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PubMed2004

A DNA microarray system for forensic SNP analysis.

Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce samples due to the high copy number of mtDNA in each cell. Due to the increasing number of complete mtDNA genome sequences available, the limited discrimination power of an mtDNA analysis, may be increased by analysis of coding region polymorphisms in addition to the non-coding variation. Since sequence analysis of the coding region would require more material than generally present in forensic samples, an alternative SNP analysis approach is required. We have developed a one-colour microarray-based SNP detection system for limited forensic materials. The method is based on minisequencing in solution prior to hybridisation to universal tag-arrays. In a first outline of a forensic chip, a combination of 12 nuclear and 21 mitochondrial SNP markers are analysed simultaneously. The mitochondrial markers on the chip are polymorphisms within the hypervariable region as well as in the coding region. Even though the number of markers in the current system is limited, it can easily be extended to yield a greater power of discrimination. When fully developed, microarray analysis provides a promising system for efficient sensitive SNP analysis of forensic samples in the future.

Complementarity Determining Regions↗
PubMed1990

Nucleotide sequences and three-dimensional modelling of the VH and VL domains of two human monoclonal antibodies specific for the D antigen of the human Rh-blood-group system.

The nucleotide sequences were determined for the VH and VL domains of two human IgG1 antibodies, Pag-1 and Fog-B, specific for the D antigen of the Rh-blood-group system. The VH-region genes of the two antibodies were derived from separate germ-line genes within the VH-IV gene family, but both antibodies used the same JH6 gene. The D-region genes differed from each other, and no similarity was found to known D regions. The light chain of Fog-B belongs to the V lambda-I subgroup and that of Pag-1 probably belongs to the V lambda-V subgroup; both light chains used the J2/3 gene. Three-dimensional models of the variable domains were made, based on those of known crystallographic structure. The surface contours at the combining sites are clearly different, consistent with the evidence that the antibodies recognize different but overlapping epitopes. Some details of the molecular modelling of hypervariable regions have been deposited as Supplementary Publication SUP 50155 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.

Amino Acid Sequence↗
PubMed1982

The complete V domain amino acid sequences of two myeloma inulin-binding proteins.

The myeloma proteins binding inulin afford a unique opportunity to study the V region patterns of variation because of the similarity in the VL as well as the VH regions. The diversity patterns in both the VL and VH regions suggest these proteins are encoded by multiple, very similar V gene segments or that somatic mutation may repeatedly generate identical variants. Because of the close similarity in the V domains of these proteins and the extensive idiotypic analyses that have been carried out previously, several interesting conclusions can be drawn about the nature of idiotypic determinants. First, a single amino acid residue may be involved in determining multiple idiotypic determinants. Second, hapten-inhibitable idiotypes may depend on residues within and outside the hypervariable regions. Third, idiotypic similarity does not always predict a corresponding sequence similarity.

Amino Acid Sequence↗
PubMed1977

Independent expression of the gene coding for the constant domain of immunoglobulin light chain: evidence from sequence analyses of the precursor of the constant region polypeptide.

The mRNA coding for the kappa-type constant region (C(kappa)) was purified from two clones derived from the MPC-11 mouse myeloma. This mRNA directs the cell-free synthesis of a C(kappa) precursor (molecular weight, about 15,000) in which an extra piece, 17 residues long, precedes the NH(2)-terminal residue (Ala(109)) of the C(kappa) region. The partial sequence of the extra piece is: Met-X-Thr-Asp-Thr-Leu-Leu-Leu-Trp-Val-Leu-Leu-Leu-Trp-Val-Pro-X- (X is unknown). Met(1) was shown to be the initiator methionine. The sequence of the C(kappa) extra piece is completely different from any known sequence preceding residue Ala(109) in whole light (L) chains, thus establishing that the C(kappa)-region mRNA could not have originated from mRNA coding for the whole L chain. The structural features of the C(kappa) extra piece (marked hydrophobicity, size, and a methionine at the NH(2)-terminus) are identical to those characteristic of the NH(2)-terminal extra piece linked to the variable (V) region of whole L-chain precursors. In addition, the C(kappa) extra piece and the extra piece linked to the V region of MOPC-321 L chain have 70% sequence homology. These findings can be explained by the two genes-one Ig chain hypothesis, if we assume that the DNA coding for the extra piece (xp-DNA) is a constitutive part of the V gene. According to this model, the C(kappa)-region mRNA could have originated from: (i) translocation of this V gene to the C gene, deletion of the entire mature V gene, and "end-to-end" repair of the remaining xp-DNA to the C gene; (ii) translocation to the C gene only of the xp-DNA portion of the V gene. Alternatively, we may assume that the xp-DNA is not covalently linked to the mature V gene at all times, as might be the case for the DNA of hypervariable regions presumed to be in episomes. This raises the intriguing speculation that the xp-DNA represents a third distinct gene, designated xp-gene. The presumed xp-gene may be involved in the regulation of gene transcription: when linked to the mature V gene it initiates a chain of events leading to whole L-chain mRNA formation; when attached to the C gene it leads to its transcription to provide the C-region mRNA.

Amino Acid Sequence↗
PubMed1981

Complete amino acid sequence of variable domains from two monoclonal human anti-gamma globulins of the Wa cross-idiotypic group: suggestion that the J segments are involved in the structural correlate of the idiotype.

The complete amino acid sequences of the variable regions of two human IgM anti-gamma globulins are reported. The two proteins, Sie and Wol, share idiotypic determinants that are distinct from those shared by the two previously sequenced IgM anti-gamma globulins, Lay and Pom [Capra, J. D. & Kehoe, J. M. (1974) Proc. Natl. Acad. Sci. USA 71, 4032-4036; Klapper, D. G. & Capra, J. D. (1976) Ann. Immunol. (Inst. Pasteur) 127C, 261-271.] In contrast to Lay and Pom, Sie and Wol have very little homology in the complementarity-determining hypervariable regions of either their light or heavy polypeptide chains. However, there are only two amino acid differences in the framework portions of the light chains, and the J segments of both chains are homologous. These data suggest that the predominant idiotypic determinant in this cross-idiotypic system may be different than in the Po group and may be either related to their light chain framework structures or to the amino acid sequence in the J segments of these molecules.

Amino Acid Sequence↗
PubMed2004

Polymorphism and transcription at the p44-1/p44-18 genomic locus in Anaplasma phagocytophilum strains from diverse geographic regions.

A polymorphic multigene family (p44) of Anaplasma phagocytophilum encodes the immunodominant 44-kDa major outer membrane proteins. With p44-specific PCR and gene-specific probes, p44-1 was found in all human isolates from New York State but not in isolates from Minnesota, whereas p44-18 and two other p44 species were found in isolates from both regions. We therefore sequenced the genomic locus corresponding to the p44-1/p44-18 tandem locus of A. phagocytophilum HZ in 14 other geographically divergent strains from various hosts. The locus was found in all 14 strains, and p44-18 was conserved among all 13 United States isolates studied. In all nine northeastern strains, p44-1 was conserved. However, in three of the Minnesota strains and in one California strain, p44-1 was replaced at this genomic locus by the novel gene p44-61 (p44-61/18), whose hypervariable region (hv) was a chimera of p44-20hv and p44-23hv. The conserved base sequence within the hv region linked the two segments. In contrast, in the Old Sourhope strain isolated from sheep in the United Kingdom, only a single and distinct p44, p44-OS, was found in this locus. This suggests different rates of evolution of p44-1 and p44-18 at this locus and conservation of the locus within strains isolated from the same geographic region. Locus-specific reverse transcription-PCR revealed expression of p44-1 by New York and p44-61 by Minnesota strains at this locus. These p44 loci provide insight into the molecular evolution and functional divergence of p44 paralogs and may serve as markers for typing strains from different geographic regions.

Anaplasma phagocytophilum↗
PubMed1995

Capsid diversity in small round-structured viruses: molecular characterization of an antigenically distinct human enteric calicivirus.

Studies of antigenic variation between small round-structured viruses (SRSVs) using immune electron microscopy have revealed 3 antigenic types currently circulating in the UK represented by the strains SRSV/Bri/93/UK, SRSV/Sot/91/UK and SRSV/Mel/89/UK. Mel/89/UK RNA was isolated from a 1989 school outbreak of gastroenteritis. The 3'-terminal 3435 nucleotides (excluding the poly(A) tail) were determined by RT-PCR and cDNA sequencing, completing our molecular characterization of antigenically diverse SRSVs. Coding regions for the calicivirus RNA polymerase and capsid protein were found together with a 3' open reading frame of unknown function. The polymerase region was most highly conserved between Mel/89/UK and the other two SRSVs while the 3' open reading frame exhibited extreme variation. Phylogenetic analysis of SRSV capsids showed that Mel/89/UK differed significantly from Bri/93/UK and Sot/91/UK (62 and 39% identity, respectively) and was distinct from 6 other non-UK SRSVs that had been previously characterized. This was consistent with the designation of Mel/89/UK as a novel antigenic variant. Comparison of the capsid amino acid sequences of the 3 UK strains together with the antigenically distinct SRSV/Nor/68/US revealed a hypervariable region that could be surface-exposed and contain the SRSV antigenic determinants.

Amino Acid Sequence↗
PubMed1995

Novel HLA-DPB1 alleles detected in the Ethiopian population.

The number of identified HLA-DPB1 alleles increased rapidly by application of DNA-based typing techniques. PCR-SSO typing indicated the presence of possible new HLA-DPB1 variants in the Ethiopian population. The use of the SBT technique, which considers polymorphic as well as constant regions in the second exon of HLA genes, allowed direct identification of two new allelic variants. Moreover, a recently identified HLA-DPA1 variant was also present in this population. The newly defined allelic HLA-DPB1 sequences found in five individuals of the Ethiopian population were confirmed by cloning and subsequent sequencing of the cloned DNA. One of the new alleles was shown to segregate in a family and was also present in unrelated individuals. Both new DPB1 alleles represent new combinations of existing polymorphism in the hypervariable regions. In different populations the frequency of these new HLA-DP variants remains to be determined.

Alleles↗
PubMed1989

Early onset of somatic mutation in immunoglobulin VH genes during the primary immune response.

The dynamics of somatic mutation in Ig variable genes was investigated in order to define a population of B cells undergoing mutation. BALB/cJ mice were injected with PC-KLH, and splenic RNA was prepared 5, 7, and 13 d later. The mRNA was annealed to gamma constant region primers to make cDNA transcripts encoding VH genes. 103 cDNA clones corresponding to 18 different genes from the VH7183, VH3660, and VHS107 subfamilies were sequenced to identify mutation. VH genes had a low level of mutation on day 5 after immunization and accumulated more mutation by day 7 at a rate of 10(-3) mutations per nucleotide per generation. However, by day 13, the number of mutations per gene did not increase, and most of the substitutions encoded replacement amino acid changes that were clustered in the hypervariable regions, indicating that the mutational process was less active during the second week and that antigen selection had occurred. The data are consistent with a developmentally regulated mechanism in which mutation is activated during the first week of the primary immune response for a limited time period, after which selection acts to preserve the beneficial mutants.

Animals↗
PubMed2001

Evidence that intragenic recombination contributes to allelic diversity of the S-RNase gene at the self-incompatibility (S) locus in Petunia inflata.

For Solanaceae type self-incompatibility, discrimination between self and nonself pollen by the pistil is controlled by the highly polymorphic S-RNase gene. To date, the mechanism generating the allelic diversity of this gene is largely unknown. Natural populations offer a good opportunity to address this question because they likely contain different alleles that share recent common progenitors. We identified 19 S haplotypes from a natural population of Petunia inflata in Argentina, used reverse transcriptase-polymerase chain reaction to obtain cDNAs for 15 alleles of the S-RNase gene, and sequenced all the cDNAs. Phylogenetic studies revealed that five of these alleles and two previously identified alleles form a major clade, and that the 5' region of S(19) allele was derived from an ancestor allele closely related to S(2), whereas its 3' region was derived from an ancestor allele closely related to S(8). A similar evolutionary relationship was found among S(3), S(12), and S(15) alleles. These findings suggest that intragenic recombination contributed to the generation of the allelic diversity of the S-RNase gene. Two additional findings emerged from the sequence comparisons. First, the nucleotide sequence of the S(1) allele identified in this work is completely identical to that of the previously identified S(1) allele of a different origin. Second, in the two hypervariable regions HVa and HVb, thought to be involved in determining S allele specificity, S(6) and S(9) alleles differ only by four nucleotides, all in HVb, resulting in two amino acid differences. The implications of these findings are discussed.

Alleles↗
PubMed2003

Novel polymorphisms influencing transcription of the human CHRM2 gene in airway smooth muscle.

Muscarinic receptors are a functionally important family of G-protein-coupled receptors. Using a combination of rapid amplification of 5' cDNA ends and reporter gene assays, we characterized the 5' untranslated region of the CHRM2 gene as expressed in human airway smooth muscle (HASM) cells. A splice site is present 46 bp upstream from the ATG start codon. Five exons with alternative splicing patterns are present upstream of this splice site, separated by introns ranging from 87 bp to > 145 kb. There is evidence for the gene being under the control of a TATA-less promoter with Sp1, GATA, and activator protein-2 binding sites. Multiple transcription start sites (TSSs) were identified. We identified a novel 0.5-kb hypervariable region located 648 bp upstream of the most 5' TSS, a multiallelic (CA) tandem repeat 96 bp downstream of the most 5' TSS, and a common C-->A SNP located 136 bp upstream of the most 5' TSS. Functional studies in primary HASM cells and the BEAS-2B cell line demonstrated highest promoter activity to be upstream of the most 3' TSS, with potential repressor elements operating in a cell type-dependent manner, located upstream of the most 5' TSS. We present functional data to show that the CA repeat may influence the transcription of the gene in HASM and BEAS-2B cells.

5' Untranslated Regions↗
PubMed1991

Molecular analysis of HLA-DQ A alleles in coeliac disease lack of a unique disease-associated sequence.

Susceptibility to coeliac disease is strongly associated with some HLA class II antigens, encoded by the HLA-D region. Since the HLA-DQ locus seems to be primarily involved, we have analysed by polymerase chain reaction amplification and allele-specific oligonucleotide hybridization the most polymorphic region of the HLA-DQ A1 gene. No difference was observed between the 20 coeliac patients and 20 HLA-D-matched healthy controls who took part in the study. Furthermore, in patients and controls, the restriction fragment length polymorphism analysis of the HLA-DQ A gene using the restriction enzyme BglII did not disclose any specific disease-associated fragment. Our results are not consistent with a unique DQ A coeliac disease-associated sequence, but rather with the hypothesis that some polymorphic residues or allelic hypervariable regions, although found also in the normal population, can predispose to coeliac disease due to their higher frequency in this condition.

Base Sequence↗
PubMed2005

Phylogenetic analysis of the hexon loop 1 region of an adenovirus from psittacine birds supports the existence of a new psittacine adenovirus (PsAdV).

Adenovirus infections in psittacine birds have been well known. Most of these infections were caused by fowl adenoviruses (FAdV). In this study, liver samples showing typical histological signs of an adenovirus infection were collected from Poicephalus spp. with acute disease. A PCR amplifying the variable loop 1 region of the hexon gene was developed using primers located in two conserved pedestal regions. A PCR product of approximately 590 bp in size was amplified and sequenced. The sequence obtained grouped outside of the FAdV reference strains of the 12 serotypes as well as egg drop syndrome virus and turkey adenovirus 3 indicating that a new avian adenovirus was detected. In comparison to the FAdV reference strains, the percentage of identical nucleotides ranged between 60.3 and 67.0 and that of identical amino acids (aa) between 51.3 and 61.0. Furthermore, 37 unique aa exchanges were observed; out of these, 27 are located in the 4 hypervariable regions of loop 1, which encode the serotype-specific epitopes. The g/c content, the isoelectric point and the charge of the amplified fragment, however, are in the range as those of group I avian adenoviruses. It was proposed, therefore, to designate this new adenovirus as psittacine adenovirus (PsAdV).

Adenoviridae↗
PubMed1993

Analysis of genes within the HLA region affecting susceptibility to ulcerative colitis.

To clarify the molecular relationship between HLA loci and ulcerative colitis (UC) in Japanese patients, we performed HLA-DP genotyping by the PCR-RFLP method and studied tumor necrosis factor beta-chain genetic polymorphism by Southern hybridization, in addition to conventional serologic typing. Significant increase was observed in Bw52, DPw9 (DPB1*0901), and DR2 (DRB1*1502) in Japanese patients with UC. Linkage analysis indicated that A24-Bw52-DR2-DPw9 alleles constitute a common haplotype in Japanese UC patients. Among the patients not carrying Bw52, B13 was significantly increased and B44 was relatively increased. These Bw52, B13, and B44 alleles share the unique amino acids, serine and aspartic acid at positions 67 and 77, respectively. These positions are in the second hypervariable region of the alpha 1-domain of the HLA-B13, B44, Bw52, and B49 antigens (B49 is quite rare in the Japanese population). The inflammatory region in UC patients was found to vary depending on their HLA-B alleles. These results suggest that the HLA-B locus itself plays an important role in the susceptibility to Japanese UC.

Adult↗
PubMedDate not supplied

Antibodies to streptococcal carbohydrate: substitutes for the myeloma proteins.

Antibodies to streptococcal carbohydrates have been employed in a number of ways as substitutes for the myeloma proteins in studies of the structures of antibody molecules and the development of the immune response. This review concentrates on the chemical nature of the streptococcal polysaccharides and on structural studies of the variable regions of the light chains of rabbit anitbodies to the group A-variant polysaccharide produced by hyperimmunization. The chemistry of the group A-variant polysaccharide has now been clarified, and this information has explained the frequently reported difficulty in the preparation of precipitating antisera to this antigen. The group A-variant vaccine is a potent antigen, but the bulk of the antibody is nonprecipitating because it is directed against the noreducing terminal sugar of the nonbranched rhamnose polymer. Chemical studies of the light chains of homogeneous rabbit antibodies reveal two hypervariable regions and a J sequence that joins the variable region to the constant region. Although the precise topographic highlights of the antigen-binding site hae yet to be determined, these structural studies have extended our knowledge of the structure and diversity of antibody molecules.

Animals↗
PubMed2001

Genetic variation in the 3' non-coding region of dengue viruses.

The 3' non-coding region (3'NCR) of strains of dengue 1 (DEN 1), DEN 2, DEN 3, and DEN 4 viruses, isolated in different geographical regions, was sequenced and compared to published sequences of the four dengue viruses. A total of 50 DEN 2 strains was compared: 7 West African strains, 3 Indonesian mosquito strains, 1 Indonesian macaque isolate, and 39 human isolates from Southeast Asia, the South Pacific, and the Caribbean and Americas. Nucleotide sequence alignment revealed few deletions and no repeat sequences in the 3' NCR of DEN 2 viruses and showed that much of the 3' NCR was well conserved. The strains could be divided into two groups, sylvatic and human/mosquito/macaque, based on nucleotide sequence homology. A hypervariable region was identified immediately following the NS5 stop codon, which involved a 2-10 nucleotide deletion in human, mosquito, and macaque isolates compared with the sylvatic strains. The DEN 2 3'NCR was also compared with 3'NCR sequences from strains of DEN 1, DEN 3, and DEN 4 viruses. DEN 1 was found to have four copies of an eight nucleotide imperfect repeat following the NS5 stop codon, while DEN 4 virus had a deletion of 75 nucleotides in the 3'NCR. We propose that the variation in nucleotide sequence in the 3'NCR may have evolved as a function of DEN virus transmission and replication in different mosquito and non-human primate/human host cycles. The results from this study are consistent with the hypothesis that DEN viruses arose from sylvatic progenitors and evolved into human epidemic strains. However, the data do not support the hypothesis that variation in the 3'NCR correlates with DEN virus pathogenesis.

3' Untranslated Regions↗
PubMed2004

[Myiasis caused by Oestridae: serological and molecular diagnosis].

Myiasis-causing Oestridae (bot flies) infect several animal species world-wide, from palaearctic to subtropical/tropical areas. Oestrids affect livestock production causing abortion, reduced milk production, losses in weight and fertility, poor hide quality and an impairment of the host's immune system. In the last few years much research has been carried out on the immunology of these infestations, in order to acquire efficient and reliable diagnostic serological tools; the genome of the different species of Oestridae has been studied to further their molecular identification, taxonomy and phylogenesis. The immunodiagnostic methods for many myiasis causing Oestrids have proven to be a viable alternative to the clinical parasitological examination or the post-mortem examination. Numerous serological tests have been developed for the diagnosis of bovine hypodermosis caused by Hypoderma bovis and Hypoderma lineatum, and ELISAs using larval hypodermin C as the antigen are currently used on serum, individual and pooled milk samples to detect the presence of circulating anti-Hypoderma antibodies. In Italy the best period to sample the animals is November-January, since it is in this period that the antibody kinetics of the animals reaches a peak. Recently the efficacy of the ELISA test on pasteurized milk samples has been demonstrated, allowing the diagnosis of bovine hypodermosis also in areas where there is no information on the presence of the disease and the sampling of the animals is laborious. The cross-reactivity between Przhevalskiana silenus antigens and anti-Hypoderma antibodies led to assessing the usefulness of a simple and cost-effective ELISA for the diagnosis of goat warble fly infection. In particular, it has been demonstrated that infected goats display an antibody peak in November-December in blood and milk, thus making this period suitable for sampling. Although no extensive data is available on the immunology of sheep and goat oestrosis caused by Oestrus ovis, the efficacy of ELISA has been demonstrated by correlating serological results with clinical post-mortem examinations. No immunological techniques are currently used to diagnose gasterophilosis of equids and only one study reports the efficacy of ELISA for detecting anti-Gasterophilus antibodies in infected equids. Several studies have been conducted into the molecular characterization of the mitochondrial DNA (mtDNA)--in particular of the gene encoding for the cytochrome oxidase I (COI)--for many free-living and parasitic arthropods for diagnostic, taxonomic and phylogenetic purposes. As regards Oestridae causing myiasis, the first study features a PCR-RFLP assay of the most common Italian species (i.e. H. bovis, H. lineatum, Gasterophilus intestinalis, P. silenus, O. ovis), which showed clear genetic differences among the genera examined, but no inter-specific variation between the two species of Hypoderma considered. The molecular characterization of the most variable region of the COI gene (encoding for the region from E4 to the terminal COOH) was able to clearly differentiate H. bovis and H. lineatum. The E4-COOH region of the COI gene has been characterized for 18 oestrid species and from a taxonomical point of view, molecular data confirm the morphological classification, with the examined species divided into four subfamilies. New insights have also been gained on the molecular differentiation of the most common species of Hypoderma (i.e. H. bovis, H. lineatum, Hypoderma actaeon, Hypoderma diana and Hypoderma tarandi) and, in particular, the restriction enzyme BfaI, provides a diagnostic profile that can be used to simultaneously differentiate all the species examined. The characterization of the E4-COOH COI gene and the hypervariable region of the gene encoding for the ribosomal Isu revealed the identity of Hypoderma sinense as a new species, infecting cattle and yaks in China. Finally, the molecular analysis of the same mitochondrial and ribosomal regions showed that P. silenus, Przhevalskiana aegagri and Przhevalskiana crossii are morphotypes of the same species.

Animals↗
PubMed1986

Fine specificity analysis of idiotype-specific suppressor factors that block a delayed-type hypersensitivity response to M315: evidence supporting a role for somatic mutations in the variable region of the lambda 2 chain of M315.

The delayed-type hypersensitivity (DTH) response in BALB/c mice to the idiotype of M315 (alpha,lambda 2), a BALB/c myeloma protein, can be suppressed by a single i.v. injection of soluble M315. This suppression involves the generation of suppressor T cells. Mice tolerized by M315 produce subcellular factors that suppress the M315-DTH response. Such suppressor factors can be obtained by mechanical disruption of spleen cells or thymocytes from mice treated i.v. with M315. The fine specificity of the factor was studied using various immunoadsorbents such as L315 and germline V lambda 2 chain-Sepharose beads with known amino acid sequences. The results indicated that the specificity of the factor depended on three contiguous somatically mutated amino acid residues, Phe94, Arg95 and Asn96, in the third hypervariable region of the V domain of the L chain of M315.

Animals↗