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Biomedical subjects

J Kingsbury

Publications and source records attributed to J Kingsbury.

8 recordsLinked to original sources
PubMed1999

Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle.

A genetic synthetic dosage lethality (SDL) screen using CTF13 encoding a known kinetochore protein as the overexpressed reference gene identified two chromosome transmission fidelity (ctf) mutants, YCTF58 and YCTF26. These mutant strains carry independent alleles of a novel gene, which we have designated CTF19. In light of its potential role in kinetochore function, we have cloned and characterized the CTF19 gene in detail. CTF19 encodes a nonessential 369-amino acid protein. ctf19 mutant strains display a severe chromosome missegregation phenotype, are hypersensitive to benomyl, and accumulate at G2/M in cycling cells. CTF19 genetically interacts with kinetochore structural mutants and mitotic checkpoint mutants. In addition, ctf19 mutants show a defect in the ability of centromeres on minichromosomes to bind microtubules in an in vitro assay. In vivo cross-linking and chromatin immunoprecipitation demonstrates that Ctf19p specifically interacts with CEN DNA. Furthermore, Ctf19-HAp localizes to the nuclear face of the spindle pole body and genetically interacts with a spindle-associated protein. We propose that Ctf19p is part of a macromolecular kinetochore complex, which may function as a link between the kinetochore and the mitotic spindle.

Anaphase↗
PubMed1997

Photodynamic therapy and hyperthermia as an adjuvant modality in preventing tumor recurrence.

BACKGROUND AND OBJECTIVE: The objective of this study was to determine the relative efficacy in preventing tumor recurrence by photodynamic therapy (PDT), and by ablative CO2 laser surgery followed by PDT, compared to ablative surgery alone (negative control) or ablative surgery followed by a course of hyperthermia (positive control). STUDY DESIGN/MATERIALS AND METHODS: The cheek pouches of 36 hamsters were treated with 0.5% 9,10 dimethyl-1,2-benzanthracene in acetone three times a week. After 12 weeks all animals showed tumors of their cheek pouches and were divided into four groups. In groups number I, II, and III, all visible tumors were removed by aid of a CO2 laser. Animals of group I did not receive any further treatment. After tumor resection, the cheek pouches in group II were treated with hyperthermia by aid of a Nd:YAG laser and a temperature of 43 degrees C for 30 minutes. In group III after resection of the tumors, the cheek pouches were treated with PDT (75mW/cm2 175J/cm2--3mg/kg Photofrin i.p./24h). In group IV, the tumors were not excised, instead they were only treated with PDT (as above). All animals were observed for 50 days for any signs of tumor recurrence. RESULTS: In group I (CO2) all tumors (100%) recurred within 50 days. In group II (CO2 + hyperthermia) 61%, in group III (CO2 + PDT) 27.7%, and in group IV (PDT) 50% of all tumors recurred. The first signs of recurrence could be seen in group I, followed by groups II and IV. Group III was the last one presenting tumor recurrence. CONCLUSIONS: The combination of CO2 surgery and PDT produced significantly better results than CO2 surgery or PDT alone, and better than the combination of CO2 surgery and hyperthermia.

Animals↗
PubMed1996

Faithful chromosome transmission requires Spt4p, a putative regulator of chromatin structure in Saccharomyces cerevisiae.

A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified by its centromere (CEN) transcriptional readthrough phenotype, suggesting perturbed kinetochore integrity in vivo. The gene complementing the s138 mutation was found to be identical to the S. cerevisiae SPT4 gene. The s138 mutation is a missense mutation in the second of four conserved cysteine residues positioned similarly to those of zinc finger proteins, and we henceforth refer to the mutation of spt4-138. Both spt4-138 and spt4 delta strains missegregate a chromosome fragment at the permissive temperature, are temperature sensitive for growth at 37 degrees C, and upon a shift to the nonpermissive temperature show an accumulation of large budded cells, each with a nucleus. Previous studies suggest that Spt4p functions in a complex with Spt5p and Spt6p, and we determined that spt6-140 also causes missegregation of a chromosome fragment. Double mutants carrying spt4 delta 2::HIS3 and kinetochore mutation ndc10-42 or ctf13-30 show a synthetic conditional phenotype. Both spt4-138 and spt4 delta strains exhibit synergistic chromosome instability in combination with CEN DNA mutations and show in vitro defects in microtubule binding to minichromosomes. These results indicate that Spt4p plays a role in chromosome segregation. The results of in vivo genetic interactions with mutations in kinetochore proteins and CEN DNA and of in vitro biochemical assays suggest that Spt4p is important for kinetochore function.

Amino Acid Sequence↗
PubMed1995

CEP3 encodes a centromere protein of Saccharomyces cerevisiae.

We have designed a screen to identify mutants specifically affecting kinetochore function in the yeast Saccharomyces cerevisiae. The selection procedure was based on the generation of "synthetic acentric" minichromosomes. "Synthetic acentric" minichromosomes contain a centromere locus, but lack centromere activity due to combination of mutations in centromere DNA and in a chromosomal gene (CEP) encoding a putative centromere protein. Ten conditional lethal cep mutants were isolated, seven were found to be alleles of NDC10 (CEP2) encoding the 110-kD protein of yeast kinetochore. Three mutants defined a novel essential gene CEP3. The CEP3 product (Cep3p) is a 71-kD protein with a potential DNA-binding domain (binuclear Zn-cluster). At nonpermissive temperature the cep3 cells arrest with an undivided nucleus and a short mitotic spindle. At permissive temperature the cep3 cells are unable to support segregation of minichromosomes with mutations in the central part of element III of yeast centromere DNA. These minichromosomes, when isolated from cep3 cultures, fail to bind bovine microtubules in vitro. The sum of genetic, cytological and biochemical data lead us to suggest that the Cep3 protein is a DNA-binding component of yeast centromere. Molecular mass and sequence comparison confirm that Cep3p is the p64 component of centromere DNA binding complex Cbf3 (Lechner, 1994).

Amino Acid Sequence↗
PubMed1993

Centromere function on minichromosomes isolated from budding yeast.

Centromeres are a complex of centromere DNA (CEN DNA) and specific factors that help mediate microtubule-dependent movement of chromosomes during mitosis. Minichromosomes can be isolated from budding yeast in a way that their centromeres retain the ability to bind microtubules in vitro. Here, we use the binding of these minichromosomes to microtubules to gain insight into the properties of centromeres assembled in vivo. Our results suggest that neither chromosomal DNA topology nor proximity of telomeres influence the cell's ability to assemble centromeres with microtubule-binding activity. The microtubule-binding activity of the minichromosome's centromere is stable in the presence of competitor CEN DNA, suggesting that the complex between the minichromosome CEN DNA and proteins directly bound to it is very stable. The efficiency of centromere binding to microtubules is dependent upon the concentration of microtubule polymer and is inhibited by ATP. These properties are similar to those exhibited by mechanochemical motors. The binding of minichromosomes to microtubules can be inactivated by the presence of 0.2 M NaCl and then reactivated by restoring NaCl to 0.1 M. In 0.2 M NaCl, some centromere factor(s) bind to microtubules, whereas other(s) apparently remain bound to the minichromosome's CEN DNA. Therefore, the yeast centromere appears to consist of two domains: the first consists of a stable core containing CEN DNA and CEN DNA-binding proteins; the second contains a microtubule-binding component(s). The molecular functions of this second domain are discussed.

Adenosine Triphosphate↗
PubMed1991

Centromere-dependent binding of yeast minichromosomes to microtubules in vitro.

We present an in vitro assay for yeast centromere function; isolated yeast minichromosomes require a functional centromere to bind to bovine microtubules and sediment with them. Centromere-bovine microtubule complexes form at physiological microtubule concentrations. Two of the three centromere DNA elements, which are necessary for centromere function in vivo, are also necessary for centromeres to bind microtubules in vitro. However, purified centromere DNA alone does not bind to microtubules. These results suggest that microtubule binding must be mediated by the two centromere DNA elements and factors that associate with one or both of them. The percent of centromeres with microtubule-binding activity is 7- to 10-fold higher in lysates made from nocodazole-arrested G2-M cells than from alpha factor G1 cells, suggesting that this centromere activity is regulated during the cell cycle. The potential of this assay for dissecting centromere assembly, function, and regulation is discussed.

Adenosine Triphosphate↗