Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata.

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.

Genes involved in light control of sexual differentiation in Chlamydomonas reinhardtii.

Gamete formation requires the sequential action of two extrinsic cues, nitrogen deprivation and blue light. The mutants described here are specifically altered in the light-dependent step. Mutations lrg1, lrg3, and lrg4 overcome this light dependence while mutation lrg2 results in a delayed execution of the light-mediated step. The four mutations are linked. The recessive nature of the lrg1, lrg3, and lrg4 mutations implies that they encode elements of negative control in this light response pathway. Analyses of diploids suggest an interaction between the gene products of the mutated loci with a central role for lrg4. The lrg4 mutation is unique also because it overcomes the light dependence of Chlamydomonas zygote germination when present in homozygous form. These data indicate that there are common components in the signal chains that control gametogenesis and zygote germination.

Chlamydomonas mutants affected in the light-dependent step of sexual differentiation.

Sexual differentiation of Chlamydomonas reinhardtii is induced by the consecutive action of two extrinsic cues--nitrogen deprivation and blue light. The definition of a blue light-dependent step in gamete formation provided a basis for the isolation of mutants altered in the signal transduction pathway by which light controls sexual differentiation. In one mutant (lrg1), gamete formation has become light independent. In the other mutant (lrg2), perception or transduction of the light signal appears to be partially impaired. In both mutants, the expression of genes activated by light in the late phase of gamete formation is affected. Genetic analyses showed that genes LRG1 and LRG2 are linked. The recessive nature of the lrg1-1 mutation implies that the gene encodes a negative factor or a protein that controls the activity of a negative factor. In the case of lrg2-1, neither wild-type nor mutant allele was dominant. Rather, two copies of the lrg2-1 gene simulate a wild-type phenotype. The identification of genetic loci in the pathway for blue light-mediated differentiation provides a basis for the isolation of signal transduction genes in Chlamydomonas.

Comparative analysis of the PCOLCE region in Fugu rubripes using a new automated annotation tool.

The Japanese pufferfish Fugu rubripes with a genome of about 400 Mb is becoming increasingly recognized as a vertebrate model organism for comparative gene analysis (see Elgar 1996 for review). We have isolated and sequenced two Fugu cosmids spanning a genomic region of 66 kb containing the Fugu homolog to the human PCOLCE-I (Glöckner et al. 1998). We then examined if RUMMAGE-DP, a newly developed analysis tool for gene discovery which was designed for human and mouse genomic DNA, can be used for automatic annotation of Fugu genomic sequence. The exon prediction programs contained in RUMMAGE-DP performed better overall for the human sequence than for the Fugu contig. The GENSCAN program was the only exon prediction programme that performed equally well for both organisms. We show that RUMMAGE-DP is very useful in automatic analysis of Fugu sequences. Comparative analysis of the genomic structure of the PCOLCE-I genes in Fugu and human reveals that the exon/intron structure throughout the protein coding region is almost identical. We defined an additional domain based on the high degree of similarity of 26 aa between mammals and Fugu. The PCOLCE-I protein in both organisms contains two highly conserved CUB domains. Exons 6 and 7 are the only coding exons that differ in length between the two species. We assume that these exons do not code for any catalytic domain of the protein. Analysis of the remaining five Fugu genes within the 66 kb interval revealed no conserved synteny with the corresponding human 7q22 region.

Genomic structure of a novel LIM domain gene (ZNF185) in Xq28 and comparisons with the orthologous murine transcript.

Construction of a transcript map in the DXS52 region in Xq28 had previously led to the isolation of a cDNA with a LIM zinc finger domain in the carboxyl terminus. In parallel, the orthologous murine transcript was isolated from the syntenic region. The human and mouse cDNAs have been designated ZNF185 and Zfp185, respectively. By integrating the cDNA sequence with the cosmid-derived genomic sequence the exon-intron structure of the 3' end of the ZNF185 gene was resolved. Comparative sequence analyses of the human genomic sequence with the full-length murine cDNA facilitated prediction of the 5' end of the gene. The selective expression of three transcripts corresponding to the ZNF185 gene and a related gene was shown by Northern and Southern blots. In situ hybridizations revealed a nonubiquitous and stage-specific expression of Zfp185, especially in differentiating connective tissue. Since LIM proteins regulate cellular proliferation and/or differentiation by diverse mechanisms, and some have directly been associated with disease, conceivably ZNF185 may represent a candidate for a disease-causing gene linked to Xq28. Knowledge of the genomic structure will permit detailed mutation analyses.

Comparative genome sequence analysis of the Bpa/Str region in mouse and Man.

The progress of human and mouse genome sequencing programs presages the possibility of systematic cross-species comparison of the two genomes as a powerful tool for gene and regulatory element identification. As the opportunities to perform comparative sequence analysis emerge, it is important to develop parameters for such analyses and to examine the outcomes of cross-species comparison. Our analysis used gene prediction and a database search of 430 kb of genomic sequence covering the Bpa/Str region of the mouse X chromosome, and 745 kb of genomic sequence from the homologous human X chromosome region. We identified 11 genes in mouse and 13 genes and two pseudogenes in human. In addition, we compared the mouse and human sequences using pairwise alignment and searches for evolutionary conserved regions (ECRs) exceeding a defined threshold of sequence identity. This approach aided the identification of at least four further putative conserved genes in the region. Comparative sequencing revealed that this region is a mosaic in evolutionary terms, with considerably more rearrangement between the two species than realized previously from comparative mapping studies. Surprisingly, this region showed an extremely high LINE and low SINE content, low G+C content, and yet a relatively high gene density, in contrast to the low gene density usually associated with such regions.