The tumor marker human placental protein 11 is an endoribonuclease

Human PP11 (placental protein 11) was previously described as a serine protease specifically expressed in the syncytiotrophoblast and in numerous tumor tissues. Several PP11-like proteins were annotated in distantly related organisms, such as worms and mammals, suggesting their involvement in evolutionarily conserved processes. Based on sequence similarity, human PP11 was included in a protein family whose characterized members are XendoU, a Xenopus laevis endoribonuclease involved in small nucleolar RNA processing, and Nsp15, an endoribonuclease essential for coronavirus replication. Here we show that the bacterially expressed human PP11 displays RNA binding capability and cleaves single stranded RNA in a Mn(2+)-dependent manner at uridylates, to produce molecules with 2',3'-cyclic phosphate ends. These features, together with structural and mutagenesis analyses, which identified the potential active site residues, reveal striking parallels to the amphibian XendoU and assign a ribonuclease function to PP11. This newly discovered enzymatic activity places PP11-like proteins in a completely new perspective.     

Cloning and expression of cDNA encoding translationally controlled tumor protein from strawberry fruits

A cDNA encoding a putative translationally controlled tumor protein (TCTP) was isolated from a cDNA library made with mRNA isolated from red ripe strawberry fruits. This protein is highly conserved in all species analyzed. Expression of strawberry TCTP increased along the ripening of strawberry fruits, and is constitutively expressed in vegetative tissues. The putative function of this protein remains still unknown     

Cloning and expression of rat cDNA encoding corticosteroid 11 beta-dehydrogenase

Corticosteroid 11 beta-dehydrogenase (11-DH) catalyzes the conversion of cortisol to the inactive metabolite cortisone. Absence of 11-DH activity leads to a potentially fatal form of childhood hypertension termed apparent mineralocorticoid excess. As a first step in elucidating the molecular basis of this disorder, we isolated and characterized a rat cDNA clone encoding 11-DH. This clone hybridized to a single mRNA species in liver, kidney, and testis RNA but not to RNA from heart. The insert was 1265 base pairs long and included an 861-base pair open reading frame encoding 287 amino acids. A search of sequence databases revealed that 11-DH is identical in about 27% of amino acid residues to ribitol dehydrogenase from Klebsiella and to the product of the nodG gene from the nitrogen-fixing bacterium, Rhizobium meliloti, thus defining a new superfamily of genes encoding dehydrogenases. The 11-DH cDNA was expressed by transfection into Chinese hamster ovary cells under the control of an SV40 promoter. The expressed enzyme mediated both 11 beta-dehydrogenation and the reverse 11-oxoreduction reaction. Southern blot analysis of rat and human DNA suggested that additional genes related to 11-DH exist in both species.     

Molecular cloning, sequencing, and expression of cDNA encoding serine protease with fibrinolytic activity from earthworm

An earthworm, Lumbricus rubellus, produces alkaline trypsin-like proteases that are greater than trypsins in their stability and strong tolerance to organic solvents. cDNAs encoding strong fibrinolytic proteases (F-III-2 and F-III-1) in the six isozymes were cloned and sequenced to study their stability-structure relationship. The cDNAs of F-III-2 and F-III-1 comprised 1011 and 973 bp and included open reading frames that encode polypeptides of 245 and 246 amino acid residues, respectively. The deduced amino acid sequences of F-III-2 and F-III-1 have 7 and 8 activation peptides in the N-termini respectively, indicating that they are translated as proenzymes and modified to active forms by posttranslational processing. They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region. The gene encoding the native form of F-III-2 was expressed in Pichia pastoris to produce and secrete the earthworm protease in the culture medium, which dissolves an artificial fibrin plate.     

Cloning and expression of human cDNA encoding human homologue of pituitary tumor transforming gene.

Recently, a potent transforming gene which was exclusively expressed in rat pituitary tumor but not in normal pituitary had been isolated and named as pituitary tumor transforming gene (PTTG). A cDNA clone encoding human homologue of rat PTTG was isolated from human fetal liver cDNA library. It contained an open reading frame of 603 base pairs predicting a protein composed of 201 amino acids with a calculated molecular weight of 26 kDa. The deduced protein showed about 85% homology (78% identity, 7% favored substitution) with the rat PTTG. Northern blot analysis showed that the cDNA hybridized to 1.0 kb mRNA species which was expressed in fetal liver and several cancer cell lines. These results suggest that the presence of the human homologue of rat PTTG gene may not be restricted to pituitary tumor.     

Cloning, structure and expression of a cDNA encoding the human androgen receptor

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.     

Cloning and expression of a cDNA encoding a novel human neurotrophic factor

A cDNA encoding a novel human neurotrophic factor (designated nerve growth factor-2; NGF-2) was cloned from a human glioma cDNA library using a synthetic DNA corresponding to human nerve growth factor (NGF). The cloned cDNA encodes a polypeptide composed of 257 amino acid residues including a prepro-sequence of 138 residues and a mature region of 119 residues. The amino acid sequence of human NGF-2 exhibits 58% similarity with that of human NGF. Conditioned medium of COS-7 cells transfected with an expression plasmid for human NGF-2 cDNA supported the survival of sensory neurons isolated from dorsal root ganglia of embryonic chicks. A 1.5 kb of NGF-2 mRNA can be detected from an early development stage in rat brain, by Northern blotting analysis.     

Direct expression of recombinant activated human protein C, a serine protease

Human protein C, like other serine proteases, is normally secreted as an inactive zymogen. It is converted to its active form extracellularly by limited proteolysis with the thrombin-thrombomodulin complex. This activation results from the removal of a 12-residue activation peptide from the NH2 terminus of the heavy (COOH-terminal) chain. We report here a successful strategy for the activation of human protein C during post-translational cellular processing, resulting in the secretion of activated protein C from transfected mammalian cells. Deletion of the nucleotides encoding the activation peptide resulted in the expression of a protease with less than 5% of the expected activity. However, the replacement of the activation peptide with an 8-residue sequence (Pro-Arg-Pro-Ser-Arg-Lys-Arg-Arg) involved in the proteolytic processing of the human insulin receptor precursor resulted in the direct expression of fully activated protein C. The mutant protein was shown to be correctly processed by NH2-terminal sequence analysis. This strategy for successful expression of an activated form of protein C may apply to the expression of active forms of other proteases which are naturally expressed as zymogens.     

Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes

Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.     

Cloning and characterization of a cDNA encoding serine palmitoyltransferase in Arabidopsis thaliana

The first and committed step in de novo sphingolipid synthesis is catalysed by serine palmitoyltransferase (EC 2.3.1.50), which condenses serine and palmitoyl-CoA to form 3-ketosphinganine in a pyridoxal-5'-phosphate-dependent reaction. We have isolated and characterized a cDNA clone from Arabidopsis thaliana that is homologous to yeast and mammalian LCB2. For a functional identification, the A. thaliana homologous cDNA was expressed in Escherichia coli, which resulted in significant production of new sphinganine in E. coli cells.     

Cloning and expression in Escherichia coli of a human cDNA encoding the DNA repair protein N-methylpurine-DNA glycosylase

A 871-base pair cDNA encoding the human N-methylpurine-DNA glycosylase (MPG) was cloned from a HeLa S3 cDNA expression library in a pUC vector by phenotypic screening of MPG-negative (tag- alkA-) Escherichia coli cells exposed to methylmethane sulfonate. The active MPG is expressed as a 31-kDa fusion protein. The human cDNA-encoded MPG releases 3-methyladenine, 7-methylguanine, and 3-methylguanine from DNA and thus has a substrate range similar to that of the indigenous enzyme and the E. coli AlkA protein. The cDNA hybridizes with distinct restriction fragments of mammalian DNAs but not with E. coli or yeast DNA. A search in the GenBank data bank failed to show any other cloned DNA with a similar sequence. Although the human protein has 62% sequence homology with the corresponding rat enzyme, only a few amino acid residues are conserved between the human protein and the E. coli and yeast MPGs. However, a conserved glutamine residue in all MPGs that release 3-alkyladenine and an arginine residue in eukaryotic MPGs and E. coli AlkA that act additionally on N-alkylguanines suggest that these residues are involved in recognition of adenine and guanine adducts in DNA, respectively. Although the 1.1-kilobase mRNAs of MPG from human and rodents are similar in size, liver and cultured cells of rat have much lower levels of MPG mRNA than do human and mouse cells. A hamster cell line variant isolated as being resistant to methylmethane sulfonate does not have a higher level of MPG mRNA than the parent cell line.     

Cloning, sequence analysis, and expression of a cDNA encoding a plastid-localized heat shock protein in maize

We have cloned and characterized a cDNA encoding a maize (Zea mays L.) heat shock protein (HSP), HSP26. The mRNA of HSP26 is present as a single mRNA species of 1.1 kilobase pairs in size and is detectable when maize seedlings are treated at 40°C but not at 28°C. Accumulation of HSP26 mRNA was detected after 10 minutes of incubation at 40°C, reaching the maximum level after 1 hour. Comparison of the deduced amino acid sequence of maize HSP26 to other HSPs indicated a strong homology to the sequences of two nuclear encoded HSPs that are transported into the chloroplasts during heat shock: pea HSP21 and soybean HSP22. Maize HSP26 was also found to cross-react with anti-pea chloroplast HSP21 antibodies. Because of the sequence homology between maize HSP26, soybean HSP22, and pea HSP21, in vitro chloroplast protein import experiments were conducted. The in vitro synthesized maize HSP26 is specifically imported to the soluble fraction of the chloroplast and processed to a smaller polypeptide. The sequence homology and antibody cross-reactivity between maize HSP26 and pea HSP21 have allowed us to conclude that maize HSP26 is a nuclear-encoded, plastid-localized protein in maize.     

Cloning and expression of cDNA encoding human basic fibroblast growth factor

A cDNA encoding human basic fibroblast growth factor (bFGF) was isolated from a human foreskin fibroblast cDNA library. The cDNA, 4 kilobases in size, had a coding sequence, 5' and 3' untranslated regions, and a poly(A) chain. Isolation of additional cDNA clones that had a short 3' untranslated region suggested the presence of multiple mRNA forms. By Northern blot analysis, at least five bFGF mRNA species were detected in cultured fibroblast cells. Transfection of the cDNA to COS cells resulted in the detection of mitogenic activity in the culture medium of the transfected cells, suggesting that a part of the synthesized protein might be secreted from cells despite its unusual short signal sequence.     

粘虫核糖体蛋白S7cDNA的克隆和表达分析

运用RT—PCR和RACE技术克隆了粘虫Mythimnaseparata（Walker）核糖体蛋白s7基因（RPS7）的全长eDNA序列（GenBank登录号：JN582331）,并对其进行生物信息学分析。结果表明,粘虫RPS7全长eDNA序列为762bp,包括5’非编码区32bp和3’非编码区67bp。其开放阅读框（573bp）编码190氨基酸肽链,具有核糖蛋白S7e蛋白家族典型特征。该肽链理论分子量为21．924ku,等电点为9．82,富含4种类型的特定功能位点。该蛋白序列与其他动物RPS蛋白序列具有96．8％-98．2％高度同源性。应用荧光实时定量技术建立了粘虫脑部胚后发育RPS7表达模式。RPS7表达量随胚后发育脑部重建呈现出动态变化,这一结果显示RPS7是在转录水平上呈现发育性调节。     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.