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PubMed1999

Molecular characterization of catalytic-subunit cDNA sequences encoding protein phosphatases 1 and 2A and study of their roles in the gibberellin-dependent Osamy-c expression in rice.

To understand the molecular mechanism of gibberellin-dependent gene regulation, the effect of three phosphatase inhibitors on the germination of rice seeds and the expression of a target gene, the alpha-amylase gene, Osamy-c, were measured. We found that okadaic acid, microcystin-LR, and calyculin A, which are known to specifically inhibit Ser/Thr phosphatases 1 and 2A, strongly inhibit the expression of the Osamy-c and may be involved in the germination of rice seeds. The protein phosphatase enzyme activity assays showed that there is no obvious effect of GA3 on total PP1/PP2A activities. To further understand the possible role of protein phosphatases 1 and 2A in the GA-dependent expression of Osamy-c, we isolated cDNA clones encoding protein phosphatase 1 and protein phosphatase 2A from a rice aleurone cDNA library. These were designated OsPP1c and OsPP2Ac, respectively. Comparison of the deduced amino acid sequences of OsPP1c and OsPP2Ac with the catalytic subunits of PP1 or PP2A of rabbit skeletal muscle, Arabidopsis thaliana, maize and Brassica napus showed that the catalytic subunit sequences of PP1 or PP2A among these organisms are highly conserved (73% to 90% similarity). Genomic Southern blot analysis indicated that there are only one or two copies of OsPP1c genes and more than two copies of OsPP2Ac genes in the rice genome. Northern blot analysis showed that OsPP1c and OsPP2Ac genes are expressed in several organs of rice, including seed, shoot and root. We also showed by using 3' gene-specific probes of OsPP1c and OsPP2Ac cDNA, that the expression of neither gene is regulated by GA. Taken together, our results suggest that protein phosphatases PP1 or PP2A are involved in the GA-dependent expression of the rice Osamy-c gene, though the PP1 or/and PP2A enzymatic activities as well as mRNA levels do not increase upon GA3 treatment.

Amino Acid Sequence↗
PubMed2002

[Molecular cloning and analysis of bovine prolactin full-long genomic as well as cDNA sequences].

In this report, a full-length sequence of bovine prolactin (bPRL) genomic DNA with 9388 bp, which has been accepted by GenBank (Accession Number: AF426315), was firstly cloned by Long PCR procedures. This sequence consists of 5 exons, 4 introns, 854 bp of 5' upstream regulatory region and 69 bp of 3' UTR. Accession number of protein encoded by AF426315 sequence in GenBank is AAL28075 that is composed of 229 amino acid residues, in which signal sequence resides in 1-30 sites and mature polypeptide consists of 199 amino acid residues. The recombinant plasmid containing bPRL genomic DNA was then transfected into eukaryotic cells (COS-7), followed by RT-PCR procedure. An 804 bp of bPRL cDNA containing all the encoding region was obtained, indicating that the bPRL genomic DNA reported herein had its biological function at the transcriptional level. Results derived from information searching by Blast program revealed that there were various SNP sites in the sequences of bPRL mRNAs and ESTs collected in GenBank, which located mainly in downstream encoding region and 3' UTR. These SNP sites did not alter the related amino acids encoded. In addition, mRNA sequences encoding 5' signal sequence of bPRL gene was highly conserve.

Animals↗
PubMed1989

Variable region cDNA sequences and antigen binding specificity of mouse monoclonal antibodies to isomaltosyl oligosaccharides coupled to proteins. T-dependent analogues of alpha(1----6)dextran.

Four mouse hybridomas specific for alpha(1----6)dextran, 16.4.12E (IgA kappa, C57BL/6), 28.4.10A (IgM kappa, BALB/c), 35.8.2H (IgG1 kappa, BALB/c), and 36.1.2D (IgM kappa, BALB/c) were obtained by immunization with the T-dependent Ag isomaltohexaose or isomaltotriose coupled to keyhole limpet hemocyanin or to BSA. Immunochemical characterization of the hybridoma antibodies showed that 16.4.12E and 36.1.2D had cavity-type combining sites, recognizing the terminal non-reducing end of alpha(1----6)dextran, whereas 28.4.10A and 35.8.2H had groove-type sites, recognizing internal linear segments of the dextran. The V region cDNA of the H and L chains of the antibodies were cloned and sequenced. VH of 16.4.12E and VH of 36.1.2D belonged to the X24 and Q52 germ-line gene families, respectively. The VH and V kappa sequences of 16.4.12E and V kappa sequence of 36.1.2D were highly homologous to those of W3129, the only anti-alpha(1----6)dextran mAb with a cavity-type site thus far sequenced; 16.4.12E differed from W3129 in the D, JH, and J kappa. VH genes of 28.4.10A and 35.8.2H were homologous to those of several anti-alpha(1----6)dextrans with groove-type sites, but belonged to the J558 germ-line gene family, differed from the other J558 anti-alpha(1----6)dextrans, probably representing a different germ-line subfamily. The L chain sequence of 28.4.10A encoded by V kappa-Ars and J kappa 2 was almost identical to other groove-type anti-alpha(1----6)dextrans obtained by immunizing with the T-independent glycolipid Ag, stearyl-isomaltotetraose. Use of T-dependent Ag such as isomaltosyl oligosaccharide-protein conjugates provides an additional parameter for probing the fine structure of antibody combining sites and evaluating the V-gene repertoire of anti-alpha(1----6)dextrans.

Amino Acid Sequence↗
PubMed1990

cDNA sequence of the human integrin beta 5 subunit.

A novel integrin receptor involved in cell adhesion to the matrix protein vitronectin has recently been described from a human lung epithelial-derived cell line (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69). This receptor has an alpha subunit that appears identical to the alpha v of the vitronectin receptor alpha v beta 3 expressed in melanoma and endothelial cells, but is complexed with a distinct beta subunit, beta 5. cDNA clones coding for beta 5 have been isolated and used to determine the mRNA and amino acid sequence of this new subunit. A 3.3-kilobase mRNA was found to code for a mature protein of 775 amino acid residues with a hydrophobic leader sequence of 24 amino acids. A 56% identity was found between the beta 5 and beta 3 protein sequences, making them the most closely related of the integrin beta subunits. Polymerase chain reaction abundance analysis revealed that alpha v and beta 5 mRNAs were found in seven very different cell lines, compared with beta 3 mRNA which was found in only three of the them, indicating that this new integrin receptor may be widely distributed.

Amino Acid Sequence↗
PubMed1983

Mouse sn-glycerol-3-phosphate dehydrogenase: molecular cloning and genetic mapping of a cDNA sequence.

The isozymes of glycerol-3-phosphate dehydrogenase (GPDH; sn-glycerol-3-phosphate:NAD+2-oxidoreductase, EC 1.1.1.8) in tissues of the mouse are coded for by two structural genes, Gdc-1 and Gdc-2, located on chromosomes 15 and 9, respectively. In order to investigate the expression of these genes, we isolated a GPDH cDNA clone from a mRNA preparation isolated from brown adipose tissue. The GPDH cDNA clone was identified by colony hybridization and hybrid selection of a mRNA that was translated in vitro to produce immunoprecipitable GPDH protein. In blot analysis, the GPDH cDNA hybridized to a single mRNA species that migrated at the position of 23S ribosomal RNA. This GPDH cDNA clone was mapped to the Gdc-1 locus by identification of a restriction enzyme polymorphism present in genomic DNA isolated from Gdc-1 congeneic lines of mice.

Adipose Tissue↗
PubMed1994

The bilin-binding protein of Pieris brassicae. cDNA sequence and regulation of expression reveal distinct features of this insect pigment protein.

The bilin-binding protein (BBP) is a blue pigment protein which is abundant in the butterfly Pieris brassicae. In an attempt to clarify the physiological role of this member of the lipocalin family of proteins, its complete cDNA was cloned and expression of the BBP gene in P. brassicae was investigated. It was found that synthesis of the BBP mRNA is highly regulated during the insect's ontogenesis. In larvae after the third ecdysis as well as in pupae and adults, large amounts of the mRNA are present. Each of these stages itself displays a distinct time course of mRNA synthesis. In addition, BBP is expressed tissue specifically, with the fat body being the major source of this secretory protein in the larvae. Hence, the expression pattern of BBP in this organism is markedly different from the closely related pigment protein insecticyanin in Manduca sexta. Finally, the bacterial expression of BBP in a functional state was established as a basis for the future analysis of its ligand-binding functions by protein engineering.

Amino Acid Sequence↗
PubMed1995

A novel cDNA sequence encoding the precursor of the D-amino acid-containing neuropeptide fulicin and multiple alpha-amidated neuropeptides from Achatina fulica.

Fulicin (Phe-D-Asn-Glu-Phe-Val-NH2) is an endogenous neuropeptide containing a D-amino acid from ganglia of the African giant snail Achatina fulica. We have cloned a novel cDNA (1,995 nucleotides) encoding a fulicin precursor from the snail cerebral and subesophageal ganglia. The fulicin precursor protein (357 amino acids) contains one copy of fulicin and at least nine other putative alpha-amidated neuropeptides composed of four to six amino acid residues. Seven of the nine neuropeptides were novel, and the other two had the same structures as Mytilus inhibitory peptide-related peptides previously isolated from the ganglia of Helix pomatia. All sequences of 10 peptides are flanked by Lys-Arg(Lys) at the N-terminus and by Gly-Lys-Arg(Lys) at the C-terminus. Nucleotide sequence analysis revealed that D-Asn present in fulicin is encoded by the usual L-Asn codon (AAT). Although fulicin has as yet only been isolated from the central ganglia. RNA blot analysis revealed that single transcripts of approximately 2.0 kb in size also exist in the ventricles and atria. These results suggest that fulicin and related peptides are produced in neurons and the heart by the processing of a ribosomally made precursor, although the mechanism of in-chain epimerization remains unclear.

Amino Acid Sequence↗
PubMed1987

cDNA sequence for human bcr, the gene that translocates to the abl oncogene in chronic myeloid leukaemia.

The hallmark of human chronic myeloid leukaemia is a 9;22 chromosome translocation that fuses most of the c-abl oncogene to the 5' portion of the breakpoint cluster region (bcr) gene, such that a hybrid bcr-abl mRNA and polypeptide are generated. To clarify further the nature of this translocation, we have analysed the structure of normal human bcr mRNA by isolating large cDNA clones that collectively span the entire coding region and extend 2.6 kb upstream of those previously described. The 3150-bp nucleotide sequence reported here includes 534 bp of a GC-rich 5' non-coding segment and indicates, in conjunction with published sequences, that the bcr polypeptide comprises 1271 amino acid residues. The predicted polypeptide is unrelated to serine or tyrosine kinases, or indeed to any previously published sequence; its structure provides no evidence of a transmembrane region. Since probes from throughout the 4.8-kb cloned region hybridized to both the 4.5 and 6.7 kb normal bcr transcripts, both RNAs contain most or all of that region.

Amino Acid Sequence↗
PubMed1998

Two diverged complement factor B/C2-like cDNA sequences from a teleost, the common carp (Cyprinus carpio).

Mammalian complement components factor B and C2 act as proteolytic subunits of the C3 convertases in the alternative and the classical activation pathways, respectively, and are believed to have diverged from a common ancestor by gene duplication. However, it is unclear when the B/C2 duplication occurred. Here, we describe two diverged B/C2-like cDNA clones (B/C2-A and B/C2-B) isolated from a bony fish, the common carp (Cyprinus carpio). B/C2-A shares the same domain structure as the factor B and C2 complement components of vertebrates reported so far and shows a close similarity to zebrafish B and medaka fish B/C2. These teleost sequences show almost the same degree of similarity to C2 and B of higher vertebrates. In contrast, B/C2-B has a novel structural feature in that it contains four short consensus repeat modules and does not have a close relative upon phylogenetic analysis. Northern blotting revealed the presence of two transcripts with different sizes for both the B/C2-A and B/C2-B in the hepatopancreas of the carp. Southern blotting suggested the presence of multiple genes for B/C2-A and a single gene for B/C2-B. Although structural features of B/C2-B are slightly more C2-like than B-like, B/C2-B has a crucial amino acid substitution in the serine protease domain, which makes it unlikely that B/C2-B functions as a C3 convertase. A possible phylogenetic relationship between the two carp sequences and mammalian C2 and B is discussed.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of cDNA sequences encoding two novel alpha-like-toxin precursors from the Chinese scorpion Buthus martensii Karsch.

According to a relative conserved fragment of alpha-scorpion toxins, a degenerate primer was designed and synthesized. Two full-length cDNAs encoding the precursors of two novel putative alpha-like-toxins were then amplified from the total cDNAs of venomous glands of the Chinese scorpion Buthusmartensi Karsch using 3' and 5' RACE (rapid amplification of cDNA ends). The precursors were both composed of 85 amino acid residues, including a putative signal peptide of 19 residues and a mature toxin of 66 residues, respectively. The predicted amino acid sequences of these two toxins show a homology of 82% with each other, and of 55-70% with other BmK-originated alpha-like-toxins. Interestingly, it is rarely seen in other alpha or alpha-like-toxins that: (1) Met residue but not a basic amino acid residue (Arg or Lys) is located on position 58 for BmKalpha2; (2) both toxins are ended with double Gly in the C-terminus.

Amino Acid Sequence↗
PubMed1991

The cDNA sequence and the deduced amino acid sequence of human transcobalamin II show homology with rat intrinsic factor and human transcobalamin I.

The cellular uptake of cobalamin (Cbl, vitamin B12) is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TCII and, therefore, served as the source of the complementary DNA (cDNA) library from which the TCII cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5'-untranslated segment of 37 nucleotides, and a 3'-untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R-IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene.

Amino Acid Sequence↗
PubMed1989

Molecular phenotypes in cultured maple syrup urine disease cells. Complete E1 alpha cDNA sequence and mRNA and subunit contents of the human branched chain alpha-keto acid dehydrogenase complex.

The activity of the branched-chain alpha-keto acid dehydrogenase complex is deficient in patients with the inherited maple syrup urine disease (MSUD). To elucidate the molecular basis of this metabolic disorder, we have isolated three overlapping cDNA clones encoding the E1 alpha subunit of the human enzyme complex. The composite human E1 alpha cDNA consists of 1783 base pairs encoding the entire human E1 alpha subunit of 400 amino acids with calculated Mr = 45,552. The human E1 alpha and the previously isolated human E2 cDNAs were used as probes in Northern blot analysis with cultured fibroblasts and lymphoblasts from seven unrelated MSUD patients. The results along with those of Western blotting have revealed five distinct molecular phenotypes according to mRNA and protein-subunit contents. These consist of type I, where the levels of E1 alpha mRNA and E1 alpha and E1 beta subunits are normal in cells, but E1 activity is deficient; Type II, where the E1 alpha mRNA is present in normal quantity, whereas the contents of E1 alpha and E1 beta subunits are reduced; Type III, where the level of E1 alpha mRNA is markedly reduced with a concomitant loss of E1 alpha and E1 beta subunits; Type IV, where the contents of both E2 mRNA and E2 subunits are markedly reduced; and Type V, where the E2 mRNA is normally expressed, but the E2 subunit is markedly reduced or completely absent. Type V includes thiamin-responsive (WG-34) and certain classical MSUD cells. These molecular phenotypes have demonstrated the complexity of MSUD and identified the affected gene in different patients for further characterization.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2001

Rat adrenoleukodystrophy-related (ALDR) gene: full-length cDNA sequence and new insight in expression.

X-linked adrenoleukodystrophy (X-ALD) is an inherited demyelinating disorder due to mutations in the ALD gene, which encodes a peroxisomal ABC half-transporter (ALDP). It has been suggested that ALDP assembles with ALDRP (adrenoleukodystrophy-related protein), a close homologous half-transporter, to form a functional heterodimer. For the first time full-length ALDRP cDNA (5.5 kb) was cloned, and 5' and 3' RACE analysis revealed that alternative usage of polyadenylation sites generates the two transcripts of 3.0 and 5.5 kb observed in the rat in Northern blot analysis. Southern blotting and chromosomal mapping demonstrated one ALDR locus in the rat genome. Characterisation of the 3' flanking region suggested that an ID sequence might be responsible for high expression of the 5.5 kb ALDRP transcript in rat brain. ALDR gene expression was found to be high in the liver of rats before weaning and very low in adult rats; the reverse developmental regulation was observed in the brain. Fenofibrate, which is a potent inducer of the ALDR gene in the liver of adult rats, could not induce the ALDR gene in suckling rats. The exact significance of this result with regard to development of an efficient pharmacological gene therapy for X-ALD is discussed.

3' Untranslated Regions↗
PubMed2002

Pituitary adenylate cyclase-activating polypeptide and growth hormone-releasing hormone-like peptide in sturgeon, whitefish, grayling, flounder and halibut: cDNA sequence, exon skipping and evolution.

To better understand the evolution of pituitary adenylate cyclase-activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH), we isolated the cDNAs encoding these peptides from the brains of five species of fish: sturgeon, whitefish, grayling, flounder and halibut. Both hormones are encoded in tandem in full-length cDNAs. We compared the phylogenetic relationship among these and other known sequences encoding PACAP. In closely related species, transcripts encoding PACAP and GHRH are strongly conserved in the hormone coding regions, moderately conserved in the signal peptide, cryptic peptide and 3'-untranslated regions, but are most varied in the 5'-untranslated regions.Next, we compared the deduced amino acid sequences for the peptides to known sequences. Sturgeon and whitefish have a PACAP(38) peptide sequence that is 92% conserved compared to human PACAP(38), the highest for a fish reported to date. GHRH is the lesser conserved of the two peptides with only 39% to 45% conservation between fish and human.Each of the five fish species had a second cDNA encoding a short precursor lacking GHRH(1-32), the bioactive portion of GHRH. This suggests that exon skipping in GHRH-PACAP transcripts may be an important mechanism for regulating the ratio of PACAP to GHRH peptides.

Alternative Splicing↗
PubMed1997

cDNA sequence and expression of a gene encoding a pyruvate dehydrogenase kinase homolog of Drosophila melanogaster.

Pyruvate dehydrogenase is a catalyst for an irreversible step in the degradation of glucose and its activity is regulated by a highly specific protein kinase, pyruvate dehydrogenase kinase (PDK). PDK belongs to a family of mitochondrial protein kinases unique from other eukaryotic protein kinases. We cloned a cDNA encoding a putative PDK from Drosophila melanogaster (DmPDK). The deduced DmPDK consists of 413 amino acids and shares up to 57.8% homology with human and rat PDK isoenzymes. Developmental Northern blot analysis revealed two major transcripts of 2.1 kb and 2.7 kb. The 2.7-kb transcript was expressed throughout ontogeny, whereas the 2.1-kb transcript was specific to embryos and adult females. Whole-mount in situ hybridization revealed that PDK mRNA is ubiquitously distributed in the embryo. The DmPdk gene was cytologically mapped to the 45CD region on the right arm of the second chromosome.

Amino Acid Sequence↗
PubMed1994

Molecular cloning, cDNA sequence, and chromosomal localization of the human phosphatidylinositol 3-kinase p110 alpha (PIK3CA) gene.

Phosphatidylinositol (PI) 3-kinase is a heterodimeric enzyme comprising a 110-kDa catalytic subunit and an 85-kDa regulatory subunit that binds to tyrosine phosphopeptide sites linked directly or indirectly to receptors serving diverse signal functions. Knowledge of the structure and function of PI 3-kinase was greatly advanced by the purification, cDNA cloning, and subsequent expression of the bovine enzyme. Here the cloning of the cDNA for the human p110 alpha subunit of PI 3-kinase (PIK3CA), encoding a protein 99% identical to the bovine p110, and of its gene in YAC is described. The chromosomal localization of the gene for PIK3CA is shown to be at 3q21-qter as determined using somatic cell hybrids. In situ hybridization performed using Alu-PCR from the YAC DNA located the gene in 3q26.3.

Animals↗
PubMed1995

Complete cDNA-sequence of the receptor responsible for arylphorin uptake by the larval fat body of the blowfly, Calliphora vicina.

In late larvae of the blowfly Calliphora vicina the storage protein arylphorin is selectively taken up by the fat body cells from the haemolymph. We have demonstrated that arylphorin incorporation is mediated by a specific membrane-bound receptor which is activated by 20-hydroxyecdysone. In this study we report the construction of a cDNA expression library from mRNA isolated from 8-day-old Calliphora larvae. Using affinity-purified antibodies against the arylphorin receptor we identified two cDNA clones of 4044 and 4043 bp, respectively. Both clones code for a 1253 amino acid protein with a deduced molecular mass of 142 kDa, the potential arylphorin receptor precursor, as confirmed by immunoprecipitation of translation products in vitro. Southern blot analysis indicated a single copy gene with multiple alleles; Northern blotting revealed a single mRNA of about 4.5 kb that appeared stage specifically from day 4 to day 8 during larval development. The deduced amino acid sequence exhibits significant similarities to arylphorin, the ligand, as well as to Fbp 1, the product of an ecdysteroid-inducible gene in Drosophila melanogaster larval fat body.

Amino Acid Sequence↗
PubMedDate not supplied

Expression and cDNA sequence of helix destabilizing protein (HDP) in rat lung.

The current study was undertaken to investigate genes that show alteration in expression during late fetal rat lung development. Using the differential display (DD) method, an initially unknown transcript was found to be abundant on day 18 compared to day 22 of gestation. This finding was confirmed by RNA blot analysis. Subsequent screening of a rat lung cDNA library with the unknown DD cDNA as a probe revealed a clone that encodes helix destabilizing protein (HDP), similar to that reported in rat brain (J Biol Chem. 1986, 261:3536-3543). Although there were several differences between lung and brain cDNAs at the 3' untranslated region (UTR), the 5' end was well conserved. There was a gradual decrease in HDP mRNA content beyond day 18 of gestation, as lung maturation increased. Similarly, a lower content of HDP mRNA was observed in maternal heart and brain tissues than in fetal heart and brain tissues from day 18 of gestation. Maternal dexamethasone treatment that promotes lung maturation did not have an impact on HDP expression. Because of the much higher abundance of HDP mRNA during fetal organ development compared to adult tissues, we speculate that HDP plays an important role during lung development. A portion of this work was previously presented (FASEB J. 1996;10:A26).

Animals↗