Two C or not two C: recurrent disruption of Zn-ribbons, gene duplication, lineage-specific gene loss, and horizontal gene transfer in evolution of bacterial ribosomal proteins

Background

Ribosomal proteins are encoded in all genomes of cellular life forms and are, generally, well conserved during evolution. In prokaryotes, the genes for most ribosomal proteins are clustered in several highly conserved operons, which ensures efficient co-regulation of their expression. Duplications of ribosomal-protein genes are infrequent, and given their coordinated expression and functioning, it is generally assumed that ribosomal-protein genes are unlikely to undergo horizontal transfer. However, with the accumulation of numerous complete genome sequences of prokaryotes, several paralogous pairs of ribosomal protein genes have been identified. Here we analyze all such cases and attempt to reconstruct the evolutionary history of these ribosomal proteins.

Results

Complete bacterial genomes were searched for duplications of ribosomal proteins. Ribosomal proteins L36, L33, L31, S14 are each duplicated in several bacterial genomes and ribosomal proteins L11, L28, L7/L12, S1, S15, S18 are so far duplicated in only one genome each. Sequence analysis of the four ribosomal proteins, for which paralogs were detected in several genomes, two of the ribosomal proteins duplicated in one genome (L28 and S18), and the ribosomal protein L32 showed that each of them comes in two distinct versions. One form contains a predicted metal-binding Zn-ribbon that consists of four conserved cysteines (in some cases replaced by histidines), whereas, in the second form, these metal-chelating residues are completely or partially replaced. Typically, genomes containing paralogous genes for these ribosomal proteins encode both versions, designated C+ and C-, respectively. Analysis of phylogenetic trees for these seven ribosomal proteins, combined with comparison of genomic contexts for the respective genes, indicates that in most, if not all cases, their evolution involved a duplication of the ancestral C+ form early in bacterial evolution, with subsequent alternative loss of the C+ and C- forms in different lineages. Additionally, evidence was obtained for a role of horizontal gene transfer in the evolution of these ribosomal proteins, with multiple cases of gene displacement 'in situ', that is, without a change of the gene order in the recipient genome.

Conclusions

A more complex picture of evolution of bacterial ribosomal proteins than previously suspected is emerging from these results, with major contributions of lineage-specific gene loss and horizontal gene transfer. The recurrent theme of emergence and disruption of Zn-ribbons in bacterial ribosomal proteins awaits a functional interpretation.     

Characteristics of the rat behavior following single and multiple exposures of the hypoglycemic insulin doses]

Rats behaviour in the "open field" changed in 2 hrs after a single episode of hypoglycaemia was abolished with glucose whereas no changes occurred in their stereotyped behaviour and intraspecies interaction. In 24 hrs quantitative parameters of the "open field" behaviour normalised although the behaviour still had an altered structure. At the same time, amphetamine-induced stereotyped behaviour's indices became reduced. These and other findings suggest some deeper behavioural changes in rats during their recovery from repeated hypoglycaemic episodes and absence of synchronisation of the insulin effects.     

Polymorphic Genes of Xenobiotic-Metabolizing Enzymes Associated with Predisposition to Bronchial Asthma in Hereditarily Burdened and Nonburdened Children

The frequencies of theCYP1A1valine allele, homozygous deletions of GSTM1 and GSTT1, and two point mutations of the NAT2 gene, NAT2: S1(C⁴⁸¹ T) and S2 (G⁵⁹⁰ A), were compared in healthy children and children having bronchial asthma. The S1 mutation was associated with resistance, and all of the other traits, with predisposition to the disease. In families of patients with diseased progenitors and in those with healthy progenitors, the estimates of the asthma risk were similar. In both groups, parameters of the trait association with the disease depended on passive smoking. At passive smoking, a trend to an overrepresentation (high odds ratio, OR) of the GSTM1 null genotype and S2 mutation of theNAT2 gene was observed, whereas the odds ratio of the GSTT1 null genotype decreased, and those of the CYP1A1 and S1 mutation of the NAT2 gene remained unchanged. The highest OR = 36.25 (P < 0.01) was characteristic of theGSTT1 null genotype in nonsmoking hereditary burdened patients. The results obtained suggest an important role of xenobiotic-metabolizing enzymes in development of bronchial asthma.     

Connected gene neighborhoods in prokaryotic genomes

A computational method was developed for delineating connected gene neighborhoods in bacterial and archaeal genomes. These gene neighborhoods are not typically present, in their entirety, in any single genome, but are held together by overlapping, partially conserved gene arrays. The procedure was applied to comparing the orders of orthologous genes, which were extracted from the database of Clusters of Orthologous Groups of proteins (COGs), in 31 prokaryotic genomes and resulted in the identification of 188 clusters of gene arrays, which included 1001 of 2890 COGs. These clusters were projected onto actual genomes to produce extended neighborhoods including additional genes, which are adjacent to the genes from the clusters and are transcribed in the same direction, which resulted in a total of 2387 COGs being included in the neighborhoods. Most of the neighborhoods consist predominantly of genes united by a coherent functional theme, but also include a minority of genes without an obvious functional connection to the main theme. We hypothesize that although some of the latter genes might have unsuspected roles, others are maintained within gene arrays because of the advantage of expression at a level that is typical of the given neighborhood. We designate this phenomenon ‘genomic hitchhiking’. The largest neighborhood includes 79 genes (COGs) and consists of overlapping, rearranged ribosomal protein superoperons; apparent genome hitchhiking is particularly typical of this neighborhood and other neighborhoods that consist of genes coding for translation machinery components. Several neighborhoods involve previously undetected connections between genes, allowing new functional predictions. Gene neighborhoods appear to evolve via complex rearrangement, with different combinations of genes from a neighborhood fixed in different lineages.     

Involvement of chloroplasts in the programmed death of plant cells

The effect of cyanide, an apoptosis inducer, on pea leaf epidermal peels was investigated. Illumination stimulated the CN^–-induced destruction of guard cells (containing chloroplasts and mitochondria) but not of epidermal cells (containing mitochondria only). The process was prevented by antioxidants (-tocopherol, 2,5-di-tret-butyl-4-hydroxytoluene, and mannitol), by anaerobiosis, by the protein kinase C inhibitor staurosporine, and by cysteine and serine protease inhibitors. Electron acceptors (menadione, p-benzoquinone, diaminodurene, TMPD, DCPIP, and methyl viologen) suppressed CN^–-induced apoptosis of guard cells, but not epidermal cells. Methyl viologen had no influence on the removal of CN^–-induced nucleus destruction in guard cells under anaerobic conditions. The light activation of CN^–-induced apoptosis of guard cells was suppressed by DCMU (an inhibitor of the electron transfer in Photosystem II) and by DNP-INT (an antagonist of plastoquinol at the Q_o site of the chloroplast cytochrome b ₆ f complex). It is concluded that apoptosis initiation in guard cells depends on the simultaneous availability of two factors, ROS and reduced quinones of the electron transfer chain. The conditions for manifestation of programmed cell death in guard and epidermal cells of the pea leaf were significantly different.     

Selection of alternative 5' splice sites: role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.     

SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories

Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.     

Molecular cloning and characterization of Pals, proteins associated with mLin-7

In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologues of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins that can bind to the amino terminus of mLin-7 and cloned the genes encoding these proteins using bacterial expression cloning. We call these proteins Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, we have identified the conserved residues among these proteins crucial for mLin-7 binding. Two of these proteins, Pals1 and Pals2, are newly described. Pals1 consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7.     

Specific Expansion of Protein Families in the Radioresistant Bacterium Deinococcus Radiodurans

Computer analysis of the complete genome of Deinococcus radioduransR1 reveals a number of protein families, which are over-represented in this organism, compared to most other bacteria with known genome sequences. These families include both previously characterized and uncharacterized proteins. Most of the families whose functions are known or could be predicted seem to be related to stress-response and elimination of damage products (cell-cleaning). The two most prominent family expansions are the Nudix (MutT) family of pyrophosphohydrolases and a previously unnoticed family of proteins related to Bacillus subtilisDinB that could possess a metal-dependent enzymatic activity whose exact nature remains to be determined. Several proteins of the expanded families, particularly the Nudix family, are fused to other domains and form multidomain proteins that are so far unique for Deinococcus. The domain composition of some of these proteins indicates that they could be involved in novel DNA-repair pathways. Such unique proteins are good targets for knock-out and gene expression studies, which are aimed to shed light on the unusual features of this interesting10.6pt bacterium.     

A cryoultratomy method and its use in immunocytochemistry]

A method of frozen ultrathin sectioning for tissues and suspension material is presented. Practical aspects and results in the field of cryoultratomy are observed in detail. An example of a good preservation of tissue ultrastructure and application of cryosections to immunocytochemical investigations is given.