A novel crystallin from octopus lens

Lens crystallins were isolated from cephalopods, octopus and squid. Two protein fractions were obtained from the octopus in contrast to only one crystallin from the squid. The native molecular mass for these purified fractions and their polypeptide compositions were determined by gel filtration, sedimentation analysis, and SDS-gel electrophoresis. Octopod and decapod lenses share one common major squid-type crystallin of 29 kDa, with one additional novel crystallin present only in the octopus lens. This newly-characterized crystallin (termed omega-crystallin) exists as a tetrameric protein of 230 kDa, consisting of 4 identical subunits of approx. 59 kDa. It is distinct from the previously known crystallins both in amino acid composition and subunit structure. N-terminal sequence analysis indicated that the omega-crystallin is N-terminally blocked, whereas the major octopus crystallin is identical to the reported squid crystallin with regard to the first 25 residues of protein sequence. Sequence similarity between this major cephalopod crystallin and glutathione S-transferase were found, which suggested some enzymatic role of crystallins inside the cephalopod lens.     

Structure and targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Photosystem I reaction center of the cyanobacterium Synechocystis sp. PCC 6803 contains seven different polypeptide subunits. The subunit with a molecular mass of about 8 kDa was isolated, and the sequence of its amino-terminal residues was determined. Oligonucleotide probes corresponding to this sequence were used to isolate the gene encoding this subunit. The gene, termed as psaE, codes for a polypeptide with a mass of 8075 Da. It is present as a single copy in the genome and is transcribed as a monocistronic messenger. The amino acid sequence of the 8-kDa subunit deduced from the gene sequence shows high homology with the deduced amino acid sequence of subunit IV of photosystem I from spinach. The DNA fragment sequenced in these studies also contains two other unidentified major open reading frames. A stable deletion mutation for the psaE gene was generated by transforming Synechocystis sp. PCC 6803 with a cloned DNA in which the psaE gene for 8-kDa subunit was replaced by a gene conferring resistance to kanamycin. The mutant strain shows minor differences in growth under photoautotrophic conditions and in the photosystem I activity in comparison to the wild type.     

Human N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase cDNA is related to human B cell recombination activating gene-associated gene

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO(4))) in chondroitin sulfate and dermatan sulfate. We have previously purified the enzyme to apparent homogeneity from the squid cartilage. We report here cloning and characterization of human GalNAc4S-6ST. The strategy for identification of human GalNAc4S-6ST consisted of: 1) determination of the amino acid sequences of peptides derived from the purified squid GalNAc4S-6ST, 2) amplification of squid DNA by polymerase chain reaction, and 3) homology search using the amino acid sequence deduced from the squid DNA. The human GalNAc4S-6ST cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 561 amino acid residues. The recombinant protein expressed from the human GalNAc4S-6ST cDNA transferred sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the nonreducing terminal and internal GalNAc(4SO(4)) residues contained in chondroitin sulfate A and dermatan sulfate. When a trisaccharide and a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine residues were used as acceptors, only nonreducing terminal GalNAc(4SO(4)) residues were sulfated. The nucleotide sequence of the human GalNAc4S-6ST cDNA was nearly identical to the sequence of human B cell recombination activating gene-associated gene.     

Isolation and characterization of a cDNA clone encoding an 18-kDa hydrophobic photosystem I subunit (PSI-L) from barley (Hordeum vulgare L.)

Photosystem I in barley contains a polypeptide with an apparent molecular mass of 14 kDa. The polypeptide is N-terminally blocked to amino acid sequencing, but partial amino acid sequences have been determined from three fragments obtained by chemical and enzymatic cleavage. Using an oligonucleotide probe specifying this amino acid sequence, a full length cDNA clone was isolated. The deduced amino acid sequence does not correspond to any previously identified photosystem I subunit. We designate the novel photosystem I subunit PSI-L and the corresponding nuclear gene PsaL. The cDNA clone encodes a precursor polypeptide of 209 amino acid residues with a deduced molecular mass of 22,210 Da. The precursor has a transit peptide typical of proteins imported into chloroplasts. Based on a putative maturation site, the deduced molecular mass of the mature protein is 18 kDa. The PSI-L polypeptide is hydrophobic and predicted to have at least two membrane-spanning alpha-helices. Northern blot analysis shows that the expression of the PsaL gene is light-induced similar to other of the barley photosystem I genes. Southern blot analysis indicates that PsaL is a single copy gene. Partial amino acid sequences of an N-terminally blocked 9-kDa polypeptide show high sequence similarity to the PSI-G polypeptide of spinach and Chlamydomonas reinhardtii. The gene product of PsaG in spinach has previously been assigned as subunit V (Steppuhn, J., Hermans, J., Nechushtai, R., Ljungberg, U., Thümmler, F., Lottspeich, F., and Herrmann, R. G. (1988) FEBS Lett. 237, 218-224). The present study suggests that PSI-L is equivalent to subunit V and that PSI-G is a subunit migrating closely to PSI-H (subunit VI) and PSI-C (subunit VII).     

Purification of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83 and characterization of the gene (bphC).

The 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) of Pseudomonas putida OU83 was constitutively expressed and purified to apparent homogeneity. The apparent molecular mass of the native enzyme was 256 kDa, and the subunit molecular mass was 32 kDa. The data suggested that 2,3-DBPD was an octamer of identical subunits. The nucleotide sequence of a DNA fragment containing the bphC region was determined. The deduced protein sequence for 2,3-DBPD consisted of 292 amino acid residues, with a calculated molecular mass of 31.9 kDa, which was in agreement with data for the purified 2,3-DBPD. Nucleotide and amino acid sequence analyses of the bphC gene and its product, respectively, revealed that there was a high degree of homology between the OU83 bphC gene and the bphC genes of Pseudomonas cepacia LB400 and Pseudomonas pseudoalcaligenes KF707.     

Molecular cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was 85 degrees C and the enzyme exhibited a high level of heat stability.     

The primary structure of a cDNA for PsaN,encoding an extrinsic lumenal polypeptide of barley photosystem I

A cDNA clone encoding a 15.501 Da photosystem I (PSI) subunit of barley was isolated using an oligonucleotide based on the NH₂-terminal amino acid sequence of the isolated protein. The polypeptide, which migrates with an apparent molecular mass of 9.5 kDa on denaturing SDS-PAGE, has been designated PSI-N, and the corresponding gene is PsaN. Analysis of the deduced protein sequence indicates a mature protein of 85 amino acid residues and a molecular mass of 9818 Da. PSI-N is a hydrophilic, extrinsic protein with no predicted membrane-spanning regions. The transit peptide of 60 residues (5683 Da) contains a predicted hydrophobic -helix, suggesting that the protein is routed into the thylakoid lumen. Thus, PSI-N is the second known lumenal protein component associated with PSI, together with PSI-F.     

Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I

The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass of 24,563Da. The uncommon start codon TTG was identified by matching the N-terminal amino acid sequence of purified Nocardia GCH with the deduced amino acid sequence. A likely ribosomal binding site was identified 9bp upstream of the translational start site. The 3' end flank region encodes a peptide that shares high homology with dihydropteroate synthases. Nocardia GCH has 73 and 60% identity to the proteins encoded by the putative gch of Mycobacterium tuberculosis and Streptomyces coelicolor, respectively. Nocardia GCH was highly expressed in Escherichia coli cells carrying a pHAT10 based expression vector, and moderately expressed in Mycobacterium smegmatis cells carrying a pSMT3 based expression vector. Enterokinase digestion of recombinant Nocardia GCH, and in-gel digestion of Nocardia GCH and recombinant GCH followed by MALDI-TOF-MS analysis, confirmed that the actual subunit size of the enzyme was 24.5kDa. Thus, we conclude that the active form of native Nocardia GCH is a decamer. Our earlier incorrect conclusion was that the native enzyme was an octamer derived from the anomalous SDS-PAGE migration of the subunit.     

The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase.

We report characterization of the component proteins and molecular cloning of the genes encoding the two subunits of the carboxyltransferase component of the Escherichia coli acetyl-CoA carboxylase. Peptide mapping of the purified enzyme component indicates that the carboxyltransferase component is a complex of two nonidentical subunits, a 35-kDa alpha subunit and a 33-kDa beta subunit. The alpha subunit gene encodes a protein of 319 residues and is located immediately downstream of the polC gene (min 4.3 of the E. coli genetic map). The deduced amino acid composition, molecular mass, and amino acid sequence match those determined for the purified alpha subunit. Six sequenced internal peptides also match the deduced sequence. The amino-terminal sequence of the beta subunit was found within a previously identified open reading frame of unknown function called dedB and usg (min 50 of the E. coli genetic map) which encodes a protein of 304 residues. Comparative peptide mapping also indicates that the dedB/usg gene encodes the beta subunit. Moreover, the deduced molecular mass and amino acid composition of the dedB/usg-encoded protein closely match those determined for the beta subunit. The deduced amino acid sequences of alpha and beta subunits show marked sequence similarities to the COOH-terminal half and the NH2-terminal halves, respectively, of the rat propionyl-CoA carboxylase, a biotin-dependent carboxylase that catalyzes a similar carboxyltransferase reaction reaction. Several conserved regions which may function as CoA-binding sites are noted.     

Cloning and sequencing of the gene, fmtC, which affects oxacillin resistance in methicillin-resistant Staphylococcus aureus

Two Tn551 insertional mutants with reduced methicillin resistance were isolated from methicillin-resistant Staphylococcus aureus KSA8. These two mutants showed increased susceptibility to beta-lactam antibiotics and bacitracin, but not to fosfomycin and vancomycin. Tn551 in these mutants was inserted into the same gene, termed fmtC. The fmtC gene has an open reading frame of 840 amino acid residues with an estimated molecular mass of 96.9 kDa. The N-terminal half of the deduced FmtC protein is very hydrophobic, implying that this protein is a membrane-associated protein.     

The ribophorin I from Penaeus monodon shrimp: cDNA cloning, expression and phylogenetic analysis

Ribophorin I, a 67 kDa subunit of the oligosaccharyl transferase complex, is involved in facilitating N-linked glycosylation of polypeptides. We have isolated a full length Penaeus monodon cDNA encoding an insect/mammalian ribophorin I homologue by screening a lymphoid cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction of lymphoid RNA. The cDNA clone of shrimp ribophorin I (PmRibI) consists of 2263 nucleotides encoding 601 amino acid residues. Primary structure analysis of PmRibI indicated that it is a type I transmembrane protein, comprising a cleavable signal sequence of 23 residues at the amino terminus, preceding 434 residues of the luminal domain, 17 residues of the transmembrane domain, and 150 residues of the cytoplasmic domain at the carboxy terminus. The protein has a calculated molecular mass of 67.98 kDa with a pI of 6.05. This putative PmRibI cDNA clone was also expressed as PmRibI-6His in Sf9 cells. The recombinant PmRibI has an apparent molecular weight of 70 kDa, similar to the MW calculated from the deduced cDNA sequence. The inferred protein sequence of PmRibI has 52% identity with that of Strongylocentrotus purpuratus, 49% identity with that of Danio rerio, and 47% identity with mammalian ribophorin I. Phylogenetic analysis showed that PmRibI is most closely related to the echinoderm ribophorin I. The expression of the ribophorin I gene is tissue specific, with its mRNA highly abundant in hemocytes, gill, lymphoid organ and hepatopancreas.     

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.     

Purification,characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17

A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37 degrees C, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date.     

Molecular analysis of a Leptospira borgpetersenii gene encoding an endoflagellar subunit protein

A flagellin gene, flaB, from Leptospira borgpetersenii (formerly L. interrogans) serovar hardjo was cloned and expressed in Escherichia coli. Expression of the 32 kDa FlaB protein was dependent upon the lacZ promoter from pUC18. Nucleotide sequence data showed an open reading frame encoding 283 amino acid residues, corresponding to a protein of molecular mass 31.3 kDa. The G + C content of the flaB gene was 54.7 mol%. Comparison of the deduced FlaB amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum FlaB proteins.     

Granulin-like peptide in the mid-gut gland of the bivalve mollusk, Patinopecten yessoensis

A cysteine-rich polypeptide, termed CRP1, with a molecular mass of 5829 Da was found to occur in the mid-gut gland of the scallop Patinopecten yessoensis. CRP1 was purified by reverse phase and cation-exchange chromatographies. The amino acid sequence of CRP1 was deduced from its N-terminal amino acid sequence, amino acid composition and the sequence of a partial cDNA, indicating that CRP1 is a 57-amino-acid polypeptide containing 12 cysteine residues with a calculated molecular mass of 5841 Da (5829 Da when oxidized to form six disulfide bridges). A homology search of databases revealed that the deduced amino acid sequence of CRP1 displays significant similarity to those of granulin/epithelins, a family of growth-modulating factors; all cysteine residues in CRP1 are located at the same positions as those conserved characteristically in other known granulin/epithelins. Purified CRP1 inhibited the proliferation of mouse embryo cells. The results suggest that CRP1 functions as a growth-modulating factor in the scallop, and that granulin/epithelin family polypeptides and their precursors play physiologically important roles in invertebrates.     

嗜铁钩端螺旋菌(Leptospirillum ferriphilum)gyrB基因的PCR扩增、克隆与序列分析

根据GenBank和ICB数据库中gyrB蛋白氨基酸序列的两个保守区域TPGMYIG和QRY(F)KGLGEM设计简并引物,以L.ferriphilum菌株YSK基因组DNA为模板,PCR扩增出获得大小约为2.2kb的DNA片段.序列分析表明,菌株YSK的扩增片断的开放阅读框(ORF)能够推导出一个编码分子量约为82....     

Isolation and sequence analysis of a cDNA clone encoding a type-1 protein phosphatase catalytic subunit: homology with protein phosphatase 2A

A 1.5 kb clone containing the full-length coding sequence of a type-1 protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The protein sequence deduced from the cDNA contains 311 residues and has a molecular mass of 35.4 kDa. A single mRNA species at 1.6 kb was visualized by Northern blotting. The type-1 protein phosphatase was strikingly homologous to protein phosphatase 2A, 49% of the amino acids between residues 11 and 280 being identical. The first 10 and last 31 residues were dissimilar. Residues 1-101 of the type-1 protein phosphatase also showed 21% sequence identity with a region of mammalian alkaline phosphatases.