Cloning of mRNA nucleotide sequences amplified during DNA replication in the regenerating liver

A library of double-stranded cDNA has been constructed using the mRNA of regenerating rat liver 20 hr after partial hepatectomy. The differential screening of the library with the regenerating liver specific and the resting-liver-specific single-stranded cDNA probes has identified 11 cDNA clones which sequences are preferentially expressed in regenerating rat liver. The RNA dot blot hybridization has shown that levels of RNA complementary to these clones are 3 to 8-fold higher in dividing cells as compared with resting cells.     

Identification and characteristics of mRNA's showing increased levels during DNA replication

A library of double-stranded cDNA was prepared using poly (A) + RNA from regenerating rat liver 20 h after partial hepatectomy. Differential screening of 350 recombinant clones with cDNA-G0 and cDNA-S identified eleven cDNA clones (pRL), the sequences of which were preferentially expressed during the DNA replication period. Levels of mRNAs complementary to these clones were 2--10-fold higher in the S-period, than in G0. Using plasmid cDNAs to different mRNA, pRL we have investigated the changes in the levels of mRNA pRL during liver regeneration. The level of mRNA mRL2 and pRL79 was increased just before DNA replication. mRNA pRL35 accumulates after partial hepatectomy with the maximum at 6 h. The augment of two other mRNA concentrations was expressed to a lesser extent. Northern-blot analysis allowed to determine the individual dual mRNAs corresponding to each of the three clones with their sizes ranging from about 1650 to 3900 bases. Three mRNAs (pRL35, 67 and 79) were shown (by hybrid-selected translation) to code for proteins of about 100, 140 and 120 kDa, respectively.     

Cloning and expression in Escherichia coli of cDNA for serine: pyruvate aminotransferase of rat liver

Cloned cDNAs for rat liver serine: pyruvate aminotransferase were obtained by screening of a cDNA expression bank of rat liver with an antibody against the enzyme. Nineteen clones were isolated from 33 000 transformants and most of them had common fragments of cDNA on analysis by digestion with some restriction enzymes. These clones were identified as those containing cDNA for serine:pyruvate aminotransferase by the following criteria. (a) At the nucleic acid level, a 500-base-pair fragment of cDNA prepared by digestion of cDNAs with EcoRI and PstI hybridized with the mRNA coding for serine:pyruvate aminotransferase as judged by hybrid-selected and hybrid-arrested translations. (b) Specific proteins were detected in nine bacterial clones, a 40-kDa protein in one clone and a 39-kDa protein in eight clones. Among them only the 40-kDa protein was found to be solubilized from the cell by sonication, and this protein was immunoprecipitated with an antibody against serine:pyruvate aminotransferase of rat liver. (c) High activity of serine:pyruvate aminotransferase was expressed both in whole cell suspension and sonicated extract prepared from the transformant producing the 40-kDa protein, and 99% of the activity was immunoreactive with the antibody. Two types of mRNA for serine:pyruvate aminotransferase were detected on the RNA blot analysis by using cloned cDNA fragment as a probe. The larger mRNA (approximately 1600 nucleotides) was glucagon-inducible while the smaller one (approximately 1500 nucleotides) was not affected by the hormone.     

应用抑制性消减杂交技术克隆大鼠肝再生过程中特异表达基因

应用抑制性消减杂交技术成功地构建了高消减效率的正向消减cDNA文库,从随机挑取的50个克隆中有31个均检出了60~400bp插入片段,对这些插入cDNA片段进行测序后经Genbank同源性检索,表明其中7个片段为未知新序列。大鼠肝切除后肝再生cDNA正向消减文库的建立为进一步大批量筛选、克隆肝再生特异性表达的未知新基因奠定了基础,初步筛选出的特异性表达的序列标记为进一步研究肝再生中基因的功能提供了依据。 Abstract:The cDNA from rat regenerating liver tissue was used as the tester and that from normal liver was used as the driver.A highly efficient subtractive cDNA library was constructed by suppression subtractive hybridization(SSH).After screening,31 clones from 50 clones which were derived from the cDNA library were inserted by 60~400bp cDNA fragments.24 cDNA fragments corresponded to known genes and 7 cDNA fragments were unknown sequences(GenBank accession number:BG447490~447496).     

Characterisation of cDNA clones for rat muscle carbonic anhydrase III

cDNA clones for rat muscle carbonic anhydrase III have been isolated from a gt-11 library and sequenced. Comparison with human CAIII cDNA showed about 90% homology to rat. The rat clones were used to estimate mRNA from liver and muscle on Northern blots and showed that the sexual dimorphism of CAIII in rat liver relates to a difference in mRNA levels.     

Molecular cloning of cDNA for rat mitochondrial 3-oxoacyl-CoA thiolase

Messenger RNA of rat 3-oxoacyl-CoA thiolase (acetyl-CoA acyltransferase), a mitochondrial matrix enzyme involved in fatty acid beta-oxidation, was enriched by immunoprecipitation of rat liver free polysomes and recombinant plasmids were prepared from the enriched mRNA by a modification of the vector-primer method of Okayama and Berg. The transformants were initially screened for 3-oxoacyl-CoA thiolase cDNA sequences by differential colony hybridization with [32P]cDNAs, synthesized from the immunopurified and unpurified mRNAs. The cDNA clones for 3-oxoacyl-CoA thiolase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pT1-1, contained a 700-base insert and hybridized to a mRNA species of 1.6 X 10(3) bases in rat liver. The transformants were rescreened using the cDNA insert of pT1-1 as a hybridization probe and a clone (pT1-19) with a 1.5 X 10(3)-base insert was obtained. Activity and concentration of 3-oxoacyl-CoA thiolase mRNA were quantified by in vitro translation and dot-blot analysis using the cDNA insert as a hybridization probe. The level of translatable and hybridizable mRNA in rat liver was increased about 5.1-fold and 4.6-fold, respectively, after administration of di-(2-ethylhexyl)phthalate, a potent inducer of the enzyme. The 3-oxoacyl-CoA thiolase mRNA levels thus determined correlated closely with levels of the activity and amount of this enzyme.     

Cloning and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.

Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.     

Isolation and characterization of a novel liver-specific gene, hepassocin, upregulated during liver regeneration

By differential cDNA cloning coupled with Xenopus oocyte expression screening, we isolated a cDNA encoding a novel protein, termed 'hepassocin', the expression of which is upregulated in the regenerating rat liver. The cDNA contained a single open reading frame encoding a protein of 314 amino acids (ca. 34 kDa), including 24 amino acids of signal sequence. The protein expressed from the cDNA in Verots cells had activity to stimulate DNA synthesis in primary rat hepatocytes and was of 66 kDa or 34 kDa, under non-reducing or reducing conditions, respectively. Using an affinity column conjugated with the antibody raised against a peptide in a hydrophilic region, we purified hepassocin from the rat liver: it had a DNA synthesis-stimulating activity in hepatocytes. The hepassocin obtained here was 66 kDa, and the 34 kDa protein obtained under reducing conditions contained five cysteine residues, indicating that hepassocin is active as a homodimer. Northern blot analysis revealed that hepassocin mRNA (1.4 kb in length) occurred only in the liver, and in situ hybridization studies revealed its presence in parenchymal hepatocytes but not in endothelial cells. Furthermore, the expression of hepassocin mRNA was upregulated during compensatory hyperplasia after partial hepatectomy and regeneration after galactosamine treatment in the rat liver. These results suggest that hepassocin plays an important role in stimulating liver cell growth, through an autocrine mechanism.     

The 3-methylcholanthrene-inducible UDP-glucuronosyltransferase deficiency in the hyperbilirubinemic rat (Gunn rat) is caused by a -1 frameshift mutation

The Gunn rat is a mutant strain of Wistar rat which has unconjugated hyperbilirubinemia as a result of the absence of hepatic UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. The Gunn rat is also deficient in a 3-methylcholanthrene (MC)-inducible UDPGT isoenzyme that has high activity toward phenolic substrates. We have isolated and sequenced a cDNA, designated 4-NP UDPGT, which encodes an MC-inducible UDPGT from normal Wistar rat livers (Iyanagi, T., Haniu, M., Sogawa, F., Fujii-Kuriyama, Y., Watanabe, S., Shively, J.E., and Anan, K.F. (1986) J. Biol. Chem. 261, 15607-15614). In the present study, we found that this cDNA detected MC-inducible UDPGT mRNA in the MC-treated homozygous Gunn rat liver. The level of this mRNA, however, was significantly lower than that of normal Wistar livers. The size of mRNA in Gunn rats was identical to that of the functionally mature UDPGT mRNA in Wistar rats, but the MC-inducible UDPGT protein was absent from homozygous Gunn rat microsomes. We therefore made a cDNA library from MC-treated Gunn rat liver mRNA and isolated cDNA clones, using the 4-NP UDPGT cDNA as a probe. Sequencing analysis of these cDNA clones revealed a single base deletion in the coding region. Northern blot analysis of mRNAs from normal Wistar and heterozygous and homozygous Gunn rats livers was performed using specific oligonucleotide probes, and the results confirmed the presence of mRNA containing the single base deletion in heterozygous and homozygous Gunn rats. These data suggested that the defect of the MC-inducible isoenzyme in Gunn rats arises from a -1 frameshift mutation that removes 115 amino acids from the COOH terminus.     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.     

Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant parac     

Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.     

Molecular cloning of cDNA for rat and human carbamyl phosphate synthetase I

Recombinant plasmids with inserts complementary to the mRNA for carbamyl phosphate synthetase I were identified from a rat liver cDNA library by hybrid-selected mRNA translation. Four clones, the largest being 3100 base pairs, were identified for the rat liver enzyme. Using the rat liver cDNA as a probe, two homologous recombinant plasmids of approximately 1200 base pairs in length were isolated from a human liver cDNA library. Northern blot analysis of rat liver mRNA and baboon liver mRNA revealed the presence of a 5000-base mRNA homologous to both rat and human cDNA probes. No homologous mRNA was observed in mRNA from rat heart or rat kidney as is consistent with the known tissue distribution of this enzyme. The induction of carbamyl phosphate synthetase and argininosuccinate synthetase mRNA during the fetal and postnatal development of the rat was studied by dot blot analysis of isolated mRNA. The mRNA for both enzymes appeared between 17 and 19 days of fetal life and reached approximately 40% of adult levels during this period. This initial increase was followed by a rapid decline just prior to birth. The mRNA levels slowly increased during postnatal life, not reaching adult levels until after the 20th day of neonatal life. Using the human cDNA clones, the human carbamyl phosphate synthetase gene was mapped to chromosome 2 utilizing a panel of Chinese hamster X human somatic cell hybrids. Analysis of one hybrid with a human-Chinese hamster translocation provided a provisional assignment to the short arm of chromosome 2.     

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.