The fission yeast ortholog of eIF3a subunit is not functional in Saccharomyces cerevisiae.

The Schizosaccharomyces pombe eIF3a ortholog (SpeIF3a) was shown to be unable to substitute for S. cerevisiae eIF3a (SceIF3a) in its essential function in the initiation of translation. Overproduction of SpeIF3a altered the distribution of SceIF3a but formation of the endogenous eIF3 complex was not affected. SpeIF3a was found to be more tightly bound to S. cerevisiae ribosomes than SceIF3a and other eIF3 subunits (eIF3g, eIF3i, eIF3j). The host cells displayed aberrant morphology and altered chitin deposition. SpeIF3a probably competes with SceIF3a for binding to either ribosomes or yet to be identified substrates.

Preparation of human recombinant kinesin heavy chain and epitope mapping of its structural domains.

The isolation of the cDNA sequence encoding the human neuronal kinesin (a force-generating motor protein which transports various membrane organelles along microtubules in an ATP-dependent manner) heavy chain (nKHC) and the construction of expression vectors to produce the full-length nKHC and its domains in Escherichia coli is described. By tuning up the conditions for the expression of nKHC, a sufficient amount of the soluble protein intragenously tagged with 6xHis tag was obtained and purified by nickel chromatography. The recombinant structural domains of nKHC, including the motor domain (FKHC1--amino acids 1-330), the microtubule binding domain (FKHC2--amino acids 174-315) and the coiled-coil stalk domain (FKHC3--amino acids 331-906) were used to determine the epitope location for monoclonal antibodies KN-01, KN-02, and IB II raised against different kinesin heavy chains. The antibodies were shown to recognize epitopes located in the stalk domain of nKHC and represent thus useful probes for this domain.

The W303 genetic background affects the isw2 delta mutant phenotype in Saccharomyces cerevisiae.

We performed detailed phenotypic analysis of the isw2 delta strains of the W303 genetic background and compared its results with those obtained previously in BY-derived genetic background. Shmoolike morphology was observed in the isw2 delta strain of alpha-mating type of the BY strains, but not in its W303-derived counterpart. On the other hand, derepression of a-specific genes in the isw2 delta (MAT alpha) strain was observed in both genetic backgrounds, although to a different extent. Unlike in BY-derived strain hyperactivation of the Ras2/cAMP pathway reduced invasiveness of the isw2 delta strain (MAT alpha) of the W303 background. Sensitivity to Calcofluor White indicating a cell wall-integrity defect was significantly increased in the isw2 delta strains of the W303 background in contrast to BY-derived strains. Our data indicate that the effects of the isw2 deletion strongly depend on the background in which the deletion, is made.

ARS sequences in homologous and heterologous ADE2 loci are capable of promoting autonomous replication of plasmids in Schwanniomyces occidentalis.

We have analyzed ARS elements linked to homologous and heterologous ADE2 loci functioning in Schwanniomyces occidentalis. We have identified a region of the ADE2 locus of S. occidentalis which promotes autonomous replication of plasmids in S. occidentalis cells. This region is within 385 bp preceding the ATG codon of the S. occidentalis ADE2 gene. It contains sequences similar to ARS core consensus sequences, ARS boxes, and a potential transcription activator binding site characterized in Saccharomyces cerevisiae. The ADE2 gene of S. cerevisiae was found to complement the ade2 mutation in S. occidentalis cells and the 5' UTR region of this gene is capable of supporting autonomous replication of plasmids in S. occidentalis. Furthermore, we confirmed that the origin of replication of the 2 microm plasmid and the ARS1 sequence of S. cerevisiae are also functional in S. occidentalis cells. Plasmids carrying either ARS, the SwARSA element of S. occidentalis, the ARS linked to the ADE2 gene of S. cerevisiae, and the ARS1 sequence or the 2 microm ori, were found to be maintained in S. occidentalis cells as episomal monomers or oligomers. However, their stability was low as already reported for the ARS in S. occidentalis.

Saccharomyces cerevisiae gene ISW2 encodes a microtubule-interacting protein required for premeiotic DNA replication.

A molecular genetic characterization of the ORF YOR304W (ISW2), identified in a screen of a yeast lambdagt11 library using a monoclonal antibody that reacts with a 210 kDa mammalian microtubule-interacting protein, is presented in this paper. The protein encoded by the ORF YOR304W is 50% identical to the Drosophila nucleosome remodelling factor ISWI and is therefore a new member of the SNF2 protein family and has been recently entered into SDG as ISW2. Although not essential for vegetative growth, we found that the ISW2 gene is required for early stages in sporulation. The isw2 homozygous deletant diploid strain was blocked in the G(1) phase of the cell cycle, unable to execute the premeiotic DNA replication and progress through the nuclear meiotic division cycle. ISW2 expression from a multicopy plasmid had the same effect as deletion, but ISW2 expression from a centromeric plasmid rescued the deletion phenotype. In vegetatively growing diploid cells, the Isw2 protein was preferentially found in the cytoplasm, co-localizing with microtubules. An accumulation of the Isw2 protein within the nucleus was observed in cells entering sporulation. Together with data published very recently by Tsukiyama et al. (1999), we propose a role for the Isw2 protein in facilitating chromatin accessibility for transcriptional factor(s) that positively regulate meiosis/sporulation-specific genes.

Postpolio syndrome: poliovirus persistence is involved in the pathogenesis.

The pathogenesis of the postpolio syndrome (PPS) remains unclear. In this study we looked for poliovirus (PV) persistence in the CSF of 20 patients with PPS, in a control group including 20 patients with unrelated neurological diseases, and in 7 patients with stable poliomyelitis sequelae. CSF samples and sera were examined using reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of PV or other enterovirus genomes; this assay allows the detection from as little as 1 fg viral RNA. Sequencing of amplified products from 5 patients was performed. PV genomic sequences were detected in the CSF of 11 of 20 patients with PPS and in none of the control group. Sequencing in the 5' untranslated region confirmed the presence of mutated PV sequences. These findings suggest that PPS is related to the persistence of PV in the central nervous system.

Evidence of presence of poliovirus genomic sequences in cerebrospinal fluid from patients with postpolio syndrome.

The postpolio syndrome (PPS) is characterized by new neuromuscular symptoms occurring 30 to 40 years after the acute episode of poliomyelitis paralysis. The presence of the poliovirus RNA genome in the cerebrospinal fluid from 10 patients with PPS and from 23 control patients was sought by using reverse transcription and a PCR specific for polioviruses and/or other enteroviruses. Poliovirus-specific genomic sequences in the 5' untranslated region and in the capsid region (VP1) were detected by reverse transcription PCR in 5 of 10 patients with PPS but in none of the control patients. Sequencing confirmed the presence of mutated poliovirus sequences. This finding suggests persistent viral infection in the central nervous system related to the presence of poliovirus genomes.

Sequence analysis of the ADE2 gene coding for phosphoribosylaminoimidazole carboxylase in Schwanniomyces occidentalis.

We have determined the nucleotide sequence of a 3.3 kb fragment containing the gene (ADE2) encoding phosphoribosylaminoimidazole carboxylase (AIRC) from the yeast Schwanniomyces occidentalis. Translation of a 1671 bp open reading frame predicts a protein of 557 amino acids which has significant homology to AIRC from Saccharomyces cerevisiae and Schizosaccharomyces pombe. The 5' untranslated region of the S. occidentalis gene contains a sequence corresponding to the consensus binding site of the S. cerevisiae transcription regulatory proteins GCN4, BAS1 and BAS2.

Isolation of auxotrophic mutants of the methylotrophic yeast Candida boidinii and determination of its ploidy.

Auxotrophic mutations in the methylotrophic yeast strain Candida boidinii 11Bh were induced by different mutagens and their combinations (nitrosoguanidine, UV light, HNO2+UV). Majority of the mutants obtained carried defects in histidine, arginine, proline and/or adenine biosynthetic pathways. His- mutants were distributed into four complementation groups using the protoplasts fusion technique. Ploidy determination of Candida boidinii 11Bh was performed by measuring its DNA content and by following its survival after chemical mutagens treatment. The DNA content of this strain was found to be similar to that of a Saccharomyces cerevisiae diploid strain. Also the kinetics of survival of Candida boidinii cells indicate that Candida boidinii 11Bh is a diploid.

Cloning of Candida boidinii DNA fragments promoting autonomous replication of plasmids in Saccharomyces cerevisiae.

Fragments of Candida boidinii chromosomal DNA were inserted into the integrative vector YIp-kanr and examined for the presence of sequences promoting autonomous replication of plasmids in Saccharomyces cerevisiae. Restriction maps of two plasmids, designated S6/4 and S6/5, originating from the same S. cerevisiae transformant, were constructed. Southern hybridization data confirmed that the plasmids carry sequences from the C. boidinii chromosome. Both plasmids transform S. cerevisiae strains at 4-5-fold higher frequency than cloning vectors based on the replication origin of the 2 microns plasmid. Mitotic stability of the constructed plasmids is similar to that of the 2 mu-based vector pNF2 in S. cerevisiae.