Parastremmatic dwarfism

The number of ribosomal RNA cistrons has been measured in the total DNA extracted from L2 juvenile and adult stages of the free-living nematode Panagrellus silusiae. Saturation hybridization studies with homologous rRNA indicate that both stages have about 275 ribosomal genes per haploid equivalent. Using homologous 125I-labelled rRNA for in situ hybridization, the mean number of silver grains per DNA content for oocyte, hypodermis and gut nuclei was similar. The mean DNA contents of maturing oocyte, hypodermis and gut nuclei are about 20C, 2C, and 10C respectively. We conclude that rDNA amplication alone is insufficient to account for the variation in DNA content of oocytes and that postembryonic development in this eutelic organism occurs without a significant differential increase in the number of ribosomal cistrons per worm.     

Bonded amalgam sealants: two-year clinical results

The results of molecular investigations of blood mononuclears from 120 clean-up workers after 7-9 years of Chernobyl accident with the total exposure radiation doses ranging from 5 to 76 cGr are presented. Structural polymorphism of the leukemia associated bcr and ribosomal RNA (rRNA) genes were studied using Southern blot hybridization. Allelic polymorphism of bcr gene with characteristic for leukemia allele distribution was detected in 16.6%. Rearrangements of rRNA genes were observed in 13% of Chernobyl accident clean-up workers.     

Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [gamma-32P]ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.     

The evaluation by indirect immunofluorescence of the incidence of respiratory syncytial virus in acute respiratory disorders in infants]

Diversity based on ribosomal RNA gene-restriction endonuclease digest patterns was detected amongst forty-seven strains of Campylobacter made up of 38 strains of Campylobacter jejuni and 9 strains of Campylobacter coli. Restriction digests of chromosomal DNA prepared by treating with Hae III were probed with an oligonucleotide specific for Campylobacter 16S ribosomal RNA genes. Seventeen distinct hybridization patterns, each indicating the presence of 2-4 copies of the 16S rRNA gene are encoded in Campylobacter DNA. Differences in fragment patterns were observed not only between members of two species, but also between individual strains of the same species. Ribopattern fragments of 8.71, 7.56, 2.81 and 1.0 kb were characteristic of the majority of C. jejuni, whereas 7.59 and 4.68 kb fragments were commonly present in C. coli. In conclusion, Hae III ribotyping was even more discriminatory than the Penner serotyping of C. jejuni and C. coli, as strains of the same serotype were distinguished.     

In vivo and in vitro detection of canine distemper virus nucleoprotein gene with digoxigenin-labelled RNA, double-stranded DNA probes and oligonucleotides by in situ hybridization

A single-stranded RNA, two double-stranded (ds) DNA probes and 10 oligonucleotides labelled with digoxigenin were comparatively evaluated for their usefulness to detect canine distemper virus (CDV) nucleoprotein RNA in in vitro infected Vero cells and in tissues of dogs with spontaneous CDV infection by in situ hybridization (ISH). In addition, results were compared to CDV nucleoprotein antigen distribution as demonstrated by immunohistochemistry. The RNA probe was derived from the virulent A75/17 strain, the DNA and oligonucleotide probes from the avirulent Onderstepoort strain of CDV. The two DNA probes were 287 and 126 base pairs long. For ISH, various factors including fixatives, proteolytic digestion, probe concentration, hybridization conditions and detection systems were compared. All probes were suitable for demonstration of CDV RNA in in vitro infected cells, regardless of the CDV strain employed. In vivo CDV nucleic acid was detected by RNA and the dsDNA probes. However, the probes varied substantially with respect to sensitivity and specificity. The CDV RNA probe was far superior in sensitivity when compared to the DNA probes. Furthermore, the shorter DNA probe displayed a higher sensitivity, indicating that length of the probe is an important parameter when selecting probes. Oligonucleotides displayed only rarely a positive signal and caused frequently hybridization signals in the nucleus, which where considered not specific for CDV. Summarized, the present study reveals that RNA probes are currently the most sensitive tool for detection of CDV RNA in tissues.     

Organization of ribosomal RNA genes in Borrelia burgdorferi sensu lato isolated from Ixodes ovatus in Japan

Borrelia burgdorferi sensu lato was obtained from adult ixodid ticks, Ixodes ovatus, collected in Nagano, Japan, and was named NT112. The genomic DNA was digested with enzymes, electrophoresed, blotted and hybridized with rRNA gene probes obtained from B. burgdorferi sensu stricto B31. The results showed that the borrelial chromosome contains a single rrs (16S rRNA gene) sequence and two copies of rrl/rrf (23S/5S rRNA genes) sequences. The rrl/rrf genes were tandemly repeated at intervals of 3.2 kb and were located separately from the rrs gene on the genome. Our findings indicate that the organization of rRNA genes in Borrelia from I. ovatus ticks is identical to that of B. burgdorferi sensu stricto.     

Combined physical and genetic map of the Pseudomonas putida KT2440 chromosome

A combined physical and genetic map of the Pseudomonas putida KT2440 genome was constructed from data obtained by pulsed-field gel electrophoresis techniques (PFGE) and Southern hybridization. Circular genome size was estimated at 6.0 Mb by adding the sizes of 19 SwaI, 9 PmeI, 6 PacI, and 6 I-CeuI fragments. A complete physical map was achieved by combining the results of (i) analysis of PFGE of the DNA fragments resulting from digestion of the whole genome with PmeI, SwaI, I-CeuI, and PacI as well as double digestion with combinations of these enzymes and (ii) Southern hybridization analysis of the whole wild-type genome digested with different enzymes and hybridized against a series of probes obtained as cloned genes from different pseudomonads of rRNA group I and Escherichia coli, as P. putida DNA obtained by PCR amplification based on sequences deposited at the GenBank database, and by labeling of macrorestriction fragments of the P. putida genome eluted from agarose gels. As an alternative, 10 random mini-Tn5-Km mutants of P. putida KT2440 were used as a source of DNA, and the band carrying the mini-Tn5 in each mutant was identified after PFGE of a series of complete chromosomal digestions and hybridization with the kanamycin resistance gene of the mini-Tn5 as a probe. We established a circular genome map with an average resolution of 160 kb. Among the 63 genes located on the genetic map were key markers such as oriC, 6 rrn loci (rnnA to -F), recA, ftsZ, rpoS, rpoD, rpoN, and gyrB; auxotrophic markers; and catabolic genes for the metabolism of aromatic compounds. The genetic map of P. putida KT2440 was compared to those of Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens SBW25. The chromosomal backbone revealed some similarity in gene clustering among the three pseudomonads but differences in physical organization, probably as a result of intraspecific rearrangements.     

Paediatric Hodgkin''s disease in Spain: association with Epstein-Barr virus strains carrying latent membrane protein-1 oncogene deletions and high frequency of dual infections

Macroscopic, histologic, ultrastructural, microbiologic, in situ hybridization (ISH) and PCR detection results in three 8-week-old pigs naturally infected with Pneumocystis carinii (PC) are described. All animals had a nonsuppurative interstitial pneumonia and intra-alveolar Pneumocystis organisms with foamy eosinophilic and PAS positive appearance. Ultrastructurally, PC trophozoites and cysts were observed in pigs No. 2 and No. 3, with the former being much more numerous. PC organisms were located on the alveolar surface or within the alveolar septa. Trophozoites had numerous filopodia and were thick-walled. Cysts had no or few filopodia, were thick-walled and contained intracystic bodies. Using non-isotopic ISH on formalin-fixed, paraffin-embedded lung tissue sections, PC DNA from pigs No. 2 and No. 3 hybridized with a probe specific for PC ribosomal RNA (rRNA). Using primers specific for mitochondrial rRNA gene (pAZ102-E/pAZ102-H), and for the internal transcriber spacers of ribosomal gene of PC, PCR methods amplified a product in the lung of pigs No. 2 and No. 3 using either frozen or formalin-fixed and paraffin-embedded lung tissue. DNA from Pig No. 1 samples did not amplify with any primer. This is the first time that molecular biology techniques (in situ hybridization and PCR) have been applied to the study of porcine pneumocystosis.     

Measurement of impaired muscle function of the gastrocnemius, soleus, and tibialis anterior muscles in spastic hemiplegia: a preliminary study

Complete sequences of cytochrome b (1,137 bases) and 12S ribosomal RNA (961 bases) genes in mitochondrial DNA were successfully determined from the woolly mammoth (Mammuthus primigenius), African elephant (Loxodonta africana), and Asian elephant (Elephas maximus). From these sequence data, phylogenetic relationships among three genera were examined. Molecular phylogenetic trees reconstructed by the neighbor-joining and the maximum parsimony methods provided an identical topology both for cytochrome b and 12S rRNA genes. These results support the "Mammuthus-Loxodonta" clade, which is contrary to some previous morphological reports that Mammuthus is more closely related to Elephas than to Loxodonta.     

Glutathione efflux associated with a low gamma-glutamyl transpeptidase activity in human melanoma cells

These experiments tested the hypothesis that a pool of PCR-derived RNA probes with defined length and even representation of the target sequences could produce more specific and intense in situ hybridization signals than randomly size-reduced, plasmid-derived RNA probes. In situ hybridization was performed with sense and anti-sense HIV-1 RNA probes that were derived from PCR products tailed with the T7 RNA polymerase promoter or from plasmid DNA. In situ hybridization using a pool of seven anti-sense or sense PCR-derived RNA probes (1805 nucleotides of HIV sequence, 257 nucleotides average probe length) was compared with hybridization using anti-sense or sense RNA probes made from a plasmid representing the HIV-1 env gene (3151 nucleotides of HIV-1 target). The pooled PCR-derived probes resulted in stronger in situ hybridization signals and less background than those produced with plasmid-derived RNA probes. This method for creating PCR-derived RNA probes improves the feasibility of synthesizing multiple, discrete RNA probes for studies of specific mRNA expression because it does not require the subcloning steps used to construct plasmids. PCR-derived RNA probes may provide a viable alternative to the use of plasmid-derived RNA probes for in situ hybridization.     

16S-23S and 23S-5S intergenic spacer regions of lactobacilli: nucleotide sequence, secondary structure and comparative analysis

Lactobacilli have been used as industrial starters for a long time, but in several cases their identification was, and still is, neither easy nor reliable. The aim of the present work was to examine whether the intergenic spacer regions could be of value in the identification of Lactobacillus species. For that purpose, the polymerase chain reaction (PCR) was used to amplify 16S-23S and 23S-5S spacer regions of Lactobacillus (L.) acidophilus, L. delbrueckii subsp. bulgaricus, L. casei, L. helveticus and L. curvatus. The PCR products were directly sequenced, and two forms of ribosomal RNA (rrn) operons were identified in each species studied: one with tandem tRNA(Ile)/tRNA(Ala) genes and the other one without tRNA genes. Our study revealed that the rrn operons of Lactobacillus species studied comprise the genes of 16S, 23S and 5S rRNA, in that order. Only the tRNA genes and the rRNA processing stems are highly conserved in spacer regions of lactobacilli. The divergence between the lactobacilli spacer region sequences arises from insertions and deletions of short sequences. These sequences could be interesting candidates for the development of species-specific probes. Theoretical RNA/RNA secondary structure models of the interaction between the two spacer region sequences were constructed. In conclusion, the two spacer region sequences may prove to be a useful alternative to 16S and 23S rDNA sequencing for designing species-specific probes and for establishing phylogenetic relationships between closely related species such as L. curvatus and L. casei or L. acidophilus and L. helveticus.     

Differentiation of Actinobacillus pleuropneumoniae strains by sequence analysis of 16S rDNA and ribosomal intergenic regions, and development of a species specific oligonucleotide for in situ detection

The aims of this study were to characterize and determine intraspecies and interspecies relatedness of Actinobacillus pleuropneumoniae to Actinobacillus lignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in situ detection of A. pleuropneumoniae. Amplification and sequence analysis of the 16S-23S rDNA ribosomal intergenic sequence (RIS) from the three species showed the existence of two RIS's, differing by about 100 bp. Both sequences contained a region resembling the ribonuclease III cleavage site found in Escherichia coli. The smaller RIS contained a Glu-tRNA gene, and the larger one contained genes encoding Ile-tRNA and Ala-tRNA. These tRNA's showed a high sequence homology to the respective tRNA genes found in E. coli. Sequence analysis of the RIS's showed a high degree of genetic similarity of 24 strains of A. pleuropneumoniae. The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A. pleuropneumoniae. These sequences showed only minor base differences, indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the target species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA probe labeled with fluorescein was used for in situ hybridization experiments to detect A. pleuropneumoniae in biopsies of diseased porcine lungs.     

DNA-dependent in vitro synthesis of fibosomal proteins, protein elongation factors, and RNA polymerase subunit alpha: inhibition by ppGpp

We have previously shown that the synthesis of ribosomal proteins (r proteins) in E. coli cells is under stringent control (Dennis and Nomura, 1974). Since guanosine tetraphosphate (ppGpp) has been implicated in stringent control, we examined the effects of ppGpp on the in vitro synthesis of r proteins directed by DNA from transducing phage lambdafus3 and lambdarifd18. lambdafus3 carries genes for protein elongation factors EF-Tu and EF-G, and RNA polymerase subunit alpha, in addition to genes for approximately 27 r proteins. lambdarifd18 carries genes for EF-Tu, RNA polymerase subunits beta and beta1, and a set of rRNAs, in addition to genes for approximately five r proteins. We have shown that low concentrations of ppGpp (0.2-0.3 mM) specifically inhibit DNA-dependent r protein synthesis in this system, and that this inhibition takes place directly, rather than as a consequence of the inhibition of rRNA synthesis by ppGpp. In addition, we have also shown that ppGpp inhibits the synthesis of EF-G, EF-Tu, and RNA polymerase subunit alpha, as well as rRNAs.     

The growing importance of the plastid-like DNAs of the Apicomplexa

Organisms in the phylum Apicomplexa possess, in addition to their mitochondrial genome, an extrachromosomal DNA that possesses significant similarities with the extrachromosomal genomes of plastids. To date, the majority of data on these plastid-like DNAs have been obtained from the human malarial organism, Plasmodium falciparum. In common with plastid DNAs, the plastid-like DNA of P. falciparum possesses genes for DNA-dependent RNA polymerase subunits beta and beta 1 and for organellar-like large- and small-subunits ribosomal RNAs. Both the polymerase subunit and ribosomal RNA gene sequences share a number of features with those from plastid DNAs. In addition, the ribosomal RNA genes are organised in an inverted repeat arrangement, reminiscent of plastid DNAs. Additional molecular features shared between the 2 genomes are discussed. Plastid-like DNAs have also been identified in other Plasmodium species as well as Toxoplasma gondii, Eimeria tenella, Babesia bovis and a number of Sarcocystis species. A cryptic organelle often observed in apicomplexans has been proposed as the organelle that harbours the plastid-like DNAs, but conclusive evidence for this has not yet been obtained. Although approximately 1/2 of the plastid-like DNA of P. falciparum has been sequenced to date, no function has yet been ascribed to this DNA or its putative organelle. Phylogenetic inferences based on sequence data from this DNA have indicated an evolutionary origin from photosynthetic organisms, but the true provenance of the plastid-like DNAs remains to be determined. Because of the specific nature of the plastid-like DNAs, they may prove useful as effective targets for chemotherapeutics.     

Application of exogenous ceramide to cultured rat spinal motoneurons promotes survival or death by regulation of apoptosis depending on its concentrations

Chronic alcoholism causes a variety of ultrastructural, biochemical and functional alterations in the myocardium, but the underlying mechanisms are not well understood. Molecular changes that developed in the left ventricles of rats fed for 1 to 24 weeks on liquid diets containing ethanol as 36% of total calories were analyzed. Total tissue RNA and DNA were chemically extracted and measured by spectroscopic methods; mitochondrial DNA and mitochondrially-coded ribosomal RNA were measured at the 12s rRNA region by a quantitative polymerase chain reaction method; mitochondrial protein and enzyme activities were assayed. Ethanol-fed rats had 83.9 +/- 2.9% (mean +/- S.E.M.) as much DNA/g tissue and 74.7 +/- 3.9% as much total left ventricle DNA as pair-fed controls (P < 0.001). The alcoholics had 71.4 +/- 1.7% as much RNA/g tissue and 64.4 +/- 2.7% as much total left ventricle RNA as controls (P < 0.001). Mitochondrially-coded 12s rRNA was a lower proportion of total left ventricle RNA in all of the alcoholics; it was only 59.9 +/- 4.6% of control values (P < 0.001). Total left ventricle 12s rRNA was < 40% of normal. There was little or no change in mitochondrial DNA levels measured at the 12s location. Mitochondrial cytochrome contents were reduced 26-38% in the ethanol-fed rats, but only after 24 weeks. This study shows that experimental alcoholism produces rapid and sustained decreases in left ventricle total RNA and DNA and mitochondrial ribosomal RNA. The observed effects would be expected to have a major impact on left ventricle structural integrity and functional capacity.     

Origin of a fungal symbiont of perennial ryegrass by interspecific hybridization of a mutualist with the ryegrass choke pathogen, Epichlo? typhina

Seed-borne fungal symbionts (endophytes) provide many cool-season grass species with biological protection from biotic and abiotic stresses. The endophytes are asexual, whereas closely related sexual species of genus Epichlo? (Clavicipitales) cause grass choke disease. Perennial ryegrass (Lolium perenne) is a host of two endophyte taxa, LpTG-1 (L. perenne endophyte taxonomic grouping one = Acremonium lolii) and LpTG-2, as well as the choke pathogen, Epichlo? typhina (represented by isolate E8). Relationships among these fungi and other Epichlo? species were investigated by analysis of gene sequences, DNA polymorphisms and allozymes. The results indicate that LpTG-2 is a heteroploid derived from an interspecific hybrid. The LpTG-2 isolates had two copies each of nine out of ten genes analyzed (the exception being the rRNA gene locus), and the profiles for seven of these were composites of those from E. typhina E8 and A. lolii isolate Lp5. Molecular phylogenetic analysis grouped the two beta-tubulin genes of LpTG-2 into separate clades. One (tub2-1) was related to that of E. typhina E8, and the other (tub2-2) to that of A. lolii. The mitochondrial DNA profile of LpTG-2 was similar to that of A. lolii, but its rRNA gene sequence grouped it with E. typhina E8. A proposed model for the evolution of LpTG-2 involves infection of a L. perenne-A. lolii symbiotum by E. typhina, followed by hybridization of the two fungi. Such interspecific hybridization may be a common and important mechanism for genetic variation in Epichlo? endophytes.