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PubMed2006

Mitogenomic evolution and interrelationships of the Cypriniformes (Actinopterygii: Ostariophysi): the first evidence toward resolution of higher-level relationships of the world's largest freshwater fish clade based on 59 whole mitogenome sequences.

Fishes of the order Cypriniformes are almost completely restricted to freshwater bodies and number > 3400 species placed in 5 families, each with poorly defined subfamilies and/or tribes. The present study represents the first attempt toward resolution of the higher-level relationships of the world's largest freshwater-fish clade based on whole mitochondrial (mt) genome sequences from 53 cypriniforms (including 46 newly determined sequences) plus 6 outgroups. Unambiguously aligned, concatenated mt genome sequences (14,563 bp) were divided into 5 partitions (first, second, and third codon positions of the protein-coding genes, rRNA genes, and tRNA genes), and partitioned Bayesian analyses were conducted, with protein-coding genes being treated in 3 different manners (all positions included; third codon positions converted into purine [R] and pyrimidine [Y] [RY-coding]; third codon positions excluded). The resultant phylogenies strongly supported monophyly of the Cypriniformes as well as that of the families Cyprinidae, Catostomidae, and a clade comprising Balitoridae + Cobitidae, with the 2 latter loach families being reciprocally paraphyletic. Although all of the data sets yielded nearly identical tree topologies with regard to the shallower relationships, deeper relationships among the 4 major clades (the above 3 major clades plus Gyrinocheilidae, represented by a single species Gyrinocheilus aymonieri in this study), were incongruent depending on the data sets. Treatment of the rapidly saturated third codon-position transitions appeared to be a source of such incongruities, and we advocate that RY-coding, which takes only transversions into account, effectively removes this likely "noise" from the data set and avoids the apparent lack of signal by retaining all available positions in the data set.

Animals↗

PubMed2005

Selective isolation of members of the Streptomyces violaceoruber clade from soil.

Large numbers of filamentous actinomycetes which formed distinctive red coloured colonies were isolated from three out of four composite soil samples using a medium designed to be selective for members of the Streptomyces violaceoruber clade, a taxon which includes the model organisms "Streptomyces coelicolor" A3(2) and "Streptomyces lividans" 66. The isolation medium, dextran-histidine-sodium chloride-mineral salts agar supplemented with antibacterial and antifungal antibiotics, also supported the growth of representatives of the S. violaceoruber clade. One hundred and ninety one representatives of the isolates that produced red colour colonies on the isolation medium were distributed into four colour groups based on their ability to form distinctive pigments and morphological properties typical of members of the S. violaceoruber clade, an assignment that was confirmed by corresponding 16S rRNA gene sequencing studies. The selective isolation and characterisation procedures used in the present investigation provide a practical means of determining the taxonomic diversity, geographical distribution and roles of representatives of the S. violaceoruber clade in natural habitats.

Bacteriological Techniques↗

PubMed2006

Mitochondrial data support an odd-nosed colobine clade.

To obtain a more complete understanding of the evolutionary history of the leaf-eating monkeys we have examined the mitochondrial genome sequence of two African and six Asian colobines. Although taxonomists have proposed grouping the "odd-nosed" colobines (proboscis monkey, douc langur, and the snub-nosed monkey) together, phylogenetic support for such a clade has not been tested using molecular data. Phylogenetic analyses using parsimony, maximum likelihood, and Bayesian methods support a monophyletic clade of odd-nosed colobines consisting of Nasalis, Pygathrix, and Rhinopithecus, with tentative support for Nasalis occupying a basal position within this clade. The African and Asian colobine lineages are inferred to have diverged by 10.8 million years ago (mya or Ma). Within the Asian colobines the odd-nosed clade began to diversify by 6.7 Ma. These results augment our understanding of colobine evolution, particularly the nature and timing of the colobine expansion into Asia. This phylogenetic information will aid those developing conservation strategies for these highly endangered, diverse, and unique primates.

Animals↗

PubMed2002

A predefined epitope-specific monoclonal antibody recognizes ELDEWA-epitope just presenting on gp41 of HIV-1 O clade.

Diverse variation of HIV-1 is a grave challenge for prevention of viral infection and immunotherapy. Monoclonal antibody (mAb) 2F5 recognizing an epitope ELDKWA (aa669-674) on HIV-1 envelope protein gp41 showed broad neutralizing activity against a lot of HIV-1 strains including primary isolates. However, viral mutation from ELDKWA to ELDEWA resulted in viral evasion from neutralization by mAb 2F5. Using ELDEWA-epitope-peptide (C-GFLDEWAGELDEWA) conjugated with carrier protein keyhole limpet hemocyanin (KLH), a mAb 14D9 (IgGl) was prepared and identified as the mAb with predefined ELDEWA-epitope specificity. The mAb 14D9 recognized the ELDEWA epitope, but not other three epitopes (ELDKWA, ELNKWA and ELEKWA). In comparison, mAb 2F5 could recognize only ELDKWA, but not three neutralization-resistant epitopes (ELDEWA, ELNKWA and ELEKWA). Interestingly, we searched several authoritative HIV sequence databases (http://hiv-web.lanl.gov) and found out that nearly all the viral isolates bearing the ELDEWA epitope belong to the O clade, the only exceptional viral isolate bearing the epitope ELDEWA has been demonstrated to be an intergroup M/O recombinant, which suggests that the ELDEWA-epitope on gp41 represents a specific epitope-marker of HIV-1 O clade. To confirm whether the mAb 14D9 recognizes gp41 of HIV-1 O clade, the rsgp41(IIIB), bearing ELDKWA-epitope was site-directed-mutated to the rsgp41 bearing ELDEWA-epitope. The mAb 14D9 could bind to rsgp41 bearing ELDEWA-epitope in immunoblotting analysis, did not bind to rsgp41 bearing ELDKWA-epitope. These experimental results suggest that the mAb 14D9 with predefined ELDEWA-epitope specificity may be applied to HIV-1 O clade identification.

AIDS Vaccines↗

PubMed2002

Induction of neutralizing antibodies and cytotoxic T lymphocytes in Balb/c mice immunized with virus-like particles presenting a gp120 molecule from a HIV-1 isolate of clade A.

We have recently developed a candidate HIV-1 vaccine based on virus-like particles (VLPs) expressing a gp120 from an Ugandan HIV-1 isolate of the clade A (HIV-VLP(A)s). In vivo immunogenicity experiments were performed in Balb/c mice, with an immunization schedule based on a multiple-dose regimen of HIV-VLP(A)s without adjuvants, showing a significant induction of both humoral and cellular immunity. The Env-specific cellular response was investigated in vitro, scoring for both the proliferative response of T helper cells and the cytolytic activity of cytotoxic T lymphocytes (CTLs). Furthermore, immune sera showed >50% neutralization activity against both the autologous field isolate and the heterologous T cell adapted B-clade HIV-1(IIIB) viral strain. This is one of the first examples of HIV-1 vaccines based on antigens derived from the A clade, which represents >25% of all isolates identified world wide. In particular, the A clade is predominant in sub-Saharan countries, where 70% of the global HIV-1 infections occur, and where vaccination is the only rational strategy for an affordable prevention against HIV-1 infection.

AIDS Vaccines↗

PubMed2001

Genetic characterization of contemporary wild-type measles viruses from Vietnam and the People's Republic of China: identification of two genotypes within clade H.

Genetic characterization was conducted on 17 wild-type measles viruses isolated near Hanoi, Vietnam, during 1998 as well as on eight viruses isolated in the Hunan, Hainan, Shandong, and Anhui provinces of the People's Republic of China during 1995, 1998, and 1999. Previous studies had shown that, compared to wild-type measles viruses found in other parts of the world, wild-type viruses from China were genetically distinct and comprised a new clade of viruses, clade H. In this study, sequence analyses of the nucleotides coding for the COOH terminal 150 amino acids of the nucleoprotein (N) and the entire hemagglutinin (H) protein indicated that although all of the viruses from Vietnam were members of clade H, they were clearly distinct from the Chinese viruses. With the exception of MVi/Beijing.China/94/1, the Vietnamese viruses differed from all of the Chinese viruses by at least 3.5 and 2.5% at the nucleotide level for the N and H genes, respectively. These data suggest that clade H should be divided into two genotypes with the Chinese viruses placed in genotype H1 and the Vietnamese viruses in genotype H2. Sequence analysis of measles viruses imported into the United States from either China or Vietnam demonstrated that this designation of genotypes will be helpful in future measles surveillance activities.

China↗

PubMed2003

Multiple new phenotypic taxa from trees and riparian ecosystems in Phytophthora gonapodyides-P. megasperma ITS Clade 6, which tend to be high-temperature tolerant and either inbreeding or sterile.

Phytophthora isolates associated with Phytophthora major ITS Clade 6 were grouped into 11 phenotypic taxa. These comprised the described morphospecies P. gonapodyides, P. megasperma s. str. and P. humicola; four previously identified but so far undescribed taxa, informally designated here P. sp. O-group, P. sp. Apple-cherry, P. taxon Pgchlamydo, and P. taxon Walnut; and four previously unknown taxa, designated P. taxon Oaksoil, P. taxon Raspberry, P. taxon Forestsoil, and P. taxon Riversoil. With the exception of P. gonapodyides, each phenotypic taxon represented an unique ITS lineage. Two isolates morphologically identical to P. gonapodyides comprised a separate lineage and probably represent another taxon, designated here P. taxon Salixsoil, P. humicola, P. sp. O-group, P. sp. Apple-cherry and P. taxon Walnut grouped together as subclade I. Within subclade II, P. taxon Oaksoil, P. taxon Raspberry, P. taxon Forestsoil, P. taxon Riversoil and P. taxon Pgchlamydo formed a cluster of closely related but phenotypically distinct lineages basal to P. gonapodyides and P. megasperma, P. taxon Salixsoil being the most basal member. The taxonomy, adaptation and breeding systems of Clade 6 taxa are discussed. They show a strong association with forests and riparian ecosystems, only a limited association with agriculture and an ability to tolerate high temperatures. Also, in contrast to most other Phytophthora clades, Clade 6 taxa are predominantly sterile or inbreeding in culture. Only one taxon, P. sp. O-group, appears classically A1/A2 heterothallic.

DNA Restriction Enzymes↗

PubMed2026

Scalable assembly of Ascaris mitogenomes from whole-genome data reveals a novel clade.

The genus Ascaris is an important group of giant parasitic roundworms, infecting over 700 million people globally and causing substantial economic losses in domestic pigs. Whilst species of Ascaris are morphologically indistinguishable, analysis of mitochondrial loci has revealed three clades (A, B, C) broadly associated with host species and geographic distribution. The diversity within these lineages may expand with the addition of further genomic data. Here, we present a bioinformatic framework for de novo assembly of complete mitochondrial genomes (mitogenomes) from low-coverage whole-genome data through host-read depletion or mtDNA read enrichment, followed by mtDNA-specific assembly. Our approach yielded 149 high-quality Ascaris mitogenome assemblies, enabling the study of population-level diversity, including the identification of a novel clade (Clade D, designated here) associated with human samples from Ethiopia. Our analysis further revealed Clade C to comprise of pig-derived samples from Europe based on characterisation of worms isolated in Germany. The methods described here provide a scalable framework for mitogenome reconstruction with insights into roundworm population-genomic and phylogenetic studies.

Animals↗

PubMed2001

Multiple causation of phylogeographical pattern as revealed by nested clade analysis of the bamboo viper (Trimeresurus stejnegeri) within Taiwan.

In order to assess the utility of nested clade analysis, both standard phylogenetic algorithms and nested clade analysis were performed on a geographically widespread survey of mitochondrial DNA haplotypes of the bamboo viper, Trimeresurus stejnegeri, within Taiwan. Gross tree topologies were congruent for all analyses and indicated the presence of two geographically overlapping clades within Taiwan. The smaller lineage was restricted to the north and east coasts, whereas the larger lineage occupied all but the northern range of the species within Taiwan including the Pacific offshore populations of Green and Orchid Islands. The phylogeographical pattern supports the existence of at least one colonization event from the continent since the initial isolation of Taiwan from the mainland in the Pliocene. However, determining the exact number of colonization events was not possible due to the simultaneous vicariant forces of hypothesized continental landbridge connections and the occurrence of dramatic in situ orogenesis throughout the Pleistocene. Nested clade analysis provided multiple temporal and spatial population historical inferences that are not possible with standard analyses and therefore should become widely applied to future phylogeographical studies.

Animals↗

PubMed1995

ndhF sequence evolution and the major clades in the sunflower family.

An extensive sequence comparison of the chloroplast ndhF gene from all major clades of the largest flowering plant family (Asteraceae) shows that this gene provides approximately 3 times more phylogenetic information than rbcL. This is because it is substantially longer and evolves twice as fast. The 5' region (1380 bp) of ndhF is very different from the 3' region (855 bp) and is similar to rbcL in both the rate and the pattern of sequence change. The 3' region is more A+T-rich, has higher levels of nonsynonymous base substitution, and shows greater transversion bias at all codon positions. These differences probably reflect different functional constraints on the 5' and 3' regions of ndhF. The two patterns of base substitutions of ndhF are particularly advantageous for phylogenetic reconstruction because the conserved and variable segments can be used for older and recent groups, respectively. Phylogenetic analyses of 94 ndhF sequences provided much better resolution of relationships than previous molecular and morphological phylogenies of the Asteraceae. The ndhF tree identified five major clades: (i) the Calyceraceae is the sister family of Asteraceae; (ii) the Barnadesioideae is monophyletic and is the sister group to the rest of the family; (iii) the Cichorioideae and its two basal tribes Mutisieae and Cardueae are paraphyletic; (iv) four tribes of Cichorioideae (Lactuceae, Arctoteae, Liabeae, and Vernonieae) form a monophyletic group, and these are the sister clade of the Asteroideae; and (v) the Asteroideae is monophyletic and includes three major clades.

Amino Acid Sequence↗

PubMed2001

Protein sequence signatures support the African clade of mammals.

DNA sequence evidence supports a superordinal clade of mammals that comprises elephants, sea cows, hyraxes, aardvarks, elephant shrews, golden moles, and tenrecs, which all have their origins in Africa, and therefore are dubbed Afrotheria. Morphologically, this appears an unlikely assemblage, which challenges-by including golden moles and tenrecs-the monophyly of the order Lipotyphla (Insectivora). We here identify in three proteins unique combinations of apomorphous amino acid replacements that support this clade. The statistical support for such "sequence signatures" as unambiguous synapomorphic evidence for the naturalness of the Afrotherian clade is reported. Using likelihood, combinatorial, and Bayesian methods we show that the posterior probability of the mammalian tree containing the Afrotherian clade is effectively 1.0, based on conservative assumptions. Presenting sequence data for another African insectivore, the otter shrew Micropotamogale lamottei, we demonstrate that such signatures are diagnostic for including newly investigated species in the Afrotheria. Sequence signatures provide "protein-morphological" synapomorphies that may aid in visualizing monophyletic groupings.

Africa↗

PubMed2004

SRP-2 is a cross-class inhibitor that participates in postembryonic development of the nematode Caenorhabditis elegans: initial characterization of the clade L serpins.

High molecular weight serpins are members of a large superfamily of structurally conserved proteins that inactivate target proteinases by a suicide substrate-like mechanism. In vertebrates, different clades of serpins distribute predominantly to either the intracellular or extracellular space. Although much is known about the function, structure, and inhibitory mechanism of circulating serpins such as alpha(1)-antitrypsin (SERPINA1) and antithrombin III (SERPINC1), relatively little is known about the function of the vertebrate intracellular (clade B) serpins. To gain a better understanding of the biology of the intracellular serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model system. A screen of the C. elegans genomic and cDNA databases revealed nine serpin genes, tandemly arrayed on chromosome V. Although the C. elegans serpins represent a unique clade (L), they share significant functional homology with members of the clade B group of intracellular serpins, since they lack typical N-terminal signal peptides and reside intracellularly. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2, was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a dual cross-class inhibitor of the apoptosis-related serine proteinase, granzyme B, and the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the intestine and hypoderm of larval and adult worms. Transgenic animals engineered to overexpress SRP-2 were slow growing and/or arrested at the first, second, or third larval stages. These data suggest that perturbations of serpin-proteinase balance are critical for correct postembryonic development in C. elegans.

Amino Acid Sequence↗

PubMed2004

Invertebrate data predict an early emergence of vertebrate fibrillar collagen clades and an anti-incest model.

Fibrillar collagens are involved in the formation of striated fibrils and are present from the first multicellular animals, sponges, to humans. Recently, a new evolutionary model for fibrillar collagens has been suggested (Boot-Handford, R. P., Tuckwell, D. S., Plumb, D. A., Farrington Rock, C., and Poulsom, R. (2003) J. Biol. Chem. 278, 31067-31077). In this model, a rare genomic event leads to the formation of the founder vertebrate fibrillar collagen gene prior to the early vertebrate genome duplications and the radiation of the vertebrate fibrillar collagen clades (A, B, and C). Here, we present the modular structure of the fibrillar collagen chains present in different invertebrates from the protostome Anopheles gambiae to the chordate Ciona intestinalis. From their modular structure and the use of a triple helix instead of C-propeptide sequences in phylogenetic analyses, we were able to show that the divergence of A and B clades arose early during evolution because alpha chains related to these clades are present in protostomes. Moreover, the event leading to the divergence of B and C clades from a founder gene arose before the appearance of vertebrates; altogether these data contradict the Boot-Handford model. Moreover, they indicate that all the key steps required for the formation of fibrils of variable structure and functionality arose step by step during invertebrate evolution.

Amino Acid Sequence↗

PubMed2003

Characterization of HIV type 1 clades in the Caribbean using pol gene sequences.

To date, 11 HIV-1 M group clades, A to K, have been characterized, displaying different distributions, prevalences, and biological properties. Approximately 90% of new HIV-1 infections occur in developing countries, including the Caribbean. However, information on HIV-1 subtypes from this region is limited. We report subtype characterization of viruses from 71 individuals, obtained during the period 2000-2002. RNA from the pol region was sequenced, generating data on subtype and drug resistance associated mutations for 71 specimens from 9 countries. Sixty-seven (94.4%) sequences were classified as clade B, three (4.2%) as D/B, and one (1.4%) as clade C. Numerous polymorphisms were observed, including some associated with drug resistance, but not signifying exposure to chemotherapy. This study adds to our knowledge of HIV-1 clades in the Caribbean, and indicates possibilities for monitoring HIV-1 chemotherapy.

Caribbean Region↗

PubMed2005

Studies in macaques on cross-clade T cell responses elicited by a DNA/MVA AIDS vaccine, better conservation of CD8 than CD4 T cell responses.

One of the unknowns faced by an HIV/AIDS vaccine is the ability of a single clade vaccine to protect against the multiple genetic subtypes and recombinant forms of HIV-1 present in the current pandemic. Here, we use a macaque model to investigate the ability of our clade B vaccine that consists of DNA priming and modified vaccinia Ankara (MVA) virus boosting to elicit T cell responses that recognize an A/G recombinant of HIV-1. To test for cross-reactive T cells, intracellular cytokine staining was conducted using five pools of Gag and six pools of Env peptides representing B or A/G sequences. Studies using the peptide pools revealed essentially complete conservation of the CD8 response but only approximately 50% conservation of the CD4 response. Thus, the ability of an HIV vaccine for one clade to protect against other clades may be more limited by the ability to provide CD4 T cell help than the ability to elicit CD8 effector functions.

AIDS Vaccines↗

PubMed2005

Cross-clade conservation of HIV type 1 Nef immunodominant regions recognized by CD8+ T cells of HIV type 1 CRF02_AG-infected Ivorian (West Africa).

Most HIV vaccine trials in the world are conducted with clade B while most circulating viral strains in Africa are non-B subtypes. We determined whether CD8+ T cells from HIV-1 intersubtype CRF02_AG-infected Ivorian individuals were able to recognize clade B epitopes. CD8+ T cell responses of nine HIV-1 intersubtype CRF02_AG-infected Ivorian patients and nine HIV-1 subtype B-infected French patients were studied using pools of HIV-1 clade B peptides (110 well-defined HIV CD8+ T cell epitopes) in an ELISPOT IFN-gamma assay. There was no difference in the number of recognized peptide pools between Ivorian and French cohorts (mean of four pools in both cases). Ivorian individuals had generated CD8+ T cell responses cross-reactive against HIV-1 subtype B and some individual peptides had been identified. Furthermore, sequence analysis of nef HIV genes of the Ivorian patients and nef cloning in two patients revealed very few variations between HIV- 1 intersubtype CRF02_AG and subtype B in nef immunodominant regions included in HIV clade B lipopeptide vaccines, currently tested in France.

Amino Acid Sequence↗

PubMed1997

CD4+ T-cell recognition of diverse clade B HIV-1 isolates.

OBJECTIVES: To evaluate the effect of sequence variation within the gp120 V3 loop on CD4 T-cell recognition. DESIGN: CD4 T-cell clones were generated using synthetic peptides to circumvent the difficulties of using polyclonal T-cell responses. Peptides based on other HIV isolates were then used to determined the influence of single and multiple sequence differences. RESULTS: Three of the panels of T-lymphocyte clones (TLC), which were all specific to diverse HIV-1 clade B gp120 V3-loop peptides differing in a limited number of residues, had heterogeneous patterns of response to peptides differing in length and sequence indicating that they recognized distinct but overlapping epitopes. The panels of TLC also differed in the extent to which they tolerated sequence differences between cell-culture-adapted or primary HIV-1 isolates. One panel responded to peptides based on several clade B and one clade D isolate. In contrast, two panels, generated from two different donors using the same peptide, only responded to a limited number of clade B isolates, whereas another only recognized HIV-1BRU. Two of the panels were also stimulated by peptides based on clinical isolates from one patient with some sequence changes enhancing T-cell recognition. CONCLUSIONS: These data are consistent with highly diverse CD4 T-cell recognition of the HIV-1 gp120 V3 loop, which is influenced by the sequence differences within the T-cell epitopic region and has implications for the pathogenesis and design of vaccines against HIV-1.

AIDS Vaccines↗

PubMed2005

Culturing and environmental DNA sequencing uncover hidden kinetoplastid biodiversity and a major marine clade within ancestrally freshwater Neobodo designis.

Bodonid flagellates (class Kinetoplastea) are abundant, free-living protozoa in freshwater, soil and marine habitats, with undersampled global biodiversity. To investigate overall bodonid diversity, kinetoplastid-specific PCR primers were used to amplify and sequence 18S rRNA genes from DNA extracted from 16 diverse environmental samples; of 39 different kinetoplastid sequences, 35 belong to the subclass Metakinetoplastina, where most group with the genus Neobodo or the species Bodo saltans, whilst four group with the subclass Prokinetoplastina (Ichthyobodo). To study divergence between freshwater and marine members of the genus Neobodo, 26 new Neobodo designis strains were cultured and their 18S rRNA genes were sequenced. It is shown that the morphospecies N. designis is a remarkably ancient species complex with a major marine clade nested among older freshwater clades, suggesting that these lineages were constrained physiologically from moving between these environments for most of their long history. Other major bodonid clades show less-deep separation between marine and freshwater strains, but have extensive genetic diversity within all lineages and an apparently biogeographically distinct distribution of B. saltans subclades. Clade-specific 18S rRNA gene primers were used for two N. designis subclades to test their global distribution and genetic diversity. The non-overlap between environmental DNA sequences and those from cultures suggests that there are hundreds, possibly thousands, of different rRNA gene sequences of free-living bodonids globally.

Animals↗