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At least 19 recordsLinked to original sources

PubMed1987

Completion of the amino acid sequence of the alpha 1 chain of human basement membrane collagen (type IV) reveals 21 non-triplet interruptions located within the collagenous domain.

The cDNA and protein sequences of the N-terminal half of human basement membrane collagen (type IV) have been determined. Overlapping cDNA clones were constructed by repeated primer extension with synthetic oligonucleotides. They cover 2953 bp, beginning at the 5' end of the corresponding mRNA. At the protein level, the sequence of the cyanogen bromide peptide CB6 adjacent to the 7S domain has been additionally elucidated. The data presented here complete the protein sequence and nearly the entire cDNA sequence of the human alpha 1(IV) chain. The amino-terminal half of the alpha 1(IV) chain contains 8 cysteine residues involved in intramolecular and intermolecular cross-links. The entire triple-helical domain of alpha 1(IV) is interrupted by 21 non-triplet regions.

Amino Acid Sequence↗

PubMed2025

Type and position of repeat interruptions as determinants of disease severity and expansion size in Friedreich ataxia.

PURPOSE: In Friedreich ataxia (FRDA) the size of the smaller GAA expansion is a major determinant of disease severity; interruption motifs were identified after the discovery of the pathogenic expansions; however, their impact is only recently investigated. METHODS: 164 patients with FRDA with biallelic expansions and 15 patients without FRDA were analyzed for interruption(s) number, position, and motif. Expansion size and age at onset of ataxia (AAO) were determined for patients with FRDA. RESULTS: Three groups of patients with FRDA were identified by the simultaneous analysis of the precise distance ("depth") between the interruptions (mostly nontriplet) and the 3' end of the expansion (P < .001), the smaller expansion size (P < .001), and AAO (P < .001). Classical FRDA corresponds to absence of interruption or interruption depth < 8 repeats, with AAO often <15 years (area under the curve [AUC] = 0.90; 95% CI, 0.84-0.96); LOFA to interruption depth of 8 to 18 repeats (AUC = 0.97; 95% CI, 0.94-1), with AAO 15 to 34 years (AUC = 1; 95% CI, 1-1); and vLOFA to interruption depth > 18 (AUC = 0.97; 95% CI, 0.92-1), with AAO > 34 years. Multiple (>5) triplet interruptions hamper further expansion. CONCLUSION: This study provides the molecular basis for a novel classification of FRDA that should be recommended for correct diagnosis.

Humans↗

PubMed1990

Identification of the cleavage sites by a hemorrhagic metalloproteinase in type IV collagen.

Type IV collagen, solubilized from Engelbreth-Holm-Swarm (EHS) tumor basement membranes is digested by a hemorrhagic metalloproteinase, Ht-e, isolated from the crude venom of the Western Diamondback rattlesnake, Crotalus atrox. The major proteolytic products have Mr 141,000, 132,000, 87,000, 71,000, 33,000 and approximately 18,000 as estimated by SDS-gel electrophoresis of pepsinized type IV collagen fragments. Sequence analysis of the digestion products reveal that the Mr 141,000, 71,000 and approximately 18,000 band are derived from the alpha 1(IV) chains and the Mr 132,000, 87,000 and 33,000 bands are derived from the alpha 2(IV) chain. The products are stable over 72-hour incubation periods. The cleavage sites on the alpha 1(IV) and alpha 2(IV) chains are not identical. The alpha 1(IV) chains are cleaved in a pepsin susceptible triplet interruption region of the triple helix at position Ala258-Gln259. The alpha 2(IV) chain is cleaved in the triple helical region near the NC2 domain at the Gly191-Leu192 peptide bond. Isolated hexameric NC1 globular domains of type IV collagen are not digested by Ht-e. The present study demonstrates that the venom hemorrhagic metalloproteinase Ht-e has type IV collagenolytic activity. The triple helix of the type IV collagen molecule is cleaved in a region located immediately carboxyl to the flexible NC2 domain. The degradation by Ht-e of type IV collagen, a major component of basement membranes which forms the scaffold of this extracellular structure, may account in part for the hemorrhagic activity of this toxin.

Amino Acid Sequence↗

PubMedDate not supplied

[Nucleotide sequence of chum salmon preproinsulin gene].

The nucleotide sequence of chum salmon preproinsulin gene isolated from the phage library of genomic sequences is presented. The transcription initiation site of the gene was experimentally determined. The resolved upstream region contains a TATA-promoter sequence and CAAT-like-box sequence. The salmon insulin gene is split and contains two introns. The first intron, 393 b.p. long, is situated in the 5'-untranslated region. The second intron, 287 b.p. long, interrupts triplet coding for the seventh amino acid residue of the C-peptide. Thus, the overall structure of the insulin gene appears to be closely related to other sequenced insulin genes, including those of birds and mammals. Both introns in the salmon insulin gene are flanked by GC...AG pairs in contrast to GT...AG, the rule common for the majority of genes. Analysis of the cloned salmon insulin gene and cDNAs reveals a high frequency of polymorphic differences, up to 6% in the translated region. The nucleotide sequence of the 3'-untranslated region of the gene is only 70% homologous to the corresponding region in the previously cloned salmon preproinsulin cDNAs. These differences may implicate for the presence of more than one insulin gene per haploid genome in salmon.

Amino Acid Sequence↗

PubMed1987

Characterization of three constituent chains of collagen type VI by peptide sequences and cDNA clones.

Pepsin-solubilized collagen VI was prepared from human placenta and used to separate three constituent chains for determining partial amino acid sequences. Antibodies raised against the chains assisted in the identification and purification of several cDNA clones from three expression lambda gt11 libraries. Most of the clones hybridized to either a 3.5-kb or 4.2-kb mRNA species which by matching peptide and nucleotide sequences could be identified as coding for the alpha 2(VI) or alpha 1(VI) chain, respectively. Other clones hybridized to either an 8.5-kb mRNA which very likely encoded the alpha 3(VI) chain or to an unknown 2.0-kb mRNA. Northern blots revealed a considerable variation in the mRNA levels for each collagen VI chain in both skin and cornea fibroblasts and in several tumor cell lines. Limited sequence data generated from peptides and cDNA clones demonstrated a characteristic cysteine pattern at the junction between N-terminal globular domain and triple helix in all three chains. In addition, the data showed occasional interruptions of triplet sequences within the triple-helical domain and the presence of two Arg-Gly-Asp sequences which are potential cell-binding structures.

Amino Acid Sequence↗

PubMed1986

Structure of mouse type IV collagen. Amino-acid sequence of the C-terminal 511-residue-long triple-helical segment of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain.

The sequence of 511 residues from the C-terminal portion of the triple helix of mouse alpha 2(IV) chain was determined by using the pepsin fragment P2 of collagen IV and two cDNA clones selected from an Engelbreth-Holm-Swarm (EHS) tumor library. The sequence contains nine interruptions of the triplet repeat Gly-Xaa-Yaa ranging in size from single insertions or deletions up to stretches of eleven amino acid residues. Five of these interruptions match those present in the homologous segment of the alpha 1(IV) chain but are otherwise different in length and/or sequence. A low homology was found for the triplet regions of the alpha 1(IV) and alpha 2(IV) chain which constitute more than 90% of the sequence. The data indicate a remote evolutionary relationship of the triple-helical sequences of the two constituent chains of basement membrane collagen.

Amino Acid Sequence↗

PubMed1991

cDNA analysis predicts a cornea-specific collagen.

In the development of chicken corneal stroma, two or more collagens often interact, either as constituents of a single heterotypic fibril or as components of the fibril surface. The latter, fibril-associated collagens, may facilitate interactions between fibrils and the surrounding extracellular matrix or between fibrils themselves. In an effort to isolate putative nonfibrillar collagens that may have such a function, we screened a 13-day embryonic cornea cDNA library under reduced stringency conditions, using a cDNA probe for a collagenous domain of type XII collagen. We isolated a 4.2-kilobase (kb) cDNA that predicts a "collagenous" protein that has three unusual, if not unique, features. (i) The putative polypeptide encoded by this cDNA has a structural arrangement in which numerous stretches of Gly-Xaa-Yaa triplets, typical of collagens, are interrupted by non-Gly-Xaa-Yaa regions. One of the potential triple-helical domains is 246 amino acids long, but most are much smaller, consisting of 15-36 amino acids. Many are very rich in the helix-stabilizing imino acid proline. (ii) Northern blot analyses demonstrated strong cDNA hybridization to a 6.8-kb mRNA whose expression is restricted to the cornea. No hybridization was observed to mRNAs from the nine other tissues used in these analyses, even with extended exposure of the film. (iii) The cDNA contains a short (less than or equal to 425-base-pair) sequence in the 3' untranslated region of the 6.8-kb mRNA that hybridizes to a 7.8-kb mRNA that has a wide tissue distribution.

Amino Acid Sequence↗

PubMed1988

Functional role of vitamin K in photosystem I of the cyanobacterium Synechocystis 6803.

The function of vitamin K1 in the primary electron-transfer processes of photosystem I (PS I) was investigated in the cyanobacterium Synechocystis 6803. A preparation of purified PS I was found to contain two vitamin K1's per reaction center. One vitamin K1 was removed by extraction with hexane, and further extraction using hexane including 0.3% methanol resulted in a preparation devoid of vitamin K1. The hexane-extracted PS I was functional in the photoreduction of NADP+, but the PS I after extraction using hexane-methanol was totally inactive. Activity was restored by using exogenous vitamin K1 plus the hexane extract. Vitamin K3 would not substitute. The room temperature recombination kinetics of the PS I extracted with hexane were not significantly modified. However, following the removal of both vitamin K1's, the 20-ms recombination between P-700+ and P-430- was replaced by a dominant relaxation (t 1/2 = 30 ns) due to recombination of the primary biradical P-700+ A0- and a slower component originating from the P-700 triplet. This kinetic behavior was consistent with an interruption of forward electron transfer to the acceptor A1. Addition of either vitamin K1 or vitamin K3 to such preparations resulted in restoration of the slow kinetic phase (greater than 2 ms), indicating significant competition by the two exogenous quinones for electron transfer from A0-. In the case of vitamin K3, this change in the kinetics induced by vitamin K1, suggesting successful reconstitution of the acceptor site A1. These data support the hypothesis that acceptor A1 is vitamin K1 and is a component of the electron-transfer pathway for NADP+ reduction.

Chlorophyll↗

PubMed1985

Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C1q of the human complement system.

Plasmid clones containing cDNA coding for the B-chain of human Clq were isolated from a liver cDNA library. The longest cDNA insert isolated contained all the coding sequence for amino acid residues B1 to B226 plus a 3' non-translated region of 264 nucleotides that extended into the poly(A) tail, thus accounting for 950 nucleotides of the mRNA. The B-chain mRNA was estimated by Northern-blot analysis to be 1.46 kb (kilobases) long, which indicated that approx. 500 bases were not accounted for in the cDNA clone. A cosmid clone containing the C1q-B chain gene was isolated from a human genomic DNA library. The precise 5' limit of gene was not established, but from the data available it appears that the gene is approx. 2.6 kb long. The coding sequence for residues B1 to B226 in the gene is interrupted by one intron, of 1.1 kb, which is located within the codon coding for glycine at position B36. This glycine residue is located in the middle of the triple-helical regions found in C1q at exactly the position where there is an unusual structural feature, i.e. a bend in each of the helical regions brought about by the interruption of the Gly-Xaa-Yaa repeating triplet sequences in the A- and C-chains and the presence of an 'extra' triplet in the B-chain. Nucleotide sequencing of the 5' end of the gene indicates the presence of a predominantly hydrophobic stretch of 29 amino acids, immediately before residue B1, which could serve as a signal peptide.

Base Sequence↗

PubMed1985

The structural organization of the chicken calmodulin gene.

The structural organization of the entire chicken calmodulin (CaM) gene was determined by analysis of overlapping genomic clones obtained from Charon 4A and cosmid DNA libraries. These clones together span 39 kilobases of chicken genomic DNA. The CaM gene is 12 kilobases long and contains 8 exons interrupted by introns of highly variable size. The first intron (A) is only 19 base pairs (bp) long and divides the 5' untranslated region. Intron B separates the ATG from the first nucleotide of the triplet which encodes the NH2-terminal amino acid of CaM (Ala) whereas intron C separates the triplets encoding amino acids 10-11. Introns D, F, and G interrupt the Ca2+ binding subdomains II, III, and IV of CaM whereas intron E is localized in the linker region between the highly homologous NH2- and COOH-terminal halves of the protein. Primer extension studies using chicken brain poly(A+) mRNA and a fragment from the 5' untranslated region of a CaM cDNA identified the presumptive transcription initiation site (cap site) of the mRNA to be 103 bp 5' from the initiation condon ATG. A consensus sequence TATTTAA was localized 29 bp 5' from this cap site while a CCAAT sequence was located further 5' at position -58 bp. The structure of the CaM gene is strikingly similar to genes that encode other Ca2+ binding proteins from sea urchin, chicken, rat, and quail. These data suggest a conservation of genome organization related to the calcium binding and regulatory domains of Ca2+ binding proteins.

Animals↗

PubMed1991

Structure of the human lipoprotein-associated coagulation inhibitor gene. Intro/exon gene organization and localization of the gene to chromosome 2.

Lipoprotein-associated coagulation inhibitor (LACI) is a multivalent, Kunitz-type proteinase inhibitor which appears to play an important role in the regulation of hemostasis. LACI directly inhibits factor Xa, and, in a Xa-dependent fashion, also inhibits the factor VIIa-tissue factor catalytic complex. Hybridization of a LACI cDNA probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes localized the human LACI gene to chromosome 2. In situ hybridization to metaphase chromosomes further mapped the gene to the region 2q31----2q32.1. Exons of the human LACI gene were cloned from genomic or chromosome 2-specific phage libraries and sequenced, including approximately 500 base pairs of 5' upstream DNA. The 5' DNA did not contain a prototypical TATAA box or CCAAT sequence, and attempts to identify a unique site for the initiation of transcription were unsuccessful in that primer extension and S1 nuclease protection analysis indicate multiple transcription initiation sites for LACI messages. Comparing the gene sequence with LACI cDNA sequences indicates that the gene contains nine exons and that alternative splicing can occur, resulting in the absence of exon 2 in the 5' untranslated region of some messages. The three Kunitz domains in LACI are encoded on separate exons. Introns which interrupt coding sequences all occur in the same codon phase interrupting the first and second bases of the codon triplets. The data are consistent with LACI evolving by a combination of gene segment duplications and exon shuffling.

Amino Acid Sequence↗

PubMed1990

[Aspiration of a gestational sac controlled by vaginal ultrasound in multiple pregnancy for the purpose of selective abortion].

A case of an infertile married couple by whom the cause of infertility was oligoasthenospermia of man is described. The couple was treated by intrauterine insemination by capacitated spermatozoa in a cycle stimulated by clomiphene. The ultrasonography revealed a pregnancy of triplets. The patient refused a multiple pregnancy and asked for its interruption. As an alternative we proposed the selective abortion by means of an ultrasound-guided aspiration of two gestational sacs. Description of this method and the result are the theme of our paper.

Abortion, Therapeutic↗

PubMed1992

Truncated phenylalanine ammonia-lyase expression in tomato (Lycopersicon esculentum).

Southern blot analyses of genomic DNA fragments suggest there are five different classes of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) genomic sequence in tomato (Lycopersicon esculentum). Isolation and subsequent sequence analysis of three examples from genomic libraries reveal highly homologous coding sequences but also a surprisingly high frequency of single point mutations which would truncate protein synthesis. The nucleotide sequence for one of the genes (PAL5) encodes a normal polypeptide of 721 amino acids, interrupted by a 710-base pair intron in the codon for amino acid 139. In contrast, premature stop codons, 363 triplets from the end in PAL1 and 304 triplets from the end in PAL3 would result in substantially (51-43%) shorter polypeptides that are consistent with the protein polymorphism, recently reported in alfalfa (Jorrin, J., and Dixon, R. A. (1990) Plant Physiol. 92, 447-445) but ascribed to protein degradation. S1 mapping of the mRNA termini and polymerase chain reaction analysis of cDNA transcripts indicate that at least one of these truncated coding sequences is expressed, strongly suggesting that at least some of the shorter polypeptides constitute original gene products with a potentially important function.

Amino Acid Sequence↗

PubMed1991

Complete primary structure of human collagen alpha 1 (V) chain.

Several cDNA clones, encoding prepropeptide of human collagen alpha 1(V) chain, have been isolated. The prepropeptide (1838 amino acids length) of the alpha 1(V) chain was composed of a putative signal peptide, a large NH2-terminal noncollagenous region, a main collagenous region, and a COOH-terminal noncollagenous region. The signal peptide contained many leucine residues. The NH2-terminal noncollagenous region was much larger than those of the other collagens and had a region homologous to the COOH-terminal domain of laminin A chain, but it did not contain a cysteine-rich region that was maintained in the region of the other collagens. This region also contained probable tyrosine sulfation sites, and short collagenous sequences that were interrupted by three noncollagenous segments. The main collagenous region of the alpha 1(V) chain consisted of 338 repeats of Gly-X-Y-triplet. This region had a high degree (82%) of homology with the amino acids of the collagen alpha 1(XI) chain. The COOH-terminal noncollagenous region resembled that of the alpha 1(XI) chain, too, and 8 residues of cysteine that were important for the formation of the triple helix structure of collagens were observed. These results suggest that the alpha 1(V) chain belongs to the fibrillar collagen relative to the alpha 1(XI) chain, but codon usage of the alpha 1(V) cDNA was clearly different from those of the other fibrillar collagens including the alpha 1(XI), while it was similar to type IV collagen. This result supposes a different evolution of the alpha 1(V) gene from those of the other fibrillar collagens.

Amino Acid Sequence↗

PubMed1987

Translation framing code and frame-monitoring mechanism as suggested by the analysis of mRNA and 16 S rRNA nucleotide sequences.

Protein coding sequences carry an additional message in the form of a universal three-base periodical pattern (G-non-G-N)n, which is expressed as a strong preference for guanines in the first positions of the codons in mRNA and lack of guanines in the second positions. This periodicity appears immediately after the initiation codon and is maintained along the mRNA as far as the termination triplet, where it disappears abruptly. Known cases of ribosome slippage during translation (leaky frameshifts, out-of-frame gene fusion) are analyzed. At the sites of the slippage the G-periodical pattern is found to be interrupted. It reappears downstream from the slippage sites, in a new frame that corresponds to the new translation frame. This suggests that the (G-non-G-N)n pattern in the mRNA may be responsible for monitoring the correct reading frame during translation. Several sites with complementary C-periodical structure are found in the Escherichia coli 16 S rRNA sequence. Only three of them are exposed to various interactions at the surface of the small ribosomal subunit: (517)gcCagCagCegC, (1395)caCacCgcC and (1531)auCacCucC. A model of a frame-monitoring mechanism is suggested based on the weak complementarity of G-periodical mRNA to the C-periodical sites in the ribosomal RNA. The model is strongly supported by the fact that the hypothetical frame-monitoring sites in the 16 S rRNA that are derived from the nucleotide sequence analysis are also the only sites known to be actually involved or implicated in rRNA-mRNA interactions.

Base Sequence↗

PubMed1992

Cloning, structural analysis, and expression of the glycogen phosphorylase-2 gene in Dictyostelium.

The glycogen phosphorylase-2 (GP2) activity that appears during the cell differentiation of Dictyostelium was purified to homogeneity. The molecular weight of the nondenatured enzyme was 200,000 as determined by Sephacryl S-300 gel filtration and was 107,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme consists of two similar subunits. The intact protein was digested with trypsin and protease V8, and the resulting peptides were purified by microbore high pressure liquid chromatography. The peptides were sequenced, and oligonucleotides were constructed for polymerase chain reaction amplification of the GP2 gene from Dictyostelium genomic DNA template. The resulting polymerase chain reaction products were sequenced directly and were confirmed to encode portions of the GP2 gene. These fragments were used to probe a partial EcoRI genomic library for the remainder of the GP2 gene. The nucleotide sequence of the GP2-selected clones revealed an open reading frame of 2975 base pairs that was interrupted by two introns of 109 and 105 base pairs, respectively. The open reading frame encoded a protein of 992 amino acids with a calculated molecular mass of 112,500 Da and an isoelectric point of 6.4. An unusual sequence within the second exon of GP2, in which the triplet CAA was repeated 11 times, resulted in 11 in-frame glutamine residues of a possible 15 amino acids coded for by this region. The CAA repeat was transcribed, as shown by the sequence of cDNA. Comparison of the amino acid sequence of Dictyostelium GP2 to the phosphorylases from other organisms revealed that the Dictyostelium protein was 50 and 44% identical to yeast and rabbit muscle phosphorylases, respectively. Northern blot analysis showed that GP2 mRNA was absent in amebas and the early stages of development, reached a maximum level of expression at the slug stage, and then decreased in the terminal stages of development. Comparison of the mRNA expression with the appearance of GP2 enzyme protein and enzyme activity revealed that gp2 mRNA and a 113-kDa GP2 enzyme peptide were expressed concurrently at 10 h of development. However, enzyme activity did not appear until 18 h, coincident with a decrease in the level of the 113-kDa peptide and a corresponding increase in the amount of a 106-kDa GP2 peptide. Addition of cAMP to aggregation-competent cells in liquid culture resulted in the induction of GP2 mRNA, GP2 protein, and GP2 enzyme activity.

Amino Acid Sequence↗

PubMed1991

Genome analysis of imperfect fungi: electrophoretic karyotyping and characterization of the nuclear gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Curvularia lunata.

The gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) has been isolated from a genomic library of the filamentous fungus Curvularia lunata. The coding region of this gene consists of 1014 nucleotides and is interrupted by four introns. The gpd gene product shows a high degree of sequence identity with the corresponding proteins of various species belonging to both taxonomically related (e.g., Aspergillus nidulans), as well as more divergent, taxa. Using contour-clamped homogeneous electric field (CHEF) gel electrophoresis eight distinct chromosomal bands have been resolved, with two bands migrating as doublets and one as a triplet. Thus, the total number of chromosomes of C. lunata appears to be 12. The size of the chromosomes ranges from about 1.4 Mb to 4.0 Mb allowing an estimation of the genome to be approximately 29.7 Mb. By hybridization of fractionated chromosomes the gpd gene and the rDNA locus have been localized on individual chromosomes.

Amino Acid Sequence↗

PubMed1985

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. 7. Continuous gene for apocytochrome b in strain EF1 (CBS 356) and sequence variation in the region of intron insertion in strain ade 7-50h.

The third BamHI fragment, containing most of gene for apocytochrome b, has been cloned and sequenced in the Schizosaccharomyces pombe strain EF1 (CBS 356). In contrast to strain ade 7-50h- (50) from the Leupold collection, in which the gene is interrupted by an intron of group II (Lang et al. 1984), the homologous gene in strain EF1 is continuous. This demonstrates that the intron in the gene for apocytochrome b is optional. Aligning the EF1 sequence with the homologous regions in strain 50, 2 base pair changes were found in the leader and 14 in the coding region. These changes led to 12 altered triplets, but 9 of them specify the same amino acid. Seven base changes were clustered within a stretch of 30 base pairs in the region in which the intron is inserted in strain 50. Five out of the resulting six triplet changes were also silent. These sequence variations around the highly conserved splice point region may be linked to the insertion or excision of the intron.

Ascomycota↗