Influence of photosynthesis and chlorophyll synthesis on polypeptide accumulation in greening euglena

Two-dimensional gel electrophoresis resolves total cellular protein from Euglena gracilis klebs var bacillaris Cori into 640 polypeptides, 79 of which are induced by light exposure. The inhibition of chloroplast translation by streptomycin, the direct inhibition of photosynthesis as well as the indirect inhibition of chlorophyll synthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and the specific inhibition of photosynthesis but not chlorophyll synthesis by DCMU in the presence of 17 millimolar ethanol failed to inhibit the accumulation of 40 polypeptides. These polypeptides appear to be synthesized on cytoplasmic ribosomes and their accumulation is independent of the developmental status of the chloroplast. Streptomycin but not DCMU completely inhibited the accumulation of six polypeptides which are undetectable in mutants lacking chloroplast DNA suggesting that these polypeptides are translated on chloroplast ribosomes. The accumulation of seven polypeptides which are detectable in mutants lacking chloroplast DNA was also inhibited by streptomycin but not by DCMU suggesting that the accumulation of these polypeptides is dependent upon stabilization by a chloroplast translation product. The accumulation of 12 polypeptides was inhibited by streptomycin and by DCMU under conditions in which chlorophyll synthesis was inhibited, but not under conditions in which chlorophyll synthesis was unaffected by DCMU. The inhibition by DCMU of the accumulation of these polypeptides appears to be due to the inhibition of chlorophyll synthesis suggesting that they are components of pigment protein complexes. The accumulation of six polypeptides was inhibited under all conditions in which photosynthesis was inhibited suggesting that the accumulation of these polypeptides is dependent upon a product of photosynthesis.     

Identification of polypeptides encoded by the replication of resistance factor R100

Several polypeptides encoded by the resistance factor R100 were synthesized in a DNA-dependent protein synthesis system using a miniplasmid derived from R100 as a template. Nine polypeptides were detected. The locations of the genes for these polypeptides were investigated by using DNA restriction fragments as templates, and also by examining the effect of restriction endonuclease digestion of these templates on the synthesis of the polypeptides. The genes for seven of the polypeptides were identified or located by comparing the results with the known nucleotide sequence and restriction map of this region. Three of the polypeptides appeared to be encoded by the repA1, repA2 and repA3 genes, which are located in the region required for the replication of R100 and the expression of incompatibility. Four of the polypeptides were encoded by regions that are not required for the autonomous replication of R100 in Escherichia coli. One is the gene product of finO, which regulates the expression of the tra genes on R100.The miniplasmid used for these experiments carried one ISI sequence that has three potential genes. However, no polypeptide was detected that could be clearly demonstrated to be encoded by ISI.     

Complexity of roles and regulation of the PMK1-MAPK pathway in mycelium development,conidiation and appressorium formation in Magnaporthe oryzae

MST50, MST11, MST7, PMK1 and GAS1/GAS2 genes are the important components in the PMK1-MAPK signal transduction pathway in fungi. Mutants with deletion of these five genes of Magnaporthe oryzae, a pathogen of the rice blast, were constructed. A cDNA array containing 4108 unique genes of M. oryzae was developed and used to analyze the gene expression profiles of these mutants against the wild type to dissect the gene expression regulation networks responsible for conidiation and appressorium formation. With this approach, differentially regulated genes by these five components were identified. The vast majority of the regulated genes were mutant-specific, while only a small proportion were in common for all of the mutants, suggesting that each of these genes has its own regulon. Functional groups and expression patterns of the regulated genes showed that (1) gene members in the PMK1-MAPK pathway are associated with multiple signaling pathways; (2) the regulation of PMK1-mediated signaling pathways is very complex and likely involved in other signaling networks; (3) glucose metabolism and signals are required in mycelium development; and (4) appressorium formation likely shares the mechanisms responsible for sexual conjugation and meiosis, which is affected by carbohydrate metabolism.     

Calcium/calmodulin-dependent protein kinase II regulates Caenorhabditis elegans locomotion in concert with a G(o)/G(q) signaling network

Caenorhabditis elegans locomotion is a complex behavior generated by a defined set of motor neurons and interneurons. Genetic analysis shows that UNC-43, the C. elegans Ca(2+)/calmodulin protein kinase II (CaMKII), controls locomotion rate. Elevated UNC-43 activity, from a gain-of-function mutation, causes severely lethargic locomotion, presumably by inappropriate phosphorylation of targets. In a genetic screen for suppressors of this phenotype, we identified multiple alleles of four genes in a G(o)/G(q) G-protein signaling network, which has been shown to regulate synaptic activity via diacylglycerol. Mutations in goa-1, dgk-1, eat-16, or eat-11 strongly or completely suppressed unc-43(gf) lethargy, but affected other mutants with reduced locomotion only weakly. We conclude that CaMKII and G(o)/G(q) pathways act in concert to regulate synaptic activity, perhaps through a direct interaction between CaMKII and G(o).     

Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm² relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.     

Spontaneous mutations in the Big Blue® transgenic system are primarily mouse derived

The Big Blue® transgenic mouse mutation detection system provides a powerful approach for measuring spontaneous and induced mutations in vivo. The observed mutations may contain a fraction of ex vivo or prokaryotic mutational events. Indeed, a modified, selectable form of the Big Blue® assay seem to generate artifactual mutants under certain circumstances. Herein we review the evidence that circular mutants (i.e., the plaque circumference is at least 50% blue) collected in the standard Big Blue® assay are derived primarily from the mouse. The most direct evidence is the similarity in the types of mutations found in jackpot and nonjackpot mutations. In addition, about half of the spontaneous mutations in the lacI transgene are transitions and transversions at CpG dinucleotides, a mammalian-specific feature. The mutation pattern observed at lacI is consistent with AT mutation pressure operating in a GC rich DNA and approaches that reported for observed germline human factor IX mutations. Furthermore, the spontaneous mutation pattern of circular Big Blue® mutants differs significantly from that of an endogenous lacI gene in E. coli. Pinpoint mutants (a dot of blue color peripherally located in a wild type plaque), which a priori were not expected to be mouse-derived, have a mutation pattern consistent with the mutation pattern of an endogenous E. coli lacI gene. Analysis of induced mutagenesis studies reveals mutation frequencies and patterns for the Big Blue® circular mutants which are comparable to endogenous genes. In reconstruction experiments, blue plaques derived from a superinfection with wild type and mutant phage produced approximately 50% blue and 50% clear plaques on replating. This phenomenon has not been seen when plaques derived from mouse were replated in the Big Blue® assay. Collectively, the evidence strongly supports a murine origin for circular mutants recovered in the standard Big Blue® assay. Validation of current assays is an essential step in determining the frequency and pattern of spontaneous murine-specific mutations. Defining this benchmark will be helpful in evaluating the next generation of transgenic mutation detection systems.     

Mutations within an intron and its flanking sites: Patterns of novel polypeptides generated by mutants in one segment of the cob-box region of yeast mitochondrial DNA

Summary We have undertaken a systematic examination of the polypeptides accumulating in thirteen (out of 23) mutants in the intron cluster box7 and its flanking clusters box2 and box9 of the cob-box (cytochrome b) region of the mitochondrial genome of Saccharomyces cerevisiae. We have subjected these polypeptides to fingerprint analysis, both sequential and in parallel, with two proteases in order to disclose sequence homologies and differences between the different novel polypeptides themselves, and between them and the wild type product of the gene, i.e. apocytochrome b. One of our aims has been to establish the existence of possible correlations between the nature of the novel polypeptides and the fine structure genetic map of that segment of the mitochondrial genome.Our results show that all box7 mutants accumulate the following set of polypeptides not seen in wild type cells: a) a characteristic set of large polypeptides consisting of three species: p56, p42 and p35 or p34.5; b) a polypeptide p23; c) a much shorter fragment (of which the apparent molecular weight varies from 12.5 to 13, according to the mutation) with the exception of two mutants; d) in addition, the majority accumulate in varying amounts a polypeptide p30 closely related to but not identical with apocytochrome b.Moreover only two box7 mutants accumulate a polypeptide in the range of mobilities corresponding to 25–27 Kd (referred to as class p26) while such a polypeptide is seen in all box9 and box2 mutants examined with one exception in box2.Only one mutant in box2 resembles box7 mutants with respect to criterion a), and no box2 or box9 mutants resemble box7 mutants with respect to criterion c); criteria b) and d) appear to apply equally well to mutants in all three clusters.Fingerprint analysis, carried out with polypeptides p56, p42, p35, p34.5, p30, p26, p23, discloses that a) The polypeptides belonging to the same class of mobility exhibit very similar if not identical sequences in various cases. b) These polypeptide classes, except p56, p42 and p26, share considerable sequence homologies with wild type apocytochrome b, perhaps encompassing 50% or more of the wild type sequences. b) Polypeptides belonging to the classes p42 and p26 exhibit less extensive but nevertheless significant homologies with the wild type sequence. c) Sequences in polypeptides belonging to class p56 are virtually indistinguishable from ones in cytochrome oxidase subunit I.The inferences from these findings are 1) one gene can produce a multiplicity of polypeptide products that share a common sequence at the promoter-proximal (N-terminal) portion, but diverge beyond these regions of homology. 2) Both the multiplicity of products in single mutants, and the protein structure found, argue against the divergent segments to be due to frameshift terminations, and instead suggest that the novel products are consequences of mRNA processing defects (excision and/or ligation) at and near intron regions. 3) Mutations at edges and the center of an intron can generate similar polypeptide patterns, i.e. produce analoguous mRNA processing defects. 4) Mutations in exons, at their boundary with introns, can produce polypeptide patterns indistinguishable from those at the neighbouring intron; they diverge and eventually become typically exonlike in mutants localized at increasing distances from the boundary. 5) Taken together these findings argue that pre-mRNA processing extends beyond the boundaries of the intron proper and that certain exonsequences participate in excision and ligation. 6) Accumulated pre-mRNAs, resulting from defects in splicing can be translated. 7) Product p56 is formally analogous to p23, as a faulty but highly conserved partial product of the wild type protein, the former of Cox I (oxi3 gene), the latter of the cob-box gene proper. Therefore both genes may utilize identical RNA processing elements which are affected by box7 mutations. 8) A small amount of product similar to p56 may accumulate even in some wild types but not in others. This observations suggests that in certain nuclear backgrounds RNA processing may be more error-prone than in others.Publication No. XXXX from the Department of Chemistry, Indiana UniversitySupported by Research Grant GM 12228 from the National Institute of General Medical Sciences, National Institutes of Health; recipient of Research Career Award K06 05060 from the same Institute.Supported by Délégation Générale à la Recherche Scientifique et Technique grant n⁰ 78-70341