transported into the cytoplasm where it may be translated, turned
over or stored in a ribonucleoprotein particle for use at a later
time.
Examples of regulation at many of these other levels have
also been found (Darnell, 1982).
Most control mechanisms demonstrated have been for tissue
specific proteins and messenger RNA since their high
intracellular levels have facilitated the isolation of both
1
2
protein and DNA probes. Since these proteins represent the
differentiation products of only one cell type and are expressed
at very high levels, mechanisms involved in regulating their gene
expression may or may not reflect those for the majority of
cellular genes.
It is important to study factors which control the
expression of genes for proteins required by all cell types
(housekeeping proteins), since these are the proteins required
for the maintainence of normal cellular metabolism.
Changes in
the expression of genes for maintainence proteins have been
suggested to result in the formation of a transformed phenotype
(VJeinberg, 1982). Housekeeping enzymes are members of this class
of proteins. These enzymes have been difficult to study because
they are present at very low levels in the cell. Although many of
these enzymes may be constitutively expressed, the expression of
some housekeeping enzymes has been found to be regulated.
The
activities of a number of enzymes have been found to increase as
cells enter the DNA synthetic phase of the cell cycle and are
known as S phase enzymes. This class includes thymidylate
synthetase, dihydrofolate reductase, cytidylate deaminase,
ribonucleotide reductase and thymidine kinase (Prescott, 1976).
Thymidylate synthetase is the enzyme involved in the
de novo synthesis of thymidylic acid
(dTMP).
It catalyzes the
reductive methylation of the 5 position of deoxyuridylate (dUMP)
to form thymidylate with concomitant oxidation of the cofactor
3
N5-N10 methylene tetrahydrofolate to dihydrofolate (figure 1)
(Walsh, 1979).
Initial studies demonstrated that thymidylate
synthetase activity was elevated in actively proliferating cells
such as found in embryonic tissues, tumor cells and regenerating
rat liver as compared to cells of nondividing tissues such as
normal liver and brain (Maley and Maley,
1959).
Changes in the
cell cycle patterns of thymidylate synthetase activity have been
investigated in many systems.
Changes in thymidylate synthetase
activity during both the culture cycle and cell cycle were
investigated in Don Chinese hamsters cells. During the culture
cycle, assays of thymidylate synthetase activity in cell extracts
demonstrated low levels of activity during the initial lag period
of growth, which increased sharply during log phase growth
(Conrad, 1971).
Cell cycle investigations, using colcemid
synchronization, demonstrated that thymidylate synthetase
activity increased approximately 2 fold, beginning at the start
of S phase.
Furthermore, this seemed to result from an increase
in enzyme content, since puromycin (an inhibitor of translation)
and actinomycin (an inhibitor of transcription) could prevent the
increase in activity (Conrad and Ruddle, 1972). When the cell
cycle regulation of thymidylate synthetase was studied in mouse
L1210 leukemia cells somewhat different results were obtained.
When these cells were synchronized by isoleucine deprivation (18
hours) followed by hydroxyurea treatment (10 hours), thymidylate
m ethylene
H^folate
H2 folate
V
dU M P'2s*— = = = - > dTMP
ATHYM IDYIATE /fv
SYNTHETASE
>
------ > dTTP
X.
5F-dUMP
/ N THYMIDINE
KINASE
5F-dUrd
I
TdR
Figure 1. The biochemistry of thymidylate synthetase.
DNA
synthetase activity was shown to increase as cells entered S
phase only when its activity was assayed in vivo.
Cytoplasmic
extracts showed no change in the specific activity of thymidylate
synthetase.
The S phase increase in thymidylate synthetase
activity was attributed to regulation by thymine nucleotide(s)
(Rode et al., 1980).
Apparent differences in regulation of
thymidylate synthetase may have resulted from the methods used to
study the cell cycle changes.
Mouse 3T6 fibroblasts provide a good model system with which
to study cell cycle events.
Cell synchronization can be achieved
without drug treatment or removal of an essential nutrient. Cells
are synchronized by lowering the concentration of serum growth
factors in the medium.
When 3T6 cells are placed in a medium
containing 0.5% serum, they stop growing when they reach
confluence and enter a reversible branch of the G1 state,
(GO),
in which there is no net synthesis of protein or RNA, and DNA
synthesis is occurring at a rate of less than 0.5% of that
occuring in exponentially growing cells.
Cultures of 3T6 cells
may be maintained in medium containing low serum concentrations
for extended periods of time.
This is a particularly useful
feature of the system because it allows for the decay of proteins
and messenger RNA that were present in the preceding cycle. Cells
can be stimulated to re-enter the cell cycle synchronously by
increasing the serum concentration in the medium to 10%.
In mcst
cases, 80-90% of the resting population can be induced to undergo
DNA. synthesis (Santiago et a l , submitted).
In these cultures,
DNA synthesis begins at about 12 hours and mitosis is observed by
24 hours.
The prolonged G0-G1 transition is another advantage of
the system because it allows for detailed examination of events
critical to DNA synthesis.
The regulation of thymidylate synthetase gene expression was
studied using mouse 3T6 fibroblasts. The specific activity of
thymidylate synthetase in growing cultures was about 20 fold
higher than that in resting cultures.
Following serum induction
of resting cultures, thymidylate synthetase activity remained low
for the first 10 hours,
and increased dramatically at the same
time the cells entered S phase. Studies using orotein and RNA
synthesis inhibitors suggested that the increase in thymidylate
synthetase activity resulted
from an increase in de novo
synthesis of the enzyme as a result of a transcriptional event.
The evidence in this system indicates that an increase in enzyme
activity is a result of an increased enzyme content (Navalgund
et a l ., 1980) .
It should be noted that cell cycle regulation of
thymidylate synthetase in this system is very similar to that
observed for other S phase enzymes such as thymidine kinase and
dihydrofolate reductase
(Johnson et a l ., 1978,
Johnson et a l .,
1982).
The fact that an increase in thymidylate synthetase content
is not observed in L1210 cells may reflect differences in the
physiological state of the cell.
For example, the presence of
hydroxyurea may stabilize thymidylate synthetase so that, even in
cells not engaged in DNA. synthesis,
its activity remains high.
Removal of hydroxyurea may result in degradation of the
accumulated enzyme which is often offset by an increase in
de novo synthesis as a result of growth stimulation.
even though de novo synthesis
is occuring,
Therefore,
it will not be
detected by enzyme- assay.
Experiments in which the in vivo activity of thymidylate
synthetase was studied have indicated that this enzyme is also
regulated (in a negative manner) at the post translational level
(Rode et a l ., 1980).
It was suggested that a decrease in
thymine nucleotides is responsible for thymidylate synthetase
activation.
However, the concentrations of either dTMP or dTTP
needed to inactivate the enzyme in vitro were much higher than
in vivo levels.
Inactivation by these nucleotides might occur
if there were some compartmentalization of dTMP and dTTP with
thymidylate synthetase. Thymidylate synthetase, along with other
S phase enzymes, has been postulated to be a member of a
replitase complex involved in DNA synthesis.
The existence of
such a complex was initially proposed to explain observations
such as the high rates of chain elongation during DNA synthesis,
the fact that pools of deoxyribonuleotide triphosphates are very
low during S phase compared to the Km of DNA polymerase for
in situ DNA synthesis,
and DNA synthesis
is initiated only
after a G1 period of RNA and protein synthesis, even though DNA
polymerase and its substrates are not limiting at this time (Prem
veer Reddy and Pardee, 1980). More recent evidence supports the
existence of a replitase (Prem veer Reddy and Pardee, 1982).
Therefore, the observed post translational control may be exerted
when the enzyme is present in this complex.
Thymidylate
synthetase activity may be high when all the enzymes are
assembled in the complex and are active.
If DNA polymerase is
inhibited, the pools of compartmentalized dTMP and dTTP become
sufficiently high to feedback inhibit thymidylate synthetase. The
total cellular pools of these nucleotides do not have to increase
dramatically to change thymidylate synthetase activity.
The
advantage of this is may be to allow the cell to utilize energy
more efficiently.
More detailed studies quantitating the rate of synthesis of
thymidylate synthetase or the amount of thymidylate synthetase
messenger RNA under various growth conditions would be extremely
difficult due to their low levels in 3T6 cells. Studies of this
sort have been facilitated by the isolation of a cell lines which
overproduce the enzyme of interest.
It has been found that cells
grown in the presence of certain enzyme inhibitors can survive by
overproducing the target enzyme.
Higher levels of overproduction
can be achieved by growing cultures in higher and higher
concentrations of the inhibitor.
This method was originally used
to isolate cultures which overproduce the enzyme dihydrofolate
reductase,
as a result of prolonged growth in methotrexate (Alt
et a l ., 1 9 7 6 ) .
It has
since been used to isolate cells which
overproduce the aspartate transcarbamylase complex (N-(phosphonacetyl)-L-aspartate resistant)
adenylate deaminase
1982),
(Kempe et al., 1 9 7 6 ) ,
(coformycin resistant)
(Debatisse et a l .,
arginosuccinate synthetase (canavanine resistant)
et a l ., 1 9 8 1 ) and metallothionein
(Hildebrand et a l ., 1 9 7 9 ) .
(Su
(cadmium resistant)
The overproduced enzyme can become
one of the most abundant proteins in the cell. In one cell line,
dihydrofolate reductase represented 30% of the total soluble
protein (Milbrandt et a l . , 1 9 8 1 ) .
far, protein overproduction
In all cases examined so
has resulted frcm a corresponding
increase in the amount of the specific messenger RNA (Kellems et
a l ., 1 9 7 6 ;
Padgett,
Palmiter, 1 9 8 1 ) .
et a l ., 1 9 7 9 ;
Su et a l ., 1 9 8 0 ;
Beach and
In all but one case (arginosuccinate synthetase)
this increase in messenger RNA is paralleled by an increase in the
number of copies of the gene
1979;
Su et a l . , 1 9 8 1 ;
( Alt,
et a l . , 1 9 7 8 ;
Wahl, et a l .,
Beach and Palimiter, 1 9 8 1 ) .
Several laboratories observed that cultures selected for
resistance to 5-fluorodeoxyuridine (5-FdU) had an elevated level
of thymidylate synthetase relative to parental cells (Baskin et
10
a l ., 1975; Wilkinson et a l ., 1977;
Priest and Ledford, 1980).
In vivo, 5-FdU can be phosphorylated by thymidine kinase into
5-fluorodeoxyuridylate (5-FdUMP), which has been found to be the
actual inhibitor of thymidylate synthetase.
In the presence of
methylene tetrahydrofolate, FdUMP forms an extremely stable,
covalent ternary complex with thymidylate synthetase and blocks
further enzyme action.
The dissociation constant for this
complex is in the range of 10
(Danenberg et a l . , 1974).
Fluorodeoxyuridine causes cell death by inhibiting thymidylate
synthetase which leads to a depletion of dTMP and inhibition of
DNA synthesis.
For this reason,
it has been used as a
chemotherapeutic agent.
In all of the resistant cell lines studied previously
(Baskin et a l ., 1975; Wilkinson et a l ., 1977;
Priest and
Ledford, 1980), the level of thymidylate synthetase
only two to eight fold over the parental cells.
was increased
A cell line
resistant to high concentrations of 5-FdU was obtained in the
following manner (Rossana et a l . , 1982).
Initially,
cultures
of M50L3 were placed in medium containing low levels of
5-FdU.
Uridine and cytidine were included in the medium
to prevent toxicity from metabolic products of this drug.
Cells
which survived this treatment were exposed to gradually increasing
concentrations of 5-FdU.
Since the inhibition of thymidylate
synthetase relies on the phosphorylation of 5-FdU, cells
11
defective in thymidine kinase would also be drug resistant.
To
eliminate this mechanism, cells were periodically grown in medium
containing hypoxanthine, aminopterin and thymidine.
In this
medium, the de novo pathway for thymidylate synthesis is
blocked.
Therefore, only cells with an active thymidine kinase
will survive.
A cell line, designated LU3-7, was isolated which was 3000
times more resistant to 5-FdU than the parental cell line. The
specific activity of thymidylate synthetase in these cells was 50
times higher than in parental cells.
These cells also contained
slightly elevated levels of dihydrofolate reductase and reduced
levels of thymidine kinase.
Thymidylate synthetase was estimated
to represent between 0.1-1% of total soluble protein. The
overproduced enzyme was partially characterized by determining
its molecular weight and its affinity for 5-FdU.
At least for
these criteria, the overproduced enzyme was found to be the same
as the wildtype enzyme
(Rossana et al., 1982).
The
overproducing cell line was found to regulate thymidylate
synthetase activity in a similar manner to the parental cell
line (Rao, Jerih, and Johnson, unpublished results).
Therefore,
this cell line is a good model system with which to study cell
cycle regulation of the thymidylate synthetase gene.
By analogy to other overproducing cell lines, the increased
amount of thymidylate synthetase should have resulted from an
increase in the level of messenger RNA.
The increase in
thymidylate synthetase messenger RNA should permit investigations
of the cell cycle metabolism of this messenger RNA.
These
experiments cannot be done without a probe with which to
quantitate these sequences.
One possible approach to isolating DN^ sequences for a
particular protein involves selection of gene sequences from a
genomic library.
This library may be constructed by obtaining a
partial restriction digestion of total cellular DMA and inserting
it into an appropriate vector, a defective
X
phage or a cosmid. To
completely represent all of the DNA sequences in the genome, a
library containing between 500,000 and 1,000,000 independent
clones is needed.
Since such a large number of colonies must be
analyzed, this approach is used only if appropriate screening
techniques or a purified nucleic acid probe are available to
identify the gene of interest. The structural gene for yeast
thymidylate synthetase was isolated in this manner (Taylor et a l .,
1982).
Total yeast DNA was cloned into a high frequency yeast
transforming vector and inserted into other yeast cells which were
defective in thymidylate synthetase
(TS ).
Cells which were able
to grow in the absence of thymidine were found to contain the gene
for yeast thymidylate synthetase.
In a similar manner, the human
gene sequences for thymidylate synthetase have been partially
purified (Ayusawa et a l . , in press).
Thymidylate synthetase
13
deficient incuse cells were transformed with human DNA, and
thymidine prototrophs were isolated.
Stable transformants were
screened and found to contain human DNA.
The isolation of the
human thymidylate synthetase gene awaits the construction of a
genomic library from rtouse prototrophs and identification of
human sequences in this library. Even when the genomic sequences
have been isolated there is difficulty in assigning the exact
boundaries of the protein coding regions (for example,
intron-exon positions) since comparisons to the messenger RNA
sequence cannot be done.
A second approach to isolating the DNA sequences for a
particular protein is the construction of a cDNA library.
Recombinant cDNA libraries are constructed using a series of
enzymatic reactions which convert messenger RNA into double
stranded DNA which can then be inserted into a bacterial plasmid.
Since these libraries are made from messenger RNA, only genomic
sequences which are expressed will be represented.
This results
in a significant enrichment of relative to genomic libraries
therefore requiring analysis of fewer colonies to isolate the
sequences of interest.
Either total poly(A)
+
rNA
or poly(A)
+
RNA enriched for a particular RNA may be used to copy into DNA.
In many of the early examples of the isolation of cDNA seqences,
the messenger RNA was easily enriched and so this was the method
used.
A disadvantage of prior enrichment of RNA is that the
resulting recombinant cDNA library may not be useful for the
isolation of other cDNA sequences,
the entire RNA population.
since it will not represent
The cDNA sequences for ovalbumin were
cloned from a fraction of purified messenger RNA.
Normally,
ovalbumin represents 60% of total soluble protein in estrogen
treated cells.
Enrichment of ovalbumin messenger RNA by sucrose
gradient fractionation resulted in a preparation of 95% pure
ovalbumin messenger RNA from which double stranded cDNA was made
(Monahan et a l ., 1976).
The cDNA sequences for dihydrofolate
reductase were also enriched prior to their insertion into a
vector. Dihydrofolate reductase coding sequences were purified by
exploiting the specific increase in dihydrofolate reductase
messenger RNA in overproducing cells (methotrexate resistant)
relative to that in methotrexate sensitive cells. Complementary
DNA was made from resistant poly(A)+ RNA which was isolated
from polysomes immunoprecioitated with anti-dihydrofolate
reductase antiserum, as a preliminary purification.
Since the
only apparent RNA difference between the two cell lines was the
amount of dihydrofolate reductase messenger RNA (220-300 fold
greater in resistant cells), this cDNA was hybridized to excess
RNA from sensitive cells. The unhybridized cDNA sequences were
found to contain 65% dihydrofolate reductase sequences.
This
cDNA was then used in the construction of recombinant plasmids
(Alt et a l . , 1978,
C h a n g et a l . , 1978).
15
Prior purification of RNA species has also been done when
it is known that the size of a particular RNA is much different
than bulk RNA.
For example, the metallothionein proteins are very-
small (61 amino acids) and their messenger RNA is much smaller
than most RNAs.
Following sucrose gradient fractionation the
messenger RNA for metallothionein could be enriched 550 fold
(Durnan et a l ., 1980).The advantage of such procedures is the
reduction in the amount of screening that has to be done to isolate
a given sequence.
Partial purification of thymidylate synthetase messenger RNA
from LU3-7 was not feasible.
A procedure comparable to that used
to enrich dihydrofolate reductase coding sequences was not
readily applicable, due to the relatively low amount of
thymidylate synthetase messenger RNA in LU3-7 cells.
Furthermore,
thymidylate synthetase has a monomeric weight of about 38,000
daltons (Rossana et a l ., 1982).
Therefore,
a messenger RNA of
at least 1150 nucleotides would be needed to code for this
protein.
Fractionation by size would not give very much
purification, since this is the average size for most messengers
RNAs.
For these reasons, the recombinant cDNA library was made
from total poly(A)+ RNA from LU3-7 cells as outlined in figure 2.
The isolation of thymidylate synthetase cDNA was therefore
dependent upon a good method with which to screen the recombinant
library.
A screening procedure, which has been used to detect
3'
mRNA
3'
c D N A C.
c
OH
JiAAA
.
I Reverrse.
gcriptase
5'
E
c o
R I
PstI
TTTT
Reverse
ranscriptase
Tet
TTTT
1
SI nuclease
D N A polymerase
PstI digestion
G
C.
Size selection
Terminal transferase
ccccc-
I
------ C T G C A
------ G
Terminal
transferase
CTGCAGGG
-CCCCC
t
G
Anneal
■G
CCC
•CTGCAGGG
cDNA
■GGGACGTC
■CCC
G
Transformation
Host repair
OACGTCCC.
.CTGCAGGG.
PstI
cDNA
GGGACGTC
CCCTGCAG
PstI
Figure 2. The strategy used to construct a cDNA library.
17
specific sequences for which a purified probe is not available,
is differential hybridization. Since certain messenger RNAs are
expressed only during some growth states, the library can be
screened with probes in which the sequences of interest are either
present or absent.
Colonies are chosen which show hybridization
under the correct conditions.
This procedure was first
used by St. John and Davis (1979) to detect galactose inducible
sequences. It has been estimated that this technique can detect
cloned messenger RNA sequences present in the original population
as low as 0.05%-0.1% of the total messenger RNA (Gergen et a l .,
1979; Crampton et a l . , 1980;
Dworkin and Dawid, 1980).
In
fact, this procedure was used for the isolation of the cDNA
sequences for arginosuccinate synthetase from cells in which this
enzyme represented only 0.6% of the soluble protein and 0.2-.03%
of total cellular protein
(Su et a l ., 1981).
Since thymidylate synthetase was estimated to represent
0.1-1% of total soluble protein in the overproducing cell line
(Rossana et a l ., 1982),
the use of differential hybridization to
screen the recombinant library seemed like a reasonable approach.
The bank was screened in triplicate with
32
P-cDNA made from resting
3T6, growing 3T6 and growing LU3-7 po l y ( A ) + RNA.
enzymatic activities,
From analysis of
as well as translatable RNA, in these different
cell lines, it was expected that colonies containing thymidylate
synthetase sequences would only be detected when hybridized with
18
"^P-cDNA of LU3-7 cells.
In this way, coding sequences for thymidylate synthetase were
isolated.
This represents the first isolation of cDNA sequences for
thymidylate synthetase.
These sequences will he useful for the
detailed examination of the gene structure and sequence, as well as
messenger RNA size and sequence.
The cell cycle regulation of
this messenger RNA can now be studied.
Finally, all this
information can be related to observations made for the
regulation of gene expression for other S phase enzymes.
This
could lead to understanding the general mechanisms used by the
cell to control the expression of this set of genes.
MATERIALS and METHODS
Cell
culture
Cultures of mouse 3T6 fibroblasts (Todaro and Green, 1963),
methotrexate-resistant M50L3 (Wiedemann and Johnson, 1979) and
5-FdU resistant LU3-7 cells
(Rossana et al., 1982) were
maintained on plastic petri dishes in the Dulbecco-Vogt
modification of Eagle’s medium supplemented with 10% calf serum
(Colorodo Serum).
Cells were incubated at 37°c in an atmosphere
of 10% carbon dioxide.
Stock cultures of M50L3 and LU3-7 were
grown in the presence of 50 uM MTX'and 3 uM 5-FdU plus ImM uridine
and cytidine,
respectively.
Routinely,
fresh cultures of cells
were thawed every 3 months.
Large quantities of exponentially growing cells were
prepared by seeding a half confluent plate (100x20mm) of cells
into roller bottles containing 100 ml of medium containing 10%
serum.
Cells were grown until they reached a density of about
3/4 confluence,
requiring 3-4 days of growth.
Cultures in roller
bottles were fed the day before use.
Large quantities of resting cells also were isolated from
roller bottles.
Roller bottles were seeded as before.
When
cells reached 3/4 confluence, the medium was replaced with medium
containing 0.5% calf serum (day 1).
Cells were fed on the third
and fifth days following medium replacement and used on the
19
20
eighth day.
Nucleic Aci d Isolation
A. RNA Isolation
Cytoplasmic RNA was isolated from polysomes by a
modification of the magnesium precipitation procedure described
by Palmiter (1974). In order to eliminate ribonuclease activity,
a few drops of diethylpyrocarbonate (Kodak) were added to all
solutions, which was inactivated either overnight at room
temperature or by autoclaving. Glassware was baked for 6 hours
before use.
All procedures were
done at 0-4°C.
Roller
bottles were rinsed five times with 50 ml of PBS. Cells were
scraped into PBS (10 ml per roller bottle) and collected by
centrifugation at 800xg for 2 minutes (min).
They were
resuspended in 2.5 volumes of a lysis buffer, transferred to a
homogenizer and lysed with several strokes of the pestle.
Nuclei
and cell debris were removed by centrifugation at 10,000 rpm for
5 min. in an SS-34 rotor (Sorvall). Polysomes were precipitated
from the cell extract by addition of 1/4 volume of a 5x
precipitation buffer.
After at least one hour, the solution was
layered on a half volume of centrifugation buffer and centrifuged
at 7000 rpm for 45 min.
in the HS-4 rotor (Sorvall). Protein was
removed using the guanidine hydrochloride extraction procedure
(Strohman et a l ., 1977).
The polysome pellet (from 2-3 roller
bottles) was resuspended in 4.5 ml of 6M guanidine hydrochloride
21
and 0.5 ml of 0.25 M EDTA (pH 7). Once the pellet was dissolved,
potassium acetate (2fl, pH 5) was added to a final concentration of
0.1M.
RNA was precipitated by adding a half volume of ethanol and
incubating the solution at -20°C for 12-16 hours.
The
precipitate was collected by centrifugation at 5000 rpm for 10
min. at -20°C in the HS3-4 rotor.
These
remove as much protein as possible.
steps were repeated to
The final precipitate was
resuspended in 1.5 ml of 0.02M EDTA (pH 7) and extracted with 3
volumes of butanol:chloroform (4:1).
The aqueous phase was
removed and the organic phase was re-extracted with 0.02M EDTA
(pH 7) to insure complete recovery of the RNA.
The aqueous
o
phases were pooled and precipitated overnight at -20 C by
addition of 4.5M sodium acetate (pH 6) to a final concentration
of 3M.
The high salt precipitate was collected by centrifugation
at -20°C for 1 hour at 7000 rpm in the HSB-4 rotor, rinsed with
-20°C ethanol (70%),
dissolved in SDS buffer and reprecipitated
with 2 volumes of ethanol. Polyadenylated RNA was isolated from
total RNA by affinity chromatography on oligodeoxythymidylic acid
cellulose (Aviv and Leder, 1972).
Purified RNA was resuspended
in elution buffer, heated to 65°C for 5 min. and cooled in
order to disrupt intermolecular RNA interactions.
This was
adjusted to 0.5M NaCl and passed through a 1 ml column of
oligodeoxythymidylic cellulose (BRL) equilibrated with binding
buffer.
This step was repeated three times.
After the column
22
was washed with 10 ml of binding buffer, polyadenylated
(poly(A)+ ) R N A was eluted with elution buffer heated to 65°C
and was precipitated by addition of NaCl to 0.2M and 2 volumes of
ethanol.
B. DNA Isolation
High molecular weight DNA was isolated from exponentially
growing cultures.
Each plate of cells was rinsed 5 times with
ice cold PBS and harvested into 1 ml of PBS.
Cells were
collected by centrifugation at 800xg for 2 min. at 0°C.
They
were resuspended in 5 volumes of RSB plus 0.5% Nonidet P-40.
After homogenization, nuclei were removed by centrifugation at
800xg for 2 min.
lysis.
This step was repeated to insure complete cell
Nuclei were resuspended in TNE buffer in the presence of
0.02mg/ml RNAse (Sigma).
Sodium dodecyl sulfate (SDS) was added
to a final concentration of 0.2%.
At this point, the solution
became very viscous and was handled gently.
at 37°C for 4-5 h o u r s .
This was incubated
Pronase and SDS were added to a final
concentration of 100 ug/ml and 0.5%, respectively.
solution was incubated for another 4-5 hours.
The DNA.
To insure complete
removal of protein, a phenol-chloroform extraction was done.
An
equal volume of a 1:1 solution of distilled phenol:chloroform
containing 1% isoamyl alcohol was mixed gently with the DNA
solution.
The phases were separated by centrifugation at 800xg.
The organic phase was removed and discarded.
The aqueous phase
23
was extracted twice more with the 1:1 mixture of
phenol-chloroform, twice more with chloroform and transferred
with a large bore pipet to' another tube.
The entire procedure
was repeated until the aqueous-organic interphase was free of
precipitated protein.
The aqueous phase was then dialysed
against 2 changes of high salt buffer and finally 2 changes of TE
buffer
at 4°C.
Construction of Recombinant cDNA Plasmids
A. Synthesis of Double Stranded cDNA
Double stranded complementary DNA was synthesized
essentially as described by Schibler et a l .(1978 ).
Poly(A) +
RNA was copied into single stranded DNA in an incubation mixture
containing 0.05M Tris-HCl
MgCCH^COO)^,
20 ug/ml
(pH 8.3 at 42°c), 0.01M
oligo dT (Miles Laboratory,
0.05M NaCl, O.Oltl dithiothreitol
(fresh),
12-18),
100 ug/ml poly(A) +
RNA, 0.5mM vanadyl ribonucleoside complexes (BRL), 85 u/ml AMV
3
reverse transcriptase (Beard), 800 uM of H dCTP and unlabelled
dCTP to a final specific activity of 1 Ci/mmole.
The first
strand synthesis was carried out in a siliconized tube in which
the labeled nucleoside triphosphate and actinomycin D (which was
dissolved in 95% ethanol) had been evaporated to dryness prior to
the addition of the other components.
volume was 0.1-0.5 ml.
Generally the reaction
The reaction was started by adding the
reverse transcriptase and allowed to continue for 1 hour at
24
42°c. The reaction was
followed by diluting a small aliquot
(1 ul) into 10% trichloroacetic acid (TCA), collecting the
precipitate on a Schleicher and Schuell BA.85 nitrocellulose
filter and determining the amount of incorporation by liquid
scintillation in a toluene based cocktail. The reaction was
terminated by addition of 5 volumes of extraction buffer
containing 300 ug/ml carrier tRNA.
This mixture was extracted
once with an equal volume of phenol:chloroform (1:1) followed by
a single chloroform extraction.
Nucleic acids were precipitated
from the aqueous phase with 2.5 volumes of ethanol at -20°C
for at least 1 hour. After centrifugation, the pellet was
resuspended in 0.2 ml of 0.3N NaOH and incubated at 50°C for 1
hour to destroy the RNA. The alkaline digest was neutralized with
0.3 ml of 1M Tris-HCl
(pH 6).
Single stranded cDNA was separated
from labeled mononucleotides by gel filtration through a 1.5x25
cm Sephadex G-50 column equilibrated with 0.00111 Tris-HCl (pH
7.4), 0.001M EDTA.
The excluded fractions were pooled (about
0.5-1 ml per fraction) and precipitated with 2.5 volumes of
ethanol following the addition of salt to 0.2M and 200 ug of
carrier tRNA.
The single stranded cDNA was converted into double
stranded DNA by a second incubation with reverse transcriptase at
42°C for 3 hours.
The reaction mixture
(same volume as
initially used) contained 0.05M Tris-HCl
(pH 8.3 at 42 °C),
0.01M Mg(CH3C O O ) 2 , 0 . 8mM unlabeled d A T P , dCTP,
d G T P , dTTP,
25
0.01M dithiothreitol (fresh) and 200 u/ml reverse transcriptase.
The reaction was stopped as described above.
An aliquot was taken
and analyzed by SI nuclease (BRL) to determine if synthesis of
the second strand was complete. This was diluted into 1 ml of SI
nuclease digestion buffer.
This was split into two (0.45 ml)
fractions and 25 units of Si nuclease were added to one of these.
Following incubation for 2 hours
at 37°C, both fractions were
precipitated with an equal volume of 10% (w:v) TCA. Precipitates
were collected by filtration on nitrocellulose filters (Scleicher
and Schuell, BA85), dried and counted in a toluene based liquid
scintillation fluid.
Once a value of about 60% resistance to SI
nuclease digestion was obtained, a bulk digestion was done to
remove the remaining single stranded regions.
The bulk reaction
was done as described above except that the SI nuclease digestion
buffer did not contain any calf thymus DNA.
The reaction was
usually done in a small volume (100 ul) to which 25-50 units of
SI nuclease was added.
The SI nuclease reaction was terminated
by addition of extraction buffer, phenol-chloroform extracted and
ethanol precipitated.
.To ensure that the population of double
stranded cDNA contained blunt ends, a final repair reaction was
carried out using the Klenow fragment of DNA polymerase I (BRL)
in a repair buffer containing 20 units of DNA polymerase.
reaction was carried out for 4-5 hours at 12-14°C.
This
DNA
polymerase was inactivated by heating the mixture to 65°C for 5
26
min.
The blunt ended double stranded cDNA was size-selected to
remove low molecular weight DMA.
The double stranded cDNA was
layered on an 11 ml, 5-15%, linear sucrose gradient in 0.1M NaCl,
0.01M Tris-HCl (pH 8), 0.001M EDTA.
The gradients were spun for
16 hours at 35000 rpm at 20°C in a Beckman SW41 rotor.
A
parallel gradient was run containing a TaqI digestion of pBR322
which was used as size markers.
Following fractionation of the
gradients, the fractions of pBR322 TaqI digestion were analyzed
by electrophoresis on a 1% agarose gel. Fractions from the double
stranded cDNA gradient corresponding to 600 base pairs or more
were pooled and precipitated overnight with 2.5 volumes of
ethanol at -20°C. No carrier was added to this sample. In order
to obtain complete recovery of the DNA, it was necessary to spin
the sample for 2 hours at 20,000 rpm at 0°C in the Beckman SW41
rotor, usually in a cellulose nitrate tube.
B. Formation of Chimeric Plasmids
The size fractionated double stranded cDNA was inserted into
the PstI site of pBR322 using the method of homopolymeric tailing
by a modification of the procedure of Roychoudhury et a l .,
(1976).
In this procedure,
Co
+2
is substituted
for Mg
+2
,
which results in the ability of terminal deoxytransferase to use
DNA molecules which contain flush ends, ends with 3' extensions
and ends with 5' extensions. Homopolymeric tails of poly(dC) were
added to the blunt ended double stranded cDNA, using terminal
deoxytransferase (BRL)
(Gordon et a l . , 1978).
Prior to use,
the terminal transferase was checked for the presence of
endogenous DNA.
The enzyme was incubated for various times with
or without substrate, and the incorporation of radioactivity into
TCA precipitable material was monitored.
Usually less than 1%,
of the total incorporation with DNA, was observed in the fraction
without DNA.
Secondly, the enzyme was checked for the presence of
nucleases by examining DNA which had been incubated with the
enzyme on an agarose gel.
The reaction mixture (50 ul) contained
0.1M potassium cacodylate (pH 6.9), O.lmM dithiothreitol, 20 uM
dCTP, 0.5 mg/ml bovine serum albumin
1 pmole of DNA ends.
listed.
(BSA), 0.001 M CoCl^ and
The components were added in the order
Prior to the addition of DNA, the reaction mixture was
incubated at 3 7°C.
This was reported to increase the rate of
reaction (McReynolds et a l ., 1977).
The DNA and terminal
transferase were added to this prewarmed reaction mixture.
The tailing reaction was followed using a parallel reaction in
which an unlabeled substrate DNA was added.
This reaction
contained the same nurriber of pmoles of ends as did the double
stranded cDNA reaction.
(The number of pmole of ends of the
double stranded cDNA was calculated assuming an average of 600
nucleotides and using the formula, #bases/end = nM dNTP in rx./nM
ends in n
x cpm/total cpm (Schleff and Wensink, 1981)).
The
reaction was terminated when the number of cytidine residues
28
incorporated per end reached about 30. Generally, the reaction
took 10-15 min.
The reaction was terminated by incubation at
65°C for 5 min, followed by phenol-chloroform extraction.
The
homopolymeric tailed double stranded cDNA was run on a Sepharose
6B column (10x0.75 cm) equilibrated with 0.001M Tris-HCl (pH 7 4),
0.001M EDTA.
Very small fractions were collected (3 drops)
and the excluded fractions pooled.
Routinely, a portion of the
final tailed double stranded cDNA was run on a 5-15% linear
sucrose gradient to insure that no breakdown of the DNA occured
during the reaction.
Poly(dG) tailed pBR322 (NEN) was annealed to the poly(dC)
tailed double stranded cDNA as described by Gordon et al .,
(1978).
Usually a 2:1 molar ratio of polydG pBR322rpolydC
ds-cDNA was mixed in annealing buffer,
min. and cooled by serial incubations
37°C, and 25°C.
heated to 65°C for 10
for 2 hours each at 45°C,
The annealed material was then placed on ice
for at least a half hour prior to use.
Bacterial
Procedures
A. Transformation
DNA transformations were done using competent Escherichia
coli, HB101.
Competent cells were prepared by a modification of
the procedure of Norgard et a l ., (1978).
A small volume
(100 ul) of an overnight culture of HB101 (prepared frcm a single
colony from a freshly streaked plate) was diluted into 100 ml of
Luria broth.
The cells were incubated at 37°C with vigorous
aeration until they reached an OD,.,-^ of 0.32 which usually took
2-3 hours, at which point they were placed on ice for 5-10 min.
All following steps were carried out at 0-4°c.
Cells were
collected by centrifugation in the GSA rotor (Sorvall) at 4000
rpm for 5 min.
This low speed of centrifugation gave a loosely
packed pellet of bacteria which facilitated resuspension.
Bacteria were washed once in 25 ml of 0. 1M M g C ^
(in 0.01M
Tris-HCl pH 7.4), collected as before and gently resuspended in 25
ml of 0.1M CaCl^
0.01M Tris-HCl pH 7.4).
The cells were
incubated in buffered 0.1M CaCl^ for 45 min. with occasional
shaking, spun down and resuspended in 5 ml of buffered 0.1M
CaCl^ and used immediately for transformation.
(Calcium
chloride causes the cells to become very fragile and so cells
were treated gently.)
For transformation, a 2:1 ratio of competent cells:DNA
solution was used.
Generally,
100 ul of competent cells were
added to 50 ul of plasmid DNA in 0.1M CaCl^ (in 0.01M Tris-HCl
pH 7.4). Samples were incubated on ice for 40 min. with
occasional gentle mixing.
Cells were then heat shocked at 37°C
for 2.5 min., diluted with prewarmed
(37°c) LB and grown
for 1 hour at 37°C, to allow for expression of drug resistance
genes.
Cells were plated onto selective plates
containing 0.05 ug/ml tetracycline)
(L plates
and grown overnight at 37°c.
30
It was found that a higher frequency of transformation
resulted when cells were plated onto agar directly and then
transferred to nitrocellulose filters.
efficiencies of 5x10
5
to 2x10
6
Routinely, transformation
colonies per ug of
covalently closed circular pBR322 were obtained.
B. Filter Replication and Storage
Bacterial colonies streaked onto selective plates were grown
until they reached about 0.1mm in diameter.
A dry nitrocellulose
filter (Schleicher and Schuell, BA85, 90mm) was then placed on
the agar and quickly removed, carrying with it the complete
array of colonies on the plate.
The nitrocellulose filters
containing the bacteria were handled as described by Hanahan and
Meselson (1980).
Prior to use nitrocellulose filters were soaked
in distilled water at 6 5°C for 1 hour,
distilled water, dried and autoclaved.
rinsed extensively with
For replication of the
filters, the original filter (template) was placed colony side up
on a stack of sterile Whatman #3 paper.
The sterile
nitrocellulose filter was placed first on a fresh plate of agar
to wet it and then directly over the template filter.
The two
filters were pressed together firmly using a velveteen covered
block.
Filters were keyed to each other by piercing holes into
them with a needle.
The replica was placed on either a selective
plate (tet plate) or a selective plate containing 5% glycerol
(G-tet plate) if the filter was to be frozen.
Replica filters
31
were grown until distinct colonies appeared.
Template filters
which were to be frozen were grown for a few hours on plates
containing glycerol.
Filters were frozen by placing another
filter, wetted on a glycerol plate, directly over the colonies as
before.
The filters were keyed, left together and placed between
sheets of sterile Whatman #1 paper.
The stack of Whatman #1 was
placed in a plastic bag containing some Whatman filters wetted
with water, in order to keep everything moist.
stored at - 8 0 ° c .
Colonies
at least 6 months.
The sealed bag was
stored in this way were stable for
When filters were needed, the plastic bag was
brought to room temperature, filters were carefully separated,
placed on plates and were incubated until colonies appeared
( 2 - 5 hours).
manner.
The recombinant cDNA library was stored in this
Each filter contained between 6 0 0 - 1 0 0 0 recombinants.
library was also maintained in solution.
The
Each filter was placed
in 3 0 0 ml of tet broth and the colonies were shaken off overnight.
Glycerol was added to a small aliquot of this suspension to a final
concentratin of 20%.
This mixture was
frozen at - 8 0 ° C until
needed.
C. Small Scale Plasmid Isolation
Small scale isolation of plasmids was done initially to
analyze recombinant cDNA clones in the library.
Plasmids were
isolated by a modification of the alkaline-SDS extraction
procedure (Birnboim and Doly, 1979).
All steps were done at
32
0-4°C.
A 10 ml overnight culture was obtained for each colony
to be analyzed.
Cells were collected from 7 ml of this suspension
by centrifugation for 5 min at 6000 rpm in the HSB-4 rotor. The
pellet was washed once in 2 ml of 25% sucrose, 0.05M Tris-HCl pH 8
and then resuspended in 2 ml of lysozyme buffer containing 0.5
mg/ml lysozyme.
After 30 min.,
(The lysozyme solution was made right before use.)
freshly made alhaline-SDS (4 ml) was added
and the mixture was incubated for 5 min.
lysis and denaturation of DNA.
This resulted in cell
The mixture was neutralized with 3M
sodium acetate pH 4.8 (3 ml) and incubated for 60 min.
During
this time, a white precipitate formed containing SDS, protein and
chromosomal DNA.
Plasmid DNA remained in solution.
The precipitate
was removed by centrifugation at 8000 rpm for 30 nin. in the HSB-4
rotor.
The supernatant was collected and plasmid DNA
was precipitated by adding 2 volumes of ethanol.
After
centrifugation, plasmid DNA was resuspended in 2 ml of TNE buffer.
RNA was removed by adding of 0.1 ml of RNAse (2 mg/ml), and
incubating the solution at 3 7°c for 60 min.
RNAse was removed
by protease treatment (pronase was added to 1 mg/ml, SDS to 0.5%) and
phenol-chloroform extraction.
ethanol precipitated twice.
Prior to use, the plasmid DNA was
For the second ethanol precipitation,
a half volume of ammonium acetate (7.5 M) was added and then two
volumes of ethanol.
Gel Electrophoresis
33
A. Agarose Gels
To separate and identify DNA samples, vertical agarose gels
(2mm thick) were run.
The percentage of agarose used was
dependent on the size of the DNA to be analyzed.
DNA fragments
of less than 1000 base pairs were either analyzed on a 2% agarose
gel or on a 3.5% polyacrylamide gel (see part C).
Agarose was
dissolved in TEA buffer by heating for 5-10 min in a boiling water
bath and cooled to 60°C before pouring.
agarose should be cooled to 70°c) .
(Solutions of 2%
A 2% agarose plug (50 ml)
was poured first, allowed to solidify and then the running gel
(75 ml) was poured.
DNA samples to be electrophoresed were mixed
with an egual volume of DNA electrophoresis sample buffer.
Electrophoresis was for approximately 300 volt-liours.
During the
run, the electrophoresis buffer was always recirculated, since the
buffering capacity was very low.
After electrophoresis, DNA
bands were detected by use of ethidium bromide. The gel was
immersed in water containing 1 ug/ml ethidium bromide.
Gels were
stained for 20 min., rinsed and examined by a transilluminator.
Pictures were taken with polaroid film.
B. Isolation of I}NA From Agarose Gels
Specific DNA fragments were isolated frcm agarose gels using
the "freeze-squeeze" method.
Usually, 75-100 ug of DNA were run
on a preparative gel, 2nm vertical gel with a long conib.
Following electrophoresis, side bands were cut from the gel,
34
stained with ethidium bromide and used to locate the DNA. in the
gel.
Ethidium bromide causes photooxidation of DNA with visible
light and molecular oxygen
(Davis et a l ., 1930).
reason, only side bands of the gel were stained.
For this
The DNA band
was cut from the gel, sliced into smaller pieces and placed in a
siliconized corex tube.
nitrogen for 1-2 min.,
The gel slices were frozen in liquid
thawed at 37°C for 15-20 min. and then
the liquid surrounding the gel was removed.
repeated three times.
These steps were
The last time, the thawed gel was
centrifuged at 7000 rpm for 15 min. to pellet the agarose.
The
pooled supernatants were passed through a 0.45 u Millipore filter
and extracted with phenol, phenol-chloroform and chloroform to
remove residual agarose.
The DNA was ethanol precipitated twice,
the second time using the ammonium acetate procedure.
Isolation
of DNA fragments in this way is very easy and results in
approximately a 50% yield of DNA.
Recovery' is greater when lower
percent agarose gels are used and smaller DNA fragments are to be
isolated.
DNA isolated in this manner can be nick translated,
however, it cannot be used as a substrate for DNA ligase probably
due to residual agarose.
C. Polyacrylamide Gel Electrophoresis of DNA
DNA fragments of less than 1000 base pairs were analyzed
using either a 3.5% or 5% polyacrylamide gel, of the same
dimensions used for agarose gels.
In this case, the appropriate
35
percentage of acrylamide solution (29 grams acrylamide:1 gram of
N, N methylene bisacrylamide) was added to 1/10 volume of 1CDC TEA
buffer.
The gel was polymerized by adding ammonium persulfate to
0.063% and tetramethylethylenediamine (TEMED) to 0.03%.
Prior to
pouring, this solution was deaerated. Polymerization was
completed within an hour.
rinsed out with water.
The comb was removed and the wells
The reservoirs were filled with TEA
buffer and the wells were flushed with electrophoresis buffer to
prevent the DNA bands from becoming wavy.
DNA samples were
loaded, electrophoresed, and analyzed as described for agarose
gels, except that the gel was stained while still attached to one
plate since these gels were very fragile.
D. Southern Blot Analysis of DNA Fragments
Specific sequences,
in DNA fragments separated by gel
electrophoresis, were identified using the Southern transfer
procedure (1975).
photographed.
Agarose gels were run, stained and
DNA standards were marked by piercing the gel with
a needle containing India ink. The gel was soaked in 200 ml of
1.5M NaCl, 0.5M NaOH to denature the DNA.
After 30 min., the gel
was rinsed several times with water and placed in 200 ml of 2M
NaCl, 1M Tris-HCl pH 5.5 for an additional 30 min. Then, the
neutralized gel was placed on the transfer apparatus.
The
transfer apparatus consisted of a stack of paper towels immersed
in a tray of 20X SSC on which 3 layers of 3MVI paper (cut slightly
36
larger than the gel) were placed-.
A.11 air bubbles between the
gel and 3MM paper were removed to insure complete transfer.
Glass rods were placed around the gel.
A nitrocellulose sheet
(Schleicher and Schuell), cut slightly larger than the gel, was
presoaked in water, and then 20X SSC and placed on top of the gel
with its edges resting on the glass rods.
Trapped air bubbles were
removed and standards were marked by piercing the nitrocellulose
filter at the location of the India ink.
A presoaked 3 MM paper
(20X SSC) was placed over the nitrocellulose, on which a large
layer of paper towels, which were cut to the same size as the 3 MM
paper, were placed.
Wrap.
The entire apparatus was covered with Saran
The transfer was for 18-24 hours.
The nitrocellulose
filter was removed, soaked in 2X SSC for 30 min. to remove excess
salt, air dried and baked under reduced pressure for 2-3 hours at
00°C.
The filter was either used immediately or stored in an
air tight bag at -20°C.
Filters were placed in plastic Seal'a Meal bags and
prehybridized in 2-3 ml of deaerated 3X SSC, 2X Denhardt's
solution,
300 ug/ml denatured,
sonicated salmon sperm DNA, 0.2%
SDS at 65°c for 12-24 hours to reduce nonspecific binding of
the probe.
Before the bag was sealed, all air bubbles were
removed to prevent high backgrounds.
Hybridization was done in
the same bag by injecting 1-2x10^ counts per min (cpm) of
denatured nick translated probe and incubating at 65°c for
37
20-24 hours.
Filters were washed for 5-6 hours at 63°C in
0.5X SSC, 0.5% SDS.
They were air dried and bands were detected
by autoradiography with Kodak X-Omat AR film placed at -80°C
with an intensifying screen.
This procedure was used when the
DNA sequences being probed represented a large percentage of the
DNA sample run on the gel.
abundance,
To detect sequences present in low
filters were processed using dextran sulfate in the
hybridisation mixture
(Wahl et a l ., 1979).
Dextran sulfate, an
anionic polymer, enhances of the hybridization signal due to an
increased rate of hybridization and formation of probe networks.
In this case, prehybridization of filters was done in 4-6 ml
of deaerated 5X SSC, 0.05M sodium phosphate pH 6.5, 250 ug/ml
denatured sonicated salmon sperm DNA, 2X Denhardt's, 50%
formamide (deionized) .
Prehybridization was at 42°C for 12-20
hours. Prehybridization solution was removed (usually by
transferring the filter) and replaced with the hybridization
solution containing 4 parts prehybridization solution and 1 part
50% (w/v) dextran sulfate (Sigma, MW 500,000).
The denatured
nick translated probe was mixed with a small amount of prewarmed
hybridization solution
facilitate mixing.
(65°c) before addition to the bag to
Hybridization was
for 20-24 hours at 42°c.
Filters were washed with 4 changes of 2X SSC, 0.1% SDS at room
temperature followed by 2 washes of 0. IX SSC, 0.1% SDS at 50°C
for 30 min.
They were dried and exposed to Xray film.
Although
38
this procedure increased the hybridization signal, it also
resulted in higher backgrounds.
E. Gel Electrophoresis of ENA
Analysis of specific RNA species electrophoresed in agarose
gels was done by two methods; either electrophoresis of
glyoxalated RNA or electrophoresis of RNA in formaldehyde gels.
The first procedure was described by Thomas (1980).
RNA samples
were denatured in 8 ul of 1M deionized glyoxal (6M solution),
50% dimethyl sulfoxide, 0.01M sodium phosphate pH 7 for 1 hour at
50°C.
Glyoxal reacts primarily with guanosine residues in RNA
and DNA interfering with base pairing (Carmichael and McMaster,
1980).
The ENA sample was prepared for electrophoresis by adding
2 ul of 50% glycerol, 0.01M sodium phosphate pH 7, and bromophenyl
blue.
RNA was run in 1.1% agarose gels containing 0.01M sodium
phosphate pH 7.
Since the phosphate buffer had a low buffering
capacity, the electrophoresis buffer was recirculation during the
run.
Glyoxalation of RNA and DNA is reversible at a pH greater
than 8 , so the pH of the electrophoresis buffer was maintained
near 7 (Carmichael and McMaster, 1980). DNA size standards may be
used to estimate molecular weights of RNA (McMaster and
Carmichael,
volt-hours.
1977).
Electrophoresis was for approximately 250
After electrophoresis, the gel, including standards,
was immediately placed on the transfer apparatus and bound to
nitrocellulose as described for DNA except that v\hen the transfer
39
was completed, RNA filters were not soaked in 2X SSC but air
dried and baked.
Prehybridization and hybridization conditions
were the same as those used for the dextran sulfate procedure.
The second method was essentially as described by Fyrberg
et al. (submitted).
RNA samples
(20 ul) to be separated were
denatured by heating for 15 min at 5 5°c in electrophoresis
buffer containing 50% formamide (deionized), 6.5% formaldehyde.
Two microliters of loading buffer were added prior to
electrophoresis.
Agarose gels (1%) were prepared by heating the
agarose in boiling water,
cooling
it to 60°c and adding a 1/4
volume of 5X electrophoresis buffer and formaldehyde to 2.2M.
Electrophoresis was for approximately 475 volt-hours.'
electrophoresis,
Following
the gel was rinsed several times with water and
blotted to nitrocellulose as described previously.
Prehybridization and hybridization of the filters were done using
the dextran sulfate conditions.
F. Polyacrylamide Gel Analysis of Proteins
Protein samples were analyzed by electrophoresis on
denaturing discontinuous polyacrylamide slab gels as described by
Laemmli (1970). The separating gel (10% acrylamide- 0.27% N,N
methylene bisacrylamide in running gel buffer) was polymerized
using TEMED and 1 mg/ml ammonium persulfate.
The upper gel (3%
acrylamide- 0.08% bisacrylamide in stacking gel buffer)
photopolymerized by 0.18mg/ml riboflavin and TEMED.
was
Protein
40
samples to be electrophoresed were dissolved in sample buffer by
heating in a boiling waterbath for 5 min.
Samples were loaded
into dry wells of the polymerized gel, over which electrophoresis
buffer was carefully layered.
Samples were electrophoresed at
25 mA for approximately 4 hours.
Gels were placed in fixing
solution for at least 3 hours, allowed, to rehydrate in destaining
solution for 3-4 hours and dried on filter paper using a Biorad
gel drier.
Proteins were identified either by staining gels in
fixing solution containing 0.25% Coomassie Brilliant Blue R for
1 hour prior to destaining or by autoradiography.
Preparation of Probes
A. cDNA Probe
The recombinant cDNA library was screened using single
stranded DNA complementary to various RNA preparations.
Single
stranded cDNA was synthesized essentially as described above,
except that the reaction volume was decreased to 15 ul in which
only
32
P-labelled dCTP
(10 uM) was present.
components were in the same concentrations.
The rest of the
The single stranded
cDNA was processed as above up to the point of second strand
synthesis. The specific activity of the probe was approximately
9
1-2x10 cpm/ug . This probe broke down rapidly and was always
used within 3-4 days of synthesis.
B. Nick Translation of DNA
DNA was nick translated in a reaction mixture
containing
41
0.05M sodium phosphate pH 7.6,
0.005M Mg^, 0.002 uM dGTP,
dATP, dTTP, 1-2 ug DNA which was added to a siliconized tube
containing
32
P labeled dCTP
(50 uCi of 800 mCi/mmole in 50%
ethanol) which had been evaporated to dryness.
E. coli DNA
polymerase I (NEN, 80 units) was added and the mixture (200 ul
total volume) was incubated at 14°C until the reaction was
completed.
The kinetics of incorporation were monitored by
TCA precipitating 1 ul aliquots in sample solution.
The
reaction was continued until the incorporation did not
increase over a 20-30 min period.
Many times, DNAse I was
not needed to get incorporation of label into the DNA.
If the
reaction proceeded slowly, dilutions of DNAse I (lOmg/ml in
0.01M HC1) were made to a final concentration of 100 ng/ml in the
reaction.
The reaction was terminated by adding EDTA to 0.015M
and phenol-chloroform extraction.
Unincorporated
32
P-dCTP was
removed by chromatography on a Sephadex G-50 column (1.5x25 cm)
equilibrated with 0.001M Tris-HCl pH 7.4, 0.001M EDTA.
Fractions
were collected, counted and the excluded volume pooled and
ethanol precipitated.
The specific activity of DNA labeled in
7
this manner was approximately 1- 2x 10 cpm/ug.
Characterization of Recombinant cDNA Library
A. Colony Filter Hybridization
A library of approximately 30,000 recombinant colonies was
obtained from transformation of HB101 with plasmids containing
42
cDNA from LU3-7 p o l y ( A ) + RNA.
These were stored on
nitrocellulose filters each containing about 1000 colonies.
Analysis of each filter was done using a modification of the
Grunstein-Hogness procedure (1975). Frozen nitrocellulose filters
were brought to room temperature, separated and placed on
selective plates (tet plates).
Bacteria were grown at 37°C
until the colony diameter was about 0 .1mm, at which time filters
were transfered to selective plates containing chloramphenicol
(200 ug/ml). This results in preferential amplification of
plasmid DNA (Hanahan and Meselson, 1980).
After 18-24 hours,
filters were processed by placing them on 3MM paper saturated
with the appropriate solution.
First, filters were placed face
up on paper saturated with 25% sucrose, 0.05M Tris-HCl pH 8
containing 1.5 mg/ml lysozyme (freshly made) for 10 min.
Bacteria
were lyzed by incubation in 0.5M NaOH, 0.2% TritonX-100 (made
fresh) for 10 min,
min.
followed by treatment in 0.5M NaOH alone for 5
At this point, DNA should be denatured and fixed to the
filter.
The filters were neutralized by sequential treatment
with 0.5M Tris-HCl pH 7.5 (twice) and 0.5M Tris-HCl pH 7.5, 1.5M
NaCl for 5 min each and air dried for 30 min.
Protein was
removed by treatment of filters with self-digested pronase.
They
were re-wet in SDS buffer, dipped in SDS buffer containing
100 ug/ml pronase and incubated on 3MM paper saturated with SDS
buffer for 30 min.
Then, filters were rinsed in SDS buffer, air
43
dried for 30 min, and baked under vacuum for 2-3 hours at 80°c.
Removal of protein was important to maintain low backgrounds•
Filters were either used immediately or stored in sealed bags at
-20°c.
Prehybridization was
for 20-24 hours
at 65°C in 5x
SET, 2x Denhardt's, 0.1% SDS, 5 ug/ml denatured pBR322,
denatured sonicated salmon sperm DNA, 100 ug/ml tRNA,
poly(A).
200 ug/ml
120 ug/ml
Hybridizations were done in the same solution
containing 1-2x10 cpm/filter.
(Filters were hybridized either
individually or in batches of 30 in a siliconized beaker which was
overlaid with mineral oil.
In both cases, hybridizations were
done using 3 ml of solution per filter).
After 20-24 hours,
filters were washed at 6 3°C for 5-6 hours
in IX SET, 0.5% SDS,
air dried and exposed to X-ray film. Each filter was hybridized
in triplicate to single stranded cDNA made to resting 3T6
poly(A)
RNA,
poly(A)+ RNA.
growing 3T6 poly(A)
"I"
RNA and growing LU3-7
Colonies which gave a signal only with cDNA from
growing LU3-7 RNA were analyzed further.
B. Hybrid Selected Translation
Plasmids from colonies giving a positive response, were
isolated by a mini-preparation and used in a hybrid-selected
translation assay
1981).
(Ricciardi et a l ., 1979;
Parnes et a l .,
Plasmid DNA (2-10 ug) was bound to nitrocellulose filters
(BA85, 13mm) as described by Hendrickson et a l ., (1980).
Poly(A)+ RNA (10-20 ug) was heated
for 10 min.
at 70°C in
44
300 ul of 65% deionized formamide, 0.02M piperazine-N-N bis(2
ethanesulfonic acid) pH 7.2, 0.2% SDS, 0.4M NaCl, 0.1 mg/ml tRNA.
DNA filters were added and incubated for 3-6 hours at 50°C.
Unbound RNA was removed by washing filters batchwise nine times
in 50 ml of 0.01M Tris-HCl pH 7.6, 0.15M NaCl, 0.001M EDTA, 0.5%
SDS at 65°C (5 min each wash),
without SDS.
and then two times in buffer
Filters were transfered to siliconized microfuge
tubes, and was RNA eluted in 300 ul of water containing 30 ug
tRNA by heating for one min in boiling water and quick freezing in
liquid nitrogen.
removed.
The solution was thawed and the filter was
Sodium acetate was added to 0.2M and the RNA was ethanol
precipitated.
RNA isolated in this manner was used as exogenous
RNA in an in vitro translation.
kit supplied by NEN.
Translations were done using a
Conditions were as suggested except that
following translation, samples were treated for 20 min. with RNAse
(80 ug/ml) to remove charged tRNAs and then run on polyacrylamide
gels.
C. Immunoprecipitation of Thymidylate Synthetase
Immunoprecipitation reactions were done by a modification of
the procedure described by Kessler (1975).
In this method,
preformed antigen-antibody complexes of IgG are precipitated by
adsorption to protein A present on the membrane of formalin-fixed
Staphylococcus aureus cells.
Extracts of UJ3-7, 3T6, or M50L3
cells or in vitro translation mixtures were diluted to 200 ul
45
by adding 1/4 volume of a 5X immunoprecipitation buffer and 1/10
volume of detergent solution.
(The immunoprecipitation buffer
was modified from that originally described by increasing
the concentration of NP-40 and BSA, while including two addition
detergents, Triton-XlOO and deoxycholate.
These changes were
important in reducing backgrounds by removing proteins which
nonspecifically adsorb to the bacterial cells).
bacterial cells (100 ul
Prewashed
of a 10% w/v suspension) were added to
the mixture, incubated at room temperature for 30 min. and
removed by centrifugation at 7000 rpm for 10 min.
further removed nonspecially adsorbed proteins.
synthetase antiserum (25 ul) was added.
This step
Thymidylate
Following incubation at
23°C for 30 min, bacterial cells were added (100 ul),
incubated for 1 hou r at 23°C,
diluted with 2 ml of
immunoprecipitation buffer and recovered by centrifugation at
7000 rpm for 5 min.
These cells were washed three additional
times in 2 ml of immunoprecipitation buffer.
After the last
centrifugation, cells were resuspended in 1 ml of 0.063M Tris-HCl
pH 7.4, transferred to a 1.5 ml polypropylene tube and spun down
in the microfuge for 1 min.
buffer.
The pellet was resuspended in sample
Antigen-antibody complexes were disrupted by heating
the sample for 5 min in a boiling water bath.
Prior to
electrophoresis, bacterial cells were removed by centrifugation
in the microfuge for 2-5 min.
46
Formalin fixed Staphylococcus aureus cells used in these
experiments were generously supplied by Rob Abbott.
These cells
were prepared as described by Kessler, except the final 10%
suspension was weight/volume.
Fixed bacterial cells were washed
just prior to use in every case.
This was primarily to remove
protein A which was no longer associated with cell membranes.
Cells were collected by centrifugation at 4000 rpm for 20 min.
They were resuspended in 1 ml of imfnunoprecipitation buffer,
incubated at 23°C for 15 min.,
followed by centrifugation
through 10 ml of immunoprecipitation buffer containing 10%
glycerol.
Bacterial cells were washed twice nore by this
procedure and finally resuspended in immunoprecipitation buffer
to the volume of the original 10 % suspension.
RESULTS
Construction of a recombinant cDNA library
The characteristics of the thymidylate synthetase
overproducing cell line indicated it could be used for the
isolation of cDNA sequences for the enzyme.
This would only be
true, however, if the content of thymidylate synthetase messenger
RNA also increased with the amount of enzyme.
This was
expected since in all other systems studied an increase in enzyme
content reflected an increase in messenger RNA content.
Analysis of the amount of thymidylate synthetase specific
messenger RNA in the sensitive and resistant cell lines was done
using an RNA dependent in vitro translation system.
Poly(A)+
RNA was added to a commercially available rabbit reticulocyte
translation system.
Proteins synthesized during a one hour
incubation were analyzed by electrophoresis on a discontinuous
SDS polyacrylamide gel (figure 3).
When the translation products
of poly(A)+ RNA from LU3-7 cells and either M50L3 or 3T6 cells
were compared no significant difference in the protein patterns was
seen except for a protein band at 38,000 daltons (figure 3,
lanes C,D,F,G,H). The pattern obtained was very similar to that
seen for in vivo labeled cell extracts
(figure 3, lanes A,B).
Thus, the amount of cytoplasmic thymidylate synthetase messenger
RNA in growing LU3-7 cells has been increased.
47
Figure 3. Comparison of in vivo and in vitro protein
synthesis from 3T6, M50L3 and LU3-7 cells. Cultures of
growing 3T6 and LU3-7 were labeled for 4 hours with
'S-methionine. Total poly(A)
mRNA (0.5ug) of growing 3T&,
M50L3, or LU3-7 cells was translated in an in vitro translating
system (NEN). A control translation (no RNA) determined the
amount of endogenous protein synthesis.
Proteins were separated
by electrophoresis on a 10% polyacrylamide gel and visualized
autoradiography.
Lane A. in vivo labeled LU3-7 cytoplasmic extract
Lane B. in vivo labeled 3T6 cytoplasmic extract
Lane C,F.- in vitro^ translation products of LU3-7
p o l y (A )
RNA
Lane D,G.- in vitro translation products of 3T6
p o l y (A)
RNA
Lane E.- No RITA
Lane H.- in vitro translation products of M50L3
poly(A)
RNA
48
l||Sf p w
ppf*'
■"J
*
'WSm
* * m
■m
n
fij't
4 i'-y <
Figure 3.
50
Initial attempts to synthesize high rrolecular weight cDNA
were only partially successful.
Although many times as much as
80% of the messenger RNA mass was converted into DNA, the modal
size of the cDNA synthesized was never greater than 200
nucleotides, as determined by alkaline sucrose gradients (figure
4,a).
For this reason, experimental conditions were varied to
increase the size of the cDNA synthesized.
Since the length of
cDNA transcripts is directly dependent on the size and purity of
the RNA preparation from which it is made, two steps were taken
to obtain high quality RNA.
polysomal preparation.
Total RNA was isolated from a
In this way,
poly(A)+ RNA sequences
which represent messenger RNA degradation products are
eliminated.
Polysomal RNA was purified using the guanidine
hydrochloride procedure.
Guanidine hydrochloride was added at
high enough concentrations to denature any nucleases.
Removal of
protein by this procedure has been reported to minimize the
nicking of RNA which sometimes occurs during phenol-chloroform
extractions (Rosen and Monahan, 1982).
In optimizing the conditions for cDNA synthesis, two criteria
were considered; the length of the cDNA and the amount of cDNA
produced per microgram of poly(A)+ RNA.
A major problem in
obtaining full length cDNA transcripts was reported to be
contamination of reverse transcriptase preparations with
ribonucleases.
Therefore,
several alterations of a basic cDNA
51
300
100
500
700
600
4001-
200
5
10
15
20
F raction Number
Figure 4. Comparison of two methods used for the synthesis of
complementary DNA.
Complimentary DNA. was synthesized from LU3-7
poly(A) RNA toy reverse t r a n s c r i p t a s e . An aliquot (not
necessarily the same number of counts) was run on a 5-11% alkaline
sucrose gradient at 40,000 r p m for 16 hours at 20 . Gradients
were fractionated (0 .5ml/fraction) and the radioactivity was
determined toy liquid scintillation.
Sizes were determined toy
computer analysis of the sucrose gradient run. Method 1 (closed
squares) contained 0.05M Tris-HCl (pH 8.2 at 37°C), 0.01M
magnesium acetate, 0.01 dithiothreitol, 0.06M NaCl, 20 ug/ml
oligo dT, 20 ug/ml RNA, 100 ug/ml Actinomycin D, ImM
deoxynucleoside triphosnhates and 60 u/ml reverse transcriptase.
Reaction proceeded at 37
for 2 hours.
Method 2 (open squares)
was as described in Materials and Methods.
52
synthesis reaction were made in an attempt to minimize this
activity.
These included (1) the use of high deoxynucleoside
triphosphate levels, (5 mM final concentration),
of sodium pyrophosphate,
the addition of tRNA,
(2) the addition
(final concentration of 4mM), and (3)
(final concentration 250 ug/ml).
None of
these modifications increased the length of single stranded cDNA
synthesized relative to the control, as judged by analysis on
alkaline sucrose gradients.
elevated temperatures
Furthermore, cDNA synthesis at
(46°C),
(to decrease the amount of
secondary structure of the RNA), failed to increase the length of
the cDNA transcript.
However, when cDNA synthesis was carried
out exactly as described by Schibler et a l . (1978), the sucrose
gradient profile changed dramatically (figure 4 ,d ).
A broad peak
was obtained at 300 nucleotides, with a large shoulder of cDNA
transcripts of higher molecular weight.
routinely gave high yields of cDNA.
This procedure also
Once Schibler's conditions
for cDNA synthesis were chosen, a final attempt was made to
minimize ribonuclease activity.
This involved the inclusion of
commercially available vanadyl ribonucleoside complexes during
the cDNA synthesis.
of ribonuclease.
These complexes are transition state analogs
Since transition state analogs have a greater
affinity for the active site of an enzyme than the substrate does,
these complexes preferentially bind to the active site of
ribonuclease, thereby inhibiting its activity (Berger and
I
j
53
Birkenmeier, 1979).
The addition of these complexes to a cDNA
synthesis reaction was found to considerably inhibit the
synthesis of low molecular weight transcripts (figure 5).
However, the total yield of cDNA was reduced.
An optimal
concentration of vanadyl ribonucleoside complexes was determined
by varying the amount of the complexes added to a reaction
mixture and examining the effect on size and yield of the cDNA
produced.
The size distribution and yield of cDNA (10-15%) was
favorable at a 0.5mM concentration.
The final cDNA synthesis
reaction conditions chosen were Schibler's conditions with the
addition of 0.5mM vanadyl ribonucleoside complexes.
Double stranded cDNA was synthesized by extension of the 3'
end of the hairpin loop by reverse transcriptase.
This enzyme
was found to require less time to complete second strand
synthesis and resulted in a higher conversion of cDNA into double
stranded DNA than when DNA polymerase I was used.
It was
reported that second strand synthesis by reverse transcriptase
resulted in the absence of intermediates between
full length cDNA and full length double stranded cDNA.
In
contrast, DNA polymerase I reactions contained large amounts of
partially double stranded cDNA (Williams, 1981).
For these
reasons, reverse transcriptase was the enzyme of choice.
Following second strand synthesis, the hairpin loop and
other single stranded regions were removed by SI nuclease.
(SI
Figure 5. Effect of vanadyl ribonucleoside complexes during cDNA
synthesis.
Complementary DNA. was synthesized by the method of
Schibler et al. (1978) wit h (circles) or without (squares) 1 m'l
vanadyl ribonucleoside complexes (BRL). An aliquot of the reaction
was run on a 5-11% alkaline sucrose gradient at 40,000 rpm for 16
hours at 20°. The gradients were fractionated and the
radioactivity determined by liquid scintillation counting.
(The same
number of counts were not run on the two gradients).
54
150
30 0
600
900
cpm
300
200
100
Fraction Number
Figure 5
Ui
Ui
56
nuclease specifically removes single stranded regions of nucleic
acids).
When an SI nuclease reaction is completed, the resulting
DNA molecules are not all blunt ended.
extension of one or two bases
Some have a 3' or 5'
(Maniatis et al., 1982).
For
this reason, a repair reaction using the Klenow fragment of
E. coli DNA polymerase I was done.
In order to prevent the construction of a recombinant
library containing predominantly plasmids with small cDNA
inserts, double stranded cDNA was size selected on a neutral
sucrose gradient.
Complementary double stranded DNA of greater
than 600 base pairs was kept. The sucrose gradient profile for
the double stranded cDNA used to construct the recombinant
library is shown in figure 6 .
Characteristics of the cDNA
synthesis are listed in Table I .
Once double stranded cDNA had been obtained, there were many
alternative methods to insert these sequences into a plasmid. They
included (1) blunt-ended ligation of the double stranded cDNA into
linear, phosphatased pBR322,
(2) blunt-ended ligation of synthetic
oligonucleotide linkers containing a specific restriction site
onto the double stranded cDNA, restriction and insertion by
staggered end ligation into pBR322 which had been linearized by
restriction at that site and phosphatased, and (3) addition of
polynucleotide tails to double stranded cDNA and annealing these
molecules with linearized pBR322 containing complementary
57
300
475
1300
120C
q
.
800
400
10
15
20
F raction number
Figure 6 . Sucrose Gradient Profile of cDNA used for the
construction of the recombinant library. A preparative cDNA
synthesis was done by a modification of the procedure of
Schibler et a l . (1978) as described in Material and Methods.
An
aliquot was run onQ a 5-15% neutral sucrose gradient at 40,000 rpm
for 16 hours at 20 C. Size standards were run on a parallel
gradient.
Fractions 12-25 were pooled and used in subsequent
steps.
58
Table 1.
Characteristics of the double stranded cDNA preparation
used to construct the recombinant library
I.
Percent conversion of RNA into DNA
22%
II. Percent double stranded after first
strand synthesis
III.
9%
Number of deoxycytosine residues added to
double stranded cDNA
IV.
15
Transformation frequency of annealed
recombinant cDNA plasmid
2.2x10
a. Calculation was based on 1 ng=550 cpm.
This
5
was estimated assuming a 30% counting efficiency,
25% of the residues are cytidine and 309 is the
average weight of a nucleotide.
b. Calculated from the equation given in Materials
and Methods, assuming an average size of cDNA to
be 600 base pairs.
c. Double stranded cDNA was annealed to pBR322 in a
1:15 (w:w) ratio.
Calculation is based on
frequency per microgram of double stranded cDNA
in mixture.
59
polynucleotide ends (figures 2,7).
The first method was chosen because the cDNA molecules were
ready for use after sucrose gradient fractionation and only a few
manipulations of pBR322 were necessary to create recombinant
molecules.
The strategy for blunt end ligation involved
restriction of pBR322 with Sail, repair of this site with the
Klenow fragment of DNA polymerase I and removal of terminal
phosphates to prevent self ligation.
The Sail site was chosen
for the following reasons. The vector, pBR322, contains a single
Sail site in the tetracycline resistance gene so that recombinant
colonies could be assessed by
their resistance to ampicillin and
sensitivity to tetracycline.
Also, repair of the Sail site
regenerates the internal TaqI site which allows removal of cDNA
sequences (figure 7).
Since TaqI recognizes a four base sequence,
the inserted sequences would have a high probablity of containing
this sequence.
Therefore, it
would probably not be possible to
isolate the intact cDNA insert from
the vector.
There were multiple technical problems with this method
which were never solved.
Blunt end ligation was difficult to
achieve since T4 ligase was extremely sensitive to minor
contaminants in the DNA preparations.
Although many different
conditions were tried in order to optimize the alkaline
phosphatase reaction, under every circumstance where the
reaction was complete, the vector was incapable of ligation.
60
F
c o RI
I
PstI
S ail (T a q l)
Sail D igestion
TCGAC*
G-
■G
■CAGC T
DNA p o l y m e r a s e
Alkaline Phosphatase
“GTCGA
CAGCT
TCGAC
AGCTG
4 Ligase
Taql
Taql
TCGAC- ----------- GTCGA
g c t g -£P n a C A GC T
Figure 7. Method for inserting double stranded cDNA into pBr322
by blunt end ligation.
pBR322 was restricted with Sail and the
ends were repaired with DNA polymerase I. The repaired pBR322 was
treated with alkaline phosphate and blunt end ligated with size
selected cDNA to form a recombinant plasmid.
61
These problems were never solved and this method was abandoned.
Insertion of cDNA sequences into a plasmid vector was done
using the homopolymeric tailing procedure (figure 2).
This method
has been used successfully in many cases to insert cDNA sequences
into plasmids.
Some advantages of this system are that (1) the
annealed plasmid can be used directly in the transformation
reaction (no repair synthesis or ligation is necessary),
(2 ) the
plasmid cannot self-anneal so backgrounds remain low, (3) the
insert can in most cases be removed since the PstI sites are
regenerated,
(4) insertion of a double stranded DNA into the PstI
site inactivates the ampicillin resistance gene and so
recombinant colonies can be screened for their tetracycline
resistance and ampicillin sensitivity, and (5) homopolymeric
tailed poly-dG pBR322 is commercially available.
A major
disadvantage of this system is that the transformation efficiency
of the annealed plasmids depends greatly on the number of
nucleotide residues added per end.
The optimal conditions are
when the plasmid and cDNA have the same number of nucleotides
added per end.
This parameter is very variable and changes with
every double stranded cDNA preparation.
When homopolymeric ends
of poly-dC were added to double stranded cDNA preparations, the
reaction was allowed to proceed until a control substrate had 30
cytidine residues added per end.
This corresponded to
roughly 15-20 cytidines per end for the double stranded cDNA
62
(assuming an average size of 600 nucleotide base pairs).
The tailed double stranded cDNA was annealed in various
weight ratios to poly-dG tailed pBR322 and the efficiency of
transformation was monitored (table 3).
A 1:1 molar ratio would
correspond to a 7.5:1 weight ratio, again assuming 600 base pairs
as the average size.
Transformation efficiences did not change
significantly between ratios of 10:1 and 20:1 (w:w).
below 10:1, the efficiency began to decrease.
At ratios
To minimize the
amount of pBR322 needed, an intermediate ratio of 15:1 (molar
ratio of approximately 2 :1 ) was chosen for all further annealing
reactions.
For the double stranded cDNA used to construct the
library, a transformation frequency of 2 .2 x 10
of cDNA was obtained.
recombinants/ug
Background contamination with pBR322 was
estimated by picking four filters randomly from the library and
determining the percentage of colonies which were both ampicillin
and tetracycline resistant.
This was determined to be less than
4% (122 out of 3130).
Transformation efficiencies were found to be affected by a
number of parameters (table 2).
It was found that amounts of
carrier tRNA which were normally added in an ethanol precipitation
resulted in a 3-5 fold reduction in transformation efficiency.
Transformation of DNA in annealing buffer, instead of 0.1M
CaC^f
also resulted in lower transformation frequencies.
For
this reason, annealing reactions were done in very small volumes
Table 2.
Effects of annealing buffer and carrier .tP.NA on the transformation efficiency of closed
circular pBR322.
F. coli HB101 was transformed with closed circular pBR322 in 0.1M CaCl^
containing various amounts of either annealing buffer or carrier tRNA.
Following
transformation bacteria were plated 'onto selective plates and incubated overnight at 37°C.
Amount of Closed
Circular pBR322
(ug)
Experiment 1
Experiment 2
Experiment 3
Final Concentration
of Annealing Buffer
Amount of tRNA
Added (ug)
0.05
Transformation
Efficiency
(colonies/ug)
1.7x1a 5
0.05
IX
-
8 .1x10^
0.05
0 .5X
-
1.2X105
0.05
0.2X
-
1.6X105
0.01
9x10^
0.01
—
50
7.2x1a 5
0.01
—
100
^xlO5
0.01
-
150
b . Oxia5
0.01
-
200
2.2x1a5
0.01
-
0.01
-
50
1.lxio6
0.01
-
100
7.5x10-’
0,01
-
200
5.5X10-5
—
1 .6xl06
O'
UJ
Table 3-
Comparisons of trans-formation frequencies obtained from cDNA annealed with different
amounts of pBR322.
Poly (dC)- double stranded cDNA was annealed with poly (dG)-pBP322
in various weight ratios.
The annealed plasmids were used to transform E. coli HB101.
Following transformation, bacteria were plated onto selective plates and were
incubated overnight at 37°C.
Amount of Double
stranded cDNA
(ng)
Experiment
1
Experiment
Experiment
3
Experiment
1.
4
Batio of PBP322:dscDNA
(w:w)
Colonies per
Plate
Trans formati on
Efficiency
colonies/ug dscDNA
10
7.5 =1
527
4
5.3*10
10
15:1
636
6 .3x10^
25
7.5:1
I2O
4.8x10^
25
15:1
400
1.6x10^
25
5:1
248
25
10 :1
570
25
15:1
710
9.9x10^
1
2 .3x10
1.
2 .3x10
5
1 0 :1
850
1.7X105
5
1.5:1
861
1 .7xl05
5
2 0 :1
1168
2 .3xl05
5
30:1
1146
2 .3*10^
O'
65
and only a portion was used in a transformation reaction.
Differential Colony Filter Hybridization
Each filter in the recombinant library was analyzed in
triplicate with
32
P-cDNA made
from either resting 3T6, growing
3T6 or growing LU3-7 p o l y ( A ) + RNA.
In this screen, sequences
complementary to thymidylate synthetase messenger RNA would give
a positive signal only when hybridized with cDNA from LU3-7
cells.
From estimates of the sensitivity of this procedure by
other investigators,
it was believed that the level of
thymidylate synthetase messenger in LU3-7 cells would give a
detectable signal in colony filter hybridizations.
However, since
the signal was not expected to be very strong, high backgrounds
could not be tolerated.
Background levels of hybridization were
determined in the following manner.
plasmid pDHFR21
Bacteria containing the
(contains the cDNA for dihydrofolate reductase)
were spotted onto filters streaked with 300 colonies containing
pBR322.
The filters were analyzed using
growing poly(A)
32
P-cDNA made from
M50L3 RNA which contains very high levels of
dihydrofolate reductase messenger RNA.
After autoradiography it
was found that these filters only showed signals where bacteria
with pDHFR21 were spotted, even after long times of exposure
(figure 8).
Therefore, background due to hybridization of
labeled cDNA to bacterial DNA or to pBR322 was not a problem.
similar experiment was done to determine if bacteria containing
A
Figure 8. Estimation of background levels in colony filter
hybridization analysis.
Bacteria containing pDHFR21 were
spotted (6 tines) on a nitrocellulose filter streaked with 300
colonies containing pBR322. The filter was hybridized v^ith
P-cDNA synthesized from p o l y ( A )
M50L3 RNA (2x10° c p m ) .
Hybridization was for 20 hours.
The filter was washed,
dried, and analyzed by autoradiography.
66
F i g u r e 8.
68
pDHFR21 could still be detected when among recombinant cDNA
colonies.
In this case, pDHFR21 was spotted onto a filter
containing 700-800 recombinants
(figure 9).
Again signals due to
. .
.
32
hybridization of
P-DHFR cDNA to pDHRF21 were strong.
Further
studies were done with replicas of these filters to determine if
the signal due to pDHFR21 was still detected when the
concentration of dihydrofolate reductase sequences in the probe
was diluted.
Since the relative percentage of dihydrofolate
reductase sequences in M50L3 cells is about 10 times greater than
for thymidylate synthetase sequences in LU3-7 cells, it was hoped
that hybidization signals would still be detected even when the
32
P-cDHFR sequences were diluted tenfold.
These sequences were
diluted by adding increasing concentrations of
from resting 3T6 poly(A)+ RNA.
32
P-cDNA made
The intensity of the pDHFR21
signals, which were initially strong, decreased when increasing
concentrations of resting
hybridization.
32
P-3T6-cDNA were present during the
These signals were still detectable when a tenfold
dilution was made.
The hybridization signals of the pDHFR21
bacteria are not uniform in intensity, even when analyzed on the
same filter.
This may result from insufficient mixing of probe
during the hybridization or differences in colony size.
a major limitation of the technique.
This is
From these experiments it
can also be seen that the overall pattern of hybridization with
32
P-cDNA from growing M50L3 poly(A)
+
RNA or resting 3T6
Figure 9. determination of the sensitivity of colony filter
hybridization.
Replica filters were made each containing
600-1000 recombinant colonies, spotted with 4 colonies of
bacteria containing pDI-IFJl^l. The filters were hybridized
with various ratios of
“P-cDNA made ^from growing M50L3
poly(A)
RNA and resting 3T6 poly (A)
RNA.
After 20 hours,
filters were washed, dried, and analyzed by autoradiography.
Arrows indicate the position of pDHFR21.
Filters were hybridized
with:
A. M50L3 cDNA
B. M50L3 cDNA:R3T6cDNA; 1:5
C .D . M50L3 cDNA:R3T6 cDNA, 1:10
E,F. R3T6 cDNA
69
70
• Jfc 5V,M» ,*
it j B u i
E
F i g u r e 9o
-t *■ *
F
poly(A)+ RNA is very similar.
This
is consistent with the
observation that only a small percentage of the total messenger
RNA sequences change in abundance in these different growth
states (Williams and Penman, 1975).
It is important to note that
even though a large nurriber of colonies show detectable signals
following hybridization,
not.
approximately half of the colonies do
This percentage is too high to be due to colonies
containing pBR322 without a cDNA insert.
Furthermore, the lack of
detectable hybridization is generally not a result of a cDNA
inert being too short (Dworkin and Dawid,
1980).
These colonies are
probably complementary to low abundance messenger RNA.
All of these experiments indicated that colony filter
hybridization was sufficiently sensitive for the detection of
thymidylate synthetase cDNA sequences.
Of the 30,000 colonies
screened, 51 showed differential hybridization, giving signals
only with
32
P-LU3-7 cDNA.
shown in figure 10.
An example of such a colony (p4) is
The 51 colonies were rescreened several times
and 9 colonies were selected for further analysis.
Identification of a Plasmid Containing Thymidylate Synthetase Coding
Sequences
To identify which,
if any, of the nine colonies contained
thymidylate synthetase cDNA sequences, the individual plasmids
were isolated.
Restriction analysis showed that all of them
contained an insert.
Next, a small amount of each plasmid (2 ug)
Figure 10.
Differential filter colony hybridization analysis
of filters containing recombinant cDNA clones. Filters
containing recombinant cDNA colonies were analyzed in
triplicate.
Each filter was hybridized with “P-cDNA
corresponding to messenger RNA frcm resting 3T6, growing 3T6,
or growing LU3-7 cells for 20 hours.
After extensive washing
the filters were dried and autoradiographed. Arrow indicates
a colony (p4) which shows differrential hybridization with the
different probes.
Filters: A. resting 3T6 cDNA probe
B. growing 3T6 cDNA probe
C. growing LU3-7 cDNA probe.
72
F i g u r e 10.
74
was pooled, bound to a nitrocellulose filter and hybridized to
poly (A)
LU3-7 RNA.
Since each cDNA was originally raade from
this messenger RNA population, it was expected that a maximum of
nine different messenger RNAs would bind to the filter.
Therefore, analysis of the in vitro translation products from
the eluted RNAs would result in the presence of a maximum of nine
polypeptides on a denaturing polyacrylamide gel.
this experiment are shown in figure 11.
The results of
When RNA which had been
selected by the nine plasmids was translated, only one
polypeptide was detected (figure 11, lane C).
(Other protein
bands are a result of endogenous protein labelling of the
in vitro translation system. )
protein band which was
This protein comigrated with the
found to be overproduced in in vitro
translation products of LU3-7 p oly (A)
poly(A)+ RNA (figure 11,
RNA relative to 3T6
lanes A, B or G,H).
When the mixture of
nine plasmids was hybridized with the same amount of 3T6 poly(A) +
RNA and the selected messenger RNA was translated, the 38,000
dalton protein was not seen (figure 11, lane E ) .
This
indicated that the selected messenger RNA was present in a
greater amount in LU3-7 cells that in 3T6 cells.
From this it was
concluded that the 38,000 dalton protein corresponded to
thymidylate synthetase and at least one of the nine plasmids
contained the cDNA sequences for this enzyme.
Since only one
protein band was detected, it was possible that all nine plasmids
•
Figure 11. In vitro translation of messenger RNA selected
by hybridiati^n to a mixture of recombinant plasmids (pl-p9).
Total poly (A) RNA (15 ug) from either 3T6 or LU3-7 cells
was hybridized to nitrocellulose filters containing a
mixture of nine recombinant cDNA plasmids (pl-p9) or pBR322.
After extensive washing, the hybridized RNA was eluted and
translated with an in vitro translation system (NEN).
Proteins were separated by SDS-oolyacrylamide gel
electrophoresis
(10%) and detected by autoradiography.
In vitro translations were of:
Lane A, G . LU3-7 poly(^)
RNA
Lane B,H. 3T6 poly(A)
RNA
Lane C. pl-p9 hybrid selected LU3-7 RNA
Lane D. pBR322 hybrid selected LU3-7 RNA
Lane E. pl-p9 hybrid selected 3T6 RNA
Lane F,H. pBR322 hybrid selected 3T6 RNA
75
Figure 11.
77
contained the thymidylate synthetase sequences.
The
hybridization of messenger RNA. to the plasmids was specific since
pBR322 failed to select any translated messenger RNA (figure 11,
lanes D,F).
Each plasmid was then screened individually.
Only
one plasmid, p4, was found to select a messenger RNA which
produced the 38,000 dalton protein.
The rest of the plasmids did
not produce proteins that were detectable in this assay.
reason for this is not understood.
The
It should be noted that under
the same conditions pDHFR21 was able to select a messenger RNA
which when translated produced a protein of the correct molecular
weight for dihydrofolate reductase (data not shown).
Further support that p4 contained thymidylate synthetase
coding sequences was obtained from immunoprecipitation
experiments.
Antiserum specific for thymidylate synthetase was
isolated by Lakshmi Rao.
This antiserum had been shown to
immunoprecipitate a protein of approximately 38,000 daltons from
cytoplasmic extracts of LU3-7 cells and not from 3T6 cell
extracts.
Messenger RNA that had been selected by hybridization
to p4 was translated in v i t r o , immunoprecipitated with anti-TS
immune serum and analyzed on a polyacrylamide gel.
As shown in
figure 12 (lane B), the antiserum was capable of precipitating
this protein, strongly indicating this protein is thymidylate
synthetase.
The specificity of this reaction was shown by the
immunoprecipitation of the in vitro translation products of
Figure 12.
In vitro translation and iipmunoprecipitation of p4
selected messenger RNA.
Total poly (A)
mRNA (15 ug) from LU3-7
cells was hybridized to a nitrocellulose filtgr containing 10 ug
of p4. Hybridization was for 4 hours at 50 . Following
hybridization, filters were washed, selected RNA was eluted and
used in an in vitro translation.
Proteins synthesized in this
way were then either analyzed directly Lanes C,D,E,F or
immunoprecipitated with anti-TS antiserum Lanes A, B.
Translations were of:
Lane A,E. total poly(A)
LU3-7 RNA
Lane B,C. p4 hybrid selected LU3-7 RNA
Lane D. poly(A)
3T6 RNA
Lane F. no RNA
78
A B
F i g u r e 12.
80
total poly(A)+ RNA of LU3-7 cells.
Once again, only one
protein was immunoprecipitated (figure 12, lane A).
controls were done and are shown in figure 13.
More extensive
A single protein
was obtained on a polyacrylamide gel only when an in vivo labeled
cytoplasmic extract of LU3-7,
an in vitro translation of total
poly(A)+ LU3-7 RNA and an in vitro translation from
messenger hybrid selected by p4 were immunoprecipitated with
anti-TS immune serum.
When pre-immune serum was used to
precipitate the in vitro translation of LU3-7 poly(A)+ RNA no
proteins were detected.
This result was also obtained when M50L3
poly(A)+ RNA was translated and immunoprecipitated.
From the
accumulated evidence it was concluded that p4 contains the coding
sequences for thymidylate synthetase.
Restriction Analysis of p4
Restriction analysis of p4 was done to determine the size of
the insert.
The cDNA sequences were removed from pBR322 by
restriction with PstI.
Size determination of the insert was
complicated by the presence of two internal PstI sites.
From
electrophoresis on a 3.5% polyacrylamide gel (figure 14) the size
was determined to be about 1180.
The cDNA sequences were found
to contain a single restriction site for Bam HI and Bgl II, and
no sites for Eco RI, Hind III, Sal I, 9na I or Xba I. A
partial restriction map of the insert is shown in figure 14.
Analysis of Thymidylate Synthetase Messenger RNA Size
Figure 13. In vitro t r a n s l a t i o n and immunoprecipitation
reactions to confirm the identity of p4.
Various messenger
RNA sanples were translated and immunoprecipitated, along with
an in vivo labeled c y t o p l a s m i c extract, with either anti-TS
antiserum (Lanes A,B,D,E) or preiimune serum (Lane C).
Following immunoprecipitation, samples were resuspended in
sample buffer and electrophoresed on a 7.5% polyacrylamide
gel.
The protein sanples which were immunoprecipitated are:
Lane A. cytoplasmic extract from LU3-7
+
Lane B,C. in v i t r o t r a n s l a t e d p o l y ( ^ )
L U 3 - 7 RNA
Lan e D. in v i t r o t r a n s l a t e d poly(A)
M 5 0 L 3 RNA
Lane E. in vitro translated p4 h y b r i d selected messenger RNA
81
A B C D E
Figure 13.
EcoRT
H in dlll
BamHI
P s tI
cDNA
In ser t
P s tI
SaLl
E -E c o R I
P -P stI
T -T a q I
B m -B a m H I
B g -B g lll
P
T P
_L -i
100 b a s e s
I_
Bm
_i—
Bg P
T P
i— i— i—I—
(j.
F i g u r e 14. R e s t r ic t io n anaLysis of p4. A . p4 w as r e s t r i c t e d with e ith e r P s tI (Lane b) or
P s t l- B a m H I (Lane c), and a n a ly z e d on a 3. 5 % poL yacrylam ide geL. A Taql d ig e s tio n
of pB R 322 w as run as a m a r k e r (Lane a). B. p4 w as r e s t r i c t e d with the e n z y m e s
in d ica ted . Shown a r e the a p p r o x im a te s i t e s fo r e a c h e n z y m e .
84
In order to determine if all of the sequences in the
thymidylate synthetase messenger RNA. were represented in the cDNA
insert, the size of the messenger RNA was determined.
Poly(A) +
RNA was denatured, electrophoresed in the presence of
formamide and formaldehyde, transferred to nitrocellulose and
hybridized with nick translated p4 (figure 15).
This analysis
showed that in LU3-7 cells, the major thymidylate synthetase
messenger species was approximately 1500 nucleotides in length.
Higher nolecular weight species (3000-4000 nucleotides) were also
present in lower amounts.
When poly(A)+ RNA was examined
from M50L3 and 3T6 cells, the thymidylate synthetase messenger RNA
also corresponded to 1500 bases.
The presence of higher molecular
weight species was not detected in these cell lines.
studies,
Fran these
it was concluded that p4 does not contain full length
cDNA.
This analysis also shows that the thymidylate synthetase
messenger RNA is overproduced since lane A contains less RNA than
lanes C,D yet the signal is much more intense.
in vitro translation results.
This confirms the
The level of overproduction is
roughly 20-30 fold, estimated by comparing the intensity of the
1500 base RNA in lanes B,C,D of figure 15, as well as from other
determinations.
The poly(A)
cells was examined.
poly(A)
fraction from LU3-7 and M50L3
While no discrete bands were apparent in
RNA from LU3-7 cells,
M50L3 p o l y (A)
RNA had three
I
Figure 15.
Analysis of the size of thymidylate synthetase
messenger RNA in LU3-7, M50L3 and 3T6 RNA.
RNA (poly(A) or
poly(A) ) was denatured and run on a formamide-formdehyde
agarose gel(l%).
The RiTA was transfered to nitrocellulose and
hybridized with nick translated p4 using the dextran sulfate
procedure.
Lane A. 0.5 ug poly (A)
LIJ3-7 RNA
Lane R. 0.05 ug poly(A^_ LU3-7 RNA
Lane C. 1.5 ug poly(A)+ 3T6 RNA
Lane D. 1.5 ug poly(A)
M50L3 RNA
Lane E. 5 ug poly(A) _LU3-7 RNA
Lane F. 10 ug polv(A)_ M50L3 RNA
Lane G.
P poly(A)
marker RNA
85
86
A B C
D E
F G
(— 5 0 0 0
Figure 15.
87
bands Whose size did not correspond to the major poly(A)
species.
RNA
The significance of these bands is unknown. However,
it
does appear that at least in LU3-7 cells, the thymidylate
synthetase messenger RNA is predominantly polyadenylated.
Structure of Thymidylate Synthetase Gene
A preliminary experiment was done to determine if the
thymidylate synthetase gene was amplified in LU3-7 cells.
High
molecular weight DNA was isolated from LU3-7, M50L3 and 3T6 cells.
This DNA was digested with restriction enzymes, electrophoresed
on a 1% agarose gel, transferred to nitrocellulose and hybridized
to nick translated p4.
Although this plasmid does not contain
the entire messenger RNA coding sequence,
large portion of the gene.
surprising.
it should detect a
The results from this experiment were
Restriction digestion of high molecular weight LU3-7
DNA resulted in the production of a number of bands not found in
the parental cell lines (figure 16).
Although some restriction
fragments were present in LU3-7 and the parental cell lines, the
relative intensity of these bands differed.
gene amplification may have occured.
This indicated some
Comparisons of M50L3 and
its parental cell line, 3T6, showed marked differences at this
locus.
These differences may represent clonal differences
between cell lines.
This data indicated that in LU3-7 cells,
gene rearrangement along with some gene amplification has
occurred which in some way is responsible for the amplification
Figure 16. Southern DNA blot analysis of genomic DNA from
sensitive and resistant cell lines. High molecular DNA (5 ug) was
digested with either EcoR (Lanes A,B) or Hind III (Lanes C,D,E) for
DNA fragments were separated by electrophoresis on a 1% agarose
gel and transferred to nitrocellulose paper. The filter was
hybridized with nick translated p4 using the dextran sulfate
procedure.
The filter was washed extensively, dried and
autoradiographed.
Lane A,C. M50L3 DNA
Lane B,D. LU3-7 DNA
Lane E. 3T6 DNA
88
89
kb
A
B
21.8
7.5—
5.9—
5.5—
4.8—
3.4—
Figure 16.
C D
E
of both the messenger RNA and protein in LU3-7 cells.
DISCUSSION
A 5-fluorodeoxyuridine resistant cell line, UJ3-7, was
isolated previously,
in order to facilitate detailed examination
of thymidylate synthetase gene expression.
This cell
line was shown to overproduce thymidylate synthetase 50 fold
compared to the parental cell
line
(Rossana et a l ., 1982).
In vitro translation of LU3-7 p o l y ( A ) + RNA has
shown this
overproduction results from an increase in the amount of
messenger RNA.
A recombinant cDNA library was constructed from
LU3-7 poly(A)+ RNA.
From this
library,
thymidylate synthetase were isolated.
evidence support this conclusion.
the cDNA sequences for
Three lines of
The isolated recombinant
plasmid, p4, was capable of hybridizing to a messenger RNA which
directed the
in vitro synthesis of a 38,000 dalton protein as
analyzed by SDS polyacrylamide gel electrophoresis.
This
corresponds to the size previously determined for thymidylate
synthetase (Rossana et a l . , 1982).
The thymidylate synthetase
cDNA plasmid was capable of selecting messenger RNA only from
LU3-7 cells, indicating that the level of this messenger is
amplified in these cells relative to 3T6.
Finally, this protein
produced by in vitro translation can be immunoprecipitated by
anti-TS immune serum.
Other studies are in progress to add
further support to this conclusion.
91
Currently, comparisons of
92
the in vitro synthesized 38,000 dalton protein and in vivo
synthesized thymidylate synthetase are being made using the
method described by Cleveland et a l . (1977).
In this procedure,
proteins recovered from one polyacrylamide gel are subjected to
partial proteolysis using a variety of proteases and the
resulting fragments are analyzed on a second gel.
The peptides
produced by each protease fingerprint the proteins.
This
technique has been used to show the identity of a number of
proteins (Little and Kleid,
et a l ., 1982).
1977;
Hanson et a l ., 1979; Hanson
Additional evidence will be obtained by
attempting to complement a TS
bacterial strain by transformation
of plasmids containing putative thymidylate synthetase cDNA
sequences.
The entire translated region of the messenger RNA
is usually required for the production of a functional protein.
In the case of thymidylate synthetase, this has been estimated to
be approximately 1150 bases.
Since the messenger RNA for
thymidylate synthetase is larger than this, it contains some
transcribed, untranslated regions.
Depending on the location of
these sequences, p4 may or may not contain enough information to
produce a functional thymidyate synthetase.
Plasmids containing
full length cDNA would be more likely to allow,for bacterial
expression of this enzyme.
Therefore,
further cDNA synthesis
will be done in order to obtain full length transcripts.
This
will be facilitated by priming the initial reverse transcriptase
reaction with internal sequences of the p4 insert.
Double
stranded cDNA made in this manner will be cloned into either the
PstI site of pBR322, the vector, pUC8.
In the case of pBR322,
expression of thymidylate synthetase sequences will rely on
initiation of transcription from the bacterial
promoter.
-lactamase
This system has been used to obtain expression of
interferon, proinsulin and dihydrofolate reductase
(Chang et a l ., ^9 7 8 ;
a l ., 1980).
Villa-Komaroff et a l ., 1978;
Nagata et
Initiation of transcription from this promoter
results in the production of a fused messenger RNA. containing
sequences of the /5-lactamase messenger RNA, as well as insert
sequences.
The formation of a functional thymidylate synthetase
protein will require that the protein coding sequences are in the
correct orientation and appropriate signals for translation
(ribosome binding site) are present.
Fulfillment of these
requirements is unpredictable due to the mode of insertion of the
cDNA sequences into this site.
Some of these problems are
alleviated by using pUC8 as the vector
(Helfman, et a l ., 1983).
In this case, cDNA is inserted into the plasmid using synthetic
linkers.
Therefore, the protein coding sequences can be correctly
oriented in relation to the promoter.
Expression relies on
transcription from the $ -galactosidase promoter.
Since the
messenger RNA produced from this promoter contains correctly
oriented translation signals, one in three cDNA inserts for a
94
particular protein should give rise to a functional protein (if
the insert is full length).
Following transformation bacteria
which can grow in the absence of thymine will be selected and
tested for the presence of the mammalian protein.
The bacterial
enzyme has a molecular weight of about 32,000 daltons (Haertle et
a l ., 1979) and so should be easily distinguished from
the mammalian enzyme by polyacrylamide gel electrophoresis.
Preliminary results have confirmed this.
The definitive
identification of any plasmid as containing thymidylate synthetase
sequences awaits both protein and cDNA sequencing.
The thymidylate synthetase messenger RNA. has been studied by
analyses of RNA blotted to nitrocellulose.
The majority of
thymidylate synthetase sequences appear to be polyadenylated.
In
each cell line examined, a predominant RNA species of
approximately 1500 nucleotides was identified as the thymidylate
synthetase messenger RNA.
This messenger RNA does not contain
very large untranslated regions, especially considering that some
of the length is contributed by the poly(A) sequences.
This
is very different from what is observed for a variety of other
messages.
For example, the messenger RNA for the aspartate
transcarbamylase complex was found to be 7900 bases, which is
2000 bases longer than necessary
(Padgett, et a l ., 1979).
In
the case of dihydrofolate reductase, mouse cells produce four
major messenger RNAs of 750, 1000, 1200, 1600 bases, although one
95
of 650 bases would be sufficient to encode the protein (Setzer et
a l ., 1980).
Also apparent in polyadenylated RNA from LU3-7 cells are
higher molecular weight thymidylate synthetase specific RNAs,
between 3-4 kilobases in length.
These RNAs appear to be present
only in the overproducing cell line.
origins for these RNAs.
There are many possible
They may represent nuclear leakage of
partially processed heterogenous nuclear RNA (hnRNA).
In this
case, their presence in LU3-7 cells might just reflect the higher
concentration of TS-hnRNA or might result from overloading a
specific processing protein for TS-hnRNA,
so that a small
proportion of unprocessed TS-hnRNA leaks from the nucleus during
isolation.
This could be tested by examining cell lines
representing earlier stages of 5FdU resistance.
If these are
unprocessed RNAs, then it is expected that in cell lines in which
there is less overproduction of thymidylate synthetase, these RNA
species will decrease in concentration or disappear frcm the
poly(A)+ RNA fraction. Alternately,
these transcripts may result
frcm changes in termination of transcription or from
transcription of altered genes in the LU3-7 cells.
It will be
interesting to determine if these RNAs are capable of
synthesizing thymidylate synthetase in vitro.
It should be
noted that these species may be associated with polysomes since
they were isolated from precipitated polysomes.
This observation
96
needs further verification, however, since ribonucleoprotein
particles are also precipitated by this method.
It has been reported that in certain cell lines which
overproduce the aspartate transcarbamylase complex, an additional
messenger RNA species for this complex was present.
This RNA was
10.2 kilobases, which is larger than the predominant messenger RNA
of 7.9 kilobases.
Further studies have indicated that this RNA
contains 3' additional sequences, which result frcm the
transcription of an amplified gene which has been altered in this
region
(Padgett et a l ., 1979;
Padgett et a l ., 1982).
Preliminary characterization of the structure of thymidylate
synthetase gene has been done.
Restriction analysis of high
molecular weight DNA from the thymidylate synthetase
overproducing cell line, as well as M50L3 and 3T6 cells, gave a
complex pattern of restriction fragments when hybridized to nick
translated p4 DNA.
In this analysis, only a portion of the gene
sequences can be analyzed since this plasmid does not represent
all of the messenger RNA sequences. The restriction pattern was
reproducible,
indicating that the multiple bands a r e not a result
of incomplete digestion.
These bands could result from the
presence of introns in the gene sequences analyzed.
Multiple
bands, such as those produced here, can also be an indication of
multiple genes.
The interpretation is complicated by the fact
that these bands change in intensity between the two parental
cell lines, M50L3 and 3T6 and that additional restriction
fragments are apparent in the digests of the LU3-7 DNA.
It is
possible that the changes in band intensity represents
differences in restriction sites between individual cells in the
population at the thymidylate synthetase locus.
The additional
fragments in LU3-7 cells may result frcm a gene rearrangement.
Gene rearrangement is also implicated by the fact that 50 fold
amplification of the gene sequences is not appearant in Southern
blot analysis of LU3-7 and 3T6 DNA.
Thus, some other alteration
in the gene must have occured in order to get an increase in the
amount of thymidylate synthetase and its messenger in these cells.
Gene rearrangements may have resulted in the production of a more
transcriptionally active gene.
This may be due to a promoter up
mutation, relocation of the thymidylate synthetase gene into a
more transcriptionally active domain in chromatin or insertion of
the gene next to sequences, such as enhancer sequences, which
cause a stimulation of transcription.
To sort out the origin of
individual restriction fragments, restriction analyses of high
molecular weight DNA will be done with clones of M50L3, 3T6 and
LU3-7.
In this way, it should be determined if differences in
relative intensities between cell lines results from clonal
polymorphisms.
Secondly, DNA frcm independently derived drug
resistant cell lines, as well as LU3-7 cells which have lost
their drug resistance, will be examined. These studies might lead
98
to the identification of particular DNA fragments which give rise
to the drug resistance phenotype.
Since the drug resistance trait has been found to be
unstable in the absence of selective pressure,
it is expected
that the genes for thymidylate synthetase are associated with
double minute chromosomes.
acentromeric chromosomes.
Double minute chromosomes are
These minichromosomes were first
identified in dihydrofolate reductase overproducing cell lines
which in the absence of selective pressure were found to be
unstably resistant to methotrexate
(Kaufman et a l ., 1979).
There is good evidence that double minute chromosomes carry the
genes for dihydrofolate reductase in these cell lines.
Since they
are not associated with spindle fibers during mitosis, they are
randomly distributed.
In the absence of selective pressure these
minichromosomes confer a growth disadvantage to the cell and are
diluted out in successive doublings.
The structure of many genes has been analyzed.
In many
cases, the amount of intron DNA far exceeds that for exon DNA.
For example, the size of the dihydrofolate reductase gene has been
estimated to be at least 31 kilobases,
and yet produces a
cytoplasmic messenger RNA of 1600 bases
(Nunberg, et a l ., 1980).
It will be interesting to see if this is also true for thymidylate
synthetase.
The isolation of a recombinant plasmid containing the
thymidylate synthetase cDNA sequences will permit 'a detailed
examination of the control mechanisms used to regulate
thymidylate synthetase gene expression, using LU3-7 cells as a
model system.
With this probe the cell cycle regulation of
thymidylate synthetase messenger RNA, as well as heterogeneous
nuclear RNA can be studied.
Furthermore, it is now possible to
isolate the gene for this enzyme and determine its organization
and sequence.
It will be important to compare the data obtained
in these studies with that known for other S phase enzymes. Since
these enzymes are coordinately expressed during the cell cycle,
it is possible that their genes share some homologous sequences
which are involved in their coordinate activation.
In this way a
cell cycle regulatory region may be identified. The function of
these regions can be tested by transformation of altered genes
into the appropriate cell line.
All of this information will
lead to further our understanding of processes involved the
regulation of gene expression in a eukaryotic cell.
APPENDIX A
A. RNA Isolation Solutions
1. Phosphate buffered saline- 0.137M NaCl, 0.003M KC1, 0.16M
N a 2 H P 0 4>
0.002M
KH2P04
2. Lysis buffer- 0.033M NaCl,
pH
7.4.
0.001M MgCl2 , 0.0033M Tris BC1
pH 8.4, 1% Nonidet P-40
3. 5X Precipitation buffer-
5mg/ml heparin, 0.5M MgCl2 , 0.125M
Tris HC1 pH 8.4, 0.125 NaCl.
4. Centrifugation buffer- 30% sucrose in 0.00375M MgCl2 , 0.025M NaCl,
0.025M Tris HC1 pH 7.5.
5. SDS buffer- 0.1M NaCl, 0.01M Tris HC1 pH 7.4, 0.001M EDTA, 0.5%
SDS.
6 . Elution buffer- 0.01M Tris HC1 pH 7.4, 0.001M EDTA.
7. Binding buffer- 0.4M Tris HC1 pH 7.4, 0.001M EDTA.
B. DNA Isolation Solutions
1. RSB- 0.01M NaCl,
0.003M M g C l 2 , 0.01M Tris HC1 pH 8.4.
2.
TNE buffer- 0.01M Tris HC1 pH 7.4, 0.1M NaCl, 0.001M
EDTA.
3.
High salt buffer- 0.25M NaCl, O.olM Tris HC1 pH 7.4,
0.001M EDTA.
4.
TE buffer- 0.001M Tris HC1 pH 7.4, 0.0001M EDTA.
C. cDNA Synthesis Solutions
1. Extraction buffer- 0.05M Tris HC1 pH 8.0, 0.1M NaCl, 0.01M EDTA,
0.5% SDS.
2. SI Nuclease digestion buffer- 0.03M Na ( C H 2C O O ) 2 pH 4.6, 0.05M
100
101
NaCl, 0.001M ZnSO^,
5% glycerol,
14 ug/ml denatured calf
thymus DNA..
3. DNA Polymerase I repair buffer- 0.04M KPO^ pH 7.0, 0.001M
mercaptoethanol, 0.0062M MgCl2 , 0.8mM dCTP, dATP, dGTP,
dTTP.
4. .Annealing buffer- 0.01H Tris HC1 pH 7.6, 0.001M EDTA, 0.15M NaCl.
D. Transformation Solutions
1. Luria broth- 1% tryptone, 0.5% yeast extract, 1% NaCl.
2. L. plates- 1% tryptone, 0.5% yeast extract, 1% NaCl, 1.4%
bactoagar.
E. Plasmid Isolation Solutions
1. Lysozyme buffer- 0.05M glucose, 0.01M EDTA, 0.025M Tris HC1 pH 8.4.
2. Alkaline SDS- 0.2N NaOH, 1% SDS.
3. TNE buffer- 0.01M Tris HC1 pH 7.4, 0.1M NaCl, 0.001M EDTA.
4.
TEA buffer- 0.04M Tris HC1 pH 7.9, 0.005M
sodium acetate,
0.001M EDTA.
5. DNA electrophoresis sample buffer- 0.05M EDTA, 50% sucrose,
0.005% bromophenol blue.
F. Hybridization Buffers
1. 20x SSC- 0.15M NaCl, 0.015M Na citrate pH
7.3.
2.
serum albumin, 0.02%
lx Denhardt's- 0.02% Ficoll, 0.02% bovine
polyvinylpyrollidine.
3. 5x SET- 0.15M NaCl, 0.03M Tris HC1 pH 8.0, 0.001M EDTA.
G . RNA Gels; Formaldehyde
102
1. Electrophoresis buffer- 0.02M Na-Mops, 0.005M NaAc, 0.001M
EDTA.
2. Loading buffer- 50% glycerol, 0.001M EDTA, broraophenol blue.
H . Nick T r a n s l a t i o n
I. Sample solution- 4 parts
parts Nal^PO^
N a 4 P 2°7
(saturated solution),
(saturatec^ solution),
4
1 part 100% TCA, 1 part
lOrrM deoxycytidine.
I . Immunoprecipitate Solution
1. 5x Immunoprecipitate buffer- 0.25M Tris HC1 pH 7.4, 0.75M NaCl,
0.1% sodium azide, 2.5% Nonidet P-40, 0.025M EDTA, 25 mg/ml
BSA.
2. lOx Detergents- 10% triton x-100, 10% deoxycholate (w/u).
J . Polyacrylamide Gel Electrophoresis
1. Running gel buffer- 0.375M Tris HC1 pH 9.2, 0.029% TEMED.
2. Stacking gel buffer- 0.06M Tris HC1 pH 7.2, 0.055% TEMED.
3. Electophoresis gel buffer- 0.025M Trizma base (pH about 8.2),
in 1.9M glycine, 0.1% SDS.
4. Fixing Solution- 9% acetic acid, 50% methanol.
5. Destaining solution- 7% acetic acid, 20% methanol.
APPENDIX B
Regulation of ribosomal protein messenger RNA content and
translation during the cell cycle of mouse fibroblasts
The ribosome is a complex organelle made up of both
ribosomal RNA and protein.
Since, ribosomes play a crucial role
in protein synthesis, they have been proposed to be involved in
the regulation of growth in both prokaryotes and eukaryotes.
In
bacteria, it has been found that the concentration of ribosomes
in the cell is proportional to the growth rate (Kjeldgaard and
Gausing).
In eukaryotic cells, the ribosome content has been
implicated as an important difference between transformed and
normal cells
(Stanners et a l ., 1979).
The mechanisms involved in regulating ribosome production
are better understood for prokaryotes than eukaryotes.
In
prokaryotes, ribosomal protein genes are organized into several
polycistronid transcriptional units.
Control of these operons
has been shown to occur autogeneously at both the transcriptional
and translational
level
(Fallon et a l . , 1979,
1980; Zengel et a l ., 1980).
In eukaryotes,
Nomura et a l .,
ribosomal protein
synthesis also appears to be coordinately regulated (Gorenstein
and Warner, 1976; Nabeshima and Ogata, 1980). Investigation of
controls responsible for this coordinate regulation have been
facilitated by the isolation of plasmids containing DNA sequences
103
104
for a number of ribosomal proteins
(Meyhaus and Perry, 1980).
Unlike bacterial cells, mouse ribosomal protein genes are present
in multiple copies Which are dispersed throughout the genome
(Monk et a l ., 1981).
Thus,
coordinate expression of this group
of protein is likely to be more complex.
The content, metabolism and translation of mouse ribosomal
protein messenger RNA. were studied to determine if any of these
processes were involved in the regulation of ribosomal protein
gene expression.
fibroblasts
These were investigated using mouse 3T6
as a model system.
resting cells,
Previous work had shown that in
the content of ribosomes is two to three times
lower than in growing cells
(Johnson et al., 1974). Serum
stimulation
of resting cultures results
in an immediate increase
in the rate
of ribosomal RNA synthesis,with an accumulation of
cytoplasmic ribosomal RNA occuring at about six hours
(Mauck and
Green, 1973 ). Since an increase in ribosomal RNA reflects an
increase in ribosome content, the amount of ribosomal proteins
must also increase during this transition.
This increase can
partly be explained by an increased rate of ribosomal protein
synthesis-.
In mouse 3T6
fibroblasts,
it has been found that
serum stimulation of resting cultures results in a greater than
50% increase in the relative rate of synthesis of at least thirty
individual ribosomal proteins (Tushinski and Warner,
1982).
An
increased rate of synthesis of a particular protein could result
105
from an increase in the amount of messenger RNA for that protein.
DNA excess filter hybridization was used to study the content of
ribosomal protein messenger RNA in resting, growing and serum
stimulated cells.
In these experiments, ribosomal protein
messenger RNA was determined using a mixture of seven different
ribosomal protein cDNA plasmids,
in order to obtain enough
hybridized radioactivity to quantitate. The relative content of
ribosomal protein poly(A)+ messenger RNA in resting cells was
found to be slightly higher than in growing 3T6 cells.
Previous
studies have shown that growing 3T6 cells have a three fold
higher poly(A)+ content of messenger RNA than resting cells
(Johnson et a l ., 1974).
Thus,
although the relative content is
lower, the absolute content of ribosomal protein messenger RNA is
two fold higher.
The content of individual ribosomal protein
messenger RNAs was also examined. This was done by spotting each
plasmid individually onto a nitrocellulose filter,
hybridizing
this with labeled RNA, and quantitating the hybrized RNA by
densitometry of the resultant autoradiograph.
experiments,
From these
it appeared that the relative amounts of the
individual ribosomal protein messenger RNA were approximately the
same in resting and growing cells, indicating that they are
controlled in a coordinant manner. This supports the validity of
using a mixture of plasmids to determine the metabolism of this
group of proteins.
In serum stimulated cells, the content of
106
ribosomal protein messenger RNA was found to remain constant for
six hours and then began increasing linearly.
It is interesting
that this pattern was also observed when the content of ribosomal
RNA was measured (Johnson et a l ., 1974).
coordinate regulation
This suggests some
of the content of these two RNAs.
The rate of synthesis and stability of ribosomal protein
messenger RNA were also examined.
The rate of synthesis of
ribosomal messenger RNA relative to total poly(A)+ was not
significantly different in resting and growing cells.
Following
serum stimulation, the relative rate of synthesis increased only
slighty (40%).
This is in constrast to
a decrease in the
relative content of this messenger at this time.
These
differences may result from many factors. The stability of the
ribosomal protein messenger RNA did not change significantly in
the different growth states examined.
Under all conditions, the
half life was about the same as that for total poly(A)+ RNA.
Following serum stimulation, it had been observed that both
ribosomal content and the rate of ribosomal protein synthesis (at
least in 3T3 cells) increased.
Since this was not due to an
increase in the content of the messenger RNA, some other
mechanism must be involved.
For this reason the translational
efficiency of ribosomal protein messenger RNA was examined.
In
resting cells, a larger percentage of the ribosomal protein
messenger RNA was associated with the nDnosomal fraction of the
107
polysomal gradient, than in either growing or serum stimulated
cells.
These results were obtained for both newly synthesized
messenger RNA (pulse-labeled) or bulk ribosomal protein messenger
RNA (long labeled).
These results indicate that the rate of
ribosomal protein synthesis during the resting to growing
transition is controlled initially at the level of initiation
ribosomal protein messenger RNA translation.
After six hours,
the increase in the content of ribosomal protein messenger RNA
may lead to further increases in this rate.
Although a large
fraction of the resting messenger RNA was isolated in the
monosomal fraction, it still remains to be determined if these
are actively being translated.
These messages could be in an
inactive RNA-protein complex, being stored by the cell for use at
a later time.
Since the translational efficiency of total
poly(A)+ RNA did not change in any of these growth states, it
appears that the increased translational efficiency of ribosomal
protein messenger RNA is species specific.
In other systems,
changes in the rate of initiation of ribosomal protein messenger
RNA translation have been implicated in controlling the rate of
ribosomal protein synthesis (Ignotz, 1981). How this
translational control is exerted is still to be to be determined.
It will be interesting to see if this is a result of autogeneous
regulation as is the case in bacteria.
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