Isolation and sequencing of mouse angiogenin DNA

The mouse genomic DNA for angiogenin, a potent blood vessel inducing protein, has been isolated from a bacteriophage library using the human angiogenin gene as a probe. The 1129 bp fragment contains 499 bp in the 5' flanking region, 192 bp in the 3' flanking region, and 438 bp coding for the mature protein (121 amino acids) and signal peptide (24 amino acids). Potential TATA box and AATAAA polyadenylation sequences are present, and a consensus sequence for an intron 3' boundary occurs 16 bp upstream of the Met-(24) codon, suggesting the presence of an intron in the 5' region. The protein sequence inferred from the DNA is 76% identical to that of human angiogenin, and matches the sequences obtained previously from tryptic peptides of a serum-derived mouse angiogenin. The critical catalytic residues of human angiogenin are conserved in the mouse protein, as are the six cysteines necessary for disulfide bond formation.     

Yeast LEU2. Repression of mRNA levels by leucine and primary structure of the gene product

It has been known that enzyme activity associated with the yeast LEU1 and LEU2 gene product (beta-isopropylmalate dehydrogenase) drops sharply when yeast is grown in the presence of leucine. RNA blot hybridizations with LEU2-specific probes establish that this is accompanied by a 5-fold repression in LEU2 mRNA levels. A similar repression was noted recently for LEU1 mRNA levels (Hsu, Y.-P., and Schimmel, P. (1984) J. Biol. Chem. 259, 3714-3719). Nuclease mapping of the 5'-end of the LEU2 mRNA shows a major start at approximately 16 nucleotides upstream of the AUG initiation codon. This initiation site in the gene is retained in an extensive LEU2 5'-noncoding region deletion which still expresses the LEU2 gene product (Erhart, E., and Hollenberg, C. P. (1983) J. Bacteriol. 156, 625-635). The primary structure of the LEU2 gene product was established from the nucleotide sequence of the gene-coding region and from fitting amino acid sequences of scattered internal peptides to the nucleotide sequence. The 364-amino acid protein has a 13-amino acid stretch which is highly homologous to the partially sequenced yeast LEU1 gene product (isopropylmalate isomerase). The homology occurs about 290 amino acids from the respective NH2 termini of the two proteins. The homology may represent residues which interact with beta-isopropylmalate, a common ligand for the enzymes.     

Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.     

紫杆柽柳与其它物种脂质转运蛋白基因编码蛋白的同源性比较

根据从柽柳cDNA文库克隆获得的脂质转运蛋白(LTP)的部分序列,用RACE技术克隆出其全长cDNA序列.基因的5'非翻译区96bp,3'非翻译区222bp,开放阅读框285bp,编码94个氨基酸,预计蛋白的分子量为9.9 kD,等电点为8.02.此基因有8个位置保守的Cys残基及26个氨基酸的信号肽,为典型的植物脂质转运蛋白基因.其基因序列数据库(GenBank)登录号为AY574218(基因)和AAS79106(蛋白).     

Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity

The complete nucleotide sequence of the Clostridium thermocellum celE gene, coding for an endo-beta-1,4-glucanase (endoglucanase E; EGE) with xylan-hydrolysing activity has been determined. The structural gene consists of an open reading frame (ORF) of 2442 bp commencing with a GTG start codon and followed by a TAA stop codon. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that derived by N-terminal amino acid sequencing of the purified protein. The EGE sequence contains a region homologous to the reiterated domain found at the C terminus of other endoglucanases from the same organism. BAL 31 deletions of the structural gene have revealed the extent to which this conserved sequence is necessary for endoglucanase and xylanase activity. A region of DNA, upstream from the structural gene has also been sequenced and a ribosome-binding site and putative promoter sequences have been identified. A second ORF which ends 349 bp 5' to the GTG start codon of the celE gene has also been identified. The encoded product contains a C terminus homologous to other C. thermocellum endoglucanases.     

Metal-binding, nucleic acid-binding finger sequences in the CDC16 gene of Saccharomyces cerevisiae.

The CDC16 gene is involved in the process of chromosome segregation in mitosis and a cdc16ts mutant accumulates the predominant microtubule-associated protein at the nonpermissive temperature. We find that the CDC16 gene open reading frame (ORF) is capable of encoding a protein whose calculated molecular weight and pI are 94,967 and 6.60, respectively. This hypothetical protein contains 16 cysteine residues; five are clustered at the N-terminal, 4 are placed about 3 residues apart in the middle of the peptide, and 3 are located close to the C-terminal. Each of these could form a metal-binding, nucleic acid-binding domain, suggesting this protein acts either as a repressor of the microtubule-associated protein gene or as a component necessary for spindle elongation, possibly interacting with the DNA. The start of the CDC16 ORF is only 95 bp downstream from the end of the MAK11 ORF. In this region there are two TATA boxes in tandem, but there is no room for a UAS or other regulatory sequences. An ATG is present 5 bp upstream of the start of the large ORF. Its frame terminates after only two amino acids.     

Sequence of a sea urchin hsp70 gene and its 5' flanking region

We report the nucleotide sequence of a 4470-bp fragment derived from a sea urchin genomic clone containing part of a heat-shock protein 70 (Hsp70)-encoding gene. This fragment, named hsp70 gene II, contains 1271 bp of the flanking region and 3299 bp of structural gene sequence interrupted by five introns and encoding the N-terminal 371 amino acids (aa) of the protein. The 5' flanking region contains a putative TATA element, two CCAAT boxes, four heat-shock consensus sequence elements (hse) and one consensus sequence for binding of Sp1. Remarkable homologies were observed for deduced aa sequence and intron-exon organization between hsp70 gene II and rat hsc73 gene.     

Degradation of Aromatics and Chloroaromatics by Pseudomonas sp. Strain B13: Cloning, Characterization, and Analysis of Sequences Encoding 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase

3-oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase carry out the ultimate steps in the conversion of benzoate and 3-chlorobenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the 3-oxoadipate pathway. This report describes the characterization of DNA fragments with the overall length of 5.9 kb from Pseudomonas sp. strain B13 that encode these enzymes. DNA sequence analysis revealed five open reading frames (ORFs) plus an incomplete one. ORF1, of unknown function, has a length of 414 bp. ORF2 (catI) encodes a polypeptide of 282 amino acids and starts at nucleotide 813. ORF3 (catJ) encodes a polypeptide of 260 amino acids and begins at nucleotide 1661. CatI and CatJ are the subunits of the 3-oxoadipate:succinyl-CoA transferase, whose activity was demonstrated when both genes were ligated into expression vector pET11a. ORF4, termed catF, codes for a protein of 401 amino acid residues with a predicted mass of 41,678 Da with 3-oxoadipyl-CoA thiolase activity. The last three ORFs seem to form an operon since they are oriented in the same direction and showed an overlapping of 1 bp between catI and catJ and of 4 bp between catJ and catF. Conserved functional groups important for the catalytic activity of CoA transferases and thiolases were identified in CatI, CatJ, and CatF. ORF5 (catD) encodes the 3-oxoadipate enol-lactone hydrolase. An incomplete ORF6 of 1,183 bp downstream of ORF5 and oriented in the opposite direction was found. The protein sequence deduced from ORF6 showed a putative AMP-binding domain signature.     

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.