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At least 1,603 records · Page 89Linked to original sources
PubMed2006

Immunogenicity of a recombinant Mycobacterium bovis bacille Calmette-Guèrin expressing malarial and tuberculosis epitopes.

Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing the malarial epitopes F2R(II)EBA and (NANP)3 as well as two T cell epitopes of the M. tuberculosis ESAT-6 antigen, generated in favour of mycobacterium codon usage elicited specific immune response against these epitopes. Immunised Balb/c mice demonstrated an increase in almost all of the IgG subclasses against both malarial epitopes and enhanced splenocyte proliferative response against the malarial epitopes as well as selected peptides of ESAT-6. Furthermore, flow cytometric analyses showed elevated numbers of CD4+ lymphocytes expressing IFN-gamma and IL-2 against the ESAT-6 peptides, suggesting a specific Th1-mediated response. This study demonstrated that expressing malarial and TB epitopes in a single rBCG construct induced appropriate humoral and cellular immune response against immunogenic epitopes from both organisms.

Amino Acid Sequence↗
PubMed2006

Ancestral and consensus envelope immunogens for HIV-1 subtype C.

Immunogens based on "centralized" (ancestral or consensus) HIV-1 sequences minimize the genetic distance between vaccine strains and contemporary viruses and should thus elicit immune responses that recognize a broader spectrum of viral variants. However, the biologic, antigenic and immunogenic properties of such inferred gene products have to be validated experimentally. Here, we report the construction and characterization of the first full-length ancestral (AncC) and consensus (ConC) env genes of HIV-1 (group M) subtype C. The codon-usage-optimized genes expressed high levels of envelope glycoproteins that were incorporated into HIV-1 virions, mediated infection via the CCR5 co-receptor and retained neutralizing epitopes as recognized by plasma from patients with chronic HIV-1 subtype C infection. Guinea pigs immunized with AncC and ConC env DNA developed high titer binding, but no appreciable homologous or heterologous neutralizing antibodies. When tested by immunoblot analysis, sera from AncC and ConC env immunized guinea pigs recognized a greater number of primary subtype C envelope glycoproteins than sera from guinea pigs immunized with a contemporary subtype C env control. Mice immunized with AncC and ConC env DNA developed gamma interferon T cell responses that recognized overlapping peptides from the cognate ConC and a heterologous subtype C Env control. Thus, both AncC and ConC env genes expressed functional envelope glycoproteins that were immunogenic in laboratory animals and elicited humoral and cellular immune responses of comparable breadth and magnitude. These results establish the utility of centralized HIV-1 subtype C Env immunogens and warrant their continued evaluation as potential components of future AIDS vaccines.

AIDS Vaccines↗
PubMed2004

Coalescent processes and relaxation of selective constraints leading to contrasting genetic diversity at paralogs AtHVA22d and AtHVA22e in Arabidopsis thaliana.

Duplicate loci offer a very powerful system for understanding the complicated genome structure and adaptive evolution of a gene family. In this study, the genetic variation at paralogs AtHVA22d and AtHVA22e, members of an ABA- and stress-inducible gene family, is examined in the selfing Arabidopsis thaliana. Population genetic analysis indicates contrasting levels of nucleotide diversity at overall exon sequence and nonsynonymous sites between AtHVA22d (pi = 0.00337, pi(rep) = 0.00158) and AtHVA22e (pi = 0.00054, pi(rep) = 0.00023). The fact of Ka/Ks ratios significantly less than 1 in all sequences indicates that both genes are functional and subjected to purifying selection. In addition, rooted at barley HVA22, accelerated evolution is detected at replacement changes in the AtHVA22d locus, indicating relaxation of purifying selection after gene duplication. However, relative rate tests reveal no deviation from the neutrality at synonymous sites between the two paralogs. Based on clock-like evolution, the rate of synonymous substitution is estimated at 1.83 x 10(-9) substitutions per site per year; and the divergence of the two paralogs is traced to 90 MYA, coinciding with a period of the diversification of angiosperms. Given no codon usage bias in both genes, natural selection alone cannot account for the 6.4-fold differences in the nucleotide variation at synonymous sites between the two paralogs. Random processes resulting in different coalescence times, 3.65 MYA at AtHVA22d vs. 1.20 MYA at AtHVA22e, may have predominantly contributed to the evident differences of the genetic diversity. Partially nonoverlapping modes of expression between the two functional paralogs suggest a subfunctionalization hypothesis for explaining the fates of duplicate loci.

Arabidopsis↗
PubMed2006

The partial mitochondrial genome of the Cephalothrix rufifrons (Nemertea, Palaeonemertea): characterization and implications for the phylogenetic position of Nemertea.

A continuous 10.1kb fragment of the Cephalothrix rufifrons (Nemertea, Palaeonemertea) mitochondrial genome was sequenced and characterized to further assess organization of protostome mitochondrial genomes and evaluate the phylogenetic potential of gene arrangement and amino acid characters. The genome is A-T rich (72%), and this biased base composition is partly reflected in codon usage. Inferred tRNA secondary structures are typical of those reported for other metazoan mitochondrial DNAs. The arrangement of the 26 genes contained in the fragment exhibits marked similarity to those of many protostome taxa, most notably molluscs with highly conserved arrangements and a phoronid. Separate and simultaneous phylogenetic analyses of inferred amino acid sequences and gene adjacencies place the nemertean within the protostomes among coelomate lophotrochozoan taxa, but do not find a well-supported sister taxon link.

Animals↗
PubMed2003

Cytochrome f and subunit IV, two essential components of the photosynthetic bf complex typically encoded in the chloroplast genome, are nucleus-encoded in Euglena gracilis.

The photosynthetic protist Euglena gracilis contains chloroplasts surrounded by three membranes which arise from secondary endosymbiosis. The genes petA and petD, encoding cytochrome f and subunit IV of the cytochrome bf complex, normally present in chloroplast genomes, are lacking from the chloroplast DNA (cpDNA) of E. gracilis. The bf complex of E. gracilis was isolated, and the identities of cytochrome f and subunit IV were established immunochemically, by heme-specific staining, and by Edman degradation. Based on N-terminal and conserved internal protein sequences, primers were designed and used for PCR gene amplification and cDNA sequencing. The complete sequence of the petA cDNA and the partial sequence of the petD cDNA from E. gracilis are described. Evidence is provided that in this protist, the petA and petD genes have migrated from the chloroplast to the nucleus. Both genes exhibit a typical nuclear codon usage, clearly distinct from the usage of chloroplast genes. The petA gene encodes an atypical cytochrome f, with a unique insertion of 62 residues not present in other f-type cytochromes. The petA gene also acquired a region that encodes a large tripartite chloroplast transit peptide (CTP), which is thought to allow the import of apocytochrome f through the three-membrane envelope of E. gracilis chloroplasts. This is the first description of petA and petD genes that are nucleus-localized.

Amino Acid Sequence↗
PubMed2002

Evidence for a wide occurrence of proton-translocating pyrophosphatase genes in parasitic and free-living protozoa.

Proton-translocating inorganic pyrophosphatases (H(+)-PPase, EC 3.6.1.1) are integral membrane proteins that have been extensively studied in higher plants, the photosynthetic bacterium Rhodospirillum rubrum and, more recently, in some human pathogenic protozoa. By using a PCR-based approach, fragments of genes coding for H(+)-PPases in a number of protists, both free-living and parasites of animals and plants, that belong to diverse taxonomic groups (trypanosomatids, ciliates, apicomplexans, euglenoids, amoeboid mycetozoa, heterokonts) have been isolated. The experimental procedure involved the use of degenerate oligonucleotides designed from protein domains conserved in H(+)-PPases from plants and bacteria. The PCR-amplified DNA fragments exhibited the characteristic genomic structure and codon usage of the corresponding protozoan group. Paralogous genes were found in some species suggesting the occurrence of protein isoforms. These results indicate that H(+)-PPases are more widely distributed among protozoa than previously thought.

Amino Acid Sequence↗
PubMed2000

Sulphur islands in the Escherichia coli genome: markers of the cell's architecture?

Two highly contrasted images depict genomes: at first sight, genes appear to be distributed randomly along the chromosome. In contrast, their organisation into operons (or pathogenicity islands) suggests that, at least locally, related functions are in physical proximity. Analysis of the codon usage bias in orthologous genes in the genome of bacteria which diverged a long time ago suggested that some physical (architectural) selection pressure organised the distribution of genes along the chromosome. The metabolism of highly reactive species such as sulphur-containing molecules must be compartmentalised to escape the deleterious actions of diffusible reagents such as gases or radicals. We analysed the distribution of sulphur metabolism genes in the genome of Escherichia coli and found a number of them to be clustered into statistically significant islands. Another interesting feature of these genes is that the proteins they encode are significantly deprived of cysteine and methionine residues, as compared to the bulk proteins. We speculate that this clustering is associated to the organisation of sulphur metabolism proteins into islands where the sensitive sulphur-containing molecules are protected from reacting with elements in the environment such as dioxygen, nitric oxide or radicals.

Chromosomes, Bacterial↗
PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 3. Methods and strategies used for sequence analysis and annotation.

The primary analysis of the sequences for our Hemiascomycete random sequence tag (RST) project was performed using a combination of classical methods for sequence comparison and contig assembly, and of specifically written scripts and computer visualization routines. Comparisons were performed first against DNA and protein sequences from Saccharomyces cerevisiae, then against protein sequences from other completely sequenced organisms and, finally, against protein sequences from all other organisms. Blast alignments were individually inspected to help recognize genes within our random genomic sequences despite the fact that only parts of them were available. For each yeast species, validated alignments were used to infer the proper genetic code, to determine codon usage preferences and to calculate their degree of sequence divergence with S. cerevisiae. The quality of each genomic library was monitored from contig analysis of the DNA sequences. Annotated sequences were submitted to the EMBL database, and the general annotation tables produced served as a basis for our comparative description of the evolution, redundancy and function of the Hemiascomycete genomes described in other articles of this issue.

Amino Acid Sequence↗
PubMed1998

The complete genome of Bacillus subtilis: from sequence annotation to data management and analysis.

The completion of the entire 4.2-Mb genome sequence of the gram-positive bacterium Bacillus subtilis has been a milestone for biological studies on this model organism. This paper describes bioinformatics work related to this joint European and Japanese project: methods and strategies for gene annotation and detection of sequencing errors, using an integrated cooperative computer environment (Imagene); construction of a specialized database for data management and a WWW server for data retrieval (SubtiList); DNA sequence analysis, yielding striking results on oligonucleotide bias, repeated sequences, and codon usage, all landmarks of evolutionary events shaping the B. subtilis genome.

Amino Acid Sequence↗
PubMed2000

Theileria parva genomics reveals an atypical apicomplexan genome.

The discipline of genomics is setting new paradigms in research approaches to resolving problems in human and animal health. We propose to determine the genome sequence of Theileria parva, a pathogen of cattle, using the random shotgun approach pioneered at The Institute for Genomic Research (TIGR). A number of features of the T. parva genome make it particularly suitable for this approach. The G+C content of genomic DNA is about 31%, non-coding repetitive DNA constitutes less than 1% of total DNA and a framework for the 10-12 Mbp genome is available in the form of a physical map for all four chromosomes. Minisatellite sequences are the only dispersed repetitive sequences identified so far, but they are limited in distribution to 13 of 33 SfiI fragments. Telomere and sub-telomeric non-coding sequences occupy less than 10 kbp at each chromosomal end and there are only two units encoding cytoplasmic rRNAs. Three sets of distinct multicopy sequences encoding ORFs have been identified but it is not known if these are associated with expression of parasite antigenic diversity. Protein coding genes exhibit a bias in codon usage and introns when present are unusually short. Like other apicomplexan organisms, T. parva contains two extrachromosomal DNAs, a mitochondrial DNA and a plastid DNA molecule. By annotating the genome sequence, in combination with the use of microarray technology and comparative genomics, we expect to gain significant insights into unique aspects of the biology of T. parva. We believe that the data will underpin future research to aid in the identification of targets of protective CD8+ cell mediated immune responses, and parasite molecules involved in inducing reversible host leukocyte transformation and tumour-like behaviour of transformed parasitised cells.

Animals↗
PubMed2003

The genome of the Cryptophlebia leucotreta granulovirus.

The genome of the Cryptophlebia leucotreta granulovirus (CrleGV) was sequenced and analyzed. The double-stranded circular genome contains 110907 bp and potentially encodes 129 predicted open reading frames (ORFs), 124 of which were similar to other baculovirus ORFs. Five ORFs were CrleGV specific and 26 ORFs were common to other granulovirus genomes. One ORF showed a significant similarity to a nonstructural protein of Bombyx mori densovirus-2. A baculovirus chitinase gene was identified, which is most likely not functional, because its central coding region including the conserved chitinase active site signature is deleted. Three gene copies (Crle20, 23, and 24) containing the Baculo PEP N domain of the polyhedron envelope protein were identified in CrleGV and other GV genomes. One of them (Crle23) appeared also to contain a p10-like sequence encoding of a number of leucine-rich heptad repeats and a proline-rich domain. Another striking feature of the genome is the presence of a hypervariable non-hr ori-like region of about 1800 bp consisting of different kinds of repeats and palindromes. Three other repeat-rich regions were identified within the genome and are considered as homologous regions (hrs). CrleGV is most closely related to the Cydia pomonella granulovirus (CpGV) as revealed by genome order comparisons and phylogenetic analyses. However, the AT content of the CrleGV genome, which is 67.6% and the highest found so far in baculoviruses, differed by 12.8% from the AT content of CpGV. This resulted in a major difference in the codon usage of both viruses and may reflect adaptive selection constraints to their particular hosts.

Amino Acid Sequence↗
PubMed2001

RT-PCR amplification of a Rhizopus oryzae lactate dehydrogenase gene fragment.

No amino acid or DNA sequence information in sequence databases was found for a fungal lactate dehydrogenase (LDH) isozyme. Highly conserved regions in the lactate dehydrogenase enzymes of all taxonomies are found to be betaalphabeta nucleotide binding and substrate binding sites, also catalysis/active site. The conserved regions were selected as PCR primer target regions. The degenerate primers were designed according to the codon usage, determined by analyzing a number of different genes of Rhizopus species. A fragment of the gene (ldh), coding for approximately 72% of the lactate dehydrogenase enzyme from Rhizopus oryzae, was amplified using degenerate primers by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The size of the amplified fragment containing betaalphabeta nucleotide binding site, substrate binding site and catalysis/active site is found to be about 700 bp. The reported degenerate PCR primers and the amplification conditions may lead to the cloning of the lactate dehydrogenase gene of R. oryzae, which is an important organism due to its utilization in lactic acid and enzyme productions in industrial scales.

Journal Article↗
PubMed2001

A phylogenetic comparison of gene trees constructed from plastid, mitochondrial and genomic DNA of Plasmodium species.

Gene trees of Plasmodium species have been reported for the nuclear encoded genes (e.g. the Small Subunit rRNA) and a mitochondrial encoded gene, cytochrome b. Here, we have analyzed a plastid gene coding for caseinolytic protease ClpC, whose structure, function and evolutionary history have been studied in various organisms. This protein possesses a 220-250 amino acid long AAA domain (ATPases associated with a variety of cellular activities) that belongs to the Walker super family of ATPases and GTPases. We have sequenced the AAA motif of this gene, encoding the protein from nine different species of Plasmodium infecting rodents, birds, monkeys, and humans. The codon usage and GC content of each gene were nearly identical in contrast to the widely varying nucleotide composition of genomic DNAs. Phylogenetic trees derived from both DNA and inferred protein sequences have consistent topologies. We have used the ClpC sequence to analyze the phylogenetic relationship among Plasmodium species and compared it with those derived from mitochondrial and genomic sequences. The results corroborate well with the trees constructed using the mitochondrially encoded cytochrome b. However, an important element distinguishes the trees: the placement of Plasmodium elongatum near the base of the plastid tree, indicating an ancient lineage of parasites in birds that branches from the tree prior to other lineages of avian malaria and the human parasite, P. falciparum.

Adenosine Triphosphatases↗
PubMed2001

Mitochondrial gene content, arrangement and composition compared in African and Asian schistosomes.

Complete sequences were obtained for the coding portions of the mitochondrial (mt) genomes of Schistosoma mansoni (NMRI strain, Puerto Rico; 14 415 bp), S. japonicum (Anhui strain, China; 14 085 bp) and S. mekongi (Khong Island, Laos; 14 072 bp). Each comprises 36 genes: 12 protein-encoding genes (cox1-3, nad1-6, nad4L, atp6 and cob); two ribosomal RNAs, rrnL (large subunit rRNA or 16S) and rrnS (small subunit rRNA or 12S); as well as 22 transfer RNA (tRNA) genes. The atp8 gene is absent. A large segment (9.6 kb) of the coding region (comprising 14 tRNAs, eight complete and two incomplete protein-encoding genes) for S. malayensis (Baling, Malaysian Peninsula) was also obtained. Each genome also possesses a long non-coding region that is divided into two parts (a small and a large non-coding region, the latter not fully sequenced in any species) by one or more tRNAs. The protein-encoding genes are similar in size, composition and codon usage in all species except for cox1 in S. mansoni (609 aa) and cox2 in S. mekongi (219 aa), both of which are longer than homologues in other species. An unexpected finding in all the Schistosoma species was the presence of a leucine zipper motif in the nad4L gene. The gene order in S. mansoni is strikingly different from that seen in the S. japonicum group and other flatworms. There is a high level of identity (87-94% at both the nucleotide and amino acid levels) for all protein-encoding genes of S. mekongi and S. malayensis. The identity between genes of these two species and those of S. japonicum is less (56-83% for amino acids and 73-79% for nucleotides). The identity between the genes of S. mansoni and the Asian schistosomes is far less (33-66% for amino acids and 54-68% for nucleotides), an observation consistent with the known phylogenetic distance between S. mansoni and the other species.

Africa↗
PubMed1998

Organisation and informational content of the Theileria parva genome.

When compared with other Apicomplexan organisms, Theileria parva has an exceptionally small, 10-12 Mbp, genome. There are only 4 chromosomes, each in the Mbp range, and a complete physical map, based on SfiI linking data, is available for each one. A number of genes and cDNAs have been mapped to specific SfiI fragments. Telomeres consist of the simple repeat sequence typical of chromosomal ends but sub-telomeric homologies do not extend beyond 5 kbp. The only dispersed repetitive sequences identified to date are minisatellites, but these are found on a subset of SfiI fragments. There are clusters of distinct multicopy sequences which contain ORFs. However, the majority of parasite protein coding genes are present in a single copy. They have a compact structure, exhibit a bias in codon usage and non-translated regions are small. Introns, if present, are unusually short. Overall, the genome contains remarkably little repetitive, non-coding DNA. The parasite mitochondrial DNA is linear in structure, has a limited protein coding capacity and fragmented rRNA genes and its telomeres contain inverted repeat sequences.

Animals↗
PubMed1998

A Plasmodium falciparum aminopeptidase gene belonging to the M1 family of zinc-metallopeptidases is expressed in erythrocytic stages.

A new single copy gene has been isolated from Plasmodium falciparum, by immunoscreening a genomic DNA expression library. The gene appears devoid of introns, displays the classical A + T richness and codon usage of P. falciparum genes, and is transcribed into a 4 kb mRNA in erythrocytic stages. The deduced amino acid sequence corresponds to a 1056 residue protein (122 kDa) containing the canonical HExxHx18E signature of zinc-metallopeptidase active sites of the M1 family at position 467-490, a downstream conserved tyrosine residue involved in catalysis in position 551, and the GAMEN conserved motif characteristic of aminopeptidases in the M1 family, at position 431-435. The greatest similarities were found with aminopeptidases N of Escherichia coli and Haemophilius influenza (more than 80% identical residues in the canonical signature of the active site) but significant similarities centred on the active site region exist with all other members of the M1 family such as other prokaryotic aminopeptidases, eukaryotic aminopeptidases A and N and leukotriene A4 hydrolases (40-50% identical residues in the canonical signature of the active site). A polyclonal serum raised to a synthetic peptide deduced from the gene labelled schizont proteins of 96 and 68 kDa purified to homogeneity and both displaying aminopeptidase activity, as well as cytoplasmic structures in schizont stages.

Amino Acid Sequence↗
PubMed1997

An Acanthamoeba polyubiquitin gene and application of its promoter to the establishment of a transient transfection system.

We have isolated and sequenced a 2388 bp polyubiquitin encoding genomic DNA from Acanthamoeba encompassing two complete and one incomplete ubiquitin units. Codon usage frequency shows extreme bias. The deduced amino acid sequences of each unit are identical to each other and the same as that deduced from a previously sequenced Acanthamoeba castellanii cDNA. The upstream region of this gene, which contained some putative regulatory modules, was recovered by PCR (polymerase chain reaction) amplification and subcloning. This upstream fragment was ligated to the CAT (chloramphenicol acetyltransferase) gene in a eukaryotic expression plasmid and successfully applied to the establishment of an Acanthamoeba transient transfection system. Transfection was performed by electroporation and the optimal voltage was 4500 volts/cm at capacitance 25 microF. DEAE-dextran (25 microg/ml) added into the electroporation buffer increased the transfection efficiency by about 45%. The CAT activity was proportional to the amount of DNA transfected and reached the peak level 48 h after transfection. CAT assays showed that the polyubiquitin gene upstream fragment contains a functional promoter which is about 2.5 times as strong as a viral RSV-LTR promoter when driving CAT expression in Acanthamoeba.

Acanthamoeba↗
PubMed1999

A possible involvement of melanocortin 3 receptor in the regulation of adrenal gland function in the chicken.

The melanocortin 3 receptor (MC3-R) in the melanocortin receptor family has been identified as a neural receptor subtype mainly expressed in the brain in mammals. We report here the isolation of the chicken gene for MC3-R, CMC3, displaying different tissue distribution from mammalian counterparts. The CMC3 gene was found to be a single copy gene encoding a 325 amino acid protein, sharing 75.3-76.8% identity with mammalian counterparts. When assessed by RT-PCR, the CMC3 mRNA was not detected in the brain but was exclusively expressed in adrenal glands, where Agouti-related protein/Agouti-related transcript (AGRP/ART), a newly identified endogenous antagonist of MC3-R, is expressed in mammals, raising the possibility that the CMC3 plays a role in complicated regulation of the gland function by melanocortins and AGRP/ART in the chicken. Noteworthy, MC1-R gene was found to be a quite unique member of the chicken MC-R family with regard to GC content and codon usage. It may reflect as yet unidentified evolutionary pressure operating specifically on the gene.

Adrenal Glands↗