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.     

Cloning and mapping of Saccharomyces cerevisiae photoreactivation gene PHR1.

The yeast Saccharomyces cerevisiae, like most organisms, is able to directly repair pyrimidine dimers by using a photoreactivating enzyme and visible light. Cells carrying the phr1 mutation were shown previously to be unable to photoreactivate dimers, but neither the map position nor the primary gene product of the PHR1 gene has been determined. We have cloned this gene and determined its map position. A plasmid containing a 6.4-kilobase yeast DNA insert has been isolated and shown to restore photoreactivation in a phr1 strain. A 3.1-kilobase subclone has also been shown to complement phr1. The original plasmid was targeted to integrate into chromosomal DNA at a site homologous to the insert by cutting within the insert. Two of these integrants have been mapped on the right arm of chromosome XV; the integrants have been further mapped at ca. 13 centimorgans from prt1. It has also been independently determined that phr1 maps at this location. Thus, we have determined the map position of PHR1 and also have shown that the plasmid contains PHR1 rather than a suppressor of the phr1 mutation.     

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.     

Cloning and mapping of the RAD50 gene of Saccharomyces cerevisiae

Summary The RAD50 gene was cloned as a 4.8 kb fragment in the 2 derived plasmid pFL1. The gene resides in a 3.9 kb segment that was subcloned into the plasmid YRp7. The cloned gene complements the deficiency caused by the rad50-1 mutation with respect to -rays, MMS resistance and UV-induced mitotic recombination. Restoration of the Rad⁺ phenotype occurs when the cloned gene is on a freely replicating multiple-copy plasmid or in the integrated form.Mapping of the cloned gene following integration of the 2 plasmid, and of the subclone in plasmid YRp7, showed it to be located on the left arm of chromosome XIV. Tetrad analysis of various crosses involving tow different strains carrying rad50-1 showed the mutation to map next to pet2 on chromosome XIV, and not on the right arm of chromsome IV, as previously published.     

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.     

Cloning of the URA1 gene of Saccharomyces cerevisiae

A 5.7-kilobase segment of Saccharomyces cerevisiae deoxyribonucleic acid which complements both the yeast ura1 and Escherichia coli pyrD mutations in dihydroorotate dehydrogenase has been cloned in plasmid YRp7.     

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.     

Cloning and restriction analysis of the hexokinase PII gene of the yeast Saccharomyces cerevisiae

Summary Carbon catabolite repression in yeast depends on catalytic active hexokinase isoenzyme PII (Entian 1980a). A yeast strain lacking hexokinase isoenzymes PI and PII was transformed, using a recombinant pool with inserts of yeast nuclear DNA up to 10 kbp in length. One hundred transformants for hexokinase were obtained. All selected plasmids coded for hexokinase isoenzyme PII, none for hexokinase isoenzyme PI, and carbon catabolite repression was restored in the transformants. Thirty-five independently isolated stable plasmids were investigated further. Analysis with the restriction enzyme EcoRI showed that these plasmids fell into two classes with different restriction behaviour. One representative of each class was amplified in Escherichia coli and transferred back into the yeast hexokinase-deficient strain with concomitant complementation of the nuclear mutation. The two types of insert were analysed in detail with 16 restriction enzymes, having 0–3 cleavage sites on transformant vector YRp7. The plasmids differed from each other by the orientation of the yeast insert in the vector. After yeast transformation with fragments of one plasmid the hexokinase PII gene was localised within a region of 1.65 kbp.     

Genetic mapping of the NYS1 gene in Saccharomyces cerevisiae

The NYS1 gene of Saccharomyces cerevisiae yeasts is linked to the centromere marker of chromosome IV - gene TRP1 and is located at 16.2 cM distance from it.     

Cloning and mapping of the sporulation gene, spoT7, in Saccharomyces cerevisiae

Summary In order to isolate a DNA fragment able to complement a sporulation-deficient mutation in Saccharomyces cerevisiae, a simple screening procedure was devised which was based on the difference in osmotic sensitivity between protoplasts and spores. A plasmid (pHT7) containing a 13 kb DNA insert that complemented the spoT7 mutation was isolated from a yeast genomic library prepared in the vector YEp13. Gene spoT7 was linked to rna1 at 1.2 cM and to mak27 at 7.2 cM on the right arm of chromosome XIII. Mapping of the cloned gene following integration into the chromosome showed that the cloned gene was allelic to spoT7 and that a part of the RNA1 gene was also cloned into the same fragment. Gene spoT7 was localized on a 5 kb DNA fragment by further subcloning.Abbreviations kb kilobase pairs - cM centiMorgans     

Restriction analysis of deletions and deletion mapping of point mutations in the ADE2 gene of Saccharomyces cerevisiae yeasts

The method of restriction analysis has been used to study the length of 10 deletion mutations in ADE2 locus of Saccharomyces cerevisiae. We showed that 7 deletions overlapped the whole transcribed region of the gene ADE2, while 3 deletions have one of the ends situated in this region. Four controlled sites were fixed on the genetic map of ADE2 locus, based on these results. Deletion mapping of great number of point mutations demonstrated non-random distribution of mutations of different types on the map of ADE2 locus.     

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 and characterization of the mitochondrial phosphate transport protein gene from the yeast Saccharomyces cerevisiae.

We have cloned the gene of the Saccharomyces cerevisiae phosphate transport protein (PTP), a member of the mitochondrial anion transport protein gene family. As PTP has a blocked N-terminus, we prepared three peptides. Oligonucleotides, based on their sequences, were used to screen a Yep24-housed genomic library. A total of 2073 bases of clone Y22 code for a 311 amino acid protein (Mr 32,814), which has similarities to the anion transport proteins: a triplicate gene structure and 6 hydrophobic segments. Typical for PTP, the triplicate gene structure possesses the X-Pro-X-(Asp/Glu)-X-X-(Lys/Arg)-X-(Arg/Lys)-X (X is an unspecified amino acid) motif and the very high homology only between the first and second repeat. The 6 hydrophobic segments harbor most of the 116 amino acids that are conserved between the yeast and the beef proteins. An N-terminal-extended signal sequence, as found in the beef protein, is absent. The yeast protein has about 33% fewer basic and acidic amino acids and five fewer Cys residues than the beef protein. The protein is insensitive to N-ethylmaleimide since Cys-42 (beef) has been replaced with a Thr. Mersalyl sensitivity has been retained and must be due to one of its three cysteines. Among these three cysteines, only Cys-28, located in the first hydrophobic segment, is conserved between the yeast and the beef protein.     

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.     

The phenomenon of predetermination of the cytoplasm upon interaction between alleles of the ADE2 and ADE13 in the yeast Saccharomyces cerevisiae

Our previous data showed that mutation ade13-1, blocking steps 8 and 12 of purine biosynthesis in the yeast Saccharomyces cerevisiae, caused the inability of strains manifesting this activity to grow on the complete nutrient medium with glucose in addition to the loss of adenylosuccinate lyase activity. It was also determined that the ade2-D mutation, inactivating aminoimidasole ribonucleotide carboxylase (the enzyme of step 6), suppressed this phenotypic manifestation of ade13-1; i.e., the ade2-D mutation restores the ability to grow on this medium. When spores of a hybrid that contained both mutations in the heterozygote were germinated on the YEPD medium, almost complete viability of segregants with genotypes ADE2 ADE13 and ade2-D ADE13 and the absence of ADE2 ade13-1 growth were observed. The number of growing segregants ade2-D ade13-1 amounted to approximately half of the possible number. In this work, a decrease in the proportion of segregants with this genotype was shown to occur only when they were obtained as a result of the segregation of hybrids with the normal allele (ADE2) in the heterozygote. The proportion of segregants with genotype ade2-D ade13-1 did not decrease upon segregation of hybrids similar in the genetic background and containing the ade2-D mutation in the homozygote and ade13-1 in the heterozygote. Spores with this genotype formed in the diheterozygous diploid were able to germinate on a medium containing glycerol and to further grow on a medium with glucose. The data suggest that, when a product of the normal ADE2 allele or of another gene, the synthesis of which is stimulated in the presence of this allele, enters spores with genotype ade2-D ade13-1 during meiosis, some of these spores lose their ability to grow on the medium with glucose; i.e., the ADE2 allele can be phenotypically expressed in the spores that did not contain this allele. This phenomenon is similar to the maternal effect known in some species of animals from various systematic groups.     

Cosntruction of the physical map of yeast（Saccharomyces cerevisiae）chromosome V~1

There are about 17 chromosomes in yeast Saccharomycescerevisiae.A middle sized chromosome,chromosome V,waschosen in this work for studying and constructing the physi-cal maps.Chromosome V from strain A364a was isolatedby pulsed-field gradient gel electrophoresis(PFGE).Gelslices containing chromosome V DNA were digestedwith two rare cutting enzymes,NotⅠand SfiⅠ,and three6-Nt recognizing enzymes,SmaⅠ,SstⅡ and ApaⅠ.Several strategies-partial or complete digestions,digestion with different sets of two enzymes,and hybrid-ization with cloned genetically mapped probes(CAN1,URA3,CEN5,PRO3,CHO1,SUP19,RAD51,RAD3)——were used to align the restriction fragments.There are 9,9,15,17,and 20 sites for NotⅠ,SfiⅠ,SmaⅠ,SstⅡ and ApaⅠrespectively in the map of the A364a chromosome V.Itstotal length was calculated to be 620 Kb(Kilo-bases).Thedistributions of the cutting sites for these five enzymesthrough the whole chromosome are not uniform.A comp-arison between the physical map and the genetic map wasalso made.