Molecular cloning and the complete nucleotide sequence of cDNA to mRNA for S-100 protein of rat brain.

The complete nucleotide sequence of mRNA for beta-subunit of rat brain S-100 protein was determined from recombinant cDNA clones. The sequence was composed of 1488 bp which included the 276 bp of the complete coding region, the 120 bp of the 5'-noncoding region and the 1092 bp of the 3'-noncoding region containing two polyadenylation signals. In addition, the poly(A) tail was also found. The amino acid sequence deduced from the nucleotide sequence was homologous to the amino acid sequence of bovine S-100 beta subunit except 4 residues showing species differences. From the viewpoint of evolutionary implications, the homology between the nucleotide sequence of S-100 and those of rat intestinal Ca-binding protein (ICaBP) and calmodulin (CaM) was examined. A dot-blot hybridization of poly(A) RNA from the developing rat brains using a labeled cDNA showed a rapid increase in S-100 mRNA at 10-20 postnatal days. The presence of S-100 mRNA in C-6 glioma cells is also described.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

GTP cyclohydrolase I from Tetrahymena pyriformis: cloning of cDNA and expression

A full-length cDNA clone for GTP cyclohydrolase I (EC 3.5.4.16) was isolated from a Tetrahymena pyriformis cDNA library by plaque hybridization. The nucleotide sequence determination revealed that the length of the cDNA insert was 1516 bp. The coding region encoded a protein of 223 amino acid residues with a calculated molecular mass of 25 416 Da. The deduced amino acid sequence of Tetrahrymena GTP cyclohydrolase I showed sequence identity with that of Escherichia coli (55%). The identity of T. pyriformis GTP cyclohydrolase I with sequences of Dictyostelium discoideum, Saccharomyces cerevisiae, Drosophila melanogaster, mouse, rat, and human enzymes was less marked and was 30, 30, 25, 28, 28, and 27%, respectively. RNA blot analysis showed a single mRNA species of 2.1 kb in this protozoan. The mRNA level of GTP cyclohydrolase I increased during synchronous cell division induced by intermittent heat treatment. The results suggest that the mRNA expression is associated with the cell cycle of T. pyriformis.     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.     

Molecular cloning and the nucleotide sequence of cDNA to mRNA for non-neuronal enolase (alpha alpha enolase) of rat brain and liver.

The nucleotide sequence for alpha alpha enolase (non-neuronal enolase: NNE) of rat brain and liver was determined from recombinant cDNA clones. The sequence was composed of 1722 bp which included the 1299 bp of the complete coding region, the 108 bp of the 5'-noncoding region and the 312 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, the poly(A) tail was also found. A potential ribosome-binding site was located 30 nucleotides upstream to the initiation codon in the 5'-noncoding region. The amino acid sequence deduced from the nucleotide sequence was 433 amino acids in length and showed very high homology (82%) to the amino acid sequence of gamma gamma enolase (neuron-specific enolase: NSE), although the nucleotide sequence showed slightly lower homology (75%). The size of NNE mRNA was approximately 1800 bases by Northern transfer analysis and much shorter than that of NSE mRNA (2400 bases) indicating a short 3'-noncoding region. A dot-blot hybridization and Northern transfer analysis of cytoplasmic RNA from the developing rat brains using a labeled 3'-noncoding region of cDNA (no homology between NSE and NNE) showed a decrease of NNE mRNA at around 10 postnatal days and then a gradual increase to adult age without changes of mRNA size. Liver mRNA did not show any significant change during development.     

Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.     

Nucleotide sequence of mouse L19 ribosomal protein cDNA isolated in screening with tre oncogene probes.

A cDNA library was prepared from cytoplasmic poly(A)RNA from mouse NIH-3T3 cells carrying a transfected human tre oncogene. Screening with tre gene probes identified a tre cDNA clone 11-4 and a co-purifying weakly hybridizing cDNA clone 11-5. The 11-5-specific RNA was expressed in both nontransfected and tre-transfected NIH-3T3 cells, showing it is of mouse rather than tre gene origin. Its nucleotide sequence was 717 bp long and contained, starting from the first nucleotide, an open reading frame of 588 bp followed by a 3' noncoding region and 26 A residues at the 3' terminus. Comparison with the GenBank data base revealed 93.7% homology with cDNA encoding the rat L19 ribosomal protein. Furthermore, the 196-amino-acid polypeptide deduced from 11-5 was of the same length and contained only one amino acid difference compared with the rat L19 protein. Comparison with the weakly hybridizing tre gene probe showed stretches of homology that were, however, too short to be taken into consideration. We conclude that the 11-5 sequence encodes the mouse L19 ribosomal protein.     

眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因的克隆及分析

从广西产眼镜王蛇（Ophiophagus hannah）毒腺中抽提总RNA,经mRNA纯化后构建眼镜王蛇毒腺cDNA文库。从所构建的cDNA文库中,随机筛选200个克隆测序,得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBank登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp,后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体;后者编码115个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明,泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白,C末端含有一个“锌指”模式结构。与16个物种比较的结果表明,眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性。     

Molecular cloning of the cDNA for two major androgen-dependent secretory proteins of 18.5 kilodaltons synthesized by the rat epididymis

cDNA clones representing two closely related androgen-dependent secretory proteins of 18.5 kDa were selected by screening a rat epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the 18.5-kDa proteins. The entire amino acid sequence of the 18.5-kDa secretory proteins and a putative signal sequence of 18 amino acids was derived from 682 base pairs of the nucleotide sequence of overlapping cDNA clones. Confirmation of the identity of the cDNA clones was obtained by matching a partial amino acid sequence obtained for the N terminus of the pure protein with that of the sequence derived from the nucleotide code of the cDNA. Evidence is presented that the difference between the two closely related proteins may be associated with differential post-translational modification of the N terminus of the protein following cleavage of the signal sequence. Northern blot analysis revealed that the mRNA for the proteins is approximately 850 nucleotides long and that the concentration of the mRNA in the tissue is androgen-dependent. The proteins and their mRNAs were restricted to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from the skin, brain, liver, kidney, heart, skeletal muscle, and testis. With the exception of a weak cross-reaction with mouse epididymis, the proteins were not detected by Western blots of extracts of guinea pig, rabbit, or bull epididymis. The two proteins account for a substantial proportion of the total protein in epididymal luminal fluid and become incorporated as components of the sperm plasma membrane where they may play a specific role in the post-testicular phase of sperm development.     

Primary structure of human preangiotensinogen deduced from the cloned cDNA sequence

Cloned cDNA sequences for human preangiotensinogen have been isolated from a human liver cDNA library by hybridization with a restriction fragment derived from a previously cloned cDNA for rat preangiotensinogen. Analyses by nucleotide sequence determination, S1 nuclease mapping, and RNA blot hybridization indicate that human preangiotensinogen is encoded by two mRNAs that differ only in the length of the 3'-untranslated region. The deduced amino acid sequence shows that the mature angiotensinogen consists of 452 amino acid residues with the angiotensin sequence at its amino-terminal portion. Two potential initiation sites have been discussed. These are the methionine codon located at the position exactly corresponding to the initiation site of rat preangiotensinogen mRNA and an additional methionine codon positioned nearest the 5' end of the mRNA. The amino acid sequences starting at either of the initiation sites and preceding the angiotensin sequence constitute a large number of hydrophobic amino acid residues, thus representing the signal peptide characteristic of the secretory proteins. Human and rat preangiotensinogens show that 63.6% of the amino acid positions of the two proteins are identical. However, the amino-terminal portions directly distal to angiotensin I diverge markedly between the two proteins and differ in their possible glycosylation sites. These structural differences may contribute to the known species specificity exhibited by renin.     

Primary structure of rat liver alkaline phosphatase deduced from its cDNA.

Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver.     

Cloned proteolipid protein and myelin basic protein cDNA. Transcription of the two genes during myelination

cDNA clones of rat brain proteolipid protein (PLP), also named lipophilin, the major integral myelin membrane protein, and of myelin basic protein (MBP), the major extrinsic myelin protein, have been isolated from a rat brain cDNA library cloned into the PstI site of pBR322. Poly(A)+ RNA from actively myelinating 18-day-old rats has been reversely transcribed. Oligonucleotides synthesized according to the established amino-acid sequence of lipophilin and the nucleotide sequence of the small myelin basic protein of the N-terminal, the central and C-terminal region of their sequences were used as hybridization probes for screening. The largest insert in one of several lipophilin clones was 2,585 base pairs (bp) in length (pLp 1). It contained 521 bp of the C-terminal coding sequence and the complete 2,064 bp long non-coding 3' sequence. The myelin basic protein cDNA insert of clones pMBP5 and pMBP6 is 2,530 bp long and that of clones pMBP2 and pMBP3 640 bp. These clones were also characterized. pMBP2 was sequenced and used together with the lipophilin cDNA clones as hybridization probes to estimate the lipophilin and myelin basic protein mRNA levels of rat brain during the myelination period. The expression of the lipophilin and myelin basic protein genes during development of the myelin sheath appears to be strictly coordinated.     

Molecular cloning and sequence analysis of the (Na+ + K+)-ATPase beta subunit from dog kidney

cDNA complementary to mRNA coding for the beta subunit of dog renal (Na+ + K+)-ATPase has been cloned into lambda gt11 and the nucleotide sequence of the DNA has been determined. The amino acid sequence of the beta subunit polypeptide has also been deduced from the DNA. The mature form of the dog kidney beta subunit contains 302 amino acids with three potential asparagine-linked attachment sites for carbohydrate. The initiation methionine is removed during processing of the polypeptide to its mature form. Although the beta subunit is an integral membrane protein there is no signal sequence for the polypeptide, and hydropathy analysis predicts that the beta subunit polypeptide spans the cell membrane only once. Secondary structure predictions and a model for the structure of the beta subunit are proposed. DNA sequencing of the 5' non-coding region of the mRNA revealed a 200 bp inverted repeat from the coding region. Blot hybridization of a fragment of the beta subunit cDNA identified a single mRNA species of 2.7 kb in dog kidney and several rat tissues. RNA from rat liver was deficient in mRNA that hybridized to the dog kidney beta subunit cDNA, although mRNA that hybridized to an alpha subunit cDNA was detected. RNA from a human hepatoma cell line, HepG2, however, contained comparable levels of mRNA for both the alpha and the beta subunits.     

Molecular Cloning of cDNA to mRNA for a Cerebellar Spot 35 Protein

The nucleotide sequence of mRNA for rat cerebellar spot 35 protein, a Ca-binding protein, was determined from recombinant complementary DNA (cDNA) clones. The sequence was composed of 1,714 base pairs (bp) which included the 783 bp of the complete coding region, the 130 bp of the 5'-noncoding region, and the 801 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, a polyadenylic acid [poly(A)] tail was also found. Because the size of spot 35 mRNA was estimated to be about 1,900 bases by Northern blot analysis, the longest insert was verified to contain a nearly full-length cDNA sequence including the poly(A) tail. The amino acid sequence of the protein deduced from the nucleotide sequence contains 261 amino acids and at least five Ca-binding domains. There was a high homology in the amino acid sequences (79%) and the nucleotide sequences (77%) between spot 35 protein and chick intestinal Ca-binding protein (28K).     

Structure of rodent helix-destabilizing protein revealed by cDNA cloning

A cDNA library of newborn rat brain poly(A+) RNA in lambda gt 11 was screened with a synthetic oligonucleotide probe corresponding to a five amino acid sequence in the N-terminal region of the calf helix-destabilizing protein, UP1. Six positive phage were isolated after testing 2 X 10(5) recombinants, and each phage was plaque purified. Four of these phage clones were positive with a second oligonucleotide probe corresponding to a 5 amino acid sequence in the C-terminal region of calf UP1; one of the clones positive with both probes was selected for detailed study. This phage, designated lambda HDP-182, contained a 1706-base pair cDNA insert corresponding to an mRNA with a poly(A) sequence at the 3' terminus and a single open reading frame starting 63 bases from the 5' terminus and extending 988 bases. The 3' untranslated region of the mRNA contained 718 bases, including an AAUAAA signal 21 bases from the poly(A) sequence and a 16-residue poly(U) sequence flanked on each side by oligonucleotide repeats. Primer extension analysis of newborn rat brain poly(A+) RNA suggested that the cDNA insert in lambda HDP-182 was full length except for about 35 nucleotide residues missing from the 5' end untranslated region, and Northern blot analysis revealed one relatively abundant mRNA species of approximately the same size as the cDNA insert. The 988-residue open reading frame in the cDNA predicted a 34,215-dalton protein of 320 amino acids. Residues 2 through 196 of this rat protein are identical to the 195-residue sequence of the calf helix-destabilizing protein, UP1. The 124-amino acid sequence in the C-terminal portion of the 34,215-dalton protein is not present in purified calf UP1. This 124-residue sequence has unusual amino acid content in that it is 11% asparagine, 15% serine, and 40% glycine and consists of 16 consecutive oligopeptide repeats. Computer-derived secondary structure predictions for the 34,215-dalton protein revealed two distinct domains consisting of residues 1 through approximately 196 and residues approximately 197 to 320, respectively.     

Isolation and characterization of a cDNA for rat liver cysteine dioxygenase

Cysteine dioxygenase is a key enzyme of cysteine metabolism in mammals. The cDNA clones for rat liver cysteine dioxygenase were isolated by immunological screening and plaque hybridization from a rat liver cDNA library. The longest clone contained an insert of 1458 bp and encoded a polypeptide of 200 amino acids. The clone included the corresponding nucleotide sequence to amino acid sequences obtained from four lysyl endopeptidase-digested fragments of purified rat liver cysteine dioxygenase. The calculated molecular weight of rat liver cysteine dioxygenase was 23,025. Northern blot analysis revealed a single cysteine dioxygenase mRNA species of about 1.7 kb. A computer homology search indicated that this protein showed no homology with any known protein.