Sequence divergence and open regions of RNA secondary structures in the envelope regions of the 17 human immunodeficiency virus isolates.

Genetic variation during the course of infection of an individual is a remarkable feature of the acquired immune deficiency syndrome (AIDS) disease. This variation has been studied for the envelope protein encoding regions of seventeen different sequences from various isolates of human immunodeficiency virus (HIV) using multiple sequence comparison and calculation of variability. The open regions with little intramolecular base pairing in these envelope sequences are predicted by a recently developed statistical method. The minimum length L for a run of hypervariable sites, conserved sites, or open regions that gives significance at the 1% (or 0.1%) level is then determined by a scan statistical method. The results show that significant clusters of open regions predicted at the RNA levels correlate with significant clusters of hypervariable sites in the HIV envelope gene. Those significant genomic variations in HIVs seem to be manifested mainly in the extracellular portion of the envelope protein. Twelve potential antigenic determinants are predicted using an antigenic index method. Interestingly, the majority of the significant hypervariable regions in the exterior envelope protein (gp120) were predicted potential epitopes.     

Hypervariable regions in the putative glycoprotein of hepatitis C virus.

A comparison of the sequences of the putative glycoprotein region in three independent cDNA clones of hepatitis C virus and of sequences of four other clones revealed extensive genetic variation clustered and interspersed with highly conserved amino acid sequences. We obtained evidence for two hypervariable regions in the putative envelope glycoprotein, one region was assumed to be a potential antigenic site, as deduced from the hydrophilicity and analyses of secondary structures. These observations suggest the existence of a large pool of antigenic variants of hepatitis C virus, in Japan.     

Size variation within the second hypervariable region of the surface envelope gene of the bovine lentivirus BIV in experimentally and naturally infected cattle.

The bovine lentivirus also known as the bovine immunodeficiency-like virus (BIV) has conserved and hypervariable regions in the surface envelope (SU) gene. Size variation between isolates can be as large as 200 bp, mostly occurring in the second hypervariable (V2) gene region of the SU gene. The V2 region was cloned and sequenced from both experimentally and naturally infected cattle. Temporal evaluation of provirus from an experimentally inoculated cow showed two different-sized variants that appeared over time. The variation appeared to result from a recombinational event resulting in an apparent direct repeat. Cloned proviral nucleotide sequence diversity increased over time. Virus that was cultured and then cloned and sequenced showed progressive change from the inoculum virus, but culturing reduced the diversity of the clones as compared with direct amplification of provirus from leukocyte samples from the cow. The quasispecies phenomenon was evident in clones sequenced from a cow naturally infected with BIV. Of 10 clones examined from the V2 region, 6 different-size clones were present with nine different patterns of sequence rearrangement. Sequence length of different clones varied by as much as 43 amino acids (aa), with 21- and 15-aa direct repeats accounting for most of the size variation. Similar to other lentiviruses, BIV appears to mutate rapidly, which may be important in viral persistence and pathogenesis.     

Estimating Protistan Diversity Using High‐Throughput Sequencing

Sequencing hypervariable regions from the 18S rRNA gene is commonly employed to characterize protistan biodiversity, yet there are concerns that short reads do not provide the same taxonomic resolution as full‐length sequences. A total of 7,432 full‐length sequences were used to perform an in silico analysis of how sequences of various lengths and target regions impact downstream ecological interpretations. Sequences that were longer than 400 nucleotides and included the V4 hypervariable region generated results similar to those derived from full‐length 18S rRNA gene sequences. Present high‐throughput sequencing capabilities are approaching protistan diversity estimation comparable to whole gene sequences.     

Recovery and altered neutralization specificities of chimeric viruses containing capsid protein domain exchanges from antigenically distinct strains of feline calicivirus

Feline calicivirus (FCV) strains can show significant antigenic variation when tested for cross-reactivity with antisera produced against other FCV strains. Previous work has demonstrated the presence of hypervariable amino acid sequences in the capsid protein of FCV (designated regions C and E) that were postulated to constitute the major antigenic determinants of the virus. To examine the involvement of hypervariable sequences in determining the antigenic phenotype, the nucleotide sequences encoding the E regions from three antigenically distinct parental FCV strains (CFI, KCD, and NADC) were exchanged for the equivalent sequences in an FCV Urbana strain infectious cDNA clone. Two of the three constructs were recovered as viable, chimeric viruses. In six additional constructs, of which three were recovered as viable virus, the E region from the parental viruses was divided into left (N-terminal) and right (C-terminal) halves and engineered into the infectious clone. A final viable construct contained the C, D, and E regions of the NADC parental strain. Recovered chimeric viruses showed considerable antigenic variation from the parental viruses when tested against parental hyperimmune serum. No domain exchange was able to confer complete recognition by parental antiserum with the exception of the KCD E region exchange, which was neutralized at a near-homologous titer with KCD antiserum. These data demonstrate that it is possible to recover engineered chimeric FCV strains that possess altered antigenic characteristics. Furthermore, the E hypervariable region of the capsid protein appears to play a major role in the formation of the antigenic structure of the virion where conformational epitopes may be more important than linear in viral neutralization.     

Application of the hypervariable region of the 16S rDNA sequence as an index for the rapid identification of species in the genus Alicyclobacillus

A comparison of the 16S rRNA gene (rDNA) sequences of seven type strains belonging to different Alicyclobacillus species (i.e., five validated species, one proposed species and one genomic species) suggested that the 5' end hypervariable region (259-273 bases in length) of 16S rDNA was specific for the respective type strains. Further phylogenetic analysis based on DNA sequences of the hypervariable region using 24 Alicyclobacillus strains revealed that the strains could be categorized into five species and the A. acidocaldarius-Alicyclobacillus genomic species 1 group. The hypervariable region was highly conserved among the five species: A. acidiphilus, A. acidoterrestris, A. cycloheptanicus, A. herbarius, and A. hesperidum. The strains in the A. acidocaldarius-Alicyclobacillus genomic species 1 group were subdivided into two clusters (Clusters I and II) based on DNA sequences in the hypervariable region. On the basis of phenotypic characteristics, chemotaxonomic and phylogenetic analyses, and DNA-DNA hybridization data, strains in Cluster I were grouped as Alicyclobacillus genomic species 1 and strains in Cluster II were re-identified as A. acidocaldarius, thereby demonstrating that the hypervariable regions were also highly conserved within these two species. These results suggest that as is the case with Bacillus, the hypervariable region is significantly species-specific in the genus Alicyclobacillus to distinguish Alicyclobacillus species by DNA sequence comparison of the hypervariable region.     

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.     

The visna virus genome: evidence for a hypervariable site in the env gene and sequence homology among lentivirus envelope proteins.

The complete nucleotide sequence of the visna virus 1514 genome was determined. Our sequence confirms the relationship of visna virus and other lentiviruses to human immunodeficiency virus (HIV) both at the level of sequence homology and of genomic organization. Sequence homology is shown to extend to the transmembrane proteins of lentivirus env genes; this homology is strongest in the extracellular domain, suggesting that close structural and functional similarities may also exist among these envelope proteins. Comparison of our data with the sequence of visna virus LV1-1, an antigenic variant derived from strain 1514, demonstrates that the rate of divergence has been about 1.7 x 10(-3) substitutions per nucleotide per year in vivo. This rate is orders of magnitude higher than that for most DNA genomes, but agrees well with estimates of the rate for HIV. A statistically significant cluster of mutations in the env gene appears to represent a hypervariable site and may correspond to the epitope responsible for the antigenic differences between 1514 and LV1-1. Analysis of the potential RNA folding pattern of the visna virus env gene shows that this hypervariable site falls within a region with little potential for intramolecular base pairing. This correlation of hypervariability with lack of RNA secondary structure is strengthened by the fact that it also holds for a hypervariable site in the env gene of HIV.     

Concurrent evolution of human immunodeficiency virus type 1 in patients infected from the same source: rate of sequence change and low frequency of inactivating mutations.

Direct sequencing of segments of the envelope gene of human immunodeficiency virus type 1 proviruses in peripheral blood mononuclear cells has revealed that a cohort of hemophiliacs who were infected after exposure to a single common batch of factor VIII share closely related virus strains. Seventy-four sequences extending from hypervariable regions V4 through V5 from nine patients yielded a mean intrapatient nucleotide distance of 5.5%, while a mean of 4.2% was observed in 39 sequences of the V3 loop (six patients). Phylogenetic analysis revealed that sequences of six Edinburgh patients were particularly closely related and those from a patient infected in the United States were very distinct. The mean nucleotide distance among these six was 8.3%, while the mean distance from the U.S.-derived sequences was 25.5% in the V4-V5 region. The rate of sequence change across this patient group has been estimated to be 0.4% per year in the V4-V5 region and 0.5% per year in the V3 region, with at least a twofold range across patients. Only two inactivating nucleotide substitutions have been observed in a total of 42 kb of sequence obtained from the env and gag genes during this study.     

Structure of the Bordetella pertussis gene coding for the serotype 3 fimbrial subunit

Bordetella pertussis strains contain at least three distinct genes coding for fimbrial subunits, designated fim2, fim3, and fimX. The sequences of the fim2 and fimX genes have been published. Here we present the sequence of the fim3 gene. Proximal and distal to the fim3 gene, regions were observed that could function as rho-independent terminators, suggesting that the gene is not part of a larger operon. Comparison of the putative promoter regions of the fim2 and fim3 genes revealed a conserved region containing a stretch of approximately 13 C's. This region may be involved in fimbrial phase variation. A comparison of the deduced amino acid sequences of the three fimbrial subunits revealed conserved, variable, and hypervariable regions. The hypervariable regions coincided with predicted antigenic determinants. Peptides derived from the conserved regions may be incorporated into a future pertussis vaccine to induce antibodies which confer protection against strains producing different fimbrial serotypes.     

Identification of a 13-kDa protein associated with the polyhydroxyalkanoic acid granules from Acinetobacter spp.

Abstract Transferrin-binding proteins from Neisseria meningitidis vary among different isolates. We have identified and studied a hypervariable region adjacent to the carboxyl-end of the transferrin-binding domain of the Tbp2 molecule. The tbp2 genes from six strains of N. meningitidis were cloned and sequenced in this particular region. Sequence analysis of these regions along with five other sequences available from pathogenic Neisseria showed a common organisation of seven highly variable nucleotide stretches interspersed with six conserved nucleotide stretches. The variable regions correlated with the location of immunoreactive epitopes in polyclonal antisera raised to transferrin-binding proteins identified by peptide pin technology. Sequence analysis suggested a mosaic-like organisation of the tbp2 genes. Taken together, these data suggest that the antigenic variation in this part of the protein may result from a strong host immune pressure.     

Phylogenetic analysis and predicted secondary structures of the rDNA internal transcribed spacers of the powdery mildew fungi (Erysiphaceae)

The nucleotide sequences of the internal transcribed spacer (ITS) regions of the ribosomal DNA including the 5.8S rRNA gene and the 5′ end of the 28S rRNA gene have been determined for 19 species in 10 genera of the powdery mildew fungi in order to analyze their phylogenetic relationship. These fungi were divided into two large groups based on the nucleotide length of the ITS regions, and this grouping was in line with that based on the morphological characters of the anamorphic stage rather than the teleomorphic stage. Although the variable ITS sequences were often ambiguously aligned, conserved sites were also found. Thus, a neighbor-joining tree was constructed using the nucleotide sequence data of the conserved sites of the ITS regions, the 5.8S rRNA gene, and the 5′ end of the 28S rRNA gene. The phylogenetic tree displayed the presence of four groups in the powdery mildews, which were distinguished by their morphology and/or host ranges. In the ITS2 region, the presence of a common secondary structure having four hairpin domains was suggested, in spite of the highly variable nucleotide sequences of this region. The predicted secondary structure was supported by the compensatory mutations as well as compensatory conserved sequences and high G+C content in the predicted stem regions. Contribution No. 142 from the Laboratory of Plant Pathology, Mie University.     

DNA variation at the rp49 gene region of Drosophila simulans: evolutionary inferences from an unusual haplotype structure.

An approximately 1.3-kb region including the rp49 gene plus its 5' and 3' flanking regions was sequenced in 24 lines of Drosophila simulans (10 from Spain and 14 from Mozambique). Fifty-four nucleotide and 8 length polymorphisms were detected. All nucleotide polymorphisms were silent: 52 in noncoding regions and 2 at synonymous sites in the coding region. Estimated silent nucleotide diversity was similar in both populations (pi = 0.016, for the total sample). Nucleotide variation revealed an unusual haplotype structure showing a subset of 11 sequences with a single polymorphism. This haplotype was present at intermediate frequencies in both the European and the African samples. The presence of such a major haplotype in a highly recombining region is incompatible with the neutral equilibrium model. This haplotype structure in both a derived and a putatively ancestral population can be most parsimoniously explained by positive selection. As the rate of recombination in the rp49 region is high, the target of selection should be close to or within the region studied.     

Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1.

Nucleotide sequences in three hypervariable regions of the human immunodeficiency virus type 1 (HIV-1) env gene were obtained by sequencing provirus present in peripheral blood mononuclear cells of HIV-infected individuals. Single molecules of target sequences were isolated by limiting dilution and amplified in two stages by the polymerase chain reaction, using nested primers. The product was directly sequenced to avoid errors introduced by Taq polymerase during the amplification process. There was extensive variation between sequences from the same individual as well as between sequences from different individuals. Interpatient variability was markedly less in individuals infected from a common source. A high proportion of amino acid substitutions in the hypervariable regions altered the number and positions of potential N-linked glycosylation sites. Sequences in two hypervariable regions frequently contained short (3- to 15-bp) duplications or deletions, and by amplifying peripheral blood mononuclear cell DNA containing 10(2) or 10(3) proviral molecules and analyzing the product by high-resolution electrophoresis, the total number and abundance of distinct length variants within an individual could be estimated, providing a more comprehensive analysis of the variants present than would be obtained by sequencing alone. Sequences from many individuals showed frequent amino acid substitutions at certain key positions for neutralizing-antibody and cytotoxic T-cell recognition in the immunodominant loop. The rates of synonymous and nonsynonymous nucleotide substitution in the region of this and flanking regions indicate that strong positive selection for amino acid change is operating in the generation of antigenic diversity.     

Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection.

Viral RNA was extracted from plasma samples collected from five individuals during the period of viremia before seroconversion in primary infection with human immunodeficiency virus type 1 (HIV-1) and amplified by polymerase chain reaction. Nucleotide sequence analysis of amplified DNA from the V3 and V4 hypervariable regions indicated that the initial virus population of each acutely infected individual was completely homogeneous in sequence. No intrasample variability was found among the 44,090 nucleotides sequenced in this region of env, contrasting with the high degree of variability normally found in seropositive individuals. Paradoxically, substantial sequence variability was found in the normally high conserved gag gene (encoding p17) in most of the preseroconversion samples. The diversity of p17 sequences in samples that were homogeneous in V3 and V4 can most readily be explained by the existence of strong selection for specific env sequences either upon transmission or in the interval between exposure and seroconversion in the exposed individual. Evidence that localizes the selected region upon transmission to V3 is provided by the similarity or identity of V3 loop sequences in five individuals with epidemiologically unrelated HIV-1 infections, while regions flanking the V3 loop and the V4 hypervariable region were highly divergent. The actual V3 sequences were similar to those associated with macrophage tropism in primary isolates of HIV, irrespective of whether infection was acquired by sexual contact or parenterally through transfusion of contaminated factor VIII. Proviral DNA sequences in peripheral blood mononuclear cells remained homogeneous in the V3 and V4 regions (and variable in p17gag) for several months after seroconversion. The persistence of HIV sequences in peripheral blood mononuclear cells identical to those found at primary infection in the absence of continued virus expression provides an explanation for the previously observed differences in the composition of circulating DNA and RNA populations in sequential samples from seropositive individuals.     

Gene Conversions Can Generate Sequence Variants in the Late Chorion Multigene Families of Bombyx Mori

The 140-kbp late chorion locus of Bombyx mori strain 703 contains 15 divergently oriented gene pairs encoding the high cysteine (Hc) eggshell proteins. Sequence homology is approximately 91% for the 2-kb region of each gene pair, including the 5' flanking region, intron and exons. The homology rapidly disappears within a few hundred basepairs of the 3' end of most genes. Here we present the results of the nucleotide sequence and genomic blot comparison of Hc genes from different races of B. mori. Comparison of the nucleotide sequences of the same gene pair in two different races reveals that most of the nucleotide differences occur in clusters or patches and correspond to sequences present in other Hc genes in the locus. The number of nucleotide differences that have accumulated in the highly conserved regions of the gene pair (2.3/100 bp), most of which are attributable to patchwork exchanges, is significantly higher than the number of differences in the poorly conserved 3' flanking regions (0.6/100 bp), due primarily to new mutations. These data are consistent with a gene conversion process, which in the short-term generates new combinations of sequence variants, but in the long-term results in concerted evolution. Genomic blot analyses of different geographical races of B. mori reveal that there is variation in the number of Hc gene pairs (14-19 gene pairs), indicating that unequal crossovers also occur in the locus.     

Phosphoprotein and nucleocapsid protein evolution of vesicular stomatitis virus New Jersey.

The entire phosphoprotein (P) and nucleocapsid (N) protein gene sequences and deduced amino acid sequences for 18 selected vesicular stomatitis virus isolates representative of the natural genetic diversity within the New Jersey serotype are reported. Phylogenetic analysis of the data using maximum parsimony allowed construction of evolutionary trees for the individual genes and the combined N, P, and glycoprotein (G) genes of these viruses. Virtually identical rates of nucleotide substitutions were found for each gene, indicating that evolution of these genes occurs at essentially the same rate. Although up to 19 and 17% sequence differences were evident in the P and N genes, respectively, no variation in gene length or evidence of recombinational rearrangements was found. However, striking evolutionary differences were observed among the amino acid sequences of vesicular stomatitis virus New Jersey N, P, and G proteins. The N protein amino acid sequence was the most highly conserved among the different isolates, indicating strong functional and structural constraints. Conversely, the P protein amino acid sequences were highly variable, indicating considerably fewer constraints or greater evolutionary pressure on the P protein. Much of the remarkable amino acid variability of the P protein resided in a hypervariable domain located between amino acids 153 and 205. The variability within this region would be consistent with it playing a structural role as a spacer to maintain correct conformational presentation of the separate active domains of this multifunctional protein. In marked contrast, the adjacent domain I of the P protein (previously thought to be under little evolutionary constraint) contained a highly conserved region. The colocalization of a short, potentially functional overlapping open reading frame to this region may explain this apparent anomaly.     

Ribosomal DNA sequence variation among sympatric strains of the Cyclotella meneghiniana complex (Bacillariophyceae) reveals cryptic diversity

Cyclotella meneghiniana Kützing is one of the most commonly found and intensively studied freshwater diatom species. However, it is considered taxonomically problematic because of its unusually wide ecological range and large frustule ultrastructural variation. As part of a study of morphological and genetic variation in this morphospecies, we surveyed nucleotide variation in the hypervariable D1/D2 regions of the 28S rDNA, in the ribosomal internal transcribed spacer region (containing ITS1, the 5.8S rDNA and ITS2) and in the 18S rDNA in a collection of 20 sympatric strains. High genetic variability and strong indications of genetic structure among the Cyclotella meneghiniana strains were found. Representatives of four genetically distinct--apparently reproductively isolated--groups were revealed among them. The random distribution of ITS variation within these four groups indicated that the genetic structure in Cyclotella meneghiniana can probably be explained by the presence of cryptic sexual species rather than by the lack of allogamous sexual reproduction. The morphological features traditionally used for species identification in this group cannot distinguish these putative cryptic species.     

Genetic diversity of the allodeterminant alr2 in Hydractinia symbiolongicarpus

Hydractinia symbiolongicarpus, a colonial cnidarian (class Hydrozoa) epibiont on hermit crab shells, is well established as a model for genetic studies of allorecognition. Recently, two linked loci, allorecognition (alr) 1 and alr2, were identified by positional cloning and shown to be major determinants of histocompatibility. Both genes encode putative transmembrane proteins with hypervariable extracellular domains similar to immunoglobulin (Ig)-like domains. We sought to characterize the naturally occurring variation at the alr2 locus and to understand the origins of this molecular diversity. We examined full-length cDNA coding sequences derived from a sample of 21 field-collected colonies, including 18 chosen haphazardly and two laboratory reference strains. Of the 35 alleles recovered from the 18 unbiased samples, 34 encoded unique gene products. We identified two distinct structural classes of alleles that varied over a large central region of the gene but both possessed highly polymorphic extracellular domains I, similar to an Ig-like V-set domain. The discovery of structurally chimeric alleles provided evidence that interallelic recombination may contribute to alr2 variation. Comparisons of the genomic region encompassing alr2 from two field-derived haplotypes and one laboratory reference sequence revealed a history of structural variation at the haplotype level as well. Maintenance of large numbers of equally rare alleles in a natural population is a hallmark of negative frequency-dependent selection and is expected to produce high levels of heterozygosity. The observed alr2 allelic diversity is comparable with that found in immune recognition molecules such as human leukocyte antigens, B cell Igs, or natural killer cell Ig-like receptors.