Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression.

The inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the "skeletal muscle" and "testis" proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKI alpha and PKI beta, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKI alpha species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)     

家兔BMP7基因的克隆及其生物信息学分析

在对已知部分编码序列(CDS)进行分析的基础上, 采用RT-PCR分步扩增以及RACE方法, 对家兔BMP7基因3′和5′末端未知序列进行了克隆与生物信息学分析。测序结果综合分析表明, 所获序列共计1 654 bp, 包括家兔BMP7近全长前肽、全长成熟肽CDS及3′非翻译序列(3′UTR), 将已有的序列向5′和3′端分别延伸了395 bp和628 bp。序列对比表明, 克隆的家兔BMP7 CDS部分与人、小鼠的对应序列的同源性分别为91.89%和89.32%, 预测的氨基酸序列同源性分别为96.51%和96.01%。家兔BMP7 3′UTR长446 bp, 与人、小鼠对应序列同源性分别为57.38%和45.57%; 具有2个转录终止信号位点。推测家兔BMP7成熟蛋白有BMPs特有的7个位置固定的半胱氨酸残基和TGF-β家族指纹。家兔BMP7 3′UTR区转录终止信号的可选择性可能与基因转录后调控有关。     

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

Molecular cloning of rat brain mitochondrial carrier protein-1 cDNA and its up-regulation during postnatal development

Brain mitochondrial carrier protein-1 (BMCP1), a new member of the mitochondrial uncoupling carrier, has been shown to be expressed predominantly in the brain of the mice and humans. We cloned rat BMCP1 cDNA and investigated its mRNA level during postnatal development and under various metabolic conditions. The nucleotide sequence of the cDNA revealed that rat BMCP1 protein was composed of 322 amino acid residues, and was 99 and 96% identical to the mouse and human proteins and 29, 33 and 35% identical to rat uncoupling protein (UCP) 1, UCP2 and UCP3, respectively. The molecular weight was predicted to be 36017 Da and the protein of this size was detectable when the cDNA was expressed in vitro. Using Northern blot analysis, the corresponding mRNA, approximately 1.8-kb in size, was found expressed predominantly in the cerebrum, cerebellum and hypothalamus. A unique developmental pattern was identified in the brain, where BMCP1 expression was low in their fetal life, but significantly elevated in the first postnatal week. Thereafter BMCP1 mRNA was maintained to be gradually increased. In 48-h fasted or insulin-induced hypoglycemic rats, BMCP1 mRNA expression in the hypothalamus slightly, but significantly, decreased compared with that in their appropriate controls. The present results indicate that BMCP1 may be involved in pathogenesis of mitochondrial dysfunction in neurons induced by aging or neurodegenerative disorders, and perhaps in energy balance in the brain.     

A second catalytic subunit of type-2A protein phosphatase from rabbit skeletal muscle

A cDNA clone encoding a second type-2A protein phosphatase catalytic subunit (2A beta) was isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The deduced protein sequence (309 residues, 35.59 kDa) was 97% identical to that of phosphatase 2A alpha (309 residues, 35.58 kDa). At the nucleotide level, the two clones showed only 82% identity in the coding region. The results indicate the presence of at least two isoforms of protein phosphatase 2A in skeletal muscle.     

Uncoupling protein 2 from carp and zebrafish, ectothermic vertebrates.

Uncoupling protein 1 (UCP1) is of demonstrated importance in mammalian thermogenesis, and early hypotheses regarding the functions of the newly discovered UCP homologues, UCP2, UCP3 and others, have focused largely on their potential roles in thermogenesis. Here we report the amino acid sequences of two new UCPs from ectothermic vertebrates. UCPs from two fish species, the zebrafish (Danio rerio) and carp (Cyprinus carpio), were identified in expressed sequence tag databases at the European Molecular Biology Laboratory. cDNAs from a C. carpio 'peritoneal exudate cell' cDNA library and from a D. rerio 'day 0 fin regeneration' cDNA library were obtained and fully sequenced. Each cDNA encodes a 310 amino acid protein with an average 82% sequence identity to mammalian UCP2s. The fish UCP2s are about 70% identical to mammalian UCP3s, and 60% identical to mammalian UCP1s. Carp and zebrafish are ectotherms--they do not raise their body temperatures above ambient by producing excess heat. The presence of UCP2 in these fish thus suggests the protein may have function(s) not related to thermogenesis.     

Cloning and sequencing of a full-length rat sucrase-isomaltase-encoding

Sucrase-isomaltase (SI) has been widely used as a marker enzyme to study cellular differentiation in the small intestine. We isolated a 6.1-kb SI cDNA clone (GC1.4) from a size-fractionated cDNA library from rat intestine. Sequencing of this cDNA clone showed 6066 nucleotides (nt) with an open reading frame (ORF) of 1841 amino acids (aa). The nt sequence correctly predicts several known aa stretches in the protein. The deduced aa sequence showed 78 and 75% overall identity with the rabbit and human SI, respectively. At the active sites of both S and I, the rat nt sequence encodes stretches of 14 and 16 aa, respectively, which show 100% identity to rabbit and human SI. In the region immediately beyond the transmembrane domain, the rat sequence encodes an extra 10 aa, as compared to rabbit and human. This 10-aa insertion consists almost entirely of Pro, Ser and Thr, and may be responsible for additional 0-glycosylations of rat SI. The cDNA contains a 3'-UTR (untranslated region) of 499 nt with polyadenylation signal sequence and a poly(A) tract. The ATG start codon was found 41 nt downstream from the 5' end of the cDNA. Primer extension experiments showed the cap site to be 61 nt upstream from the start codon. The results indicate that our cDNA clone lacks only 20 nt in the 5'-UTR. Given that this cDNA encodes the entire coding region of SI, it should be useful in elucidating the regulatory mechanisms of SI biosynthesis, localization and targeting during rat intestinal development and differentiation.     

Cloning and sequencing of a rat type II activin receptor.

A full-length cDNA for a rat type II activin receptor was cloned by hybridization from a rat ovary cDNA library. The deduced amino acid sequence (513 residues) containing a single membrane-spanning domain and an intracellular kinase domain with predicted serine/threonine specificity. The amino acid sequence is 99.8% and 99.4% identical in the coding region with the previously cloned mouse and human type II activin receptor, and only 66.7% identical in the coding region with the previously cloned rat type IIB activin receptor. We examined the effect of PMSG-hCG on the mRNA level of type II activin receptor in immature rat ovaries. Northern blot analysis of ovarian RNA revealed two mRNAs (3.0 kb and 6.0 kb).     

Molecular cloning and nucleotide sequence of cDNA encoding the human liver S-adenosylmethionine synthetase.

cDNA clone for human liver S-adenosylmethionine synthetase (liver-specific isoenzyme) was isolated from a cDNA library of human liver poly(A)+ RNA. The cDNA sequence encoded a polypeptide consisting of 395 amino acid residues with a calculated molecular mass of 43675 Da. Alignment of the predicted amino acid sequence of this protein with that of rat liver S-adenosylmethionine synthetase showed a high degree of similarity. The coding region of the human liver S-adenosylmethionine synthetase cDNA sequence was 89% identical at the nucleotide level and 95% identical at the amino acid level to the sequence for rat liver S-adenosylmethionine synthetase.     

Human debrisoquine 4-hydroxylase (P450IID1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22

The enzyme P450db1 (db1) is responsible for the common human defect in drug oxidation known as the "debrisoquine/sparteine polymorphism." Polyclonal antibody against the rat db1 protein was used to screen a human liver lambda gt11 library for the db1 cDNA clone. A cDNA containing the full protein coding sequence was isolated; the deduced NH2-terminal sequence of this cDNA was identical to that derived from direct sequencing of the purified human db1 protein. Comparison of the human db1 with rat db1 revealed 71 and 73% similarities of nucleotides and amino acids, respectively. By use of human-rodent somatic cell hybrids the db1 gene was localized to human chromosome 22 (CYP2D locus).     

Functional expression of the rat brown adipose tissue uncoupling protein in Saccharomyces cerevisiae

The uncoupling protein (UCP) from mammalian brown adipose tissue is an integral component of the mitochondrial inner membrane where it dissipates the proton electrochemical gradient. UCP is transported into mitochondria from the cytosol but lacks a cleavable targeting peptide. We have expressed the rat UCP in Saccharomyces cerevisiae and shown that this protein, which is not normally found in yeast, is targeted to the mitochondria where it disrupts mitochondrial function, probably by uncoupling oxidative phosphorylation. The observed growth defect is dependent upon the level of expression of UCP. When the unmodified UCP cDNA is expressed in yeast under the control of the GAL10 promoter no defect in growth is observed. We have inserted the UCP coding sequence behind the strong phosphoglycerate kinase promoter under the control of the GAL1-10 upstream activation site and introduced a yeast consensus sequence (ATAATG) at the translation start site. We have found that UCP expressed in S. cerevisiae is targeted to mitochondria and that its expression induces a marked growth defect on non-fermentable carbon sources in a manner dependent on induction with galactose.     

Brown adipose tissue in lean and fat selection lines of sheep identified by immunodetection of uncoupling protein in western blots of tissue homogenates

1. Uncoupling protein (UCP) was purified from perirenal adipose tissue of 2-day-old lambs by a procedure involving Triton solubilization and hydroxyapatite treatment. It has an apparent Mr of 34,000. 2. Rabbit anti-sheep UCP and rabbit anti-rat UCP each cross-reacted with both rat and sheep UCP in Western blots, indicating that the major antigenic determinants of the sheep UCP and rat UCP are similar. 3. In Western blots, the anti-sheep UCP showed tissue specificity by detecting a band corresponding to UCP only in brown adipose tissue, but not in heart or liver homogenates. 4. The Western blotting procedure was used to analyse sheep tissues. UCP was detected in samples of perirenal, omental, back and lymph node fat from 2-day-old lambs, but not in heart, liver, muscle or kidney samples. 5. UCP was not detected in any tissue samples from 34-day-old or 7-month-old lambs. 6. Comparison of the amount of UCP in perirenal fat of 2-day-old lambs from lean, fat and control selection lines, using the Western blotting procedure, showed no apparent difference.     

Molecular identification of uncoupling proteins (UCP2 and UCP3) and absence of UCP1 in the marsupial Tasmanian bettong,Bettongia gaimardi

This study has identified the expression of uncoupling proteins in a marsupial using molecular techniques. The Tasmanian bettong, Bettongia gaimardi, increases non-shivering thermogenesis (NST) in response to cold exposure and norepinephrine, although previous studies have been unable to demonstrate the presence of brown adipose tissue or uncoupling protein 1 (UCP1). This study used molecular techniques to confirm the absence of UCP1 as well as ascertain if this species expresses UCP2 and/or UCP3. Tissue samples from four B. gaimardi were taken prior to and post-cold exposure at 4-5 degrees C for 2 weeks. The tissues were then examined for UCP1, UCP2 and UCP3 expression using Western blotting. UCP2 and UCP3 were amplified through RT-PCR and subsequently sequenced to confirm molecular identity. Our work confirms that B. gaimardi does not express UCP1 and that this species expresses both uncoupling proteins 2 and 3. The sequencing of the amplified B. gaimardi UCP2 and UCP3 cDNAs have revealed a 74% homology with rat UCP2 cDNA, and 65% homology with rat UCP3 cDNA. Although this work has not yet characterised the functional properties of these proteins in the marsupial, it does suggest a possible mechanism to explain the existence of NST in B. gaimardi.     

cDNA encoding type B subunit of rat phosphoglycerate mutase: its isolation and nucleotide sequence.

A cDNA encoding the nonmuscle-specific (type B) subunit of phosphoglycerate mutase (PGAM-B) was isolated and characterized. A cDNA probe, synthesized by the polymerase chain reaction (PCR) from rat liver cell mRNA using mixed primers specific to the amino acid sequence of human PGAM-B, was used to screen a rat liver cell cDNA library. The identity of the cDNA was confirmed by amino acid sequence data for 24 peptides obtained by digesting the purified protein with three different endopeptidases. The coding region encoded a polypeptide composed of 253 amino acid (plus the initiator Met). RNA blot analysis showed a single mRNA species of 1.7 kilobases in rat liver cell. The deduced amino acid sequence of rat PGAM-B was identical to that of human PGAM-B except for only one substitution at position 251 near the carboxyl terminus (valine for the rat and alanine for the human).