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PubMed2006

Sequencing and comparative analysis of a pig bovine viral diarrhea virus genome.

In present study, we report the first complete genomic sequence of pig bovine viral diarrhea (BVD) virus, that of strain ZM-95, which is 12,220 nucleotides long and contains short 5' and 3' non-coding regions and one open reading frame encoding a large polyprotein with 28 potential N-glycosylation sites (Asn-X-Ser or Asn-X-Thr). Within the non-structural protein encoding region, no foreign nucleotide insertions was found as those usually observed for cytopathogenic BVDV-1, but close to the 3'-terminal of the capsid protein (1119-1124bp) it contains a short insertion of a six nucleotide sequence (CTCACA). Three hypervariable regions were identified in the polyprotein-encoding region, with one of them comprising a sequence motif encoding a unique five amino acid peptide HYKKK in glycoprotein E2 gene. The genomic comparison and phylogenetic analyses showed that ZM-95 should be classified into BVDV-1, but was genetically divergent from other pestiviruses sequenced to date since its highest genetic similarity was only 76.6% (with SD-1), therefore, placed as a novel subgroup of BVDV-1.

3' Untranslated Regions↗

PubMed2006

Molecular phylogeny of the Cricetinae subfamily based on the mitochondrial cytochrome b and 12S rRNA genes and the nuclear vWF gene.

Despite some popularity of hamsters as pets and laboratory animals there is no reliable phylogeny of the subfamily Cricetinae available so far. Contradicting views exist not only about the actual number of species but also concerning the validity of several genera. We used partial DNA sequences of two mitochondrial (cytochrome b, 12S rRNA) and one partial nuclear gene (von Willebrand Factor exon 28) to provide a first gene tree of the Cricetinae based on 15 taxa comprising six genera. According to our data, Palaearctic hamsters fall into three distinct phylogenetic groups: Phodopus, Mesocricetus, and Cricetus-related species which evolved during the late Miocene about 7-12MY ago. Surprisingly, the genus Phodopus, which was previously thought to have appeared during the Pleistocene, forms the oldest clade. The largest number of extant hamster genera is found in a group of Cricetus-related hamsters. The genus Cricetulus itself proved to be not truly monophyletic with Cricetulus migratorius appearing more closely related to Tscherskia, Cricetus, and Allocricetulus. We propose to place the species within a new monotypic genus. Molecular clock calculations are not always in line with the dating of fossil records. DNA based divergence time estimates as well as taxonomic relationships demand a reevaluation of morphological characters previously used to identify fossils and extant hamsters.

Animals↗

PubMed1999

Cloning and characterisation of cDNA encoding the Trypanosoma brucei ribosomal protein L24.

A cDNA encoding ribosomal protein L24 was amplified by PCR from the protozoan parasite, Trypanosoma brucei. The 621 nucleotide cDNA had an open reading frame of 375 nucleotides, predicting a highly basic protein of 125 aa. Database searches revealed 33-40% identity between the T. brucei RPL24 protein and several eukaryotic RPL24 homologues. Southern blot analysis indicated that the gene was present as a single copy, and a transcript of approximately 620 nucleotides was detected in procyclic forms of the parasite. Interestingly, T. brucei PRL24 is the smallest eukaryotic RPL24 protein described to date. It is also the most divergent of the known kinetoplastid ribosomal proteins.

Amino Acid Sequence↗

PubMed1999

Sequence, expression and genetic mapping of a rainbow trout retinoblastoma cDNA.

A full-length cDNA for retinoblastoma (RB1) has been cloned from a cDNA library prepared from 3-week-old rainbow trout (Oncorhynchus mykiss) eyed embryos. The trout RB1 cDNA encodes a predicted protein of 910 amino acids and is the most divergent cloned retinoblastoma gene sequence to date. RT-PCR studies reveal high levels of RB1 expression by the second week of embryogenesis, which remains uniformly expressed until hatching. Expression studies of adult fish tissues show the RB1 gene to be expressed in all tissues examined, including the oesophagus, eye, liver, intestine, posterior and anterior kidney, skin, stomach, muscle, spleen, gill, swim bladder, gonads and brain. The RB1 gene appears to be a single copy gene based on Southern analysis, and maps to linkage group XVI in the trout genome map. Polymorphisms in the RB1 gene and in closely linked markers should facilitate LOH analysis of RB1.

Amino Acid Sequence↗

PubMed2003

[Diagnostic and therapeutic problems in chronic progressive external ophthalmoplegia (CPEO)].

BACKGROUND: Strictly speaking, CPEO is defined as paralysis of the external ocular muscles combined with ptosis. In Kearns-Sayre syndrome additional neurological or muscular deficits can be observed, sometimes even decades after the onset of ocular symptoms. METHOD: Three patients with classical CPEO and one patient with Kearns-Sayre syndrome where included in the present study. The clinical examinations included electromyography, various serological parameters and histological biopsies. RESULTS: The onset of ocular symptoms with divergent strabism and ptosis could be dated back to the age of 7, 27, 38 and 44 years starting on one eye and progressing slowly over years to both eyes. In the Kearns-Sayre syndrome patient a general decrease in cardio-respiratory fitness and occasional collapses were noted. In three patients ptosis and strabism surgery led to the relief of the ocular symptoms. CONCLUSIONS: In CPEO respectively Kearns-Sayre syndrome the onset of the disease is variable. The final correct diagnosis is often delayed due to the mild beginning of the symptoms and the slow progression of the paralyses. Early EMG and muscle biopsy examination may facilitate the diagnosis.

Adult↗

PubMed2005

To B or Not to B a flower: the role of DEFICIENS and GLOBOSA orthologs in the evolution of the angiosperms.

DEFICIENS (DEF) and GLOBOSA (GLO) function in petal and stamen organ identity in Antirrhinum and are orthologs of APETALA3 and PISTILLATA in Arabidopsis. These genes are known as B-function genes for their role in the ABC genetic model of floral organ identity. Phylogenetic analyses show that DEF and GLO are closely related paralogs, having originated from a gene duplication event after the separation of the lineages leading to the extant gymnosperms and the extant angiosperms. Several additional gene duplications followed, providing multiple potential opportunities for functional divergence. In most angiosperms studied to date, genes in the DEF/GLO MADS-box subfamily are expressed in the petals and stamens during flower development. However, in some angiosperms, the expression of DEF and GLO orthologs are occasionally observed in the first and fourth whorls of flowers or in nonfloral organs, where their function is unknown. In this article we review what is known about function, phylogeny, and expression in the DEF/GLO subfamily to examine their evolution in the angiosperms. Our analyses demonstrate that although the primary role of the DEF/GLO subfamily appears to be in specifying the stamens and inner perianth, several examples of potential sub- and neofunctionalization are observed.

DEFICIENS Protein↗

PubMed1993

Zebrafish pou[c]: a divergent POU family gene ubiquitously expressed during embryogenesis.

We report the isolation and characterization of cDNA for a novel zebrafish (Brachyodanio rerio) POU domain gene, pou[c], which is ubiquitously expressed during embryonic development. This gene encodes a 610 amino acids long protein with a 149 amino acid POU domain ending only 8 residues before the C terminus. The 453 amino acids long region N-terminal to the POU domain contains several features typical of transcriptional activation domains such as an acidic region with a putative amphipathic alpha-helix, a glutamine-rich region, and short threonine- and/or serine-rich regions. Comparison of the POU domain of pou[c] to other known POU sequences clearly show that pou[c] has the most divergent POU domain sequence reported to date. Thus, we suggest that pou[c] should be placed as the presently sole member of a new, sixth class of POU proteins. DNA-binding studies revealed that pou[c] is not an octamer-binding transcription factor like the Oct proteins described from mammals, chicken and Xenopus. Rather, pou[c] binds with high affinity to the TAATGARAT motif found in the promoters of the herpes simplex virus immediate early genes and to degenerate octamer-TAATGA motifs. Circular permutation analyses also show that pou[c] induces DNA bending upon sequence-specific binding.

Amino Acid Sequence↗

PubMed2003

Consensus-degenerate hybrid oligonucleotide primers for amplification of priming glycosyltransferase genes of the exopolysaccharide locus in strains of the Lactobacillus casei group.

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3' degenerate core based on four highly conserved amino acids and a longer 5' consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS(-)) and EPS-producing (EPS(+)) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS(+) bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS(+) strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.

Amino Acid Sequence↗

PubMed1998

A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost F. heteroclitus.

The killifish, Fundulus heteroclitus, is a euryhaline teleost fish capable of adapting rapidly to transfer from freshwater (FW) to four times seawater (SW). To investigate osmoregulation at a molecular level, a 5.7-kilobase cDNA homologous to human cystic fibrosis transmembrane conductance regulator (hCFTR) was isolated from a gill cDNA library from SW-adapted killifish. This cDNA encodes a protein product (kfCFTR) that is 59% identical to hCFTR, the most divergent form of CFTR characterized to date. Expression of kfCFTR in Xenopus oocytes generated adenosine 3',5'-cyclic monophosphate-activated, Cl(-)-selective currents similar to those generated by hCFTR. In SW-adapted killifish, kfCFTR was expressed at high levels in the gill, opercular epithelium, and intestine. After abrupt exposure of FW-adapted killifish to SW, kfCFTR expression in the gill increased severalfold, suggesting a role for kfCFTR in salinity adaptation. Under similar conditions, plasma Na+ levels rose significantly after 8 h and then fell, although it is not known whether these changes are directly responsible for the changes in kfCFTR expression. The killifish provides a unique opportunity to understand teleost osmoregulation and the role of CFTR.

Adaptation, Physiological↗

PubMed2004

[Change in nucleotide sequence of mitochondrial DNA from cytochrome b gene in chars of Salvelinus genus].

Nucleotide sequences of two (405- and 1050-bp) regions of mitochondrial DNA (mtDNA) cytochrome c gene were established in chars of the genus Salvelinus from Russian Far East and Siberia. Based on the divergence and phylogenetic analysis of nucleotide sequences of the mtDNA cytochrome c gene, S. laecomaenis was shown to carry the most ancient mitochondrial lineage, which is close to the ancestral one. The archaic mtDNA of S. levanidovi occupied an isolated position on the phylogenetic trees. The mtDNA lineage of the southern S. malma was close to the S. alpinus-S. malma complex group. Within the S. alpinus-S. malma complex, three groups of mtDNA types having particular geographic distributions were distinguished. The Kolyma-Chukotka group includes lake S. taranetzi, S. boganidae and S. elgyticus from Chukotka, lake chars from Kolyma. The Okhotsk group is represented by northern S. malma, lake chars from northern Sea of Okhotsk, and anadromous S. taranetzi. The Siberian group is close to the Okhotsk one and consists of Taimyr and Baikal region chars as well as Arctic char from Finland. The divergence of char mitochondrial lineages was dated to the Pliocene-Pleistocene.

Animals↗

PubMed2001

Comparison of systems performance for TT virus detection using PCR primer sets located in non-coding and coding regions of the viral genome.

BACKGROUND: the heterogeneity of the TT virus (TTV) DNA prevalence values reported from comparable human cohorts suggests that diagnostic PCR protocols still require to be optimized. OBJECTIVES: to design TTV PCR primer sets with low genotype restriction and to compare their performances with commonly used amplification systems. STUDY DESIGN: we compared full length TTV genomic sequences and identified conserved nucleotide patterns in the 5' and 3' non-coding regions of the viral genome. This permitted to design two new primer sets usable for the PCR amplification of the most divergent human isolates of TTV described to date. The performances of these amplification systems were compared with those of three other PCR systems earlier used for prevalence studies. RESULTS: the primer systems P5Bx and P3Bx exhibited higher PCR scores than the other systems tested; 14 to 34% improvement values were obtained, and divergent positive results of earlier described PCR systems were confirmed systematically by our new detection assays. CONCLUSIONS: an optimized detection of TT virus DNA is a pre-requisite for the accurate epidemiological survey of viral infection and for the realization of phylogenetic studies. Such PCR systems with low genotype restriction will be helpful in the future for a better knowledge of natural history of TT virus infection.

3' Untranslated Regions↗

PubMed1984

Calibrating the molecular clock: estimates of ground squirrel divergence made using fossil and geological time markers.

According to 5-Myr-old fossil evidence, ground squirrels within the genus Spermophilus had diverged into subgenera Spermophilus and Otospermophilus by late Miocene times. Radiometric dating has also provided a precise time for the sudden onset of a geological event, occurring 0.725 Myr ago, that initiated the complete and permanent reproductive isolation of two subspecies within the subgenus Otospermophilus. Since these two subspecies (S. beecheyi beecheyi and S. b. douglasii) readily hybridize with each other under laboratory conditions, allopatric subspeciation is unlikely to have occurred prior to 0.725 Myr ago. We employed Nei's model for estimating genetic distance in units which are linear in time, calibrated on the 0.725-Myr-ago date for initiation of S. b. subspeciation, to test its ability to generate a time scale for subgeneric divergence in keeping with the minimum estimate provided by the fossil record. This represents the most valid test to date of the utility of Nei's model for estimating genetic distance in units which are linear in time. Nei's model was found to underestimate this minimum time by 1 Myr, but it approximated this date after correcting values of D for variation in rates of evolution among loci.

Animals↗

PubMed2005

The age of the angiosperms: a molecular timescale without a clock.

The age of the angiosperms has long been of interest to botanists and evolutionary biologists. Many early efforts to date the age of the angiosperms and evolutionary divergences within the angiosperm clade using a molecular clock have yielded age estimates that are grossly inconsistent with the fossil record. We investigated the age of angiosperms using Bayesian relaxed clock (BRC) and penalized likelihood (PL) approaches. Both of these methods allow the incorporation of multiple fossil constraints into the optimization procedure. The BRC method allows a range of values for among-lineage rate of substitution, from a nearly clocklike behavior to a condition in which each branch is allowed an optimal substitution rate, and also accounts for variation in molecular evolution across multiple genes. A topology derived from an analysis of genes from all three plant genomes for 71 taxa was used as a backbone. The effects on age estimates of different genes, single-gene versus concatenated datasets, and the inclusion and assumptions of fossils as age constraints were examined. In addition, the influence of prior distributions on estimates of divergence times was also explored. These results indicate that widely divergent age estimates can result from the different methods (198-139 million years ago), different sources of data (275-122 million years ago), and the inclusion of temporal constraints to topologies. Most dates, however, are between 180-140 million years ago, suggesting a Middle Jurassic-Early Cretaceous origin of flowering plants, predating the oldest unequivocal fossil angiosperms by about 45-5 million years. Nonetheless, these dates are consistent with other recent studies that have used methods that relax the assumption of a strict molecular clock and also agree with the hypothesis that the angiosperms may be somewhat older than the fossil record indicates.

Bayes Theorem↗

PubMed1989

Variations in the reporting of gestational age at induced termination of pregnancy.

Utilizing the 1980 Induced Abortion File maintained by the National Center for Health Statistics, we compared gestational age from date of last normal menses and the physician-based estimate of gestational age. An average .51 week difference between the two methods was observed. Beyond seven weeks gestation, the date of last normal menses value was underestimated by the physician-based estimate with a markedly greater divergence after 20 weeks. A relatively greater underestimation of the date of last normal menses interval by the physician estimate was apparent for Whites after 13 weeks. The data of last normal menses value for non-state residents was overestimated across the entire range of the date of last normal menses gestational age distribution until 21 weeks.

Abortion, Induced↗

PubMed2002

Slow molecular clocks in Old World monkeys, apes, and humans.

Two longstanding issues on the molecular clock hypothesis are studied in this article. First, is there a global molecular clock in mammals? Although many authors have observed unequal rates of nucleotide substitution among mammalian lineages, some authors have proposed a global clock for all eutherians, i.e., a single global rate of 2.2 x 10(-9) substitutions per nucleotide site per year. We reexamine this issue using noncoding, nonrepetitive DNA from Old World monkeys (OWMs), chimpanzee, and human. First, using the minimal date of 6 MYA for the human-chimpanzee divergence and more than 2.5 million base pairs of genomic sequences from human and chimpanzee, we estimate a maximal rate of 0.99 x 10(-9) for noncoding, nonrepetitive genomic regions for these two species. This estimate is less than half of the proposed global rate and much smaller than the commonly used rate (3.5 x 10(-9)) for eutherians. Further, using a minimal date of 23 MYA for the human-OWM divergence, we estimate a maximal rate of 1.5 x 10(-9) for both introns and fourfold degenerate sites in humans and OWMs. In addition, with the New World monkey (NWM) lineage as an outgroup, we estimate that the rate of substitution in introns is 30% higher in the OWM lineage than in the human lineage. Clearly, there is no global molecular clock in eutherians. Second, although many studies have indicated considerable variation in the mutation rate among regions of the mammalian genome, a recent study proposed a uniform rate. Using new and existing intron sequence data from higher primates, we find significant rate variation among genomic regions and a positive correlation between the rate of substitution and the GC content, refuting the claim of a uniform rate.

Animals↗

PubMed2001

The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua.

The mammalian order Xenarthra (armadillos, anteaters and sloths) is one of the four major clades of placentals, but it remains poorly studied from the molecular phylogenetics perspective. We present here a study encompassing most of the order's diversity in order to establish xenarthrans' intra-ordinal relationships, discuss the evolution of their morphological characters, search for their extant sister group and specify the timing of their radiation with special emphasis on the status of the controversial fossil Eurotamandua. Sequences of three genes (nuclear exon 28 of the Von Willebrand factor and mitochondrial 12S and 16S rRNAs) are compared for eight of the 13 living genera. Phylogenetic analyses confirm the order's monophyly and that of its three major lineages: armadillos (Cingulata), anteaters (Vermilingua) and sloths ('Tardigrada', renamed in 'Folivora'), and our results strongly support the grouping of hairy xenarthrans (anteaters and sloths) into Pilosa. Within placentals, Afrotheria might be the first lineage to branch off, followed by Xenarthra. The morphological adaptative convergence between New World xenarthrans and Old World pangolins is confirmed. Molecular datings place the early emergence of armadillos around the Cretaceous/Tertiary boundary, followed by the divergence between anteaters and sloths in the Early Eocene era. These Tertiary dates contradict the concept of a very ancient origin of modern xenarthran lineages. They also question the placement of the purported fossil anteater (Eurotamandua) from the Middle Eocene period of Europe with the Vermilingua and instead suggest the independent and convergent evolution of this enigmatic taxon.

Animals↗

PubMed2002

Genetic diversity within human erythroviruses: identification of three genotypes.

B19 virus is a human virus belonging to the genus Erythrovirus: The genetic diversity among B19 virus isolates has been reported to be very low, with less than 2% nucleotide divergence in the whole genome sequence. We have previously reported the isolation of a human erythrovirus isolate, termed V9, whose sequence was markedly distinct (>11% nucleotide divergence) from that of B19 virus. To date, the V9 isolate remains the unique representative of a new variant in the genus Erythrovirus, and its taxonomic position is unclear. We report here the isolation of 11 V9-related viruses. A prospective study conducted in France between 1999 and 2001 indicates that V9-related viruses actually circulate at a significant frequency (11.4%) along with B19 viruses. Analysis of the nearly full-length genome sequence of one V9-related isolate (D91.1) indicates that the D91.1 sequence clusters together with but is notably distant from the V9 sequence (5.3% divergence) and is distantly related to B19 virus sequences (13.8 to 14.2% divergence). Additional phylogenetic analysis of partial sequences from the V9-related isolates combined with erythrovirus sequences available in GenBank indicates that the erythrovirus group is more diverse than thought previously and can be divided into three well-individualized genotypes, with B19 viruses corresponding to genotype 1 and V9-related viruses being distributed into genotypes 2 and 3.

Antibodies, Viral↗

PubMed2004

New nuclear evidence for the oldest divergence among neognath birds: the phylogenetic utility of ZENK (i).

To date, there is little consensus concerning the phylogenetic relationships among neognath orders, which include all extant birds except ratites and tinamous. Different data sets, both molecular and morphologic, have yielded radically different and often unresolved ordinal topologies, especially within the neoaves clade. This lack of resolution and ongoing conflict indicates a need for additional phylogenetic characters to be applied to the question of higher-level avian phylogeny. In this study, sequences of a single-copy nuclear gene, ZENK, were used to reconstruct an ordinal-level phylogeny of neognath birds. Strong support was indicated for the oldest divergence within Neognathae; the chicken- and duck-like birds formed a clade that was sister to all other modern birds. In addition, many families of traditional taxonomic orders clustered together in the ZENK tree, indicating the gene's general phylogenetic reliability. However, within the neoaves clade, there was little support for relationships among orders, which is a result similar to all other recent molecular studies of higher-level avian phylogeny. This similarity among studies suggests the possibility of a rapid radiation of the major neoaves lineages. Despite the ongoing lack of neoaves resolution, ZENK's sequence divergence and base composition patterns indicate its general utility as a new phylogenetic marker for higher-level avian systematics.

3' Untranslated Regions↗