磷酸烯醇式丙酮酸羧化酶cDNA在T7 RNA多聚酶/启动子系统中专一性表达的研究

用PT7和pGP1—2质粒偶联表达系统,通过温度诱导(30～42℃),使温度敏感基因CI875失活;加入利福霉素选择性抑制E.coliRNA多聚酶的表达,从而使外源PEP羧化酶cDNA得到专一性的表达。产物经SDS—PAGE和Wesern杂交分析,表明该系统表达出两条PEP羧化酶带,分子量分别是78kD和80kD。     

Cytosolic triosephosphate isomerase is a single gene in rice.

A cDNA clone encoding rice (Oryza sativa L.) cytosolic triosephosphate isomerase (TPI), an important glycolytic enzyme, was isolated and characterized. The clone (pRTPI-6) contains an open reading frame of 759 base pairs, encoding a polypeptide chain of 253 amino acid residues (M(r) 27,060). The identity of this clone was defined by its high homology (85% nucleotide sequence and 89% amino acid sequence identical match) with a maize mRNA sequence encoding the cytosolic TPI and with TPIs from other species. Genomic DNA blot analysis using the cDNA as a probe showed that the cytosolic TPI gene is present as a single copy per haploid rice genome, as opposed to that found in maize, in which multiple TPI gene copies exist. A single TPI mRNA species of about 1100 nucleotides was detected by gel blot hybridization analysis of RNA isolated from root, culm, and leaf tissues, indicating that its expression is ubiquitous. Based on sequence comparison and molecular analysis, we propose that the chloroplast-located TPI may be encoded by divergent structural nuclear genes in rice.     

Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph

Summary Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA ^–auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35854, predicted from the deduced amino acid sequence, was similar to the 38 000 M_r determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.     

Nucleotide sequence of the yeast glutathione S-transferase cDNA

The nucleotide sequence (658 bp) of the cDNA coding for glutathione S-transferase Y-2 of yeast Issatchenkia orientalis was obtained. The cDNA clone contains an open reading frame of 570 nucleotides encoding a polypeptide comprising 190 amino acids with a molecular weight of 21,520. The primary amino acid sequence of the enzyme exhibits only 25.0% and 21.1% identity with 177 and 151 amino acid residues of maize glutathione S-transferase I and rat glutathione S-transferase Yb2, respectively.     

Characterization of recombinant Dictyostelium discoideum sepiapterin reductase expressed in E. coli

A cDNA clone (SSC801) putatively encoding sepiapterin reductase (SR) was obtained from the expressed sequence tag clones of Dictyostelium discoideum. The cDNA sequence of 878 nucleotides constituted an ORF of 265 amino acid residues but was missing a few N-terminal residues. The deduced amino acid sequence showed 29.8% identity with mouse SR sequence and a molecular mass of 29,969 Da. The coding sequence was cloned in E. coli expression vector and overexpressed. The purified His-tag recombinant enzyme was confirmed to have the genuine activity of SR to produce tetrahydrobiopterin from 6-pyruvoyltetrahydropterin in a coupled assay with 6-pyruvoyltetrahydropterin synthase as well as dihydrobiopterin from sepiapterin. However, dictyopterin was not observed in our assay condition. The enzyme was also inhibited by N-acetylserotonin and to a lesser extent by melatonin. Km values for NADPH and sepiapterin were 51.8+/-2.7 microM and 40+/-2 microM, respectively. Vmax was determined as 0.14 micromol/min/mg of protein.     

Cloning and nucleotide sequence of human liver cDNA encoding for cystathionine gamma-lyase.

We have cloned and sequenced a full-length cDNA (1083 bp) encoding the human liver cystathionine-gamma-lyase enzyme (cystathionase). The human cystathionase sequence presented a substantial deletion of 132 bases (44 amino acids) compared to that reported for rat cystathionase, and of 135 bases (45 amino acids) compared to that reported for yeast cystathionase. After re-alignment for the missing nucleotides, the human cDNA sequence shows significant amino acid homology to that for the rat enzyme (85%) and the yeast enzyme (50%). A search for an undeleted cDNA, by the polymerase chain reaction, yielded a second clone which contained the missing 132 bases. Flanking nucleotides in the latter clone were identical to those in the cDNA clone containing the deletion. The two forms of human cystathionase deduced from the two cDNA clones may be derived from two different genes or may be splice variants.     

Human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of iron sulfur (Ip) subunit of liver mitochondria

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex of both the tricarboxylic acid cycle and of the aerobic respiratory chains of mitochondria in eukaryotic cell and prokaryotic organisms. In this study, the amino acid sequence of iron sulfur-subunit in human liver mitochondria was deduced from cDNA which was isolated by immunoscreening a human liver lambda gtll cDNA library. An isolated clone contains an open reading frame of 786 nucleotides and encodes a mature protein of 252 amino acids with a molecular weight of 28,804. The amino acid sequence was highly homologous with that of bovine heart (94.1%) which has been determined from the purified peptide and that of Escherichia coli sdh B product (50.8%). Striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme. This is the first report on the cDNA sequence of mitochondrial complex II.     

A lung type prostaglandin F synthase is expressed in bovine liver: cDNA sequence and expression in E. coli.

Using the cDNA of bovine lung prostaglandin F synthase (EC 1.1.1.2) as a probe, we isolated a clone from a bovine liver cDNA library which differed in only eleven nucleotides from the probe. The corresponding protein contained three amino acid substitutions, including a leucine residue which is conserved throughout all aldo-keto reductases. We inserted the liver cDNA into expression vector pUC19 and expressed the recombinant liver enzyme in E.coli. The purified liver enzyme reduced prostaglandin H2 as well as prostaglandin D2 and various carbonyl compounds. The high relative activity against prostaglandin H2 in combination with a high Km value for prostaglandin D2 identified this liver enzyme as a lung type prostaglandin F synthase. However, the binding constant for NADPH of the liver enzyme was 3.5 fold higher than that of lung prostaglandin F synthase.     

Regulatory seryl-phosphorylation of C4 phosphoenolpyruvate carboxylase by a soluble protein kinase from maize leaves

A reconstituted system composed of purified phosphoenolpyruvate carboxylase (PEP-Case) and a soluble protein kinase (PK) from green maize leaves was developed to critically assess the effects of in vitro protein phosphorylation on the catalytic and regulatory (malate sensitivity) properties of the target enzyme. The PK was partially purified from light-adapted leaf tissue by ammonium sulfate fractionation (0-60% saturation fraction) of a crude extract and blue dextran-agarose affinity chromatography. The resulting preparation was free of PEPCase. This partially purified protein kinase activated PEPCase from dark-adapted green maize leaves in an ATP-, Mg2+-, time-, and temperature-dependent fashion. Concomitant with these changes in PEPCase activity was a marked decrease in the target enzyme's sensitivity to feedback inhibition by L-malate. The PK-mediated incorporation of 32P from [gamma-32P]ATP into the protein substrate was directly correlated with these changes in PEPCase activity and malate sensitivity. The maximal molar 32P-incorporation value was about 0.25 per 100-kDa PEPCase subunit (i.e., 1 per holoenzyme). Phosphoamino acid analysis of the 32P-labeled target enzyme by two-dimensional thin-layer electrophoresis revealed the exclusive presence of phosphoserine. These in vitro results, together with our recent studies on the light-induced changes in phosphorylation status of green maize leaf PEPCase in vivo (J. A. Jiao and R. Chollet (1988) Arch. Biochem. Biophys. 261, 409-417), collectively provide the first unequivocal evidence that the seryl-phosphorylation of the dark-form enzyme by a soluble protein kinase is responsible for the changes in catalytic activity and malate sensitivity of C4 PEPCase observed in vivo during dark/light transitions of the parent leaf tissue.     

Isolation and sequencing of a cDNA encoding for a ribonuclease from the insect Ceratitis capitata

Two overlapping clones encoding for a ribonuclease from six-day-old larvae of the insect Ceratitis capitata (Cc-RNase) have been isolated by immunoscreening a cDNA library and by 5' RACE. The sequence of the Cc-RNase cDNA contains an open reading frame of 414 nucleotides encoding for a precursor protein of 138 amino acids long with a putative signal peptide consisting of 19 amino acids. The calculated M(r) of the mature protein was found to be 13.7 kDa. Multiple alignments of the deduced amino acid Cc-RNase sequence with other ribonucleases revealed an approximate 25% average identity. Despite the low percentage of identity, histidine and lysine residues which are essential for its catalytic activity, were found to be completely conserved. Furthermore, expression of the clone in E. coli resulted in the production of a recombinant product that showed strong immunoreactivity with anti-RNase specific antibodies. These results support the hypothesis that the identified clone encodes for a protein which is a new member of the RNase superfamily.     

cDNA cloning, genomic structure and chromosomal localization of the human BUP-1 gene encoding beta-ureidopropionase.

A full-length cDNA clone encoding human beta-ureidopropionase was isolated. A 1152-nucleotide open reading frame which corresponds to a protein of 384 amino acids with a calculated molecular weight of 43? omitted?158 Da, surrounded by a 5'-untranslated region of 61 nucleotides and a 3'-untranslated region of 277 nucleotides was identified. The protein showed 91% similarity with the translation product of the rat beta-ureidopropionase cDNA. Expression of the human cDNA in an Escherichia coli and eukaryotic COS-7 expression system revealed a very high beta-ureidopropionase enzymatic activity, thus confirming the identity of the cDNA. Since human EST libraries from brain, liver, kidney and heart contained partial beta-ureidopropionase cDNAs, the enzyme seems to be expressed in these tissues, in agreement with the expression profile of this enzyme in rat. Using the human cDNA as a probe a genomic P1 clone could be isolated containing the complete human beta-ureidopropionase gene. The gene consist of 11 exons spanning approximately 20 kB of genomic DNA. Fluorescence in situ hydridization localized the human beta-ureidopropionase gene to 22q11.2.     

Bacterial expression and isolation of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase, EPSPS), an in vivo enzyme target of the herbicide glyphosate (N-phosphonomethyl glycine), was purified from a Petunia hybrida suspension culture line, MP4-G, by a small-scale high-performance chromatographic purification procedure. The cDNA encoding the mature petunia EPSPS (lacking the chloroplast transit sequence) was cloned into a plasmid, pMON342, for expression in Escherichia coli. This clone complemented the EPSPS deficiency of an E. coli aroA- mutant, and the plant enzyme constituted approximately 1% of the total extractable protein. Large-scale purification of the enzyme from E. coli cells resulted in a highly active protein which was homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino terminal sequencing. Antibodies raised against the purified enzyme also reacted with the E. coli EPSPS in Western analyses. The availability of large quantities of the plant enzyme will significantly facilitate mechanistic investigations as well as a comparative study with EPSPS from bacteria and fungi.     

Structure and properties of an engineered transketolase from maize

The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high level in E. coli and cleaved with thrombin, affording plastid TK. The protein in complex with thiamine pyrophoshate was crystallized, and its structure was solved by molecular replacement. The enzyme is a C2 symmetric homodimer closely similar to the enzyme from yeast (Saccharomyces cerevisiae). Each subunit is folded into three domains. The two topologically equivalent active sites are located in the subunit interface region and resemble those of the yeast enzyme.     

Cloning and characterization of the chloroplast elongation factor EF-Tu cDNA of Oryza sativa L

From the rice leaf cDNA library, we have cloned a cDNA encoding rice chloroplast translational elongation factor EF-Tu (tufA). The rice tufA cDNA clone contains 1678 nucleotides and codes for a 467 amino acid protein including a putative chloroplast transit peptide of 59 amino acid residues. The predicted molecular mass of the mature protein is approximately 45 kDa. This cDNA clone contains the 61 nucleotides of the 5' untranslated region (UTR) and the 213 nucleotides of 3' UTR. Amino acid sequence identity of the rice tufA with the mature chloroplast EF-Tu proteins of tobacco, pea, arabidopsis, and soybean ranges from 83% to 86%. The deduced polypeptide of the rice tufA cDNA contains GTP binding domains in its N-terminal region and chloroplast EF-Tu signature regions in the C-terminal region. The rice tufA appears to exist as a single copy gene, although its homologues of maize and oat exist as multiple copy genes. The rice tufA gene is located in chromosome 1 and is more highly expressed in the leaf than in root tissue.     

Cloning, nucleotide sequence and expression of thioltransferase (glutaredoxin) cDNA from Schizosaccharomyces pombe

Thioltransferase (TTase), also known as glutaredoxin (Grx), is an enzyme that catalyzes the reduction of a variety of disulfide compounds, including protein disulfides, in the presence of reduced glutathione. TTase acts as a cofactor for various enzymes such as ribonucleotide reductase. We previously purified a TTase from Schizosaccharomyces pombe and its molecular size was determined. In the present study, a cDNA coding TTase was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization, which was constructed in a plasmid vector pGAD GH, and its corresponding insert was confirmed by Southern hybridization. The nucleotide sequence of the 375 bp long cDNA clone reveals an open reading frame, which encodes a protein of 101 amino acids. The coding region of the original clone was transferred after the lac promoter of pUC13 vector for expression in E. coli, and simultaneously, a suitable Shine-Dalgarno (SD) sequence was added in front of the coding region by PCR. The two primers used for PCR also separately contained BamHI and HindIII restriction sites. The E. coli strain (A434) harboring the pUC13 derivative pKU10 showed a 17.3-fold increase in TTase activity compared to the strain with only the vector plasmid.     

Mammalian 5'(3')-deoxyribonucleotidase, cDNA cloning, and overexpression of the enzyme in Escherichia coli and mammalian cells

5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known. It was earlier purified to homogeneity from human placenta. We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa. The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells. The recombinant enzyme was purified to homogeneity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme. Several polymerase chain reaction-based approaches to find this sequence met with no success. A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme.     

The cDNA clone for strictosidine synthase from Rauvolfia serpentina. DNA sequence determination and expression in Escherichia coli

The cDNA clone for strictosidine synthase, the enzyme which catalyzes the stereospecific condensation of tryptamine with secologanin to form the key intermediate in indole alkaloid biosynthesis, strictosidine, has been identified with a synthetic oligodeoxynucleotide hybridization probe in a lambda gt11 cDNA library of cultured cells of Rauvolfia serpentina. The DNA has been sequenced, revealing an open reading frame of 1032 base pairs encoding 344 amino acids. The sequence of 60 nucleotides in the 5'-flanking region has been determined by primer extension analysis. The encoded protein has been expressed in E. coli DH5 as detected by immunoblotting of protein extracts with antibodies raised against the native enzyme.     

Expression of the human alpha-galactosidase A in Escherichia coli K-12

We used the prokaryotic expression vector, ptrpL1, for the expression in Escherichia coli K-12 of a cDNA clone specific for the human lysosomal hydrolase, alpha-galactosidase A. The 5' terminus of the cDNA clone was engineered so that an ATG codon precedes the first codon of the mature form of the enzyme. A clone with elevated expression of this human enzyme was constructed by increasing the distance between the Shine-Dalgarno site and the ATG start codon from 6 to 8 bp. Clones with alpha-galactosidase A specific cDNA encoding the proenzyme produce a protein of 45 kDa, the size expected for the intact proenzyme. The 45-kDa protein is specifically precipitated by antibody to alpha-galactosidase A, and its expression is repressed by tryptophan and induced by 3-beta-indoleacrylic acid as expected for this expression vector. The human enzyme is produced in E. coli in a catalytically active form at levels sufficient to support the growth of cells using alpha-galactosides as sole sources of carbon and energy. In addition, bacterial colonies that produce the human enzyme turn blue in the presence of 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside.