Analysis of the inheritance patterns of 5′-truncated copies of the German cockroach R2 retroposons in individual crosses

The inheritance patterns of the 5′-truncated copies of R2 retroposons were analyzed in individual crosses of the German cockroach. The recombination level within the cluster of ribosomal RNA genes was determined. It was demonstrated that only the frequencies of individual variants of 5′-truncated retroposon copies are appropriate for population analysis rather than the patterns characterizing individual X chromosomes. The methodical approach used in the work is convenient for studying the genetic variation in ribosomal DNA multigene families.     

Copy Number of Ribosomal Operons in Prokaryotes and Its Effect on Phylogenetic Analyses

Different aspects of the presence of multiple copies of ribosomal operons in prokaryotic genomes are reviewed. The structure of prokaryotic ribosomal operons is briefly described. The available data are summarized regarding the copy number of ribosomal genes in various prokaryotic genomes, the degree of polymorphism of their individual copies, and physiological and evolutional aspects of the presence of the multiple copies of ribosomal genes. The review also considers the influence of the presence of multiple copies of ribosomal genes on the results of identification of prokaryotic isolates and of the studies of prokaryotic diversity in environmental samples based on phylogenetic analysis of 16S rRNA gene sequences.     

Copy number of ribosomal operons in prokaryotes and its effect on phylogenic analyses

Different aspects of the presence of multiple copies of ribosomal operons in prokaryotic genomes are reviewed. Structure of prokaryotic ribosomal operons is briefly described. The available data are summarized regarding the copy number of ribosomal genes in various prokaryotic genomes, the degree of polymorphism of their individual copies, physiological and evolutionary aspects of the presence of the multiple copies of ribosomal genes. The review also considers the influence of the presence of multiple copies of ribosomal genes on the results of identification of prokaryotic isolates and of the studies of prokaryotic diversity in environmental samples based on phylogenetic analysis of 16S rRNA gene sequences.     

Relationship between the number and function of human ribosomal genes

Summary The relative number of ribosomal RNA genes of the acrocentric chromosomes in one individual was measured by counting grains after in situ hybridization of ³H-labeled human 18S rDNA to fixed metaphase chromosomes. The relative amount of ribosomal RNA gene activity of each of the same chromosomes was estimated by determining the frequency with which the chromosome's nucleolus organizer region (NOR) was silver stained, the size of the silver-stained region, and how often the chromosome was found in satellite association. Results were similar in phytohemagglutinin-stimulated T-lymphocytes, Epstein-Barr virus transformed lymphoblasts, and fibroblasts. One chromosome 21 had few gene copies and low activity. One chromosome 22 had many gene copies but low activity. Both chromosomes 14 had few gene copies but high activity. The level of expression that can be achieved by rRNA gene clusters can, therefore, be determined by factors other than the number of gene copies.     

Molecular analysis of integration sites of the drosophila retrotransposon burdock]

Data on molecular analysis of the insertion sites of nine random copies of burdock retrotransposon are presented. The 12-bp consensus sequence of the insertion sites, YNNUTUTUYAYA (Y-pyrimidine; U-purine), was determined. Homology between the burdock sequence and ribosomal genes was revealed. Three copies of this element were located within the region of ribosomal repeats: one copy in the 18S RNA gene, and two copies in the same intergenic spacer region, in the so-called Alu-repeats of Drosophila, in different copies of ribosomal genes.     

Overexpression of the crgA gene abolishes light requirement for carotenoid biosynthesis in Mucor circinelloides.

This work describes the isolation and characterization of crgA, a Mucor circinelloides gene, which has a dominant-positive effect on light-regulated carotenogenesis. The crgA gene was originally identified in a transformation experiment as a 3'-truncated open reading frame which caused carotenoid overaccumulation in the dark. The complete cloning and sequencing of crgA revealed that its putative product presented several recognizable structural domains: a RING-finger zinc binding domain near the N-terminus, a putative nuclear localization signal, two stretches of acidic amino acids, glutamine-rich regions and a putative isoprenylation motif. The expression of exogenous copies of the complete crgA gene or two different 3'-truncated versions, produced a similar dominant-positive effect on the light-inducible carotenogenesis of M. circinelloides. The presence of these exogenous sequences also caused a missregulation of the endogenous crgA gene, resulting in its overexpression. Collectively, these observations suggest that crgA is involved in the regulation of carotenoid biosynthesis by light.     

Development of SCAR markers for early identification of cytoplasmic male sterility genotype in chili pepper (Capsicum annuum L.)

We previously used Southern blot analysis to detect restriction-length polymorphisms between male fertile and cytoplasmic male sterile (CMS) cytoplasms at the coxII and atp6 loci of the mtDNA of Capsicum annuum L. Two copies of atp6 were found in each male fertile and CMS pepper lines. Interestingly, one of the copies of atp6 in CMS pepper was a 3'-truncated pseudogene. The open reading frame of the coxII gene was the same in the fertile (N-) and CMS (S-) lines. However, the nucleotide sequence in the S-cytoplasm diverged from that in the N-cytoplasm 41 bp downstream of the stop codon. To develop CMS-specific sequence-characterized amplified region (SCAR) markers, inverse PCR was performed to characterize the nucleotide sequences of the 5' and 3' flanking regions of mitochondrial atp6 and coxII from the cytoplasms of male fertile (N-) and CMS (S-) pepper plants. Based on these data, two CMS-specific SCAR markers, 607 and 708 bp long, were developed to distinguish N-cytoplasm from S-cytoplasm by PCR. The CMS-specific PCR bands were verified for 20 cultivars containing either N- or S-cytoplasm. PCR amplification of CMS-specific mitochondrial nucleotide sequences will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F1 seed lots. The strategy used in this report for identifying CMS-specific markers could be adopted for many other crops where CMS is used for F1 seed production.     

R1 and R2 retrotransposons of German cockroach Blattella germanica: comparative analysis of 5' truncated copies integrated into genome

This is the first report providing results on identification, cloning, and sequencing of extended fragments (5'-truncated copies) of R1 and R2 retrotransposons integrated into Blattella germanica genome. Comparative structural analysis of the received clones revealed two distinct subfamilies of R1 elements. However, all B. germanica R1 clones have two common features: poly(T) tails and similar target site duplications. Nucleotide structure and organization of five sequenced R2 fragments was similar. Analysis of R2 nucleotide sequences revealed typical deletions at the 3'end of target sites and lack of homopolynucleotides tails.     

Ribosomal RNA and ribosomal proteins in corynebacteria

Ribosomal RNAs (rRNAs) (16S, 23S, 5S) encoded by the rrn operons and ribosomal proteins play a very important role in the formation of ribosomes and in the control of translation. Five copies of the rrn operon were reported by hybridization studies in Brevibacterium (Corynebacterium) lactofermentum but the genome sequence of Corynebacterium glutamicum provided evidence for six rrn copies. All six copies of the C. glutamicum 16S rRNA have a size of 1523 bp and each of the six copies of the 5S contain 120 bp whereas size differences are found between the six copies of the 23S rRNA. The anti-Shine-Dalgarno sequence at the 3'-end of the 16S rRNA was 5'-CCUCCUUUC-3'. Each rrn operon is transcribed as a large precursor rRNA (pre-rRNA) that is processed by RNaseIII and other RNases at specific cleavage boxes that have been identified in the C. glutamicum pre-rRNA. A secondary structure of the C. glutamicum 16S rRNA is proposed. The 16S rRNA sequence has been used as a molecular evolution clock allowing the deduction of a phylogenetic tree of all Corynebacterium species. In C. glutamicum, there are 11 ribosomal protein gene clusters encoding 42 ribosomal proteins. The organization of some of the ribosomal protein gene cluster is identical to that of Escherichia coli whereas in other clusters the organization of the genes is rather different. Some specific ribosomal protein genes are located in a different cluster in C. glutamicum when compared with E. coli, indicating that the control of expression of these genes is different in E. coli and C. glutamicum.     

Quantitative analysis of the protein composition of yeast ribosomes

The molecular weights of the individual yeast ribosomal proteins were determined. The ribosomal proteins from the 40-S subunit have molecular weights ranging from 11 800 to 31 000 (average molecular weight = 21 300). The molecular weights of the 60-S subunit proteins range from 10 000 to 48 400 (average molecular weight = 21 800). Stoichiometric measurements, performed by densitometric scanning on ribosomal proteins extracted from high-salt dissociated subunits revealed that isolated ribosomal subunits contain, besides some protein species occurring in submolar amounts, a number of protein species which are present in multiple copies: S13, S27, L22, L31, L33, L34 and L39. The mass fractions of the ribosomal proteins which were found to be present on isolated ribosomes in non-unimolar amounts, were re-examined by using an isotope dilution technique. Applying this method to proteins extracted from mildely isolated 80-S ribosomes, we found that some protein species such as S32, S34 and L43 still are present in submolar amounts. On the other hand, however, we conclude that some other ribosomal proteins, in particular the strongly acidic proteins L44 and L45 get partially lost during ribosome dissociation. Proteins L44/L45 appears to be present on 80-S ribosomes in three copies.     

Intragenomic variation of fungal ribosomal genes is higher than previously thought

Nuclear ribosomal genes in most eukaryotes are present in multiple copies and often used for taxonomic and phylogenetic analyses. We comprehensively examined intragenomic polymorphism levels of three nuclear ribosomal loci for four important plant pathogenic fungi by polymerase chain reaction amplification and cloning. Here, we show that single nucleotide polymorphisms are present in an unexpectedly high amount. This might have implications for studies of fungal evolution, phylogenetics, and population genetics. Furthermore, our work demonstrates that the majority of all ribosomal sequences obtained from one individual and gene is identical to the majority rule consensus sequence of all detected sequence variants. Due to the large number of polymorphisms found and the fact that the polymorphism level differed markedly even between ribosomal genes of one and the same individual, we assume that nuclear ribosomal genes might not always evolve in a strictly concerted manner.     

Ribosomal DNA of Caenorhabditis elegans

We have characterized the organization of the genes coding for 18 S, 5·8 S and 26 S ribosomal RNAs in the nematode Caenorhabditis elegans. These ribosomal genes, present in about 55 copies per haploid genome, alternate in a repeating tandem array. The repeating unit is only 7000 base-pairs, containing a non-transcribed spacer of no more than 1000 base-pairs. Most of the repeating units have identical restriction maps, but one repeat contains a deletion of 2900 base-pairs, which eliminates all or part of the 18 S coding region. We have found no difference in the major ribosomal DNA restriction endonuclease cleavage patterns between two interbreeding strains of C. elegans, but found differences between C. elegans and the closely related Caenorhabditis briggsae.     

Human alpha-satellite DNA specific to chromosomes 13 and 21: use for the analysis of polymorphism of acrocentric chromosomes and the origin of the additional chromosome 21 in Down's syndrome]

Chromosomal distribution of cloned human alpha-satellite DNA alpha R1-6 has been studied by in situ hybridization technique. The sequence under study has been shown to be predominantly located in the centromeric regions of chromosomes 13 and 21. Intercellular variability of labelling patterns in every person under analysis being insignificant, there exists strong individual variability of interchromosomal distribution of the satellite. This variability leads to the differences of the chromosome labelling density (i.e. the number of satellite DNA copies) both between and within chromosome pairs. The difference in the copy number between two homologues chromosomes, 13 and 21 reaches up to 5 times. No correlation between nondisjunction and the number of copies of alpha-satellite DNA was found. Analysis of individual distribution of satellite between homologues of chromosome 21 provides new possibilities for determination of the origin of extra chromosome in the patients with trisomy 21.     

tRNA and the guanosinetriphosphatase activity of elongation factor Tu

Different sites of the tRNA molecule influence the activity of the elongation factor Tu (EF-Tu) center for GTP hydrolysis [Parlato, G., Pizzano, R., Picone, D., Guesnet, J., Fasano, O., & Parmeggiani, A. (1983) J. Biol. Chem. 258, 995-1000]. Continuing these studies, we have investigated some aspects of (a) the effect of different tRNA(Phe) species, including Ac-Phe-tRNA(Phe) and 3'-truncated tRNA-CCA in the presence and absence of codon-anticodon interaction, and (b) the effect of occupation of the ribosomal P-site by different tRNA(Phe) species. Surprisingly, we have found that 3'-truncated tRNA can enhance the GTPase activity in the presence of poly(U), in contrast to its inhibitory effect in the absence of codon-anticodon interaction. Moreover, Ac-Phe-tRNA(Phe) was found to have some stimulatory effect on the ribosome EF-Tu GTPase in the presence of poly(U). These results indicate that under specific conditions the 3'-terminal end and a free terminal alpha-NH2 group are not essential for the stimulation of the catalytic center of EF-Tu; therefore, the same structure of the tRNA molecule can act as a stimulator or an inhibitor of EF-Tu functions, depending on the presence of codon-anticodon interaction and on the concentration of monovalent and divalent cations. EF-Tu-GTP does not recognize a free ribosomal P-site from a P-site occupied by the different tRNA(Phe) species. When EF-Tu acts as a component of the ternary complex formed with GTP and aa-tRNA, the presence of tRNA in the P-site strongly increases the GTPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)