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PubMed2006

Use of sequence variation in three highly variable regions of the mitochondrial DNA for the discrimination of allogeneic platelets.

BACKGROUND: Human mitochondrial DNA (mtDNA) polymorphisms can be used to detect allogeneic transfused platelets. To increase the number of informative polymorphisms we investigated three hypervariable regions (HVR1, HVR2, and HVR3) within the displacement loop (D-loop) region of the mtDNA. STUDY DESIGN AND METHODS: mtDNA was obtained from 119 unrelated blood donors. Forward and reverse primers were designed and conditions optimized to amplify and sequence the template mtDNA by dye terminator cycle sequencing. RESULTS: We established a sequencing protocol for all three HVRs of the mtDNA. Polymorphic sites were found in all three regions: 66 in HVR1, 44 in HVR2, and 18 in HVR3. Combining the sequence information of HVR1, -2, and -3 resulted in 105 different genotypes of which 95 were unique. We were able to discriminate between two randomly chosen individuals with a random match probability of 1.2 percent. CONCLUSION: The D-loop region of mtDNA contains a wealth of informative molecular markers for chimerism and survival studies after transfusions of cellular blood components.

Base Sequence↗
PubMed1998

APO B 3' HVR polymorphism in healthy population: relationships to serum lipid levels.

We have analyzed allele frequency distribution at the hypervariable locus 3' to the apolipoprotein B gene in a healthy population sample (241 women and 246 men) from the Belgrade area. The bimodal distribution of sixteen different hypervariable region (HVR) alleles and the heterozygosity index (average 0.76) in both samples are similar to ones reported for other Caucasian populations. However, we found the hypervariable element containing 34 repeats-HVE34 allele to be the most common one in both female and male samples, and that there was a lower frequency of the HVE>36 alleles. For further analysis of 3'HVR allele frequency and its possible association with serum lipid levels, all HVR alleles were grouped into five main types (HVE<32, HVE32, HVE34, HVE36, and HVE>36), and the samples were divided into lipid quartiles. We found that (1) apo B gene 3'HVR polymorphism is associated with variation of normal lipid levels in males, (2) HVE32 alleles are associated with decreased serum levels of total cholesterol, LDL cholesterol, and triglycerides, and (3) HVR genotypes containing HVE34 and HVE36 alleles are associated with elevated serum levels of total cholesterol, low density lipoprotein (LDL) cholesterol, and triglycerides.

Adult↗
PubMed1997

Envelope glycoprotein nucleotide sequence and genetic characterization of North American ovine lentiviruses.

Ovine lentiviruses (OvLV) resemble human immunodeficiency viruses in genomic organization, viral heterogeneity, and spectrum of cytophenotypic expression. To gain a better understanding of the relationship of North American OvLV isolates with other characterized OvLV strains, the complete DNA nucleotide sequence of the env region of a highly lytic (rapid/high) OvLV strain (85/34) was determined and compared with the sequence of amplicons within env of three other OvLV strains of varying cytophenotype and isolated from the same flock of sheep. LTR and pol regions also were compared among these strains. The env region of 85/34 was 986 codons in length and the reported nucleotide sequence showed features shared by other OvLV including heavy glycosylation and conserved and hypervariable regions within the surface membrane protein region. Phylogenetic analyses of regions within LTR, reverse transcriptase, and env grouped the four virus strains together and similar to the maedi-visna OvLV strains, including visna virus, South African ovine maedi visna virus, and EV1 (British OvLV isolate), but they were distinct from caprine arthritis encephalitis virus.

Amino Acid Sequence↗
PubMed2006

Delta mtDNA4977 is more common in non-tumoral cells from gastric cancer sample.

BACKGROUND: The aim of this study was to determine the frequency of delta mtDNA4977 in tumoral cells as compared with adjacent normal cells in gastric cancer. METHODS: In order to investigate whether a high incidence of mutation exists in mitochondrial DNA of gastric cancer tissues, we screened one of common region of the mitochondrial genome by PCR amplification and Southern blot followed by DNA sequence analysis. DNA isolated from these cells was used to amplify hypervariable regions ATPase8/6, COXIII, ND3, ND4 and ND5 of delta mtDNA4977. RESULTS: In 107 cancer patients, delta mtDNA4977 was detected in 6 cases (5.60%) of the tumoral tissues and 18 cases (16.82%) of the non-tumoral tissues that were adjacent to the tumors. Levels of delta mtDNA4977 deletions were found to be more in non-tumoral tissues than in adjacent tumoral tissues. There was no correlation of patients with certain clinical parameters like age, sex, tumor location and tumor size; however, there was an obvious relationship with intestinal-type of gastric cancer. CONCLUSIONS: Unknown genetic aspects, ambiguous environmental factors and reactive oxygen species (ROS) can cause the delta mtDNA4977 mutation rate to be increased in gastric cancer. The results suggest that percentage level of delta mtDNA4977 is less common and intolerable in tumoral tissue, probably because of high metabolism and ROS generation. We supposed that the cells initially had delta mtDNA4977 transform to tumoral cells and the existed deletion conferred metabolic disadvantage; thus, cells containing such a mtDNA deletion would be overgrown by other cancer cells without this mtDNA deletion. As a result, the presence of delta mtDNA4977 will be low in tumoral cells.

Adult↗
PubMed1998

Polymorphic sites in human mitochondrial DNA control region sequences: population data and maternal inheritance.

Sequence analysis of the mitochondrial DNA (mtDNA) control region is of central importance for forensic identity testing as well as for studies of human evolution. Here we report the sequencing data of the hypervariable regions I and II from 50 unrelated individuals from a western German population (Rhine area). In regions I and II, 52 and 26 sites of sequence polymorphism, respectively, were noted. Nucleotide substitution rather than insertion/deletion was the majority of variation. The distribution showed a large bias towards transitional changes than transversional changes. Furthermore we investigated uniparental inheritance in seven CEPH families each family with 7-9 maternal descendants. Most maternal relatives shared identical mtDNA sequences. Additionally sequences were compared for father:child pairs and as expected no evidence for paternal transmission of mtDNA was observed. The high variability of mtDNA control region sequences permits utility in forensic identity investigations. The data also indicate that the neomutation rate seems to be very low from one generation to the other.

Base Sequence↗
PubMed1999

Characterization of the interaction of a TCR alpha chain variable domain with MHC II I-A molecules.

The alphabeta TCR recognizes peptides bound to MHC molecules. In the present study, we analyzed the interaction of a soluble TCR alpha chain variable domain (Valpha4.2-Jalpha40; abbreviated to Valpha4.2) with the MHC class II molecule I-Au. Valpha4.2 bound specifically to I-Au expressed on the surface of a transfected thymoma cell line. Modifications in the amino acid residues located within the three complementarity-determining regions (CDRs) of the Valpha domain did not markedly affect this interaction. However, mutation of glutamic acid to alanine at position 69 of the fourth hypervariable region (HV4alpha) significantly increased the binding. Antibody inhibition studies suggested that the binding site was partly contributed by a region of the beta chain of I-Au. Furthermore, the binding of Valpha4.2 to the MHC molecule was dependent on the nature of the peptide bound in the groove. Soluble Valpha4.2 specifically inhibited the activation of TCR transfectants by I-Au-expressing cells pulsed with an N-terminal peptide of myelin basic protein. Valpha4.2 also bound to MHC class II-expressing spleen cell populations from mice of the H-2(u) and H-2(d) haplotypes. The binding of Valpha4.2 to I-A molecules might explain the immunoregulatory effects reported previously for TCR alpha chains. This Valpha4.2 interaction may also be relevant to models of antigen presentation involving the binding of intact proteins to MHC class II molecules followed by their processing to generate epitopes suitable for T cell recognition.

Amino Acid Substitution↗
PubMed2005

Diversification of DNA sequences in the symbiotic genome of Rhizobium etli.

Bacteria of the genus Rhizobium and related genera establish nitrogen-fixing symbioses with the roots of leguminous plants. The genetic elements that participate in the symbiotic process are usually compartmentalized in the genome, either as independent replicons (symbiotic plasmids) or as symbiotic regions or islands in the chromosome. The complete nucleotide sequence of the symbiotic plasmid of Rhizobium etli model strain CFN42, symbiont of the common bean plant, has been reported. To better understand the basis of DNA sequence diversification of this symbiotic compartment, we analyzed the distribution of single-nucleotide polymorphisms in homologous regions from different Rhizobium etli strains. The distribution of polymorphisms is highly asymmetric in each of the different strains, alternating regions containing very few changes with regions harboring an elevated number of substitutions. The regions showing high polymorphism do not correspond with discrete genetic elements and are not the same in the different strains, indicating that they are not hypervariable regions of functional genes. Most interesting, some highly polymorphic regions share exactly the same nucleotide substitutions in more than one strain. Furthermore, in different regions of the symbiotic compartment, different sets of strains share the same substitutions. The data indicate that the majority of nucleotide substitutions are spread in the population by recombination and that the contribution of new mutations to polymorphism is relatively low. We propose that the horizontal transfer of homologous DNA segments among closely related organisms is a major source of genomic diversification.

DNA, Bacterial↗
PubMed1990

Idiotope structure and genetic diversity in anti-streptococcal group A carbohydrate antibodies.

Three cross-reactive idiotopes(Id), termed IdX, IdI-1, and Id5, that are present on free L chains from murine anti-group A streptococcal carbohydrate antibodies have been mapped; these Id distinguish between products of three homologous V kappa genes. For each determinant, sequence analysis of anti-streptococcal group A carbohydrate antibody V domains yielded small numbers of amino acids invariably associated with Id expression. Flow micro-fluorimetry was used to isolate three IdI-1- spontaneous mutants of the IdI-1+ hybridoma GAC 39; all had single amino acid changes in the L chain at position 60 and 77, all retained other Id, and all bound group A carbohydrate. Computer modeling was used to examine spatial relationships between Id. A number of the conserved Id5 and IdX residues cluster in the L chain framework region 1 around the first back loop connecting strands of the beta pleated sheets, and overlap at residue 15 (Id5, proline; IdX, leucine). This overlap accords with the mutually exclusive expression of Id5 and IdX. The IdI-1 loss variants have mutations of residues 60 or 77 on adjacent back loops, approximately 7.5 and 14 A from residue 15. Competitive inhibition of anti-IdX and anti-IdI-1 binding to antibodies expressing both Id can be attributed to steric hindrance. The framework back loops may be favored sites for cross-reactive Id expressed by products of a single V region gene. IdI-3a, an individual Id not associated with use of a particular gene segment, has been localized in part to residue 31 (hypervariable region 1) of the H chain.

Amino Acid Sequence↗
PubMedDate not supplied

Interspousal transmission of hepatitis C virus: application of comparing the variability of HVR1 nucleotide region.

BACKGROUND/AIMS: The interspousal transmission of hepatitis C virus (HCV) has been a controversial issue in previous studies. We thus aim to investigate the possibility of HCV transmission between spouses by comparing the variability of HCV hypervariable region 1 (HVR1) between spouses and non-spouses. METHODOLOGY: Four spouses both positive for anti-HCV antibody and HCV RNA were enrolled in our study. Reverse transcriptional polymerase chain reaction (PCR) of HVR1 region was done from each patient for blood samples. The amplified PCR products were molecularly cloned, and eight to ten clones from each patient were analyzed. In addition to the phylogenetic analysis, comparing the variability of nucleotide and deduced amino acid sequences of HVR1 and outside HVR1 from these clones by a computer software program PHYLIP version 3.57 was performed in each patient, between spouse and among non-family patients. RESULTS: All eight patients were infected with genotype Ib HCV. The sequences of eight to ten clones of HCV HVR1 in each patient showed quasispecies nature of HCV. Moreover, the variabilities of nucleotide sequence and deduced amino acid sequence of HVR1 were much higher than those outside of HVR1. Between spouses, the variabilities of HVR1 were significantly lower (p<0.001) than those of non-family members in 3 of 4 families. Clones of the same patient displayed the closest relationship in the phylogenetic tree. In addition, spouses of three families showed a closer relationship than other non-family patients. CONCLUSIONS: This study strongly suggests that sexual transmission does exist, which can be confirmed by comparing the variability of HVR1 nucleotide region of HCV.

Female↗
PubMed1995

Sequence analysis of the human alpha beta T-cell receptor CDR3 region.

Although the three-dimensional structure of the T-cell receptor (TCR) has not yet been determined, several groups have proposed that the outline structure of the TCR will closely resemble that of immunoglobulin (Ig). Hypervariable regions can be identified within the TCR variable (V) domains, and by analogy to similar regions in the Ig molecule which together form the antigen combining site these have been termed the complementarity determining regions (CDR) 1, 2, and 3. By far the greatest extent of variability occurs at CDR3 and this has led to the proposal that CDR3 is involved in interaction with the peptide bound within the cleft of a major histocompatibility complex (MHC) molecule. We have cloned and sequenced the CDR3 region of several hundred human TCRA and TCRB transcripts from different T-cell populations and studied the amino acid usage in this region. Results show that the average length of the CDR3 region is 10 amino acids with less variation in length than is seen for the Ig heavy chain. There is no difference in CDR3 length between fetal and adult T cells or between CD4 and CD8 populations. The pattern of amino acid usage in the CDR3 region is dissimilar between TCRA and TCRB transcripts. In particular there is a predominance of charged and polar residues in the region of the TCRA transcript thought to interact with peptide. These data provide information on the general pattern of CDR3 length and composition for both TCRA and TCRB.

Adult↗
PubMed1996

TCRB junctional regions from HLA-B27-restricted T cells and HLA-B27 binding peptides display conserved hydropathy profiles in the absence of primary sequence homology.

Analysis of formal amino acid sequence identity between different TCRB chain (TCRB) hypervariable regions (CDR3) is commonly used to localize relevant sites of TCR antigen interaction or to yield indirect information on unknown corresponding antigens. However, this analysis sometimes fails to demonstrate expected concordances, e.g. between CDR3 from T cell clones of identical reactivity. Since this may be due to ignorance of physico-chemical parameters, we have now use hydropathy profile analysis as an additional method to examine TCRB-CDR3 and putative peptide antigens. Superimposed hydropathy plots (SHOP) of 20 TCRB-CDR3 from HLA-B27-restricted autoreactive and Yersinia enterocolitica-specific synovial cytotoxic T lymphocytes (CTL) isolated from patients with reactive arthritis (ReA) revealed restricted distribution of polar amino acids resulting in characteristically different SHOP profiles between the two CTL groups. Similarly, Yersinia-derived and self nonapeptides known to bind HLA-B27 differed in SHOP profiles. To validate the method we have extended SHOP analysis to published TCRB sequence data from additional TCRB-CDR3 from peptide-specific CTL but not in TCRB from HLA-B27-alloreactive CTL or non-HLA-B27-restricted control CTL. We here demonstrate that SHOP may improve TCR-CDR3 sequence analysis by detection of structural constraints which remain cryptic by conventional sequence analysis. Our data suggest that electrostatic properties rather than rigid sequence motifs determine T cell specificities.

Amino Acid Sequence↗
PubMed1998

rab3 mediates cortical granule exocytosis in the sea urchin egg.

Egg activation at fertilization in the sea urchin results in the exocytosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membrane proteins modeled in the SNARE hypothesis, synaptotagmin, VAMP, and syntaxin, in addition to a small GTPase of the ras superfamily, rab3, were present on cortical granules (Conner, S., Leaf, D., and Wessel, G., Mol. Reprod. Dev. 48, 1-13, 1997). Here we report that rab3 is associated with cortical granules throughout oogenesis, during cortical granule translocation, and while docked at the egg plasma membrane. Following cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocytic origin. Because of its selective association with cortical granules in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of interfering with rab3 function by peptide competition with its effector domain, a conserved region within specific rab types. We first identified the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglocentrotus purpuratus. Then, with synthetic peptides to different regions of the rab3 protein, we find that cortical granule exocytosis is inhibited in eggs injected with effector domain peptides, but not with peptides from the hypervariable region or with a scrambled effector peptide. Additionally, effector-peptide-injected eggs injected with IP3 are blocked in their ability to exocytose cortical granules, suggesting that the inhibition is directly on the membrane fusion event and not the result of interference with the signal transduction mechanism leading to calcium release. We interpret these results to mean that rab3 functions in the regulation of cortical granule exocytosis following vesicle docking.

Amino Acid Sequence↗
PubMed1998

Automated classification of antibody complementarity determining region 3 of the heavy chain (H3) loops into canonical forms and its application to protein structure prediction.

A computer-based algorithm was used to cluster the loops forming the complementarity determining region (CDR) 3 of the heavy chain (H3) into canonical classes. Previous analyses of the three-dimensional structures of CDR loops (also known as the hypervariable regions) within antibody immunoglobulin variable domains have shown that for five of the six CDRs there are only a few main-chain conformations (known as canonical forms) that show clear relationships between sequence and structure. However, the larger variation in length and conformation of loops within H3 has limited the classification of these loops into canonical forms. The clustering procedure presented here is based on aligning the Ramachandran-coded main-chain conformation of the residues using a dynamic algorithm that allows the insertion of gaps to obtain an optimum alignment. A total of 41 H3 loops out of 62 non-identical loops, extracted from the Brookhaven Protein Data Bank, have been automatically grouped into 22 clusters. Inspection of the clusters for consensus sequences or intra-loop interactions or invariant conformation led to the proposal of 13 canonical forms representing 31 loops. These canonical forms include a consideration of the geometry of both the take-off region adjacent to the bracing beta-strands and the remaining loop apex. Subsequently a new set of 15 H3 loops not included in the initial analysis was considered. The clustering procedure was repeated and nine of these 15 loops could be assigned to original clusters, including seven to canonical forms. A sequence profile was generated for each canonical form from the original set of loops and matched against the sequences of the new H3 loops. For five out of the seven new H3 loops that were in a canonical form, the correct form was identified at first rank by this predictive scheme.

Amino Acid Sequence↗
PubMed2001

The TolC protein of Escherichia coli serves as a cell-surface receptor for the newly characterized TLS bacteriophage.

The TolC protein of Escherichia coli is implicated in a variety of diverse cellular functions, including antibiotic efflux and alpha-hemolysin secretion. An incidental role of TolC is to facilitate the entry of the bacteriophage TLS and colicin E1 into the bacterial cell. Despite the resolution of TolC's atomic structure, the roles of specific residues in its diverse functions are unknown. Here, we describe a genetic strategy for isolating missense tolC mutations that abolish the bacteriophage receptor activity of the TolC protein without influencing its role in antibiotic efflux. These spontaneous mutations affected two regions of the TolC protein and included base-pair substitutions, insertions, and deletions. Comparison of the TolC sequence with those of its homologues revealed two hypervariable stretches that were predicted to represent loops. Interestingly, all but one of the TolC alterations preventing phage binding were located in these two hypervariable regions, which are likely to be exposed on the cell surface. This was substantiated by the recently solved three-dimensional structure of TolC. Curiously, all the phage-resistant TolC mutants showed varying degrees of resistance to colicin E1, suggesting the involvement of overlapping regions of TolC in colicin E1 import and phage binding. The phage used in this study, TLS, was earlier reported as a strain of U3. However, we show here that, unlike the previously reported lipopolysaccharide-specific U3 phage, this phage displays a distinctly different host range and discrete morphological features and, in addition to utilizing TolC as receptor, it requires the inner core of a lipopolysaccharide.

Adsorption↗
PubMed1996

Comparison of German population data on the apoB-HVR locus with other Caucasian, Asian and black populations.

A population study of 505 unrelated individuals from Southwestern Germany was carried out on the 3'-apoB hypervariable region (HVR). After amplification via polymerase chain reaction (PCR) and agarose gel electrophoresis, 15 different alleles and 47 genotypes were observed. The most common alleles were hypervariable elements (HVE) 37 and 35 with an allele frequency of 0.374 and 0.244, respectively. The heterozygosity index was calculated to be 78.4%. Allele frequencies of this study are compared with results from other databases obtained from a French, a Spanish, an Asian and an American (Black) population.

Alleles↗
PubMed1980

Amino acid sequence of homogeneous antibodies to dextran and DNA rearrangements in heavy chain V-region gene segments.

The complete variable region sequences from ten antibodies and two myeloma proteins binding alpha-1,3 dextran have been determined. The diversity patterns of these homogeneous antibody molecules suggest that the variable regions of heavy chains are encoded by separate variable (V) and joining (J) gene segments. The most striking feature of these data is the extensive sequence variability of a region that we denote the D (diversity) segment which is located at the junction between the V and J segments in the centre of the third hypervariable region. The D segment diversity may arise from a novel somatic mutational mechanism or may be encoded by multiple D gene segments. For the first time, the amino acid sequence correlates of several V region idiotypes are determined.

Amino Acid Sequence↗
PubMed1986

Antigenic variation of gonococcal pilus involves assembly of separated silent gene segments.

The pilus is a major outer-membrane protein of Neisseria gonorrhoeae that undergoes phase and antigenic variation. In strain MS11 pilus expression is regulated at two expression loci on the chromosome, pilE1 and pilE2, although many other regions contain silent pilin information. A comparison of variant pilin sequences has revealed that the gene can be divided into a constant, a semivariable, and a hypervariable region. We report here that complete pilin genes are found only at the expression loci. Silent constant and variable region pilin gene segments are located on separate and distinct restriction fragments, and the generation of a complete pilin gene within the expression loci is the result of multiple recombination events. Conserved sequences within and flanking the pilin gene are proposed to act as recombination sites during the gene conversion events needed to produce a functional pilin gene.

Antigens, Bacterial↗
PubMed1991

Excess nonsynonymous substitution of shared polymorphic sites among self-incompatibility alleles of Solanaceae.

The function of the self-incompatibility locus (S locus) of many plant species dictates that natural selection will favor high levels of protein diversity. Pairwise sequence comparisons between S alleles from four species of Solanaceae reveal remarkably high sequence diversity and evidence for shared polymorphism. The level of amino acid constraint was found to be significantly heterogeneous among different regions of the gene, with some regions being highly constrained and others appearing to be virtually unconstrained. In some regions of the protein, there was an excess of nonsynonymous over synonymous substitution, consistent with the strong diversifying selection that must operate on this locus. These hypervariable regions are candidates for the sites that determine functional allelic identity. Simple contingency table tests show that sites that have polymorphism shared between species have more nonsynonymous substitution than polymorphic sites that do not exhibit shared polymorphism. This is consistent with the idea that adaptive evolution favoring amino acid replacement is occurring at sites with shared polymorphism. Tests of clustered polymorphism reveal that an unusually low rate of recombination must be occurring in this locus, allowing very ancient alleles to preserve their identity.

Alleles↗