A Drosophila gene encoding a DEAD box RNA helicase can suppress loss of wee1/mik1 function in Schizosaccharomyces pombe

We describe a screen to isolate cDNAs encoding Drosophila mitosis inhibitors capable of suppressing the mitotic catastrophe phenotype resulting in Schizosaccharomyces pombe from the combination of the weel-50 mutation with either a deletion allele of mil1, or with overexpression of cdc25 ⁺. One plasmid was isolated which could suppress the temperature sensitive lethality of both these strains. The cDNA in this plasmid encodes a protein highly homologous to the DEAD-box family of ATP-dependent RNA helicases, rather than to protein kinases as might be expected. It is possible that the RNA helicase described here may regulate entry into mitosis by down regulating the expression of other genes whose activity may be rate-limiting for entry into mitosis.     

Theste13+ gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeastSchizosaccharomyces pombe

When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13 ⁺ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13 ⁺ gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13 ⁺ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13 ⁺ functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development.     

Response of root mRNA population to sulphate shortage in maize seedlings

Roots of ten-days-old seedlings obtained from a maize hybrid grown in complete or in sulphate-deprived medium were used to extract Poly(A)+RNA. The response to sulphate deprivation, which is known to increase the uptake capacity up to ten times, was manifested also by the expression of three mRNA species, as shown by the in vitro translation of the mRNA population. One hour after transfer to complete nutrient medium all three mRNAs were still present.Abbreviations BSA Bovine Serum Albumine - DTT 1,4-Dithio-DL-Threitol - EDTA Ethylenediaminetetraacetic-acid - MES 2-(N-Morpholino) ethane sulfonic acid - SDS Sodium Dodecyl Sulfate - TCA Trichloracetic acid - TRIS Tris(hydroxy-methyl)-aminomethane     

Presence of double‐stranded RNAs and virus‐like particles in the entomopathogenic fungus Metarhizium anisopliae

Nucleic acids from 41 strains of Metarhizium anisopliae, obtained from different parts of the world were extracted and examined by electrophoresis. Strong bands of double‐stranded RNA (dsRNA) were detected in two isolates from Brazil, V215 and V291, which had, respectively, 13 and 9 distinct bands ranging in size from ca. 0.75 to 3.5 kb. Icosahedral virus‐like particles (VLPs) (ca. 33 nm in diameter) were observed by transmission electron microscopy in extracts of these isolates. The VLPs and dsRNA were both absent from a clone of the isolate V291 which had been subcultured successively on solid medium. Bioassays against the aphid Myzus persicae showed no detectable difference in virulence between the clone of V291 which contained dsRNA and the clone that did not.     

Reduced lignin in transgenic plants containing a caffeic acidO-methyltransferase antisense gene

Lignin is a major structural polymer of secondarily thickended plant vascular tissue and fibres, imparting mechanical strength to stems and trunks and hydrophobicity to conducting vessels. Constitutive expression of a lucerne caffeic acid 3-O-methyltransferase antisense RNA in transgenic tobacco leads to a significant reduction in lignin content, particularly in the younger parts of the stems, without apparent alterations in lignin monomer composition. These observations open up the possibility of genetically manipulating plants with reduced lignin for improved processing and biomass digestibility.     

Recovery of transgenic trees after electroporation of poplar protoplasts

Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 10⁵ to 10⁴ protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 M paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 M phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated.Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.     

Comparison of phylogenetic relationships based on phospholipid fatty acid profiles and ribosomal RNA sequence similarities among dissimilatory sulfate-reducing bacteria

Abstract Twenty-five isolates of dissimilatory sulfate-reducing bacteria were clustered based on similarity analysis of their phospholipid ester-linked fatty acids (PLFA). Of these, 22 showed that phylogenetic relationships based on the sequence similarity of their 16S rRNA directly paralleled the PLFA relationships. Desulfobacter latus and Desulfobacter curvatus grouped with the other Desulfobacter spp. by 16S rRNA comparison but not with the PLFA analysis as they contained significantly more monoenoic PLFA than the others. Similarly, Desulfovibrio africanus clustered with the Desulfovibrio spp. by 16S rRNA but not with them when analyzed by PLFA patterns because of higher monoenoic PLFA content. Otherwise, clustering obtained with either analysis was essentially congruent. The relationships defined by PLFA patterns appeared robust to shifts in nutrients and terminal electron acceptors. Additional analyses utilizing the lipopolysaccharide-lipid A hydroxy fatty acid patterns appeared not to shift the relationships based on PLFA significantly except when completely absent, as in Gram-positive bacteria. Phylogenetic relationships between isolates defined by 16S rRNA sequence divergence represent a selection clearly different from the multi-enzyme activities responsible for the PLFA patterns. Determination of bacterial relationships based on different selective pressures for various cellular components provides more clues to evolutionary history leading to a more rational nomenclature.