Biomedical subjects

K M Tuffo

Publications and source records attributed to K M Tuffo.

6 recordsLinked to original sources

PubMed2000

Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren.

In vitro studies suggest that the Barren protein may function as an activator of DNA topoisomerase II and/or as a component of the Xenopus condensin complex. To better understand the role of Barren in vivo, we generated conditional alleles of the structural gene for Barren (BRN1) in Saccharomyces cerevisiae. We show that Barren is an essential protein required for chromosome condensation in vivo and that it is likely to function as an intrinsic component of the yeast condensation machinery. Consistent with this view, we show that Barren performs an essential function during a period of the cell cycle when chromosome condensation is established and maintained. In contrast, Barren does not serve as an essential activator of DNA topoisomerase II in vivo. Finally, brn1 mutants display additional phenotypes such as stretched chromosomes, aberrant anaphase spindles, and the accumulation of cells with >2C DNA content, suggesting that Barren function influences multiple aspects of chromosome transmission and dynamics.

Cell Cycle Proteins↗

PubMed1999

Dominant mutations in three different subunits of replication factor C suppress replication defects in yeast PCNA mutants.

To identify proteins that interact with the yeast proliferating cell nuclear antigen (PCNA), we used a genetic approach to isolate mutations that compensate for the defects in cold-sensitive (Cs(-)) mutants of yeast PCNA (POL30). Because the cocrystal structure of human PCNA and a p21(WAF1/CIP1) peptide shows that the interdomain region of PCNA is a site of p21 interaction, we specifically looked for new mutations that suppress mutations in the equivalent region of yeast PCNA. In independent screens using three different Cs(-) mutants, we identified spontaneously arising dominant suppressor mutations in the RFC3 gene. In addition, dominant suppressor mutations were identified in the RFC1 and RFC2 genes using a single pol30 mutant. An intimate association between PCNA and RFC1p, RFC2p, and RFC3p is suggested by the allele-restricted suppression of 10 different pol30 alleles by the RFC suppressors. RFC1, RFC2, and RFC3 encode three of the five subunits of the replication factor C complex, which is required to load PCNA onto DNA in reconstituted DNA replication reactions. Genomic sequencing reveals a common region in RFC1p, RFC2p, and RFC3p that is important for the functional interaction with PCNA. Biochemical analysis of the wild type and mutant PCNA and RFC3 proteins shows that mutant RFC3p enhances the production of long DNA products in pol delta-dependent DNA synthesis, which is consistent with an increase in processivity.

Alleles↗

PubMed1997

Structural and functional complementation of an inactive Bcl-2 mutant by Bax truncation.

Interactions among proteins in the Bcl-2 family regulate the onset of programmed cell death. Previous work has shown that the death-inhibiting family members Bcl-2 and Bcl-xL form heterodimers with the death-promoting homologue Bax and that certain site-directed mutants of Bcl-2 and Bcl-xL lose both biological activity and the ability to bind Bax. To better understand the structural basis of heterodimer formation, we have used a yeast two-hybrid assay to screen for mutants of Bax that regain the ability to bind to these inactive Bcl-2(G145A) and Bcl-xL(G138A) mutants. This screen identified a series of C-terminally truncated Bax molecules that contain complete BH3 (Bcl-2 homology domain 3) domains but that have lost BH1 and BH2 sequences. These results indicate that while the Bcl-2 and Bcl-xL mutants fail to bind full-length Bax, they still retain a binding site for the critical BH3 domain. This suggests that conformational constraints in full-length Bax regulate its ability to bind to other Bcl-2 family members. Furthermore, we demonstrate that the normally inert Bcl-2(G145A) mutant effectively blocks apoptosis induced by a C-terminally truncated Bax molecule, but does not block apoptosis induced by wild-type Bax. This demonstrates that cell protection can be effected by directly binding pro-apoptotic members of the Bcl-2 family.

Amino Acid Sequence↗

PubMed1996

The large subunit of replication factor C (Rfc1p/Cdc44p) is required for DNA replication and DNA repair in Saccharomyces cerevisiae.

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.

Base Sequence↗

PubMed1994

Modified facelift incision for parotidectomy.

The most commonly used incision for parotidectomies is the modified Blair incision. We have successfully used an alternative incision which allows good exposure, and leaves no neck scar. Between 1 March 1989 and 1 August 1991, 18 parotidectomies were performed using a modified facelift incision. Fifteen parotidectomies were done for similar indications during the same period using a modified Blair incision. The mean age in both groups of patients was 40.3 years. The pathology and incidence of complications was similar in the two groups. The difference in mean (+/- SD) time of surgery between the two groups was not statistically significant: 3.14 +/- 0.75 hours in patients with a modified facelift incision and 3.25 +/- 1.27 hours in patients with a modified Blair incision (p > 0.1). The modified facelift incision is an alternative approach to parotidectomy for selected patients. It provides adequate exposure, even for a total parotidectomy and mastoidectomy and it results in improved patient satisfaction without additional risk of complications.

Adenoma, Pleomorphic↗

PubMed1994

Mandible fracture repair: specific indications for newer techniques.

Mandible fracture repair is commonly undertaken by otolaryngologists. Although the essential principles of reduction and immobilization are undisputed, the approach used to obtain these goals varies considerably. We performed a critical evaluation of all mandible fractures treated at the Santa Clara Valley Medical Center by the otolaryngology service between January 1988 and February 1992, with the purpose of better defining the indications for plate fixation and for the use of more traditional techniques. One hundred eighty-three fractures in 112 patients were evaluable. Thirty-six (32.1%) of these patients had at least one plate placed (group A); 39 (34.8%) underwent an open procedure, with interosseous wire fixation (group B); and 37 (33.0%) were treated with closed techniques (group C). The severity of fracture (indexed by comminution, presence of infection, teeth in the fracture line, interval to repair, and whether the fracture was open or closed) was similar in plated and nonplated mandibles. Mean (+/- standard deviation) operative times for the three groups were 3.2 +/- 1.6 hours for group A, 3.0 +/- 0.9 hours for group B, and 1.4 +/- 0.5 hours for group C. The number of follow-up visits required was not statistically different (group A, 5.6 +/- 3.8 visits; group B, 5.2 +/- 2.5 visits; and group C, 5.3 +/- 2.0 visits). The overall incidence of major complications was 14.3% (16 of 112), including 11 of 36 (30.6%) in group A, 4 of 39 (10.3%) in group B, and 1 of 37 (2.7%) in group C.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗