Comparisons of the complete sequences of two collagen genes from Caenorhabditis elegans

Several collagen genes have been isolated from the nematode Caenorhabditis elegans. The complete nucleotide sequences of two of these genes, col-1 and col-2, have been determined. These collagen genes differ from vertebrate collagen genes in that they contain only one or two introns, their triple-helical regions are interrupted by nonhelical amino acid sequences and they are smaller. A high degree of nucleotide and amino acid homology exists between col-1 and col-2. In particular, the regions around cysteines and lysines are most highly conserved. The C. elegans genome contains 50 or more collagen genes, the majority of which probably encode cuticle collagens; col-1 and col-2 apparently are members of this large family of cuticle collagen genes.     

Characteristics of the new plasmid,pMTB1, from the metagenome of the microbial community of underground thermal waters of Western Siberia

A new circular 4935-bp long plasmid pMTB1 has been identified in sequences of the metagenome. The nucleotide sequence of pMTB1 is highly similar to that of plasmids, pME2001 and pME2200, from the methanogenic archaeon Methanothermobacter marburgensis. One of six putative protein-coding genes encodes a protein containing helix-turn-helix and ATP/GTP-binding motifs and, probably, functioning as a replication initiator protein. Homologs of other genes have been found only in the plasmids of M. marburgensis, but their functions are unknown. Comparison of the complete nucleotide sequences of the plasmids pMTB1, pME2001, and pME2200 has revealed that they have a common origin but differ from each other by the presence of several inserts flanked by nearly perfect direct repeats within regions not essential for replication.     

Genetic characterization of toxigenic Vibrio cholerae non-O1/non-O139 strains, isolated in the Middle Asia

Here, we report on the characterization of 22 clinical toxigenic V. cholerae non-O1/non-O139 strains isolated in the Middle Asia (Uzbekistan) in 1971–1990. PCR analysis has revealed that these strains contain the main virulence genes such as ctxA, zot, ace (CTXφ); rstC (RS1φ); tcpA, toxT, aldA (pathogenicity island VPI), but they lack both pandemic islands VSP-I and VSP-II specific to epidemic strains of O1 serogroup of El Tor biotype and O139 serogroup. Only two of the twenty two toxigenic strains have tcpA gene of El Tor type, one strain has tcpA gene of classical type, while nineteen other strains carry a new variant of this gene, designated as tcpA ^uzb. Nucleotide sequences analysis of virulence genes in toxigenic V. cholerae non-O1/non-O139 strains from Uzbekistan showed that they differ significantly from the sequences of these genes in epidemic O1 and O139 strain indicating that they belong to a separate line of evolution of virulent V. cholerae strains. For the first time it is shown that V. cholerae non-O1/non-O139 toxigenic strains of different serogroups may belong to the same clone.     

Cloning,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

Identification and Characterization of ATP/ADP Isopentenyltransferases (ATP/ADP PpIPTs) Genes in Peach

ATP/ADP isopentenyltransferase (IPTs) genes encode key enzymes involved in cytokinin synthesis. In this study, the functions of ATP/ADP PpIPTs in peach were investigated. According to the genome sequence, we have found and verified that there are four members of this gene family in peach, namely, PpIPT1, PpIPT3, PpIPT5, and PpIPT7. Overexpression of each of these genes in Arabidopsis resulted in increased levels of cytokinins in the transgenic plants, confirming their roles in cytokinin synthesis. Numerous altered phenotypes were observed in the transgenic plants, including vigorous growth and enhanced salt resistance. ATP/ADP PpIPTs were expressed in tissues throughout the plant, but the expression patterns differed between the genes. Only PpIPT3 was upregulated within 2 h after the application of nitrate to N-deprived peach seedlings, and the increase was resistant to pre-treatment of a specific nitrate metabolism inhibitor. Results showed that ATP/ADP PpIPT expression levels decreased significantly in pulp within 2 weeks after flowering and remained low. However, pulp cytokinin levels were quite high during this time. Only PpIPT5 in seed increased significantly within 2 weeks after flowering, which was consistent with cytokinin levels during early fruit development, suggesting that PpIPT5 in seed is the key gene for cytokinin biosynthesis during early fruit development. ATP/ADP PpIPT expression also increased significantly during later fruit development in seed.

     

5 S DNAs of Xenopus laevis and Xenopus mulleri: evolution of a gene family

The 5 S DNA which contains the genes for 5 S RNA has been purified from the frog Xenopus mulleri and compared with the 5 S DNA of Xenopus laevis. Both DNAs contain highly repetitive sequences in which the gene sequence that codes for 5 S RNA alternates with a spacer sequence. The 5 S DNAs of X. laevis and X. mulleri comprise about 0.7% of the total DNA or about 24,000 and 9000 repeating sequences, respectively. The average repeat length within native X. laevis and X. mulleri 5 S DNA is about 0.5 to 0.6 and 1.2 to 1.5 × 10⁶ daltons, respectively, each repeat of which contains a single gene sequence for 5 S RNA (0.08 × 10⁶ daltons). The two DNAs differ in the average length of their spacers and no cross homology can be detected by heterologous hybridization of the two DNAs, except within the 5 S RNA gene regions. Despite their differences, the spacer sequences of X. laevis and X. mulleri 5 S DNA resemble each other enough to conclude that they have diverged from a common ancestral sequence.The multiple repeating sequences of 5 S DNA in each species have evolved as a family of similar, but not identical sequences. It is known that 5 S DNA is located at the ends (telomeres) of the long arms of most, if not all, X. laevis chromosomes. It is proposed that multiple gene sequences located on the ends of many chromosomes can evolve together as a family if there is extensive and unequal exchange of DNA sequences between homologous and non-homologous chromosomes at their ends.     

Characterization of the two H1°-encoding genes from Xenopus laevis

We have analyzed the promoter and the coding sequences of the two homologous histone H1°-encoding genes from Xenopus laevis, here termed H1°-1 and H1°-2. Both genes encode proteins of 193 amino acids and differ at just 16 amino-acid residues. Putative regulatory sequences identified in the promoter region are the same and are highly conserved. However, significant differences exist in the 5′ untranslated regions (UTR) of the transcribed sequences of these two genes, such as several deletions in the 5′-UTR of the H1°-2 gene in comparison with the H1°-1 gene 5′-UTR. The 3′-UTR is a short sequence of about 200 bp which is unexpected compared with the long 3′-UTR of mammalian H1° mRNA, but it is in the same size range as in avian H5 mRNA. Thus, the main differences between these two genes are observed in sequences potentially involved in the regulation of the H1° gene expression such as the 5′-UTR. The two genes are expressed during embryogenesis and in several adult tissues. We discuss these findings in terms of the evolution of histone H1° genes in vertebrates and the appearance of histone H5 in avian species.© 1997 Elsevier Science B.V. All rights reserved.     

Identification of a tRNA isopentenyltransferase gene from Arabidopsis thaliana

The tRNA of most organisms contain modified adenines called cytokinins. Situated next to the anticodon, they have been shown to influence translational fidelity and efficiency. The enzyme that synthesizes cytokinins on pre-tRNA, tRNA isopentenyltransferase (EC 2.5.1.8), has been studied in micro-organisms like Escherichia coli and Saccharomyces cerevisiae, and the corresponding genes have been cloned. We here report the first cloning and functional characterization of a homologous gene from a plant, Arabidopsis thaliana. Expression in S. cerevisiae showed that the gene can complement the anti-suppressor phenotype of a mutant that lacks MOD5, the intrinsic tRNA isopentenyltransferase gene. This was accompanied by the reintroduction of isopentenyladenosine in the tRNA. The Arabidopsis gene is constitutively expressed in seedling tissues.     

Noncognate Mycobacterium tuberculosis Toxin-Antitoxins Can Physically and Functionally Interact

The Mycobacterium tuberculosis genome harbors a striking number (>40) of toxin-antitoxin systems. Among them are at least seven MazF orthologs, designated MazF-mt1 through MazF-mt7, four of which have been demonstrated to function as mRNA interferases that selectively target mRNA for cleavage at distinct consensus sequences. As is characteristic of all toxin-antitoxin systems, each of the mazF-mt toxin genes is organized in an operon downstream of putative antitoxin genes. However, only one of the seven putative upstream antitoxins (designated MazE-mt1 through MazE-mt7) has significant sequence similarity to Escherichia coli MazE, the cognate antitoxin for E. coli MazF. Interestingly, the M. tuberculosis genome contains two independent operons encoding E. coli MazE orthologs, but they are not paired with mazF-mt-like genes. Instead, the genes encoding these two MazE orthologs are each paired with proteins containing a PIN domain, indicating that they may be members of the very large VapBC toxin-antitoxin family. We tested a spectrum of pair-wise combinations of cognate and noncognate Mtb toxin-antitoxins using in vivo toxicity and rescue experiments along with in vitro interaction experiments. Surprisingly, we uncovered several examples of noncognate toxin-antitoxin association, even among different families (e.g. MazF toxins and VapB antitoxins). These results challenge the “one toxin for one antitoxin” dogma and suggest that M. tuberculosis may enlist a sophisticated toxin-antitoxin network to alter its physiology in response to environmental cues.     

Comprehensive cloning of Schizosaccharomyces pombe genes encoding translation elongation factors

In the course of the Schizosaccharomyces pombe cDNA project, we succeeded in cloning all the genes encoding translation elongation factors EF-1α, EF-1β, EF-1γ, EF-2 and EF-3. With the exception of the EF-1γ gene, the nucleotide (nt) sequence of S. pombe elongation factors has not been previously reported. For EF-1α, we found three genes whose amino acid (aa) sequences are quite homologous each other (99.5%), but whose 3′ untranslated regions (UTRs) are completely different. Southern blot indicated that those three EF-1α genes are located at different loci. Northern analysis indicated that one of three EF-1α genes was inducible with UV-irradiation, while the level of expression for another of three EF-1α genes was repressed by UV and heat-shock (HS) treatments. The aa sequence predicted from the nt sequence of the S. pombe EF-1β cDNA clone covered almost all the coding sequence (CDS) of EF-1β except the first methionine which has 55.4% identity with that of S. cerevisiae. We also identified two copies of S. pombe EF-2 genes. Their aa sequences deduced from nt sequences are identical (100%), but they have different 3′ UTRs. The location of these two EF-2 genes in different loci was proved by Southern analysis. The S. pombe EF-3 cDNA clone encoded only a third of the CDS from the C-terminal and its deduced aa sequence has a 76% identity with those of other yeasts and fungi.     

Gain and loss of multiple genes during the evolution of Helicobacter pylori

Sequence diversity and gene content distinguish most isolates of Helicobacter pylori. Even greater sequence differences differentiate distinct populations of H. pylori from different continents, but it was not clear whether these populations also differ in gene content. To address this question, we tested 56 globally representative strains of H. pylori and four strains of Helicobacter acinonychis with whole genome microarrays. Of the weighted average of 1,531 genes present in the two sequenced genomes, 25% are absent in at least one strain of H. pylori and 21% were absent or variable in H. acinonychis. We extrapolate that the core genome present in all isolates of H. pylori contains 1,111 genes. Variable genes tend to be small and possess unusual GC content; many of them have probably been imported by horizontal gene transfer. Phylogenetic trees based on the microarray data differ from those based on sequences of seven genes from the core genome. These discrepancies are due to homoplasies resulting from independent gene loss by deletion or recombination in multiple strains, which distort phylogenetic patterns. The patterns of these discrepancies versus population structure allow a reconstruction of the timing of the acquisition of variable genes within this species. Variable genes that are located within the cag pathogenicity island were apparently first acquired en bloc after speciation. In contrast, most other variable genes are of unknown function or encode restriction/modification enzymes, transposases, or outer membrane proteins. These seem to have been acquired prior to speciation of H. pylori and were subsequently lost by convergent evolution within individual strains. Thus, the use of microarrays can reveal patterns of gene gain or loss when examined within a phylogenetic context that is based on sequences of core genes.     

Variations and evolution of polyubiquitin genes from ciliates

Polyubiquitin genes from seven ciliate species were amplified, cloned and sequenced. It is estimated that Strombidium sulcatum, Euplotes vannus, E. rariseta and Anteholosticha manca have a polyubiquitin gene of 3 repeats, and A. parawarreni, Paramecium caudatum and Pseudokeronopsis flava 4 repeats. The newly obtained ubiquitins mostly differ from that of humans by 1–5 residues in amino acid sequences. A neighbor-joining tree constructed based on monomeric ubiquitin genes supports the monophyly of an assemblage comprising the litostomateans and some oligohymenophoreans, but not the class Spirotrichea. The monomers from the same species are generally placed together and highly supported for the class Litostomatea, the genera Paramecium and Ichthyophthirius, but not for other species. The non-synonymous/synonymous rate ratio (dN/dS) at the protein level are less than 1, and the synonymous nucleotide differences per synonymous site (p_S) from intraspecific comparisons are fairly high (0.02–0.72). These results indicate that ciliates have not only the conserved, but also some quite divergent, polyubiquitin genes and confirm that the polyubiquitin genes in ciliates evolve according to the birth-and-death mode of evolution under strong purifying selection.     

Intracellular neutralization of SV40 tumor antigens following microinjection of specific antibody

We present the nucleotide sequences of the ^Gγ- and ^Aγ-globin genes from one chromosome (A) and of most of the ^Aγ gene from the other chromosome (B) of the same individual. All three genes have a small, highly conserved intervening sequence (IVS1) of 122 bp located between codons 30 and 31 and a large intervening sequence (IVS2) of variable length (866–904 bp) between codons 104 and 105. A stretch of simple sequence DNA occurs in IVS2 which appears to be a hot spot for recombination. On the 5′ side of this simple sequence, the allelic ^Aγ genes differ considerably in IVS2 whereas the nonallelic ^Gγ- and ^Aγ genes from chromosome A differ only slightly. Yet on the 3′ side of the simple sequence, the allelic genes differ only slightly whereas the nonallelic genes differ considerably. We hypothesize that the 5′ two thirds of the ^Aγ gene on chromosome A has been “converted” by an intergenic exchange to become more like the ^Gγ gene on its own chromosome A than it is like the allelic ^Aγ gene on the other chromosome B. Our sequence data suggest that intergenic conversions occur in the germ line. The DNA sequence differences between two chromosomes from a single individual strongly suggest that DNA sequence polymorphisms for localized deletions, additions and base substitutions are very common in human populations.     

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii: Evolutionary and functional implications

The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoë, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.     

Complete Mitochondrial Genome of Echinostoma hortense (Digenea: Echinostomatidae)

Echinostoma hortense (Digenea: Echinostomatidae) is one of the intestinal flukes with medical importance in humans. However, the mitochondrial (mt) genome of this fluke has not been known yet. The present study has determined the complete mt genome sequences of E. hortense and assessed the phylogenetic relationships with other digenean species for which the complete mt genome sequences are available in GenBank using concatenated amino acid sequences inferred from 12 protein-coding genes. The mt genome of E. hortense contained 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 non-coding region. The length of the mt genome of E. hortense was 14,994 bp, which was somewhat smaller than those of other trematode species. Phylogenetic analyses based on concatenated nucleotide sequence datasets for all 12 protein-coding genes using maximum parsimony (MP) method showed that E. hortense and Hypoderaeum conoideum gathered together, and they were closer to each other than to Fasciolidae and other echinostomatid trematodes. The availability of the complete mt genome sequences of E. hortense provides important genetic markers for diagnostics, population genetics, and evolutionary studies of digeneans.     

Identification and characterization of a novel chitinase-like gene cluster (AgCht5) possibly derived from tandem duplications in the African malaria mosquito, Anopheles gambiae

Insect chitinase 5 (Cht5), a well-characterized enzyme found in the molting fluid and/or integument, is classified as a group I chitinase and is usually encoded by a single gene. In this study, a Cht5 gene cluster consisting of five different chitinase-like genes (AgCht5-1, AgCht5-2, AgCht5-3, AgCht5-4 and AgCht5-5) was identified by a bioinformatics search of the genome of Anopheles gambiae. The gene models were confirmed by cloning and sequencing of the corresponding cDNAs and gene expression profiles during insect development were determined. All of these genes are found in a single cluster on chromosome 2R. Their open reading frames (ORF) range from 1227 to 1713 bp capable of encoding putative proteins ranging in size from 409 to 571 amino acids. The identities of their cDNA sequences range from 52 to 66%, and the identities of their deduced amino acid sequences range from 38 to 53%. There are four introns for AgCht5-1, two for AgCht5-2 and AgCht5-3, only one for AgCht5-4, but none for AgCht5-5 in the genome. All five chitinase-like proteins possess a catalytic domain with all of the conserved sequence motifs, but only AgCht5-1 has a chitin-binding domain. Phylogenetic analysis of these deduced proteins along with those from other insect species suggests that AgCht5-1 is orthologous to the Cht5 proteins identified in other insect species. The differences in expression patterns of these genes at different developmental stages further support that these genes may have distinct functions. Additional searching of the genomes of two other mosquito species led to the discovery of four Cht5-like genes in Aedes aegypti and three in Culex quinquefasciatus. Thus, the presence of a Cht5 gene cluster appears to be unique to mosquito species and these genes may have resulted from gene tandem duplications.     

Immune gene discovery by expressed sequence tag (EST) analysis of hemocytes in the ridgetail white prawn Exopalaemon carinicauda

The ridgetail white prawn Exopalaemon carinicauda is one of the most important commercial species in eastern China. However, little information of immune genes in E. carinicauda has been reported. To identify distinctive genes associated with immunity, an expressed sequence tag (EST) library was constructed from hemocytes of E. carinicauda. A total of 3411 clones were sequenced, yielding 2853 ESTs and the average sequence length is 436 bp. The cluster and assembly analysis yielded 1053 unique sequences including 329 contigs and 724 singletons. Blast analysis identified 593 (56.3%) of the unique sequences as orthologs of genes from other organisms (E-value < 1e-5). Based on the COG and Gene Ontology (GO), 593 unique sequences were classified. Through comparison with previous studies, 153 genes assembled from 367 ESTs have been identified as possibly involved in defense or immune functions. These genes are categorized into seven categories according to their putative functions in shrimp immune system: antimicrobial peptides, prophenoloxidase activating system, antioxidant defense systems, chaperone proteins, clottable proteins, pattern recognition receptors and other immune-related genes. According to EST abundance, the major immune-related genes were thioredoxin (141, 4.94% of all ESTs) and calmodulin (14, 0.49% of all ESTs). The EST sequences of E. carinicauda hemocytes provide important information of the immune system and lay the groundwork for development of molecular markers related to disease resistance in prawn species.