Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.
ABSTRACT The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 184.108.40.206) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.
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ABSTRACT: The budding yeast Saccharomyces cerevisiae is characterized by asymmetric cell division and the asymmetric inheritance of spindle components during normal vegetative growth and during certain specialized cell divisions. There has been a longstanding interest in the possibility that yeast chromosomes segregate non-randomly during mitosis and that some of the differences between mother and daughter cells could be explained by selective chromatid segregation. This review traces the history of the experiments to determine if there is biased chromatid segregation in yeast. The special aspects of spindle morphogenesis and behavior in yeast that could accommodate a mechanism for biased segregation are discussed. Finally, a recent experiment demonstrated that yeast chromatids segregate randomly without mother-daughter bias in a common laboratory strain grown under routine laboratory conditions.Chromosome Research 05/2013; 21(3):193-202. · 2.69 Impact Factor
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ABSTRACT: Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae reduce the levels of the NUC2 endo-exonuclease by approximately 90% compared to the levels in wild-type strains. To examine the potential role of this nuclease in the induction of mitochondrial 'petite' mutations, congenic RAD52 and rad52-1 haploids were subjected to treatment with ethidium bromide, a well-known inducer of these mutations. The rad52 strain showed a much higher resistance to ethidium bromide-induced petite formation than the corresponding wild-type strain. Two approaches were taken to confirm that this finding reflected the nuclease deficiency, and not some other effect attributable to the rad52-1 mutation. First, a multicopy plasmid (YEp213-10) carrying NUC2 was transformed into a RAD52 strain. This resulted in an increased fraction of spontaneous petite mutations relative to that seen for the same strain without the plasmid and sensitized the strain carrying the plasmid to petite induction by ethidium bromide treatment. Second, a strain having a nuc2 allele that encodes a temperature-sensitive nuclease was treated with ethidium bromide at the restrictive and permissive temperatures. Petite induction was reduced under restrictive conditions. Enzyme assays revealed that the RAD52 (YEp213-10) strain had the highest level of antibody-precipitable NUC2 endo-exonuclease whereas the nuc2 and rad52 mutants had the lowest levels. Furthermore, addition of ethidium bromide to the reaction mixture stimulated the activity of the nuclease on double-stranded DNA. Petite induction by antifolate-mediated thymine nucleotide depletion was also inhibited by inactivation of RAD52 indicating that the effect of reduced NUC2 endo-exonuclease was not restricted to ethidium bromide treatment.(ABSTRACT TRUNCATED AT 250 WORDS)Current Genetics 08/1991; 20(1-2):39-44. · 1.71 Impact Factor
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ABSTRACT: Received 3November 1983/Accepted 13February 1984 Deoxycytidylate deaminase activity inSaccharomyces cerevisiae hasbeenpartially characterized. The yeast enzymewasfound toexhibit properties similar tothose ofdCMPdeaminases isolated fromhigher eucaryotes. A mutant strain completely deficient indCMPdeaminase activity wasisolated byselection for resistance to5-fluoro-2'-deoxycytidylate followed byscreening forcross sensitivity to5-fluoro-2'-deoxyuri- dylate, apotent inhibitor oftheyeast thymidylate synthetase. We havedesignated this newallele dcdl. A strain exhibiting anauxotrophic requirement fordUMP wasisolated after mutagenesis ofadcdltup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted fromacombination ofthedcdl allele andasecond, unlinked, nuclear mutation thatwe designated dmpl.Thisallele, whichbyitself conveys noreadily discernable phenotype, presumably impairs efficient synthesis ofdUMPfromUDP.The auxotrophic requirement ofdcdldmpltup7strains alsocanbesatisfied byexogenous dTMPbutnot deoxyuridine. Inrecent years numerous studies, inbothprocaryotic and eucaryotic systems, haveshownthat disturbances indeoxy- nucleotide biosynthesis arecapable ofinducing various kinds ofgenetic change (15). Previous workinourlaboratory hasshownthat inhibition ofdTMPsynthesis inSaccharomy- cescerevisiae results inenhanced frequencies ofmitotic recombination events, bothgeneconversion andmitotic crossing over(1,16). Inviewofthese findings, particularly those involving pyrimidine deoxynucleotides,
MOLECULAR AND CELLULAR BIoLoGY, Apr. 1982, p. 437-442
Vol. 2, No. 4
Isolation of the Thymidylate Synthetase Gene (TMPJ) by
Complementation in Saccharomyces cerevisiae
G. R. TAYLOR, B. J. BARCLAY,t R. K. STORMSJJ. D. FRIESEN,§ AND R. H. HAYNES*
Department ofBiology, York University, Toronto, Ontario, Canada M3J IP3
Received 21 May 1981/Accepted 20 October 1981
The structural gene (TMPI) for yeast thymidylate synthetase (thymidylate
synthase; EC 220.127.116.11) was isolated from a chimeric plasmid bank by genetic
complementation in Saccharomyces cerevisiae. Retransformation of the dTMP
auxotroph GY712 and a temperature-sensitive mutant (cdc2l) with purified
plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the
plasmid was tested for the ability to complement a bacterial thyA mutant that
lacks functional thymidylate synthetase. Although it was not possible to select
Thy' transformants directly, it was found that all pTL1 transformants were
phenotypically Thy' after several generations of growth in nonselective condi-
tions. Thus, yeast thymidylate synthetase is biologically active in Escherichia
coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure
liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3HJdTMP.
In protein extracts from the thymidylate auxotroph (tmpl-6) enzymatic conver-
sion ofdUMP to dTMP was barely detectable. Lysates ofpTL1 transformants of
this strain, however, had thymidylate synthetase activity that was comparable to
that of the wild-type strain.
Previous studies have shown that perturba-
tions in thymine nucleotide pools in yeast,
provoked either by blockage of thymidylate
synthetase (thymidylate synthase; N5,N10-meth-
ase; EC 18.104.22.168) activity or provision of excess
product of the thymidylate synthetase reaction
(dTMP), have profound genetic consequences.
Among the various findings, two of the most
striking are the extreme mutagenicity of exoge-
nous thymidylate and the high mitotic recombi-
nation frequencies observed in dTMP-starved
cells. These experiments have suggested a possi-
ble role for pyrimidine nucleotide metabolism in
the control of mitotic recombination (18) and
mutation (3). In addition to these results, an
intriguing finding is that the growth ofvegetative
yeast cells in limiting concentrations ofthymidy-
late results in the rapid appearance of a nuclear
dense body. The formation ofthis nuclear dense
body occurs in both haploid and diploid cells
that are starved for thymidylate in nutrient broth
medium. This finding was somewhat surprising,
as this structure had previously been observed
only in diploid meiotic cells. A possible explana-
tPresent address: Department of Biological Sciences,
Brock University, St. Catharines, Ontario, Canada L2S 3A1.
Present address: Department of Biology, Concordia Uni-
versity, Montreal, Quebec, Canada H3G 1M8.
0Present address: Department of Medical Genetics, Facul-
ty of Medicine, University of Toronto, Toronto, Ontario,
Canada MSS 1A8.
tion for this observation is that thymine nucleo-
tides or their metabolic derivatives may be im-
plicated in regulating the meiotic pathway in
yeasts (25). These studies have been greatly
facilitated by the availability of yeast mutants
defective (7, 20) or conditionally defective (5,
13, 16) in thymidylate synthetase.
Thymidylate synthetase catalyzes the methyl-
ation of dUMP to dTMP. The cofactor in this
(6, 12). During the reaction the cofactor donates
a methyl group to the 5 position of the pyrimi-
dine ring ofdUMP; concomitantly, the cofactor
is oxidized to dihydrofolate.
Little is known about the control and regula-
tion of thymidylate synthetase in eucaryotes, in
spite of the fact that in addition to its possible
involvement in mediating the genetic effects
described above, it is a common target enzyme
in cancer chemotherapy (10, 27). As a prelude to
the investigation of thymidylate synthetase
expression in yeasts at the molecular level, we
report here the isolation of the structural gene
(TMPI) for yeast thymidylate synthetase.
MATERIALS AND METHODS
Strains. The haploid Saccharomyces
strain GY712 (a leu2-3 leu2-112 tmpl-6 tup-), kindly
provided by G. B. Y. Kiss, was derived from a cross of
LL20 (a leu2-3 leu2-112 his3-11 his3-15), from G. R.
Fink, with 308/6C (a tmpl-6 tup- lysi trpS ilvl-92
hisl,7), which has been previously described (2, 20).
The cdc2l strain gt7/14 (a cdc2l leu2-3 leu2-112) was
TAYLOR ET AL.
derived from a cross of LL20 with the cdc2l strain
obtained from J. C. Game. The wild-type S. cerevisiae
strain used was S288C (a mal gao, obtained from the
stock collection of the University of California at
Berkeley. The Escherichia coli strains used were RP31
(F- Alac thyA argFpro relA Rif' Su-) and JF1754 (lac
gal metB leuB hisB436 hsdR).
Media and buffers. For routine growth, bacterial
strains were cultured in complete medium (LB) as
described previously (19). Yeast thymidylate auxo-
trophs were grown at 34°C in YPD medium (20)
supplemented with 1.5 mg of KH2PO4 per ml and
dTMP (100 ,ug/ml), in accordance with the conditions
described by Little and Haynes (20). Temperature-
sensitive thymidylate synthetase mutants were grown
at 25°C. The defined medium used for the growth ofE.
coli was M9 plus 0.2% glucose (1), and that used for
the growth of yeast strains was SD (20). KET buffer
contained 1 M KCI, 100 mM EDTA, and 50 mM Tris-
hydrochloride (pH 7.5).
transferred from agarose gels to nitrocellulose mem-
branes as described by Southern (29). 32P-labeled
plasmid DNA for probes was prepared by the nick
translation method with E. coli DNA polymerase (23).
Restriction endonudeases. The restriction endonu-
cleases were purchased from Bethesda Research Lab-
oratories (Rockville, Md.) and were used as recom-
mended by the supplier. DNA fragments were
analyzed by 0.7% agarose gel electrophoresis as de-
scribed elsewhere (28). Restriction fragment sizes
were determined with reference to lambda DNA frag-
ments of known size.
Transformation. The transformation of E. coli was
done by the method of Mandel and Higa (22). The
transformation of yeasts was carried out essentially as
described by Hinnen et al. (17), with the modifications
reported by McNeil et al. (24).
DNA preparations. Covalently closed circular plas-
mid DNA and nuclear DNA were isolated from E. coli
as described by Clewell and Helinski (9). Yeast DNA
for the Southern analysis was isolated by the method
of Cryer et al. (11). Rapid isolation of yeast DNA for
the transformation of E. coli was done essentially as
described by Livingston and Hahne (21).
Thymidylate synthetase assay. Logarithmic-phase
cells were harvested by centrifugation, washed in
KET buffer, and resuspended in the same buffer
containing 5 mg ofzymolyase (Kirin Breweries, Japan)
per ml and 0.25% (vol/vol) P-mercaptoethanol. The
cell suspension was incubated in the presence of the
enzyme until the majority of cells were spheroplasts,
as determined by lysis in 1% sodium dodecyl sulfate.
Spheroplasts were washed twice in 1 M sorbitol,
suspended in water, and stored at -60°C. Immediately
before use the spheroplasts were lysed by repeated
freezing at -60°C and thawing to room temperature.
As reported previously by Bisson and Thorner (5), we
found that the enzyme activity remained stable under
these conditions for several weeks. Protein concentra-
tions were determined by the biuret reagent method
(14). The assay conditions used were those described
by Bisson and Thorner (5), except that a commercial
preparation of tetrahydrofolic acid obtained from ICN
Pharmaceuticals (Cleveland, Ohio) was used. Conver-
sion of [6-'H]dUMP (Moravek Chemicals, Brea, Cal-
if.) to [63-H]dTMP was measured by high-pressure
conditions. DNA fragments were
liquid chromotography in a Beckman Altex C"8 ion
pair ultrasphere column (Beckman Instruments, Inc.,
Fullerton, Calif.). The mobile phase was 20%o (vollvol)
methanol-water (high-pressure liquid chromatography
grade; J. T. Baker Chemical Co., Phillipsburg, N.J.)
containing 5 mM tetrabutylammonium phosphate
(PicA; Waters Associates, Mississauga, Ontario) col-
lected at a flow rate of 1 ml/min. Radioactive samples
were counted in 5 ml of Phase Combining System
Liquid (Amersham Corp., Arlington Heights, Ill.) in a
Searle model 6892 liquid scintillation system (G. D
Searle and Co., of Canada Ltd., Oakville, Ontario).
Isolation of the yeast thymidylate synthetase
gene. The yeast thymidylate synthetase gene
was isolated directly in yeast. This was accom-
plished by the transformation of yeast strain
GY712 (leu2 tmpl) followed by simultaneous
selection for Leu+ and Tmp+ clones in regenera-
tion media containing limiting leucine (2% YPD
medium supplemented with 1.5 mg of KH2PO4
per ml) and thymidylate (1 ,ug/ml). Limiting
requirements were included in the regeneration
media to allow for expression of the LEU2 and
TMPI genes after transformation. GY712 was
transformed with pYF91, a high-frequency
transforming vector (Fig. 1) carrying HindIII
fragments of total yeast DNA (30). After trans-
formation, the plates were incubated at 34°C for
5 to 6 days; putative Leu+ Tmp+ transformants
were picked and scored for dTMP prototrophy.
FIG. 1. Restriction endonuclease map ofthe isolat-
ed plasmid (pTL1) containing the yeast thymidylate
synthetase gene. The thick segment represents the 9.6-
kb fragment isolated from a HindlIl digest of total
yeast DNA. The thin segment represents the cloning
vehicle pYF91 constructed by Storms et al. (30). Ap,
Ampicillin resistance; 2pL, 2- Lm plasmid DNA; Tc',
fragment of the tetracycline resistance gene.
MOL. CELL. BIOL.
CLONING OF YEAST THYMIDYLATE SYNTHETASE
Of 600 colonies tested, only 2 were found to be
Plasmid DNA was isolated from the two
GY712 Tmp+ transformants by a modification of
the method described by Livingston and Hahne
(21). These rapid plasmid preparations were
used to transform E. coli strain JF1754 to ampi-
cillin resistance. Plasmid DNA was then purified
from these transformants by cesium chloride-
ethidium bromide density gradient centrifuga-
tion and analyzed by restriction endonuclease
digestion and agarose gel electrophoresis. This
analysis showed that the plasmids retrieved
from the two independent GY712 transformants
contained the same 9.6-kilobase (kb) insert
cloned in the same orientation. The restriction
endonuclease map of the plasmid (pTL1) is
shown in Fig. 1.
The 9.6-kb Hindlll insert was recloned into
pACYC184 (8). This plasmid (pTL3) was puri-
fied, labeled by nick translation (23), and used to
probe Southern blots (29) of HindIII and PstI
digests of pTL1 and S288C DNA. This analysis
(Fig. 2) showed that pTL1 and S288C both
contain a common 9.6-kb HindIll fragment
(lanes 1 and 2). The upper band (lane 2), showing
weaker hybridization, resulted from the homolo-
gy of pACYC184 to sequences found on
pBR322, the E. coli plasmid contained in the
original chimenc plasmid pYF91. The PstI-di-
gested pTL1 and yeast DNA samples both con-
tained two major bands of the same size (Fig. 2,
lanes 3 and 4). The weaker hybridization bands
present in Fig. 2 (lane 1 and most ofthose in lane
3) arose because the 9.6-kb yeast fragment con-
tained some sequences that were repeated sev-
eral times within the yeast genome. Hybridiza-
tion of 32P-labeled pTL3 DNA to Southern blots
of total E. coli DNA revealed no homology
between E. coli DNA and pTL3 DNA under
Purified plasmid DNA was used to transform
the original yeast host strain GY712 and a yeast
strain (gt7/14) that is temperature sensitive at the
thymidylate synthetase locus (cdc2l). As shown
in Table 1, both of these strains were trans-
formed to dTMP prototrophy at a high frequen-
cy by the pTL1 plasmid.
Growth of the dTMP auxotroph containing the
pTL1 plasmid was vigorous under selective con-
ditions. However, during permissive growth
conditions we found that after approximately
nine cell doublings, 50%o of the transformants
were phenotypically Tmp- Leu-. This instabil-
ity is characteristic of yeast cells that have been
transformed with autonomously replicating plas-
mids (30, 33).
It has been shown previously that several
eucaryotic genes complement analogous mutant
FIG. 2. Southern analysis ofpTL1 and S288C. Restric-
tion endonuclase-digested total yeast DNA and puified
pTL1 plasmid DNA were transferred after agarose gel
electrophoresis to nitrocellulose filters by the method
of Southern (29). One-tenth microgram of 32P-labeled
pTL3, a plasmid consisting of the 9.6-kb yeast thymi-
dylate synthetase-encoding HindlIl fragment cloned
into the HindIII site of pACYC184 (8), at a specific
activity of 8.7 x 106 cpm per Fjg ofDNA, was used as
the probe. Lane 1, 2.5 g ofHindIll-digested total yeast
DNA; lane 2, 1.3 x 1O-3 j.g of HindIll-digested pTL1
DNA; lane 3, 2.5"zgofPstI-digested yeast DNA; lane
4, 1.3 x 1O-3 ,ug of PstI-digested pTL1 DNA. The
intense band present in lane 1 and also present in lane
2 is 9.6 kb. The two intense bands in lane 3 and also
present in lane 4 are the 5.1-kb upper band and the 2.5-
kb lower band.
alleles in E. coli (26, 31, 32, 34). It was of
interest, therefore, to determine whether yeast
thymidylate synthetase, the activity of which is
known to be cell cycle dependent in vivo (C.
McLaughin, personal communication), was bio-
logically active in a procaryote. pTL1 trans-
formed E. coli strain RP31 to ampicillin resist-
ance at a frequency of 1.1 x 104 per microgram
of DNA. No transformants were obtained when
Thy+ clones were selected directly. However,
all of the Ampr transformants were capable of
growth after replication on Thy- medium (Table
Enzyme assay. To quantitate thymidylate syn-
thetase activity in pTL1 transformants, we de-
veloped a modification of the enzyme assay
published previously (5). We wished to incorpo-
rate into the procedure a more direct measure-
VOL. 2, 1982
TAYLOR ET AL.
TABLE 1. Transformation of S. cerevisiae and E.
Transformantsb per micro-
8.5 x 103
4.1 x 103
4.8 x 103
2.5 x 102
2.6 x 102
3.2 x 102
RP31 No DNA
1.1 x l04
6.2 x 104
5.8 x 104
aYeast transformation was carried out essentially
as described by Hinnen et al. (17) and modified by
McNeil et al. (24). Transformants of gt7/14 were kept
at 25°C for 48 h and then shifted to 37C for 4 days to
score Leu+ Tmp+ transformants. E. coli strain RP31
was transformed as previously described (22). Trans-
formants were spread on solid ampicillin-selective
medium containing thymidine (50 ,ug/ml). Colonies
grown overnight were then replica plated on solid
thymidine-selective medium. It was found that direct
selection of Thy' colonies yielded no transformants.
b For yeasts, the transformants were Leu+ (column
A) and Leu' Tmp+ (column B). For bacteria, the
transformants were Ampr (column A) and Ampr Thy'
ment of the conversion of the substrate (dUMP)
to the product (dTMP) by exploiting the sensitiv-
ity and relative ease of high-pressure liquid
experiments revealed that deoxyribonucleotide
monophosphates separated well after elution
from a reversed phase column with 20%o metha-
nol-water as the mobile phase, in combination
with 5 mM tetrabutylammonium phosphate (see
Materials and Methods). Under these conditions
deoxyribonucleotide monophosphates could be
easily separated within 20 min in the following
order: dCMP < dUMP < dGMP < dTMP <
dAMP. For the enzyme assay, samples were
incubated for 60 min at 30°C, combined with a
mixture of 100 ,umol of deoxyribonucleotide
monophosphates, and then run through the col-
umn. After fractionation, the dUMP and dTMP
radioactive peaks were identified by comparison
with 254-nm detector traces.
As shown in Fig. 3, extracts from wild-type
yeast cells had thymidylate synthetase activity
that was dependent on the presence ofa reduced
folate cofactor. The activity was strongly inhib-
ited by 5-fluoro-2'-deoxyuridine 5'-monophos-
phate (FdUMP). These results are substantially
in agreement with data previously reported by
Bisson and Thorner (5). In cell lysates ofa tmpl-
6 mutant, enzyme levels were barely detectable.
However, when this dTMP auxotroph was
transformed with plasmid pTL1, the specific
activity of the enzyme was similar to that ob-
served in the wild-type strain (Table 2 and Fig.
FIG. 3. Thymidylate synthetase activity in cell ly-
sates. Cell lysates from spheroplasts were assayed for
thymidylate synthetase activity under the reaction
conditions described by Bisson and Thorner (5). Con-
version of [6-3H]dUMP to [6-3H]dTMP was monitored
by high pressure liquid chromatography (Beckman
Alex C"8 ion pair ultrasphere column, model 332;
liquid phase, 20% methanol plus 5mM tetrabutylam-
monium phosphate). The reaction mixtures were incu-
bated at 30°C for 60 min. (A) No enzyme; (B) LL20
(wild type); (C) LL20 minus tetrahydrofolate; (D)
LL20 plus FdUMP; (E) GY712 (tmpl-6 mutant); (F)
pTL1 transformant of GY712.
/0 2030 4050
A dUMPd dMP
dUMPdrMP4F dU/MP dTrMP
MOL. CELL. BIOL.
CLONING OF YEAST THYMIDYLATE SYNTHETASE
TABLE 2. Thymidylate synthetase assay
a tup- tmpl-
aReaction conditions were as described in the leg-
end to Fig. 3.
We have isolated the thymidylate synthetase
gene (TMPI) by using a high-frequency yeast-
transforming vector containing the LEU2 gene
and random inserts of a partial HindIII digest of
the total yeast genome. Leu+ Tmp+ transfor-
mants were selected by genetic complementa-
tion in a leu2 tmpl-6 host. The two GY712
transformants obtained during the initial screen-
ing segregated Leu- Tmp- cells at a high fre-
quency. This is evident because 50%o of the
GY712 transformants became Leu- Tmp- after
growth under nonselective conditions for about
nine generations. This instability is characteris-
tic of yeast strains containing chimeric plasmids
like pYF91 (4). Southern analysis and restriction
endonuclease mapping demonstrated that the
two original GY712 transformants contained the
same plasmid (pTL1). Southern analysis showed
major bands ofhybridization between the 9.6-kb
insert and S288C or pTL1 DNA which were
consistent with those predicted from the restric-
tion map (see Fig. 1). This suggests that the
cloned fragment encoding thymidylate synthe-
tase activity is present in the yeast genome. No
homology was detectable with E. coli chromo-
somal DNA under similar hybridization condi-
During the initial screening of the clone bank,
limiting exogenous thymidylate was provided
during the selection procedure. As yeasts lack a
functional thymidine kinase (15), we assumed
that plasmid thymidylate synthetase expression
would be necessary to supply thymine nucleo-
tides for DNA replication in dTMP auxotrophs.
Thus, provision of dTMP might be necessary
early in the transformation episode to maintain
balanced DNA precursor pools. However, in
subsequent experiments in which purified pTL1
plasmid was used to transform the original yeast
host, we found that we were able to select Tmp+
transformants directly at a high frequency with-
out the dTMP supplement. In contrast, we found
that it was not possible to select Thy' transfor-
mants in E. coli without prior growth of cells
under nonselective conditions. One interpreta-
tion ofthese data is that yeasts contain sufficient
pools of thymine nucleotides to allow for repli-
cation of plasmid DNA in newly transformed
cells. In bacteria, however, these deoxyribonu-
cleotide pools may be much smaller. Thus,
before a gene is established as an autonomously
replicating unit in the bacterial host, the plasmid
DNA could be damaged during attempted DNA
replication at low intracellular concentrations of
could lose viability as a result of the "thymine-
Transformation of a cdc2l strain, shown pre-
viously by Game (13) to be allelic with the tmpl-6
mutants used in this study, yielded transfor-
mants that no longer had a temperature-sensitive
phenotype. In spite of the fact that the transfor-
mants were incubated at the permissive tem-
perature for 48 h to allow for the expression of
the cloned gene, the transformation frequency
during these experiments was somewhat lower
than that obtained in the tmpl-6 auxotroph.
Although there are considerable differences in
transformation frequencies in yeasts which de-
pend upon the genetic background of the host
strain, another consideration is that cdc2l
strains are defective in thymidylate synthetase
activity at 25°C (5). Thus, the conditions for
DNA replication may not be ideal in these
strains because of DNA precursor imbalances,
even at the permissive temperature.
Thymidylate synthetase activity measured in
crude cell lysates was found to have those
characteristics previously reported by Bisson
and Thorner (5); i.e., the enzyme had an abso-
lute requirement for a reduced folate cofactor
and was strongly inhibited by FdUMP. In pro-
tein extracts from the thymidylate auxotroph
(tmpl-6), enzymatic conversion of dUMP to
dTMP was barely detectable. Lysates of pTL1
transformants of this strain, however, had thy-
midylate synthetase activity comparable to that
of the wild-type strain. One interpretation of the
latter finding is that the plasmid-encoded thymi-
dylate synthetase activity may be regulated in
the host cell.
VOL. 2, 1982
442 TAYLOR ET AL.
We thank Kathryn Pepper for expert technical assistance,
G. An for preparation of the clone bank DNA, and Christina
Sutherland for help in preparing the manuscript. We also
thank J. G. Little and E. M. McIntosh for helpful discussion.
This work was supported by grants from the Natural
Sciences and Engineering Research Council of Canada.
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