Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

RNA editing of wheat mitochondrial ATP synthase subunit 9: direct protein and cDNA sequencing.

RNA editing of subunit 9 of the wheat mitochondrial ATP synthase has been studied by cDNA and protein sequence analysis. Most of the cDNA clones sequenced (95%) showed that editing by C-to-U transitions occurred at eight positions in the coding region. Consequently, 5 amino acids were changed in the protein when compared with the sequence predicted from the gene. Two edited codons gave no changes (silent editing). One of the C-to-U transitions generated a stop codon by modifying the arginine codon CGA to UGA. Thus, the protein produced is 6 amino acids shorter than that deduced from the genomic sequence. Minor forms of cDNA with partial or overedited sequences were also found. Protein sequence and amino acid composition analyses confirmed the results obtained by cDNA sequencing and showed that the major form of edited atp9 mRNA is translated.     

Isolation and amino acid sequence analysis of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase.

3 beta-Hydroxysteroid dehydrogenase/steroid isomerase has been purified to homogeneity from bovine adrenal glands. A single protein of molecular weight 42,090 +/- 40 containing both enzyme activities has been isolated. Approximately 86% of the amino acid sequence of the bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase has been obtained by sequencing peptides isolated from digests with trypsin and lysyl endopeptidase and by chemical cleavage with CNBr. The sequence obtained is identical with that of the deduced amino acid sequence of the bovine ovarian 3 beta-hydroxysteroid dehydrogenase/steroid isomerase [Zhao et al. (1989) FEBS Lett. 259, 153-157], with the exception that the N-terminal methionine residue found in the bovine ovarian sequence is not present in the mature bovine adrenal enzyme. On the basis of the primary structure and comparisons with other NAD+ binding proteins, we propose a structural model of the bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase localizing the NAD+ binding site as well as the membrane-anchoring segment.     

Cloning and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.     

DNA sequencing of the eta gene coding for staphylococcal exfoliative toxin serotype A

We report the nucleotide sequence of a 1.45 kb segment containing the eta gene, coding for staphylococcal exfoliative toxin A (ETA), isolated from the recombinant plasmid pETA-J3. The coding region of 840 bp specified a polypeptide of 280 amino acid residues which included a putative 38 residue signal sequence. The amino acid composition deduced from the structural gene was in agreement with the results of peptide analysis of the ETA molecule reported by others. The sequence of the 35 N-terminal amino acid residues of ETA derived from Staphylococcus aureus strain ZM was also consistent with that deduced from the DNA sequencing.     

地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增、克隆及序列测定

在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3％左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。     

The nucleotide sequence of a cDNA clone encoding the wheat Em protein.

The nucleotide sequence of a cDNA clone isolated from developing wheat embryos and encoding the Em protein is reported. The entire coding region for Em and the 3' non-translated flank are contained within this clone. The amino acid sequence deduced for Em is very rich in glycine (18 mol%) as well as both basic and acidic residues. The molecular weight of the protein is ca. 9,900 daltons. The deduced sequence is supported by direct amino acid sequencing of cyanogen bromide cleavage fragments obtained from purified Em protein. Em is shown by Southern blots to be a product of a gene family of approximately ten members.     

西瓜八氢番茄红素合成酶基因片段的克隆和序列分析

以西瓜品种ZXG00152为材料,提取瓜瓤总RNA,进行反转录,依据植物八氢番茄红素合成酶(PSY)的氨基酸保守序列设计引物,以cDNA第一链为模板,扩增得到长约750 bp的cDNA片段。将该片段克隆到pMD19-T载体,测序结果表明该基因片段长748 bp,编码249个氨基酸,Blast搜索结果表明,由该片段推导出的氨基酸序列与其它植物的八氢番茄红素合成酶有较高的同源性,其中与甜瓜的一致性达到97.8%。该片段已在GenBank中登录(登录号:DQ494214)。     

Isolation and sequence of a cDNA clone for the rat pulmonary surfactant-associated protein (PSP-A)

Pulmonary surfactant is composed mainly of phospholipid and two groups of apoproteins. One of these apoproteins is a family of glycoproteins (pulmonary surfactant-associated protein A, PSP-A). We have isolated and sequenced a cDNA clone encoding for rat PSP-A and the full amino acid sequence has been deduced from the nucleotide sequence. The sequence of 56 amino acids at the N-terminus of PSP-A isolated from rats treated with silica was determined independently, and there is complete agreement with the sequence deduced from the cDNA. Isolated rat alveolar type II cells contain two species of mRNA for this protein.     

Nucleotide sequence and linkage map position of the secX gene in maize chloroplast and evidence that it encodes a protein belonging to the 50S ribosomal subunit

The nucleotide sequence of the segment of maize chloroplast DNA lying between the map coordinate positions 32.59 and 32.98 Kb and containing the secX gene has been determined. The derived amino acid sequence of maize chloroplast secX is 95%, 87% and 62% identical to the corresponding derived amino acid sequences from two plant chloroplasts and Escherichia coli, respectively. It is also 70% identical to the experimentally determined amino acid sequence of a protein isolated from Bacillus stearothermophilus ribosomes. Separation of the 50S ribosomal subunit proteins of E. coli by reversed phase HPLC gave a peak which contained pure secX protein, as determined by N-terminal amino acid sequencing. Spinach chloroplast 50S subunit proteins separated by HPLC also gave a peak corresponding to pure secX protein. From these results we conclude that the secX gene in E. coli and in plant chloroplasts encodes a small (37-38 amino acid residues) ribosomal protein belonging to the 50S subunit. The same conclusion has been reached recently by A. Wada with respect to E. coli secX. In agreement with Wada, we name the secX protein L36. Its chloroplast gene is designated rpL36.     

Primary structure of maize pyruvate, orthophosphate dikinase as deduced from cDNA sequence

We have isolated two overlapping cDNA clones that encompass the entire structural gene for pyruvate, orthophosphate dikinase from maize. The analysis of the nucleotide sequence has revealed that the cDNA clones include an insert of a total of 3,171 nucleotides without a poly(A) tail and encode a polypeptide that contains 947 amino acid residues and has a molecular weight of 102,673. Comparison of the N-terminal amino acid sequence of purified pyruvate, orthophosphate dikinase protein with that deduced from the nucleotide sequence shows that the mature form of pyruvate, orthophosphate dikinase in the maize chloroplast consists of 876 amino acid residues and has a molecular weight of 95,353. The amino acid composition of the deduced sequence of pyruvate, orthophosphate dikinase is in good agreement with that of the purified enzyme. The region that contains the active and regulatory sites of pyruvate, orthophosphate dikinase can be found in the deduced sequence of amino acids. We have predicted the secondary structure and calculated the hydropathy pattern of this region. The extra 71 residues at the N terminus of the deduced sequence of amino acid residues corresponds to the transit peptide which is indispensable for the transport of the precursor protein into chloroplasts. We have compared the primary structure of the pyruvate, orthophosphate dikinase transit peptide to those of other proteins and found sequences similar to the consensus sequences found in other transit peptides.     

The primary structure of the Chloroflexus aurantiacus reaction-center polypeptides

The complete nucleotide sequence of two Chloroflexus aurantiacus reaction-center genes has been obtained. The amino acid sequence deduced from the first gene showed 40% similarity to the L subunit of the Rhodobacter sphaeroides reaction center. This L subunit was 310 amino acids long and had an approximate molecular mass of 35 kDa. The second gene began 17 bases downstream from the first gene. The amino acid sequence deduced from it (307 amino acids; 34950 Da) was 42% similar to the M subunit of the Rhodobacter sphaeroides reaction center. 20% of the deduced primary structure were confirmed through automated Edman degradation of cyanogen bromide peptide fragments or N-chlorosuccinimide peptide fragments isolated from the purified reaction-center complex or from the individual subunits. The peptides were isolated by preparative gel electrophoresis combined with molecular sieve chromatography in the presence of a mixture of formic acid, acetonitrile, 2-propanol and water. This method appeared to be applicable to the isolation of other hydrophobic proteins and their peptides.     

Unusual and Strongly Structured Sequence Variation in a Complex Satellite DNA Family from the Nematode Meloidogyne chitwoodi

An AluI satellite DNA family has been isolated in the genome of the root-knot nematode Meloidogyne chitwoodi. This repeated sequence was shown to be present at approximately 11,400 copies per haploid genome, and represents about 3.5% of the total genomic DNA. Nineteen monomers were cloned and sequenced. Their length ranged from 142 to 180 bp, and their A + T content was high (from 65.7 to 79.1%), with frequent runs of As and Ts. An unexpected heterogeneity in primary structure was observed between monomers, and multiple alignment analysis showed that the 19 repeats could be unambiguously clustered in six subfamilies. A consensus sequence has been deduced for each subfamily, within which the number of positions conserved is very high, ranging from 86.7% to 98.6%. Even though blocks of conserved regions could be observed, multiple alignment of the six consensus sequences did not enable the establishment of a general unambiguous consensus sequence. Screening of the six consensus sequences for evidence of internal repeated subunits revealed a 6-bp motif (AAATTT), present in both direct and inverted orientation. This motif was found up to nine times in the consensus sequences, also with the occurrence of degenerated subrepeats. Along with the meiotic parthenogenetic mode of reproduction of this nematode, such structural features may argue for the evolution of this satellite DNA family either (1) from a common ancestral sequence by amplification followed by mechanisms of sequence divergence, or (2) through independent mutations of the ancestral sequence in isolated amphimictic nematode populations and subsequent hybridization events. Overall, our results suggest the ancient origin of this satellite DNA family, and may reflect for M. chitwoodi a phylogenetic position close to the ancestral amphimictic forms of root-knot nematodes. Received: 23 April 1997 / Accepted: 9 July 1997     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.     

cDNA cloning, genomic structure, chromosomal mapping, and expression analysis of parotid secretory protein in pig

A novel cDNA has been isolated from pig parotid glands by 3' and 5' rapid amplification of cDNA ends and designated parotid secretory protein (PSP). The open reading frame of this cDNA covers 714 bases, encoding 238 amino acids, which show 56% identity with human PSP at the level of the primary protein structure. The PSP genomic sequence comprises eight exons and seven introns, is approximately 22 kb in size, determined by sequencing, and maps to pig chromosome 17q21-q23. RT-PCR, dot blot, and Northern blot analyses demonstrated that PSP is strongly expressed in parotid glands, but is not present in heart, liver, lung, kidney, muscle, or stomach. A search for functionally significant protein motifs revealed consensus sequences for casein kinase II phosphorylation and N-myristoylation. We observed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu-X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is similar to the leucine zipper.     

EST sequencing of Onychophora and phylogenomic analysis of Metazoa

Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873 5' cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs. BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than 10(-4). Most ESTs had the best hit with proteins from either Chordata or Arthropoda (approximately 40% respectively). The ESTs included sequences of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases, including other EST projects. A concatenated amino acid alignment comprising 5021 positions was constructed, which covers 4259 positions when problematic regions were removed. Bayesian and maximum likelihood methods place Epiperipatus within the monophyletic Ecdysozoa (Onychophora, Arthropoda, Tardigrada and Nematoda), but its exact relation to the Euarthropoda remained unresolved. The "Articulata" concept was not supported. Tardigrada and Nematoda formed a well-supported monophylum, suggesting that Tardigrada are actually Cycloneuralia. In agreement with previous studies, we have demonstrated that random sequencing of cDNAs results in sequence information suitable for phylogenomic approaches to resolve metazoan relationships.