Characterization of Eimeria tenella unsporulated oocyst-specific cDNA clones.

A cDNA library was constructed with poly(A)+ RNA from unsporulated oocysts of Eimeria tenella in pUC18. After screening, 4 cDNA clones that hybridized to RNA of unsporulated and sporulating oocysts but not to RNA of either sporulated oocysts or second generation merozoites were isolated and characterized. Each of the cDNA clones is unique. The loci for 2 of the clones are on E. tenella chromosome 7, the site of the third is located on chromosome 6 and the last clone hybridizes, for the most part, to chromosome 5 but also to other E. tenella chromosomes. The cognate RNAs for each of the cDNA clones show differential patterns of hybridization during oocyst sporulation with the levels of RNA being low at the start of sporulation (0 hr), increasing to peak levels between 6.5 and 23 hr after the onset of sporulation and, in each case, decreasing to low hybridization levels at 48 hr after initiation of sporulation. These results establish that specific mRNA levels are differentially regulated during sporulation.     

Selective gene expression during sporulation of Physarum polycephalum.

The two-dimensional gel electrophoresis of polypeptides synthesized in vitro from poly(A)+ RNA showed that mRNA populations change during sporulation of Physarum polycephalum. The differential hybridization of a cDNA library prepared from poly(A)+ RNA isolated from sporulating cells revealed that of 846 clones, 64 corresponded to sporulation-specific mRNAs. Further analysis demonstrated that these clones contained seven different sequences: three abundant sequences composing 3.2, 1.8, and 1.2% of the library and four other less abundant sequences. It is probable that all the major mRNAs specifically expressed in early stages of sporulation were identified. The most abundant mRNA from this group coded for a hydrophobic protein that contained a signal peptide. This protein is 47% similar to another Physarum protein, which was encoded by the most abundant plasmodium-specific mRNA. The plasmodial mRNA was degraded during sporulation and was replaced by the sporulation mRNA. These two proteins are thus encoded by members of a gene family whose expression is developmentally regulated.     

Role of the Y-Family DNA Polymerases YqjH and YqjW in Protecting Sporulating Bacillus subtilis Cells from DNA Damage

The role played by the Y-family DNA polymerases YqjH and YqjW in protecting sporulating cells of Bacillus subtilis from DNA damage was determined. The absence of either yqjH and/or yqjW not only reduced sporulation efficiency but also sensitized the sporulating cells to hydrogen peroxide, tert-butylhydroperoxide (t-BHP), mitomycin-C (M-C), and UV-C radiation. Moreover, these DNA-damaging agents increased the mutation frequency of wild-type sporulating cells to 4-azaleucine, but the production of mutants was YqjH- and YqjW-dependent. In conclusion, the results presented here indicate that YqjH/YqjW-dependent-translesion synthesis (TLS) operates in sporulating B. subtilis cells and contributes in processing spontaneous and artificially induced genetic damage, which is apparently required for an efficient sporulation process.     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

Two polypeptides associated with the ribonucleic acid polymerase core of Bacillus subtilis during sporulation.

The ribonucleic acid (RNA) polymerase from log-phase and sporulating cells of Bacillus subtilis was analyzed to determine whether any structural changes occurred during sporulation. The elution pattern of RNA polymerase from a deoxyribonucleic acid (DNA)-cellulose column revealed that sporulating cells at stages III and IV contained a new RNA polymerase fraction in addition to the vegetative holoenzyme (alpha2betabeta'sigma). Stage III cells contained the vegetative holoenzyme and a new enzyme with the composition alpha2betabeta'delta1; the molecular weight of delta1 was 28,000. Stage IV cells contained the vegetative holoenzyme, the delta1-containing enzyme, and another enzyme with the composition alpha2betabeta'delta2. The delta2 factor had a molecular weight of around 20,000. The delta-containing enzymes have a higher affinity for the DNA-cellulose column and a higher specific activity on various templates than vegetative holoenzyme. The simultaneous appearance of these enzymes with vegetative holoenzymes in sporulating cells is consistent with the data found previously with DNA-RNA hybridization studies, which showed that sporulating cells contained both vegetative and sporulation messenger RNAs.     

Sporulation genes in members of the low G+C Gram-type-positive phylogenetic branch (Firmicutes)

Endospore formation is a specific property found within bacteria belonging to the Gram-type-positive low G+C mol% branch (Firmicutes) of a phylogenetic tree based on 16S rRNA genes. Within the Gram-type-positive bacteria, endospore-formers and species without observed spore formation are widely intermingled. In the present study, a previously reported experimental method (PCR and Southern hybridization assays) and analysis of genome sequences from 52 bacteria and archaea representing sporulating, non-spore-forming, and asporogenic species were used to distinguish non-spore-forming (void of the majority of sporulation-specific genes) from asporogenic (contain the majority of sporulation-specific genes) bacteria. Several sporulating species lacked sequences similar to those of Bacillus subtilis sporulation genes. For some of the genes thought to be sporulation specific, sequences with weak similarity were identified in non-spore-forming bacteria outside of the Gram-type-positive phylogenetic branch and in archaea, rendering these genes unsuitable for the intended classification into sporulating, asporogenic, and non-spore-forming species. The obtained results raise questions regarding the evolution of sporulation among the Firmicutes.This paper is dedicated to Prof. H.G. Schlegel in honor of his 80th birthday.     

Characteristics of pulse-labeled ribonucleic acid in relation to medium pH and acetate concentration during meiosis of yeast

Summary Cellular impermeability associated with sporulating cells of Saccharomyces cerevisiae is caused by a rapid increase in the medium pH. Three factors have been identified as being important in regulating the rise in medium pH: 1) the cell density, 2) the potassium acetate concentration of the sporulation medium, and 3) and initial pH below 6.0. Sporulation conditions were established for strain 4579 which resulted in optimum uptake of ³H-adenine at T₇, a period when the cells would be normally impermeable. Pulse-labeled polysomal RNA was characterized at T₄ in naturally permeable cells of strain SK-1 and impermeable cells which required manipulation of the medium pH to facilitate uptake. Transfer ribonucleic acid (RNA), poly A-containing RNA and ribosomal RNA were synthesized in both cultures during the 20 min pulse. Furthermore, the rate of ribosomal RNA synthesis and processing into functional ribosomes approached the rate reported for vegetative cells. Initial sporulation conditions which caused a prolonged delay in the rise in medium pH adversely affected the kinetics of appearance and number of ascospores. The affect was shown to be on meiotic events since a reduction of sporulation was always accompanied by a reduction in the amount of intragenic recombination.     

Physiological suppression of Bacillus subtilis conditional sporulation phenotypes: RNA polymerase and ribosomal mutations

Summary The temperature-sensitive sporulation phenotype (Spo^ts) of Bacillus subtilis RNA polymerase, ribosomal and protein synthesis elongation factor G mutations can be corrected by supplementing the growth medium with carbohydrates such as ribose or glycerol, or with synthetic lipids such as Tween 40. The data suggest that these mutations affect a single common aspect of developmental cell function. It is proposed that these lesions prevent sporulation by disturbing the regulation of sporulating cell metabolic balance.     

The disease resistance response in pea is associated with increased levels of specific mRNAs

A cDNA library was constructed using poly(A)⁺RNA fromPisum sativum which had been treated for 8 h with the fungusFusarium solani f. sp.phaseoli. Two thousand four hundred recombinant colonies were screened by differential colony hybridization using³²P-labelled cDNAs prepared from RNA extracted from either noninoculated or inoculated pea tissue. cDNA clones were then selected, which showed greater hybridization with cDNA prepared from pea RNA 8 h post-inoculation than with a cDNA probe from 0 h. Seven distinct hybridization classes were chosen for further study. Northern blot analyses of total cellular RNAs inoculated for 16 h with eitherF. solani phaseoli or water demonstrated that each cDNA clone selected represents an mRNA species which increases substantially in abundance during infection. Results of³H-uridine pulse-labelling experiments suggested that enhanced synthesis is at least partially responsible for the accumulation of the fungus-inducible mRNAs which hybridized with the clones.     

Sulfur Compounds in RNA Fraction from Sporulating Cells of Bacillus subtilis

As we reported previously, in the sporulating cells of Bacillus subtilis about 20% of intracellular sulfur is found in the nucleic acid fraction. In the present work further characterization of sulfur compounds in this fraction was made using tracer technique and MAK column chromatography, and changes in pattern of the sulfur compounds during sporulation was observed.

It was found that the greater part of sulfur in the nucleic acid fraction was present as methionine and cysteine, which were associated with tRNA throughout the growth and sporulation. The amount of methionine as methionine tRNA was larger than that of cysteine as cysteine tRNA in the vegetative cells and vice versa in the sporulating cells.     

Molecular cloning of lupin leghemoglobin cDNA

Poly(A)⁺RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences specific for nodules were selected by differential colony hybridization using³²P-labeled cDNA synthesized either from nodule poly(A)⁺RNA or from poly(A)⁺RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)⁺RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules.     

Correlation among turnover of nucleic acids,ribonuclease activity and sporulation ability of Saccharomyces cerevisiae

The turnover of nucleic acids and changes in ribonuclease activity during sporulation of Saccharomyces cerevisiae were studied. In the sporulating strains, 37–58% of vegetatively synthesized RNA were degraded during the sporulation process. The degree of degradation of vegetative RNA was proportional to the sporulation ability. In the non-sporulating strains, the degradation of vegetative RNA was less than 28% in the sporulation medium. Accompanied by the degradation of vegetative RNA, a ribonuclease activity increased several times during sporulation. We have found a close relation among the sporulation rate, the degree of the degradation of vegetative RNA and the increase in ribonuclease activity in the sporulation medium, using cells of which sporulation ability was repressed by changing the age or carbon source in various degrees.     

The isolation of plasmids containing dna complementary to messenger rna for variant surface glycoproteins of Trypanosoma brucei

We have isolated poly(A)⁺ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)⁺ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs.The total translation products from the four poly(A)⁺ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins.We have made duplex DNA copies of these poly(A)⁺ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)⁺ RNA showed that 7–10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)⁺ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation.Poly(A)⁺ RNA from each of the variants only hybridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.