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J H Shero

Publications and source records attributed to J H Shero.

11 recordsLinked to original sources
PubMed1995

Cis-acting determinants affecting centromere function, sister-chromatid cohesion and reciprocal recombination during meiosis in Saccharomyces cerevisiae.

We have employed a system that utilizes homologous pairs of human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to assess the specific role(s) of conserved centromere DNA elements (CDEI, CDEII and CDEIII) in meiotic chromosome disjunction fidelity. Thirteen different centromere (CEN) mutations were tested for their effects on meiotic centromere function. YACs containing a wild-type CEN DNA sequence segregate with high fidelity in meiosis I (99% normal segregation) and in meiosis II (96% normal segregation). YACs containing a 31-bp deletion mutation in centromere DNA element II (CDEII delta 31) in either a heterocentric (mutant/wild type), homocentric (mutant/mutant) or monosomic (mutant/--) YAC pair configuration exhibited high levels (16-28%) of precocious sister-chromatid segregation (PSS) and increased levels (1-6%) of nondisjunction meiosis I (NDI). YACs containing this mutation also exhibit high levels (21%) of meiosis II nondisjunction. Interestingly, significant alterations in homolog recombination frequency were observed in the exceptional PSS class of tetrads, suggesting unusual interactions between prematurely separated sister chromatids and their homologous nonsister chromatids. We also have assessed the meiotic segregation effects of rare gene conversion events occurring at sites located immediately adjacent to or distantly from the centromere region. Proximal gene conversion events were associated with extremely high levels (60%) of meiosis I segregation errors (including both PSS and NDI), whereas distal events had no apparent effect. Taken together, our results indicate a critical role for CDEII in meiosis and underscore the importance of maintaining sister-chromatid cohesion for proper recombination in meiotic prophase and for proper disjunction in meiosis I.

Base Sequence↗
PubMed1992

Multifunctional yeast high-copy-number shuttle vectors.

A set of four yeast shuttle vectors that incorporate sequences from the Saccharomyces cerevisiae 2 mu endogenous plasmid has been constructed. These yeast episomal plasmid (YEp)-type vectors (pRS420 series) differ only in their yeast selectable markers, HIS3, TRP1, LEU2 or URA3. The pRS420 plasmids are based on the backbone of a multifunctional phagemid, pBluescript II SK+, and share its useful properties for growth in Escherichia coli and manipulation in vitro. The pRS420 plasmids have a copy number of about 20 per cell, equivalent to that of YEp24. During non-selective yeast growth, pRS420 plasmids are lost through mitotic segregation at rates similar to other YEp vectors and yeast centromeric plasmid (YCp) vectors, in the range of 1.5-5% of progeny per doubling. The pRS420 series provides high-copy-number counterparts to the current pRS vectors [Sikorski and Hieter, Genetics 122 (1989) 19-27].

Cloning, Molecular↗
PubMed1991

Yeast artificial chromosome vectors for efficient clone manipulation and mapping.

The yeast artificial chromosome (YAC) cloning system allows the cloning of exogenous DNA several hundred kilobases in length. To enhance the usefulness of this technology, yeast artificial chromosome vectors have been designed for efficient clone characterization, manipulation, and mapping. The vectors contain a polylinker with unique EcoRI, BglII, NotI, EagI, SacII, SalI, NruI, NheI, and ClaI cloning sites and T7 bacteriophage promoters positioned to allow the generation of riboprobes from the exogenous DNA ends. Centric and acentric vector arms were constructed as separate plasmids to allow the recovery of both ends of the YAC insert DNA directly in Escherichia coli. In addition, YACs generated using this vector system contain a yeast gene (SUP 11) that allows visual monitoring of YAC stability and copy number.

Animals↗
PubMed1991

A suppressor of a centromere DNA mutation encodes a putative protein kinase (MCK1).

A new approach to identify genes involved in Saccharomyces cerevisiae kinetochore function is discussed. A genetic screen was designed to recover extragenic dosage suppressors of a CEN DNA mutation. This method identified two suppressors, designated MCK1 and CMS2. Increased dosage of MCK1 specifically suppressed two similar CEN DNA mutations in CDEIII, but not comparably defective CEN DNA mutations in CDEI or CDEII. A strain containing a null allele of MCK1 was viable under standard growth conditions, had a cold-sensitive phenotype (conditional lethality at 11 degrees C), and grew slowly on Benomyl (a microtubule-destabilizing drug). Furthermore, when grown at 18 degrees C or in the presence of Benomyl, the null mutant exhibited a dramatic increase in the rate of mitotic chromosome loss. The allele-specific suppression and chromosome instability phenotypes suggest that MCK1 plays a role in mitotic chromosome segregation specific to CDEIII function. The MCK1 gene encodes a putative protein-serine/threonine kinase, which suggests a possible role for the MCK1 protein in regulating the activity of centromere-binding proteins by phosphorylation. MCK1 was identified and cloned independently for its involvement in the induction of meiosis and is identical to a gene that encodes a phosphotyrosyl protein with protein kinase activity.

Amino Acid Sequence↗
PubMed1989

Construction of human chromosome 21-specific yeast artificial chromosomes.

Chromosome 21-specific yeast artificial chromosomes (YACs) have been constructed by a method that performs all steps in agarose, allowing size selection by pulsed-field gel electrophoresis and the use of nanogram to microgram quantities of DNA. The DNA sources used were hybrid cell line WAV-17, containing chromosome 21 as the only human chromosome and flow-sorted chromosome 21. The transformation efficiency of ligation products was similar to that obtained in aqueous transformations and yielded YACs with sizes ranging from 100 kilobases (kb) to greater than 1 megabase when polyamines were included in the transformation procedure. Twenty-five YACs containing human DNA have been obtained from a mouse-human hybrid, ranging in size from 200 to greater than 1000 kb, with an average size of 410 kb. Ten of these YACs were localized to subregions of chromosome 21 by hybridization of RNA probes (corresponding to the YAC ends recovered in Escherichia coli) to a panel of somatic cell hybrid DNA. Twenty-one human YACs, ranging in size from 100 to 500 kb, with an average size of 150 kb, were obtained from approximately equal to 50 ng of flow-sorted chromosome 21 DNA. Three were localized to subregions of chromosome 21. YACs will aid the construction of a physical map of human chromosome 21 and the study of disorders associated with chromosome 21 such as Alzheimer disease and Down syndrome.

Cell Line↗
PubMed1989

A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae.

Saccharomyces cerevisiae centromeres contain a conserved region ranging from 111 to 119 base pairs (bp) in length, which is characterized by the three conserved DNA elements CDEI, CDEII, and CDEIII. We isolated a 125-bp CEN6 DNA fragment (named ML CEN6) containing only these conserved elements and assayed it completely separated from its chromosomal context on circular minichromosomes and on a large linear chromosome fragment. The results show that this 125-bp CEN6 DNA fragment is by itself sufficient for complete mitotic and meiotic centromere functions.

Base Sequence↗
PubMed1988

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae.

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

Base Sequence↗
PubMed1987

Multiple tandemly repeated binding sites for cellular nuclear factor 1 that surround the major immediate-early promoters of simian and human cytomegalovirus.

We show that the large DNA genomes of human and simian cytomegaloviruses (HCMV and SCMV, respectively) each contain multiple binding sites for purified cellular nuclear factor 1 (NF1) protein. Examination of the major immediate-early (IE) gene region in the HindIII H fragment of SCMV (Colburn) by filter binding assays showed that it competed 45-fold better than the single adenovirus type 2 binding site for NF1 protein and that it contained at least two distinct binding loci. Direct DNase I footprinting analyses of the 5' upstream locus detected at least 20 adjacent NF1-binding sites located between positions -600 and -1300 relative to the IE94 mRNA start site. DNA sequence analysis of the region revealed a conserved consensus NF1 recognition element (T)TGG(C/A)N5GCCAA embedded within each of 23 highly diverged 30-base-pair tandem repeats, together with a second downstream cluster of five consensus NF1-binding sites between positions +470 and +570 in the large first intron. Two separate NF1-binding loci were also found in the equivalent IE68 gene of HCMV(Towne) DNA, but in this case the DNA sequence and competition filter binding experiments indicated a maximum of only four to five consensus binding sites encompassing the promoter-enhancer region. In transient expression assays, neither the isolated upstream IE94 tandem repeats nor a synthetic single-copy consensus NF1-binding site acted as transcriptional cis activators or enhancers when placed adjacent to the simian virus 40 minimal early region promoter. We conclude that the large and complex 5' upstream promoter-regulatory region for the SCMV IE94 gene comprises two distinct domains. The previously described four sets of 13- to 18-base-pair interspersed repeat elements between -55 and -580 provide most of the high basal transcriptional strength, whereas the arrangement of further upstream tandemly repeated NF1-binding sites may contribute significantly to the expanded biological host range for expression of SCMV IE94 compared with HCMV IE68.

Animals↗
PubMed1986

High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients.

Patients with rheumatic diseases often have circulating autoantibodies to nuclear components. The clinical significance of the antibodies is controversial, although in some cases they are valuable in the diagnosis of the disease. This report presents results of a study of Scl-70, an autoantigen recognized by sera of many patients with the most severe form of progressive systemic sclerosis. It was possible to show, by three independent criteria, that Scl-70 is the abundant nuclear enzyme DNA topoisomerase I. Therefore, antibody probes of high titer and high affinity are now available for the study of this important nuclear enzyme.

Antibody Specificity↗