A gene encoding the tryptophan synthase beta subunit of Arabidopsis thaliana.

The DNA sequence of a tryptophan synthase gene and the flanking 5' and 3' regions has been determined for Arabidopsis thaliana. The sequence encodes only the beta subunit domain, indicating that alpha and beta subunits are specified by separate genes. The gene contains four introns and encodes 470 amino acid residues. The plant amino acid sequence is highly conserved with respect to corresponding microbial sequences. The NH2-terminal amino acid sequence is characteristic of chloroplast transit peptides. Identity of the sequences of the genomic exons and a cDNA clone and the presence of cellular RNA corresponding in size and 5' sequence to the gene indicate that the gene is expressed. Analysis of Arabidopsis genomic DNA suggests the presence of a second gene for the beta subunit.     

Organization of the gene for human erythrocyte membrane protein 4.2: structural similarities with the gene for the a subunit of factor XIII.

Human erythrocyte band 4.2 is a major membrane-associated protein with an important, but still undefined, role in erythrocyte survival. We previously sequenced the complete cDNA for band 4.2 and showed that the protein has a strong sequence identity with the transglutaminase family of proteins but lacks transglutaminase activity. Here we have analyzed the genomic organization of band 4.2. The band 4.2 gene is approximately 20 kilobases, consisting of 13 exons and 12 introns. Reticulocytes contain two different sized messages for band 4.2, and our results show that the major, smaller, message is produced by alternative splicing within band 4.2 exon I. The upstream region of the gene has several prospective promoter elements arranged in a pattern similar to that of two other erythroid genes, beta-globin and porphobilinogen deaminase. Alignment of the band 4.2 amino acid sequence with that of the a subunit of human coagulation factor XIII and division of the sequences into exons reveal a remarkable correspondence, and in most cases identity, in the sizes of the paired exons. Moreover, each corresponding intron of the two genes is of an identical splice junction class. These and other similarities suggest that the gene for band 4.2 is closely related to and possibly derived from that for the a subunit of factor XIII and that the proteins may share common structural and functional properties.     

Individual exons encode the integral membrane domains of human myelin proteolipid protein.

The gene encoding human proteolipid protein (PLP) was isolated from a human genomic library by hybridization with labeled DNA of a PLP-specific cDNA clone. The entire PLP gene spans approximately 17 kilobases. Restriction and sequence analysis revealed seven exons and six introns. The entire nucleotide sequences of the exons and of the exon-intron transitions were determined, and the intron lengths were measured. Exon I includes only ATGG of the translated region, the N-terminal methionine codon and G of glycine, the first amino acid of mature PLP. Each hydrophobic trans- and cis-membrane domain of PLP together with its adjacent hydrophilic sequence correlates closely with one exon of the gene except for the C-terminal transmembrane helix that is encoded by two exons. The amino acid sequence of human PLP derived from the nucleic acid sequence is highly conserved. Human and rat PLP are completely homologous, whereas only four amino acid residues are exchanged in bovine PLP sequence derived from protein sequencing and a partial cDNA clone. Homology search on the nucleic acid level among human, bovine, and rat brain PLPs indicates an unusually high homology in the coding regions. Hybridization analysis with DNA of human-rodent hybrid clones revealed that the gene encoding PLP segregates with human X chromosome in the region q13-q22.     

Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana.

We have isolated a gene coding for a G protein alpha subunit from the flowering plant Arabidopsis thaliana. This gene, named GPA1, was isolated by using a DNA probe generated by polymerase chain reaction based on protein sequences from mammalian and yeast G protein alpha subunits. The sequences of genomic and cDNA clones indicate that GPA1 has 14 exons, and the deduced amino acid sequence shows that the GPA1 gene product (GP alpha 1) has 383 amino acid residues (44,582 Da). The GP alpha 1 protein exhibits similarity to all known G protein alpha subunits--36% of its amino acids are identical and 73% are similar (identical and conservative changes) to mammalian inhibitory guanine nucleotide-binding regulatory factor alpha subunits and transducins. Furthermore, the GP alpha 1 protein has all of the consensus regions for a GTP-binding protein. The GPA1-encoded mRNA of 1.55 kilobases is most abundant in vegetative plant tissues, as determined by RNA blot analysis. Restriction fragment length polymorphism mapping experiments show that GPA1 is approximately 1.2 centimorgans from the visible marker er on chromosome 2.     

Isolation of genomic DNA clones spanning the entire fibronectin gene.

Overlapping recombinant clones that appear to encompass the entire fibronectin gene have been isolated by step-wise screening of a library of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned fibronectin cDNA hybridization probe. The remaining clones were obtained by using defined fragments of this and successive genomic clones as probes. Their relationships and overlaps were determined by electron microscopy, restriction mapping, and heteroduplex analysis. Based on electron microscopic analysis of hybrids between these clones and fibronectin mRNA, the gene is approximately 48 kilobases long, more than 5 times larger than the corresponding mRNA. This large gene contains at least 48 exons interrupted by introns of highly variable size. The total exon size as estimated by R-loop analysis is 8 kilobases, similar to the mRNA for fibronectin. With the exception of the 3'- and 5'-terminal exons, the exons are small and roughly similar in size. The average exon size is 147 +/- 37 base pairs, corresponding to a protein unit of 50 amino acids. The nucleotide sequence of one of these exons was determined. The deduced amino acid sequence has marked homologies with one type of repetitive protein sequence unit known to exist in bovine fibronectin. These results suggest that the gene for fibronectin may have arisen by multiple gene duplications of a primordial gene or genes approximately equal to 150 base pairs long.     

Molecular cloning and characterization of the gene coding for human complement protein factor B.

Four cosmid clones, each with an average insert size of 40 kilobase pairs and containing the factor B gene, were isolated from a human genomic DNA library. The clones were identified by hybridization with a 515-base-pair cDNA probe isolated by using a unique 17-base synthetic oligonucleotide probe from a human liver cDNA library. The cosmid clones were characterized by restriction endonuclease digestion and Southern blotting, and a partial restriction map of the DNA represented in the cosmids was constructed. The Bb portion of the factor B gene is about 4 kb in length. DNA sequence analysis has resulted in the determination of 3.3 kb of sequence at the 3' end of the gene. This region codes for amino acids 87-505 of Bb and includes the whole of the serine proteinase domain of the protein. The three active site residues of histidine, aspartic acid, and serine found at positions 267, 317, and 440 of the Bb sequence, respectively, lie on separate exons. Other functional regions within the serine proteinase domain are separated also by intervening sequences.     

Identification of a second insulin-like growth factor in a fish species.

An internal portion of insulin-like growth factor (IGF) amplified from the total cDNA of rainbow trout (Oncorhynchus mykiss) liver by a PCR was used to screen a rainbow trout liver cDNA library, and recombinant clones encoding two distinct IGFs were isolated. On the basis of a 98.7% nucleotide and 98.3% predicted amino acid identity to coho salmon IGF-I, one cDNA sequence was identified as rainbow trout preproinsulin-like growth factor I (rtIGF-I). The second cDNA sequence shared 46.1% and 43.3% identity with rtIGF-I at the nucleotide and predicted amino acid levels, respectively, and was identified as rainbow trout preproinsulin-like growth factor II (rtIGF-II). Predicted amino acid sequence comparisons of rtIGFs with those of human IGFs indicate that rtIGF-I is more similar to human IGF-I than to human IGF-II, and that rtIGF-II is more similar to human IGF-II than to human IGF-I. Southern blot analysis of rainbow trout genomic DNA probed with rtIGF-I and -II cDNA suggests that these two forms of IGF originate from separate genes. The presence of a teleost IGF-II suggests that the divergence of IGFs occurred early in vertebrate evolution.     

Structure of the human hexabrachion (tenascin) gene.

The structure of the gene encoding human hexabrachion (tenascin) has been determined from overlapping clones isolated from a human genomic bacteriophage library. The genomic inserts were characterized by restriction mapping, Southern blot analysis, PCR, and DNA sequencing. The coding region of the hexabrachion gene spans approximately 80 kilobases of DNA and consists of 27 exons separated by 26 introns. The exon-intron structure supports a hypothesis based on the cDNA sequence that the hexabrachion gene is an assembly of DNA modules that are also found elsewhere in the genome. Single exons may encode a module, a portion of a module, or a group of modules. The 15 type III units similar to those found in fibronectin are each encoded either by a single exon or by two exons interrupted by an intron. All type III units known to be spliced out of the smaller forms of the protein are encoded by one exon. The fibrinogen-like domain of 210 amino acids is encoded by five exons. The 14.5 epidermal growth factor-like repeats are all encoded by a single exon.     

Molecular cloning and characterization of cDNA encoding the GTP-binding protein alpha i and identification of a related protein, alpha h.

We have cloned and characterized cDNA encoding alpha i, the GTP-binding subunit of Gi, a protein that mediates hormonal inhibition of adenylate cyclase and hormonal regulation of other membrane functions. We have also identified cDNA encoding a putative protein, which we have named alpha h, that is highly homologous to alpha i but different from other known GTP-binding proteins. Both cDNAs were isolated from a bovine pituitary library. The cDNA encoding alpha i was identified by finding that the amino acid sequence determined for two tryptic peptides from alpha i agreed exactly with amino acid sequences deduced from the cDNA. We also determined the amino acid sequence of peptides derived from alpha o, a related 39-kDa protein purified from bovine brain. These sequences are approximately 75% identical to the sequence determined for alpha i. Southern blot analysis of bovine genomic DNA, using as probes radiolabeled cDNAs for alpha i, alpha h, and the alpha subunit of a related protein, transducin, showed that each probe recognized different genomic DNA fragments. Our results suggest a further level of complexity in the organization of the G-protein gene family, with multiple G proteins of very similar structural properties likely to be identified as products of distinct genes.     

Cloning and nucleotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.

A 537-base cDNA encoding a portion of Saccharomyces cerevisiae dihydrolipoamide acetyltransferase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) was isolated from a lambda gt11 yeast cDNA library by immunoscreening. This cDNA was subcloned and used as a probe to screen a lambda gt11 yeast genomic DNA library. Two overlapping clones were used to determine the complete sequence of the acetyltransferase gene. The composite sequence has an open reading frame of 1446 nucleotides encoding a presequence of 28 amino acids and a mature protein of 454 amino acids (Mr = 48,546). The deduced amino acid sequence contains the experimentally determined amino acid sequences of the amino terminus and two internal peptide fragments of the acetyltransferase. Hybridization analysis of yeast genomic DNA showed that the gene has a single copy. A 915-base segment of the acetyltransferase gene hybridized to a yeast mRNA of approximately equal to 1.6 kilobases. Analysis of the deduced amino acid sequence of the dihydrolipoamide acetyltransferase revealed a multidomain structure similar to those reported for the corresponding acetyltransferases from Escherichia coli and rat liver, and extensive sequence similarity among the three enzymes. However, the yeast enzyme contains only one lipoyl domain, in contrast to three lipoyl domains reported for the E. coli enzyme and apparently two for the rat liver enzyme.     

Molecular cloning and characterization of cDNA encoding the alpha subunit of the rat protein synthesis initiation factor eIF-2B.

Eukaryotic initiation factor 2B (eIF-2B) is an essential component of the pathway of peptide-chain initiation in mammalian cells, yet little is known about its molecular structure and regulation. To investigate the structure, regulation, and interactions of the individual subunits of eIF-2B, we have begun to clone, characterize, and express the corresponding cDNAs. We report here the cloning and characterization of a 1510-bp cDNA encoding the alpha subunit of eIF-2B from a rat brain cDNA library. The cDNA contains an open reading frame of 918 bp encoding a polypeptide of 305 aa with a predicted molecular mass of 33.7 kDa. This cDNA recognizes a single RNA species approximately 1.6 kb in length on Northern blots of RNA from rat liver. The predicted amino acid sequence contains regions identical to the sequences of peptides derived from bovine liver eIF-2B alpha subunit. Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2B alpha in SDS/polyacrylamide gels. The predicted amino acid sequence exhibits 42% identity to that deduced for the Saccharomyces cerevisiae GCN3 protein, the smallest subunit of yeast eIF-2B. In addition, expression of the rat cDNA in yeast functionally complements a gcn3 deletion for the inability to induce histidine biosynthetic genes under the control of GCN4. These results strongly support the hypothesis that mammalian eIF-2 alpha and GCN3 are homologues. Southern blots indicate that the eIF-2B alpha cDNA also recognizes genomic DNA fragments from several other species, suggesting significant homology between the rat eIF-2B alpha gene and that from other species.     

Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA.

Poly(A)+ RNA, isolated from a single 7-mo fetal human aorta, was used to synthesize cDNA by the RNase H method, and the cDNA was inserted into lambda gt10. Recombinant phage containing elastin sequences were identified by hybridization with cloned, exon-containing fragments of the human elastin gene. Three clones containing inserts of 3.3, 2.7, and 2.3 kilobases were selected for further analysis. Three overlapping clones containing 17.8 kilobases of the human elastin gene were also isolated from genomic libraries. Complete sequence analysis of the six clones demonstrated that: the cDNA encompassed the entire translated portion of the mRNA encoding 786 amino acids, including several unusual hydrophilic amino acid sequences not previously identified in porcine tropoelastin, exons encoding either hydrophobic or crosslinking domains in the protein alternated in the gene, and a great abundance of Alu repetitive sequences occurred throughout the introns. The data also indicated substantial alternative splicing of the mRNA. These results suggest the potential for significant variation in the precise molecular structure of the elastic fiber in the human population.     

日本血吸虫RNA聚合酶Ⅱ转录因子SⅢ-p15亚单位的分离和序列分析

目的　分离和鉴定日本血吸虫 (Schistosomajaponisum ,Sj)新基因。 方法　应用表达序列标签 (Expressedse quencetags ,ESTs)法随机筛选SjcDNA文库 ,通过PCR直接序列测定技术和同源性比较对阳性插入片段进行鉴定。采用亚克隆技术和生物信息学分析对日本血吸虫RNA聚合酶Ⅱ转录延长因子SⅢ -p15亚单位进行结构和功能的鉴定。结果　我们从SjcDNA文库中随机筛选到一个cDNA克隆 ,它含有一个 371个碱基组成的阅读框 ,编码 118个氨基酸 ,与哺乳动物等的RNA聚合酶Ⅱ转录因子SⅢ - p15亚单位具有较高的同源性 ,且具有该因子所特有的氨基酸序列 ,表明我们已经获得了日本血吸虫RNA聚合酶Ⅱ转录因子SⅢ - p15亚单位。 结论　获得了日本血吸虫RNA聚合酶Ⅱ转录因子SⅢ - p15亚单位的全长cDNA序列 ,为该基因功能的实验性鉴定工作奠定基础     

The nucleotide sequence of the gene for human protein C.

A human genomic DNA library was screened for the gene for protein C by using a cDNA probe coding for the human protein. Three different overlapping lambda Charon 4A phage were isolated that contain inserts for the gene for protein C. The complete sequence of the gene was determined by the dideoxy method and shown to span about 11 kilobases of DNA. The coding and 3' noncoding portion of the gene consists of eight exons and seven introns. The eight exons code for a preproleader sequence of 42 amino acids, a light chain of 155 amino acids, a connecting dipeptide of Lys-Arg, and a heavy chain of 262 amino acids. The preproleader sequence and the connecting dipeptide are removed during processing, resulting in the mature protein composed of a heavy and a light chain held together by a disulfide bond. The heavy chain also contains the catalytic region for the serine protease. Two Alu sequences and two homologous repeats of about 160 nucleotides were found in intron E. The seven introns in the gene for protein C are located in essentially the same positions in the amino acid sequence as the seven introns in the gene for human factor IX, while the first three introns in protein C are located in the same positions as the first three in the gene for human prothrombin.