Isolation of the ARO1 cluster gene of Saccharomyces cerevisiae.

The AROl cluster gene was isolated by complementation in Saccharomyces cerevisiae after transformation with a comprehensive yeast DNA library of BamHI restriction fragments inserted into the shuttle vector YEp13. Most of the transformants exhibited the expected episomal inheritance of the ARO+ phenotype; however, one stable transformant has been shown to be an integration of the AROl fragment and the vector YEp13 at the arol locus. The insert containing AROl is a 17.2-kilobase pair (kbp) BamHI fragment which complements both nonsense and missense alleles of arol. Subcloning by Sau3AI partial digestion further locates the AROl segment to a 6.2-kbp region. An autonomously replicating sequence (ars) was found on the 17.2-kbp fragment. Yeast arol mutants transformed with the AROl episome express 5 to 12 times the normal level of the five AROl enzyme activities and possess elevated amounts of the AROl protein. The yeast AROl fragment also complemented aroA, aroB, aroD, and aroE mutants of Escherichia coli. The expression of AROl in both S. cerevisiae and E. coli was independent of the orientation of the fragment with respect to the vector.     

Transcription of the his3 gene region in Saccharomyces cerevisiae

The dodecamer d(CpGpCpGpApApTpTpCpGpCpG) or C-G-C-G-A-A-T-T-C-G-C-G crystallizes as slightly more than one full turn of right-handed B-DNA. It is surrounded in the crystal by one bound spermine molecule and 72 ordered water molecules, most of which associate with polar N and O atoms at the exposed edges of base-pairs. Hydration within the major groove is principally confined to a monolayer of water molecules associated with exposed N and O groups on the bases, with most association being monodentate. Waters hydrating backbone phosphate oxygens tend not to be ordered, except where they are immobilized by 5-methyl groups from nearby thymines. In contrast, the minor groove is hydrated in an extensive and regular manner, with a zigzag “spine” of first- and second-shell hydration along the floor of the groove serving as a foundation for less-regular outer shells extending beyond the radius of the phosphate backbone. This spine network bridges purine N-3 and pyrimidine O-2 atoms in adjacent base-pairs. It is particularly regular in the A-A-T-T center, and is disrupted at the C-G-C-G ends, in part by the presence of the N-2 amino groups on guanine residues. The minor groove hydration spine may be responsible for the stability of the B form of polymers containing only A · T and I · C base-pairs, and its disruption may explain the ease of transition to the A form of polymers with G · C pairs.     

Identification of the ureidoglycolate hydrolase gene in the DAL gene cluster of Saccharomyces cerevisiae.

This report describes the isolation of the genes encoding allantoicase (DAL2) and ureidoglycolate hydrolase (DAL3), which are components of the large DAL gene cluster on the right arm of chromosome IX of Saccharomyces cerevisiae. During this work a new gene (DAL7) was identified and found to be regulated in the manner expected for an allantoin pathway gene. Its expression was (i) induced by allophanate, (ii) sensitive to nitrogen catabolite repression, and (iii) responsive to mutation of the DAL80 and DAL81 loci, which have previously been shown to regulate the allantoin degradation system. Hybridization probes generated from these cloned genes were used to analyze expression of the allantoin pathway genes in wild-type and mutant cells grown under a variety of physiological conditions. When comparison was possible, the patterns of mRNA and enzyme levels observed in various strains and physiological conditions were very similar, suggesting that the system is predominantly regulated at the level of gene expression. Although all of the genes seem to be controlled by a common mechanism, their detailed patterns of expression were, at the same time, highly individual and diverse.     

Physical evidence for a Saccharomyces cerevisiae transposable element which carries the his4C gene.

A Saccharomyces cerevisiae transposable element which carries the his4C structural gene and which is capable of transposition, excision, and mutator activity is described. Physical evidence is presented for transposition of the his4C deoxyribonucleic acid sequences to a new location in the genome and for precise excision of these transposed deoxyribonucleic acid sequences in spontaneous his4C- segregants.     

GAL3 gene product is required for maintenance of the induced state of the GAL cluster genes in Saccharomyces cerevisiae.

The activities of the first three enzymes for galactose catabolism normally become detectable within 15 min after the addition of galactose into a culture of the yeast Saccharomyces cerevisiae. In S. cerevisiae with a recessive mutation termed gal3, a longer-than-normal lag is observed before the appearance of the enzyme activities (O. Winge and C. Roberts, C. R. Trav. Lab. Carlsberg Ser. Physiol. 24:263-315, 1948). I isolated two S. cerevisiae mutants with temperature-sensitive defects in the GAL3 gene. Temperature shift experiments with one of those mutants led to the conclusion that the GAL3 function is required not only for the initiation of enzyme induction but also for the maintenance of the induced state in galactose-nonfermenting S. cerevisiae because of a defect in any of the genes for the galactose-catabolizing enzymes, such as gal1 or gal10. In contrast, the GAL3 function is phenotypically dispensable in galactose-metabolizing S. cerevisiae. Thus, the normal catabolism of galactose can substitute for the GAL3 function.     

DNA binding properties of the Saccharomyces cerevisiae DAT1 gene product.

The DAT1 gene of Saccharomyces cerevisiae encodes a DNA binding protein (Dat1p) that specifically recognizes the minor groove of non-alternating oligo(A).oligo(T) tracts. Sequence-specific recognition requires arginine residues found within three perfectly repeated pentads (G-R-K-P-G) of the Dat1p DNA binding domain [Reardon, B. J., Winters, R. S., Gordon, D., and Winter, E. (1993) Proc. Natl. Acad. Sci. USA 90, 11327-1131]. This report describes a rapid and simple method for purifying the Dat1p DNA binding domain and the biochemical characterization of its interaction with oligo(A).oligo(T) tracts. Oligonucleotide binding experiments and the characterization of yeast genomic Dat1p binding sites show that Dat1p specifically binds to any 11 base sequence in which 10 bases conform to an oligo(A).oligo(T) tract. Binding studies of different sized Dat1p derivatives show that the Dat1p DNA binding domain can function as a monomer. Competition DNA binding assays using poly(I).poly(C), demonstrate that the minor groove oligo(A).oligo(T) constituents are not sufficient for high specificity DNA binding. These data constrain the possible models for Dat1p/oligo(A).oligo(T) complexes, suggest that the DNA binding domain is in an extended structure when complexed to its cognate DNA, and show that Dat1p binding sites are more prevalent than previously thought.     

Organization of the ribosomal RNA gene cluster in the yeast Saccharomyces cerevisiae

The chromosomal organization of the ribosomal RNA gene cluster from Saccharomyces cerevisiae was investigated. 18 S rRNA R-loops were formed with unfractionated high molecular weight DNA crosslinked once per 2.7 × 10³ bases with trioxsalen and observed in the electron microscope. Almost all the R-loops were found in very long continuous 9.34 ± 0.18 × 10³ base repeating units. In addition, molecules were found at a frequency of one to two per genome equivalent of rDNA where several rRNA genes were linked to long stretches of non-rDNA. These results suggest that rDNA is arranged in a single tandem repetitive cluster of 100 to 140 genes flanked on one or both sides by non-rDNA.     

Interaction between the Saccharomyces cerevisiae CDC25 gene product and mammalian ras.

In order to characterize the interaction between the Saccharomyces cerevisiae Cdc25 protein and Harvey-ras (p21H-ras), we have constructed a yeast strain disrupted at the RAS1 and RAS2 loci, expressing both p21H-ras and the catalytic domain of the bovine GTPase activating protein (GAP) and containing the cdc25-2 mutation. Such a strain exhibits a temperature-sensitive phenotype. The shift to the nonpermissive temperature is accompanied by the loss of guanyl nucleotide-dependent activity of adenylylcyclase in vitro. The temperature-sensitive phenotype can be rescued by CDC25 itself, as well as by a plasmid containing a truncated SDC25 gene. In addition, wild type CDC25 significantly improves the guanyl nucleotide response observed in the background of the cdc25ts allele at the permissive temperature in a dosage-dependent manner and restores the guanyl nucleotide response at the restrictive temperature. Both CDC25 and a truncated SDC25 also restored p21H-ras-dependent guanyl nucleotide response in a strain isogenic to the one described above but containing a disrupted CDC25 locus instead of the temperature-sensitive allele. These results suggest that the S. cerevisiae Cdc25 protein interacts with p21H-ras expressed in yeast by promoting GDP-GTP exchange. It follows that the yeast system can be used for characterizing the interaction between guanyl nucleotide exchangers of Ras proteins and mammalian p21H-ras.     

Potential DNA-binding domains in the RAD18 gene product of Saccharomyces cerevisiae

The RAD18 gene of Saccharomyces cerevisiae is involved in the error-prone DNA repair. Its nucleotide sequence, as reported here, predicts an open reading frame of 1461 nt which corresponds to a protein of 487 amino acids, with an Mr of 55,237. This protein has three putative zinc fingers, two acidic regions and a nucleotide-binding domain, suggesting that it is a nucleic acid-binding protein with a possible regulatory role.     

Positive regulatory interactions of the HIS4 gene of Saccharomyces cerevisiae.

The role of cis- and trans-acting elements in the expression of HIS4 has been examined by using HIS4-lacZ fusions in which lacZ expression is dependent upon the HIS4 5' noncoding region. The cis-acting sequences involved in regulation were defined by studying the effects of the wild-type and various deletions and their revertants on regulation via the general control of amino acid biosynthesis. The role of trans-acting genes was analyzed by studying the regulation of the HIS4-lacZ fusions in strains carrying mutations in the GCN (AAS) or GCD (TRA) genes and in strains carrying the GCN genes on high-copy-number plasmids. These studies have led to the following conclusions. (i) HIS4 is positively regulated by the general control. (ii) At least one copy of the 5'TGACTC3' repeat at -136 is required in cis for this regulation. (iii) Both the GCN4 gene and at least one copy of the repeated sequence are required for expression at the repressed level. (iv) The open reading frames in the 5' noncoding region are not required in either cis or trans for the regulation of HIS4.     

The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA.

The SPS4 gene of Saccharomyces cerevisiae, a sporulation-specific gene identified previously in a differential hybridization screen of a genomic yeast DNA library, has been characterized further. The protein encoded by this gene was inferred from its nucleotide sequence to be 38,600 daltons with an isoelectric pH of 8.2. Consistent with this, two-dimensional polyacrylamide gel electrophoresis of the in vitro translation products of RNA purified by hybridization with the cloned SPS4 DNA indicated that the SPS4 gene product is a 39-kilodalton, basic protein. This protein was found to be identical in size and charge to a major, sporulation-specific protein identified in a two-dimensional polyacrylamide gel electrophoretic comparison of the in vitro translation products of total RNA from sporulating MATa/MAT alpha cells and asporogenous MAT alpha/MAT alpha cells. A MATa/MAT alpha strain homozygous for a partial deletion of the SPS4 gene appeared, however, to be unaffected in its ability to form viable ascospores.