Human diadenosine 5′,5′′′-P1,P4-tetraphosphate pyrophosphohydrolase is a member of the MutT family of nucleotide pyrophosphatases

The cDNA and derived amino acid sequence of human diadenosine 5′,5‴-P1,P4-tetraphosphate pyrophosphohydrolase have been determined with the aid of the GenBank Expressed Sequence Tag database. This enzyme possesses a modification of the MutT sequence motif found in certain nucleotide pyrophosphatases. It is unrelated to the enzymes of diadenosine tetraphosphate catabolism found in prokaryotes and fungi.

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In silico characterization and expression analyses of sugarcane putative sucrose non-fermenting-1 (SNF1) related kinases

Genetics and Molecular Biology ◽

10.1590/s1415-47572001000100006 ◽

2001 ◽

Vol 24 (1-4) ◽

pp. 35-41 ◽

Cited By ~ 4

Author(s):

Dirce Maria Carraro ◽

Marcio R. Lambais ◽

Helaine Carrer

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Protein Kinases ◽

Catalytic Domain ◽

Expressed Sequence Tag ◽

Higher Plants ◽

Expression Patterns ◽

Amino Acid Sequences ◽

Cdna Libraries ◽

Expressed Sequence

Sucrose non-fermenting-1-related protein kinases (SnRKs) may play a major role in regulating gene expression in plant cells. This family of regulatory proteins is represented by sucrose non-fermenting-1 (SNF1) protein kinase in Saccharomyces cerevisiae, AMP-activated protein kinases (AMPKs) in mammals and SnRKs in higher plants. The SnRK family has been reorganized into three subfamilies according to the evolutionary relationships of their amino acid sequences. Members of the SnRK subfamily have been identified in several plants. There is evidence that they are involved in the nutritional and/or environmental stress response, although their roles are not yet well understood. We have identified at least 22 sugarcane expressed sequence tag (EST) contigs encoding putative SnRKs. The amino acid sequence alignment of both putative sugarcane SnRKs and known SnRKs revealed a highly conserved N-terminal catalytic domain. Our results indicated that sugarcane has at least one member of each SnRK subfamily. Expression pattern analysis of sugarcane EST-contigs encoding putative SnRKs in 26 selected cDNA libraries from the sugarcane expressed sequence tag SUCEST database has indicated that members of this family are expressed throughout the plant. Members of the same subfamily showed no specific expression patterns, suggesting that their functions are not related to their phylogenic relationships based on N-terminal amino acid sequence phylogenetic relationships.

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Molecular cloning and functional expression of human deoxyhypusine synthase cDNA based on expressed sequence tag information

Biochemical Journal ◽

10.1042/bj3150429 ◽

1996 ◽

Vol 315 (2) ◽

pp. 429-434 ◽

Cited By ~ 16

Author(s):

Yong Ping YAN ◽

Yong TAO ◽

Kuang Yu CHEN

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Recombinant Protein ◽

Amino Acid Sequence ◽

Reverse Genetics ◽

Expressed Sequence Tag ◽

Initiation Factor ◽

Universal Primers ◽

Deoxyhypusine Synthase ◽

Expressed Sequence

Deoxyhypusine synthase is an NAD+-dependent enzyme that catalyses the formation of a deoxyhypusine residue on the eukaryotic initiation factor 5A (eIF-5A) precursor by transferring an aminobutyl moiety from spermidine to the ε-amino group of a unique lysine residue. We have recently cloned and characterized the Neurospora crassa deoxyhypusine synthase cDNA using a reverse genetics approach. A GenBank search showed that a stretch of the deduced amino acid sequence (96 amino acids) of Neurospora deoxyhypusine synthase matches a short human expressed sequence tag (EST), Z25337, with greater than 70% amino acid identity. Gene-specific primers based on this EST were used together with universal primers to obtain 1219 bp and 1078 bp cDNAs from a human cDNA library. The 1219 bp and 1078 bp sequences, each containing an open reading frame, encode polypeptides of respectively 368 and 321 amino acids. The short sequence is identical to the long one except that it is missing a stretch of 47 amino acids spanning residues 261–307. The 368-amino-acid sequence of human deoxyhypusine synthase shares a high degree of identity (> 50%) and similarity (> 60%) with that of the Neurospora and yeast deoxyhypusine synthases. After cloning into an expression vector, the 368-amino-acid recombinant protein exhibits high deoxyhypusine synthase activity. In contrast, the 321-amino-acid recombinant protein shows no detectable activity.

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Beta-centractin: characterization and distribution of a new member of the centractin family of actin-related proteins.

Molecular Biology of the Cell ◽

10.1091/mbc.5.12.1301 ◽

1994 ◽

Vol 5 (12) ◽

pp. 1301-1310 ◽

Cited By ~ 28

Author(s):

S W Clark ◽

O Staub ◽

I B Clark ◽

E L Holzbaur ◽

B M Paschal ◽

...

Keyword(s):

Amino Acid ◽

Cdna Clone ◽

Expressed Sequence Tag ◽

Full Length ◽

Northern Analysis ◽

Related Protein ◽

Two Dimensional Gel Electrophoresis ◽

Full Length Cdna ◽

Dynactin Complex ◽

Expressed Sequence

An examination of human-expressed sequence tags indicated the existence of an isoform of centractin, an actin-related protein localized to microtubule-associated structures. Using one of these tags, we isolated and determined the nucleotide sequence of a full-length cDNA clone. The protein encoded represents the first example of multiple isoforms of an actin-related protein in a single organism. Northern analysis using centractin-specific probes revealed three species of mRNA in HeLa cells that could encode centractin isoforms. One mRNA encodes the previously-identified centractin (now referred to as alpha-centractin). The full-length cDNA clone isolated using the expressed sequence tag encodes a new member of the centractin family, beta-centractin. A probe specific for alpha-centractin hybridized to the third species of mRNA observed (referred to as gamma-centractin). Comparisons of Northern blots of human tissues indicated that alpha-centractin and beta-centractin mRNAs are equally distributed in all populations of mRNA examined, whereas the expression of gamma-centractin appears to be tissue specific. The amino acid sequence of beta-centractin, deduced from the cDNA, indicates a 91% identity with alpha-centractin, increasing to 96% similarity when conservative amino acid changes are taken into account. As antibodies previously raised against alpha-centractin reacted only poorly with beta-centractin, new antibodies were produced and combined with two-dimensional gel electrophoresis to discriminate the two isoforms. Using this system, the subcellular distribution of the alpha- and beta-isoforms were determined. Both isoforms were found predominantly in the cytosolic fraction as a part of a previously identified 20S complex (referred to as the dynactin complex) with no evidence for a free pool of either isoform. The isoforms were found in a constant ratio of approximately 15:1 (alpha:beta) in the dynactin complex.

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Genetic Organization and Molecular Analysis of the EcoVIII Restriction-Modification System of Escherichia coli E1585-68 and Its Comparison with Isospecific Homologs

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2638-2650.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2638-2650 ◽

Cited By ~ 10

Author(s):

Iwona Mruk ◽

Tadeusz Kaczorowski

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Start Codon ◽

Sequence Motif ◽

Specific Sequence ◽

Modification System ◽

Acid Protein ◽

Restriction Modification ◽

Amino Acid Protein

ABSTRACT The EcoVIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the EcoVIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E. coli genomic DNA (50.8%). The difference suggests that there is a possibility that the EcoVIII R-M system was recently acquired by the genome. The 921-bp EcoVIII endonuclease (R · EcoVIII) gene (ecoVIIIR) encodes a 307-amino-acid protein with an M r of 35,554. The convergently oriented EcoVIII methyltransferase (M · EcoVIII) gene (ecoVIIIM) consists of 912 bp that code for a 304-amino-acid protein with an M r of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5′-AAGCTT-3′. Preparations of EcoVIII R-M enzymes purified to homogeneity were characterized. R · EcoVIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5′ protruding ends. M · EcoVIII functions as a monomer and modifies the first adenine residue at the 5′ end of the specific sequence to N 6-methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the HindIII (Haemophilus influenzae Rd) and LlaCI (Lactococcus lactis subsp. cremoris W15) R-M systems. This finding is reflected by the levels of homology of M · EcoVIII with M · HindIII and M · LlaCI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m6 N-adenine β-class methyltransferases. The deduced amino acid sequence of R · EcoVIII shows weak homology with its two isoschizomers, R · HindIII (26%) and R · LlaCI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R · EcoVIII (D108X12DXK123), as well as in the primary structures of R · LlaCI and R · HindIII. Polyclonal antibodies raised against R · EcoVIII did not react with R · HindIII, while anti-M · EcoVIII antibodies cross-reacted with M · LlaCI but not with M · HindIII. R · EcoVIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M · EcoVIII enzyme. The biological implications of this finding are discussed.

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Identification and characterization of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium

Biochemical Journal ◽

10.1042/bj3530635 ◽

2001 ◽

Vol 353 (3) ◽

pp. 635-644 ◽

Cited By ~ 9

Author(s):

Hidekazu KUWAYAMA ◽

Helena SNIPPE ◽

Mari DERKS ◽

Jeroen ROELOFS ◽

Peter J. M. VAN HAASTERT

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Catalytic Domain ◽

Expressed Sequence Tag ◽

Sequence Similarity ◽

Second Messengers ◽

Kinetic Properties ◽

Intracellular Camp ◽

Catalytic Domains ◽

Dual Specificity

In Dictyostelium cAMP and cGMP have important functions as first and second messengers in chemotaxis and development. Two cyclic-nucleotide phosphodiesterases (DdPDE 1 and 2) have been identified previously, an extracellular dual-specificity enzyme and an intracellular cAMP-specific enzyme (encoded by the psdA and regA genes respectively). Biochemical data suggest the presence of at least one cGMP-specific phosphodiesterase (PDE) that is activated by cGMP. Using bioinformatics we identified a partial sequence in the Dictyostelium expressed sequence tag database that shows a high degree of amino acid sequence identity with mammalian PDE catalytic domains (DdPDE3). The deduced amino acid sequence of a full-length DdPDE3 cDNA isolated in this study predicts a 60kDa protein with a 300-residue C-terminal PDE catalytic domain, which is preceded by approx. 200 residues rich in asparagine and glutamine residues. Expression of the DdPDE3 catalytic domain in Escherichia coli shows that the enzyme has Michaelis–Menten kinetics and a higher affinity for cGMP (Km = 0.22µM) than for cAMP (Km = 145µM); cGMP does not stimulate enzyme activity. The enzyme requires bivalent cations for activity; Mn2+ is preferred to Mg2+, whereas Ca2+ yields no activity. DdPDE3 is inhibited by 3-isobutyl-1-methylxanthine with an IC50 of approx. 60µM. Overexpression of the DdPDE3 catalytic domain in Dictyostelium confirms these kinetic properties without indications of its activation by cGMP. The properties of DdPDE3 resemble those of mammalian PDE9, which also shows the highest sequence similarity within the catalytic domains. DdPDE3 is the first cGMP-selective PDE identified in lower eukaryotes.

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An amino acid sequence motif observed amongst enzymes of the shikimate pathway

Biochemical Journal ◽

10.1042/bj2760841 ◽

1991 ◽

Vol 276 (3) ◽

pp. 841-843 ◽

Cited By ~ 2

Author(s):

T D H Bugg ◽

P R Alefounder ◽

C Abell

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Shikimate Pathway ◽

Sequence Motif ◽

Amino Acid Sequence Motif

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Peptides with More than One 106-amino Acid Sequence Motif Are Needed to Mimic the Structural Stability of Spectrin

Journal of Biological Chemistry ◽

10.1074/jbc.271.48.30410 ◽

1996 ◽

Vol 271 (48) ◽

pp. 30410-30416 ◽

Cited By ~ 23

Author(s):

Nick Menhart ◽

Tracy Mitchell ◽

Denise Lusitani ◽

Nancy Topouzian ◽

W.-M.L. Fung

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Structural Stability ◽

Sequence Motif ◽

Amino Acid Sequence Motif

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Molecular Cloning of a Novel Mouse Testis-specific Spermatogenic Cell Apoptosis Inhibitor Gene mTSARG7 as a Candidate Oncogene

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00057.x ◽

2005 ◽

Vol 37 (6) ◽

pp. 396-405 ◽

Cited By ~ 6

Author(s):

Xiao-Jun Tan ◽

Xiao-Wei Xing ◽

Lu-Yun Li ◽

Zhao-Di Wu ◽

Chang-Gao Zhong ◽

...

Keyword(s):

Amino Acid ◽

Mrna Expression ◽

Expressed Sequence Tag ◽

Mouse Testis ◽

Control Group ◽

Amino Acid Residues ◽

Apoptosis Inhibitor ◽

Expressed Sequence ◽

Candidate Oncogene

Abstract A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and 11 introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFP-tagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(–)/mTSARG7 plasmid was constructed and introduced into GC-1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.

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