Plasmid 68 (2012) 186–194
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Plasmid
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Short Communication
New multi-purpose high copy number vector with greater mitotic
stability for diverse applications in fission yeast Schizosaccharomyces pombe
Hemant Kumar Verma, Jagmohan Singh ⇑
Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Sector 39 A, Chandigarh 160036, India
a r t i c l e
i n f o
Article history:
Received 7 June 2012
Accepted 5 July 2012
Available online 15 July 2012
Communicated by Dr. Dhruba K. Chattoraj
Keywords:
Schizosaccharomyces pombe
Copy number
Mitotic stability
Vector
a b s t r a c t
We have constructed a pUC19-based multipurpose ATG vector in Schizosaccharomyces
pombe with higher copy number and mitotic stability possible with commonly used vectors. The vector, having an NdeI site in its polylinker to provide ATG site for expression, carries a greatly truncated version of URA3 gene, URA3m, of Saccharomyces cerevisiae as a
selection marker. In addition, it contains the mat2P-right flank region (mat2P-RF) of S. pombe as an autonomous replicating sequence (ARS) and a polylinker with wider choice of
restriction sites. While URA3m confers an increase in plasmid copy number up to 200 copies/cell, mat2P-RF imparts greater mitotic stability than the standard ars1 element of S.
pombe. Finally, the vector also includes the transcription termination signal of the nmt1
gene (Tnmt1). This basic vector should serve as a versatile tool for studies of gene function
in S. pombe.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
Vectors are fundamental tools in molecular biology and
genetics that can be used to study different physiological
and gene functions within the organism. Vectors can be
used for mutagenesis of genes as well as overexpression
studies. Schizosaccharomyces pombe has been extensively
used for studies in cell biology, genetics and molecular
biology and numerous vectors are in use today (Siam
et al., 2004; Adams et al., 2005; Van Driessche et al., 2005).
Vectors have great impact on protein productivity,
which is governed by factors like plasmid copy number,
plasmid stability and segregation efficiency (Clyne and
Kelly, 1997). Plasmid copy number determines the gene
dosage available for expression and thus generally leads
to higher productivity (Friehs, 2004). Thus, both for obtaining high level commercial scale expression of proteins and
studying gene function at high expression level, achieving
higher copy number should be helpful.
Auxotrophic markers such as ura4, his3, arg6, and ade6
have been commonly used in episomal vectors of S. pombe.
⇑ Corresponding author. Fax: +91 172 2690585.
E-mail address: [email protected] (J. Singh).
0147-619X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.plasmid.2012.07.001
The LEU2 and URA3 genes of Saccharomyces cerevisiae are
also useful markers, which complement leu1-32 and
ura4D18 mutations, respectively in S. pombe (Grimm
et al., 1988; Apolinario et al., 1993; Waddell and Jenkins,
1995; Adams et al., 2005). It is reported that plasmid copy
number can be enhanced by truncation of promoter elements of the selection marker. In S. cerevisiae, the use of
promoter-less URA3 marker and LEU2-d promoter created
by removal of 50 -flanking sequence of LEU2 gene substantially increased the plasmid copy number (Loison et al.,
1989; Steinborn et al., 2007).
The commonly used DNA sequences required for autonomous replication of vectors as episomes include 2lm ori
and ars1. The 2l ori, replication origin of the 2l plasmid
of S. cerevisiae, provides approximately 5 copies/cell in S.
pombe (Heyer et al., 1986; Wright et al., 1986). On the
other hand, ars1 (autonomous replicating sequence) of S.
pombe promotes extra-chromosomal autonomous replication and episomal maintenance of the plasmid. Plasmids
containing ars1 are present in multiple copies per cell
(15–80) but are mitotically unstable (Losson and Lacroute,
1983; Wright et al., 1986; Maundrell et al., 1988). In S.
pombe, pREP series vectors are general purpose episomal
vectors that contain the ars1 replication origin and ura4
H.K. Verma, J. Singh / Plasmid 68 (2012) 186–194
or LEU2 as the selectable marker (Forsburg, 1993). Therefore, there is still a need to develop a vector with greater
copy number and mitotic stability that will ultimately result in increases in the gene copy number and thereby a
higher level of expression of proteins.
In this study, we report the construction of a multi-purpose ATG vector with higher copy number and mitotic stability than the existing vectors that can be used as
expression vector after cloning of promoters and inserting
genes from homologous and heterologous origin downstream, for constructing genomic and cDNA expression libraries and other important genes of interest into the
multiple cloning sites provided in this vector.
2. Materials and methods
2.1. Strains and media
E.coli strain Top10F’ (Invitrogen, USA) and S. pombe wild
type strain SPJ25 (Msmto leu1-32 ura4D18 ade6-210 his2)
were used in this study. Yeast growth medium (YEA) and
selective minimal medium (PMA) supplemented with
appropriate amino acids (Moreno et al., 1991), and bacterial LB and TB media (Sambrook et al., 1989) were used
for growth and maintenance of cultures.
2.2. Design of mitotically stable and high copy number vector
As a part of this study, we intended to generate an ATG
vector with higher copy number as well as greater mitotic
stability. First, we constructed the pUC19-based shuttle
vector, pJS1 (Fig. 1A), which contained ura4 as the selection
marker, ars1 as the origin of replication and multiple cloning sites. To construct a vector with desired complete features, we cloned new selection marker, ARS element and a
polylinker containing additional restriction sites. A schematic picture of the vector series is shown in Fig. 1. The
steps involved in the construction are described below:
2.2.1. Insertion of new multiple cloning sites (MCS)
The NdeI site located in the vector backbone was destroyed by Klenow-fill-in reaction, as we propose to include an NdeI site in the MCS to provide start codon ATG
for any gene of interest cloned downstream of the promoter. The NdeID vector was designated as pJHI (Fig. 1B).
A newly synthesized polylinker to provide a wider choice
of restriction sites in the order SphI-NdeI-XbaI-KpnI-PstISalI-AfeI-XhoI-StuI-BamHI-SmaI-SacI was inserted into
vector pJHI while replacing the existing polylinker (SphIPstI-XhoI-SalI-BamHI-SmaI-SacI) at sites SphI/ SacI. The
resulting vector was named as pJH2 (Fig. 1C).
2.2.2. Selection marker
We inserted the URA3 gene of S. cerevisiae with its minimal regulatory sequences in the vector. We amplified the
918 bp region, consisting of 45 bp upstream to 69 bp
downstream of the URA3 gene, by PCR, using primers 50 ATGCAAGCTTACCCAACTGCACAGAAC-30 (forward) and 50 ATGCAAGCTTCTGATATAATTAAATTGAAGC-30
(reverse),
having HindIII restriction sites, using S. cerevisiae genomic
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DNA as the template and named it as URA3m. The resulting
PCR product was sub-cloned in the pJET1.2 vector (CloneJET PCR Cloning Kit, Fermentas, USA) and sequenced to
confirm the correctness of sequence. The URA3m fragment
was excised with HindIII from the pJET1.2 vector and
cloned at HindIII site in the vector pJH2 by replacing the
ura4 selectable marker. The resulting vector with URA3m
marker was named as pJH3 (Fig. 1D).
2.2.3. ARS element
As a putative ARS element (Olsson et al., 1993), we excised the 1.3 Kb NcoI/HindIII fragment of right flank of
mat2-P (mat2P-RF) from the plasmid pSP10 (Kelly et al.,
1988) containing the 6.4 Kb HindIII region of mat2-P locus.
The fragment was blunt-ended by Klenow-fill-in reaction
and inserted into the blunt-ended EcoRI site of vector
pJH3, thus replacing the EcoRI fragment containing ars1
in the vector pJH3 with the mat2P-RF ARS to yield the vector pJH4 (Fig. 1E).
2.3. Quantitation of plasmid copy number
To check the plasmid copy number, vectors pWH5,
pJH2, pJH3 and pJH4 were introduced into the S. pombe
wild type strain SPJ25 by the DNA-mediated transformation procedure (Moreno et al., 1991). The vector pWH5
containing 2l ori and LEU2 selection marker, which exists
at 5 copies per cell in S. pombe (Heyer et al., 1986; Wright
et al., 1986) served as a copy number control. Total genomic DNA of the transformants was isolated and quantitated visually in 0.8% agarose gel on UV transilluminator
or spectrophotometrically on nanodrop (ND-1000, USA).
To plot a standard curve for quantitation, a known amount
of genomic DNA (ranging from 0.5 to 4 lg) of the strain
harboring the plasmid pWH5 was hybridized in duplicate
on nylon membrane (Amersham Biosciences, USA). To
check the copy number of vectors pJH2, pJH3 and pJH4,
varying amounts of genomic DNA of the respective transformants required to achieve radioactive band intensity,
upon hybridization, in the linear range of the pWH5 standard, were standardized. Accordingly, 100 ng of genomic
DNA of the strains having plasmids pJH2, pJH3 and pJH4
was hybridized in quadruplicate. Furthermore, 25 ng of linearized 2.6 Kb pUC19 DNA was radio-labeled with [a-32P]
dCTP by random priming and hybridized with the blotted
DNA. Band intensities were measured in PSL/mm2 by densitometric analysis on Phosphoimager (FUJIFILM FLA-9000,
Japan). A standard curve was plotted between radioactive
band intensity (in PSL/mm2) and the amount of genomic
DNA for the vector pWH5. The copy number of plasmids
pJH2, pJH3 and pJH4 was calculated by comparing the
average of the band intensities (PSL/mm2) with the standard curve.
2.4. Determination of ARS activity/transformation efficiency in
S. pombe
To determine the transformation efficiency of the vectors with different ARS elements 0.1 lg DNA of each plasmid (pWH5 containing 2l ori of S. cerevisiae, pJH2, pJH3
both containing ars1 and pJH4 containing mat2P-RF of S.
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Fig. 1. Schematic representation of vector series described in this study. (A) Vector pJS1 was modified to generate vector with increased copy number and
mitotic stability. (B) To generate ATG vector, the NdeI site located in the vector was destroyed by Klenow-fill-in reaction, yielding the vector pJH1. (C) Newly
synthesized polylinker was inserted by replacing existing MCS at SphI/SacI sites, resulting in vector pJH2. (D) To increase the vector copy number S.
cerevisiae URA3 auxotrophic marker with its minimal promoter sequence, URA3m, was PCR amplified and cloned at HindIII site by replacing existing S.
pombe ura4 marker, resulting in vector pJH3. (E) Furthermore, S. pombe ars1 was replaced by mat2P-RF resulting in the vector pJH4. The 1.3 Kb mat2P-RF
(NcoI/HindIII) fragment was excised out from pSP10 (vector having 6.4 Kb HindIII region of mat2P), blunt-ended by Klenow-fill-in reaction and ligated into
the blunt-ended vector, generated after ars1 removal by EcoRI digestion followed by Klenow-fill-in reaction.
H.K. Verma, J. Singh / Plasmid 68 (2012) 186–194
pombe) was transformed into S. pombe wild type strain,
which was auxotrophic for leu1 and ura4 genes, using lithium acetate method (Moreno et al., 1991). Approximately
1 108 cells were used for each transformation and plated
on the selective plates (PMA-leu or PMA-ura). These
plates were incubated at 30 °C and after 5 days, transformants were counted. Three such individual transformations were carried out and colony counts (transformants/
lg of plasmid DNA) were averaged.
2.5. Determination of mitotic stability of plasmids
The mitotic stability of plasmids was determined
according to Clyne and Kelly (1997). Transformants were
grown in selective media at 30 °C. After 24 h of growth,
these cultures were diluted to final cell density of
1 103 cells/ml (based on 1 OD = 2 107 cells/ml; Clyne
and Kelly, 1997) and plated on complete plates (PMA)
and selective plates (PMA-leu or PMA-ura). Plates were
incubated at 30 °C for 3–4 days and the percentage of plasmid containing cells (F0) on selective plates was determined. Each value plotted was an average of at least
three independent experiments.
2.6. Determination of plasmid loss rate in S. pombe
Plasmid loss rate was determined according to Clyne
and Kelly (1997). Cultures grown for 24 h at 30 °C in selective media were diluted to 1:1000 into non selective/complete media (PMA) and grown further for 15 generations at
30 °C. After two days these cultures were diluted to
1 103 cells/ml and plated onto selective plates (PMA
leu/ura) and nonselective/complete plates (PMA) and
incubated for 2–3 days at 30 °C. The percentage of plasmid-containing cells (F1) under non-selective conditions
was determined. Plasmid loss rate per generation was calculated by using the following equation according to Longtine et al. (1992): Loss rate = 2{1exp [(1/n) ln (F1/F0)]},
where n is the number of generations between measurements F0 and F1. Loss rate was plotted an average of at least
three independent experiments.
3. Results
3.1. Study of ARS activity/transformation efficiency in S.
pombe
We first compared the transformation efficiency of different plasmids constructed by us. We found that plasmid
pWH5 (2l) was not as efficient as plasmids pJH2 and pJH3
containing ars1, which showed almost 2-fold higher transformation efficiency. Among ars1-containing plasmids,
pJH3 showed slightly higher transformation efficiency than
pJH2. This small difference could be due to the effect of
selection marker (URA3m). Furthermore, transformation
efficiency was approximately 2-fold higher in case of the
vector pJH4 having mat2P-RF as compared to the vectors
pJH2 and pJH3 and almost 4-fold higher as compared to
the vector pWH5 (Fig. 2).
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3.2. Mitotic stability of plasmids
Next we compared the mitotic stability of plasmids.
Interestingly, the mitotic stability of plasmid pJH4 (containing mat2P-RF) was estimated to be approximately 2fold higher than the plasmid pWH5 and approximately
1.4-fold higher than that of ars1-containing plasmids. A
slight difference was observed in the stability of plasmids
pJH2 and pJH3: the latter shows a slightly but consistently
greater mitotic stability (Fig. 3).
3.3. Plasmid loss rate in S. pombe
Plasmid stability can be affected both by the copy number and the segregation efficiency of the vector. However,
this difference should not be problem when culture is
grown in selective media. We found that plasmid pWH5
(2l ori) showed an almost 2-fold greater rate of plasmid
loss as compared to the ars1-containing plasmids pJH2
and pJH3, indicating lack of efficient segregation of 2l
plasmids in S. pombe. The ars1-containing plasmids
showed lower plasmid loss rate probably indicating their
improved segregation function and stability (Fig. 4). Surprisingly, although plasmid pJH4 containing mat2P-RF
showed maximum mitotic stability, it also showed a
slightly higher loss rate as compared to plasmids having
ars1.
3.4. S. cerevisiae URA3m marker causes increase in plasmid
copy number in S. pombe
DNA hybridization experiment showed that the plasmid pJH2 with ars1 was present at 50 copies/cell, 10-fold
higher than pWH5. Interestingly, the copy number of vectors pJH3 and pJH4 having URA3m was estimated to be
200 copies/cell (Fig. 5). Thus, copy number increased by
at least 4-fold when S. pombe ura4 was replaced with
URA3m (S. cerevisiae marker with its minimal promoter sequence). The vector pJH4 showed no significant increase in
copy number as compared to pJH3 indicating an almost
similar replication efficiency of ars1 and mat2P-RF elements of S. pombe (Fig. 5) although the latter has greater
mitotic stability.
Based on greater mitotic stability demonstrated above,
we constructed the basic high copy ATG vector with truncated selectable marker URA3m and ARS element mat2PRF.
3.5. Insertion of nmt1 terminator sequences (Tnmt1)
Transcription terminator is required for efficient transcription termination and polyadenylation of the mRNA
of any gene of interest that one wishes to express. Therefore, we inserted terminator sequences of nmt1 gene of S.
pombe into our basic vector pJH4. We isolated the 1 Kb
SmaI/SacI fragment containing nmt1 transcription terminator and polyadenylation signal from the vector pREP3X
(Maundrell, 1993) and inserted into the MCS of pJH4 plasmid at SmaI/SacI sites. The resulting vector with nmt1 terminator was designated as pJH5 (Fig. 6).
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Fig. 2. Comparison of transformation efficiency of different plasmids. One hundred nanograms of each plasmid pWH5 (2l, LEU2), pJH2 (ura4, ars1), pJH3
(URA3m, ars1) and pJH4 (URA3m, mat2P-RF) was transformed into S. pombe wild type strain with leu1–32, ura4D18 mutations. Number of transformants
obtained from three different experiments were averaged and plotted (upper panel). Lower panel represents photographs of transformants, grown on
respective media plates.
4. Discussion
The broad objective of our study was to construct a
multi-purpose ATG vector with greater mitotic stability
and copy number in comparison with the available vectors
of S. pombe. While ATG codon provides translational start
site, mitotic stability and high copy number of vectors
are basic requirements for high level expression of pombe
genes and for achieving higher and stable product yield
of heterologous proteins. Several reports suggest that copy
number can be increased using a defective promoter element for expression control of a selection marker (Loison
et al., 1989; Kjaerulff and Jensen, 2005; Steinborn et al.,
2007). For example, an attenuated version of URA3, named
URA3d, containing some of the proximal mRNA start site
sequences showed a 1.5–2-fold increase in copy number
as compared to the vectors containing wild type URA3
and ura4 (Kjaerulff and Jensen, 2005). In the present study,
in order to develop a vector with even higher copy number,
we tested an even more severely truncated URA3 gene,
URA3m, with only the minimal possible promoter element
of URA3 gene. By using URA3m we have successfully increased the copy number of an ars1/mat2P-RF-containing
vector by at least 2.5–13-fold (200 copies/cell) as compared to plasmid containing ura4, as the reported copy
number of the currently used ars1-vectors in S. pombe lies
in range of 15–80 copies/cell (Giga-Hama et al., 1994).
It has been reported that mat2P-RF (right flank) has high
ARS-like activity (Olsson et al., 1993). Therefore, we tested
it by using different parameters like transformation efficiency, mitotic stability and plasmid loss rate. We found
that mat2P-RF confers greater transformation efficiency
(Fig. 2, compare pJH3 and pJH4) and mitotic stability
(Fig. 3), while it confers slightly greater plasmid loss rate
than ars1 (Fig. 4). On the other hand, we found no significant difference between mat2P-RF and ars1 of S. pombe in
H.K. Verma, J. Singh / Plasmid 68 (2012) 186–194
191
Fig. 3. Mitotic stability of plasmids containing different ARS elements. Transformants containing different plasmids (as indicated) were grown at 30 °C in
selective minimal media (PMA-leu or PMA-ura). After 24 h of growth, 1 103 cells/ml were plated on complete (PMA) and selective plates (PMA-leu or
PMA-ura). After 2–3 days, percentage of leu+ or ura+ colonies were calculated and plotted. Each value is an average of at least three measurements.
Fig. 4. Loss rate of plasmids containing different ARS elements. Cultures of strains containing the indicated plasmids were grown in selective media for
24 h, diluted to 1:1000 before being inoculated into non-selective complete media (PMA) and grown for 15 generations. Approximately 1 103 cells/ml
were plated on complete (PMA) and selective plates (PMA-leu or PMA-ura). Loss rate per generation was calculated according to Longtine et al. (1992).
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Fig. 5. Determination of vector copy number. (A) Standard graph of pWH5. A known copy number plasmid pWH5 (5 copies/cell) was taken as reference.
0.5–4 lg of genomic DNA of S. pombe wild type strain harboring pWH5 was hybridized in duplicates on nylon membrane with radio-labeled probe of linear
pUC19 vector. Band intensities were calculated by densitometric analysis on phosphoimager. Average of band intensity in PSL/mm2 on Y-axis was plotted
against the genomic DNA of the S. pombe strain having pWH5. Total DNA concentration was taken in log2 scale at X-axis. (B) To quantitate the vector copy
number dot-blot assay was done. 100 ng of genomic DNA was hybridized as slot-blot on nylon membrane in quadruplets. 25 ng of linear pUC19 vector was
radio-labeled and used as probe. The radio-active band intensity was calculated by densitometric analysis on phosphoimager and average of bands intensity
(PSL/mm2) was taken to estimate the copy number.
conferring high copy number, which seems to be mainly
due to the URA3m marker with minimal promoter. Previously, a G418-based selection system was developed to select the cells with high plasmid copy number up to
170 copies/cell (Giga-Hama et al., 1994). This system allowed selection for increasing copy number with increasing concentration of the antibiotic G418. However, both
from industrial point of view and carrying out basic physiological experiments, the use of costly antibiotics is not
cost-effective. Our approach to increase the copy number
avoids the use of antibiotics. Thus, our high copy vector
should be useful for experiments involving high level
expression of proteins. Furthermore, besides being more
cost-effective, commercial use of this vector would be in
better compliance with the guidelines for pharmaceutical
protein production.
The vector could potentially cause derepression of the
chromosomal mat2P locus in the strain having stable
mat1 M locus, owing to sequestration of silencing factors
by the mat2P-RF on a high copy plasmid. However, lack
of haploid meiosis phenotype in the transformants rules
out that possibility (not shown).
The new vector has some additional advantages. The
transformation efficiency of plasmid pJH4-mat2P-RF is almost 2-fold higher than the ars1-containing plasmids
(pJH3 and pJH4) and 4-fold higher than the plasmid
pWH5-2l. Although high-frequency transformation is a
desirable characteristic of an ARS element, transformation
frequency may be affected by factors other than the intrinsic replication activity of an ARS element (Clyne and Kelly,
1997). Other assays have been developed in an attempt to
obtain more quantitative measure of ARS function. The
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193
Fig. 6. Construction of the ATG vector pJH5 having greater mitotic stability and copy number. The Tnmt1 fragment containing the 1 Kb transcription
terminator and polyadenylation signal of nmt1 gene of S. pombe was isolated from pREP3X vector by restriction digestion with SmaI/SacI and inserted into
SmaI/SacI sites in the MCS of the plasmid pJH4 by standard ligation procedure. Resulting vector with Tnmt1 terminator was designated as plasmid pJH5.
most commonly employed assays are mitotic stability and
plasmid loss rate. Our data suggest that plasmid pWH5-2l
was more unstable in S. pombe as compared to ars1 (pJH2
and pJH3) and mat2P-RF (pJH4) containing plasmids. Interestingly, mat2P-RF containing plasmid pJH4 showed more
than twofold greater stability in comparison with plasmid
pWH5 (2l ori) and approximately 1.4-fold higher than
plasmid pJH2 (ars1) of S. pombe. The slight difference in mitotic stability of vector pJH3 and pJH2, while both plasmids
contain ars1, might be due to the selection marker URA3m
(pJH3); the defective promoter may augment the plasmid
copy number by selection and thus enhance the apparent
plasmid stability indirectly.
In addition, we found that plasmid loss rate per generation was maximum, approximately 2-fold higher, in case
of plasmid pWH5-2l as compared to plasmids containing
ars1 (pJH2 and pJH3). We observed an almost similar rate
of plasmid loss in case of vectors pJH2 and pJH3 although
they have different copy numbers. It has been noticed that
plasmids with different copy number may have same plasmid loss rate and increasing the plasmid copy number does
not necessarily decrease plasmid loss rate (Longtine et al.,
1992). We have made similar observations in our study.
We observed that the loss rate was also slightly higher in
case of plasmid pJH4 (mat2P-RF) in comparison with
ars1-bearing plasmids pJH2 and pJH3 but the two plasmids
had comparable level of mitotic stability. In S. pombe, difference in the rate of loss of plasmids containing various
ARS elements is due to unequal partitioning of newly replicated plasmids to the daughter cells during cell division.
The rate of plasmid loss with different ARS elements is largely a function of the efficacy of its replication. However,
even plasmids containing efficient ARS elements display
mitotic stability <100%. In budding yeast, only 43% cells
on average retained ARS1-plasmids in plasmid stability assay (Marahrens and Stillman, 1992). Moreover, such assay
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can provide more sensitive measure of ARS activity than
the standard transformation assay. It is reported, that certain mutations in the budding yeast ARS1 do not dramatically affect transformation frequency, but significantly
increase the rate of plasmid loss per generation (Marahrens and Stillman, 1992). Thus, we conclude that we have
obtained new vector pJH4 containing URA3m selection
marker and mat2P-RF as an ARS element, having higher
transformation frequency and mitotic stability. The final
vector pJH5 was derived by inserting the nmt1 terminator
sequence into pJH4 as described in Section 2.
The vector pJH5 can serve as a platform for multiple
applications. First, it can be used to construct high copy
genomic and cDNA libraries that could be useful for complementation studies and to study the effect of high level
over-expression of genes on physiology of S. pombe cells
and for cloning genes by complementation. Second, we
can generate a high copy promoter library between SphI
and NdeI sites, while suitable reporters can be cloned using
NdeI site to provide the first ATG codon. Such a library
could be useful for screening for new pombe promoters.
Lastly, given the hitherto highest reported copy number
of any plasmid without any drug marker, these vectors
could be developed for heterologous gene expression by
cloning of strong promoters between SphI and NdeI sites
and the gene of interest between NdeI (for providing ATG
codon) and suitable downstream sites in the polylinker.
One limitation of this study is that we have not cloned
regulatable promoters in the polylinker. Given the fact that
the existing promoters, like nmt1, do not provide high enough expression of heterologous proteins and are not userfriendly, we propose to isolate new strong promoters.
These promoter elements will then be cloned to develop
new expression vectors, which could provide cost-effective
and high level expression of useful heterologous proteins.
Acknowledgments
Council of Scientific and Industrial Research (CSIR) is
acknowledged for fellowship to Hemant Kumar Verma.
We thank S. Kumaran for discussion.
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