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B K Tye

Publications and source records attributed to B K Tye.

At least 37 records · Page 2Linked to original sources
PubMed1991

Both activation and repression of a-mating-type-specific genes in yeast require transcription factor Mcm1.

Mcm1 is a yeast transcription factor with homologs throughout the metazoa. MCM1 was first identified as a gene involved in maintenance of artificial minichromosomes in yeast. More recently Mcm1 has been shown to serve as a transcriptional regulator of mating-type-specific genes. Biochemical data suggest that Mcm1 coactivates alpha-specific genes and corepresses a-specific genes by binding to a 10-base-pair dyad symmetry element in their upstream regions. We reported previously that an mcm1 point mutation reduced activation of alpha-specific genes but had little effect on the expression of a-specific genes. We now show that another mcm1 allele, which depletes the Mcm1 protein, affects both activation and repression of a-specific genes. The mutant strain remains capable of high levels of pheromone induction of a-specific genes, although with retarded kinetics. Mcm1 joins an increasing number of transcription factors involved in both positive and negative regulation of gene expression.

Alleles↗
PubMed1991

A yeast protein that binds to vertebrate telomeres and conserved yeast telomeric junctions.

We have identified three yeast proteins that bind to poly(C.A)/poly(T.G) repeats characteristic of telomeric sequences from yeast to human. TBF alpha binds to the telomeric sequences of yeast, Tetrahymena, and vertebrates. In contrast, TBF beta binds only to yeast telomeric sequences. Also identified was RAP1, the transcriptional silencer protein, which binds to a sequence motif found in upstream activating sequences (UASs) of a number of genes; the sequence motif also occurs frequently in yeast telomeric sequences. Because poly(C.A)/poly(T.G) sequences from a wide range of organisms will serve as the primer for the in vivo extension of telomeres in yeast, TBF alpha is of particular interest. DNase I footprinting analysis indicated that TBF alpha binds to the junction between the subtelomeric X sequence and poly(C1-3A) in a cloned yeast telomere. Examination of the junctions of known X sequences indicated that they all contain one or more repeats of CCCTAA, a sequence that is repeated in vertebrate telomeres. Earlier, Murray et al. (1988) reported that heterologous telomeric sequences positioned as far as several hundred base pairs from the termini of linear molecules can allow the addition of yeast telomeric sequences from nontelomeric termini in vivo. A possible function for TBF alpha might be to serve as an anchoring protein for the yeast telomerase by binding to the conserved junction sequence at a distance from the terminus to allow addition of an irregular repeating sequence at the chromosome end.

Animals↗
PubMed1991

Functional domains of the yeast transcription/replication factor MCM1.

MCM1 is an essential yeast DNA-binding protein that affects both minichromosome maintenance, in a manner suggesting that it has DNA replication initiation function, and gene expression. It activates alpha-specific genes together with MAT alpha 1, and represses a-specific genes together with MAT alpha 2. Alone, MCM1 can activate transcription. To determine whether different domains of the protein mediate these diverse functions, we constructed and analyzed several mcm1 mutants. The gene expression and minichromosome maintenance phenotypes of these mutants suggest that the role of MCM1 in DNA replication initiation may not involve transcriptional activation. However, both transcription and replication activities require only the 80-amino-acid fragment of MCM1 homologous to the DNA-binding domain of the serum response factor (SRF). This small fragment is also sufficient for cell viability and repression of a-specific genes. A polyacidic amino acid stretch immediately adjacent to the SRF homologous domain of MCM1 was found to be important for activation of alpha-specific genes in alpha cells. Mutants lacking the acidic stretch confer higher expression from an alpha-specific UAS in a cells in addition to lower expression in alpha cells, suggesting that negative regulation at this site occurs in a cells, in addition to the well-documented positive regulation in alpha cells.

Base Sequence↗
PubMed1991

Mcm2 and Mcm3, two proteins important for ARS activity, are related in structure and function.

MCM2 and MCM3 are essential genes believed to play important roles in the initiation of DNA replication in Saccharomyces cerevisiae. Mutants defective in Mcm2 or Mcm3 are remarkably similar in phenotype. They both show an autonomously replicating sequence (ARS)-specific minichromosome maintenance defect, although their ARS specificities are not identical. In addition, these mutants exhibit a premitotic cell cycle arrest and an increase in chromosome loss and recombination. Genetic studies suggest that the two MCM gene products play interacting or complementary roles in DNA replication. Double mutants of mcm2-1 and mcm3-1 are inviable at the permissive growth temperature (23 degrees C) for each of the single mutants. Furthermore, overproduction of Mcm3 accentuates the deleterious effect of the mcm2-1 mutation, whereas overproduction of Mcm2 partially complements the mcm3-1 mutation. MCM2 encodes a protein of 890 amino acids containing a putative zinc-finger domain that is essential for Mcm2 function. Mcm2 shows striking homology to Mcm3 and three other proteins, Cdc46 of S. cerevisiae, and Nda4 and Cdc21 of Schizosaccharomyces pombe. The phenotypes of mutants defective in these proteins suggest that they belong to a protein family involved in the early steps of DNA replication.

Amino Acid Sequence↗
PubMed1990

The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation of the left arm of chromosome V approximately 3 kb centromere proximal of anp1. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 protein may play interacting roles in DNA replication.

Amino Acid Sequence↗
PubMed1989

A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes.

The Saccharomyces cerevisiae MCM1 gene product is a protein with multiple functions. It is a transcription factor necessary for expression of mating-type-specific genes and is also required for the maintenance of minichromosomes. MCM1 shows DNA-binding specificities similar to those of two previously reported DNA-binding factors, pheromone/receptor transcription factor (PRTF) and general regulator of mating type (GRM); like PRTF, its activity can be modulated by the alpha 1 protein. MCM1 binds to the dyad symmetry element 5'-CCTAATTAGG and related sequences, which we refer to as MCM1 control elements (MCEs). MCEs are found within the regulatory regions of a- and alpha-specific genes. Direct and indirect DNA binding assays suggest that a conserved 5'-ATTAGG in one-half of the dyad symmetry element is important for MCM1 binding whereas variants in the other half are tolerated. We have used a novel DNase I 'nicking interference' assay to investigate the interaction of MCM1 with its substrate. These data suggest that MCM1 binds as a dimer, interacting symmetrically with the ATTAGG residues in each half of the binding site. MCM1 contains striking homology to the DNA-binding domain of the human serum response factor (SRF) which mediates the transient transcriptional activation of growth-stimulated genes by binding to the serum response element (SRE). We have shown that MCM1 binds to the human c-fos SRE in vitro and, like other MCEs, the c-fos SRE exhibits MCM1-mediated upstream activating sequence (UAS) activity in vivo.

Base Sequence↗
PubMed1989

The OBF1 protein and its DNA-binding site are important for the function of an autonomously replicating sequence in Saccharomyces cerevisiae.

The autonomously replicating sequence ARS121 was cloned as a 480-base-pair (bp) long DNA fragment that confers on plasmids autonomous replication in Saccharomyces cerevisiae. This fragment contains two OBF1-binding sites (sites I and II) of different affinities, as identified by a gel mobility shift assay and footprint analysis. Nucleotide substitutions (16 to 18 bp) within either of the two sites obliterated detectable in vitro OBF1 binding to the mutagenized site. Linker substitution (6 bp) mutations within the high-affinity site I showed effects similar to those of the complete substitution, whereas DNA mutagenized outside the binding site bound OBF1 normally. We also tested the mitotic stability of centromeric plasmids bearing wild-type and mutagenized copies of ARS121. Both deletion of the sites and the extensive base alterations within either of the two OBF1-binding sites reduced the percentage of plasmid-containing cells in the population from about 88% to 50 to 63% under selective growth and from about 46% to 15 to 20% after 10 to 12 generations of nonselective growth. Furthermore, linker (6 bp) substitutions within site I, the high-affinity binding site, showed similar deficiencies in plasmid stability. In contrast, plasmids containing linker substitutions in sequences contiguous to site I displayed wild-type stability. In addition, plasmid copy number analysis indicated that the instability probably resulted not from nondisjunction during mitosis but rather from inefficient plasmid replication. The results strongly support the notion that the OBF1-binding sites and the OBF1 protein are important for normal ARS function as an origin of replication.

Base Sequence↗
PubMed1989

Meiotic segregation of normal and deletion chromosomes in Saccharomyces cerevisiae.

We explored the behavior of meiotic chromosomes in Saccharomyces cerevisiae by examining the effects of chromosomal rearrangements on recombination and disjunction. Chromosome III derivatives in which the entire left arm or the entire right arm was deleted (telocentric) segregated with fidelity from a normal chromosome III. Recombination between either of these two deletion chromosomes and the intact chromosome also appeared normal. In a strain containing a right arm telocentric, a left arm telocentric and one normal chromosome both telocentrics disjoined from the normal chromosome. Homology on one arm was sufficient for proper recombination and segregation of these chromosomes. In strains containing two normal chromosomes and one telocentric chromosome the two normal chromosomes preferentially disjoined. In a few cases however, the two normal chromosomes cosegregated. Recombination between the two normal chromosomes or between one normal chromosome and the deletion chromosome increased the probability that they would disjoin, although cosegregation of recombinants was observed. A chromosome III derivative which contained a large centromeric deletion and an insertion of the centromere from chromosome V into a nonhomologous position segregated with fidelity from a normal chromosome III. These studies demonstrate that it is not pairing of the centromeres, but pairing and recombination along the arms of the homologs that directs meiotic chromosome disjunction.

Chromosome Deletion↗
PubMed1988

Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MAT alpha cells.

We previously reported the isolation of yeast mutants that seem to affect the function of certain autonomously replicating sequences (ARSs). These mutants are known as mcm for their defect in the maintenance of minichromosomes. We have now characterized in more detail one ARS-specific mutation, mcm1-1. This Mcm1 mutant has a second phenotype; MAT alpha mcm1-1 strains are sterile. MCM1 is non-allelic to other known alpha-specific sterile mutations and, unlike most genes required for mating, it is essential for growth. The alpha-specific sterile phenotype of the mcm1-1 mutant is manifested by its failure to produce a normal amount of the mating pheromone, alpha-factor. In addition, transcripts of the MF alpha 1 and STE3 genes, which encode the alpha-factor precursor and the alpha-factor receptor, respectively, are greatly reduced in this mutant. These and other properties of the mcm1-1 mutant suggest that the MCM1 protein may act as a transcriptional activator of alpha-specific genes. We have cloned, mapped and sequenced the wild-type and mutant alleles of MCM1, which is located on the right arm of chromosome XIII near LYS7. The MCM1 gene product is a protein of 286 amino acid residues and contains an unusual region in which 19 out of 20 residues are either aspartic or glutamic acid, followed by a series of glutamine tracts. MCM1 has striking homology to ARG80, a regulatory gene of the arginine metabolic pathway located about 700 base-pairs upstream from MCM1. A substitution of leucine for proline at amino acid position 97, immediately preceding the polyanionic region, was shown to be responsible for both the alpha-specific sterile and minichromosome-maintenance defective phenotypes of the mcm1-1 mutant.

Alleles↗
PubMed1988

Specific interaction between a Saccharomyces cerevisiae protein and a DNA element associated with certain autonomously replicating sequences.

We have isolated a protein from Saccharomyces cerevisiae that binds specifically to a nucleotide sequence associated with the autonomously replicating sequence (ARS) ARS120, located in the telomeric region of a yeast chromosome. "Footprinting" analysis revealed that a 26-base-pair DNA sequence, 5'-CAAGTGCCGTGCATAATGATGTGGGT-3', was protected by this protein from DNase I digestion. A plasmid containing 48 direct tandem repeats of this oligonucleotide was constructed and used to affinity-purify the binding activity. The purified protein, OBF1 (origin binding factor), showed specific binding to ARS120. The 26-base-pair OBF1-protected sequence was sufficient for the recognition and binding of the protein, since the mobility of a DNA fragment containing the synthetic binding site was retarded in agarose gels when incubated with OBF1. By performing competition experiments with a number of different ARSs, we showed that OBF1 binds tightly to some but not all ARSs. Interestingly, OBF1 does not appear to have a discernible affinity for ARS1 or the ARSs associated with mating type loci, HML alpha and HMRa, which are substrates for a DNA-binding activity reported by others. Since OBF1 appears to bind to DNA associated with a number of ARSs, we suggest that this protein may have a function related to ARS activity, perhaps in the initiation of DNA replication at selected ARSs.

DNA Replication↗
PubMed1988

Meiotic disjunction of homologs in Saccharomyces cerevisiae is directed by pairing and recombination of the chromosome arms but not by pairing of the centromeres.

We explored the behavior of meiotic chromosomes in Saccharomyces cerevisiae by examining the effects of chromosomal rearrangements on the pattern of disjunction and recombination of chromosome III during meiosis. The segregation of deletion chromosomes lacking part or all (telocentric) of one arm was analyzed in the presence of one or two copies of a normal chromosome III. In strains containing one normal and any one deletion chromosome, the two chromosomes disjoined in most meioses. In strains with one normal chromosome and both a left and right arm telocentric chromosome, the two telocentrics preferentially disjoined from the normal chromosome. Homology on one arm was sufficient to direct chromosome disjunction, and two chromosomes could be directed to disjoin from a third. In strains containing one deletion chromosome and two normal chromosomes, the two normal chromosomes preferentially disjoined, but in 4-7% of the tetrads the normal chromosomes cosegregated, disjoining from the deletion chromosome. Recombination between the two normal chromosomes or between the deletion chromosome and a normal chromosome increased the probability that these chromosomes would disjoin, although cosegregation of recombinants was observed. Finally, we observed that a derivative of chromosome III in which the centromeric region was deleted and CEN5 was integrated at another site on the chromosome disjoined from a normal chromosome III with fidelity. These studies demonstrate that it is not pairing of the centromeres, but pairing and recombination along the arms of the homologs, that directs meiotic chromosome segregation.

Centromere↗
PubMed1986

A mutant that affects the function of autonomously replicating sequences in yeast.

We previously reported the isolation of a series of mcm mutants that are defective in the maintenance of minichromosomes in yeast. These minichromosomes are circular plasmids, each containing an autonomously replicating sequence (ARS) and a centromere. One of the mcm mutants, mcm2, has the following phenotype: at room temperature it affects the stability of only some minichromosomes depending on the ARS present, while at high temperature it affects all minichromosomes tested irrespective of the ARS present. Here we show that the mcm defect as well as its temperature-dependent specificity for ARSs can be demonstrated with circular as well as linear plasmids that do not contain centromeric sequences. Larger chromosomes containing multiple ARSs are also unstable in this mutant. Further analyses indicate that the mcm2 mutation causes the loss, rather than the aberrant segregation, of the circular minichromosomes. In addition, this mutation appears to stimulate mitotic recombination frequencies. These properties of the mcm2 mutant are consistent with the idea that the mcm2 mutation results in a defect in the initiation of DNA replication at ARSs, the putative chromosomal replication origins in yeast.

Centromere↗
PubMed1986

Host factors in nuclear plasmid maintenance in Saccharomyces cerevisiae.

In yeast, the initiation of DNA replication on chromosomes is believed to occur at specific sequences known as autonomously replicating sequences (ARSs). We previously isolated a number of mutants that are defective in the maintenance of minichromosomes. Analysis of these mutants suggests that although ARSs differ greatly from one another in their primary sequences, they appear to share a common enzyme complex for the initiation of DNA replication. However, this initiation enzyme complex probably binds with differential affinity to different ARSs. This idea is corroborated by our identification of an ARS-binding protein that binds to different ARSs with different efficiencies.

Cell Nucleus↗
PubMed1986

Identification of a telomere-binding activity from yeast.

In yeast, the ends of the chromosomes (telomeres) terminate in repeated poly(C1-3A) sequences. We have identified a yeast activity that binds specifically to these poly(C1-3A) repeats. An agarose gel binding assay was used to detect and characterize this activity in cell extracts using both cloned telomere DNA and yeast genomic DNA as substrates. The activity appears to bind specifically to poly(C1-3A) sequences, despite their different primary sequences, yet does not bind specifically to telomeric repeats, such as poly(C4A2), poly(C4A4), and poly (C1-8T) from other lower eukaryotes.

Base Sequence↗
PubMed1986

The mitotic stability of deletion derivatives of chromosome III in yeast.

We have constructed a series of deletion derivatives of chromosome III in yeast. Two telocentric chromosomes, one with a deletion of about 100 kilobases (kb) from the left arm and another with a deletion of about 240 kb from the right arm, are mitotically stable, showing only a 2- to 3-fold decrease in stability compared to a normal chromosome III. Chromosomes as small as 100 kb with deletions on both the left and right arms show only slight decreases in mitotic stability. Slight decreases in size in chromosomes smaller than 100 kb produce dramatic decreases in mitotic stability. In general, deletion chromosomes of similar size but different structure display similar stabilities. We find no evidence for the existence of any new cis-acting elements [besides the centromere, autonomously replicating sequences (ARS elements) and telomeres] essential for the stabilization of chromosome III.

Chromosome Deletion↗
PubMed1985

Construction of telocentric chromosomes in Saccharomyces cerevisiae.

We describe a simple method for the construction of large chromosomal deletions in yeast. Diploid yeast cells were transformed with DNA fragments that replace large regions of the chromosomes by homologous recombination. Using this method, we have constructed a telocentric chromosome III in which approximately equal to 100 kilobases (kb) of DNA has been removed from the left arm of the chromosome, so that the centromere is 12 kb from the left telomere. This telocentric chromosome is mitotically stable. Its rate of loss in a diploid strain is 2.5-7.4 X 10(-4) per cell division compared to a rate of loss of 0.36-1.8 X 10(-4) per cell division for a normal chromosome III. It also segregates 2+:2- with fidelity during meiosis. The construction of systematic deletions in a chromosome should be useful in determining the essential features for proper chromosomal segregation and replication.

Base Sequence↗