Molecular & general genetics : MGG最新文献

We previously used a genetic approach to identify a new class of Schizosaccharomyces pombe genes (chromosome loss when overexpressed; clo genes) that, when present in elevated dosage, cause the loss of an otherwise stable cen1 linear minichromosome at high rates. Here we report the identities of two clo genes; one encodes histone H3.3 and the other, designated clo2, encodes a novel protein with significant homology to fission yeast Swi6p, human and Drosophila HP1 heterochromatin proteins, and other chromo domain-containing proteins. Members of this group have been shown to localize to heterochromatic DNA, including centromeres, and to play roles in chromatin formation and organization. The S. pombe Clo2 protein localizes to centromere DNA in vivo, and overexpression of clo2 leads to a dramatic increase in the rate of mitotic loss of an artificial chromosome. Clo2p is not essential for mitotic growth, however, even in cells that also lack Swi6p. Thus, fission yeast appears to utilize multiple, functionally redundant, HP1-related proteins for heterochromatin-associated activities at centromeres and perhaps elsewhere in the genome.

The extent of conservation of RNA editing sites in the plastid genome of rice was determined by comparing the genomic sequence with that of the cDNA. The presence of a T in the cDNA predicted to be a C by the DNA sequence of the plastid genome, indicated C to U editing. In the 11 plastid transcripts of rice a total of 21 editing sites were found. In maize, a closely related grass species, 26 editing sites have been reported in 13 plastid transcripts. Most editing sites are conserved between the two species, although differences in RNA editing were found at eight sites. In seven cases the T was already encoded at the DNA level, eliminating the requirement for RNA editing. In one case (rpoB, codon 206) the RNA sequence was conserved between the two species, but the mRNA is still not edited in rice. It appears that, although evolutionarily conserved, RNA editing is essential only for a few plastid editing sites. Information about RNA editing in rice plastids will facilitate the design of plastid vectors with broad applicability in grass species.

Protein phosphatase 2A is ubiquitous among eukaryotes and exists as a family of holoenzymes in which the catalytic subunit. PP2Ac, binds a variety of regulatory subunits. Using the yeast Saccharomyces cerevisia, we have investigated the role of the phylogenetically invariant C-terminal leucine residue of PP2Ac, which, in mammalian cells, undergoes reversible methylation and modulates binding of the PR55/B subunit. In S. cerevisiae, the C-terminal Leu-377 residue of Pph22p (equivalent to human PP2Ac Leu-309) was dispensable for cell growth under optimum conditions and its removal, or substitution by alanine, did not inhibit PP2A activity in vitro. However, Leu-377 is required for binding of the yeast PR55/B subunit, Cdc55p, by Pph22p, though apparently not for the binding of Rts1p, the yeast PR61/B' subunit. Furthermore, mutation of this leucine enhanced the sensitivity of cells to microtubule destabilization, a defect characteristic of cdc55delta mutant cells, which are impaired for spindle checkpoint function. These results demonstrate that the regulation of PP2A, mediated by PR55/B binding to the highly conserved PP2Ac C-terminus, is critical for cell viability under conditions of microtubule damage and support a role for PP2A in exit from mitosis.

The glutamine synthetase II (GSII, encoded by glnII) activity detectable in crude extracts from Streptomyces coelicolor is low compared to the activity of glutamine synthetase I (GSI, encoded by glnA) and to that of GSII from S. viridochromogenes. We have identified and sequenced a 3.9-kb BglII-BamHI fragment carrying the glutamine synthetase II gene (glnII) from S. coelicolor. Besides glnII, this region contains four ORFs (orf1-orf4). While homologues of orf1 and orf2 were also found in the glnII region of the S. viridochromogenes chromosome, this was not the case for orf3 and orf4, which encode a putative hydrolase and a transcriptional regulator (Ptr) of the MarR family, respectively. High-resolution S1 nuclease mapping showed that the S. coelicolor glnII gene is expressed from two overlapping promoters. The first comprises a vegetative promoter sequence and the second contains sequence elements that are recognized by Esigma31. Similar promoter structures were found upstream of the S. viridochromogenes glnII gene. The involvement of ptr in glnII regulation was studied by gel retardation assays. Recombinant Ptr interacted with the upstream region of ptr, but not with the promoter region of glnII. A ptr gene replacement mutant (S. coelicolor IP) was also constructed. RT-PCR analysis of RNA from wild-type S. coelicolor and the IP mutant demonstrated that expression of orf3 depends on Ptr. Thus, the difference in gene organization between S. coelicolor and S. viridochromogenes is not responsible for the difference in GSII activity.

The SHL gene from Arabidopsis thaliana encodes a small nuclear protein that contains a BAH domain and a PHD finger. Both domains are found in numerous (putative) transcriptional regulators and chromatin-remodeling factors. Different sets of transgenic lines were established to analyze the physiological relevance of SHL. SHL expression driven by the CaMV 35S promoter results in reduced growth, early flowering, early senescence, and impaired flower and seed formation. Antisense inhibition of SHL expression gives rise to dwarfism and delayed development. In-frame N-terminal fusion of the SHL protein to beta-glucuronidase (GUS) directs GUS to the nucleus of stably transformed Arabidopsis plants. Thus, SHL encodes a novel putative regulator of gene expression, which directly or indirectly influences a broad range of developmental processes.

Aflatoxins (AF) are polyketide-derived mycotoxins that frequently contaminate food and feed crops, causing health risks to animals and humans. The fluP gene was cloned by screening an Aspergillus parasiticus genomic DNA library with a cDNA probe encoding part of a polyketide synthase (PKS), the 6-methylsalicylic acid synthase (MSAS) from Penicillium patulum. FluP was hypothesized to function as a PKS in AF biosynthesis. The predicted amino acid sequence of FluP demonstrated a high degree of identity to MSAS (55%), moderate identity to another fungal PKS protein encoded by wA from A. nidulans (22%) and low identity (<5%) to fungal fatty acid synthase (FAS) proteins. Disruption of fluP in A. parasiticus resulted in the loss of fluP transcript, a 3- to 4-fold reduction in hyphal growth rate, the appearance of a fluffy, cotton-like hyphal morphology, reduction or elimination of asexual spores and spore-bearing structures, and a twofold reduction in aflatoxin accumulation. Removal of selective pressure on fluP knockout transformants resulted in frequent reversion (10%) to the wild-type genotype and phenotype, establishing a direct link between gene disruption and the associated phenotype. The data suggest that fluP encodes a novel PKS associated with hyphal growth and cell development (sporulation), whose activity indirectly influences aflatoxin accumulation in A. parasiticus.

Of the actin-related proteins, Arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. Dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. The role of Arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. Using the filamentous fungus Neurospora crassa, we have identified genes encoding subunits of cytoplasmic dynein and dynactin. In this study, we describe a genetic screen for N. crassa Arp1 (ro-4) mutants that are defective for dynactin function. We report that the ro-4(E8) mutant is unusual in that it shows alterations in the localization of cytoplasmic dynein and dynactin and in microtubule organization. In the mutant, dynein/dynactin complexes co-localize with bundled microtubules at hyphal tips. Given that dynein transports membranous cargo from hyphal tips to distal regions, the cytoplasmic dynein and dynactin complexes that accumulate along microtubule tracts at hyphal tips in the ro-4(E8) mutant may have either reduced motor activity or be delayed for activation of motor activity following cargo binding.

A striking characteristic of the centromeric heterochromatin of Drosophila melanogaster is that each chromosome carries different satellite DNA sequences. Here we show that while the major component of the 1.688 satellite DNA family expands across the centromere of the X chromosome the rest of the minor variants are located at pericentromeric positions in the large autosomes. Immunostaining of prometaphase chromosomes with the kinetocore-specific anti-BUB1 antibody reveals the transient presence of this centromeric protein in all the regions containing the 1.688 satellite.

We isolated a Neurospora crassa cDNA that encodes a Rad52 homologue (ncRAD52) by PCR, using degenerate primers. RFLP mapping demonstrated that the cloned gene is located close to the ro-4 locus on the right arm of linkage group V (LGVR). In a second experiment, we used sib selection to identify a cosmid clone containing the mus-11 gene in a N. crassa genomic library. Fine-scale mapping of the mus-11 mutant showed the gene order on LGVR near ro-4 to be: ad-7 - (9.5 mu) - pab-2 (7.8 mu) - mus-11 - (3.7 mu) - inv. The nucleotide sequence of the mus-11 gene matched that of the ncRAD52 cDNA. Thus, the mus-11 gene encodes the Rad52 homologue. The deduced amino acid sequence of the MUS11 protein shows 32.0% and 27.5% overall identity to the Schizosaccharomyces pombe Rad22 protein and the human hRad52 protein, respectively, and a higher level of identity (55-66%) within the conserved N-terminal region (141 residues). The MUS11 protein shows homology to Rad52 from budding yeast only within the N-terminal region (53.2% identity over 141 amino acids) which is conserved among Rad52 homologues. Yeast two-hybrid analysis reveals that the MUS11 protein binds to both the MEI-3 protein, a Rad51 homologue, and to itself in vivo. An ncRAD52 mutant obtained by the RIPping procedure showed the same sensitivity as the original mus-11 mutant to the following mutagens and chemicals: UV light, 4NQO (4-nitroquinoline 1-oxide), MMS (methyl methanesulfonate), EMS (ethyl methanesulfonate), MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), TBHP (tert-butyl hydroperoxide), HU (hydroxyurea) and histidine. Unlike the RAD52 transcript in Saccharomyces cerevisiae, the mus-11 transcript could not be detected in mycelium under normal growth conditions, but expression of the gene was induced by UV irradiation or treatment with MMS.

Transgenic rice cell lines transformed with a heterologous cDNA derived from the arginine decarboxylase gene of oat, in an antisense orientation, exhibited significant (P < 0.05) down-regulation of the activity of the endogenous arginine and ornithine decarboxylases, compared to wild type and controls transformed only with the selectable marker (hpt). Changes in enzyme activity were reflected in a marked decrease in the level of putrescine (P < 0.001) and spermidine (P < 0.01) but not spermine (P > 0.05) in the majority of cell lines analyzed. In agreement with previous results, we confirmed that cell lines with low levels of polyamines exhibited normal morphogenic responses. In vegetative tissue at the whole plant level no significant variation (P > 0.05) in polyamine levels was observed. However, we measured significant reductions (P < 0.001) in putrescine levels in seeds derived from three out of five plants analyzed in detail. Thus, simultaneous reduction of the activity of the two alternative enzymes in the early steps of the polyamine pathway results in significant reduction in end-product accumulation in the seeds of transgenic plants.