Cloning and sequencing of a full length cDNA coding for human retinol-binding protein.

We have isolated and sequenced a cDNA clone coding for human Retinol Binding Protein. The sequence indicates that Retinol Binding Protein is synthesized as a single polypeptide chain precursor which is then matured to the secreted protein by removal of a leader peptide. Southern and Northern blot analysis suggest that the gene is present in one or few copies per haploid genome and is transcribed in a single mRNA species.     

Cloning and sequencing of a Porifera partial cDNA coding for a short-chain collagen

Collagen is present in Porifera, the lowest multicellular animals, but there is no information available on the primary structure of the collagen chains in this phylum. Developing fresh-water sponges have been used to extract total RNA in order to study in vitro translation products and to construct a cDNA library. Four translated proteins were collagenase-sensitive (200 kDa, 160 kDa, 81 kDa and 48 kDa). The cDNA library was screened with a human collagen probe and a clone, EmC4, covering 1.2 kb was isolated. Nucleotide sequencing of EmC4 revealed a conceptual open reading frame coding for 366 amino acids terminated by a stop codon TGA with 103 nucleotides downstream. The presumed translation product encoded contained several domains: a non-collagenous C-terminal domain of 156 amino acids with 9 cysteines, an uninterrupted collagenous domain of 171 amino acids, a non-collagenous domain of 16 amino acids with 3 cysteines and a probably incomplete N-terminal collagenous domain of 23 amino acids. Comparison with other sequences suggested that this collagen chain might belong to a non-fibrillar collagen family which evolved into several sub-families giving rise to nematode cuticular collagens, and type IV collagens.     

Cloning and expression of cDNA coding for bouganin.

Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.     

Cloning and sequencing of chloroperoxidase cDNA.

An oligod-d(T) 12-18 primed cDNA library has been prepared from Caldariomyces fumago mRNA. A clone containing a full-length insert was sequenced on the supercoiled plasmid, pBR322. The complete primary sequence of chloroperoxidase has been derived. We have also determined about 73% of the peptide sequence by amino acid sequencing. The DNA sequence data matches all of the available known peptide sequences. The mature polypeptide contains 300 amino acids having a combined molecular weight of 32,974 daltons. A putative signal peptide of 21 amino acids is proposed from DNA sequence data. The chloroperoxidase gene encodes three potential glycosylation sites recognized as Asn-X-Thr/Ser sequences. Three cysteine residues are found in the protein sequence. A small region around Cys87 bears a minimal homology to the active site of cytochrome P450cam. No other heme protein homologues can be detected. We propose that Cys87 serves as a thiolate ligand to the iron of heme prosthetic group. A rare arginine codon, AGG, is used three times out of twelve in contrast to the very infrequent use of this codon in E. coli or yeast.     

Cloning and sequencing of a human cDNA coding for dihydroorotate dehydrogenase by complementation of the corresponding yeast mutant.

Dihydroorotate dehydrogenase (DHOdehase, EC 1.3.3.1) catalyses the fourth enzymatic step in de novo pyrimidine biosynthesis. A truncated human cDNA encoding this enzyme was isolated from a HeLa cell cDNA library by functional complementation of a corresponding deletion mutant from the yeast, Saccharomyces cerevisiae. The complementing clone contained a 1.5-kb poly(A)(+)-tailed insert with a 1191-bp open reading frame, hybridising with a unique human mRNA of 1.6 kb. The deduced amino acid sequence has 54%, 46% and 42% identity with Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli DHOdehases, respectively. In contrast, it has only 21% identity with the S. cerevisiae enzyme, which probably reflects the cytosolic location of the enzyme in the latter organism.     

Cloning and overproduction of biodegradative threonine deaminase from Escherichia coli W strain.

We have cloned the structural gene (tdcB) of biodegradative threonine deaminase from Escherichia coli W strain by utilizing the polymerase chain reaction. The JM109/pUCTDA strain, which was obtained by transforming E. coli JM109 with a vector plasmid (pUCTDA) containing the cloned tdcB gene, produced a large amount of the enzyme corresponding to more than 5% of the total soluble protein. Amino acid sequence analysis of this recombinant enzyme showed that the amino acid sequence is identical to the nucleotide-deduced sequence of biodegradative threonine deaminase from E. coli K-12.     

Cloning and sequencing of human FSH receptor cDNA.

We have isolated and sequenced a cDNA encoding the follicle stimulating hormone (FSH) receptor. The deduced amino acid sequence (678 residues) containing seven putative transmembrane segments which displays sequence similarity to G protein-coupled receptors. The receptor consists of 359 residue extracellular domain which contains four N-linked glycosylation sites. While the protein is 89% identical overall with the previously cloned rat FSH receptor, the most highly conserved regions are the putative transmembrane segments (95% similarity).     

Cloning and sequencing of papain-encoding cDNA

Messenger RNA extracted from Carica papaya fruit was converted to cDNA and cloned into the PstI restriction site of plasmid pBR322. Subclones of the approximately 1.4-kb fragment were sequenced. The nucleotide sequence matched that expected, based on the amino acid (aa) sequence for papain, with the following exceptions: at aa positions 47, 118 and 135 the codon for glutamate was found instead of glutamine; at aa position 169 the codon for asparagine was found instead of glycine; at aa positions 86-88, a difference in the order of the aa codons was observed, namely tyr-pro-tyr instead of the published pro-tyr-tyr. The upstream sequence revealed that papain is probably synthesized with a 133-aa prosegment, suggesting that the enzyme is synthesized as an inactive zymogen. The downstream segment revealed an unusual (AT)9AGAA sequence beginning 26 bp from the double TGA stop codon.     

人TRALL基因cDNA的克隆与序列测定

为研究人 TRALL的基因组结构 ,生物学性能和用于肿瘤生物治疗的可能性 ,利用反转录聚合酶链反应 (RT- PCR)从人急性早幼粒白血病细胞系 HL - 6 0细胞总 PNA中扩增出人 TRALL基因编码区 c DNA序列 ,将其克隆至 p GEM- T载体中 ,序列测定表明 ,克隆片段与文献报道的人TRALL基因编码区 c DNA序列完全一致。     

人TRAIL基因cDNA的克隆与序列测定

为研究人TRAIL的基因组结构,生物学性能和用于肿瘤生物治疗的可能性,利用反转录聚合酶链反应(RT-PCR)从人急性早幼粒白血病细胞系HL-60细胞总RNA中扩增出人TRAIL基因编码区cDNA序列,将其克隆至pGEM-T载体中,序列测定表明,克隆片段与文献报道的人TRAIL基因编码区cDNA序列完全一致.