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.

Max and inhibitory c-Myc mutants induce erythroid differentiation and resistance to apoptosis in human myeloid leukemia cells.

We have used the human leukemia cell line K562 as a model to study the role of c-myc in differentiation and apoptosis. We have generated stable transfectants of K562 constitutively expressing two c-Myc inhibitory mutants: D106-143, that carries a deletion in the transactivation domain of the protein, and In373, that carries an insertion in the DNA-interacting region. We show here that In373 is able to compete with c-Myc for Max binding and to inhibit the transformation activity of c-Myc. K562 cells can differentiate towards erythroid or myelomonocytic lineages. K562 transfected with c-myc mutants showed a higher expression of erythroid differentiation markers, without any detectable effects in the myelomonocytic differentiation. We also transfected K562 cells with a zinc-inducible max gene. Ectopic Max overexpression resulted in an increased erythroid differentiation, thus reproducing the effects of c-myc inhibitory mutants. We also studied the role of c-myc mutants and max in apoptosis of K562 induced by okadaic acid, a protein phosphatases inhibitor. The expression of D106-143 and In373 c-myc mutants and the overexpression of max reduced the apoptosis mediated by okadaic acid. The common biochemical activity of D106-143 and In373 is to bind Max and hence to titrate out c-Myc to form non-functional Myc/Max dimers. Similarly, Max overexpression would decrease the relative levels of c-Myc/Max with respect to Max/Max. The results support a model where a threshold of functional c-Myc/Max is required to maintain K562 cells in an undifferentiated state and to undergo drug-mediated apoptosis.

Exploiting the complete yeast genome sequence.

The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings. This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest. In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.

Establishing genetic interactions by a synthetic dosage lethality phenotype.

We have devised a genetic screen, termed synthetic dosage lethality, in which a cloned "reference" gene is inducibly overexpressed in a set of mutant strains carrying potential "target" mutations. To test the specificity of the method, two reference genes, CTF13, encoding a centromere binding protein, and ORC6, encoding a subunit of the origin of replication binding complex, were overexpressed in a large collection of mutants defective in either chromosome segregation or replication. CTF13 overexpression caused synthetic dosage lethality in combination with ctf14-42 (cbf2, ndc10), ctf17-61 (chl4), ctf19-58 and ctf19-26. ORC6 overexpression caused synthetic dosage lethality in combination with cdc2-1, cdc6-1, cdc14-1, cdc16-1 and cdc46-1. These relationships reflect specific interactions, as overexpression of CTF13 caused lethality in kinetochore mutants and overexpression of ORC6 caused lethality in replication mutants. In contrast, only one case of dosage suppression was observed. We suggest that synthetic dosage lethality identifies a broad spectrum of interacting mutations and is of general utility in detecting specific genetic interactions using a cloned wild-type gene as a starting point. Furthermore, synthetic dosage lethality is easily adapted to the study of cloned genes in other organisms.

Urethroscopy and urethral cytology in men with external genital condyloma.

OBJECTIVE: To develop guidelines as to which asymptomatic male patients with genital human papillomavirus (HPV) infection need further evaluation of the urethra, we studied two screening methods: urethroscopy and voided urethral cytology. METHODS: In a four-year period, 135 asymptomatic men underwent complete screening for HPV infection. They were evaluated because of HPV-related genital disease in their female sex partners or visible genital lesions, or both. RESULTS: Of the 135 patients, 21 (16%) had no clinical, subclinical, cytologic, or urethroscopic evidence of disease, and 114 (84%) had biopsy-proven HPV infection. Of these 114 patients, only 14 (12.3%) had intraurethral condyloma. All of these 14 patients had current or historical evidence of meatal or perimeatal "sentinel" lesions. They constituted 29.8 percent of 47 such patients with sentinel lesions. In 5 patients (4%), results of voided urine cytology were positive for condyloma cells, but only 1 of these had visible intraurethral disease. Of the 14 patients with urethral disease, only 1 (7%) had positive results of urine cytology. CONCLUSIONS: These observations suggest that any asymptomatic male patient undergoing screening for condyloma acuminatum who has a history of or demonstrable subclinical or grossly visible perimeatal or meatal HPV infection should undergo urethroscopy and that voided urine cytology is not a reliable or cost-effective test for the detection of visible intraurethral disease.

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures.

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.

Nutrition broadcasting.

The use of radio by the dietitians of the Whittington Hospital and their guest speakers to advise and educate hospital patients on varying aspects of human nutrition, reaching a much larger section of the local population than they could in their usual work, is described.