Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region.

To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CEN1-ADE1-CDC15 region from chromosome I was isolated and characterized. Starting with the previously cloned ADE1 gene (J. C. Crowley and D. B. Kaback, J. Bacteriol. 159:413-417, 1984), a series of recombinant lambda bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization. The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CEN1). The CDC15 gene was located by isolating plasmids from a YCp50 S. cerevisiae genomic library that complemented the cdc15-1 mutation. S. cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization. The cdc15-1-complementing plasmids all shared only one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene. This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1. CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S. cerevisiae plasmid vectors. Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CEN1 to CDC15. In addition to the transcribed regions corresponding to the ADE1 and ADC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Ty1 transposable element. Notably, a higher than average proportion of the DNA examined was transcribed into RNA.     

Molecular cloning of the ADE1 gene of Saccharomyces cerevisiae and stability of the transformants

Plasmid YEp(ADE1)1a, containing a 2.7-kb Sau3A fragment of Saccharomyces cerevisiae DNA inserted at the BamHI site of the yeast shuttle vector pBTI-1 (Morris et al., 1981), results in high frequency, unstable transformation of ade1 yeast strains. A second plasmid, YRp(ADE1)2, containing adjacent 0.5-kb and 3.0-kb BamHI fragments in pBR322 gave three types of yeast transformants: (1) transformants carrying extrachromosomal copies of the plasmid which indicate the presence of a functional ars sequence, (2) transformants indistinguishable from ade1 strains by hybridization analyis, and (3) a transformant carrying a multimeric form of YRp(ADE1)2. Cells transformed with either of the plasmids are free of the red pigment characteristic of ade1 mutants and indicate potential for direct colour-based selection of yeast transformants using ADE1 plasmids.     

Molecular cloning of the actin gene from yeast Saccharomyces cerevisiae.

Two overlapping DNA fragments from yeast Saccharomyces cerevisiae containing the actin gene have been inserted into pBR322 and cloned in E.coli. Clones were identified by hybridization to complementary RNA from a plasmid containing a copy of Dictyostelium actin mRNA. One recombinant plasmid obtained (pYA102) contains a 3.93-kb Hindlll fragment, the other (pYA208) a 5.1-kb Pstl fragment, both share a common 2.2-kb fragment harboring part of the actin gene. Cloned yeast actin DNA was identified by R-loop formation and translation of the hybridized actin mRNA and by DNA sequence analysis. Cytoplasmic actin mRNA has been estimated to be about 1250 nucleotides long. There is only one type of the actin gene in S.cerevisiae.     

Molecular cloning and sequencing of genomic DNA encoding aminopeptidase I from Saccharomyces cerevisiae

Yeast aminopeptidase I is a vacuolar enzyme, which catalyzes the removal of amino acids from the NH2 terminus of peptides and proteins (Frey, J., and Rohm, K-H. (1978) Biochim. Biophys. Acta 527, 31-41). A yeast genomic DNA encoding aminopeptidase I was cloned from a yeast EMBL3A library and sequenced. The DNA sequence encodes a precursor protein containing 514 amino acid residues. The "mature" protein, whose NH2-terminal sequence was confirmed by automated Edman degradation, consists, based only on the DNA sequence, of 469 amino acids. A 45-residue presequence contains positively and negatively charged as well as hydrophobic residues, and its NH2-terminal residues could be arrayed in an amphiphilic alpha-helix. This presequence differs from the signal sequences which direct proteins across bacterial plasma membranes and endoplasmic reticulum or into mitochondria. It remains to be established how this unique presequence targets aminopeptidase I to yeast vacuoles and how this sorting utilizes classical protein secretory pathways. Further, the aminopeptidase I gene, localized previously by genetic mapping to yeast chromosome XI and called the LAP4 gene (Trumbly, R. J., and Bradley, G. (1983) J. Bacteriol. 156, 36-48), was determined by DNA blot analyses to be a single copy gene located on chromosome XI.     

Participation of the HIM1 gene from Saccharomyces cerevisiae in correction of heteroduplex DNA. Molecular cloning of the gene]

A group of Saccharomyces cerevisiae mutants deficient in repair of induced premutation lesions (him mutants) were previously isolated in our laboratory. Recessive him1 mutant had enhanced level of spontaneous and induced mutagenesis as well as specific altered mitotic conversion. This HIM1 gene was supposed to be involved in the process of mismatch correction of heteroduplexes. In this paper the correction efficiency of in vitro constructed heteroduplex DNA in wild-type cells and him1 mutant was studied. In the former cells heteroduplex DNA was repaired highly efficiently (about 90%), this repair efficiency being reduced in him cells approx. two times as compared with the wild-type cells. Molecular cloning of yeast chromosomal DNA fragments containing HIM1 gene was carried out. The clones were selected from the bank of yeast DNA fragments by complementing him1-1 mutation which enhances conversion frequency in ADE2 gene. One of the DNA fragments was analysed by restriction endonuclease digestion and shown to contain an insert of 6 Kb. Chromosomal integrants were obtained by homologous recombination between the plasmid and chromosomal gene him1.     

Molecular cloning of the Candida maltosa ADE1 gene.

The structural gene (ADE1) encoding phosphoribosyl-aminoimidazole-succinocarboxamide synthetase (SAICAR synthetase; EC 6.3.2.6) in Candida maltosa has been isolated by functional complementation of an ade1 strain of Saccharomyces cerevisiae. The gene was localized on a 2.5-kb BamHI DNA fragment. Nucleotide sequence analysis of the cloned gene has revealed an open reading frame encoding a protein (SAICAR synthetase) with an Mr of 32,751. The codon bias index, 0.68, indicates that the ADE1 gene is a moderately highly expressed gene. The cloned gene shows 63.5% nt identity and 65.2% deduced amino acid identity with the S. cerevisiae ADE1 gene which encodes the same enzymatic activity. The gene may be used as a convenient genetic marker for construction of a new host-vector system for C. maltosa.     

Molecular cloning and nucleotide sequence analysis of the Saccharomyces cerevisiae RAD1 gene.

We have screened a yeast genomic library for complementation of the UV sensitivity of mutants defective in the RAD1 gene and isolated a plasmid designated pNF1000 with an 8.9-kilobase insert. This multicopy plasmid quantitatively complemented the UV sensitivity of two rad1 mutants tested but did not affect the UV resistance of other rad mutants. The location of the UV resistance function in pNF1000 was determined by deletion analysis, and an internal fragment of the putative RAD1 gene was integrated into the genome of a RAD1 strain. Genetic analysis of several integrants showed that integration occurred at the chromosomal RAD1 site, demonstrating that the internal fragment was derived from the RAD1 gene. A 3.88-kilobase region of pNF1000 was sequenced and showed the presence of a small open reading frame 243 nucleotides long that is apparently unrelated to RAD1, as well as a 2,916-nucleotide larger open reading frame presumed to encode RAD1 protein. Depending on which of two possible ATG codons initiates translation, the size of the RAD1 protein is calculated at 110 or 97 kilodaltons.     

A directed DNA sequencing strategy based upon Tn3 transposon mutagenesis: application to the ADE1 locus on Saccharomyces cerevisiae chromosome I.

We have developed a directed DNA sequencing strategy based upon the Escherichia coli transposon Tn3. This transposon displays little sequence specificity for transposition and is thus well suited to this task. Both mini-Tn3 transposons and sequencing vectors bearing the phage f1 single stranded origin of replication have been constructed. Upon mutagenesis of a target sequence, a population is produced in which each clone has two f1 origins of replication, one of which is at a variable position depending upon the transposon insertion site. When helper phage is added to the mutagenised population, the two f1 origins present on each clone are nicked, dividing the packaged strand into two segments, each of which is packaged into a separate phage particle. One of these segments contains no resistance markers and is lost, whilst the other is recovered as a deleted clone with a single chimeric f1 origin. A unidirectionally, variably-deleted set of sequencing clones is produced, and appropriately sized clones are sequenced using a primer complimentary to the transposon end. In addition to being inexpensive, the method does not require the same degree of technical expertise needed for many in vitro, enzymatically based methods. The strategy has been used to determine 2.6 kilobases of nucleotide sequence in the Saccharomyces cerevisiae ADE 1 locus.     

Molecular cloning and primary structure of the uracil-DNA-glycosylase gene from Saccharomyces cerevisiae

The structural gene for the Saccharomyces cerevisiae repair enzyme uracil-DNA-glycosylase (UNG1) was selected from a yeast genomic library in the multicopy vector YEp24 by complementation of the ung1-1 mutant in in vitro enzyme assays. The sequenced gene has an open reading frame which codes for a protein with molecular weight of 40,471. The measured size of the mRNA of 1.25 kb is in agreement with the predicted molecular weight of the protein. The gene product was overproduced about 100-fold in strains carrying an UNG1 gene containing plasmid at 100-200 copies/cell. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cleared lysates from such an overproducing strain, followed by renaturation of enzyme activity from individual gel slices showed the presence of two enzymatic activities in comparable quantities with Mr values of 39,500 and 33,000, indicating that the full size protein is either readily degraded in vivo or is very sensitive to proteolytic digestion in vitro. The carboxyl-terminal two-thirds of the yeast uracil-DNA-glycosylase is highly homologous to the entire Escherichia coli enzyme (50% amino acid identity). Genetic mapping experiments have localized the UNG1 gene on the left arm of chromosome XIII at 17 cM from the GAL80 locus proximal to the centromer. Deletions of the UNG1 gene are viable.     

Molecular cloning and genetic mapping of the DNA topoisomerase II gene of Saccharomyces cerevisiae

The structural gene for DNA topoisomerase II from the yeast Saccharomyces cerevisiae has been cloned. The clones were selected from a YEp13 plasmid bank of yeast DNA by complementing a temperature-sensitive mutation (top2-1) in the topoisomerase II gene, TOP2. Chromosomal integrants of the clone were derived by homologous recombination in strains lacking the 2 mu circle plasmid. Genetic analysis of these integrants indicates that we have cloned the TOP2 gene and not an extragenic suppressor. A YEp13-TOP2 hybrid plasmid integrant was used to localize the TOP2 gene to the left arm of chromosome XIV by the 2 mu circle-directed marker loss method. Results from standard meiotic mapping experiments indicate that TOP2 is about 16 centi-Morgans to the centromere proximal side of MET4. Northern blot analysis of TOP2 RNA isolated from a wild-type strain and from an rna2 mutant shows the RNA to be 4.5 kb long in both cases, thus indicating that the TOP2 gene has no large introns.     

Molecular cloning of the RAD10 gene of Saccharomyces cerevisiae

We have cloned the RAD10 gene of Saccharomyces cerevisiae and physically mapped it to a 1.0-kb DNA fragment. Strains containing disruptions of the RAD10 gene were found to show enhanced UV sensitivity compared with the previously characterized rad10-1 or rad10-2 mutants. The UV sensitivity of the disruption mutant is comparable to the highly UV sensitive rad1-19, rad2-delta, and rad3-2 mutants.     

Nucleotide sequence of the ADE 1 gene of the yeast Saccharomyces cerevisiae

The yeast ADE 1 gene has been cloned and sequenced. The primary structure deduced from the nucleotide sequence demonstrated that phosphoribosylaminoimidazole-succinocarboxamide synthetase is a protein with molecular weight of 34 500 D.     

The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors

We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevisiae. A DNA fragment of 2241 bp capable of complementing ade2 mutations was modified so it is available as a single BglII fragment for use in yeast vectors or for gene disruptions. The minimal fragment codes for a putative protein which is highly similar to the protein encoded by the ADE6 gene from Schizosaccharomyces pombe and to the proteins encoded by the purEK operon of Escherichia coli.     

Molecular nature of the mutations in gene ADE2 in Saccharomyces cerevisiae yeasts. III. Determination of the correlation of the GC AT pairs in genes ADE1 and ADE2

Lethal and mutagenic effects and the nature of mutations induced by decay of 32P incorporated into yeast cell DNA as 32P-deoxyguanosine monophosphate (32PdGMP) and 32P-thymidine monophosphate (32P-TMP), were studied. The lethal efficiency per 32P decay is independent of a labelled nucleotide incorporated into DNA. However, the mutagenic efficiency in ADE1, ADE2 genes per 32P decay is approximately 3 times greater for 32PdGMP than for 32P-TMP. This suggests that ADE1, ADE2 genes contain about 3 times more GC base pairs than AT pairs. Variations in a relative frequencies of GC leads to AT and AT leads to GC transitions were obtained depending upon a nucleotide labelled.     

Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae.

We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective in the RAD1, RAD2, RAD4, and RAD14 loci was not affected by transformation with these plasmids. The 4.5-kb fragment was subcloned into the integrating yeast vector YIp5, and the resultant plasmid was used to transform the rad3-1 mutant to UV resistance. Both genetic and physical studies showed that this plasmid integrated by homologous recombination into the rad3 site uniquely. We conclude from these studies that the cloned DNA that transforms the rad3-1 mutant to UV resistance contains the yeast chromosomal RAD3 gene. The 4.5-kb fragment was mapped by restriction analysis, and studies on some of the subclones generated from this fragment indicate that the RAD3 gene is at least 1.5 kb in size.     

Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.