scholarly journals Isolation and characterization of cDNA clones encoding pig gastric mucin

1995 ◽  
Vol 308 (1) ◽  
pp. 89-96 ◽  
Author(s):  
B S Turner ◽  
K R Bhaskar ◽  
M Hadzopoulou-Cladaras ◽  
R D Specian ◽  
J T LaMont
Keyword(s):  
Amino Acid ◽  
Tandem Repeats ◽  
Consensus Sequence ◽  
Gastric Gland ◽  
Repeat Sequence ◽  
Gastric Mucin ◽  
Gastric Epithelium ◽  
Cdna Clones ◽  
Repeat Region ◽  
Isolation And Characterization

Polyclonal antibodies raised to deglycosylated pig gastric mucin were used to screen a cDNA library constructed with pig stomach mucosal mRNA. Immunocytochemistry indicated that the antibody recognizes intracellular and secreted mucin in surface mucous cells of pig gastric epithelium. A total of 70 clones producing proteins immunoreactive to this antibody were identified, two of which (PGM-2A,9B) were fully sequenced from both ends. Clone PGM-9B hybridized to a polydisperse mRNA (3-9 kb) from pig stomach, but not liver, intestine or spleen, nor to mRNA from human, mouse, rabbit or rat stomach. Sequence analysis indicated that PGM-9B encodes 33 tandem repeats of a 16-amino-acid consensus sequence rich in serine (46%) and threonine (17%). Using the restriction enzyme MwoI, which has a single target site in the repeat, it was demonstrated that PGM-9B consists entirely of this tandem repeat. Southern-blot analysis indicated that the repeat region is contained in a 20 kb HindIII-EcoRI fragment, and BamHI digestion suggested that most of the repeats are contained in a 10 kb fragment. In situ hybridization with an antisense probe to PGM-9B showed an intense signal in the entire gastric gland. Clone PGM-2A also contains the same repeat sequence as 9B, but, in addition, has a 64-amino-acid-long non-repeat region at its 5′ end. Interestingly the non-repeat region of PGM-2A has five cysteine residues, the arrangement of which is identical with that reported for human intestinal mucin gene MUC2.

scholarly journals Mouse gastric mucin: cloning and chromosomal localization

1995 ◽  
Vol 311 (3) ◽  
pp. 775-785 ◽  
Author(s):  
L L Shekels ◽  
C Lyftogt ◽  
M Kieliszewski ◽  
J D Filie ◽  
C A Kozak ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
Dna Analysis ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Repeat Sequence ◽  
Gastric Mucin ◽  
Gastric Epithelium ◽  
Chromosome 11P15 ◽  
Mucin Gene ◽  
Intestinal Mucin

Mucins protect gastric epithelium by maintaining a favourable pH gradient and preventing autodigestion. The purpose of this study was to clone a mouse gastric mucin which would provide a foundation for analysis of mucin gene regulation. Mucin was purified from the glandular portion of gastric specimens and deglycosylated by HF solvolysis. Antibodies against native and deglycosylated mouse gastric mucin (MGM) were raised in chickens. Screening of a mouse stomach cDNA library with the anti-(deglycosylated MGM) antibody yielded partial clones containing a 48 bp tandem repeat and 768 bp of non-repetitive sequence. The 16-amino-acid tandem repeat has a consensus sequence of QTSSPNTGKTSTISTT with 25% serine and 38% threonine. The MGM tandem repeat sequence bears no similarity to previously identified mucins. The MGM non-repetitive region shares sequence similarity with human MUC5AC and, to a lesser extent, human MUC2 and rat intestinal mucin. Northern blot analysis reveals a polydisperse message beginning at 13.5 kb in mouse stomach with no expression in oesophagus, trachea, small intestine, large intestine, caecum, lung or kidney. Immunoreactivity of antibodies against deglycosylated MGM and against a synthetic MGM tandem repeat peptide was restricted to superficial mucous cells, antral glands and Brunner's glands in the pyloric-duodenal region. DNA analysis shows that MGM recognizes mouse and rat DNA but not hamster, rabbit or human DNA. The MGM gene maps to a site on mouse chromosome 7 homologous to the location of a human secretory mucin gene cluster on human chromosome 11p15. Due to sequence similarity and predominant expression in the stomach, the MGM gene may be considered a MUC5AC homologue and named Muc5ac.

Isolation and characterization of some novel genes of the apolipoprotein A-I family in Japanese eel, Anguilla japonica

2011 ◽  
Vol 6 (4) ◽  
pp. 545-557 ◽  
Author(s):  
Malay Choudhury ◽  
Takahiro Oku ◽  
Shoji Yamada ◽  
Masaharu Komatsu ◽  
Keita Kudoh ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Consensus Sequence ◽  
Structural Features ◽  
Lipid Binding ◽  
Japanese Eel ◽  
Amino Acid Sequences ◽  
Novel Genes ◽  
Isolation And Characterization

AbstractApolipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1–11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family.

scholarly journals cDNA cloning and characterization of the protein encoded by RD, a gene located in the class III region of the human major histocompatibility complex

1993 ◽  
Vol 294 (2) ◽  
pp. 589-593 ◽  
Author(s):  
J Cheng ◽  
K J Macon ◽  
J E Volanakis
Keyword(s):  
Major Histocompatibility Complex ◽  
Amino Acid ◽  
Tandem Repeats ◽  
Cdna Clones ◽  
Major Histocompatibility ◽  
Class Iii ◽  
Human Major Histocompatibility Complex ◽  
Calculated Molecular Mass ◽  
Mouse Cdna ◽  
Histocompatibility Complex

The RD gene, initially defined in the mouse, has been mapped between the Bf and C4A genes in the human major histocompatibility complex class III region. Using the mouse cDNA as a probe, we isolated and sequenced human RD cDNA clones. The composite nucleotide sequence consisted of 1301 nucleotides, excluding a poly(A) tail at the 3′ end. It contained a single open reading frame encoding a polypeptide of 380 amino acid residues with a calculated molecular mass of 42274 Da. The most striking structural feature of the deduced amino acid sequence is a region consisting entirely of 24 tandem repeats of an Arg-Asp (or Glu) dipeptide. The human RD cDNA was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and used to produce antisera in rabbits. Western blot analysis and immunoprecipitation of lysates of biosynthetically labelled HeLa cells indicated that RD is a 44 kDa nuclear protein.

scholarly journals A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts.

1989 ◽  
Vol 109 (6) ◽  
pp. 2575-2587 ◽  
Author(s):  
S Piñol-Roma ◽  
M S Swanson ◽  
J G Gall ◽  
G Dreyfuss
Keyword(s):  
Hela Cell ◽  
Rna Binding ◽  
Consensus Sequence ◽  
Cdna Clones ◽  
Cell Nuclei ◽  
Intense Staining ◽  
L Protein ◽  
Isolation And Characterization ◽  
Hnrnp Protein ◽  
Nascent Transcripts

Immediately after the initiation of transcription in eukaryotes, nascent RNA polymerase II transcripts are bound by nuclear proteins resulting in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. hnRNP complexes from HeLa cell nuclei contain greater than 20 major proteins in the molecular mass range of 34,000-120,000 D. Among these are the previously described A, B, and C groups of proteins (34,000-43,000 D) and several larger, and as yet uncharacterized, proteins. Here we describe the isolation and characterization of a novel hnRNP protein termed the L protein (64-68 kD by mobility in SDS-polyacrylamide gels). Although L is a bona fide component of hnRNP complexes, it also appears to be a different type of hnRNP protein from those previously characterized. A considerable amount of L is found outside hnRNP complexes, and monoclonal antibodies to the L protein also strongly stain unidentified discrete nonnucleolar structures, in addition to nucleoplasm, in HeLa cell nuclei. Interestingly, the same antibodies stain the majority of nonnucleolar nascent transcripts from the loops of lampbrush chromosomes in the newt, but the most intense staining is localized to the landmark giant loops. The L protein is the first protein of giant loops identified so far, and antibodies to it thus provide a useful tool with which to study these unique RNAs. In addition, isolation and sequencing of cDNA clones for the L protein from human cells predicts a glycine- and proline-rich protein of 60,187 D, which contains two 80 amino acid segments only distantly related to the RNP consensus sequence-type RNA-binding domain. The L protein, therefore, is a new type of hnRNP protein.

scholarly journals Comparison of Ambisense M RNA of Watermelon Silver Mottle Virus with Other Tospoviruses

1998 ◽  
Vol 88 (4) ◽  
pp. 351-358 ◽  
Author(s):  
Fang-Hua Chu ◽  
Shyi-Dong Yeh
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Consensus Sequence ◽  
Chenopodium Quinoa ◽  
Distinct Species ◽  
Mottle Virus ◽  
Impatiens Necrotic Spot Virus ◽  
Cdna Clones ◽  
Genomic Rnas ◽  
Watermelon Silver Mottle Virus

Double-stranded genomic RNAs (dsRNAs) extracted from Chenopodium quinoa infected with watermelon silver mottle virus (WSMV) were similar to those of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), except that the S dsRNA of WSMV is 0.75 and 0.6 kbp longer than those of TSWV and INSV, respectively. The complete nucleotide sequence of the genomic M RNA of WSMV was determined from cDNA clones generated from separated M dsRNA. The M RNA is 4,880 nucleotides in length with two open reading frames (ORFs) in an ambisense organization. The M RNA-encoded nonstructural (NSm) ORF located on the viral strand encodes a protein of 312 amino acids (35 kDa), and the G1/G2 ORF located on the viral complementary strand encodes a protein of 1,121 amino acids (127.6 kDa). The RNA probe corresponding to the NSm or G1/G2 ORF of WSMV failed to hybridize with the M dsRNAs of TSWV and INSV. Comparison of M and S RNAs of WSMV, TSWV, INSV, and peanut bud necrosis virus (PBNV, serogroup IV) revealed a consensus sequence of eight nucleotides of 5′-AGAGCAAU…-3′ at their 5′ ends and 5′-…AUUGCUCU-3′ at their 3′ ends. The low overall nucleotide identities (56.4 to 56.9%) of the M RNA and the low amino acid identities of the NSm and G1/G2 proteins (30.5 to 40.9%) with those of TSWV and INSV indicate that WSMV belongs to the Tospovirus genus but is phylogenetically distinct from viruses in serogroups I and III. The M RNA of WSMV shares a nucleotide identity of 79.6% with that of PBNV, and the two viruses share 83.4 and 88.7% amino acid identities for their NSm and G1/G2 proteins, respectively. It is concluded that they are two related but distinct species of serogroup IV. In addition to the viral or viral complementary full-length M RNA, two putative RNA messages for the NSm gene and the G1/G2 gene, 1.0 and 3.4 kb, respectively, were detected from the total RNA extracted from WSMV-infected tissue of Nicotiana benthamiana. The 1.0- and 3.4-kb RNAs were also detected in the viral RNAs extracted from purified nucleocapsids, suggesting that the putative messages of the M RNA of WSMV can also be encapsidated by the nucleocapsid protein.

Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

scholarly journals Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen.

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230 ◽  
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

scholarly journals Suggestive evidence for two different mucin genes in rat intestine

1993 ◽  
Vol 294 (2) ◽  
pp. 391-399 ◽  
Author(s):  
I A Khatri ◽  
G G Forstner ◽  
J F Forstner
Keyword(s):  
Tandem Repeats ◽  
Hydrophobic Interactions ◽  
Present Report ◽  
Consensus Sequence ◽  
Cdna Probe ◽  
Cdna Clones ◽  
Rat Intestine ◽  
Hydrophobic Region ◽  
Unique Region ◽  
Mucin Genes

In the present report we describe the isolation and sequence of a partial cDNA (M2-798) for a rat intestinal mucin designated M2. A rat intestinal lambda ZAP II cDNA library was screened using a polyclonal antiserum which was prepared against deglycosylated high-molecular-mass glycopeptides of the purified mucin. Mucin cDNA clones were found to contain tandem repeats of 18 nt which encoded a threonine- and proline-rich peptide having a consensus sequence of TTTPDV. This is the same sequence reported recently by Gum, Hicks, Lagace, Byrd, Toribara, Siddiki, Fearney, Lamport and Kim [(1991) J. Biol. Chem. 266, 22733-22738] for a rat intestinal cDNA called RMUC 176. A novel feature present in the cDNA M2-798 is a 246 nt unique region at the 3′ end which encodes a hydrophobic sequence of 82 amino acids. RNA blots probed with M2-798 cDNA produced a single hybridization band between 7.5 and 9.0 kb in rat small intestine and colon. An identical hybridization pattern was obtained with a PCR-generated cDNA probe corresponding solely to the unique hydrophobic region of M2-798, demonstrating that this region is encoded by the authentic M2 mRNA. Our data suggest that the unique region of M2 has the potential to be either a transmembrane region, or a domain which mediates hydrophobic interactions of the mucin with other molecules. Since we have previously reported another rat intestinal cDNA which encodes the C-terminus of a mucin-like peptide (MLP) [Xu, Wang, Huan, Cutz, Forstner and Forstner (1992) Biochem. J. 286, 335-338], we wished to discover whether M2 was encoded by the same gene. RNA blotting experiments with probes specific for M2 and MLP showed different mRNAs for each. The message for M2 (7.5-8.5 kb) was smaller than that for MLP (> 9.5 kb) and, unlike MLP, gave no signal in human colonic LS174T cells. The results of DNA blots probed with M2-798 and an MLP-probe suggest that M2 and MLP are likely to be single-copy genes. It would appear therefore that normal rat intestine, like human intestine, may express two different mucin genes.

scholarly journals Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana ecotype Columbia.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 921-939 ◽  
Author(s):  
J Callis ◽  
T Carpenter ◽  
C W Sun ◽  
R D Vierstra
Keyword(s):  
Arabidopsis Thaliana ◽  
Amino Acid ◽  
Tandem Repeats ◽  
Synonymous Substitution ◽  
Coding Region ◽  
Coding Regions ◽  
Isolation And Characterization ◽  
Genes Encoding ◽  
Polyubiquitin Gene ◽  
Polyubiquitin Genes

Abstract The Arabidopsis thaliana ecotype Columbia ubiquitin gene family consists of 14 members that can be divided into three types of ubiquitin genes; polyubiquitin genes, ubiquitin-like genes and ubiquitin extension genes. The isolation and characterization of eight ubiquitin sequences, consisting of four polyubiquitin genes and four ubiquitin-like genes, are described here, and their relationships to each other and to previously identified Arabidopsis ubiquitin genes were analyzed. The polyubiquitin genes, UBQ3, UBQ10, UBQ11 and UBQ14, contain tandem repeats of the 228-bp ubiquitin coding region. Together with a previously described polyubiquitin gene, UBQ4, they differ in synonymous substitutions, number of ubiquitin coding regions, number and nature of nonubiquitin C-terminal amino acid(s) and chromosomal location, dividing into two subtypes; the UBQ3/UBQ4 and UBQ10/UBQ11/UBQ14 subtypes. Ubiquitin-like genes, UBQ7, UBQ8, UBQ9 and UBQ12, also contain tandem repeats of the ubiquitin coding region, but at least one repeat per gene encodes a protein with amino acid substitutions. Nucleotide comparisons, Ks value determinations and neighbor-joining analyses were employed to determine intra- and intergenic relationships. In general, the rate of synonymous substitution is too high to discern related repeats. Specific exceptions provide insight into gene relationships. The observed nucleotide relationships are consistent with previously described models involving gene duplications followed by both unequal crossing-over and gene conversion events.

The human insulin gene-linked polymorphic region adopts a G-quartet structure in chromatin assembled in vitro

1993 ◽  
Vol 10 (2) ◽  
pp. 121-126 ◽  
Author(s):  
M C U Hammond-Kosack ◽  
M W Kilpatrick ◽  
K Docherty
Keyword(s):  
Human Insulin ◽  
Tandem Repeats ◽  
Consensus Sequence ◽  
Repeat Sequence ◽  
Insulin Gene ◽  
Polymorphic Region ◽  
Dna Structures ◽  
Nuclease P1 ◽  
Human Insulin Gene

ABSTRACT The insulin gene-linked polymorphic region (ILPR), located 363 bp upstream of the human insulin gene, is composed of tandem repeats of the consensus sequence ACAGGGGT(G/C)(T/C)GGGG. It has previously been shown that an insulin gene fragment containing the ILPR adopts an altered DNA structure in vitro. Furthermore, oligonucleotides containing the consensus repeat sequence exhibit multiple quadriplex DNA structures. The present study was undertaken to determine whether such altered DNA structures existed within the ILPR when the insulin gene was assembled into chromatin in vitro. Chromatin assembly was achieved using histones and an extract from unfertilized eggs from Xenopus laevis. The presence of altered DNA conformations within the 5′ region of the human insulin gene was investigated using the structural probe nuclease P1. Nuclease P1 recognized multiple distinct sites in the 5′ flanking region of the human insulin gene in naked DNA. Most of these sites disappeared when the recombinant plasmid DNA was treated with histones and unfertilized egg extract. In the assembled DNA, the ILPR appeared as the major site of nuclease P1 hypersensitivity. Fine-mapping of the multiple reactive sites within the ILPR showed a pattern characteristic of G-quartet foldback structures similar to those that have been observed for telomeric DNA.

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