Cloning and nucleotide sequence of ovine prolactin cDNA

A cDNA expression library was constructed in the lambda gt 11 phage vector using ovine (o) pituitary mRNA. The clone, pOP1, carrying a 934-bp insert contains an open reading frame beginning with the first nucleotide (nt) and ending with the stop codon TAA at nt position 781. Two potential translation start codons (ATGs) are present in the 5' region of this cDNA. Translation initiation could occur at the 5' proximal ATG at nt position 61. The nucleotide sequence around this ATG (TCCATGG), resembles the optimum sequence context for translation initiation by the eukaryotic ribosomes, as defined by mutational analysis [Kozak, Cell 44 (1986) 283-292)], with its substitution of the A at -3 of the consensus sequence by a T residue in this clone. Translation initiated at this codon could potentially code for the entire pre-prolactin (pre-PRL) molecule. The 3'-untranslated region is 154 nt long and contains a polyadenylation signal AATAAA. The deduced amino acid sequence agrees in totality with the published amino acid sequence of the mature hormone. The present study reports on the nucleotide sequence of o-PRL mRNA and the deduced amino acid sequence in the signal peptide of the hormone.     

Complete nucleotide sequence coding for rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase derived from a cDNA clone

cDNA clones for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were isolated from rat liver expression libraries in lambda gt11 by antibody, oligonucleotide, and cDNA screening. One 1860 bp long clone contained a full-length nucleotide sequence coding for the 470 amino acids of each of the two identical subunits of the bifunctional enzyme. This clone also contained untranslated sequences, one 173 bp long upstream from the ATG start codon and one 271 bp long downstream from the TGA stop codon. The clone was terminated by a poly(A) tail of 29 nucleotides.     

Isolation and analysis of a cDNA coding for human C1 inhibitor

A cDNA coding for C1 inhibitor was isolated from a human liver lambda gt11 expression library and sequenced by the dideoxy method. The amino acid sequence deduced from the cDNA indicated that the insert was a partial clone coding for 310 amino acids including the reactive site present at the carboxyl end of the molecule. The reactive site corresponds to that previously reported by Salvesen et al. (J. Biol. Chem. 260, 2432, 1985). The cDNA also contained a stop codon of TGA, 264 nucleotides at the 3' noncoding region, and a polyadenylation signal sequence of AATAAA 15 nucleotides upstream from the poly(A) tail. The amino acid sequence flanking the reactive site of the inhibitor is homologous to other members of the superfamily of plasma serine protease inhibitors.     

Characterization by cDNA cloning of the mRNA of a new growth factor from bovine seminal plasma: acidic seminal fluid protein.

A cDNA expression library in lambda gt11 prepared from cDNA derived of seminal vesicle tissue was screened by means of monospecific rabbit anti-aSFP IgG. The sequence of clone pTF21, containing an insert of 668 bp comprised an open reading frame from position 7 to 411 terminated by two stop codons. From this sequence a protein of 134 amino acid residues can be deduced. The mature aSFP was preceded by a signal peptide of 20 amino acids length. The protein sequence contains no signal for N-glycosylation. The molecular weight calculated from the amino acid sequence is 12922 Da. The start codon ATG is part of the sequence AAGATGA which fulfills the criteria of an initiation consensus sequence. The coding region was followed by 257bp of the complete 3'-untranslated region (3'UTR). A putative polyadenylation signal AATAAT, although not of the standard type, is observed at position 650. According to Northern analysis, aSFP mRNA is expressed in seminal vesicle tissue, ampulla and weakly in tissue of epididymis, but not in testis or other bovine tissue. aSFP is specified by a single copy gene. Attempts to detect homologies to known protein sequences were not successful.     

The primary structure of hemoglobin D from the Aldabra giant tortoise,Geochelone gigantea

The complete primary structures of alpha D-2- and beta-globin of hemoglobin D (Hb D) from the Aldabra giant tortoise, Geochelone gigantea, have been constructed by amino acid sequencing analysis in assistance with nucleotide sequencing analysis of PCR fragments amplified using degenerate oligonucleotide primers. Using computer-assisted sequence comparisons, the alpha D-2-globin shared a 92.0% sequence identity versus alpha D-globin of Geochelone carbonaria, a 75.2% versus alpha D-globin of Aves (Rhea americana) and a 62.4% versus alpha A-globin of Hb A expressed in adult red blood cells of Geochelone gigantea. Additionally, judging from their primary structures, an identical beta-globin was common to the two hemoglobin components, Hb A and Hb D. The alpha D-2- and beta-globin genes contained the three-exon and two-intron configurations and showed the characteristic of all functional vertebrate hemoglobin genes except an abnormal GC dinucleotide instead of the invariant GT at the 5' end of the second intron sequence. The introns of alpha D-2-globin gene were both small (224-bp/first intron, 227-bp/second intron) such that they were quite similar to those of adult alpha-type globins; the beta-globin gene has one small intron (approximately 130-bp) and one large intron (approximately 1590-bp). A phylogenetic tree constructed on primary structures of 7 alpha D-globins from Reptilia (4 species of turtles, 2 species of squamates, and 1 species of sphenodontids) and two embryonic alpha-like globins from Aves (Gullus gullus) and Mammals (Homo sapiens) showed the following results: (1) alpha D-globins except those of squamates were clustered, in which Sphenodon punctatus was a closer species to birds than turtles; (2) separation of the alpha A- and alpha D-globin genes occurred approximately 250 million years ago after the embryonic alpha-type globin-genes (pi' and zeta) first split off from the ancestor of alpha-type globin gene family.     

大腹园蛛Avg1 cDNA的克隆和序列分析

组织蛋白酶(cathepsin)是一大类裂解肽键的蛋白水解酶,根据其催化中心不同,组织蛋白酶B(CB)属于半胱氨酸蛋白酶,易被巯基试剂抑制,因此又是巯基酶类,隶属于木瓜蛋白酶族[1 ] .CB可分解胶原蛋白、层粘连蛋白等基底膜的主要成分,近年来发现与癌细胞的浸润和转移有密切的关系[2 ]     

The araBAD operon of Salmonella typhimurium LT2. II. Nucleotide sequence of araA and primary structure of its product, L-arabinose isomerase

The nucleotide sequence of gene araA of Salmonella typhimurium LT2 has been determined. The gene encodes an L-arabinose isomerase (EC 5.3.1.4) of 500 amino acid residues with a calculated Mr of 55814. The ATG start codon of araA is 10 bp distal to the TAA termination codon of araB. A presumed ribosome-binding site (RBS) "TAAGGA" 7 bp from the ATG codon overlaps the stop codon of araB. L-Arabinose isomerase was purified and the amino acid composition is in agreement with that predicted from the DNA sequence. The NH2-terminus of the protein is modified as the sequence cannot be analyzed by the automated Edman degradation. Amino acid composition analyses of both NH2-terminal and C-terminal cyanogen bromide (CNBr) cleaved peptides and partial amino acid sequence of the C-terminal peptide are consistent with the deduced amino acid sequence.     

Cloning and selective overexpression of an alkaline protease-encoding gene from Aspergillus oryzae.

The gene alpA encoding Aspergillus oryzae alkaline protease (ALP) was isolated from a genomic library of an industrial strain used in Thailand by using oligodeoxyribonucleotide probes based on the published cDNA sequence [Tatsumi et al., Agric. Biol. Chem. 52 (1988) 1887-1888]. The entire nucleotide sequence of the genomic clone obtained was determined. By comparison with the published cDNA sequence, it was found that ALP is encoded by four exons of 314, 445, 89 and 351 bp. Three introns, which interrupt the coding sequence, are 50, 59 and 56 bp in length. The gene contains a typical TATA box 103 bp upstream from the start codon, and a consensus polyadenylation signal, AATAAA, 189 bp from the stop codon. The alpA gene, introduced into a protease deficient strain (A. oryzae U1638) by cotransformation, directed the secretion of enzymatically active ALP into the culture medium. Cotransformants of the high-level ALP-producing strain U212 containing multiple copies of the alpA gene were able to secrete up to five times more ALP than the parental strain.     

圈卷产色链霉菌尼可霉素生物合成基因-sanK的克隆及功能研究

利用染色体步移策略,以尼可霉素生物合成相关的基因片段为探针,从圈卷产色链霉菌中克隆到了一个大约１０ｋｂ的ＤＮＡ片段。对其中１．８ｋｂ的ＰｖｕⅡ－ＳａｃⅡ片段进行了序列分析,结果表明：此片段中含有一个具有１１７０个核苷酸的完整开放阅读框,起始密码子为４４７位的ＡＴＧ,终止密码子为１６１４位的ＴＧＡ,推测其编码一个３８９个氨基酸的蛋白质产物。利用ＢＬＡＳＴＸ程序进行了分析揭示,此基因编码一个肌氨酸单体