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PubMed1986

[Lipases of the digestive system].

Studies on gastrointestinal lipolysis have underestimated several important points. In view of recent in vitro data obtained in our laboratories, this review focuses on the role of gastric lipolysis during fat digestion. Polyclonal antibodies generated from purified rat lingual lipase were used to screen a cDNA library prepared from mRNA isolated from the serous glands of rat tongue cloned in E. coli expression vectors. A cDNA clone was isolated and the nucleotide and predicted amino acid sequences obtained. Comparison with the N-terminal amino acid sequence of the purified enzyme confirmed the identity of the cDNA. The amino acid sequence of rat lingual lipase consisted of 377 residues and showed little homology with porcine pancreatic lipase, apart from a short region containing a serine residue at an analogous position to the Ser 152 of the porcine enzyme. Human gastric lipase activity on tributyrin emulsion was detected only in the presence of amphiphiles. This behaviour was in sharp contrast with the strong inhibitory effect of amphiphiles observed on pure pancreatic lipase. To reveal human gastric lipase activity, amphiphiles must be added to human gastric lipase in order to prevent irreversible interfacial denaturation. Human gastric lipase activity was found to be restricted to triacylglycerol/water surface tensions ranging from 8 to 13 dynes/cm. All amphiphiles which decrease interfacial tension to less than 8 dynes/cm act as irreversible inhibitors of human gastric lipase in the absence or presence of bile salts. Our results confirm that human gastric lipase is capable of hydrolysing triacylglycerol in the presence of the bile salts concentration prevailing in the upper small intestine and in the presence of alimentary proteins. These observations could explain the high dietary lipid absorption observed under pancreatic lipase deficiency.

Amino Acid Sequence↗
PubMed1987

Effect of estrogen on the expression of mRNAs of different actin isoforms in immature rat uterus. Cloning of alpha-smooth muscle actin message.

Cytoplasmic beta- and gamma-actin mRNAs as well as smooth muscle actin mRNAs have been shown to be transiently increased in rat uterus after treatment with the steroid hormone estradiol. A clone isolated as an estradiol-induced message from a lambda-gt10 cDNA library prepared from the mRNA of estrogen-stimulated immature rat uterus was identified as alpha-smooth muscle actin. A single-stranded RNA probe composed mainly of the 3'-untranslated region of this clone, as well as DNA probes derived from the 3'-untranslated regions of other actin genes, were used to study the induction kinetics of different actin isoforms in rat uterus after being stimulated by estradiol. The beta- and gamma-cytoskeletal actins showed an induction peak at 4 h after estradiol administration with 1.4- and 1.8-fold increases, respectively. The smooth muscle actin was maximally increased 2.1-fold at 8-12 h. Messages of alpha-skeletal and alpha-cardiac actins were neither expressed nor induced by estradiol in this tissue. The different induction kinetics of the cytoplasmic and smooth muscle actins suggest that they are regulated by different mechanisms and possibly in different cell types of the uterus.

Actins↗
PubMed1987

Cloning and identification of lupin nodule specific cDNA sequences.

Yellow lupin nodule specific sequences were selected by screening of cDNA library prepared from lupin nodule poly(A)+RNA. From about 3,000 clones containing fragments of lupin DNA 150-1,500 base pair long, 7% of clones carrying nodule specific sequences were identified. Among them the most abundant sequence species, represented by 32% clones, encodes leghemoglobin. Another abundant species designated pLN13 is represented by 13% clones. The Northern blot analysis of lupin mRNA confirmed nodule specificity of the cloned sequences. The nucleotide sequence of one clone, pLN281 of 225 bp, is presented.

Base Sequence↗
PubMed1987

Cloning and sequence of rat myoadenylate deaminase cDNA. Evidence for tissue-specific and developmental regulation.

Myoadenylate deaminase is the muscle-specific isoform of AMP deaminase (EC 3.5.4.6), an enzyme which plays a special role in energy metabolism in skeletal muscle. A 2.3-kilobase cDNA encoding this enzyme has been cloned from a lambda gt10 library prepared from rat skeletal muscle using oligonucleotide probes designed from AMP deaminase peptide sequences. This cDNA was sequenced, and the amino acid sequence of this isoform of AMP deaminase was deduced. Sequences homologous to this cDNA are identified in the genome of eukaryotes as diverse as yeast and man. Tissue-specific expression of a 2.5-kilobase AMP deaminase transcript is demonstrated, and the abundance of this transcript as well as the 80-kDa adult, muscle-specific peptide of AMP deaminase increase in parallel during postnatal skeletal muscle development. In the adult animal, the abundance of this transcript and AMP deaminase activity are differentially expressed in various skeletal muscle fiber types. We conclude that AMP deaminase sequences have been highly conserved during evolution, and in mammals there is developmental and tissue-specific control of expression of this gene.

AMP Deaminase↗
PubMed1987

Isolation and sequencing of a cDNA clone for a prolactin-inducible protein (PIP). Regulation of PIP gene expression in the human breast cancer cell line, T-47D.

We have initiated an analysis of the action of prolactin in a human breast cancer cell line (T-47D) by isolating and sequencing cDNA clones for a prolactin-inducible protein (PIP). PIP cDNA (approximately 600 base pairs) was isolated from a lambda gt11 expression library prepared from mRNA extracted from T-47D cells treated with prolactin and hydrocortisone. The identity of PIP cDNA was confirmed by hybrid-selected translation. PIP mRNA is a single mRNA species of approximately 900 bases which responds to lactogenic hormones (human prolactin and growth hormone) in a time- and dose-dependent manner. Treatment of T-47D cells with human growth hormone (hGH) increased PIP mRNA levels by 4.6-fold, while the combination of hGH and hydrocortisone or dihydrotestosterone had a more dramatic, 12.4-fold, effect. The latter steroid was the most potent. Dihydrotestosterone plus hGH increased transcription of the PIP gene by 3.7-fold. Dihydrotestosterone alone was as effective as dihydrotestosterone plus hGH in increasing the transcription rate, while hGH alone had no effect. Thus, lactogen may regulate PIP gene expression at a post-transcriptional step. PIP mRNA was expressed at low levels in other human breast cancer cell lines which were prolactin receptor-positive; MCF-7 and EFM-19 lines were exceptions. PIP cDNA had an open reading frame of 146 amino acids, sufficient to encode the precursor form of the secreted PIP protein (approximately 14.5 kDa). The similarity of the predicted amino acid sequence of PIP to the sequence of a protein encoded by a gene expressed in the mouse submaxillary gland prompted us to look for PIP in human saliva. Western blot analysis confirmed the presence of PIP in human saliva. Therefore, the PIP gene is useful in studying the molecular actions of the prolactin/growth hormone polypeptide hormone family and the interaction with androgen, in mammary and other potential target cells.

Amino Acid Sequence↗
PubMed1985

Isolation of cDNAs encoding human and gibbon GM-CSF.

We have used a mammalian cell expression cloning system to identify cDNA clones encoding human granulocyte-macrophage colony stimulating factor. The human clone was used as a hybridization probe to identify the corresponding sequence from a cDNA library prepared from a gibbon T-cell line. The human cDNA has been used to produce recombinant GM-CSF in monkey COS-1 cells. The purified protein from COS cells is very similar to the GM-CSF isolated from a continuous human T-cell line.

Amino Acid Sequence↗
PubMed1985

Complete structure of a rat RT1 E beta chain: extensive conservation of MHC class II beta chains.

The rat major histocompatibility complex (RT1 complex) encodes two sets of class II molecules, A alpha A beta and E alpha E beta. A cDNA clone encoding a RT1 E beta chain was isolated from a cDNA library prepared from Wistar (RT1 mu) spleen poly(A) RNA by screening with DNA fragments encoding a RT1 A beta chain. The nucleotide sequence of this cDNA insert was determined and was shown to encode a complete RT1 E beta chain. The RT1 E beta chain was highly homologous in terms of sequence identities to H-2 E beta and HLA-DR beta chains. Comparisons of predicted protein sequences of RT1, H-2, and HLA E beta and A beta chains showed that class II beta-chains are highly conserved with 105 out of 278 residues identical in all six beta-chains. The divergences in terms of the number of silent and replacement nucleotide substitutions between class II E beta or A beta sequences showed that the number of replacement substitutions reached a plateau after 10 million yr. Replacement substitutions therefore may be under selective pressure during evolution. The accumulation of silent and replacement substitutions in RT1 and H-2 class II beta-chains was faster than that in HLA beta-chain sequences. Thus, it appears that class II beta chains may be evolving at different rates in different species.

Amino Acid Sequence↗
PubMed1984

Structure of a precursor to human pancreatic polypeptide.

We have isolated mRNA from a human pancreatic islet cell tumor and have identified among the cell-free translation products a precursor of pancreatic polypeptide with an approximate Mr = 11,000. Recombinant DNA molecules encoding this precursor were selected from a cDNA library prepared from the islet tumor mRNA. From the nucleotide sequences of cDNAs encoding the precursor, we have deduced the complete amino acid sequence of pre-propancreatic polypeptide. These sequences encode a protein consisting of 95 amino acid residues with a Mr = 10,432. The sequence of human pancreatic polypeptide occurs in the middle of the precursor and is flanked at its carboxyl terminus by a 27-amino acid sequence which is similar to a peptide previously isolated from canine pancreatic islets. At the amino terminus of the precursor is a probable leader sequence which is rich in hydrophobic residues. A smaller pancreatic polypeptide-related protein was generated in cell-free translations of mRNA supplemented with microsomal membranes. Sequential Edman degradations of this smaller peptide indicate that the sequence of pancreatic polypeptide is located at the amino terminus of the prohormone.

Amino Acid Sequence↗
PubMed1984

Serendipitous cloning of a mitochondrial cDNA and its polymorphism.

A cDNA clone p5x23 was isolated from a library prepared from membrane bound mRNA from a human lymphoblastoid cell line. This clone was shown to detect a restriction fragment length polymorphism with the enzyme MspI, with frequency for the minor variant of about 10% in a Caucasian population. The clone apparently only detected homozygotes, and did not hybridize to any of a panel of human-mouse somatic cell hybrids containing all human chromosomes in various combinations. Sequence analysis of the clone showed that it encoded a part of the 3' end of the human mitochondrial cytochrome b gene.

Amino Acid Sequence↗
PubMed1984

Isolation and sequence of a rat chymotrypsin B gene.

A cDNA clone encoding part of chymotrypsin B was isolated from a cDNA library prepared from rat pancreatic mRNA and used as a probe to isolate the chymotrypsin B gene. The nucleotide sequence of this gene is presented. The 4709-base pair transcribed portion of the isolated gene was inferred from the cDNA and gene sequence, and the 5' border was determined by primer extension on pancreatic polyadenylated RNA. The coding portion of the gene is interrupted by six introns. The active site residues His 57, Asp 102, and Ser 195 are encoded by separate exons. Moreover, two regions of the enzyme which form the substrate-binding pocket are also encoded by separate exons. Thus, the substrate specificity and catalytic activity of the enzyme are produced by joining several exons encoding protein segments that are intrinsically catalytically inactive. The number and location of the intron/exon junctions of the chymotrypsin gene as compared to those of other serine protease genes, as well as the location of the genes on separate chromosomes, suggest that the duplication that resulted in the formation of the chymotrypsin gene was an ancient evolutionary event.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequence analysis of a proline-rich protein cDNA and peptide homologies of rat and human proline-rich proteins.

Plasmids containing cDNAs for proline-rich proteins have been isolated from a cDNA library prepared from parotid glands of isoproterenol-treated rats. The nucleotide sequence of one of these cDNAs (pRP33) has been determined, and it encodes an acidic proline-rich protein. The N-terminal 13 amino acids are highly hydrophobic and may be part of a signal peptide. The next 66 amino acids constitute an acidic domain which is interrupted by a short (8 residue) basic region. The sequence beginning at proline 80 contains six tandemly repeated regions of 18 to 19 amino acids in length with the prototype sequence PPPQGGPQXPPQPGXPQG. Two pairs of these repeats (amino acid residues 116 to 152 and 153 to 189) are 93% homologous in nucleotide sequence. The high homology in both nucleotide and amino acid sequences in the repeat region suggests that this protein may have evolved by duplication of internal portions of a progenitor gene.

Amino Acid Sequence↗
PubMed1983

Structure and expression of a cloned cDNA for mouse interferon-beta.

A unique sequence in the mouse genome which cross-hybridized to a cloned human interferon-beta 1 gene was detected by DNA blot analysis. Taking advantage of this, a cDNA library prepared from partially purified mRNA for mouse interferon-beta was screened using human interferon-beta 1 DNA as a probe. One of the positive clones, pM beta-3, contained a 680-base pair cDNA insert, whose base sequence contained a single large open reading frame for 182 amino acids. The coding sequences of the cDNA showed homologies of 63% at the nucleotide and 48% at the amino acid level with respect to human interferon-beta 1 cDNA (Taniguchi, T., Ohno, S., Fujii-Kuriyama, Y., and Muramatsu, M. (1980) Gene 10, 11-15). The first 21 amino acids, considered to be the signal peptide, were followed by 24 amino acids, whose sequence was identical with the NH2-terminal sequence that had been reported for mouse interferon-beta from Ehrlich ascites tumor cells (Taira, H., Broeze, R. J., Jayaram, B. M., Lengyel, P., Hunkapiller, M. W., and Hood, L. E. (1980) Science (Wash. D.C.) 207, 528-530). The complete primary sequence of mature interferon-beta polypeptide consisting of 161 amino acids (Mr = 19,700) was deduced. There are three N-glycosylation sites, and this offers an explanation for the larger molecular size (Mr = 26,000-40,000) of natural mouse interferon-beta in comparison to the deduced interferon polypeptide. The cDNA, when fused to a SV40 promoter sequence and then introduced into COS-7 cells, directed the synthesis and secretion of a protein product indistinguishable from the authentic mouse interferon-beta.

Amino Acid Sequence↗
PubMed1995

Identification of lysyl oxidase and other platelet-derived growth factor-inducible genes in vascular smooth muscle cells by differential screening.

BACKGROUND: Atherosclerosis and arterial injury are characterized by vascular smooth muscle cell (VSMC) migration and growth and an increase in synthesis of extracellular matrix. Platelet-derived growth factor (PDGF) has been implicated in these processes. This study was designed to identify additional PDGF-regulated genes in VSMC. EXPERIMENTAL DESIGN: A cDNA library prepared from PDGF-stimulated rat aortic VSMC was screened by differential hybridization to identify clones representing PDGF-inducible genes. The time course of growth factor-induced changes in gene expression was examined by RNA blot hybridization. Assays of protein activity were also performed for selected gene products. RESULTS: Four PDGF-regulated cDNA clones were identified by DNA sequencing. These encoded the extracellular matrix proteins lysyl oxidase (LO), thrombospondin, and osteopontin and the intracellular enzyme lactate dehydrogenase (LDH). Levels of mRNA corresponding to all four genes were low in quiescent VSMC and were markedly induced by PDGF, angiotensin II, and 10% calf serum. The regulation of LO and LDH mRNA by these agonists in VSMC has not been previously reported. LO enzymatic activity in the culture media was increased by approximately equals to 700% after exposure to PDGF. In contrast, LDH activity was not increased by PDGF treatment. CONCLUSIONS: The induction of LO mRNA and its secretion by VSMC is an early event accompanying growth factor stimulation and may contribute to organization of the extracellular matrix.

Angiotensin II↗
PubMed1995

Chicken acidic ribosomal phosphoprotein PO: isolation and molecular characterization of cDNA clones.

Differential screening of a cDNA library prepared from chicken mitochondrial DNA-less cells led to the isolation of the acidic phosphoprotein PO, as inferred from similar sequences from human, rat and mouse deposited in databases. The chicken PO protein is highly conserved among vertebrates. Nucleotide identities between the chicken and mammalian cDNA sequences are greater than 94%. Amino acid sequence identities greater than 92% are observed between the chicken protein and its mammalian counterparts, and when changes to conservative amino acids are considered, similarities range from 99.4 to 100%. The gene evolved mainly by silent transitions occurring at the third codon position. The highly antigenic carboxy-terminal sequences of mammalian PO proteins are identical to that of the chicken. Southern blot analysis of restricted DNA from chicken embryo fibroblasts suggests that only one gene encoding PO exists in the chicken genome. By Northern analysis, the PO probe detects a major RNA species 1.1-kilobases long, and a minor species 4.0-kilobases long which has no equivalent thus far in mammals.

Amino Acid Sequence↗
PubMed1995

Cloning and stable expression of a cDNA encoding a rat liver UDP-glucuronosyltransferase (UDP-glucuronosyltransferase 1.1) that catalyzes the glucuronidation of opioids and bilirubin.

A chicken anti-rat polyclonal antibody to a purified rat liver UDP-glucuronosyltransferase (UGT) with catalytic activity toward opioid substrates was used to screen a liver cDNA library prepared from phenobarbital-treated Wistar rats. A number of positive clones were obtained, and one of these clones, pM1, was further characterized. Clone pM1 was found to be a full length cDNA coding for a member of the rat UGT1 gene family. Specifically, pM1 represents the full length homologue of the Gunn rat liver pseudo-gene product UGT1.1P and, therefore, has been designated UGT1.1r. The cDNA insert has an open reading frame of 1605 base pairs, which codes for a protein of 535 amino acids and is flanked by 2 and 632 base pairs of 5' and 3' noncoding sequence, respectively. The deduced amino acid sequence of pM1 contains amino acid sequences identical to the amino-terminal and internal peptides of the purified rat liver opioid UGT and to sequences reported for a rat liver bilirubin UGT [FEBS Lett. 299:183-186 (1992)]. Stable expression of UGT1.1r in human embryonic kidney 293 cells showed that a protein with a subunit molecular mass (56 kDa) identical to that of the purified protein was produced. Expressed UGT1.1r protein catalyzed the glucuronidation of buprenorphine and bilirubin at high rates. Other opioids, such as nalorphine and morphine, were also substrates for the expressed UGT1.1r protein. These results show that bilirubin and opioids can be conjugated by the same rat liver UGT.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and characterization of S-adenosyl-L-methionine:scoulerine-9-O-methyltransferase from cultured cells of Coptis japonica.

S-Adenosyl-L-methionine:scoulerine-9-O-methyltransferase (SMT) catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine to the 9-hydroxyl group of scoulerine during the biosynthesis of berberine. We have isolated functionally active cDNA clones (pCJSMTs) from a cDNA library prepared from cultured cells of Coptis japonica. The longest cDNA insert (pCJSMT1) had an open reading frame that encoded 351 amino acids, but the calculated molecular mass (38,364 Da) of the deduced product was slightly lower than the experimentally determined molecular mass of purified SMT. Rapid amplification of the 5' end of the cDNA indicated that the full-length cDNA of SMT consisted of 1,458 nucleotides that encoded 381 amino acids. When the full-length cDNA was expressed in E. coli, the molecular mass of the expressed SMT was greater than that of native SMT in Coptis cells. This result suggests that SMT might be produced in a pre-mature form and processed post-translationally. SMT was also found to exhibit sequence homology to other O-methyltransferases from plants and N-terminal region of the SMT polypeptide appeared to be necessary for enzymatic activity.

Amino Acid Sequence↗
PubMed1994

Identification of a novel variant form of fibroblast growth factor receptor 3 (FGFR3 IIIb) in human colonic epithelium.

Although several tyrosine kinase-type growth factor receptors have been demonstrated in human colonic epithelial cells, the full spectrum of growth factor receptors has not been identified. Low stringency screening of a complementary DNA library prepared from the human colon cancer-derived cell line HT-29 with a probe containing the tyrosine kinase domain of human c-src kinase led to the identification and isolation of a clone containing a receptor class tyrosine kinase. This putative receptor was found to be identical to the human fibroblast growth factor receptor 3 (FGFR3) except for a region of 150 nucleotides (50 amino acids) encoding the presumptive ligand-binding domain, where it exhibited only 32% homology with the previously described FGFR3. The variant domain corresponded precisely to the splicing junctions of the exon encoding the carboxyl terminal half of the third immunoglobulin-like domain, suggesting that two forms of FGFR3 result from splicing of alternate exons in a manner similar to that previously found for FGFR1 and FGFR2. By prior convention, the previously reported from of FGFR3 was designated IIIc due to its high degree of homology with the IIIc domain of FGFR1 (83% homology) and the IIIc domain of FGFR2 (81% homology). However, the ligand-binding domain of FGFR3 found in the HT-29 cell line was more highly divergent from all previously reported FGFR immunoglobulin-like domain IIIs than any other two members of this receptor family. Therefore, we propose to designate the newly reported form as the FGFR3 IIIb variant. Genomic polymerase chain reaction confirmed that the IIIb-containing exon occupies a position 5' relative to the IIIc-containing exon within the FGFR3 gene. Northern blot analysis using a probe encompassing sequences unique to the FGFR3 IIIb mRNA confirmed the expression of a 4.4-kilobase transcript in two colon cancer-derived cell lines as well as normal human colonic mucosa. Using a technique combining reverse transcriptase polymerase chain reaction with restriction endonuclease digestion, cell lines, primary cells, and tissues were assessed for IIIb and IIIc transcripts; expression of the IIIb variant was associated with an epithelial lineage, while the IIIc variant was expressed predominantly in nonepithelial cells and tissues.

Amino Acid Sequence↗
PubMedDate not supplied

[Cloning and sequencing the cDNA for the beta-subunit of Baikal omul gonadotropin].

Two types of cDNA encoding gonadotropin beta subunits (GTH beta) were isolated from a cDNA library prepared from pituitary gland of Baikal omul (Coregonus autumnalis migratorius Georgi). The nucleotide sequences of cDNA were determined. The CTHI beta and GTHII beta cDNAs code for polypeptides of 137 and 142 amino acids, respectively. Both of them include a putative signal peptide of 24 amino acids. The predicted amino acid structures of omul gonadotropins were compared with those of other vertebrate species.

Amino Acid Sequence↗