Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus.

We have isolated a cDNA clone encoding a novel calcium-binding protein of the recoverin family from rat brain cDNA library. This clone (PCB11) has 588 nucleotides in the open reading frame including the termination codon, 174 nucleotides of the 5' leader and 800 nucleotides of the 3' noncoding region. The complete amino acid sequence deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22,574 Daltons, and contains three putative calcium-binding domains of the EF-hand structure. The deduced amino acid sequence has a striking sequence homology to those of the retinal recoverin family (recoverin, visinin, P26, 23kD protein, S-modulin) and the brain-derived recoverin family (P23k, 21-kDa CaBP and neurocalcin). Northern blot, in situ hybridization, immunoblot and immunohistochemical analyses revealed that the protein is exclusively expressed in pyramidal layer of the hippocampus. The protein was therefore designated hippocalcin.     

Neurocalcin: a novel calcium-binding protein from bovine brain.

A novel calcium-binding protein (molecular weight 23,000-24,000, pI 5.3-5.5), which we term neurocalcin, was identified in bovine brain. Using calcium-dependent drug affinity chromatography ((S)-P-(2-aminoethyloxy)-N-[2-(4-benzyloxycarbonylpiperazinyl++ +)-1-(P- methoxybenzyl)ethyl]-N-methylbenzene-sulfonamide dihydrochloride, W-77, -coupled Sepharose 6B), we purified neurocalcin from bovine brain. The partial amino acid sequence of neurocalcin revealed it to be an as yet unidentified protein with three putative calcium binding sites (EF-hands). Further purification and sequence analysis demonstrated the presence of four isoprotein forms designated alpha, beta, gamma 1, and gamma 2. When the 165 sequenced residues of neurocalcin beta are compared with sequences of other proteins, neurocalcin beta has a 38.2% sequence homology with visinin and 45.5% with recoverin (Yamagata, K., Goto, K., Kuo, C.-H., Kondo, H., and Miki, N. (1990) Neuron 2, 469-476; Dizhoor, A. M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A., Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918). Both visinin and recoverin are expressed specifically in retinal photoreceptors and are not found in brain. Unlike visinin and recoverin, neurocalcin is purified not only from retina but also from bovine brain. Our results suggest that neurocalcin is a recoverin-like protein expressed in bovine brain.     

Molecular cloning, mapping and characterization of the human neurocalcin delta gene (NCALD)

We identified a new human gene that encodes a cognate of the bovine neurocalcin delta from a human fetal brain cDNA library; hence we named it human neurocalcin delta (NCALD) gene. The deduced polypeptide product of the cDNA is 22 kDa in size, and its amino acid sequence is 100% and 99% identical to that of the bovine and chicken neurocalcin, respectively. Northern blots showed that the NCALD gene is more abundantly expressed in brain, testis, ovary and small intestine. Tissue in situ hybridization confirmed the existence of the NCALD mRNA in the adult human testis. Radiation hybrid panel mapping localized the gene to chromosome 8 between molecular markers D8S270 and D8S257.     

The nucleotide sequence and derived amino acid sequence of cDNA coding for mouse carbonic anhydrase II

The nucleotide sequence of a clone containing mouse carbonic anhydrase (CA) cDNA in pBR322 has been determined. The cloned cDNA contains all of the coding region except for nucleotides specifying the first eight amino acids, and all of the 3' noncoding region, which consists of 700 nucleotides. A cDNA clone was identified which contains an additional 54 bp at the 5' end, so that the complete amino acid sequence of mouse CA could be deduced. This sequence showed a 73-81% homology with other mammalian CA form II isozymes, 56-63% with form I isozymes, and 52-56% with form III isozymes. By examination of the amino acids which are unique and invariant for each isozyme, the mouse amino acid sequence was found to contain 16 of the 23 residues that are unique and invariant to mammalian CA form II isozymes, but only one or no residue for forms I and III, respectively.     

Amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum

The amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum has been determined. It is composed of 260 amino acid residues and its N-terminus is acetylated. The molecular mass is calculated to be 29 851 Da. The presence of six calcium-binding sites (I-VI) has been proposed, two of them (sites II and VI) have lost their calcium-binding function through amino acid replacements, and the other four are able to bind calcium. Six calcium-binding domains are supposed to be derived from two gene duplications of the two ancestral calcium-binding domains. In comparison with the sequence of chick intestinal calcium-binding protein deduced from a cDNA sequence [(1985) Nucleic Acids Res. 13, 8867-8881], the bovine calcium-binding protein is two amino acid residues shorter at the N-terminus and the other parts show 78.5% identity.     

Nucleotide sequence of hamster S-adenosylmethionine decarboxylase cDNA.

The nucleotide sequence of a cDNA encoding the proenzyme of hamster S-adenosylmethionine decarboxylase including 169 nucleotides of the 5' untranslated region has been determined. The deduced amino acid sequence shows a remarkable similarity to the human proenzyme with only seven differences out of 334 amino acids. The nucleotide sequence of the 5' untranslated region showed 93% homology with the corresponding rat and human sequences suggesting that this region may play an important role in the regulation of S-adenosylmethionine decarboxylase expression.     

Molecular cloning and sequence of the gene for p9Ka. A cultured myoepithelial cell protein with strong homology to S-100, a calcium-binding protein

The gene for p9Ka, a protein of molecular weight 9000 that is expressed in cultured rat mammary myoepithelial cells, has been isolated from a normal rat genomic library in bacteriophage lambda, by its ability to hybridize to a cloned complementary DNA corresponding to p9Ka mRNA. The cloned rat genomic DNA fragment hybridized to translatable p9Ka mRNA. A nucleotide sequence of 2340 base-pairs of genomic DNA surrounding the p9Ka cDNA sequence has been obtained; the gene contains one intervening sequence of 675 nucleotides. The 3' end of the p9Ka mRNA has been identified on the gene sequence to be 13 nucleotides downstream from a poly(A) addition signal AATAAA. The gene contains an open reading frame of 101 amino acid residues, which is the only open reading frame in the entire gene long enough to encode a protein of molecular weight at least 9000. This hypothetical protein sequence shows greater than 40% homology to rat or bovine S-100 protein and over 30% homology to bovine intestinal calcium-binding protein. The results suggest that p9Ka may be related to a family of low molecular weight calcium-binding proteins.     

Complete nucleotide sequence of the neuraminidase gene of human influenza virus A/WSN/33.

The complete nucleotide sequence of the neuraminidase (NA) gene of WSN/33 (H1N1) virus was determined. The entire sequence was derived from the insert of cDNA clones, except the last 20 nucleotides, which were determined by primer extension. The WSN NA gene contained 1,409 nucleotides beginning at the 5' end (sense strand), with an untranslated region of 19 nucleotides followed by 1,359 nucleotides coding for 453 amino acids and finally ending with a 31-nucleotide sequence of untranslated region at the 3' termini. The amino acid sequence of WSN NA, as deduced from the DNA sequence, showed the presence of a stretch of 29 amino acids (7 to 35) enriched in hydrophobic amino acids, which may anchor the protein into the viral or cellular membrane. When compared with the PR8 NA sequence, WSN NA appeared to possess a similar structure, including the identical location of all cysteine and proline residues. However, WSN NA contained only three of the five potential glycosylation sites present in PR8 NA. Additionally, WSN NA contained a substitution of a five-amino acid sequence for a six-amino acid sequence in PR8 NA. The possible significance of these sequence changes in the primary structure of WSN NA in the unique role of WSN NA as a virulence factor in mouse brain and MDBK cells is discussed.     

Molecular cloning and characterization of hNP22: a gene up-regulated in human alcoholic brain

An improved differential display technique was used to search for changes in gene expression in the superior frontal cortex of alcoholics. A cDNA fragment was retrieved and cloned. Further sequence of the cDNA was determined from 5' RACE and screening of a human brain cDNA library. The gene was named hNP22 (human neuronal protein 22). The deduced protein sequence of hNP22 has an estimated molecular mass of 22.4 kDa with a putative calcium-binding site, and phosphorylation sites for casein kinase II and protein kinase C. The deduced amino acid sequence of hNP22 shares homology (from 67% to 42%) with four other proteins, SM22alpha, calponin, myophilin and mp20. Sequence homology suggests a potential interaction of hNP22 with cytoskeletal elements. hNP22 mRNA was expressed in various brain regions but in alcoholics, greater mRNA expression occurred in the superior frontal cortex, but not in the primary motor cortex or cerebellum. The results suggest that hNP22 may have a role in alcohol-related adaptations and may mediate regulatory signal transduction pathways in neurones.     

Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family.

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.     

The complete nucleotide sequence of murine beta-glucuronidase mRNA and its deduced polypeptide

The complete nucleotide sequence of murine beta-glucuronidase (GUS) mRNA has been compiled from three overlapping cloned cDNAs and a single GUS-specific genomic clone. The sequence is composed of 2455 nucleotides, exclusive of the poly(A) tail. The 5' and 3' untranslated regions contain 12 and 499 bases, respectively, with the open reading frame encoding a polypeptide of 648 amino acids (74.2 kDa), including a 22 amino acid signal sequence. The nucleotide and deduced amino acid sequences of murine GUS are compared to those published for rat and human GUS and the results are presented. Murine GUS also shares amino acid sequence identity with Escherichia coli GUS and beta-galactosidase. The complete sequences of murine GUS mRNA and its deduced polypeptide provide a basis from which to study the mechanisms responsible for the well-characterized variation in GUS expression among inbred mouse strains.     

Isolation and sequence characterization of mammary derived growth inhibitor gene of riverine buffalo (Bubalus bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5' UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3' UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI/fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Neurocalcin, a novel calcium binding protein with three EF-hand domains, expressed in retinal amacrine cells and ganglion cells.

Neurocalcin (molecular weight 23,000 and 24,000) is a newly identified Ca2+ binding protein with three EF-hand domains and has a strong amino acid sequence homology with visinin and recoverin (Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. J. Biol. Chem. In press). We produced antibody against neurocalcin. Immunoblotting showed the presence of neurocalcin in bovine retina as well as brain, suggesting that neurocalcin was a neuron specific Ca2+ binding protein. Immunohistochemistry revealed the expression of neurocalcin in retinal amacrine cells and ganglion cells but not in the photoreceptor layer. This distribution of neurocalcin was quite different from that of visinin and recoverin. Our results suggest that neurocalcin may play an important role in a Ca2+ signal pathway of the nervous system.     

Molecular cloning of beta-galactosidase from Japanese pear (Pyrus pyrifolia) and its gene expression with fruit ripening

We have cloned a cDNA fragment encoding a beta-galactosidase from Japanese pear (Pyrus pyrifolia) fruit (JP-GAL). It contained an untranslated sequence of 182 nucleotides at the 5' end, a presumptive coding sequence of 2,193 nucleotides and an untranslated sequence of 268 nucleotides including a polyadenylation signal and a poly (A) tail at the 3' end. It encoded a protein with a calculated molecular weight of 80.9 kDa which consists of 731 amino acids. Both the nucleotide and the deduced amino acid sequences showed a 98% sequence identity with that obtained from the apple beta-galactosidase cDNA. The peptide sequence obtained from the purified Japanese pear beta-galactosidase III matched the deduced amino acid sequence of SVSYDHKAIIINGQKRILISG (amino acid 25-45). Northern blot analysis showed that the probe derived from JP-GAL hybridized to a single 2.6 kb RNA. The mRNA was detected solely in the fruit; none was detected in the buds, leaves, roots or shoots of the Japanese pear. The steady-state level of the beta-galactosidase mRNA was measured during fruit ripening in three cultivars, Housui, Kousui (early ripening) and Niitaka (late ripening). The results showed that regardless of the cultivar, no JP-GAL mRNA was detected in the immature fruit. Increment of the mRNA level with fruit ripening coincided with the increase in the beta-galactosidase III activity. Our results showed that the expression of JP-GAL correlated with fruit softening and JP-GAL may be beta-galactosidase III.     

Nucleotide sequence of a Sendai virus genome region covering the entire M gene and the 3'' proximal 1013 nucleotides of the F gene.

We determined the sequence of the 2,138 nucleotides in the Sendai virus genome just following the 3' proximal 3,686 nucleotides which we had previously reported (Nucleic Acids Res. 11, 7317-7330, 1983). This covers the entire third gene of 1,173 nucleotides and the 3' proximal 1,013 nucleotides of the fourth gene. Like the NP and P+C genes, both the third and fourth genes start from consensus sequence R1 (3'-UCCCAC(or UA)UUUC) at the 3' end and the third gene terminates with consensus sequence R2 (3'-AUUCUUUUU) at the 5' end. The third gene was identified as M, and the deduced 348 amino acids indicated that the M protein is rich in basic residues and has hydrophobic domains near the C-terminal. The fourth gene, although sequencing is not complete yet, was identified as F, since a large open reading frame found in the gene contains the characteristic sequence of 20 amino acids located at the N-terminal of the F1 protein. Analyses of the amino acid sequence suggested that the structure of the F gene product is NH2-signal peptide-F2-F1-COOH.     

Nucleotide sequence of hamster spermidine/spermine-N1-acetyltransferase cDNA.

The nucleotide sequence of a cDNA encoding hamster spermidine/spermine-N1-acetyltransferase, a key enzyme in polyamine degradation and excretion, has been determined. The cDNA consists of a 1016 base pair insert including 120 nucleotides of the 5' untranslated region and the complete 3' untranslated region. The deduced amino acid sequence is very similar to the human spermidine/spermine-N1-acetyltransferase with only 8 differences in 171 amino acids and the corresponding nucleotide sequence shows 91% identity. The 5' untranslated regions are even more closely related with 97% identity suggesting that this region may play a role in the regulation of acetyltransferase activity. Translation of the acetyltransferase mRNA in a reticulocyte lysate was not altered by the addition of N1,N12-bis(ethyl)spermine.     

稻瘟病菌(Magnaporthe grisea)诱导的水稻早期反应基因ER1的5' 端cDNA片段克隆及序列分析

研究高等生物基因表达与调控的一个重要方面是分离基因的编码区及其上游的调控序列（ＤｅＶｅｅｒ等１９９７）,这需要获得一个基因的ｃＤＮＡ全长及从植物基因组获取全基因。在前文（周建明等１９９９）中曾经分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１的ｃＤＮＡ片段,但是运用ｍＲＮＡ差异显示技术分离的ｃＤＮＡ片段往往只有近ｍＲＮＡ３’端的一部分,难以反映基因的结构及功能特点,因此,必须进一步分离其５’端的部分才有可能比较全面地了解此基因的特点。ＲＡＣＥ（ｒａｐｉｄａｍｐｌｉｆｉｃａｔｉｏｎｏｆｃＤＮＡｅｎ…     

Amino acid sequence of human respiratory syncytial virus nucleocapsid protein.

Amino acid sequence of the human respiratory syncytial (RS) virus nucleocapsid (NC) protein, deduced from the DNA sequence of a recombinant plasmid, is presented. The cDNA plasmid (pRSB11) has 1412 bp of RS viral NC sequence and lacks six nucleotides of the 5' end of mRNA. There is a single long open reading frame encoding 467 amino acids. This 51540 dal protein is rich in basic amino acids and has no homologies with other known viral capsid proteins.     

Nucleotide sequence encoding the precursor of the small subunit of ribulose 1,5-bisphosphate carboxylase from Lemna gibba L.G-3

We have sequenced a cDNA clone, pLgSSU, which encodes the small subunit of ribulose 1,5-bisphosphate carboxylase of Lemna gibba L.G-3 a monocot plant. This clone contains a 832 basepair insert which encodes the entire 120 amino acids of the mature small subunit polypeptide (Mr = 14,127). In addition this clone encodes 53 amino acids of the amino terminal transit peptide of the precursor polypeptide and 242 nucleotides of the 3' non-coding region. Comparison of the nucleotide sequence of pLgSSU with Lemna gibba genomic sequences homologous to the 5' end of the cDNA clone suggests that nucleotides encoding four amino-terminal amino acids of the transit peptide are not included in the cDNA clone. The deduced amino acid sequence of the Lemna gibba mature small subunit polypeptide shows 70-75% homology to the reported sequences of other species. The transit peptide amino acid sequence shows less homology to other species. There is 50% homology to the reported soybean sequence and only 25% homology to the transit sequence of another monocot, wheat.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).