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PubMed2005

Mitochondrial DNA typing screens with control region and coding region SNPs.

Mitochondrial DNA (mtDNA) analysis has found an important niche in forensic DNA typing. It is used with highly degraded samples or low-copy number materials such as might be found from shed hair or bones exposed to severe environmental conditions. The primary advantage of mtDNA is that it is present in high copy number within cells and therefore more likely to be recovered from highly degraded specimens. A major disadvantage to traditional forensic mtDNA analysis is that it is time-consuming and labor-intensive to generate and review the 610 nucleotides of sequence information commonly targeted in hypervariable regions I and II (HVI and HVII) of the control region. In addition, common haplotypes exist in HVI/HVII mtDNA sequences that can reduce the ability to differentiate two unrelated samples. In this report we describe the utility of two newly available screening assays for rapid exclusion of non-matching samples. The LINEAR ARRAY mtDNA HVI/HVII Region-Sequencing Typing Kit (Roche Applied Science, Indianapolis, IN) was used to type 666 individuals from U.S. Caucasian, African American, and Hispanic groups. Processing of the LINEAR ARRAY probe panels "mito strips" was automated on a ProfiBlot workstation. Observable variation in 666 individuals is reported and frequencies of the mitotypes within and between populations are presented. Samples exhibiting the most common Caucasian mitotype were subdivided with a multiplexed amplification and detection assay using eleven single nucleotide polymorphisms in the mitochondrial genome. These types of screening assays should enable more rapid evaluation of forensic casework samples such that only samples not excluded would be subjected to further characterization through full HVI/HVII mtDNA sequence analysis.

Automation↗
PubMed1990

DNaseI hypersensitive sites at the 3' end of the human apolipoprotein B gene.

The chromatin structure of the 3' end of the human apolipoprotein B gene has been examined. Two DNaseI hypersensitive sites were present in nuclei from liver-derived HepG2 cells and intestine-derived CaCo-2 cells, in which the apo-B gene is transcriptionally active, but were absent from HeLa cells, where the gene is not expressed, and from free DNA. The region in a segment enriched in recognition sites for topoisomerase II and known to participate in anchoring the 3' end of the gene to the nuclear matrix. The second DNaseI hypersensitive site resided in the 3' untranslated portion of the gene. Furthermore, nucleosomes were present along a 1.4-kilobase (HindIII-BamHI) segment of DNA containing the two 3' DNaseI hypersensitive sites, and a static array of nucleosomes was present along the A/T-rich hypervariable region.

Apolipoproteins B↗
PubMed1997

Enhancement of humoral immunity to SIVenv following simultaneous inoculation of mice by three recombinant adenoviruses encoding SIVenv/poliovirus chimeras, Tat and Rev.

A means of inducing gene expression by simultaneous infection with three recombinant adenoviruses (Ad) is described. The simian immunodeficiency virus (SIV) envelope-coding region was placed under the control of the human immunodeficiency virus type 1 (HIV-1) Tat and Rev proteins provided in trans by distinct Ad vectors (Ad-tat; Ad-rev). Coinfection of cells with the three recombinant adenoviruses led to induction of high levels of SIV env mRNA and protein synthesis, while inoculation of mice elicited anti-Env antibodies. Insertion of the poliovirus VP1 neutralization epitope (C3) in the V1 hypervariable region of SIV envelope not only proved to be highly immunogenic for the poliovirus epitope but also enhanced the kinetics of anti-SIV Env antibody production. By contrast, insertion in V4 elicited no anti-C3 response and only normal anti-Env responses.

Adenoviridae↗
PubMed2002

Genetic evolution of structural region of hepatitis C virus in primary infection.

AIM: To investigate the dynamics of hepatitis C virus (HCV) variability through putative envelope genes during primary infection and the mechanism of viral genetic evolution in infected hosts. METHODS: Serial serum samples prospectively collected for 12 to 34 months from a cohort of acutely HCV-infected individuals were obtained, and a 1-kb fragment spanning E1 and the 5' half of E2, including Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined a single-stranded conformational polymorphism (SSCP) and heteroduplex analysis (HDA) method to determine the number of clonotypes hypervariable region, was amplified by reverse transcriptase PCR and cloned. Nonsynonymous mutations per nonsynonymous site (dn), synonymous mutations per synonymous site (ds), dn/ds ratio and genetic distances within each sample were evaluated for intrahost evolutionary analysis. RESULTS: Quasispecies complexity and sequence diversity were lower in early samples and a further increase after seroconversion, although ds value in the envelope genes was higher than dn value during primary infection. The trend, pronounced in most of samples, toward lower ds values in the E1 than in the 5' portion of E2. Quasispecies complexity was higher and E2 dn/ds ratio was a trend toward higher value in later samples during persistent viremia. We also found individual features of HCV genetic evolution in different subjects who were infected with different HCV genotypes. CONCLUSION: Mutations of actively replicating virus arise stochastically with certain functional constaints. A complexity quasispecies exerted by a combination of either neutral evolution or selective forces shows clear differences in individuals, and associated with HCV persistence.

Adult↗
PubMed1989

Photo-CIDNP studies of Bence Jones proteins, immunoglobulins, and their proteolytic fragments.

The results of photo-CIDNP measurements of Bence Jones proteins, immunoglobulins, and their proteolytic fragments are reported. The CIDNP spectra of constant region CL fragments, which were derived from lambda-type Bence Jones proteins with three different isotypes, gave polarizations originating from two Tyr and one Trp residues. From comparisons of the results obtained by using lambda-type Bence Jones proteins that possess the known amino acid sequences with those for the CL fragments, it was concluded that the CIDNP signals observed in the case of Bence Jones proteins are all due to the hypervariable region. On the basis of the results obtained by using IgG1, IgG2, IgG3, and IgG4 myeloma proteins along with their Fab, Fc, and pFc' fragments, it was concluded that CIDNP signals for the Fab region all originate from the variable region. The Fc region gives two Tyr polarizations, one of which has been assigned to Tyr-296. We have also shown that the second Tyr signal is due to Tyr-373. Interaction of Fc and staphylococcal protein A has also been examined by the CIDNP technique.

Amino Acid Sequence↗
PubMed1980

An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH.

We have determined the sequences of separate germline genetic elements which encode two parts of a mouse immunglobulin heavy chain variable region. These elements, termed gene segments, are heavy chain counterparts of the variable (V) and joining (J) gene segments of immunoglobulin light chains. The VH gene segment encodes amino acids 1-101 and the JH gene segment encodes amino acids 107-123 of the S107 phosphorylcholine-binding VH region. This JH gene segment and two other JH gene segments are located 5' to the mu constant region gene (Cmu) in germline DNA. We have also determined the sequence of a rearranged VH gene encoding a complete VH region, M603, which is closely related to S107. In addition, we have partially determined the VH coding sequences of the S107 and M167 heavy chain mRNAs. By comparing these sequences to the germline gene segments, we conclude that the germline VH and JH gene segments do not contain at least 13 nucleotides which are present in the rearranged VH genes. In S107, these nucleotides encode amino acids 102-106, which form part of the third hypervariable region and consequently influence the antigen-binding specificity of the immunoglobulin molecule. This portion of the variable region may be encoded by a separate germline gene segment which can be joined to the VH and JH gene segments. We term this postulated genetic element the D gene segment, referring to its role in the generation of heavy chain diversity. Essentially the same noncoding sequences are found 3' to the VH gene segment and as inverse complements 5' to two JH gene segments. These are the same conserved nucleotides previously found adjacent to light chain V and J gene segments. Each conserved sequence consists of blocks of seven and ten conserved nucleotides which are separated by a spacer of either 11 or 22 nonconserved nucleotides. The highly conserved spacing, corresponding to one or two turns of the DNA helix, maintains precise spatial orientations between blocks of conserved nucleotides. Gene segments which can join to one another (VK and JK, for example) always have spacers of different lengths. Based on these observations, we propose a model for variable region gene rearrangement mediated by proteins which recognize the same conserved sequences adjacent to both light and heavy chain immunoglobulin gene segments.

Animals↗
PubMed2002

Non-homogeneous infinite sites model under demographic change: mathematical description and asymptotic behavior of pairwise distributions.

We developed a mathematical model, which makes possible to predict joint distributions of numbers of mismatches in two or more linked regions of the genome, based on the Infinite Sites Models, under mutation-drift equilibrium as well as under various patterns of population growth. With mutation rates varying in the region, one of the predictions is different correlation between numbers of mismatches in the two regions, depending on the pattern of the past population growth (constant, slowly growing, or rapidly growing). Also, for slower growth patterns of population sizes, the coalescence tree is not necessarily 'starlike'. Thus, the joint distribution of mismatches, predicted by the model, provides additional insights into the demographic history of the populations. We also developed expectations and variances of sample statistics under different growth scenarios. As an application we used a sample of mitochondrial sequences from hypervariable regions 1 and 2 (HV1 and HV2), representing major world populations (Europeans, Asians and Africans). The patterns of joint distributions of numbers of mismatches differ markedly from one population to another. In addition, there is a considerable variability in the proportion of numbers of mismatches between HV1 and HV2 sequences. The patterns of bivariate distributions from the HV1 and HV2 data in these data are consistent with those generated by the model involving a stepwise change in population size.

Asian People↗
PubMed1991

Characterization of variable regions in the envelope and S3 open reading frame of equine infectious anemia virus.

The polymerase chain reaction was used to amplify and clone parts of the envelope gene and overlapping S3 open reading frame, thought to encode rev, of the virulent in vivo-derived Th-1 isolate of equine infectious anemia virus (EIAV). The results indicated that EIAV consists of a heterogeneous mixture of genotypes present at the first febrile cycle after initial infection. We showed that the Th-1 isolate apparently contains nondefective genotypes as well as types which have transmembrane protein truncations or are rev deficient. Furthermore, we could confirm the presence of a hypervariable region in the gp90 envelope glycoprotein. Taken together with earlier data on the heterogeneity of the regulatory motifs present in the long terminal repeat sequences of viruses from the same in vivo isolate (S. Carpenter, S. Alexandersen, M. J. Long, S. Perryman, and B. Chesebro, J. Virol. 65:1605-1610, 1991), our findings indicate that EIAV uses a complex system of diversity in biological phenotypes together with variation in regulatory and antigenic makeup to evade host response and to cause persistent infection and recurrent chronic disease.

Amino Acid Sequence↗
PubMedDate not supplied

CDR substitutions of a humanized monoclonal antibody (CC49): contributions of individual CDRs to antigen binding and immunogenicity.

One of the major obstacles in the successful clinical application of monoclonal antibodies has been the development of host immune responses to murine Ig constant and variable regions. While the CDR grafting of MAbs may alleviate many of these problems, the potential remains that one or more murine CDRs on the human Ig backbone of a "humanized" MAb may still be immunogenic. Studies were undertaken employing a MAb of potential clinical utility, CC49, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We previously developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto frameworks of human MAbs. To identify those CDRs essential for binding, a panel of variant HuCC49 MAbs was generated here by systematically replacing each of the murine CDRs with their human counterparts. The relative affinity constant of each variant was determined. Serum from a patient who received murine CC49 was used to determine the potential immunogenicity of each CDR in humans. The serum was shown to react with the anti-CC49 variable region. Results showed that patients' anti-idiotypic responses are directed mainly against LCDR3 and moderately against LCDR1 and HCDR2. These studies demonstrate for the first time that variants containing individual CDR substitutions of a humanized MAb can be constructed, and each CDR can be defined for the two most important properties for potential clinical utility: antigen binding and immunogenicity.

Amino Acid Sequence↗
PubMed2001

Multiple regions of the murine coronavirus spike glycoprotein influence neurovirulence.

The spike (S) glycoprotein of mouse hepatitis virus (MHV) is a major determinant of neurovirulence. Using targeted recombination we previously demonstrated that the S gene of the highly neurovirulent MHV-4 conferred a dramatic increase in neurovirulence to the mildly neurovirulent MHV-A59. To identify the genetic determinants of neurovirulence within the MHV-4 spike, we generated isogenic recombinant viruses containing various MHV-4/MHV-A59 chimeric spike genes, and studied their phenotypes in vivo. The MHV-4/MHV-A59 chimeric spike genes consisted of either reciprocal exchanges between the S1 and S2 spike subunits, or smaller exchanges specifically in the hypervariable region (HVR) of S1. The chimeric spike gene containing recombinants all exhibited efficient replication in vitro, yet many were severely attenuated for virulence in vivo. Furthermore, these attenuated recombinants exhibited decreased titers of infectious virus in the brain relative to the parental recombinant viruses containing the full-length MHV-4 or MHV-A59 spike genes. This is the first report that compares the neurovirulence and pathogenesis of isogenic viruses with defined alterations in the MHV spike protein. From these studies, it appears that the interactions of multiple regions of the MHV spike, including the HVR, act in concert to allow for efficient infection of and virulence in the murine central nervous system.

Animals↗
PubMed2003

The complete nucleotide sequence, genome organization, and origin of human adenovirus type 11.

The complete DNA sequence and transcription map of human adenovirus type 11 are reported here. This is the first published sequence for a subgenera B human adenovirus and demonstrates a genome organization highly similar to those of other human adenoviruses. All of the genes from the early, intermediate, and late regions are present in the expected locations of the genome for a human adenovirus. The genome size is 34,794 bp in length and has a GC content of 48.9%. Sequence alignment with genomes of groups A (Ad12), C (Ad5), D (Ad17), E (Simian adenovirus 25), and F (Ad40) revealed homologies of 64, 54, 68, 75, and 52%, respectively. Detailed genomic analysis demonstrated that Ads 11 and 35 are highly conserved in all areas except the hexon hypervariable regions and fiber. Similarly, comparison of Ad11 with subgroup E SAV25 revealed poor homology between fibers but high homology in proteins encoded by all other areas of the genome. We propose an evolutionary model in which functional viruses can be reconstituted following fiber substitution from one serotype to another. According to this model either the Ad11 genome is a derivative of Ad35, from which the fiber was substituted with Ad7, or the Ad35 genome is the product of a fiber substitution from Ad21 into the Ad11 genome. This model also provides a possible explanation for the origin of group E Ads, which are evolutionarily derived from a group C fiber substitution into a group B genome.

Adenoviruses, Human↗
PubMed1986

Structural studies of a carbohydrate-containing immunoglobulin-lambda-light-chain amyloid-fibril protein (AL) of variable subgroup III.

The amino acid sequence of the variable region of a carbohydrate-containing amyloid-fibril protein MOL of immunoglobulin-light-chain type (AL) was elucidated. The sequence determination involved cleaving the protein with CNBr, BNPS-skatole, thermolysin and trypsin. The sequenced protein consisted of about 130 amino acid residues; however, gel-filtration and N-terminal analysis studies revealed AL proteins ranging in Mr from about 10,000 to 25,000. The oligosaccharide chain was found to be bound in the hypervariable region. By sequence homology to other lambda chains the AL protein MOL was shown to be of the V lambda III subgroup.

Aged↗
PubMed1989

Structural characterization of antiidiotypic antibodies. Evidence that Ab2s are derived from the germline differently than Ab1s.

We have found that syngeneic Ab2s in the antiarsonate system are serologically and structurally similar to one another. In contrast, the allogeneic Ab2 response is heterogeneous and derives from a large number of unrelated germline gene segments. The Ab2 response of the BALB/c strain to polyclonal A/J Ars A molecules can probably best be compared with a response to a foreign protein and might have been predicted in a strain that completely lacks the H chain V region gene from which the Ab1 derives. Partial variable region sequences of Ab2s from three other systems in addition to previously reported Ab2 structures indicates that this difference in allogeneic vs. syngeneic Ab2s may be a general phenomena. These data support Jerne's hypothesis of complementary V region genes existing in the germline. However, there is good evidence that these antiidiotypic antibodies are not derived directly from the germline, as somatic processes most likely play an important role in their generation. The D segments of Ab2s in the arsonate system as well as in other systems, are novel in structure and cannot easily be explained by previously described germline D segments. D-D fusion may play a role in the generation of the third hypervariable region in these antibodies.

Amino Acid Sequence↗
PubMed1999

Genetic basis for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common disabling disorder of unknown etiology. In the past 2 decades, a number of studies have examined the genetic basis for RA. One major focus of these studies has been to identify genes within the MHC class II (HLA-DR) chromosomal region, which confer susceptibility/resistance to RA. A strong association between HLA-DR4 and adult seropositive RA has been observed in majority of populations. In addition, there is evidence of a positive association between HLA-DR1 and RA. On the basis of prevalence of DR1 (B1*0101) and of subtypes of DR4 (B1*0401, B1*0404 and B1*0405), it has been suggested that a five amino acid sequence motif (QKRAA/QRRAA) from position 70 to 74 in the third hypervariable region of DRbeta1 molecules is associated with susceptibility to RA. These associations between RA and HLA-DR genes are however incomplete in that about 1/4 of patients do not carry RA-susceptibility DRB1 epitope. Since MHC class III region contains genes that are involved in immune response, we have recently examined the role of a number of microsatellites (D6S273, Bat2, TNFa) and HSP70 promoter region alleles in susceptibility to RA. The results demonstrate that two regions in MHC, class II (DRbeta1) and class III (D6S273, HSP70, Bat2, TNFa) more completely define the risk for development of RA.

Alleles↗
PubMed1990

Structural characterization of human monoclonal cold agglutinins: evidence for a distinct primary sequence-defined VH4 idiotype.

Cold agglutinins that bind the developmentally regulated I red cell determinant occur naturally among human monoclonal IgM proteins. These autoantibodies are known to use light chains that derive mainly from the minor kappa III (kappa III) variable region subgroup. The kappa III subgroup is also highly expressed in monoclonal rheumatoid factors. However, while most monoclonal rheumatoid factors use structurally homologous heavy chains that derive from the VH1 family, information regarding the structure of the cold agglutinin heavy chains remains fragmentary. We demonstrate here that the kappa III cold agglutinin autoantibodies exclusively use heavy chains that derive from the VH4 family. Furthermore, these autoantibody heavy chains all express the same primary sequence-defined idiotype, corresponding to the second hypervariable region. These data indicate that cold agglutinins use a remarkably homogeneous subset of heavy chain variable regions. Moreover, unique patterns of preferential VH and VL pairing clearly distinguish the anti-I cold agglutinins from all other known monoreactive autoantibodies.

Agglutinins↗
PubMed1985

The amino acid sequence of a carbohydrate-containing immunoglobulin-light-chain-type amyloid-fibril protein.

The amino acid sequence of an amyloid-fibril protein Es492 of immunoglobulin-lambda-light-chain origin (AL) was elucidated. The amyloid fibrils were obtained from the spleen of a patient who died from systemic amyloidosis. The amino acid sequence was elucidated from structural studies of peptides derived from digestion of the protein with trypsin, thermolysin, chymotrypsin and Staphylococcus aureus V8 proteinase and from cleavage of the protein with CNBr and BNPS-skatole. A heterogeneity in the length of the polypeptide was seen in the C-terminal region. The protein was by sequence homology to other lambda-chains shown to be of the V lambda II subgroup. Although an extensive homology was seen, some amino acid residues in positions 26, 31, 32, 40, 44, 93, 97, 98 and 99 have not previously been reported in these positions of V lambda II proteins. The significance of these residues in the fibril formation is unclear. The protein was found to contain carbohydrate, with glycosylation sites in two of the hypervariable regions.

Amino Acid Sequence↗
PubMed1996

Cultivation of hepatitis C virus in primary hepatocyte culture from patients with chronic hepatitis C results in release of high titre infectious virus.

To investigate the viral replication cycle and genomic heterogeneity of hepatitis C virus (HCV), we established an HCV cultivation system by using a primary hepatocyte culture from patients with chronic hepatitis C. Liver tissue was obtained by needle biopsy or surgery, then hepatocytes were isolated by collagenase digestion. After several weeks, we determined the HCV RNA titre of the cultured cells and supernatant by a competitive polymerase chain reaction (PCR) method. A significant amount of HCV RNA was observed in the cells and supernatant during cultivation. Negative-strand RNA, regarded as a marker of viral replication, could be detected by a strand-specific reverse transcription PCR method and the HCV core protein could be detected by immunofluorescence microscopy. Many HCV particles released into the supernatant were infectious. In addition, we compared the nucleotide sequences in the E2/NS1 region of pre- and post-cultivation hepatocytes for 8 weeks. At the beginning of the culture period, three major HCV types containing two subtypes were isolated. Following cultivation, the same types were isolated from the cultured hepatocytes in the same ratio as prior to cultivation. We could detect the same clones in this patient's serum, but in vivo we observed genetic variability over a 6 month interval. One clone detected throughout the 6 month period mutated extensively in the hypervariable region. These results indicated that HCV can replicate in cultured hepatocytes, and that infectious virions are released into the supernatant. This cultivation system should facilitate the study of HCV genomic heterogeneity, infection and replication.

Adult↗
PubMed1997

Mutation rate of GB virus C/hepatitis G virus over the entire genome and in subgenomic regions.

A patient on maintenance hemodialysis was infected with a recently discovered putative non-A to -E hepatitis virus designated GB virus C (GBV-C) or hepatitis G virus (HGV) by transfusion. The viral isolate was recovered from the patient soon after she turned positive for GBV-C/HGV RNA in serum (GS185) and 8.4 years thereafter (GS193), and the entire nucleotide sequences were determined. They both had a genomic length of 9391 nucleotides with a defective C gene made of only 42 nucleotides. Between GS185 and GS193, 31 (0.33%) nucleotides were different, which changed 5 (0.18%) of the encoded 2842 amino acids. Thus, GBV-C/HGV was estimated to have a mutation rate of 3.9 x 10(-4) base substitutions per site per year. Nucleotide conversions were distributed over subgenomic regions, except in the 5' untranslated region of 552 nucleotides and a defective short C gene, which were conserved in sequence. The change in the putative envelope genes (E1 and E2) was no different from that in the entire genome with only 6 (0.35%) nucleotide substitutions among the 1730, just 1 of which induced an amino acid conversion. Taken along with the comparison of the two isolates with the reported five GBV-C or HGV isolates, these results indicate that GBV-C/HGV would not have hypervariable regions and would use a strategy for viral persistence that is different from immune escape.

Amino Acid Sequence↗