Isolation and sequencing of cDNAs for the XL-I and XL-III forms of bovine liver xenobiotic-metabolizing medium-chain fatty acid:CoA ligase

The XL-I form of xenobiotic-metabolizing medium-chain fatty acid:CoA ligase was previously purified to apparent homogeneity from bovine liver mitochondria, and the amino acid sequence of a short segment of the enzyme was determined. This sequence was used to develop a probe for screening a bovine cDNA library from which a 1.6 kb cDNA was isolated. This cDNA was sequenced and found to contain the code for the known amino acid sequence. The complete open reading frame was not present in this cDNA, but it was estimated to code for approximately 75% of the XL-I sequence. The XL-III ligase was purified to apparent homogeneity from bovine liver mitochondria. The enzyme eluted from a gel filtration column as a single peak with an apparent molecular weight of ca. 55,000. It ran as a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 62 kDa. N-Terminal sequence analysis of the enzyme gave no sequence, which indicates a blocked N-terminus. The enzyme was chemically cleaved using CNBr. The resulting peptides were separated by SDS-PAGE. The cleavage pattern revealed two large peptides of ca. 21 and 25 kDa, plus several smaller peptides including a prominent 6 kDa peptide. The N-terminus of the 6, 21, and 25 kDa peptides was sequenced and the 21 and 25 kDa sequences were identical indicating incomplete cleavage. The sequences were used to design probes for screening a bovine liver cDNA library. This resulted in the isolation of a 2,065 bp cDNA. This cDNA was sequenced and found to contain the initiation and termination codons, as well as the requisite amino acid sequences. The open reading frame coded for a 64,922 Da protein. The sequence of XL-III cDNA was markedly different from that of XL-I, indicating the genetic uniqueness of the two ligases. They are, however, 64% homologous, which suggests a common evolutionary origin.     

Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits

Near-full-length cDNA clones to the small and large subunit of the heterotetrameric potato tuber ADP-glucose pyrophosphorylase have been isolated and characterized. The missing amino terminal sequence of the small subunit has also been elucidated from its corresponding genomic clone. Primary sequence comparisons revealed that each potato subunit had less identity to each other than to their homologous subunit from other plants. It also appeared that the smaller subunit is more conserved among the different plants and the larger subunit more divergent. Amino acid comparisons of both potato tuber sequences to theEscherichia coli ADP-glucose pyrophosphorylase sequence revealed conserved regions important for both catalytic and allosteric function of the bacterial enzyme.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: I. Sequence of trpG encoding the glutamine amidotransferase subunit

We have determined the DNA sequence of the distal 148 codons of trpE and all of trpG in Pseudomonas aeruginosa. These genes encode, respectively, the large and small (glutamine amidotransferase) subunits of anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. The sequenced region of trpE is homologous with the distal portion of E. coli and Bacillus subtilis trpE, whereas the trpG sequence is homologous to the glutamine amidotransferase subunit genes of a number of bacterial and fungal anthranilate synthases. The two coding sequences overlap by 23 bp. Codon usage in these Pseudomonas genes shows a marked preference for codons ending in G or C, thereby resembling that of trpB, trpA, and several other chromosomal loci from this species and others with a high G + C content in their DNA. The deduced amino acid sequence for the P. aeruginosa trpG gene product differs to a surprising extent from the directly determined amino acid sequence of the glutamine amidotransferase subunit of P. putida anthranilate synthase (Kawamura et al. 1978). This suggests that these two proteins are encoded by loci that duplicated much earlier in the phylogeny of these organisms but have recently assumed the same function. We have also determined 490 bp of DNA sequence distal to trpG but have not ascertained the function of this segment, though it is rich in dyad symmetries.      

The subunit interfaces of oligomeric enzymes are conserved to a similar extent to the overall protein sequences.

It is well established that, within families of homologous enzymes, amino acid residues that are involved in the chemistry of the reaction are highly conserved. To determine if residues at the subunit interface of oligomeric enzymes with shared active sites are also conserved, comparative analysis of five enzyme families was undertaken. For the chosen enzyme families, sequence data were available for a large number of proteins and a three-dimensional structure was known for at least two members of each family. The analysis indicates that the subunit interface and the hydrophobic core of proteins from all five families have diverged to a similar extent to the overall protein sequences.     

Cloning and characterization of the bovine testicular PH-20 hyaluronidase core domain

The core nucleotide sequence of bovine (Bos taurus) testicular PH-20 hyaluronidase was cloned using one step RT-PCR. The 5' and 3' regions were cloned separately and a sequence overlap of 124 bp facilitated the fusion of these two fragments by overlapping PCR, resulting in a concatenated sequence of 1422 bp. This nucleotide sequence and its deduced amino acid sequence were compared to homologous sequences from eight other mammal species. The bovine sequences were most similar to those of the pig, Sus scrofa (swine Spam1: 79.1% nucleotide and 70.1% amino acid similarity) and least similar to sequences from the Norway rat, Rattus norvegicus (murine Spam1: 61% nucleotide and 53.3% amino acid similarity). A phylogenetic analysis joined the red fox (Vulpes vulpes) sequence as sister to the bull-pig pair. Twelve cysteine residues were conserved among all nine aligned amino acid sequences and five proposed glycosylation sites have been identified. The feasibility of developing an effective, low-cost bovine PH-20 expression system is discussed in light of these new data.     

ATP synthase of yeast mitochondria. Characterization of subunit d and sequence analysis of the structural gene ATP7.

The subunit analogous to the d-subunit of ATP synthase from bovine heart mitochondria was isolated from the purified yeast enzyme. Partial protein sequences were determined by direct methods. From this information, two oligonucleotide probes were constructed and used for screening a DNA genomic bank of Saccharomyces cerevisiae. The sequence of yeast subunit d was deduced from the DNA sequence of ATP7 gene. Mature yeast subunit d is 173 amino acids long. Its NH2-terminal serine is blocked by an N-acetyl group, and the protein has no processed NH2-terminal sequence other than the removal of the initiator methionine. The protein is predominantly hydrophilic. The amino acid sequence is 22% identical and 44% homologous to bovine subunit d. A null mutant was constructed. The mutant strain was unable to grow on glycerol medium. The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity, and the catalytic sector F1 was loosely bound to the membranous part. The mutant mitochondria did not contain subunit d, and the mitochondrially encoded hydrophobic subunit 6 was not present.     

Heterogeneity in human soluble guanylate cyclase due to alternative splicing.

Two forms of the smaller subunit of the human soluble guanylate cyclase enzyme have been cloned by using PCR. One of the clones (HSGC-1) is identical to bovine and rat lung smaller subunit cyclase. However, the other (HSGC-2) is lacking 33 amino acids. Comparison of its sequence with published partial genomic sequences of bovine guanylate cyclase indicates that HSGC-2 is formed due to alternative splicing.     

牛卵巢PRSS35基因CDS序列分析

为测定牛卵巢丝氨酸蛋白酶35 (PRSS35)的CDS序列并进行生物信息学分析。试验根据NCBI上已公布牛PRSS35基因的mRNA序列设计特异性引物,使用RT-PCR技术扩增牛卵泡中PRSS35的CDS序列。结果显示,牛PRSS35基因CDS区序列全长为1 239 bp,共编码412个氨基酸,PRSS35与其他10个物种的同源序列相似性较高,且该蛋白具有一个长度为20个氨基酸的信号肽,具有11个O-糖基化位点和2个N-糖基化位点,以及3个磷酸化位点,并发现有一个典型的Tryp_Spc结构域,即胰蛋白酶样丝氨酸蛋白酶结构域。为进一步研究该基因及其编码蛋白在卵泡发育过程中所起的作用提供了一定的理论依据。     

Legumain from bovine kidney: its purification, molecular cloning, immunohistochemical localization and degradation of annexin II and vitamin D-binding protein

Legumain (asparaginyl endopeptidase) was purified to homogeneity from bovine kidneys. The molecular mass of the purified enzyme was calculated to be 34000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of beta-mercaptoethanol. The enzyme rapidly hydrolyzed the substrate Z-Ala-Ala-Asn-MCA and was strongly inhibited by N-ethylmaleimide, p-chloromercuribenzene-sulfonic acid, Hg(2+) and Cu(2+). The amino acid sequence of the first 26 residues of the enzyme was Gly-Gly-Lys-His-Trp-Val-Val-Ile-Val-Ala-Gly-Ser-Asn-Gly-Gln-Tyr-Asn-Tyr-Arg-His-Gln-Ala-Phe-Ala-Asp-His-. This sequence is highly homologous to the sequences in the N-terminal of pig kidney legumain. We screened a bovine kidney cortex cDNA library using a DNA probe that originated from rat legumain, and we determined the bovine kidney cDNA structure and deduced the amino acid sequence. The cDNA is composed 1934 bp and encodes 433 amino acids in the coding region. The enzyme was strongly stained in the proximal tubules of the rat kidney in an immunohistochemical study. Vitamin D-binding protein which is known to be a ligand to megalin existing in the proximal tubules, was cleaved in a limited proteolytic manner by bovine kidney legumain. These results suggested that legumain contributes to the processing of macromolecules absorbed by proximal tubule cells. The enzyme also cleaved an N-terminal synthetic peptide of bovine annexin II (Gly(24)-Ser-Val-Lys-Ala-Tyr-Thr(30)-Asn-Phe-Asp-Ala-Glu(35)-Arg-Asp(37)) at a position between Asn(31) and Phe(32). The amino-terminal domain of annexin II has p11 subunit binding sites and phosphorylation sites for both pp60(src) and protein kinase C. This suggests that legumain plays an important role in inactivation and degradation of annexin II, which is abundant in the receptor-recycling compartments of endosomes/lysosomes.     

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.     

Sequence analysis of the catalytic subunit of H(+)-ATPase from porcine renal brush-border membranes.

The catalytic subunit of the H(+)-ATPase from brush-border membranes of porcine renal proximal tubules was labeled with the hydrophobic SH-group reagent 10-N-(bromoacetyl)amino-1-decyl-beta-glucopyranoside (BADG) which irreversibly inhibits proton pump activity in the absence but not in the presence of ATP. The labeled protein was purified and digested with proteinases. After isolation and sequencing of proteolytic peptides two BADG-labeled cysteines were identified. The amino acid sequences of the obtained proteolytic peptides were homologous to the catalytic subunit of V-ATPases. From mRNA of porcine kidney cortex a catalytic H(+)-ATPase subunit was cloned. 181 of the 183 amino acids which overlap in the sequence derived from the cDNA and the proteolytic peptides were identical, and the two deviations are due to single base exchanges. A comparison of the amino acid sequence derived from the cloned cDNA with sequences of catalytic H(+)-ATPase subunits communicated by other laboratories revealed 98%, 96% and 94% identity with sequences from bovine adrenal medulla, from bovine kidney medulla and from clathrin-coated vesicles of bovine brain. Between 64% and 69% identity was obtained with sequences from fungi and plants. The data show that the catalytic subunit of V-ATPases is highly conserved during evolution. They indicate organ and species specificity in mammalians.     

Cloning and nucleotide sequence of the gene coding for a subunit of the respiratory NADH dehydrogenase from Bacillus stearothermophilus

The putative gene coding for a subunit of the respiratory NADH dehydrogenase from Bacillus stearothermophilus was cloned in Escherichia coli and the nucleotide sequence was determined. A large open reading frame (ORF1) was recognized, which was composed of 879 bp corresponding to 293 amino acids and a molecular weight of 33,600. Possible promoter and Shine-Dalgarno sequences were found upstream from the initiation codon. The deduced amino acid sequence of the gene was homologous to the NADH dehydrogenase of Paramecium aurelia.     

羊草光合作用相关基因的克隆与分析

在构建了羊草叶片cDNA文库的基础上,利用M13载体通用引物筛选其亚文库,挑选阳性克隆进行测序,将测序结果在NCBI基因库中进行比对,得到一个Rubisco大亚基基因全长序列和Rubisco小亚基基因部分序列,并对其核苷酸及其编码的氨基酸序列进行分析。结果显示,Rubisco大亚基基因长度为1 796 bp,与禾本科大麦、小麦、野雀麦、粗山羊草、旱麦草、异形花草、黑麦等的核苷酸序列同源性达98%以上;羊草的Rubisco小亚基基因部分序列含有一个开放阅读框,其长度为186 bp,编码61个氨基酸,与禾本科的小麦、大麦、燕麦、黑麦以及扁穗雀麦Rubisco小亚基基因氨基酸序列的同源性分别为93%、93%、91%、91%、92%。羊草Rubisco基因的克隆与分析有利于进一步研究其光合作用效率。     

A-type ATP binding consensus sequences are critical for the catalytic activity of the calmodulin-sensitive adenylyl cyclase from Bacillus anthracis

Analysis of the predicted amino acid sequence of Bacillus anthracis adenylyl cyclase revealed sequences with homology to consensus sequences for A- and B-type ATP binding domains found in many ATP binding proteins. Based on the analysis of nucleotide binding proteins, a conserved basic amino acid residue in the A-type consensus sequence and a conserved acidic amino acid residue in the B-type consensus sequence have been implicated in the binding of ATP. The putative ATP binding sequences in the B. anthracis adenylyl cyclase possess analogous lysine residues at positions 346 and 353 within two A-type consensus sequences and a glutamate residue at position 436 within a B-type consensus sequence. The two A-type consensus sequences overlap each other and have the opposite orientation. To determine whether Lys-346, Lys-353, or Glu-436 of the B. anthracis adenylyl cyclase are crucial for enzyme activity, Lys-346 and Lys-353 were replaced with methionine and Glu-436 with glutamine by oligonucleotide-directed mutagenesis. Furthermore, Lys-346 was also replaced with arginine. The genes encoding the wild type and mutant adenylyl cyclases were placed under the control of the lac promoter for expression in Escherichia coli, and extracts were assayed for adenylyl cyclase activity. In all cases, a 90-kDa polypeptide corresponding to the catalytic subunit of the enzyme was detected in E. coli extracts by rabbit polyclonal antibodies raised against the purified B. anthracis adenylyl cyclase. The proteins with the Lys-346 to methionine or arginine mutations exhibited no adenylyl cyclase activity, indicating that Lys-346 in the A-type ATP binding consensus sequence plays a critical role for enzyme catalysis. Furthermore, the enzyme with the Lys-353 to methionine mutation was also inactive, suggesting that Lys-353 may also directly contribute to enzyme catalysis. In contrast, the protein with the Glu-436 to glutamine mutation retained 75% of enzyme activity, suggesting that Glu-436 in the B-type ATP binding consensus sequence may not be directly involved in enzyme catalysis. It is concluded that Lys-346 and Lys-353 in B. anthracis adenylyl cyclase may interact directly with ATP and contribute to the binding of the nucleotide to the enzyme.     

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.     

Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei

In Trypanosoma brucei (stock 427) genes encoding the glycolytic enzyme pyruvate kinase are present on two homologous chromosomes. We have cloned and characterized one of the alleles. Two large, tandemly arranged open reading frames were found, each coding for a pyruvate kinase polypeptide of 498 amino acids. The gene sequences differ at 15 positions, resulting in five amino acid substitutions. The calculated molecular masses of the polypeptides are 54,378 Da and 54,363 Da. These values are somewhat smaller than those reported for the subunit molecular mass of the purified protein, which is 57-59 kDa. However, in vitro translation of the DNA region corresponding to the open reading frame, and translation of the RNA in a wheat-germ lysate, yielded a product that comigrated exactly with the native polypeptide in SDS/PAGE. The overall identity between the sequences of the trypanosomal enzyme and the enzymes from other sources is 41-51%. The conserved residues are not equally distributed over the polypeptide. The primary structure of domains A and, to a lesser extent, B, which constitute the active site, are rather well conserved. In contrast, the sequence of domain C, which supposedly is involved in the regulation of the enzyme activity, is much more variable. The cytosolically located pyruvate kinase of T. brucei lacks the specific features found in the majority of the glycolytic enzymes of this organism that are sequestered in a microbody-like organelle, the glycosome. It has neither a relatively high subunit molecular mass, due to unique insertions or terminal extensions, nor a high excess of positively charged amino acids. The polypeptide is shorter than that of most other pyruvate kinases and the calculated net charge is only +3.     

Identification of the goldfish 20S proteasome beta6 subunit bound to nuclear matrix

Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most of the regulated intracellular protein breakdown in eukaryotic cells. Proteasomes are present in both the nucleus and cytoplasm. When we analyzed the molecular composition of protein constituents of the nuclear matrix preparation of goldfish oocytes by two-dimensional polyacrylamide gel electrophoresis followed by sequence analysis, we found a 26 kDa spot identical in amino acid sequence to the beta6 subunits of the 20S proteasome. No spot of other subunits of 20S proteasome was detected. Here we describe the cloning, sequencing and expression analysis of Carassius auratus, beta6_ca, which encodes one of the proteasome beta subunits from goldfish ovary. From the screening of an ovarian cDNA library, two types of cDNA were obtained, one 941 bp and the other 884 bp long. The deduced amino acid sequences comprise 239 and 238 residues, respectively. These deduced amino acid sequences are highly homologous to those of beta6 subunits of other vertebrates. Immunoblot analysis of nuclear matrix using anti-proteasome antibodies showed only a spot of beta6_ca. These results suggest that the beta6 subunit of the goldfish 20S proteasome, beta6_ca, is responsible for anchoring proteasomes in the nucleus.     

cDNA clones encoding bovine gamma-crystallins

We have determined the nucleotide sequence of two bovine lens gamma-crystallin cDNA clones, pBL gamma II-1 and pBL gamma III-1. The 644 bp cDNA insert of pBL gamma II-1 contains coding information for the entire amino acid sequence of bovine gamma II-crystallin. The 497 bp cDNA insert of pBL gamma III-1 encodes a homologous but different gamma-crystallin polypeptide, and appears to lack the coding information for the C-terminal 17 amino acid residues. While the nucleotide and predicted amino acid sequences of the coding regions of the clones show a high degree of homology, the untranslated leader sequences are relatively dissimilar. The leader sequence of pBL gamma III-1 is strikingly homologous to a portion of a rabbit immunoglobulin alpha-heavy chain mRNA.     

Bovine hexokinase type I: Full-length cDNA sequence and characterisation of the recombinant enzyme

This study reports the revised and full-length cDNA sequence of bovine hexokinase type I obtained from bovine brain. Since dissimilarities have been observed between the published bovine hexokinase type I coding sequence (GenBank accession no. M65140) (Genomics 11: 1014-1024, 1991) and an analysed portion of bovine hexokinase type I gene, the entire open reading frame was re-sequenced and the ends of cDNA isolated by rapid amplification of cDNA ends. The coding sequences, when compared with the published bovine hexokinase type I, contained a large number of mismatches that lead to changes in the resulting amino acid sequence. The revisions result in a hexokinase type I cDNA of 3619 bp that encodes a protein of 917 amino acids highly homologous to human hexokinase type I. The expression of the recombinant full-length enzyme demonstrated that it was a catalytically active hexokinase. When characterised for its kinetic and regulatory properties, it displayed the same affinity for glucose and MgATP as the human hexokinase type I and was inhibited by glucose 6-phosphate competitively versus MgATP. The production of the N- and C-terminal recombinant halves of the enzyme followed by comparison with the full-length hexokinase indicated that the catalytic activity is located in the C-terminal domain. (Mol Cell Biochem 268: 9–18, 2005)