Molecular evolution of Mycoplasma capricolum subsp. capripneumoniae strains, based on polymorphisms in the 16S rRNA genes

Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP.     

The structure underlying physical performance measures for older adults in the community

Nucleotide sequence variations in a region of the mitochondrial cytochrome c oxidase subunit I (COI) gene (391 bp) were examined within seven species of the genus Taenia and two species of the genus Echinococcus, including ten isolates of T. taeniaeformis and six isolates of E. multilocularis. More than a 12% rate of nucleotide differences between taeniid species was found, allowing the species to be distinguished. In E. multilocularis, no sequence variation was observed among isolates, regardless of the host (gray red-backed vole, tundra vole, pig, Norway rat) or area (Japan, Alaska) from which each metacestode had been isolated. In contrast, six distinct sequences were detected among the ten T. taeniaeformis isolates examined. The level of nucleotide variation in the COI gene within T. taeniaeformis isolates except for one isolate from the gray red-backed vole (TtACR), which has been proposed as a distinct strain or a different species, was about 0.3%-4.1%, whereas the COI gene sequence for TtACR differed from those of the other isolates, with levels being 9.0%-9.5%. Phylogenetic trees were then inferred from these sequence data using two different algorithms.     

Sequence variation within three important cytomegalovirus gene regions in isolates from four different patient populations

We determined the nucleotide (nt) and amino acid (aa) heterogeneities of three distinct regions of the human cytomegalovirus (CMV) genome for 46 low-passage CMV isolates from four different patient populations (congenitally infected infants, children attending day-care centers, renal transplant recipients, and human immunodeficiency virus-infected individuals) and for two laboratory strains (CMV Ad169 and Towne). The gene regions for the major immediate-early (MIE) exon 4 gene (nt positions 1702 to 1982, aa positions 152 to 244), the DNA polymerase gene (nt positions 2797 to 3046, aa positions 713 to 795), and the glycoprotein B (gB) gene (nt positions 1698 to 1884, aa positions 567 to 628) were sequenced. The sequence information was used to design sets of nested PCR primers directed against the most highly conserved regions identified. MIE was the most variable gene region compared to the variability of the DNA polymerase and gB gene regions. Comparison of the sequences of all 46 isolates with that of Ad169 revealed nt and aa sequence homologies of 87.9 and 87.2%, respectively, within the MIE gene compared to 92.8 and 100% homologies, respectively, within the DNA polymerase gene and 93 and 95.2% homologies, respectively, within the gB gene. Within the MIE gene, compared to the Ad169 nt sequence the homology at the nt level among isolates obtained from children attending day-care centers was high (96.4%), while it was lower (90%) among isolates obtained from the other three patient populations. Preliminary results of a nested PCR with oligonucleotide primers selected from the DNA polymerase gene region with a low level of nt sequence variation indicates that primers selected from this region might be more powerful for use in PCR than primers selected from the MIE gene region.     

Analysis of the type 1 pilin gene cluster fim in Salmonella: its distinct evolutionary histories in the 5' and 3' regions

The type 1 pilin encoded by fim is present in both Escherichia coli and Salmonella natural isolates, but several lines of evidence indicate that similarities at the fim locus may be an example of independent acquisition rather than common ancestry. For example, the fim gene cluster is found at different chromosomal locations and with distinct gene orders in these closely related species. In this work we examined the fim gene cluster of Salmonella, the genes of which show high nucleotide sequence divergence from their E. coli counterparts, as well as a different G+C content and codon usage. DNA hybridization analysis revealed that, among the salmonellae, the fim gene cluster is present in all isolates of S. enterica but is absent from S. bongori. Molecular phylogenetic analyses of the fimA and fimI genes yield an estimate of phylogeny that is in satisfactory congruence with housekeeping and other virulence genes examined in this species. In contrast, phylogenetic analyses of the fimZ, fimY, and fimW genes indicate that horizontal transfer of this region has occurred more than once. There is also size variation in the fimZ, fimY, and fimW intergenic regions in the 3' region, and these genes are absent in isolate S2983 of subspecies IIIa. Interestingly, the G+C contents of the fimZ, fimY, and fimW genes are less than 46%, which is considerably lower than those of the other six genes of the fim cluster. This study demonstrates that horizontal transmission of all or part of the same gene cluster can occur repeatedly, with the result that different regions of a single gene cluster may have different evolutionary histories.     

Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences

Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis and feminization. While it is known that the bacterial strains responsible for these different host phenotypes form a single clade within the alpha-Proteobacteria, until now it has not been possible to resolve the evolutionary relationships between different Wolbachia strains. To address this issue we have cloned and sequenced a gene encoding a surface protein of Wolbachia (wsp) from a representative sample of 28 Wolbachia strains. The sequences from this gene were highly variable and could be used to resolve the phylogenetic relationships of different Wolbachia strains. Based on the sequence of the wsp gene from different Wolbachia isolates we propose that the Wolbachia pipientis clade be initially divided into 12 groups. As more sequence information becomes available we expect the number of such groups to increase. In addition, we present a method of Wolbachia classification based on the use of group-specific wsp polymerase chain reaction (PGR) primers which will allow Wolbachia isolates to be typed without the need to clone and sequence individual Wolbachia genes. This system should facilitate future studies investigating the distribution and biology of Wolbachia strains from large samples of different host species.     

Antigenic heterogeneity and molecular analysis of CopB of Moraxella (Branhamella) catarrhalis

Outer membrane protein (OMP) CopB, an iron-repressible 81-kDa major OMP of Moraxella (Branhamella) catarrhalis has been a major focus of investigation. To assess CopB as a potential vaccine antigen, we elucidated the degree of antigenic and sequence heterogeneity in this protein among strains of M. catarrhalis. Two monoclonal antibodies, 1F5 and 2.9F, which bind to surface-exposed epitopes on CopB recognized 60 and 70% of the strains, respectively. The degree of sequence heterogeneity in CopB was assessed by cloning and sequencing the CopB gene from two different strains of M. catarrhalis and comparing with the published sequence. There was 92 to 96% homology between the sequences at the nucleotide level and 90 to 95% homology at the amino acid level. The variability in the protein sequence is confined mainly to three moderately variable regions. Restriction fragment length polymorphism (RFLP) analysis of the CopB genes obtained from 20 diverse strains by PCR was performed. Ninety percent of the potential restriction sites in the constant regions and 47% of the potential restriction sites in the variable regions were present in the 20 strains, indicating that the pattern of variable and constant areas in the CopB gene is a general pattern among strains of M. catarrhalis. We conclude that the CopB gene is largely conserved among strains of M. catarrhalis and contains discrete regions which show moderate heterogeneity among strains.     

Comparison of circumsporozoite proteins from avian and mammalian malarias: biological and phylogenetic implications

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.     

Interferon treatment of chronic hepatitis C in human immunodeficiency virus infected patients]

There are no reports to date of entire gene sequences coding for chitinolytic enzymes from entomopathogenic fungi, even though these enzymes act synergistically with proteolytic enzymes to solubilize insect cuticle during the key step of host penetration, having considerable importance in the biological control of some insect pests. This paper reports the complete nucleotide sequence and analysis of the chromosomal and full-length cDNA copies of the regulated gene (chit1) coding one of the chitinases produced by the biocontrol agent Metarhizium anisopliae. Degenerated primers, encompassing conserved regions of other fungal chitinases, were used to amplify a 650-bp DNA fragment, which was used to isolate genomic and cDNA clones from M. anisopliae. Albeit at least two different chitinases are characterized in this fungus, only one chit gene was isolated. The chit1 gene is interrupted by three short typical fungal introns and has a 1,521-bp ORF, which encodes a protein of 423 amino acids with a stretch of 35 amino acid residues displaying characteristics of signal peptide. The deduced sequence of the mature protein predicts a 42-kDa protein with pI of 5.8. Southern analysis of genomic DNA indicates a single copy of chit1 in the M. anisopliae genome.     

The mouse Vcs2 gene is a composite structure which evolved by gene fusion and encodes five distinct salivary mRNA species

Genes of the VCS (variable coding sequence) family are characterized by an extensive evolutionary divergence in the protein-coding sequence. The VCS family has been characterized by cDNA cloning from submandibular glands in the rat, mouse and humans. At the genomic level, the sequences of two members of this family are known in the rat Rattus norvegicus: the VCSA1 gene, encoding the prohormone-like polypeptide SMR1, and the VCSB1 gene, encoding a salivary Pro-rich polypeptide. No genomic data were available for the VCS genes of other species. To understand the evolution of the VCS gene family better, we have now sequenced 23 kilobases (kb) of the mouse Vcs2 gene. The Vcs2 sequence reveals numerous genomic reorganizations such as an inversion, insertions of short elements and an unusually high number of long interspersed repeated elements (LINEs), which make up 42% of this region. Interestingly, Vcs2 is composed of three different VCS-like regions. The first of these regions contains all the exons necessary to encode the previously described mouse submandibular gland polypeptide MSG2alpha. This region aligns with the entire genomic sequences of rat VCSA1 and VCSB1 genes. The two other regions align with fragments of these rat sequences. The three regions are arrayed in tandem and flanked by LINEs. In particular, the third region also contains exons that were found in mRNA species from the submandibular gland. In total, we have characterized five mRNAs from mouse submandibular glands which have in common their first exon, and are produced by alternative splicing. Vcs2 is thus a single gene that arose by the fusion of three genes (or pseudogenes) of the VCS multigene family.     

Tolerance, compliance and psychological consequences of post-exposure prophylaxis in health-care workers

We have cloned, from an oribatid mite, a gene homologous to the zerknült (zen) genes of insects and the Hox 3 genes of vertebrates. Hox genes specify cell fates in specific regions of the body in all metazoans studied and are expressed in antero-posteriorly restricted regions of the embryo. This is true of the vertebrate Hox 3 but not of the zen genes, the insect homologs, and it has been proposed that the zen genes have lost their Hox-like function in the ancestor of the insects. We studied expression of a mite Hox 3/zen homolog and found that it is expressed in a discrete antero-posterior region of the body with an anterior boundary coinciding with that of the chelicerate homolog of the Drosophila Hox gene, proboscipedia, and propose that its loss of Hox function in insects is due to functional redundancy due to this overlap with another Hox gene.     

Evolutionary analysis of the hisCGABdFDEHI gene cluster from the archaeon Sulfolobus solfataricus P2

While sequencing the genome of the archaeon Sulfolobus solfataricus P2, we found an 8,313-bp sequence containing a cluster of nine histidine biosynthesis genes in an order different from that of any known his operon. Results of phylogenetic analysis of the coding regions in the putative operon give conflicting evolutionary histories for individual his genes.     

ALS and SAD-like nicotinic acetylcholine receptor subunit genes are widely distributed in insects

Segments of nicotinic acetylcholine receptor alpha subunit genes have been isolated from a panel of insect species by polymerase chain reaction, using degenerate oligonucleotide primers designed to recognize conserved regions of the Drosophila melanogaster ALS and SAD genes. The amplified segments encode elements of typical alpha-subunits anticipated to play roles in ligand binding and ion channel formation. Each is also clearly either ALS or SAD-like. The predicted protein sequences display extremely high levels of conservation (over 85% for each subtype) even though derived from very distantly related insect species.     

Avian IFN-gamma genes: sequence analysis suggests probable cross-species reactivity among galliforms

Little is known about the evolution of cytokines in non-mammalian systems. To address this problem, we attempted to clone the gene for interferon-gamma (IFN-gamma) from a variety of avian species using oligonucleotide primers based on the sequence of the chicken IFN-gamma gene. The coding sequence and partial intron sequences were determined for four species, namely guinea fowl, ring-necked pheasant, Japanese quail, and turkey. To obtain sequence information on the gene extremities, a modified 5' and 3' RACE protocol was used. The sequence information showed that the coding regions of the IFN-gamma gene are highly conserved among the species studied (93.5%-96.7% and 87.8%-97.6% at the nucleotide and peptide levels, respectively) and are more conserved at the amino-terminal region (exons 1 and 2) than the carboxyl-terminal (exons 3 and 4). This high degree of overall identity at the predicted primary amino acid sequence level of the protein, including the deduced IFN-gamma receptor binding motifs, suggests that IFN-gamma may be cross-reactive among these species. Phylogenetic analysis shows that the similarity of the avian IFN-gamma sequences parallels the presumed evolutionary relationships between the species.     

Evolutionary shifts in three major structural features of the mitochondrial genome among iguanian lizards

A phylogenetic tree for major lineages of iguanian lizards is estimated from 1,488 aligned base positions (858 informative) of newly reported mitochondrial DNA sequences representing coding regions for eight tRNAs, ND2, and portions of ND1 and COI. Two well-supported groups are defined, the Acrodonta and the Iguanidae (sensu lato). This phylogenetic hypothesis is used to investigate evolutionary shifts in mitochondrial gene order, origin for light-strand replication, and secondary structure of tRNACys. These three characters shift together on the branch leading to acrodont lizards. Plate tectonics and the fossil record indicate that these characters changed in the Jurassic. We propose that changes to the secondary structure of tRNACys may destroy function of the origin for light-strand replication which, in turn, may facilitate shifts in gene order.     

Phylogenetic positions of Clostridium chauvoei and Clostridium septicum based on 16S rRNA gene sequences

The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined. After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers. A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C. chauvoei and C. septicum in Clostridium cluster I (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani. We found that 99.3% of the nucleotides in the genes of C. chauvoei and C. septicum are identical.     

Reduced growth hormone (GH) responsiveness to combined GH-releasing hormone and pyridostigmine administration in the Prader-Willi syndrome

Pea aphid (Acyrthosiphon pisum) clones have been shown to be adapted to particular host plant species but it is unknown whether there are host races. A 1101 base pair region of the mitochondrial cytochrome oxidase I gene (COI) was sequenced for 21 pea aphid clones that had been collected from different host plants in Canada and the U.S.A. Only five closely related mitochondrial haplotypes were found. A maximum likelihood phylogeny was estimated for these five haplotypes and four related aphid species: Acyrthosiphon macrosiphum, A. kondoi, Fimbriaphis fimbriata, and Macrosiphum creelii. Pea aphids from the same host plant species were no more likely to have the same mitochondrial haplotype than aphids from different host plant species. In addition, aphids from the same geographical regions were no more likely to have the same mitochondrial haplotype than aphids from different geographic regions. I therefore reject the hypothesis that there are monophyletic host races of the pea aphid.     

Occurrence of a sequence in marine cyanophages similar to that of T4 g20 and its application to PCR-based detection and quantification techniques

Viruses are ubiquitous components of marine ecosystems and are known to infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus Synechococcus. A conserved region from the cyanophage genome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant similarity to a gene encoding a capsid assembly protein (gp20) from the enteric coliphage T4. The results of a comparison of gene 20 sequences from three cyanomyoviruses and T4 allowed us to design two degenerate PCR primers, CPS1 and CPS2, which specifically amplified a 165-bp region from the majority of cyanomyoviruses tested. A competitive PCR (cPCR) analysis revealed that cyanomyovirus strains could be accurately enumerated, and it was demonstrated that quantification was log-linear over ca. 3 orders of magnitude. Different calibration curves were obtained for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial inaccuracies in estimates of phage abundance in natural assemblages. Further sequence analysis of cyanomyovirus gene 20 homologs would be necessary in order to design primers which do not exhibit phage-to-phage variability in priming efficiency. It was demonstrated that PCR products of the correct size could be amplified from seawater samples following 100x concentration and even directly without any prior concentration. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophages in natural assemblages. Further optimization of procedures may ultimately lead to a sensitive assay which can be used to analyze natural cyanophage populations both quantitatively (by cPCR) and qualitatively following phylogenetic analysis of amplified products.