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PubMed1996

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

Cell Line↗

PubMed1998

Compilation of human mtDNA control region sequences.

This paper describes the organisation of a database for human mitochondrial control-region sequences. The data are divided into three ASCII files that contain aligned sequences from the hypervariable region I (HVRI), from the hypervariable region II (HVRII), and the available information about the individuals, from whom the sequences stem. The current collection comprises 4079 HVRI and 969 HVRII sequences. From 728 individuals sequences of both HVRI and HVRII are available. For easy access, the collection is made available to the scientific community via World Wide Web at URL http://www.zi.biologie.uni-muenchen.de/[symbol: see text]meyers/mtdna.html

Computer Communication Networks↗

PubMed1984

Gene conversion between murine class II major histocompatibility complex loci. Functional and molecular evidence from the bm 12 mutant.

The experiments presented in this study define the molecular basis of the bm 12 mutation. Initial characterization of an alloreactive T cell clone, 4.1.4, showed this clone to recognize an allodeterminant present on the E beta b and A beta bm12 chains, but not on the bm 12 parent A beta b chain. To define the extent of sequence shared between the I-E beta product and the mutant I-A beta product, we isolated a cDNA clone of the E beta b gene and determined its nucleotide sequence. Comparison of the nucleotide sequences of E beta b, A beta b, and A beta bm12 shows the the A beta bm12 gene to be identical to the E beta b gene in the region where it differs from its A beta b parent. We predict that the bm 12 mutation arose by gene conversion of this region, which spans 14 nucleotides between amino acid residues 67-71 of the mature A beta chain, from the E beta b locus to the corresponding position at the A beta b locus. Recognition of this region, which spans one of the previously defined E beta allelic "hypervariable" regions, by an alloreactive T cell clone provides the first direct evidence of the functional importance of these hypervariable regions in T cell stimulation. The identification of a gene conversion event involving one of these allelic variable regions implicates conversion as a mechanism that acts on class II beta genes to create sequence diversity in regions of Ia molecules that interact with foreign antigen or a T cell receptor, regions where protein sequence polymorphism would presumably be selected for by the expanded ability it affords the organism to mount effective immune responses against a wider variety of foreign antigens.

Animals↗

PubMedDate not supplied

Structure, expression and divergence of T-cell receptor beta-chain variable regions.

Analysis of three new T-cell receptor beta-chain variable regions together with those in the literature indicates that they have both remarkable similarities and differences with those of immunoglobulin. Less than 10 V regions appear to predominate in the thymus. V beta sequences are much more heterogeneous at the amino acid level than are immunoglobulin V regions and they appear to diverge between species much more quickly, apparently the result of additional hypervariable regions. Three of these putative new hypervariable regions lie outside of the classical immunoglobulin binding site, an indication that important interactions may be occurring in these regions with polymorphic MHC determinants.

Amino Acid Sequence↗

PubMed1994

A rapid and simple DNA fingerprinting method using RFLP and SSCP analysis of the hypervariable noncoding region of human mitochondrial DNA.

A simple method for identification of individuals and maternity testing has been developed. This technique includes PCR amplification of the 199-bp hypervariable portion of the noncoding region of human mtDNA, digestion with RsaI and subsequent SSCP analysis of restriction fragments with DNA silver staining. Using this approach we have analysed the DNA fingerprint patterns of the family members. The fingerprints of maternally related individuals appeared to be identical in three generations, while maternally unrelated members of the family showed differences in their fingerprints, either in SSCP or both RFLP and SSCP patterns. Sequencing data have confirmed the results obtained. Further DNA fingerprinting analysis of 19 unrelated mother-child pairs by means of the method described revealed complete identity of the fingerprint patterns within the pairs. The probability of a random fingerprint match for two maternally unrelated individuals was estimated as 8%.

Adult↗

PubMed1993

A structurally flexible and antigenically variable N-terminal domain of the hepatitis C virus E2/NS1 protein: implication for an escape from antibody.

Hepatitis C virus persists in most infected hosts, and causes chronic hepatitis, liver cirrhosis, and/or hepatocellular carcinoma in humans. During the infection the RNA genome of hepatitis C virus undergoes frequent missense mutations at one or two "hypervariable" regions within a presumptive envelope gene (Okamoto et al., 1992, Virology 190, 894-899; Ogata et al., 1991, Proc. Natl. Acad. Sci. USA 88, 3392-3396). In the present study, we analyzed three cases of hepatitis C virus infection, two in chimpanzees and one in humans, for the antigenicity of peptides predicted from the hypervariable region of viral RNA obtained during the follow-up. Our results showed a successive appearance of hepatitis C virus mutants with antigenically distinct amino acid sequence within the domain; and the amino acid replacement was associated with an alteration of predicted local secondary structure of the epitope region. Hence, the hypervariable domain of the hepatitis C virus envelope appeared to be structurally flexible and antigenically variable, providing the virus a way to escape from host immunity.

Amino Acid Sequence↗

PubMed2002

HVI and HVII mitochondrial DNA data in Apaches and Navajos.

Most mtDNA studies on Native Americans have concentrated on hypervariable region I (HVI) sequence data. Mitochondrial DNA haplotype data from hypervariable regions I and II (HVI and HVII) have been compiled from Apaches (N=180) and Navajos (N=146). The inclusion of HVII data increases the amount of information that can be obtained from low diversity population groups. Less mtDNA variation was observed in the Apaches and Navajos than in major population groups. The majority of the mtDNA sequences were observed more than once; only 17.8% (32/180) of the Apache sequences and 25.8% of the Navajo sequences were observed once. Most of the haplotypes in Apaches and Navajos fall into the A and B haplogroups. Although a limited number of haplogroups were observed, both sample populations exhibit sufficient variation for forensic mtDNA typing. Genetic diversity was 0.930 in the Apache sample and 0.963 in the Navajo sample. The random match probability was 7.48% in the Apache sample and 4.40% in the Navajo sample. The average number of nucleotide differences between individuals in a database is 9.0 in the Navajo sample and 7.7 in the Apache sample. The data demonstrate that mtDNA sequencing can be informative in forensic cases where Native American population data are used.

Complementarity Determining Regions↗

PubMed2001

Investigation of infection with Campylobacter jejuni in a man with hypogammaglobulinaemia using PCR-single-stranded conformational polymorphism (PCR-SSCP) typing.

This study investigated several episodes of infection of Campylobacter jejuni in an immunocompromised male with hypogammaglobulinaemia, presenting with diarrhoea and bacteraemia over a 16-month period, by employing three phenotyping and four genotyping schemes, including the single-stranded conformational polymorphism (SSCP) technique to establish if infection was reinfection or persistent infection. Four isolates from blood culture and two faecal isolates of Campylobacter jejuni were obtained from the patient by direct selective plating on Skirrow Selective agar. Isolates were characterised at the sub-species level by Penner serology, Preston biotyping, Preston phage-typing, as well as E3CJC2 restriction fragment length polymorphism (RFLP), 16S ribotyping, flagellin (flaA) RFLP and single-stranded conformational polymorphism (SSCP) analyses. Phenotyping and genotyping sub-species analyses demonstrated that the patient was infected with at least two different strains of Campylobacter jejuni, i. e. one strain that persisted throughout the 16-month period and another strain that was transient suggesting reinfection from a different source. SSCP analysis was the most discriminatory of all the typing schemes examined and demonstrated an altered genotype of the persistent strain, whereby there were subtle modifications to the hypervariable regions of the flaA gene. Overall, as SSCP examines the hypervariable region of the flaA gene and as this technique can detect point mutations, differences between SSCP banding patterns may represent markers and thus examine mutations that occur under immune selection, thereby permitting the C. jejuni to evade the host immune response. In conclusion, this study describes the novel use of SSCP genotyping of C. jejuni and demonstrated that this method is a highly discriminatory technique which may be beneficial in outbreak characterisation, but which is not suitable to examine the clonal patterns of C. jejuni over a long period of time.

Agammaglobulinemia↗

PubMed2001

Antigenic variation of Anaplasma marginale: major surface protein 2 diversity during cyclic transmission between ticks and cattle.

The rickettsial pathogen Anaplasma marginale expresses a variable immunodominant outer membrane protein, major surface protein 2 (MSP2), involved in antigenic variation and long-term persistence of the organism in carrier animals. MSP2 contains a central hypervariable region of about 100 amino acids that encodes immunogenic B-cell epitopes that induce variant-specific antibodies during infection. Previously, we have shown that MSP2 is encoded on a polycistronic mRNA transcript in erythrocyte stages of A. marginale and defined the structure of the genomic expression site for this transcript. In this study, we show that the same expression site is utilized in stages of A. marginale infecting tick salivary glands. We also analyzed the variability of this genomic expression site in Oklahoma strain A. marginale transmitted from in vitro cultures to cattle and between cattle and ticks. The structure of the expression site and flanking regions was conserved except for sequence that encoded the MSP2 hypervariable region. At least three different MSP2 variants were encoded in each A. marginale population. The major sequence variants did not change on passage of A. marginale between culture, acute erythrocyte stage infections, and tick salivary glands but did change during persistent infections of cattle. The variant types found in tick salivary glands most closely resembled those present in bovine blood at the time of acquisition of infection, whether infection was acquired from an acute or from a persistent rickettsemia. These variations in structure of an expression site for a major, immunoprotective outer membrane protein have important implications for vaccine development and for obtaining an improved understanding of the mechanisms of persistence of ehrlichial infections in humans, domestic animals, and reservoir hosts.

Anaplasma↗

PubMed1993

Analysis of feline calicivirus capsid protein genes: identification of variable antigenic determinant regions of the protein.

Three isolates of feline calicivirus (FCV) designated NADC, KCD and CFI/68 were compared for biochemical, serological and genetic variation within the capsid protein gene. The M(r) of the capsid protein from purified virions was approximately 66,000 for the NADC virus isolate, which differed slightly from the relative mobilities of the purified capsid proteins of the KCD and CFI/68 isolates. Polyclonal antisera from either cats infected or rabbits hyperimmunized with the CFI/68 isolate cross-reacted with all three isolates by Western blot analysis. However, these polyclonal antisera to CFI/68 varied considerably in their virus-neutralization titres to the KCD and NADC isolates. Nucleotide sequence data confirmed the genetic variability among these FCV isolates. Comparison of the predicted amino acid sequence of the capsid protein among isolates revealed two regions of sequence divergence that probably contain the antigenically variable determinants. These hypervariable regions may vary by as much as 55% among isolates of FCV. The amino acid sequence diversity in the hypervariable regions of the KCD and NADC isolates correlated well with the virus-neutralization data and suggests that polyvalent vaccines may be more protective than the commonly used monovalent vaccines.

Amino Acid Sequence↗

PubMed1975

Primary structure of a deleted human lambda type immunoglobulin light chain containing carbohydrate: protein Sm lambda.

An internal molecular deletion occurring in a human lambda type immunoglobulin light (L)-chain (Sm lambda) has been defined by sequence analysis. The Sm protein was isolated from the urine of a patient with a plasma cell dyscrasia involving the synthesis of an IgG molecule with both deleted gamma and lambda subunits. The Sm lambda polypeptide chain has an approximate molecular weight of 15,000 and contains 135 amino-acid residues. The constant (C) region is fully intact, comprising 105 residues, whereas the variable region (V) has only 30 residues. The V-region segment represents residues 1 through 30 of normal lambda chains and possesses considerable homology (87%) to lambda chains of subgroup II. Since lambdaII proteins normally contain 216 amino-acid residues, the defect represents an intramolecular deletion of 81 residues, which is entirely confined to the carboxyterminal three-quarters segment of the V-region, with a resumption of normal synthesis at a glutaminyl residue at position 110, the initiation point of the C-region. Carbohydrate is attached to an Asx residue at position 25, in the first hypervariable region, associated with the sequence triplet Asx-Ser-Ser, which is postulated to be a common recognition site for glycosylation of immunoglobulins. The carbohydrate moiety is a complex oligosaccharide with a branched chain structure containing sialic acid, fucose, mannose, N-acetylglucosamine, and galactose. These structural studies and other findings suggest that restricted areas in the DNA of immunoglobulin genes, such as the hinge regions of heavy (H) and light (L) chains and the hypervariable regions, are particularly susceptible to breakage and reunion. We postulate that the genetic defect of protein Sm could have originated from a somatic mutational event in the plasmacyte precursor during or after the integration of the V and C genes. These studies provide additional support for the hypothesis and two distinct structural genes encode a single immunoglobulin polypeptide chain.

Amino Acid Sequence↗

PubMed1997

Detection of somatic mutations in the mitochondrial DNA control region of colorectal and gastric tumors by heteroduplex and single-strand conformation analysis.

Each entire hypervariable region of the mitochondrial DNA control region was screened for mutations from paired normal and tumor DNA corresponding to a group of 21 patients (13 colorectal and 8 gastric adenocarcinomas) using both heteroduplex analysis and single-strand conformation analysis. These two mutation scanning strategies allowed the identification of sequence alterations in 3/13 (23%) colorectal tumors and in 3/8 (37%) gastric tumors. Heteroduplex analysis showed the heteroplasmic state of the majority of these tumor mutations. Sequence analysis revealed two A:T/G:C transitions (nucleotide positions: 16241 and 16166) in hypervariable region 1 (HV1) and two C:G/T:A transitions (nucleotide positions: 76 and 312), one A:T/G:C transition (nucleotide position: 93), a 1-basepair C:G deletion (nucleotide position: 309), and a 2-base-pair CC:GG insertion (nucleotide position: 309) in the HV 2 region. A considerable proportion of these mutations was found in homopolymeric regions which are highly polymorphic among humans. Different mechanisms (clonal expansion, increased oxidative damage, and nuclear mutator mutations) were suggested to explain the increased mitochondrial DNA mutation rate observed in cancer.

Adenocarcinoma↗

PubMed2003

Clinical applications of denaturing high-performance liquid chromatography-based genotyping.

AIM: To develop and evaluate heteroduplex forming templates (HFTs) as a common set of molecular standards for genotyping by denaturing high-performance liquid chromatography (DHPLC) using hypervariable regions of human mitochondrial DNA (mtDNA) as a model system. METHODS: Hypervariable regions 1 and 2 from the mtDNA D-loop of 22 maternally related and unrelated human volunteers were amplified by polymerase chain reaction (PCR) and individually mixed with each of three HFTs. Following denaturation and reannealing of the mixture, the resulting hetero- and homoduplicies were separated by DHPLC using temperature-modulated heteroduplex analysis. RESULTS: Each of three HFTs, when cross-hybridized with a target mtDNA amplicon, induced the formation of an assemblage hetero- and homoduplex peaks, which were uniquely characteristic of a given mtDNA sequence variant. The mtDNA DHPLC profiles obtained in the current study were identical between maternal relatives and different between unrelated individuals--consistent with uniparental maternal inheritance of mtDNA in humans. CONCLUSION: DHPLC in combination with a common set of HFTs targeted to a locus of interest can be used as a reliable means of genotyping. DHPLC profiles can be readily stored as a bit-coded string of hetero- and homoduplex peak retention times to form a searchable database. This approach to DHPLC genotyping will have immediate utility in extended pedigree analyses, where it will allow rapid sorting and/or confirmation of maternal lineages. Additional applications of DHPLC profiling include the discovery and scoring of clinically relevant nuclear and mitochondrial loci.

Chromatography, High Pressure Liquid↗

PubMed1994

Antigenic diversity within a family of M proteins from group A streptococci: evidence for the role of frameshift and compensatory mutations.

The genes (emm) encoding M proteins, from isolates of group-A streptococci (GAS) serotyped as M52, M53, M80 and M nontypeable (MNT; serologically related to M53 and M80), were examined. Characterization of emm from these GAS revealed some discrepancies with serotyping, illustrating the difficulty in serotype determination when cross-reactions occur. DNA sequences corresponding to the N-terminal region of M proteins from the isolates showed considerable similarity both in the hypervariable region and the repeat regions. We propose that these serotypes form a family of closely related M types. Frameshift mutations in the hypervariable region followed by a corrective (compensatory) frameshift were observed. This may be an effective mechanism for generating antigenic diversity in the M protein.

Amino Acid Sequence↗

PubMed1976

Rabbit antibody light chains: selective breeding narrows variability in framework and complementarity-determining residues.

The amino acid sequence of 5 light (L) chain (b4) variable (Vl) regions and the partial sequence of VL (kappa) regions from 12 anti-streptococcal group A-varant polysaccharide (Av-CHO) and 2 anti-streptococcal group C polysaccharide (C-CHO) antibodies was determined. These sequences contain 70 invariant positions as opposed to 50 invariant positions in other rabbit VL regions. Variability within the framework residues lacks randomness, and parent offspring relationship or otherwise close familial relationship is apparent in several instances. Variability in the complementarity-determining regions is reduced by 2.3-5.5-fold in comparison with other rabbit L-chains with several identical first and third hypervariable regions. Residue positions 50-56, known to mark the second hypervariable region in human kappa-chains, are not hypervariable in L-chains from Av-CHO rabbit antibodies. Considering the 67 rabbit L-chain sequences, completely or partially known today, for counting the number of V region germ line genes, it is concluded that the species rabbit has at least 27 VL germ line genes available.

Amino Acid Sequence↗

PubMed2002

Phylogenetic analysis of Brassiceae based on the nucleotide sequences of the S-locus related gene, SLR1.

Nucleotide sequences of orthologs of the S-locus related gene, SLR1, in 20 species of Brassicaceae were determined and compared with the previously reported SLR1 sequences of six species. Identities of deduced amino-acid sequences with Brassica oleracea SLR1 ranged from 66.0% to 97.6%, and those with B. oleracea SRK and SLR2 were less than 62% and 55%, respectively. In multiple alignment of deduced amino-acid sequences, the 180-190th amino-acid residues from the initial methionine were highly variable, this variable region corresponding to hypervariable region I of SLG and SRK. A phylogenetic tree based on the deduced amino-acid sequences showed a close relationship of SLR1 orthologs of species in the Brassicinae and Raphaninae. Brassica nigra SLR1 was found to belong to the same clade as Sinapis arvensis and Diplotaxis siifolia, while the sequences of the other Brassica species belonged to another clade together with B. oleracea and Brassica rapa. The phylogenetic tree was similar to previously reported trees constructed using the data of electrophoretic band patterns of chloroplast DNA, though minor differences were found. Based on synonymous substitution rates in SLR1, the diversification time of SLR1 orthologs between species in the Brassicinae was estimated. The evolution and function of SLR1 and the phylogenetic relationship of Brassiceae plants are discussed.

Journal Article↗

PubMed1997

Hydropathic characteristics of adenovirus hexons.

The complete nucleotide sequence and the predicted amino acid sequence of the adenovirus type 7 hexon gene were determined. The hydropathy of the hexon proteins from human adenovirus types 2, 3, 4, 5, 7, 12, 16, 40, 41, and 48, bovine adenovirus type 3, murine adenovirus type 1, and avian adenovirus types 1 and 10 was analysed. The presence of purines and pyrimidines in the second position of the codons was correlated to hydrophilicity and hydrophobicity, respectively. Comparison of the hydrophilicity plots of eight hexons showed seven hypervariable regions to be distributed on the surface. A large portion of the hypervariable regions manifests hydrophilicity. The strength of the surface charge accumulated on the hydrophilic and hydrophobic regions correlated to the tissue tropism of the different adenovirus types. Analysis of codon usage for adenovirus hexons showed that among synonymous codons those with cytidine in the third position were preferably used to a great extent. Analysis of the nucleotide and amino acid sequence pair distances and the phylogenetic tree of 14 hexon proteins showed members of subgenera B, D and E to be closely related, especially Ad4 and Ad16, and subgenus A to be closely related to subgenus F.

Adenoviruses, Human↗

PubMed1986

Identification and characterization of conserved and variable regions in the envelope gene of HTLV-III/LAV, the retrovirus of AIDS.

To determine the extent and nature of genetic variation present in independent isolates of HTLV-III/LAV, the nucleotide sequences of the entire envelope gene and parts of gag and pol were determined for two AIDS viruses. The results indicated that variation throughout the viral genome is extensive and that the envelope gene in particular is most highly variable. Within the envelope, changes were most prevalent within the extracellular region where clustered nucleotide substitutions and deletions/insertions were evident. Based on predicted secondary protein structure and hydrophilicity, these hypervariable regions represent potential antigenic sites. In contrast to the hypervariable regions, other sequences in the extracellular envelope and the overall envelope structure (including 18 of 18 cysteine residues), as well as most of the transmembrane region, were highly conserved.

Acquired Immunodeficiency Syndrome↗