Mitotic chromosome condensation in the rDNA requires TRF4 and DNA topoisomerase I in Saccharomyces cerevisiae.

DNA topoisomerase I (topo I) is known to participate in the process of DNA replication, but is not essential in Saccharomyces cerevisiae. The TRF4 gene is also nonessential and was identified in a screen for mutations that are inviable in combination with a top1 null mutation. Here we report the surprising finding that a top1 trf4-ts double mutant is defective in the mitotic events of chromosome condensation, spindle elongation, and nuclear segregation, but not in DNA replication. Direct examination of rDNA-containing mitotic chromosomes demonstrates that a top1 trf4-ts mutant fails both to establish and to maintain chromosome condensation in the rDNA at mitosis. We show that the Trf4p associates physically with both Smclp and Smc2p, the S. cerevisiae homologs of Xenopus proteins that are required for mitotic chromosome condensation in vitro. The defect in the top1 trf4-ts mutant is sensed by the MAD1-dependent spindle assembly checkpoint but not by the RAD9-dependent DNA damage checkpoint, further supporting the notion that chromosome structure influences spindle assembly. These data indicate that TOP1 (encoding topo I) and TRF4 participate in overlapping or dependent steps in mitotic chromosome condensation and serve to define a previously unrecognized biological function of topo I.

Cloning, mapping, and in vivo localization of a human member of the PKCI-1 protein family (PRKCNH1).

We report here the complete cDNA sequence, genomic mapping, and immunolocalization of the first human member of the protein kinase C inhibitor (PKCI-1) gene family. The predicted human protein (hPKCI-1) is 96% identical to bovine and 53% identical to maize members, indicating the great evolutionary conservation of this protein family. The hPKCI-1 gene (HGMV-approved symbol PRKCNH1) maps to human chromosome 5q31.2 by fluorescence in situ hybridization. Indirect immunofluorescence shows that hPKCI-1 localizes to cytoskeletal structures in the cytoplasm of a human fibroblast cell line and is largely excluded from the nucleus. The cytoplasmic localization of hPKCI-1 is consistent with a postulated role in mediating a membrane-derived signal in response to ionizing radiation.

The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.

Identification of novel regions of altered DNA copy number in small cell lung tumors.

Identification of the genetic alterations that occur in tumors is an important approach to understanding tumorigenesis. We have used comparative genomic hybridization (CGH), a novel molecular cytogenetic method, to identify the gross DNA copy number changes that commonly occur in small cell lung cancer (SCLC). We analyzed ten SCLC tumors (seven primary tumors and three metastases) from eight patients. We found frequent increases in DNA copy number on chromosome arms 5p, 8q, 3q, and Xq and frequent decreases in copy number on chromosome arms 3p, 17p, 5q, 8p, 13q, and 4p. The increase in copy number at 8q24 (MYC) and decreases at 17p13 (TP53), 13q14 (RB), and 3p have previously been identified in SCLC with other methods. Many of the other regions in which we detected common copy number changes have not been reported to be regions of common alteration in SCLC tumors. Comparison of copy number changes between a primary tumor and a metastasis from the same patient showed that they were more closely related to each other than to any of the other tumors. The results of direct CGH analysis of SCLC tumors reported here confirm the existence of copy number changes that we identified previously by using cell lines.

Frequent novel DNA copy number increase in squamous cell head and neck tumors.

We have undertaken a study of DNA copy number changes in head and neck squamous cell carcinomas to identify novel DNA copy number changes and to determine the significance of previous findings of cytogenetic alterations in cultured cells. Comparative genomic hybridization was performed on genomic DNA extracted from ten tumors. A novel copy number gain on chromosome 3q26-27 and a loss of chromosome 3p were found at high frequency (> or = 50% of tumors). Many other novel chromosomal copy number changes were identified but occurred at a lower frequency. In addition, our data confirm that DNA copy number changes that frequently occur in cultured cells, such as loss of chromosome 3p, also occur in tumors. Frequently altered loci may encode oncogenes or tumor suppressor genes involved in head and neck squamous cell carcinoma tumorigenesis.

lpsZ, a lipopolysaccharide gene involved in symbiosis of Rhizobium meliloti.

lpsZ+ is an allele that allows exo (exopolysaccharide-deficient) mutants of Rhizobium meliloti to invade nodules by modifying rhizobial lipopolysaccharide. We have cloned and sequenced the lpsZ gene. The predicted LpsZ protein has a molecular weight of 48,589 and is probably localized in the cytoplasm. A beta-glucuronidase fusion in the lpsZ gene indicates that lpsZ is not regulated by oxygen or nitrogen.

Rhizobium meliloti chromosomal loci required for suppression of exopolysaccharide mutations by lipopolysaccharide.

Mutants of alfalfa symbiont Rhizobium meliloti SU47 that fail to make extracellular polysaccharide (exo mutants) induce the formation of nodules that are devoid of bacteria and consequently do not fix nitrogen. This Fix- phenotype can be suppressed by an R. meliloti Rm41 gene that affects lipopolysaccharide structure. Here we describe mutations preventing suppression that map at two new chromosomal loci, lpsY and lpsX, present in both strains. Two other lps mutations isolated previously from SU47 also prevented suppression.