SEARCH · PubMed Health

Results for “germline sequencing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources

PubMed2006

Mutation of RET proto-oncogene in Hirschsprung's disease and intestinal neuronal dysplasia.

AIM: To investigate the genetic relationship between Hirschsprung's disease (HD) and intestinal neuronal dysplasia (IND) in Chinese population. METHODS: Peripheral blood samples were obtained from 30 HD patients, 20 IND patients, 18 HD/IND combined patients and 20 normal individuals as control. Genomic DNA was extracted according to standard procedure. Exons 11,13,15,17 of RET proto-oncogene were amplified by polymerase chain reaction (PCR). The mutations of RET proto-oncogene were analyzed by single strand conformational polymorphism (SSCP) and sequencing of the positive amplified products was performed. RESULTS: Eight germline sequence variants were detected. In HD patients, 2 missense mutations in exon 11 at nucleotide 15165 G-->A (G667S), 2 frameshift mutations in exon 13 at nucleotide 18974 (18974insG), 1 missense mutation in exon 13 at nucleotide 18919 A-->G (K756E) and 1 silent mutation in exon 15 at nucleotide 20692 G-->A(Q916Q) were detected. In HD/IND combined patients, 1 missense mutation in exon 11 at nucleotide 15165 G-->A and 1 silent mutation in exon 13 at nucleotide 18888 T-->G (L745L) were detected. No mutation was found in IND patients and controls. CONCLUSION: Mutation of RET proto-oncogene is involved in the etiopathogenesis of HD. The frequency of RET proto-oncogene mutation is quite different between IND and HD in Chinese population. IND is a distinct clinical entity genetically different from HD.

Asian People↗

PubMed1994

The human T-cell receptor TCRAC/TCRDC (C alpha/C delta) region: organization, sequence, and evolution of 97.6 kb of DNA.

We sequenced and analyzed 97.6 kb of new DNA sequence containing the human TCRAC (C alpha) and TCRDC (C delta) genes as well as the TCRDV3 (V delta 3) and 61 different TCRAJ (J alpha) gene segments and compared its organization and structure to the previously described mouse T-cell receptor TCRAC/TCRDC (C alpha/C delta) region. A comprehensive nomenclature, consistent with the IUIS nomenclature committee recommendations, for both human and mouse TCRAJ gene segments is presented. In the human sequence, we identified 20 new TCRAJ gene segments and obtained the germline sequence for 23 additional TCRAJ gene segments known from cDNA clones. Using the sequence data obtained from the human TCRAC/TCRDC region, we have extended a polymerase chain reaction-based assay to test for the expression of the individual TCRAJ gene segments. At least five TCRAJ pseudogene segments were identified by sequence criteria. Like the murine TCRAC/TCRDC sequence, this sequence contains a high level of coding sequence, with over 6.6% of the total sequence being transcribed. Comparison of the human sequence with the previously reported mouse DNA sequence reveals homologous counterparts for the variable and joining (J) gene segments and both constant genes. Eleven new J pseudogene segments have been identified in the mouse TCRAC/TCRDC sequence through the use of human and mouse sequence comparisons. In terms of structure and organization, this region of the human and mouse genome appears to be remarkably conserved.

Amino Acid Sequence↗

PubMed1986

Structure and expression of a human subgroup II immunoglobulin kappa gene.

We have determined the entire nucleotide sequence of an active immunoglobulin V kappa gene of the human subgroup II. Comparison of the main coding sequence of this gene to another member of this subgroup reveals germline sequence differences that occur not only in complementarity determining regions but also in framework regions. We find that at least two regions commonly associated with active immunoglobulin genes--the TATAA box and the decanucleotide TGATT-TCAT--are represented in this gene. The gene was expressed by permanent and transient DNA-mediated transfection in various cell lines. We have determined the precise site of transcriptional initiation using S1 analysis of gene transcripts and show that this site is the same in the transfected cell lines and in the cell line from which the gene was isolated.

Animals↗

PubMed2004

Genomic organization of the chicken T-cell receptor beta chain D-J-C region.

We examined overlapping genomic clones containing the chicken T cell receptor (TCR) Dbeta-Jbeta-Cbeta complex, which contains a single diversity segment, four joining segments and four exons that encode the constant region. This sequence comprised 18.3 kb. All four Jbeta sequences possessed typical recombination signal sequences (RSS) with intervening 12-bp spacers at their 5'-ends and splice sites at their 3'-ends. No Jbeta-pseudogenes were identified. TGTG sequences in the RSS heptamer sequences were well conserved, as is the case in mammals. A chicken repeat 1-like sequence was found in the intron region between Jbeta-1336 and Cbeta, and several small repeat sequences were identified in intron regions throughout this cloned genome. As germline sequences revealed complete Jbeta sequences, the CDR3 (complementarity-determining region) sequences of TCRbeta from non-immunized splenocytes were analyzed. Non-coding (N) and palindromic (P) nucleotides were frequently observed at the Dbeta-Jbeta recombination sites. There were differences in length of deletion at the 5'-end of each Jbeta. Deletion of the 5'-end of Jbeta-1280 was particularly short when compared with that of Jbeta-1336, but there were no changes in the length of the CDR3 using any of the four Jbeta sequences.

Amino Acid Sequence↗

PubMed2000

Precursor B-lymphoblastic transformation of grade I follicle center lymphoma.

Part of the natural history of follicle center lymphoma (FCL) is transformation to a more aggressive neoplasm, almost always a diffuse large B-cell lymphoma. We describe a rare example of a precursor B-lymphoblastic transformation of grade I FCL occurring in a 45-year-old woman 12 years after initial presentation and 3 years after successful treatment for a diffuse large cell transformation. The lymphoblastic lymphoma shared the same immunoglobulin heavy chain gene rearrangement as the FCL as assessed by polymerase chain reaction amplification and direct sequencing, as well as identical kappa light chain gene rearrangements by Southern blot analysis. The immunoglobulin heavy chain variable gene sequences of both tumors showed numerous identical base substitutions compared with germline sequences and 3 additional mutations in the lymphoblastic lymphoma not present in the low-grade FCL. These results indicate origin of the lymphoblastic process from the mature follicle center B-cell clone, rather than divergent origin of the 2 tumors from a common immature B-cell precursor.

Adult↗

PubMed1999

Phenotypic and molecular analysis of six human cell lines derived from patients with plasma cell dyscrasia.

Cell lines RPMI 8226, JJN3, U266 B1, NCI-H929 (all EBV-) and ARH77 and HS-Sultan (both EBV+) have been extensively characterized in this study. EBV- lines expressed the phenotype (CD138-, CD19+, CD20+) whereas EBV+ were (CD138+, CD19-, CD20-). CD56 expression was restricted to EBV- cell lines, with the exception of U266 B1, whereas PCA-1 was strongly expressed on five of the six cell lines. Only EBV+ cell lines bound peanut-agglutinin (PNA). However, all cell lines bound the lectin Jacalin that binds the same receptor as PNA, irrespective of the receptors sialylation status. By RT-PCR and direct sequencing of their IgH V/D/J domains, ARH77 was demonstrated to use the germline sequence VH4-34/dm1/JH6b, whereas no arrangement was demonstrated for RPMI 8226, suggesting IgH gene deletion or mutation. HLA class I and II antigens were detected using HLA typing on all cell lines warranting their use as suitable targets for HLA-restricted cytotoxic T cells. By sensitive RT-PCR, mRNA for IL-6, IL-6R and TNFbeta was found expressed in all cell lines. IL-1 mRNA expression was predominantly associated with the EBV+ phenotype. Although mRNA for IL-3 and GM-CSF was never detected, transcripts for c-kit ligand and, more commonly, its receptor were. Likewise GM-CSF, M-CSF and erythropoietin mRNA transcripts were detected in the majority of cell lines.

Base Sequence↗

PubMed1998

Multiple sites of V lambda diversification in cattle.

Ig repertoire diversification in cattle was studied in the ileal Peyer's patch (IPP) follicles of young calves and in the spleens of late first-trimester bovine fetuses. To investigate follicular diversification, individual IPP follicles were isolated by microdissection; VA diversity was examined by RT-PCR and subsequent cloning and sequencing. When 52 intrafollicular sequences from a 4-wk-old calf were determined and compared, two major groups, one of 23 members and the other of 25, could be delineated. An examination of these groups revealed clear genealogic relationships that implicated in situ diversification of V lambda sequences within the confines of an IPP follicle. V lambda expression was also examined in early (95 and 110 gestational day) fetal bovine spleens. Although earlier studies in cattle and sheep implicated the IPP as a likely site of Ab diversification, a close investigation of V lambda sequences in late first-trimester fetal calves revealed that diversity appears in the early fetal spleen before the establishment of a diverse repertoire in the ileum. When the sequences for the fetal spleen were compared with an existing pool of germline sequences, we found evidence of possible gene conversion events and possible untemplated point mutations occurring in sequences recovered from fetal spleens. We conclude that IPP is not the sole site of VA diversification in cattle. Also, as suggested for rabbits, cattle may use both gene conversion and untemplated somatic point mutation to diversify their primary VA repertoire.

Animals↗

PubMed1994

Sequence analysis and fine specificity of two human monoclonal antibodies to histone H1.

Two human IgM lambda monoclonal antibodies (MAb) derived from the splenic lymphocytes of patients with idiopathic thrombocytopenia (Ben) and systemic lupus erythematosus (Wri) were studied. BEN-27 and WRI-170 hybridoma supernatants were screened for binding to ssDNA, dsDNA, poly (ADP-ribose), cardiolipin, histone subclasses and Klebsiella K30 cell wall antigen. Of this panel of antigens, BEN-27 and WRI-170 antibodies reacted only with histone H1. Their fine specificity was defined by direct and inhibition ELISA with synthetic peptides of the major human H1b variant. Antibody WRI-170 was shown to bind to both the N- and C-terminal peptides encompassing residues 1-16 and 204-218 of H1b whereas BEN-27 reacted only with peptide 204-218. To analyse the genetic origin of these autoantibodies, we determined the nucleotide sequence of the heavy (H) and light (L) chain variable regions of these two hybridomas. BEN-27 and WRI-170 MAbs were found to use VH1-DN1-JH4/V lambda 3-J lambda 2 and VH3-DIR2-D21/9-JH1/V lambda 2-J lambda 2 gene segment combinations respectively. Between 70 and 95% homology was demonstrated when the mRNA sequences for BEN-27 and WRI-170 were compared with published VH and V lambda germline sequences. This finding suggests that BEN-27 heavy and light chains and WRI-170 light chain use unidentified VH and V lambda germline gene segments whereas WRI-170 heavy chain derives from a VH gene segment recently identified. It is noteworthy that the CDRs of the two MAbs contain several negatively charged amino acids which are assumed to be of critical importance in antigen binding. Moreover, striking similarities are observed between BEN-27 heavy chain CDR2 and a previously described murine anti-H1 Ab heavy chain CDR2.

Amino Acid Sequence↗

PubMed1992

Regulatory aspects of clonally expanded B-1 (CD5+ B) cells.

B-1 (CD5+ B) cells appear early in ontogeny, produce mainly unmutated polyreactive antibodies, and are capable of self-renewal. B-1 cells clonally expand with age and are the malignant cell in chronic lymphocytic leukemia. In this report immunological analysis of B-1 malignancies in NZB mice, a murine model of chronic lymphocytic leukemia, is related to current information on B-1 cells. B-1 clones from NZB mice produce high levels of interleukin-10, detected at the RNA level by semi-quantitative polymerase chain reaction. In addition, the B-1 malignant clones in NZB mice and their hybrids, are negative for B220/6B2 expression, the B-specific antigenic form of CD45 which is a membrane-associated phosphatase involved in lymphocyte activation. Both the autocrine production by B-1 cells of interleukin-10 and altered CD45 expression may be responsible for the clonal expansion of these cells, as well as the accompanying T cell expansion. We report the establishment of an in vitro cytotoxic CD8+ T cell line derived from an NZB with a B-1 malignancy. The effect of B-1 cell-derived interleukin-10 on subsets of T lymphocytes may account for the immunoregulatory properties of B-1 cells. In addition, the NZB malignancies were also characterized for immunoglobulin variable region sequence and antigen specificity. The B-1 malignancies produced immunoglobulin derived from unmutated germline sequences with no N base substitutions. It appears that both the immunoglobulin and interleukin-10 produced by the B-1 malignant cell in NZB mice may have immunoregulatory properties. A study of B-1 malignancies may shed light on the immunoregulatory properties of non-clonally expanded normal B-1 cells.

Animals↗

PubMed1993

Molecular characterization of extrachromosomal circular DNAs from an embryonal carcinoma cell line induced to differentiate into neuron-like cells in vitro.

Extrachromosomal circular DNAs isolated from a P19 embryonal carcinoma cell line were induced to differentiate into neuron-like cells by retinoic acid and cloned into an EcoRI site of a phage vector. Of the 26 DNA inserts (2.1 kb in average length) analyzed, 16 contained repetitive sequences. Out of 10 DNA inserts with unique sequence, 6 carried linear chromosomal sequences and 4 showed chromosomal rearrangements in Southern blots. Two unique fragments with germline configuration were enriched in circular DNA clone libraries. We assigned the breakpoints of 3 circular DNA fragments to positions in the germline sequence. Patchy short inverted repeats were found in the vicinity of breakpoints. An intrastrand loop structure between such inverted short homology region may be required for the circularization of excised DNA.

Animals↗

PubMed1998

Molecular and genetic characterization of two anti-DNA antibodies derived from patients with systemic lupus erythematosus.

Anti-double stranded(ds) DNA antibody is one of markers of systemic lupus erythematosus (SLE). Two human monoclonal anti-DNA antibody-producing cell lines were established from two SLE patients. One cell line secreted IgG isotype antibody (KSUG) and the other secreted IgM isotype antibody (KSUN). The light chains of the two immunoglobulins were lambda chains. The nucleotide sequences for the immunoglobulin variable region genes of the two antibodies were determined and compared to germline sequences. The heavy and lambda light chains of KSUG were VH3 family and V lambda IIIb, respectively. The heavy and lambda light chains of KSUN were VH4 family and V lambda IX, respectively. Antibody KSUG, IgG isotype, showed somatic mutations, whereas KSUN, IgM isotype, used the germline gene without mutation. These findings reconfirm the current paradigms that IgM anti-DNA antibodies are produced by utilizing germline genes whereas IgG anti-DNA antibodies are produced by somatic mutations.

Adult↗

PubMed1994

Phage display of a human antibody against Clostridium tetani toxin.

9F12, a human antibody capable of neutralising tetanus toxin in an animal model, has been expressed as a Fab fragment in a phagemid system. The nucleotide sequence for the variable domain of both chains has been determined and compared to germline sequences.

Amino Acid Sequence↗

PubMed1992

IGM kappa/lambda EBV human B cell clone: an early step of differentiation of fetal B cells or a distinct B lineage?

In agreement with the clonal theory, one B lymphocyte synthesizes one antibody due to allelic and isotypic exclusion. We analyzed an EBV B-cell clone, E29.1, derived from an 11 week-old embryo, and secreting both IgM kappa and IgM lambda. Structural analysis of produced IgM, indicated that lambda-containing pentamers could be considered hybrid molecules, expressing both the kappa and lambda. chains, with a kappa/lambda ratio between 5 and 10. It was also found that 60% of the lambda chains were secreted in free form, presumably as a result of a better affinity of mu chains for kappa chains. The sequence of the three transcripts had an entirely ORF (Open Reading Frame), and were very close to germline sequences, with, however, an additional codon between V kappa and J kappa gene which has never been described in adult myeloma protein or cDNA human sequence. This observation is suggestive of N diversity taking place in kappa chains. The possible role of Kde (kappa deleting element) recombination onto kappa/lambda locus activation was analyzed on a collection of 23 lambda clones. The status of rearrangement of kappa genes indicated that 35% of these clones had retained, at least, one kappa allele without the Kde recombination, four lambda clones had one kappa allele in germline configuration. Different hypotheses of maturation from pre-B cell to B cell with activation of light chain genes are discussed.

Aging↗

PubMed2000

Germline repertoire of the immunoglobulin V(H)3 family in rhesus monkeys.

To facilitate molecular studies of antibody responses in rhesus monkeys (Macaca mulatta), we cloned and sequenced germline segments from its largest and most diverse immunoglobulin heavy-chain gene family, V(H)3. Using a PCR-based approach, we characterized 29 sequences, 20 with open reading frames (ORFs) and 9 pseudogenes. The leader sequences, introns, exons, and recombination signal sequences of M. mulatta V(H)3 gene segments are not strictly identical to those of humans, but the mature coding regions demonstrate, on average, greater than 90% sequence similarity. Although the framework regions are more highly conserved, the complementarity-determining regions (CDRs) also show strong similarities, and their predicted three-dimensional structures resemble those of their human homologues. In one instance, homologous macaque and human CDR1 sequences were 100% identical at the nucleotide level, and some CDR2s shared nucleotide identity as high as 96.5%. However, some rhesus V(H)3 ORFs have unusual structural features, including atypical CDR lengths and uncommon amino acids at structurally crucial positions. The similarity of rhesus and human V(H)3 homologues reinforces the notion that humoral immunity in this nonhuman primate species is an appropriate system for modeling human antibody responses.

Amino Acid Sequence↗

PubMed1996

Ongoing mutation in MALT lymphoma immunoglobulin gene suggests that antigen stimulation plays a role in the clonal expansion.

Indirect antigenic stimulation by H. pylori-specific T cells is implicated in the development of low-grade gastric lymphoma of mucosa-associated lymphoid tissue (MALT), however, the role of direct antigen stimulation is unknown. To study the role of direct antigen stimulation in MALT lymphomagenesis and its relationship with the pathogenesis of distinct pathological lesions, which represent different stages of the tumour progression, we cloned and sequenced the rearranged immunoglobulin (Ig) heavy chain gene in three low-grade (two from the lung, one from the stomach) and one high-grade (from the stomach) cases. In the low-grade gastric case, we studied the Ig sequence in primary as well as its disseminated and recurrent tumours. In the high-grade gastric case, we analysed the Ig sequence in tumour cell populations microdissected from the residual diffuse low-grade lesions, diffuse high-grade areas from follicles colonized by high-grade blasts. Compared with the published germline sequences, the heavy chain variable (VH) genes of three MALT lymphomas, in which the putative germline was identified, contained frequent somatic mutations, showing a much higher ratio of replacement/silent mutations in the complementarity determining regions (CDRs) than the framework regions (FRs). Ongoing mutation as indicated by intraclonal variation of the Ig sequence clearly existed in low-grade tumour including its dissemination and recurrence, but was not evident in high-grade tumour cell populations including those microdissected from independent colonized follicles. In addition, the germlines of VH genes used by the three MALT lymphomas are frequently found in autoreactive antibodies. Our results suggest that MALT lymphoma derives from postgerminal centre memory B cells, possibly autoreactive B cell clones, and that direct antigen stimulation may play an important role in the clonal expansion of low-grade MALT lymphoma.

Animals↗

PubMed1994

Humanization of 60.3, an anti-CD18 antibody; importance of the L2 loop.

Monoclonal antibody 60.3 binds to the CD18 component of the beta 2 integrin family of adhesion molecules. 60.3 has potential clinical application in blocking the neutrophil-mediated organ damage which occurs following a myocardial infarct or hemorrhagic shock. Analysis of the nucleotide and deduced amino acid sequences of murine 60.3 shows that the light chain contains no amino acid substitutions relative to the closest germline sequence, while the heavy chain is heavily substituted. We report here the humanization of 60.3. The humanized antibody binds to CD18-bearing cells with approximately 4-fold less affinity than the murine or chimeric antibody. We have shown that modification of amino acid L50 in the L2 loop of the humanized antibody results in loss of binding, while modification of a structural determinant (H71) for the H2 loop has no effect.

Amino Acid Sequence↗

PubMed2002

Molecular cloning and chimerisation of an inhibitory anti-cathepsin B antibody and its expression in Chinese hamster ovary cells.

The lysosomal cysteine protease cathepsin B is one of several proteases that have been linked to tumour progression. Its increased expression and secretion in tumour cells may facilitate the degradation of extracellular matrix proteins, leading to tumour cell invasion and metastasis. Specific inhibitory monoclonal antibodies are a possible alternative to synthetic inhibitors as a therapeutic tool for cancer treatment. An inhibitory monoclonal antibody, which binds to an epitope near the active site of cathepsin B and inhibits its proteolytic activity, was prepared and its effect on invasion of ras-transformed MCF-10A neoT cells was tested in vitro. Here we present the nucleotide sequences of the heavy and light chains of the inhibitory antibody and compare them to the murine immunoglobulin germline sequences for possible somatic hypermutations. Since no harmful mutations were found, a mouse/human chimeric antibody was constructed by fusing murine V(H) and V(L) variable regions of the inhibitory antibody with human gamma 1 and K constant regions, respectively. Chinese hamster ovary K1 cells were co-transfected with expression vectors pcD-NA3L and pcDNA3H and the reactivity of the isolated chimeric antibody was tested by ELISA and Western blotting. We could demonstrate an inhibitory effect of the chimeric antibody.

Amino Acid Sequence↗

PubMed2000

Accumulation of clonally related B lymphocytes in the cerebrospinal fluid of multiple sclerosis patients.

The accumulation of B lymphocyte clones in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and patients with other neurological disorders was investigated using PCR technologies. Oligoclonal B cell accumulations were detected in 10 of 10 MS patients, but only in 3 of 10 of the patients with other neurological disorders. Analyses of the Ig V(D)J sequences on the CSF from MS patients disclosed that VH3 and VH4 genes were extensively mutated compared with germline sequences. Moreover, a substantial proportion of the molecular clones analyzed shared the same third CDR of the H chain variable region gene (HCDR3) and the same VH genes, albeit with different numbers and locations of point mutations, thus indicating an ongoing process of intraclonal diversification. A larger number of clonally related VH sequences could be obtained by using a VH3 gene-specific PCR so that genealogical trees depicting the process of diversification could be drawn. Analyses of the Ig V(D)J from the CSF of a patient with viral meningitis and oligoclonal B cell accumulations revealed that VH3 genes were extensively mutated. However, no intraclonal diversification could be observed even using VH3 gene-specific PCR methodologies. Clone-specific PCR and sequencing was used to detect the V(D)J found in the CSF of one MS patient in the PBL of the same patient. Only 1/3 of the V(D)J sequences investigated could be demonstrated in the PBL, indicating that the V(D)J genes utilized by B cells in the CSF are much less represented in the PBL. Collectively, the data suggest that in MS there is a compartmentalized clonal expansion.

Adolescent↗