PubMed Health⌕ Search

Biomedical subjects

K Tatchell

Publications and source records attributed to K Tatchell.

At least 55 records · Page 3Linked to original sources
PubMed1985

RAS2 of Saccharomyces cerevisiae is required for gluconeogenic growth and proper response to nutrient limitation.

Saccharomyces cerevisiae contains two genes with remarkable homology to members of the ras oncogene family. These two genes, RAS1 and RAS2, constitute an essential gene family since spores with disruptions of both genes fail to grow. We report here that strains containing RAS2 disruptions have three distinct phenotypes. First, they fail to grow efficiently on nonfermentable carbon sources. Second, they hyperaccumulate the storage carbohydrates glycogen and trehalose. Third, diploid cells homozygous for the RAS2 disruptions sporulate on rich media. Extragenic suppressors have been isolated that suppress the gluconeogenic defect. These suppressors fall into at least three complementation groups, mutations in two of which bypass the normal requirement of RAS for cell viability, allowing cells containing neither RAS gene to grow. The phenotype of the RAS2 mutant and extragenic suppressors implicate RAS with some function in the normal response to nutrient limitation.

Carbohydrate Metabolism↗
PubMed1985

Molecular cloning and characterization of the STE7 and STE11 genes of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, haploid cells occur in one of the two cell types, a or alpha. The allele present at the mating type (MAT) locus plays a prominent role in the control of cell type expression. An important consequence of the elaboration of cell type is the ability of cells of one mating type to conjugate with cells of the opposite mating type, resulting in yet a third cell type, an a/alpha diploid. Numerous genes that are involved in the expression of cell type and the conjugation process have been identified by standard genetic techniques. Molecular analysis has shown that expression of several of these genes is subject to control on the transcriptional level by the MAT locus. Two genes, STE7 and STE11, are required for mating in both haploid cell types; ste7 and ste11 mutants are sterile. We report here the molecular cloning of STE7 and STE11 genes and show that expression of these genes is not regulated transcriptionally by the MAT locus. We also have genetically mapped the STE11 gene to chromosome XII, 40 centimorgans from ura4.

Alleles↗
PubMed1984

Transcription and regulatory signals at the mating type locus in yeast.

The mating type locus with two alleles (MATa and MAT alpha) determines cell type in yeast by activating and repressing sets of cell-type-specific genes. The two genes at MAT alpha, alpha 1 and alpha 2, are transcribed divergently from a central promoter region. Deletions in this intergenic region have been used to map DNA sequences involved in the transcription and regulation of the MAT alpha genes. A single promoter region, essential for transcription of both alpha 1 and alpha 2, is found in the region between alpha 1 and alpha 2. Deletions removing the alpha 1 or alpha 2 TATA box are still transcribed but the transcripts fail to initiate properly. A separate regulatory region is also found between alpha 1 and alpha 2. Deletions of this region lead to the constitutive expression of these genes. These regulatory mutants synthesize alpha 1 mRNA in diploids, but this is not sufficient to activate the alpha-specific genes.

Base Sequence↗
PubMed1984

Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.

Cloning, Molecular↗
PubMed1981

A position effect in the control of transcription at yeast mating type loci.

The two mating type loci MATa and MAT alpha each produce two mRNAs that are transcribed in opposite and diverging directions from central promoters. Silent copies of MATa (HMRa) and MAT alpha (HML alpha) contain identical DNA sequences throughout the transcribed region, yet are not transcribed. It is concluded that sequences to the left of HMRa (and probably HML alpha) must somehow affect transcription initiated at the centre of each locus 700 to 1,400 base pairs away. A possible mechanism for this position effect is discussed.

Base Sequence↗
PubMed1981

Isolation of a gene from Drosophila by complementation in yeast.

Transformation of mutant yeast cells by cloned genomic DNA from a higher eukaryote has made it possible to isolate a Drosophila DNA sequence that complements a yeast adenine-8-mutation. A 0.8-kilobase poly(A)-containing RNA is transcribed from the cloned Drosophila segment in transformed yeast cells and can account for functional expression of the gene.

Adenine↗
PubMed1981

The sequence of the DNAs coding for the mating-type loci of Saccharomyces cerevisiae.

The complete sequences of the yeast a mating-type locus, MATa, and of the silent alpha cassette, HML alpha, have been determined. A segment of 642 nucleotides is unique to MATa, and a corresponding segment of 747 nucleotides is unique to MAT alpha. The major mRNAs (a1, a2, alpha 1 and alpha 2) encoded by MATa and MAT alpha have been aligned with the DNA sequence. The a1 mRNA is encoded entirely within the a-specific DNA sequence. The a2 mRNA, which is transcribed divergently from a1 mRNA, is encoded in a region common to both Mata and Mat alpha. The alpha 1 and alpha 2 mRNAs are also transcribed divergently and have their 5' starts about 240 nucleotides apart within the alpha-specific sequence. The amino acid sequences of the MAT proteins have been predicted from the DNA sequences. An unanticipated conclusion is that the a1 protein, containing 148 amino acids, results from readthrough of a UGA at codon 45. Polymorphic forms of the homologous outer segments of the HML alpha, MAT alpha, MATa and HMRa sequences suggest that the boundaries of the segments involved in mating-type switching are immediately adjacent to the a-specific and alpha-specific sequences.

Base Sequence↗
PubMed1981

In vitro mutation analysis of the mating-type locus in yeast.

The mating-type locus (MAT) of Saccharomyces cerevisiae is a complex locus that codes for the regulators of cell type. Two unique messages are transcribed from each MAT allele. Using the in vitro mutagenesis technique whereby synthetic oligonucleotides containing restriction sites (linkers) were inserted into plasmids, we have constructed a series of mutations in cloned DNA containing either the MATa or MAT alpha locus. The new restriction site associated with each "linker" mutation has allowed the mutation to be mapped and sequenced. We have complemented genetically defined mutations (mata1, mat alpha 1 and mat alpha 2) with plasmids containing these in vitro mutations by yeast transformation, thereby mapping the genes onto the DNA sequence. MATa1 has been localized to the MATa unique region (Ya) from which the a1 message is transcribed. We find no function for the other MATa message by using our complementation assay. MAT alpha 1 maps to the MAT alpha unique (Y alpha) and adjacent (Z) region from which the alpha 1 message is transcribed. MAT alpha 2 maps to the other major message found in the common (X) region of the MAT alpha loci. Although most linker mutations that have a mutant phenotype appear to disrupt the translated portion of each gene, two mutations may disrupt transcription.

Chromosome Mapping↗
PubMed1981

Deletion mapping of sequences essential for in vivo transcription of the iso-1-cytochrome c gene.

The 5' termini of yeast CYC1 RNA molecules have been mapped, by nuclease S1 digestion of mRNA . DNA duplexes, to seven locations from 29 to 93 base pairs upstream from the initiating ATG codon. When the CYC1 gene is introduced into yeast in plasmid YEp13, substantially the same transcripts are made. Using this system to study in vivo gene expression, we measured the capacity of enzymatically produced DNA deletions to form the normal set of RNAs. Four regions of 5'-flanking DNA were identified as functional. Sequences within the region -242 to -139 are required for maximal CYC1 transcript formation; their deletion reduces transcription by a factor of 15 but does not change the pattern of 5' ends observed. Deletion of the sequence between -242 and -99 does not further change the overall transcript level but does affect the specificity of CYC1 mRNA starting. A deletion that extends from -242 to -75 causes both an additional shift in the pattern of 5' ends observed and a further large decrease (factor of 10--20) in CYC1 RNA level. Deletions that extend from -242 to -43, and particularly two deletions that extend still closer to the initiating ATG, cause the appearance of an abundant transcript which starts upstream of position -1078 and of minor transcripts starting in the region -325 to -245.

Base Sequence↗
PubMed1980

DNA-histone interactions in nucleosomes.

We have utilized micrococcal nuclease digestion and thermal denaturation studies to investigate the binding of DNA to the histone core of the nucleosome. We conclude that a total of approximately 168 base pairs (bp) of DNA can interact with the histone core under appropriate solution conditions, even in the absence of lysine-rich histones. The interactions in this total length of DNA can be divided into three classes: (a) approximately 22 bp at the ends is bound only at moderate ionic strength. It is easily displaced, and its removal yields the 146 bp core particle. (b) approximately 46 bp near the ends of the core DNA are quite weakly bound to the core, and are displaced at quite moderate temperatures. (c) The remaining central 100 bp are strongly bound, and interact with all of the sites on the histones which strongly protect DNA against DNAse I digestion. A theoretical analysis of the cleavage of nucleosomal DNA by DNAse I has been used to develop evidence that the pattern of protection offered by the histone core is very similar in nuclei to that in isolated core particles.

Animals↗
PubMed1980

The structure of transposable yeast mating type loci.

A recombinant plasmid containing a MAT alpha mating type locus of Saccharomyces cerevisiae has been isolated by its ability to complement a sterile mat alpha mutation. The plasmid hybridizes to restriction fragments containing both active mating type loci (MATa and MAT alpha) and both silent mating type loci (HMRa and HML alpha). All loci therefore have common sequences. Recombinant lambda clones of the locihave been isolated by plaque hybridization and their structures have been compared by a heteroduplex analysis. At its center, each locus contains one of two apparently nonhomologous sequences. Loci concerned with the alpha phenotype (MAT alpha and HML alpha) contain and 850 bp alpha-specific sequence, whereas loci concerned with the a phenotype (MATa and HMRa) contain a 700 bp a-specific sequence. The a- or alpha-specific sequences are surrounded by DNA sequences that are common to all loci. These homologous sequences extend for 230 bp on the left and 700 bp on the right. They appear to be unrelated to each other. Surprisingly, HML alpha and HMRa differ in their extent of homology to MATa and MAT alpha outside the above regions. HMRa lacks an extensive (700 bp) DNA sequence to the right of the large right-hand homologous region, and possibly also a small (90 bp) sequence to the left of the small left-hand homologous region, both of which are present at HML alpha, MATa and MAT alpha. Hybridization studies have shown that the 700 bp sequence is present at HMLa but absent at HMR alpha alleles. It is therefore characteristic of HML, irrespective of whether it contains a- or alpha-specific sequences. The results imply that mating type interconversion is effected by transposition of DNA sequences from HML or HMR to MAT, as predicted by the controlling element model of Oshima and Takano (1971) and the Cassette model of Hicks, Strathern and Herskowitz (1977).

Base Sequence↗
PubMed1979

Nucleosome reconstitution: effect of DNA length on nuclesome structure.

Core histones (H2A, H2B, H3, and H4) are reconstituted by salt gradient dialysis with DNA molecules ranging in length from 177 bp down to 50 bp. While reconstituted particles containing 125 bp are very similar to native particles, those particles containing a single piece of shorter DNA tend to aggregate. The aggregation depends on the ionic strength and DNA length. The DNA placement on the histone core is not random as determined by pancreatic DNase I digestions of particles containing 32P 5'-end-labeled DNA. Rather, it is found that all DNA molecules, up to 161 bp in length, reassociate with core histones in such a way as to produce defined patterns of DNase I cutting with respect to the 5' ends. Particles were made that contained two pieces of 65-bp DNA. These particles are very similar to native particles under most conditions but tended to dissociation results in the production of two half-nucleosomes (hemisones).

Animals↗
PubMed1978

Compact oligomers and nucleosome phasing.

Micrococcal nuclease (EC 3.1.4.7) digestion of histone H1- and H5-depleted chicken erythrocyte chromatin yields, in addition to 140-base-pair (bp) core particles, a series of nucleosome oligomers containing about 260 bp (compact dimer), 380 bp (compact trimer), etc. of DNA. These are postulated to represent members of a class of oligomers in which the DNA is tightly wound on stacked protein cores. The physical properties (melting, circular dichroism) as well as DNase I (EC 3.1.4.5) digestion patterns support this view. DNase I digestion of tight oligomers in which the 5' ends of the DNA have been labeled yields results consistent with this model and inconsistent with some other possible models. Several classes of such particles are postulated to exist, differing in DNA length by 10-bp increments. This may be an explanation of the 10-bp nucleosome "phasing" that has been observed in some nuclei.

Chromatin↗
PubMed1978

Thermal denaturation of nucleosomal core particles.

Thermal denaturation of very homogeneous preparations of core particles from chicken erythrocyte chromatin is studied by several techniques. The change in absorbance, which is very closely paralleled by changes in heat capacity, which is very closely paralleled by changes in heat capacity, is a biphasic process with inflexions at 60 degrees C and 74 degrees C. In contrast, isolated DNA of the same length denatures in a single transition around 44 degrees C. Monitoring the circular dichroism of the cores during thermal denaturation reveals biphasic changes in the secondary structure of the DNA, preceding the base unstacking by 10 degrees C in the first and 3 degrees C in the second phase. However, measurable alterations in the secondary structure of the histones are confined to the second phase with a melting temperature at 71 degrees C. Increase in the ionic strength of the buffer from 1 mM to 10 mM leads to almost monophasic melting curves as measured by absorbance and CD, while not causing any measurable conformational changes at room temperature. The melting of core particles is interpreted as a denaturation of about 40 base pairs in the first phase, followed by a massive breakdown of the native structure of a tight histone-DNA complex, which frees the remaining 100 base pairs for unstacking.

Animals↗
PubMed1977

Reconstitution of chromatin core particles.

Chromatin core particles, containing 140 base pairs (bp) of DNA plus the inner histones, can be nearly quantitatively formed either by reassociation from 2 M NaCl or by reconstitution from salt extracted histones and DNA. The reassociated or reconstituted particles appear to be identical with the native particles in all physical properties examined (sedimentation velocity, histone content, circular dichroism, and melting) as well as in their patterns of digestion by micrococcal nuclease, DNase I, and trypsin. In the presence of excess DNA, no "half-particles" are formed. In the presence of excess histone, aggregated structures are formed in addition to 11S core particles.

Animals↗
PubMed1977

On the occurrence of nucleosome phasing in chromatin.

We have found that DNAase I digestion of yeast, HeLa and chicken erythrocyte nuclei produces a pattern of DNA fragments spaced 10 bases apart and extending to at least 300 bases. This "extended ladder" of DNA fragments is most clearly seen with yeast, and least clearly with chicken erythrocytes. The appearance of regular and discrete bands at sizes much larger than the repeat size shows that the core particles (140 bp of DNA + H2A, H2B, H3 H4) in at least some fraction of chromatin are spaced in a particular fashion, by discrete lengths of spacer DNA, and not randomly. Based on the abundance of small repeats in yeast and from experiments with nucleosome oligomers, we conclude that the extended ladder and nucleosomal phasing probably arise mainly from regions in the chromatin in which nucleosome cores are closely packed or closely spaced (140-160 bp X n). Contributions from less closely packed but still accurately phased nucleosomes, however, cannot be entirely excluded.

Animals↗
PubMed1977

Comparative subunit structure of HeLa, yeast, and chicken erythrocyte chromatin.

We have compared the chromatin subunit structure of yeast, HeLa, and chicken erythrocyte by analyzing the DNA fragments produced by in situ digestion with staphylococcal nuclease (EC 3.1.4.7) and DNase I(EC 3.1.4.5). The repeat size of the chromatin varies among (and within two of) the three organisms but the size and the structure of the most nuclease-resistant "core" of the repeat is the same. Thus, the interspecies differences in repeat size are due to different lengths of nuclease-sensitive "spacer" DNA between the cores. There also seems to be a difference in the manner of spacing of cores; the transcriptionally active (yeast and HeLa) chromatins have spacings of variable length while the transcriptionally inactive (chicken erythrocyte) has a more regular spacing of cores.

Animals↗