A 9.6 kb intervening sequence in D. virilis rDNA, and sequence homology in rDNA interruptions of diverse species of Drosophila and other diptera.

A large proportion of the 28S ribosomal RNA genes in Drosophila virilis are interrupted by a DNA sequence 9.6 kilobase pairs long. As regards both its presence and its position in the 28S gene (about two thirds of the way in), the D. virilis rDNA intervening sequence is similar to that found in D. melanogaster rDNA, but lengths differ markedly between the two species. Degrees of nucleotide sequence homology have been detected bewteen rDNA interruptions of the two species. This homology extends to putative rDNA intervening sequences in diverse higher diptera (other Drosophila species, the house fly and the flesh fly), but hybridization of cloned D. melanogaster and D. virilis rDNA interruption segments to DNA of several lower diptera has been negative. As is the case with melanogaster rDNA interruptions, segments of the virilis rDNA intervening sequence hybridize with non-rDNA components of the virilis genome, and interspecific homology may involve these non-rDNA sequences as well as rDNA interruptions. There is, however, evidence from buoyant density fractionation of DNA that the distributions of interruption-related sequences are distinct in D. melanogaster and D. virilis genomes. Moreover, thermal denaturation studies have indicated differing extents of homology between hybridizable sequences in D. virilis DNA and different segments of the D. melanogaster rDNA intervening sequence. We infer from our studies that rDNA intervening sequences are prevalent among higher diptera; that in the course of the evolution of these organisms, elements of the intervening sequences have been moderately to highly conserved; and that this conservation extends in at least two distantly related species of Drosophila to similar sequences found elsewhere in the genomes.     

A novel arrangement of the 18S and 28S sequences in a repeating unit of Drosophila melanogaster rDNA.

The sequences corresponding to the 18S and 28S rRNAs have been mapped within a cloned 17 kilobase (kb) fragment formed by Eco R1 cleavage of Drosophila melanogaster rDNA. This fragment, Dm103, represents the longer of two major types of repeating units that are present in the rDNA of this fly, and was cloned as a hybrid plasmid, pDm103, consisting of Dm103 inserted at the Eco R1 site of the pSC101 vector (Glover et al., 1975). Mapping of the 18S and 28S rDNA in Dm103 was accomplished by quantitative determination of the amount of these rDNAs in each member of an ordered set of restriction fragments obtained by Hind III and Eco R1 ccleavage of pDm103. The amounts of 18S and 28S rDNAs were determined by hybridization of the rRNAs to fragments that were purified by cloning, and an unambiguous order of the fragments within pDm103 was established by heteroduplex mapping and from the stoichiometry of the fragment lengths. The resulting map revealed that the 4 kb of 28S rDNA within the long repeating unit represented by Dm103 is divided into two blocks that are separated by 5.4 kb of DNA of unknown function. It is this unusual arrangement of the 28S rDNA that distinguishes the long repeating units (17 kb) from the short units (11.5) kb), whose 4 kb of 28S rDna is confined to a single block, as is shown in the accompanying paper (White and Hogness, 1977). The remainder of the DNA in this long unit appears to be typically arranged, with the 2 kb of 18S rDNA confined to a single block that is separated by about 1 kb from the closest block of 28S rDNA.     

Paenibacillus macerans possesses two types of 16S rDNA copies in a genome with a length difference of twelve base pairs

Two Paenibacillus macerans strains, JCM 2500T and MCRI 12, exhibited two types of 16S rDNA copies in their genomes, accompanied by a length difference of 12 bp at positions 203 to 214 (Escherichia coli numbering). The long-type sequences were newly identified for P. macerans 16S rDNA, and the copy numbers were different between the two strains. Both types of 16S rRNA were expressed in each strain, and it was predicted that the polymorphism at this position is located in helix H10, based on a comparison with the E. coli 16S rRNA secondary structure model.     

The heat-shock reaction is disturbed in a Drosophila virilis strain incapable of a neurohormonal stress reaction

Cellular stress response was investigated in two lines of D. virilis: wild type and line with disturbed neurohormonal stress-reaction. Analysis of proteins, synthesized in salivary glands of larvae of both lines under heat stress, revealed malfunction in heat shock reaction of mutant specimen. This malfunction expresses in decreased level of heat shock protein synthesis. Analysis of electrophoretic spectra of proteins from homogenates of imagoes of both lines maintained under normal conditions and those exposed to heat (38 degrees C, 60 min.) revealed correlation between protein spectrum and physiological state of the organism. Interlinear differences by proteins spectra in normal condition, controlled by a single gene (or by block of closely linked genes), were found. The question if there is a common genetic control for the neurohormonal stress-reaction and cellular stress response is discussed.     

The family Pleosporaceae: intergeneric relationships and phylogenetic perspectives based on sequence analyses of partial 28S rDNA

The Pleosporaceae is an important loculoascomycete family. There has been disagreement, however, regarding the taxonomic placement of many genera within this family. This study investigates phylogenetic relationships among the genera Cochliobolus, Kirschsteiniothelia, Leptosphaerulina, Macroventuria, Pleospora, Pyrenophora, and Wettsteinina. Partial 28S rDNA sequences from taxa within these genera were analyzed with maximum parsimony, likelihood and Bayesian methods. Cochliobolus can be segregated broadly into two groups as previously proposed. Pleospora is polyphyletic in its current sense. Taxa with Stemphylium anamorphs are closely related to Cochliobolus and fit within the Pleosporaceae, whereas the affinities of Pleospora herbarum and P. ambigua are still ambiguous. Pyrenophora constitutes a monophyletic group within the Pleosporaceae, whereas Leptosphaerulina and Macroventuria appear to share phylogenetic affinities with the Leptosphaeriaceae and Phaeosphaeriaceae. Phylogenies indicate that Wettsteinina should be excluded from the Pleosporaceae. Similar findings are reported for Kirschsteiniothelia, which is probably polyphyletic. Anamorphic characters appear to be significant (especially in Cochliobolus) while ascospore morphologies, such as shape and color and substrate occurrence are poor indicators of phylogenetic relationships among these loculoascomycetes.     

The dopamine system stress reaction in adults of Drosophila virilis is controlled by genes of Chromosome 6

A linkage group of the gene responsible for changes of DA titer under stress in adults of D. virilis was determined. Line 160 of D. virilis, all autosomes of which bear visible recessive mutations, was used as an analyzer. Flies of lines 160 and 147 were shown to differ in DA content under normal conditions and in the way DA metabolic system reacts to stress. Among the offspring of the analyzing cross, groups of flies were found with one testable autosome from line 147 and all other chromosomes from line 160. Results of the DA titer measurements in flies of these groups under stress conditions have proved that the gene in question is linked to chromosome 6.     

Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element

The pigmentation (Pgm+) phenotype of Yersinia pestis encompasses a variety of different physiological traits, all of which are missing in Pgm- mutants. We have previously shown that loss of the Pgm+ phenotype is accompanied by the spontaneous deletion of at least 45 kb of chromosomal DNA, referred to as the pgm locus. Using chromosomal walking, we have now mapped the full extent of the pgm locus in Y. pestis strain KIM6+. Our results indicate that the locus spans 102 kb of DNA which is absent in the spontaneous Pgm- mutant, KIM6. Yersinia pseudotuberculosis PB1/0 contains sequences homologous to the entire pgm locus while only part of this region hybridized to Yersinia enterocolitica WA-LOX DNA. Restriction enzyme mapping and hybridization studies revealed the presence of a repetitive element at both ends of the pgm locus and in multiple copies elsewhere in the Y. pestis genome. This element may be responsible for generating the deletion.     

The I-CeuI endonuclease recognizes a sequence of 19 base pairs and preferentially cleaves the coding strand of the Chlamydomonas moewusii chloroplast large subunit rRNA gene.

The I-CeuI endonuclease is a member of the growing family of homing endonucleases that catalyse mobility of group I introns by making a double-strand break at the homing site of these introns in cognate intronless alleles during genetic crosses. In a previous study, we have shown that a short DNA fragment of 26 bp, encompassing the homing site of the fifth intron in the Chlamydomonas eugametos chloroplast large subunit rRNA gene (Ce LSU.5), was sufficient for I-CeuI recognition and cleavage. Here, we report the recognition sequence of the I-CeuI endonuclease, as determined by random mutagenesis of nucleotide positions adjacent to the I-CeuI cleavage site. Single-base substitutions that completely abolish endonuclease activity delimit a 15-bp sequence whereas those that reduce the cleavage rate define a 19-bp sequence that extends from position -7 to position +12 with respect to the Ce LSU.5 intron insertion site. As the other homing endonucleases that have been studied so far, the I-CeuI endonuclease recognizes a non-symmetric degenerate sequence. The top strand of the recognition sequence is preferred for I-CeuI cleavage and the bottom strand most likely determines the rate of double-strand breaks.     

The integrated forms of the S1 and S2 DNA elements of maize male sterile mitochondrial DNA are flanked by a large repeated sequence.

The mitochondrial DNA of maize was cloned using the cosmid, Homer I. Recombinants carrying sequences homologous to the S1 and S2 DNA elements of male sterile maize have been analysed. Restriction endonuclease maps for Sac II, Sma I and Bam HI have been constructed. The S1 and S2 sequences are single copy sequences occurring at unique sites; each is flanked by a 26 kb repeated sequence. The repeated sequence has been shown not to contain the mitochondrial ribosomal RNA genes.     

Identification of causative pathogens in mouse eyes with bacterial keratitis by sequence analysis of 16S rDNA libraries

The clone library method using PCR amplification of the 16S ribosomal RNA (rRNA) gene was used to identify pathogens from corneal scrapings of C57BL/6-corneal opacity (B6-Co) mice with bacterial keratitis. All 10 samples from the eyes with bacterial keratitis showed positive PCR results. All 10 samples from the normal cornea showed negative PCR results. In all 10 PCR-positive samples, the predominant and second most predominant species accounted for 20.9 to 40.6% and 14.7 to 26.1%, respectively, of each clone library. The predominant species were Staphylococcus lentus, Pseudomonas aeruginosa, and Staphylococcus epidermidis. The microbiota analysis detected a diverse group of microbiota in the eyes of B6-Co mice with bacterial keratitis and showed that the causative pathogens could be determined based on percentages of bacterial species in the clone libraries. The bacterial species detected in this study were mostly in accordance with results of studies on clinical bacterial keratitis in human eyes. Based on the results of our previous studies and this study, the B6-Co mouse should be considered a favorable model for studying bacterial keratitis.     

Construction of a double-stranded deoxyribonucleotide sequence of 45 base pairs designed to code for S-peptide 2-14 of bovine ribonuclease A.

An artificial DNA duplex, each strand consisting of 45 monomers, is constructed from chemically synthesized deoxyriboöligonucleotides. The resulting bihelical polymer may code for a modified S-peptide of Ribonuclease A. This is the first synthetic duplex designed to code for a eukaryotic message.     

The 5S ribosomal genes in the Drosophila melanogaster species subgroup. Nucleotide sequence of a 5S unit from Drosophila simulans and Drosophila teissieri

The 5S genes of the eight species of the D. melanogaster subgroup have been mapped. The spacers, in contrast with coding regions, differ markedly between most species. One 5S gene unit has been sequenced for both D. simulans and D. teissieri. The mature 5S RNA region in these two species is identical to the corresponding region of D. melanogaster. Only 5 nucleotide variations occur between the D. melanogaster and D. simulans 5S gene spacers. The spacer in D. teissieri is very different. Only two segments, located one at each side of the coding region, are clearly homologous to corresponding sequences of D. melanogaster and D. simulans.     

The direct repeat sequence upstream of Bacillus chitinase genes is cis-acting elements that negatively regulate heterologous expression in E. coli

To explore the influence of the direct repeat sequence (DRS) in Bacillus chitinase genes on heterogonous expression in Escherichia coli, we cloned and sequenced the entire open reading frame (ORF) and upstream sequences of the chitinase B (chiB) and chitinase MY75 (chiMY75) from Bacillus thuringiensis and Bacillus licheniformis. A pair of 8-bp DRS was found upstream of each chi gene. Chi ORFs with a series of truncated DRS were cloned and transformed into E. coli XL-Blue. The activity of the transformants without the DRS were significantly higher in chitinase assays than transformants containing the DRS. SDS-PAGE showed that part and full deletion of the DRS increased chi gene expression by approximately 1.7 and 3.8-fold, respectively. Northern blotting revealed deletion of the DRS regions increased chiB and chiMY75 mRNA expression. Specific binding of DNA-binding factors in the E. coli cell lyaste was observed to both the chiB and chiMY75 promoter regions and DRS elements. This is the first investigation to demonstrate that heterologous expression of Bacillus chi genes in E. coli is negatively regulated by their upstream DRS regions, which act as cis-acting elements.     

The intervening sequence of the ribosomal RNA precursor is converted to a circular RNA in isolated nuclei of Tetrahymena

The Tetrahymena thermophila ribosomal RNA gene contains an intervening sequence (IVS), which is transcribed as part of the precursor RNA and subsequently removed by splicing. We have found previously that the IVS is excised as a 0.4 kb RNA in isolated nuclei. We now report the finding of a novel RNA molecule, which is an electrophoretic variant (EV) of this 0.4 kb IVS RNA. The EV was identified as a form of the IVS RNA by Southern hybridization, RNA fingerprinting and R-loop mapping. A pulse-chase experiment established that in vitro the excised IVS RNA is converted to the EV by a post-splicing event. This conversion is enhanced at 39 degrees C compared to 30 degrees C and is irreversible under our experimental conditions. The EV of the IVS is a circular RNA. This structure was first suggested by its anomalous electrophoretic mobility on denaturing compared to nondenaturing gels. When the EV was prepared for electron microscopy under totally denaturing conditions, 0.4 kb circular molecules were observed. Furthermore, we have converted the circular form to a linear form by limited T1 RNAase digestion. The circular RNA survived treatment with DNAase, protease, glyoxal and various denaturants, which suggests that it is a covalently closed RNA circle.     

Localization of the Huntington's disease gene to a small segment of chromosome 4 flanked by D4S10 and the telomere

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of late onset, characterized by progressive motor disturbance, psychological manifestations, and intellectual deterioration. The HD gene has been genetically mapped by linkage to the DNA marker D4S10, but the exact physical location of the HD defect has remained uncertain. To delineate critical recombination events revealing the physical position of the HD gene, we have identified restriction fragment length polymorphisms for two recently mapped chromosome 4 loci, RAF2 and D4S62, and determined the pattern of segregation of these markers in both reference and HD pedigrees. Multipoint linkage analysis of the new markers with D4S10 and HD establishes that the HD gene is located in a very small physical region at the tip of the chromosome, bordered by D4S10 and the telomere. A crossover within the D4S10 locus orients this segment on the chromosome, providing the necessary information for efficient application of directional cloning strategies for progressing toward, and eventually isolating, the HD gene.