Sequence of a yeast DNA fragment containing a chromosomal replicator and a tRNA Glu 3 gene.

The sequence of a 1.9 kb Bam x Hind III fragment from yeast has been determined. This fragment is part of a yeast 6.7 kb Hind III segment cloned into pBR322 (pY20). The fragment carries a single gene for a glutamate tRNA which has no intron. According to genetic analyses [1] this fragment also contains a yeast chromosomal replicator. We have analyzed the sequence for potential open reading frames and for several structural features which are thought to be involved in the initiation of DNA replication. Hybridization studies have revealed that portions of this sequence are repeated within the yeast genome.     

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.     

Cloning of the ADE2 gene of Saccharomyces cerevisiae and localization of the ARS-sequence

Mutational changes in ADE2 result in the accumulation of red pigment in cells, which serves as an indicator for the selection of mutants. This easily detectable phenotype of red-coloured colonies can account for the wide use of ade2 mutants in yeast genetics. ADE2 gene was cloned in a shuttle vector by complementing the ade2 mutation in the yeast. It was shown that the 2.2 kbp HindIII fragment of yeast DNA contains structural sequences of the ADE2 gene as well as the ARS sequence. Deletion analysis of the 5' end of the ADE2 gene showed the ARS sequence to be situated at the distal end of the 1 kbp HindIII fragment. Removal of the ARS sequence does not influence ADE2 gene complementation ability. Transformants containing the ADE2 gene comprised in their plasmids form white colonies. Loss of the plasmids results in colour change of colonies.     

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme.

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.     

Transformation of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts by a plasmid carrying the ADE2 gene of Saccharomyces cerevisiae

Red adenine-dependent mutants of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts have been transformed by the plasmid pYE (ADE2) 2 DNA containing ADE2 gene from Saccharomyces cerevisiae. The analysis of two P. guilliermondii Ade+-transformants has revealed the integration of pYE (ADE2)2 sequence into the recipient strain genome and partial restoration of the deficient function.     

"Illegal" transformation of red adenine-dependent mutants of the yeast Pichia pinus by the pYE(ADE2)2 plasmid

The red adenine-dependent mutants ade1 of the yeast Pichia pinus blocked in the VI step of adenine biosynthesis (lack of AIR-carboxylase) and ade2 mutants blocked in the VII step of adenine biosynthesis (lack of SAIKAR-synthase) were transformed with the plasmid pYE(ADE2)2 containing ADE2 gene of Saccharomyces cerevisiae encoding AIR-carboxylase. The appearance of white Ade+ clones with the frequency 2-7.10(-8) (which is ten-fold higher than reversion frequency) was only observed in the case of ade2 transformation. Genetic analysis points to connection of the "illegitimate" transformants' appearance with the change in the mutant ade2 locus or in a locus closely linked to the former. Ade+ phenotype was stable during 20 generations of mitotic budding. Southern blotting assay of transformant chromosomal DNA indicates that reconstitution of ade2 defective gene is related with its "correction", owing to integration of pYE(ADE2)2 sequence in the vicinity of the mutant locus.     

The Saccharomyces cerevisiae ADE1 gene: structure, overexpression and possible regulation by general amino acid control.

The ADE1 gene of the yeast Saccharomyces cerevisiae has been cloned by complementation of the ade1 mutation. The nucleotide sequence has been determined for the 918-bp coding region, 240-bp 5'-noncoding region and 292-bp 3'-noncoding region. The sequenced region includes a single large open reading frame coding for a protein of 306 amino acid (aa) residues. The promoter of the ADE1 gene contains a copy of the 5'-TGACTC hexanucleotide, a feature characteristic of promoters under general aa control. Subsequent search of other published purine biosynthesis gene sequences revealed that all of them also contain general aa control signals in their promoter regions. An expression plasmid containing the ADE1 coding region under control of the PHO5 promoter produced N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthetase in yeast cells at a level of 40% of total cellular protein. One-step purification resulted in an almost homogeneous preparation of SAICAR synthetase.     

Chromosome polymorphisms close to the cm-ADE1 locus of Candida maltosa

The imperfect yeast Candida maltosa has an ill-defined genetic constitution; it is nominally diploid, but probably highly aneuploid, in nature. We report on polymorphisms specifically affecting those chromosomes which bear the cm-ADE1 gene. This gene encodes phosphoribosylaminoimidazole-succinocarboxamide synthetase, an enzyme in the adenine biosynthetic pathway. By electrophoretic karyotype analysis, three differently sized chromosomes were demonstrated to carry cm-ADE; the size (but not the number) of these chromosomes was also found to vary, both between strains and during the mitotic growth of a single strain. Four different alleles of cm-ADE1 have been cloned and sequenced from one prototrophic strain. DNA sequence divergence between these different alleles is as high as 8%, with the greatest divergence being found in the upstream region. Mitotic recombination events that led to changes in the karyotype were followed by using cm-ADE1 DNA as an hybridization probe. A recombination hot-spot in the neighbourhood of the gene appears to be responsible for the instability of the chromosomes on which it resides.     

Chromosome polymorphisms close to the cm-ADE1 locus of Candida maltosa

The imperfect yeast Candida maltosa has an ill-defined genetic constitution; it is nominally diploid, but probably highly aneuploid, in nature. We report on polymorphisms specifically affecting those chromosomes which bear the cm-ADE1 gene. This gene encodes phosphoribosylaminoimidazole-succinocarboxamide synthetase, an enzyme in the adenine biosynthetic pathway. By electrophoretic karyotype analysis, three differently sized chromosomes were demonstrated to carry cm-ADE; the size (but not the number) of these chromosomes was also found to vary, both between strains and during the mitotic growth of a single strain. Four different alleles of cm-ADE1 have been cloned and sequenced from one prototrophic strain. DNA sequence divergence between these different alleles is as high as 8%, with the greatest divergence being found in the upstream region. Mitotic recombination events that led to changes in the karyotype were followed by using cm-ADE1 DNA as an hybridization probe. A recombination hot-spot in the neighbourhood of the gene appears to be responsible for the instability of the chromosomes on which it resides.     

Structural comparison of two yeast tRNA Glu 3 genes.

DNA sequences in a 1.7 kb Pst fragment from yeast have been determined. This fragment is part of a yeast 7.4 kb Hind III segment cloned ino pBR322 (pY 5). The fragment carries a single gene for a glutamate tRNA. The coding portion of this gene is identical in sequence to that of the tRNA Glu 3 gene from pY 20 [1]. The flanking regions differ in their sequences, but possible secondary structures within the 5'-flanking regions bear similar features. Sequence homologies between pY 5 and pY 20 were detected far outside the tRNA genes. More surprisingly, extended sequence homologies were seen between the flanking regions of the pY 20 tRNA Glu 3 gene and a tRNA Ser gene [2,3]. We have also checked the known tRNA genes for structural similarities. Hybridization studies indicate that portions of the Pst fragment are repeated within the yeast genome.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the ADE1 gene.

The ADE1 gene of Saccharomyces cerevisiae was isolated by complementation in S. cerevisiae from a yeast genomic DNA library carried on plasmid YEp13. Electron microscopy of R-loop-containing DNA indicated the location of the ADE1 gene on the plasmid insert. Gene disruption and gene replacement were used to demonstrate that the ade1-complementing sequence was the actual ADE1 gene that maps on chromosome I. ade1 strains which normally form red colonies form white ones when transformed with the cloned ADE1 gene. This property should be very useful, since it enables detection of plasmids carrying this gene under nonselective conditions.     

Isolation and characterization of a gene coding for glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

A yeast glyceraldehyde-3-phosphate dehydrogenase gene has been isolated from a collection of Escherichia coli transformants containing randomly sheared segments of yeast genomic DNA. Complementary DNA, synthesized from partially purified glyceraldehyde-3-phosphate dehydrogenase messenger RNA, was used as a hybridization probe for cloning this gene. The isolated hybrid plasmid DNA has been mapped with restriction endonucleases and the location of the glyceraldehyde-3-phosphate dehydrogenase gene within the cloned segment of yeast DNA has been established. There are approximately 4.5 kilobase pairs of DNA sequence flanking either side of the glyceraldehyde-3-phosphate dehydrogenase gene in the cloned segment of yeast DNA. The isolated hybrid plasmid DNA has been used to selectively hybridize glyceraldehyde-3-phosphate dehydrogenase messenger RNA from unfractionated yeast poly(adenylic acid)-containing messenger RNA. The nucleotide sequence of a portion of the isolated hybrid plasmid DNA has been determined. This nucleotide sequence encodes 29 amino acids which are at the COOH terminus of the known amino acid sequence of yeast glyceraldehyde-3-phosphate dehydrogenase.     

Genetic control of purine biosynthesis in Pichia methanolica yeast. The ADE5 (PUR4) gene, controlling 5'-phosphoribosylformyl glycineamide synthetase

By comparing published and experimental data on spontaneous mutability of early genes controlling biosynthesis of purine nucleotides (BPN) in different yeast species in the system "from red to white," it was shown that the PUR4 gene encoding 5'-phosphoribosylformyl glycinamidine synthetase (FGAM-synthetase) (EC 6.3.5.3) is the most mutable gene in yeast Saccharomyces cerevisiae (the ADE6 gene), Schizosaccharomyces pombe (the ade3 gene), and Pichia methanolica (the ADE5 gene). This correlates with a considerably large size of the FGAM-synthetase polypeptide, as compared to the products of other genes belonging to this group. Study of characteristics of spontaneous mutations in early BPN genes of P. methanolica demonstrated that the vast majority of unstable ade5sU alleles (mutations with a high reversion frequency ranging from 0.2 x 10(-6) to 2 x 10(-6)) appeared solely among mutants for the ADE5 gene. Based on these results, it was assumed that there are two independent mechanisms responsible for reversions of spontaneous mutations in this gene. The DNA sequence that can compensate for the P. methanolica ade5 mutation and probably is the structural P-ADE5 gene, was cloned from a genomic library of P. methanolica by the ade6 mutation complementation in the recipient S. cerevisiae strain.     

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.