Biomedical subjects

D B Kaback

Publications and source records attributed to D B Kaback.

At least 19 recordsLinked to original sources

PubMed1992

Cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the FUN52 gene, whose product has homology to protein kinases.

A gene whose product has homology to protein kinases and is closely related to the Aspergillus nidulans nimA cell-cycle gene was identified on chromosome I of the yeast, Saccharomyces cerevisiae. This gene has been temporarily designated FUN52, where FUN is the acronym for 'function unknown now'. In A. nidulans, nimA is required to enter mitosis. In addition, overexpression of nimA causes premature onset of mitosis and cell cycle arrest. In contrast, S. cerevisiae cells that were either deleted for FUN52 or were overexpressing it had no detectable growth phenotypes. FUN52 proved to be the same as the previously identified KIN3 gene [Jones and Rosamond, Gene 90 (1990) 87-92] that was reported to map on chromosome VI.

Amino Acid Sequence↗

PubMed1992

Chromosome size-dependent control of meiotic recombination.

Smaller chromosomes have higher rates of meiotic reciprocal recombination (centimorgans per kilobase pair) than larger chromosomes. This report demonstrates that decreasing the size of Saccharomyces cerevisiae chromosomal DNA molecules increases rates of meiotic recombination and increasing chromosome size decreases recombination rates. These results indicate that chromosome size directly affects meiotic reciprocal recombination.

Chromosome Mapping↗

PubMed1992

Identification of a Saccharomyces cerevisiae homolog of the SNF2 transcriptional regulator in the DNA sequence of an 8.6 kb region in the LTE1-CYS1 interval on the left arm of chromosome I.

The DNA sequence of an 8.6 kb region of the left arm of chromosome I has been determined. This region, between the LTE1 and CYS1 loci, is approximately 40 kb from the centromere. There are six potential open-reading frames (ORFs), provisionally named YAL001-006 within this fragment of chromosome I. Four of these ORFs can be aligned with previously identified FUN transcripts: FUN28 with YAL006, FUN29 with YAL004, FUN30 with YAL001 and FUN31 with YAL002. The YAL001 ORF shows significant homology to the SNF2 transcriptional regulator. A region of the DNA contains an extensive repeat of the bases C-A-T positioned in the 5' terminus of the YAL004 promoter region.

Adenosine Triphosphatases↗

PubMed1992

The CCR4 protein from Saccharomyces cerevisiae contains a leucine-rich repeat region which is required for its control of ADH2 gene expression.

The CCR4 gene from Saccharomyces cerevisiae is required for the transcription of the glucose-repressible alcohol dehydrogenase (ADH2). Mutations in CCR4 also suppress the transcription at the ADH2 and his4-912delta loci caused by defects in the SPT10 (CRE1) and SPT6 (CRE2) genes. The CCR4 gene was mapped to the left arm of chromosome I and cloned by complementation of function using previously isolated segments of chromosome I. DNA sequence analysis of the cloned gene defined CCR4 as a 2511 bp open reading frame that would encode a polypeptide of 837 amino acids. The CCR4 mRNA was found to be 2.8 kb in size and Western analysis identified CCR4 as a 95,000 D protein. Disruption of the CCR4 gene resulted in reduced levels of ADH2 expression under both glucose and ethanol growth conditions and in temperature sensitive growth on nonfermentative medium, phenotypes essentially indistinguishable from previously identified mutations in CCR4. The amino terminus of the CCR4 protein was found to be rich in glutamine residues similar to a number of genes which are required for transcription. More importantly, CCR4 showed similarity to a diverse set of proteins sharing a leucine-rich tandem repeat motif, the presence of which has been implicated in mediating protein-protein interactions. Deletions of several of the five leucine-rich repeats in CCR4 were shown to produce nonfunctional proteins indicating the importance of the repeats to CCR4 activity. This leucine-rich repeat region may mediate the contact CCR4 makes with another factor.

Alcohol Dehydrogenase↗

PubMed1991

Distributive disjunction of authentic chromosomes in Saccharomyces cerevisiae.

Distributive disjunction is defined as the first division meiotic segregation of either nonhomologous chromosomes that lack homologs or homologous chromosomes that have not recombined. To determine if chromosomes from the yeast Saccharomyces cerevisiae were capable of distributive disjunction, we constructed a strain that was monosomic for both chromosome I and chromosome III and analyzed the meiotic segregation of the two monosomic chromosomes. In addition, we bisected chromosome I into two functional chromosome fragments, constructed strains that were monosomic for both chromosome fragments and examined meiotic segregation of the chromosome fragments in the monosomic strains. The two nonhomologous chromosomes or chromosome fragments appeared to segregate from each other in approximately 90% of the asci analyzed, indicating that yeast chromosomes were capable of distributive disjunction. We also examined the ability of a small nonhomologous centromere containing plasmid to participate in distributive disjunction with the two nonhomologous monosomic chromosomes. The plasmid appeared to efficiently participate with the two full length chromosomes suggesting that distributive disjunction in yeast is not dependent on chromosome size. Thus, distributive disjunction in S. cerevisiae appears to be different from Drosophila melanogaster where a different sized chromosome is excluded from distributive disjunction when two similar size nonhomologous chromosomes are present.

Blotting, Southern↗

PubMed1990

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation, characterization and regulation of the SPO7 sporulation gene.

SPO7 is one of several previously identified genes from the yeast Saccharomyces cerevisiae that is required for sporulation but not for vegetative growth. The SPO7 gene has been cloned by functional complementation and physically mapped 15-16 kb to the left of CEN1. Gene-disruption experiments confirmed that the cloned gene was the bona fide SPO7 gene. SPO7 codes for a 0.95-kb transcript that is expressed at approximately the same level in both vegetative and sporulating cells. The gene was sequenced and has the capacity to encode a 259-amino acid protein that does not appear to be related to other known proteins.

Amino Acid Sequence↗

PubMed1989

Cloning of chromosome I DNA from Saccharomyces cerevisiae: mutational analysis of the FUN2 transcribed region.

To increase the number of mutationally defined genes on chromosome I from Saccharomyces cerevisiae, most of the FUN2 (Function Unknown Now) transcribed region was deleted by gene replacement. Strains containing the deletion were viable, but grew with a 20% longer generation time. The mutation was recessive. Mutant haploids were able to mate, and homozygous mutant diploids were able to sporulate, giving asci containing four viable ascospores. These results indicate FUN2 is dispensable for the life cycle of S. cerevisiae, but required for an optimal growth rate.

Chromosome Deletion↗

PubMed1989

Meiotic segregation of circular plasmid-minichromosomes from intact chromosomes in Saccharomyces cerevisiae.

Distributive disjunction is defined by first meiotic division segregation of either two nonhomologous chromosomes that lack homologous pairing partners, or of two homologous chromosomes that have failed to undergo crossing-over. In the yeast Saccharomyces cerevisiae, plasmid minichromosomes, synthetic linear chromosomes and a fragment of a real chromosome have been observed to segregate from nonhomologous DNA species at the first meiotic divisions. Suggesting that this organism may have a distributive mechanism for chromosome segregation. However, it is not known whether intact chromosomes also participate in a distributive process. To determine whether intact, full length, S. cerevisiae chromosomes could segregate from nonhomologous chromosomal species, the meiotic behavior of an unpaired intact copy of chromosome I has been analyzed with respect to several centromere-containing circular plasmid minichromosomes. Strains monosomic or trisomic for chromosome I were transformed with centromere plasmids containing either homologous or nonhomologous inserts, sporulated, and analyzed genetically both for the presence of plasmid and for the number of copies of chromosome I. Each plasmid segregated from an intact unpaired copy of chromosome I at the first meiotic division in a significant majority (63-93%) of the asci examined. These results suggest that intact chromosomes from S. cerevisiae are capable of distributive disjunction.

Centromere↗

PubMed1989

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: localization of a repeated sequence containing an acid phosphatase gene near a telomere of chromosome I and chromosome VIII.

A 17 kb region from near the right end of chromosome I of Saccharomyces cerevisiae was isolated on recombinant lambda bacteriophages. This region contained the PHO11 gene which was located only 3.4 kb from the right end of the chromosome. We found that this region also was repeated approximately 13 kb from the end of the chromosome VIII DNA molecule. The chromosome VIII sequence appears to be a previously unnamed acid phosphatase gene that we propose to call PHO12. Thus, similar to the repeated SUC, MAL, X and Y' sequences, some members of the repeated acid phosphatase gene family also appear near the termini of yeast chromosomes.

Acid Phosphatase↗

PubMed1989

Enhanced meiotic recombination on the smallest chromosome of Saccharomyces cerevisiae.

Chromosome I is the smallest chromosome in Saccharomyces cerevisiae and contains a DNA molecule that is only 250 kilobases (kb). Approximately 75% of this DNA molecule has been cloned. A restriction map for the entire DNA molecule from chromosome I was determined and most of its genetically mapped genes were located on this physical map. Based on the average rate of recombination (centimorgans/kb) found for other S. cerevisiae chromosomes, the outermost markers on the genetic map of chromosome I were expected to be close to the ends of the DNA molecule. While the rightmost genetic marker was 3 kb from the end, the leftmost marker, CDC24, was located near the middle of the left arm, suggesting that the genetic map would be much longer. To extend the genetic map, a copy of the S. cerevisiae URA3 gene was integrated in the outermost cloned region located 32 kb centromere distal to CDC24, and the genetic map distance between these two genes was determined. The new marker substantially increased the genetic map length of chromosome I. In addition, we determined the relationship between physical and genetic map distance along most of the length of the chromosome. Consistent with the longer genetic map, the average rate of recombination between markers on chromosome I was greater than 50% higher than the average found on other yeast chromosomes. Owing to its small size, it had been estimated that approximately 5% of the chromosome I homologues failed to undergo meiotic recombination. New measurements of the zero-crossover class indicated that the enhanced rate of recombination ensures at least one genetic exchange between virtually every pair of chromosome I homologues.

Chromosome Mapping↗

PubMed1987

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures.

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16-1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M1 replication defects of mak16-1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade 1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1.2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20 degrees C, 30 degrees C, or 37 degrees C, but could not grow at 8 degrees C. The deleted region is thus essential only at 8 degrees C, and we name this gene LTE1 (low temperature essential).

Base Sequence↗

PubMed1987

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.

Centromere↗

PubMed1986

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.

Chromosome Mapping↗

PubMed1986

Isolation and functional analysis of sporulation-induced transcribed sequences from Saccharomyces cerevisiae.

Strains of the yeast Saccharomyces cerevisiae that are heterozygous for the mating-type locus (MATa/MAT alpha) undergo meiosis and spore formation when they are starved for nitrogen and are provided with a nonfermentable carbon source such as potassium acetate. Haploids and diploids homozygous for the mating-type locus (MAT alpha/MAT alpha or MATa/MATa) are asporogenous and undergo neither meiosis nor spore formation when incubated under the same conditions. A small number of genes produce transcripts that appear to be induced specifically in sporulating cells. These transcripts either are not found or are present at much lower levels both in vegetatively growing cells and in cells from asporogenous strains that have been incubated in sporulation medium. Several genes complementary to these MATa/MAT alpha-dependent sporulation-induced transcripts were isolated from a gene-size insert yeast-lambda recombinant DNA library, by differential-plaque filter hybridization. An attempt was made to determine the function of three of these genes by mutating them in the yeast genome with in vitro mutagenesis and one-step gene replacement techniques. One gene was extensively disrupted by both a 0.3-kilobase deletion and the insertion of two large DNA sequences at different sites within the gene. Surprisingly, this compound mutation did not appear to affect meiosis or the production of viable ascospores, indicating that this gene was dispensable for differentiation. The other two genes were disrupted by simple insertion mutations at a site where it was possible that they might still possess some gene activity. These mutations also did not appear to affect sporulation. These results suggest that not all sporulation-induced genes are essential for meiosis and the production of viable ascospores under the conditions examined.

Chromosome Mapping↗

PubMed1985

Saccharomyces cerevisiae exhibits a sporulation-specific temporal pattern of transcript accumulation.

Cultures of the yeast Saccharomyces cerevisiae that are heterozygous for the mating type (MATa/MAT alpha) undergo synchronous meiosis and spore formation when starved for nitrogen and supplied with a nonfermentable carbon source such as acetate. Haploid and homozygous MAT alpha/MAT alpha and MATa/MATa diploid cells incubated under the same conditions fail to undergo meiosis and are asporogenous. It has not yet been firmly established that gene expression during sporulation is controlled at the level of transcript accumulation. To examine this question, we used cloned genes that encode a variety of "housekeeping" functions to probe Northern blots to assay the appearance of specific transcripts in both sporulating and asporogenous S. cerevisiae. In sporulating cells, each transcript showed a characteristic pattern of accumulation, reaching a maximum relative abundance at one of several different periods. In contrast, in both asporogenous haploid MATa and diploid MAT alpha/MAT alpha cells, all transcripts accumulated with similar kinetics. These results suggest a sporulation-specific pattern for transcript appearance. During these studies, high levels of several different transcripts were observed at unexpected times in sporulating cells. Histone (H)2A and (H)2B1 transcripts, although most abundant during premeiotic DNA synthesis, remained at one-third to one-half maximal levels after its end and were found in mature ascospores. Their appearance at this time is in sharp contrast to vegetative cells in which these histone transcripts are only found just before and during the period of DNA synthesis. Furthermore, transcripts from GAL10 and CDC10 genes, which are believed to be dispensable for sporulation, were much more abundant in sporulating cells than in asporogenous cells and vegetative cells grown on glucose or acetate. The presence of these transcripts did not appear to be due to a general activation of transcription because each accumulated with different kinetics. In addition, the transcript for at least one gene, HO, that is also dispensable for sporulation was not detected. The increased abundance of transcripts from some genes not required for sporulation leads us to propose that genes preferentially expressed during sporulation need not be essential for this differentiation.

Cell Differentiation↗

PubMed1979

Improved methods for the formation and stabilization of R-loops.

Improved methods for the formation and stabilization of R-loops for visualization in the electron microscope are presented. The two complementary strands of a duplex DNA are photochemically crosslinked once every 1 to 3 kb using 4, 5', 8 trimethylpsoralen. R-loops are then formed by incubation with RNA in 70% formamide at a temperature above the DNA melting temperature. Finally, the R-loops are stabilized by modifying the free single strand of DNA with glyoxal, thus minimizing the displacement of the hybridized RNA by branch migration. In this manner R-loops can be formed and visualized at a high frequency irrespective of the base composition of the nucleic acid of interest.

Base Sequence↗

PubMed1979

Isolation of yeast histone genes H2A and H2B.

Analysis of cloned sequences for yeast histone genes H2A and H2B reveals that there are only two copies of this pair of genes within the haploid yeast genome. Within each copy, the genes for H2A and H2B are separated by approximately 700 bp of spacer DNA. The two copies are separated from one another in the yeast genome by a minimum distance of 35-60 kb. Sequence homology between the two copies is restricted to the genes for H2A and H2B; the spacer DNA between the genes is nonhomologous. In both copies, the genes for H2A and H2B are divergently transcribed. In addition, both plasmids code for other nonhistone proteins. Sequences coding for histones H3 and H4 have not been detected in the immediate vicinity of the genes for H2A and H2B.

Base Sequence↗

PubMed1979

Immunoglobulin heavy chain gene organization in mice: analysis of a myeloma genomic clone containing variable and alpha constant regions.

We have isolated a myeloma genomic DNA clone containing the variable and constant regions of a mouse alpha chain. Restriction enzyme analyses and electron microscopic R loop mapping have demonstrated that the variable region is separated from the constant region by 6.8 kilobases of intervening DNA. In addition, two intervening DNA sequences of 100--200 bases separate the constant region into three approximately equal units. These intervening sequences may separate each of the segments coding for the three constant region domains of the alpha heavy chain. Southern blot analysis of embryo and myeloma DNA suggests that DNA rearrangement of heavy chain variable and constant regions occurs during the differentiation of antibody-producing cells.

Animals↗