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At least 109 records · Page 6Linked to original sources
PubMed1995

A novel point mutation (R243Q) in exon 7 of the c-erbA beta thyroid hormone receptor gene in a family with resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) is characterized by variable tissue hyporesponsiveness to thyroid hormones. Recently, a large number of different point mutations have been identified in the c-erbA beta thyroid hormone receptor (TR beta) in subjects with RTH. We describe a Japanese family with RTH with a novel point mutation in exon 7 of the TR beta gene. A single nucleotide substitution, guanine for adenine, was identified at the second position of codon 243 located in the hinge domain between the ligand binding and DNA binding domains in one of the two alleles of the proband and his mother, resulting in the substitution of the normal arginine (CGG) with a glutamine (CAG). Except for one family, point mutations so far described in RTH are clustered at exons 8-10 of the TR beta gene. This report presents a novel mutation in the characteristic portion in exon 7 of the TR beta.

Amino Acid Sequence↗
PubMed1990

Structure of a tobacco endochitinase gene: evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain.

The endochitinases (E.C. 3.2.1.14, chitinase) are a structurally diverse group of enzymes believed to be important in the biochemical defense of plants against potential pathogens. The gene for a chitinase of Nicotiana tabacum L. cv. Havana 425 has been cloned and sequenced. The major transcription start is 11 bp upstream of the ATG codon and 28 bp downstream of the TATA box. The gene contains two introns and encodes a basic chitinase of 329 amino acids with a 23 amino acid N-terminal signal peptide followed by a 43 amino acid, cysteine-rich domain, which is linked by a hinge region to the main structure of the enzyme. This gene appears to be expressed because the exons are identical to the coding sequence of a cDNA which was isolated. Comparison of chitinase amino acid sequences from different plants indicates there are at least three classes of these enzymes: class I, basic chitinases with an N-terminal cysteine-rich domain and a highly conserved main structure; class II, chitinases similar to the main structure of class I chitinases but lacking the cysteine-rich domain; and, class III, chitinases with conserved sequences different from those of the class I and II enzymes. The sequences encoding the cysteine-rich domain in class I chitinases are flanked by 9-10 bp imperfect direct repeats suggesting that these domains arose from a common ancestral gene and were introduced into genes for class I enzymes by transposition events.

Amino Acid Sequence↗
PubMedDate not supplied

Partial structure of a rat IgD molecule with a deletion in the heavy chain.

The isolation, purification and characterization of the rat IR-731 monoclonal immunoglobulin is reported. The molecule is IgD-like, as appreciated from immunochemical and biochemical characteristics. H and L chains, Fab and Fc tryptic fragments have been isolated, analyzed, and partial sequence data have been obtained, including most cysteyl-containing peptides. The heavy chain contains a deletion which encompasses most of the CH2 domain and the beginning of the CH2 domain, and observation which does not reflect the intron-exon organization depicted for some murine heavy chains at the gene level. Basic structural features of the hinge region of the IgD molecule (extreme susceptibility to proteolytic enzymes, presence of basic amino acids near its COOH-terminus) have been found for the IR-731 molecule. In addition, partial sequence of the light chains points to the existence of K subgroups in the rat. Very limited amino acid changes have been identified in the K constant region, suggesting a possible--but limited--polymorphism which might be isotypic and/or allotypic in nature.

Amino Acid Sequence↗
PubMed1997

Evidence for a dystrophin missense mutation as a cause of X-linked dilated cardiomyopathy.

BACKGROUND: X-linked dilated cardiomyopathy (XLCM) has previously been shown to be due to mutations in the dystrophin gene, which is located at Xp21. Mutations in the 5' portion of the gene, including the muscle promoter, exon 1, and the exon 1-intron 1 splice site, have been reported previously. The purpose of this study was to analyze the originally described family with XLCM (and other) for dystrophin mutations. METHODS AND RESULTS: Polymerase chain reaction (PCR) was used to amplify genomic DNA, and reverse-transcriptase PCR amplified cDNA from RNA obtained from heart and lymphoblastoid cell lines. Primers to the muscle promoter, brain promoter, and Purkinje cell promoter were designed, in addition to the exon 1 to exon 14 regions of dystrophin. Single-strand conformation polymorphism analysis was used for mutation detection, and DNA sequencing defined the mutation. Protein modeling was used for amino acid and secondary structure analysis. A missense mutation in exon 9 at nucleotide 1043 was identified that causes an alanine to be substituted for threonine, a highly conserved amino acid, at position 279 (T279A). This mutation results in a change in polarity in the evolutionarily conserved first hinge region (H1) of the protein and substitution of a beta-sheet for alpha-helix in this portion of the protein, destabilizing the protein. CONCLUSIONS: A novel missense mutation in exon 9 of dystrophin causing an abnormality at H1 leads to the cardiospecific phenotype of XLCM.

Amino Acid Sequence↗
PubMed1993

Estrogen receptor variant messenger RNA lacking exon 4 in estrogen-responsive human breast cancer cell lines.

We have identified a messenger RNA coding for a variant estrogen receptor jointly expressed with the normal mRNA in the estrogen receptor-positive, -responsive mammary carcinoma cell lines MCF-7 and ZR 75-1 by means of reverse transcription polymerase chain reaction. This variant mRNA was not observed in estrogen receptor-negative, -unresponsive MDA-MB 231 cells. Partial sequence analysis of the variant complementary DNA revealed identity to sequences of the estrogen receptor exons 3, 5, and 6, but the absence of the entire exon 4. We suggest that this variant receptor messenger is created by alternative splicing. The variant protein is expected to lack most of the hinge domain and part of the hormone binding domain, and it might have a cellular distribution and estrogen-binding affinity different from that of the normal receptor protein.

Amino Acid Sequence↗
PubMed1995

Characterization of a class I chitinase gene and of wound-inducible, root and flower-specific chitinase expression in Brassica napus.

Complementary and genomic DNAs coding for a Brassica napus chitinase have been cloned and sequenced. The genomic DNA contains one intron and encodes a 322-amino acid basic chitinase with a 20-amino acid N-terminal signal peptide followed by a 40-amino acid cysteine-rich domain, linked by a hinge region to the main domain of the enzyme. The sequence of the cDNAs is identical to the exon sequence deduced from the genomic DNA. A probe derived from this gene identified a 1.2-kb transcript present in high amount in roots, moderate in floral tissues and low in stems and leaves. The synthesis of these transcripts is regulated during development and is induced in roots by wounding and ethephon. This type of chitinase is encoded by two sequences in Brassica napus, as shown either by Southern hybridizations or by genomic amplification and sequencing using the polymerase chain reaction. These genes are homologous to one sequence found in the Brassica oleracea genome.

Amino Acid Sequence↗
PubMed2006

Hybrid spectrin type repeats produced by exon-skipping in dystrophin.

Dystrophin is the protein whose defect underlies Duchenne Muscular Dystrophy, DMD, a common (1:3500 male births) and fatal condition in which muscle tissue deteriorates leading to death in the second or third decade of life. Dystrophin is coded for by the largest human gene, and one of the most complex. It is translated from at least 7 distinct promoters, with the largest transcripts (which are the ones involved in DMD) containing 79 exons over >2.5 Mbp [K.F. O'Brien, L.M. Kunkel, Dystrophin and muscular dystrophy: past, present, and future, Mol. Genet. Metab. 74 (2001) 75-88, H.M. Sadoulet-Puccio, L.M. Kunkel, Dystrophin and its isoforms, Brain Pathol. 6 (1996) 25-35]. Exacerbating this complexity, it has recently been shown that dystrophin is subject to extensive alternative RNA processing, potentially producing a wide variety dystrophin variants [M. Sironi, R. Cagliani, U. Pozzoli, A. Bardoni, G.P. Comi, R. Giorda, N. Bresolin, The dystrophin gene is alternatively spliced throughout its coding sequence FEBS Lett 517 (2002) 163-166]. The structure of the dystrophin protein is highly modular, with the most common module being a motif termed the spectrin type repeat, or STR, of which there are 24. Each STR is roughly coded for by two exons, and the most common type of multiple exon-skipping events start and end at introns in the middle of STRs [R.G. Roberts, A.J. Coffey, M. Bobrow, D.R. Bentley, Exon structure of the human dystrophin gene Genomics 16 (1993) 536-538, M. Koenig, L.M. Kunkel, Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility, J. Biol. Chem. 265 (1990) 4560-4566]. This would produce fractional STR modules, however, the concept of STRs as proteins domains makes the viability of such fractional motifs questionable. However, certain of these events produce pairs of potentially complementary fractional domain that might reassemble into a hybrid STR motif. We have constructed model fragment corresponding to one such exon-skipping event, and show that the hybrid STR so produced is viable, and furthermore that some of the properties of the protein containing it differ substantially of the native, un-skipped parent.

Amino Acid Motifs↗
PubMed1994

Characterization of a C alpha gene of swine.

The cDNA sequence encoding the constant region of the porcine IgA heavy chain as well as the exon-intron structure of the germline gene, have been determined. A cDNA clone (1A1) spanning the CH3 domain and part of the CH2 domain was isolated from a porcine mesenteric lymph node cDNA library. Clone 1A1 was aligned with a PCR-generated DNA fragment encompassing the CH1 domain through the 5' end of the CH3 domain to derive the complete cDNA sequence. Comparison with other mammalian C alpha heavy chains (hinge regions excluded) indicated that the deduced amino acid sequence of porcine C alpha is most homologous with the human C alpha subclasses (> 70%), followed by mouse C alpha (61%) and a consensus sequence of the 13 rabbit C alpha heavy chains (59%). The greatest sequence homology was found among the CH3 domains in all species. A striking feature of porcine C alpha is its short six amino acid hinge which like other mammalian IgAs, is encoded with the CH2 domain. Sequence analysis of germline C alpha, generated by PCR from liver or sperm DNA, revealed an exon-intron organization similar to other mammalian C alpha genes. Genomic Southern blot data are consistent with the presence of a single C alpha gene within the porcine genome. Data obtained in these studies will be valuable in pursuing the use of swine as a model in immunological research.

Amino Acid Sequence↗
PubMed1999

Sheep CD4(+) alphabeta T cells express novel members of the T19 multigene family.

The sheep T19 multigene family contains at least 50 genes which are thought to be expressed exclusively on gammadelta T cells. The archetypal T19 molecule (represented by a full-length cattle cDNA clone termed WC1) is thought to have a relative molecular mass of about 220 000 and to contain 11 scavenger receptor cysteine rich (SRCR) repeats and a long cytoplasmic tail. In this study, purified CD4(+) and gammadeltaTCR+ sheep lymphocytes were examined by reverse transcriptase-polymerase chain reaction for the expression of T19 molecules. As expected, gammadelta T cells were found to express T19 molecules which closely resembled the archetypal form. However, CD4(+) alphabeta T cells were found to express at least two different types of T19 molecules; one resembled the previously described T19 molecules of gammadelta T cells which possessed the archetypal WC1-like structure, but a novel type of T19 variant which lacked SRCR domains 10 and 11 was also found in CD4(+) T cells but not gammadelta T cells. This novel molecule exhibited an unusual, incomplete SRCR repeat 9 joined directly to a hinge region. The transmembrane and cytoplasmic domains of this unusual T19 variant resembled the cattle T19 clone WC1, except that a complete exon within the cytoplasmic region was missing. These results, in contradistinction to existing serological data, suggest that expression of the T19 gene family is not confined to gammadelta T cells. Selected T19 genes are apparently expressed within CD4(+) T cells and possibly other lymphocytes as well.

Amino Acid Sequence↗
PubMed1999

Expression of estrogen receptor alpha mRNA and protein variants in human endometrial carcinoma.

Breast cancer tissue has been shown to contain alternatively spliced estrogen receptor alpha (ER-alpha) mRNA variants, which have altered biological activities compared to the full-length ER-alpha. The development of endometrial cancer, as well as drug resistance in breast cancer patients undergoing tamoxifen therapy, may represent altered ER-alpha function secondary to specific exon deletions. While the literature is replete with ER mRNA variant data, little information is available regarding the presence and function of endometrial ER variant proteins. We evaluated the presence of human ER-alpha mRNA and protein variants in six premenopausal, six postmenopausal, and six endometrial carcinoma samples. Reverse transcription-polymerase chain reaction, DNA hybridization, and sequencing techniques identified exon 4, exon 5, and exon 7 mRNA splice variants in all patients as well as MCF-7 and Ishikawa cell lines. Presence of translated proteins for full-length ER-alpha, as well as splice variants, was investigated by Western blot analysis using antibodies directed against the N-terminus, hinge region, and C-terminus portions of the ER. These experiments confirmed the presence of immunopositive protein bands of approximately 64-66 kDa in all patients corresponding to wild-type ER-alpha. A protein band migrating at 41 kDa, consistent with an exon 5 splice variant, was only seen in endometrial adenocarcinoma samples. Premenopausal and postmenopausal endometrial samples did not contain detectable amounts of ER splice variant protein. Human ER-alpha mRNA variants are present in all human endometrial samples, but detectable levels of variant proteins are only observed in patients with endometrial adenocarcinoma.

DNA, Neoplasm↗
PubMed2002

Evolution of the six horse IGHG genes and corresponding immunoglobulin gamma heavy chains.

It is generally assumed that the different mammalian IgG isotypes have developed during evolution by duplications of a common ancestor gamma heavy chain constant region gene (IGHG). In contrast to other species studied so far, which express between one and four IGHG genes, the horse (Equus caballus) genome contains six IGHG genes, and it has been postulated that they all can be expressed. For determination of the evolutionary history of the six horse IGHG genes, genomic DNA and cDNA of the IGHG genes were sequenced. The structure of these genes with reference to exons and introns was determined. Comparison of the deduced amino acid sequences of the horse IGHG genes revealed the greatest divergences in the hinge regions, and in the proximal CH2 domains. A phylogenetic comparison of the amino acid sequences of the six horse IGHG genes to those of other species shows that the horse IGHG genes form a distinct cluster. This indicates that the mammalian species included in this study probably share only one common ancestor IGHG gene with the horse. The six horse IGHG genes probably then evolved by gene duplication after species separation. In addition, various segmental exchanges were found between the horse IGHG genes, which might be the result of unequal crossing over and/or gene conversion events during the evolution of the six horse IGHG genes.

Amino Acid Sequence↗
PubMed2004

A novel 1297-1304delGCCTGCCA mutation in the exon 10 of the thyroid hormone receptor beta gene causes resistance to thyroid hormone.

INTRODUCTION: Resistance to the thyroid hormone (RTH) is an inherited syndrome of reduced tissue responsiveness to hormonal action caused by mutations located in the ligand-binding domain and adjacent hinge region of the thyroid hormone receptor beta (TRbeta) gene. PATIENT: The patient in this study, a 42-year-old Caucasian male, came to medical attention because he experienced atrial fibrillation. Clinical evaluation showed a small and diffuse goiter and biochemical tests revealed markedly elevated concentrations of total T4, total T3, and free T4, normal thyroid-stimulating hormone (TSH) values and slightly increased I131 thyroid uptake at 24 hours. The thyroperoxidase, thyroglobulin, and TSH receptor antibodies were positive. He was treated with cabergoline plus methimazole. This treatment was stopped because of the inconsistent response, monotherapy with tri-iodothyroacetic acid (TRIAC) was then prescribed after molecular diagnosis confirmed RTH syndrome. METHODS: The exons 9 and 10 of the TRbeta gene, including splicing signals and the flanking intronic regions of each intron, were amplified with PCR. DNA sequences from each amplified fragment were performed with the Taq polymerase-based chain terminator method and using the specific TRbeta forward and reverse primers. RESULTS: Direct sequence analysis of the exons 9 and 10 of the TRbeta gene revealed an eight basepair deletion, 1297-1304delGCCTGCCA in exon 10. The mutation produces a frameshift at amino acid 433 and introduces a stop codon TGA at position 461, 85 nucleotides downstream from deletion. This alteration was not detected in either the father or mother of the patient, suggesting a de novo mutation that was confirmed by DNA fingerprint analysis. CONCLUSIONS: In the present study we have identified a novel sporadic mutation corresponding to 1297-1304delGCCTGCCA deletion in the activating function 2 (AF-2) region of TRbeta. To our knowledge, this is the first time that the presence of a partial deletion of eight nucleotides in the TRbeta has been reported.

Adult↗
PubMed1998

Giant dystrophin deletion associated with congenital cataract and mild muscular dystrophy.

We report a patient with a large intragenic dystrophin deletion of exons 17-51 inclusive associated with congenital cataract and mild Becker muscular dystrophy. The cataract was similar to the congenital cataract described in the mdx mouse. The loss of 68% of the rod domain including hinge 2 and 3 regions did not adversely affect the correct localization of the dystrophin and the association with the dystrophin-associated glycoprotein complex. This observation may have implications for minigenes suitable for gene therapy.

Cataract↗
PubMed1995

The N-terminal half of dystrophin is protected from proteolysis in situ.

Using a panel of "exon-specific" monoclonal antibodies, we have examined the products of degradation of dystrophin by endogenous proteases in post-mortem human muscle. Four main sites of dystrophin digestion were identified, all of them in the C-terminal half of the molecule. Two of them correspond to "hinges" in the central rod region and a third in the C-terminal domain follows the dystroglycan binding site. The results support the Koenig and Kunkel model for the tertiary structure of dystrophin (J. Biol. Chem. 265 (1990) 4560-4566), but suggest that much of the N-terminal half of dystrophin is protected from proteolysis, possibly by interaction with the sub-sarcolemmal cytoskeleton. Although the results seem inconsistent with an anti-parallel dimer model of dystrophin in which hinge 2 and hinge 3 are close together, possible ways of reconciling them with such a model are also considered.

Antibodies, Monoclonal↗
PubMed1999

Domain identification of hormone-sensitive lipase by circular dichroism and fluorescence spectroscopy, limited proteolysis, and mass spectrometry.

Structure-function relationship analyses of hormone-sensitive lipase (HSL) have suggested that this metabolically important enzyme consists of several functional and at least two structural domains (Osterlund, T., Danielsson, B., Degerman, E., Contreras, J. A., Edgren, G., Davis, R. C., Schotz, M. C., and Holm, C. (1996) Biochem. J. 319, 411-420; Contreras, J. A., Karlsson, M., Osterlund, T., Laurell, H., Svensson, A., and Holm, C. (1996) J. Biol. Chem. 271, 31426-31430). To analyze the structural domain composition of HSL in more detail, we applied biophysical methods. Denaturation of HSL was followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event. Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one corresponding exactly to the proposed N-terminal domain. Major cleavage sites were found in the predicted hinge region between the two domains and in the regulatory module of the C-terminal, catalytic domain. Analyses of a hinge region cleavage mutant and calculations of the hydropathic pattern of HSL further suggest that the hinge region and regulatory module are exposed parts of HSL. Together, these data support our previous hypothesis that HSL consists of two major structural domains, encoded by exons 1-4 and 5-9, respectively, of which the latter contains an exposed regulatory module outside the catalytic alpha/beta-hydrolase fold core.

Animals↗
PubMed1992

Control of human coagulation by recombinant serine proteases. Blood clotting is activated by recombinant factor XII deleted of five regulatory domains.

The availability of engineered serine proteases allows one to study the activation, substrate specificity and regulation of human coagulation and fibrinolytic activities. Human coagulation factor XII is composed of the protease catalytic region at the C-terminus, a hinge proline-rich region and regulatory domains at the N-terminus. From cDNA clones coding for factor XII, two DNA molecules were constructed, one being full length and the other being deleted of exons coding for the regulatory domains. Engineered factor-XII cDNA species were inserted by a homologous recombination technique into vaccinia viruses, which were used to infect the human hepatoma cell line HepG2. Two recombinant proteins were prepared from the culture media and identified by their antigenic properties and electrophoretic mobilities. The recombinant protein of larger size was identified as the full-length factor XII of 80 kDa and its specific activities and activation patterns, determined both by the coagulation and the amidolytic assays, are very similar to these of native human factor XII. The recombinant protein of smaller size was identified as a 319-amino-acid-deleted factor-XII protein of 32 kDa, containing only the entire protease region and part of the proline-rich hinge. This protein was expected to be the 'minimal' portion of factor XII able to sustain protease but unable to recognize substrates and surfaces necessary to activate the contact phase of coagulation. However, this 'minimal' factor-XII protein displays a marked protease activity and, although lacking five regulatory domains of factor XII, is bound and activated by negative charges and promotes coagulation with high efficiency.

Base Sequence↗
PubMed1993

Additional sequence complexity in the muscle gene, unc-22, and its encoded protein, twitchin, of Caenorhabditis elegans.

Null mutations of the Caenorhabditis elegans unc-22 gene cause a pronounced body surface twitch associated with impaired movement and disruption of muscle structure. Partial sequence analysis of unc-22 has previously revealed that its encoded polypeptide, named twitchin, consists of a single protein kinase domain and multiple copies of both an immunoglobulin-like domain and a fibronectin type III-like domain. This paper reports additional DNA sequence information that has revealed the transcription start of unc-22, the N terminus of twitchin, and an explanation for the weak phenotype of a transposon insertion allele. These new data indicate that the unc-22 gene is 18 kb larger than previously reported and has a transcription unit of 38,308 bp. These data add 791 amino acids to the twitchin N terminus for a complete polypeptide size of 6,839 amino acids and a predicted molecular weight of 753,494. This new polypeptide sequence includes four additional copies of the above-mentioned immunoglobulin-like domains and also includes a glycine-rich sequence that might form a flexible hinge. The additional coding sequence reveals that the insertion of the Tc1 transposon, in the unc-22 allele, st139, should disrupt twitchin structure because it is located in an exon. However, cDNA sequencing has revealed that several cryptic splice donors and acceptors adjacent to the Tc1 insertion site are used to splice the transposon out of unc-22(st139) mRNA. One of these splicing events produces a near wild-type mRNA that deletes only six amino acids from twitchin, and this might explain the unusually mild phenotype associated with this mutation.

Alleles↗