The Bacillus subtilis small cytoplasmic RNA gene and 'dnaX' map near the chromosomal replication origin.

Summary TheBacillus subtilis small cytoplasmic RNA (scRNA) has an important, although not yet defined function in protein biosynthesis. Here we describe the mapping of the single copy scRNA gene and the flanking homolog todnaZX ofEscherichia coli, termed dnaX. The scRNA gene region of aB. subtilis wild-type strain was marked with acat gene and mapped by scoring chromosomal co-transformation rates of various mutant strains to chloramphenicol resistance and loss of the mutant phenotypes, respectively. This analysis, together with anEcoRI map comparison, places the scRNA gene anddnaX in the vicinity ofrecM near the replication origin region ofB. subtilis.     

σs-dependent overexpression offtsZ in anEscherichia coli K-12 rpoB mutant that is resistant to the division inhibitors DicB and DicF RNA

TheEscherichia coli genesdicF anddicB encode division inhibitors, which prevent the synthesis and activity, respectively, of the essential division protein FtsZ. A mutation at the C-terminal end of the RNA polymerase subunit renders cells resistant to both inhibitors. In the mutant strain the level of theftsZ gene product is higher than in the wild type. Disruption ofrpoS, which encodes the stationary phase sigma factor ^S, lowers FtsZ protein levels in the mutant, and partially restores sensitivity to the inhibitors.     

Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity

The pheromone signal in the yeastSaccharomyces cerevisiae is transmitted by the and subunits of the mating response G-protein. TheSTE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G mutation. The same genetic screen identifiedBEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designatedMDG1 (multicopy suppressor ofdefectiveG-protein). TheMDG1 gene was independently isolated in a search for multicopy suppressors of abem1 mutation. TheMDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins. A fusion of Mdg1p with the green fluorescent protein fromAequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein. Deletion ofMDG1 causes sterility in cells in which the wild-type G has been replaced by partly defective G derivatives but does not cause any other obvious phenotypes. The mating defect of cells deleted forSTE20 is partially suppressed by multiple copies ofBEM1 andCDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity. Elevated levels ofSTE20 andBEM1 are capable of suppressing a temperature-sensitive mutation inCDC42. This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating.     

Recombination protein A gene,recA, fromSpirulina platensis IAM-M135

We report the cloning and sequencing of therecA gene fromSpirulina platensis. A genomic library ofSpirulina was constructed in pUC19 and screened by PCR using oligonucleotides corresponding to the conserved amino acid sequences ofAnabaena variabilis andSynechococcus RecA proteins. TheSpirulina recA gene consists of an open reading frame (ORF) of 1095 nucleotides encoding a protein (365 residues) which shares an identity of 79%, 70% and 57% with the RecA proteins ofAnabaena variabilis, Synechococcus andEscherichia coli respectively. TherecA gene is located close to one end of the clonedBglII fragment and has only 53 bp of 5 nucleotides. The isolation of this gene has implications for the development of gene transfer system(s) forSpirulina.     

Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of<Emphasis Type="Italic"> PHGPX</Emphasis> and<Emphasis Type="Italic"> URE2</Emphasis> genes in aluminum resistance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 , phgpx2 , and phgpx3), were tested for their sensitivity to Al. The triple mutant, phgpx1 /2/3, was more sensitive to Al (55% reduction in growth at 300 M Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3 to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3 and phgpx1/2/3 mutants were sensitive to oxidative stress (exposure to H₂O₂ or diamide). Lipid peroxidation was also increased in the phgpx1/2/3 mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1 and gtt2), glutathione biosynthesis (gsh1 and gsh2), glutathione reductase (glr1) and a glutathione transporter (opt1) did not show hypersensitivity to Al relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to Al. The hypersensitivity of the ure2 mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 M Al. Intracellular oxidation levels in the ure2 mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H₂O₂) increase compared to BY4741; however, the ure2 mutant showed no change in lipid peroxidation compared to the control. The phgpx1/2/3 and ure2 mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.Communicated by D.Y. Thomas     

Identification of hypomorphic and null alleles of<Emphasis Type="Italic"> swallow</Emphasis> via molecular and phenotypic analyses

The swallow gene of Drosophila is required for the localization of two messenger RNAs, bicoid and hu-li tai shao, to the anterior pole of oocytes during the later stages of oogenesis. In addition, swallow appears to play a role in early embryogenesis, as swallow mutant embryos have defects in early nuclear cleavage and migration. In an effort to identify regions of the Swallow protein that are essential for function, we have initiated a molecular characterization of seven existing alleles of swallow. All seven alleles have been sequenced, and comparison to wild-type swallow indicates that the seven alleles include single amino acid substitutions that identify critical residues, as well as lesions that result in truncated proteins. Western blots using affinity-purified antibodies agree well with the DNA sequence data, and identify a probable null protein. In order to determine the extent to which each allele affects swallow function, females homozygous or hemizygous for each allele were tested for the range and abundance of (1) RNA localization defects, and (2) embryonic cuticular defects. Swallow alleles can be grouped into two categories: those that retain partial function, and those indistinguishable from the putative null allele. Some swallow mutant alleles partially rescue the dominant female sterility of mutations in the atypical 67C -tubulin gene, supporting other studies that suggest a link between RNA localization and the microtubule cytoskeleton.Edited by C. Desplan     

Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 M phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K _m and k _cat values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K _m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K _m and k _cat values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.     

Characterization of a dwarf gene in<Emphasis Type="Italic"> Brassica rapa</Emphasis>, including the identification of a candidate gene

Dwarf genes have been valuable for improving harvestable yield of several crop plants and may be useful in oilseed Brassica. We evaluated a dwarf gene, dwf2, from Brassica rapa in order to determine its phenotypic effects and genetic characteristics. The dwf2 mutant was insensitive to exogenous GA₃ for both plant height and flowering time, suggesting that it is not a mutation in the gibberellin biosynthesis pathway. The dwarf phenotype was controlled by a semidominant allele at a single locus. Near-isogenic lines that were homozygous or heterozygous for dwf2 had 47.4% or 30.0% reduction in plant height, respectively, compared to the tall wild-type line, and the reduction was due to reduced internode length and number of nodes. The dwf2 homozygous and heterozygous lines had the same or significantly higher numbers of primary branches than the wild-type line, but did not differ in flowering time. The DWF2 gene was mapped to the bottom of linkage group R6, in a region having homology to the top of Arabidopsis thaliana chromosome 2. The map position of DWF2 in comparison to markers in A. thaliana suggests it is a homolog of RGA (repressor of ga1-3), which is a homolog of the wheat Green Revolution gene. This dwarf gene could be used to gain more insight on the gibberellin pathway and to reduce lodging problems in hybrid oilseed Brassica cultivars.Communicated by J.S. Heslop-Harrison     

Glucose-6-phosphate isomerase from the hyperthermophilic archaeon<Emphasis Type="Italic"> Methanococcus jannaschii:</Emphasis> characterization of the first archaeal member of the phosphoglucose isomerase superfamily

ORF MJ1605, previously annotated as pgi and coding for the putative glucose-6-phosphate isomerase (phosphoglucose isomerase, PGI) of the hyperthermophilic archaeon Methanococcus jannaschii, was cloned and functionally expressed in Escherichia coli. The purified 80-kDa protein consisted of a single subunit of 45 kDa, indicating a homodimeric (₂) structure. The K _m values for fructose 6-phosphate and glucose 6-phosphate were 0.04 mM and 1 mM, the corresponding V _max values were 20 U/mg and 9 U/mg, respectively (at 50 °C). The enzyme had a temperature optimum at 89 °C and showed significant thermostability up to 95 °C. The enzyme was inhibited by 6-phosphogluconate and erythrose-4-phosphate. RT-PCR experiments demonstrated in vivo expression of ORF MJ1618 during lithoautotrophic growth of M. jannaschii on H₂/CO₂. Phylogenetic analyses indicated that M. jannaschii PGI was obtained from bacteria, presumably from the hyperthermophile Thermotoga maritima.