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Query: UNIPROT:P06889 (Mol)
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Endonuclease III (endoIII; nth gene product) of Escherichia coli is known to be a DNA repair enzyme having a relatively broad specificity for damaged pyrimidine bases of DNA. Here, we describe the cloning and characterization of the cDNA and the gene for a mouse homologue (mNthl1/mNth1) of endoIII. The cDNA was cloned from a mouse T-cell cDNA library with a probe prepared by PCR using the library and specific PCR primers synthesized based on the reported information of partial amino acid sequences of bovine NTHL1/NTH1 and of EST Data Bases. The cDNA is 1025 nucleotides long and encodes a protein consisting of 300 amino acids with a predicted molecular mass of 33.6 kDa. The amino acid sequence exhibits significant homologies to those of endoIII and its prokaryotic and eukaryotic homologues. The recombinant mNthl1 with a hexahistidine tag was overexpressed in a nth::cmr nei::Kmr double mutant of E. coli, and purified to apparent homogeneity. The enzyme showed thymine glycol DNA glycosylase, urea DNA glycosylase and AP lyase activities. Northern blot analysis indicated that mNthl1 mRNA is about 1 kb and is expressed ubiquitously. A 15 kb DNA fragment containing the mNthl1 gene was cloned from a mouse genomic library and sequenced. The gene consists of six exons and five introns spanning 6.09 kb. The sequenced 5' flanking region lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors such as Ets, Sp1, AP-1 and AP-2. The mNthl1 gene was shown to lie immediately adjacent to the tuberous sclerosis 2 (Tsc2) gene in a 5'-to-5' orientation by sequence analysis and was assigned to chromosome 17A3 by in situ hybridization.
J Mol Biol 1998 Oct 02
PMID:Cloning and characterization of a mouse homologue (mNthl1) of Escherichia coli endonuclease III. 974 25

The availability of large EST (Expressed Sequence Tag) databases has led to a revolution in the way new genes are cloned. Difficulties arise, however, due to high error rates and redundancy of raw EST data. For these reasons, one of the first tasks performed by a scientist investigating any EST of interest is to gather contiguous ESTs and assemble them into a larger virtual cDNA. The REX (Recursive EST eXtender) algorithm described in this paper completely automates this process by finding ESTs that can be clustered on the basis of overlapping bases, and then assembling the contigs into a consensus sequence. By combining the clustering and assembly steps, REX can quickly generate assemblies from EST databases that are frequently updated without having to preprocess the data. A consensus assembly method is used to correct miscalled bases and remove indel errors. A unique feature of this method is that it addresses the issues of splice variants and unspliced cDNA data. Since REX is a fast greedy algorithm, it can address the problem of generating a database of assembled sequences from very large collections of EST data. A procedure is described for creating and maintaining an Assembled Consensus EST database (ACE) that is useful for characterizing the large body of data that exists in EST databases.
Proc Int Conf Intell Syst Mol Biol 1998
PMID:Automated clustering and assembly of large EST collections. 978 26

The dietary phytoestrogen, daidzein, produced a biphasic response in cell proliferation of cultured, estrogen-responsive human breast carcinoma MCF-7 cells. Cell growth was stimulated at a daidzein concentration of 0.25 microg/ml whereas the addition of daidzein at concentrations >25 microg/ml significantly inhibited cell growth in a dose-dependent fashion, resulting in an IC50 value of 50 microg/ml. Upon exposure to 50 microg/ml of daidzein, cell morphology was severely altered, cell volume decreased, and condensation of the chromosomes was clearly noticeable. To identify genes whose expression were inhibited by daidzein, a differential display reverse transcriptase polymerase chain reaction assay (DD-RT-PCR) was performed and the cDNA fragments of several daidzein-regulated genes were visualized. The sequence of one of the cloned cDNA fragments that showed differential mRNA expression level in response to daidzein at a concentration of 50 microg/ml had a high homology with a cDNA expressed in fetal human brain, EST 06411.
Mol Reprod Dev 1999 Feb
PMID:Differential display screening for specific gene expression induced by dietary nonsteroidal estrogen. 989 Jul 44

Sequences encoding proteins with homology to protein tyrosine phosphatases have been identified in Arabidopsis, soybean and pea. Each contains a predicted catalytic domain containing sequence motifs characteristic of tyrosine-specific protein phosphatases (PTPs) which play an important role in signal transduction in other eukaryotes and are distinct from dual-specificity, cdc25 or low-molecular-weight protein tyrosine phosphatases. Their identity as PTPs was confirmed by characterising the soybean PTP expressed as a recombinant His-tagged fusion protein. The enzyme had phosphatase activity towards p-nitrophenolphosphate (pNPP) and phosphotyrosine, but did not hydrolyse phosphoserine or phosphothreonine at a measureable rate. Phosphotyrosine containing peptides also served as substrates, with Km values in the micromolar range. Activity was abolished by inhibitors specific for tyrosine phosphatases (vanadate, dephostatin) but was unaffected by inhibitors of serine/threonine protein phosphatases (fluoride, cantharidin, metal-chelating agents). Gel filtration chromatography showed that the recombinant enzyme was a monomer. The Arabidopsis PTP sequence was isolated both as a genomic clone and as a partial EST, whereas the pea and soybean sequences were isolated as cDNAs. Southern analysis suggested a single gene in Arabidopsis and a small gene family in pea and soybean. In pea, PTP transcripts were present in embryos, and decreased in level with development; transcripts were also detectable in other tissues. The plant PTPs all contain a similar N-terminal domain which shows no similarity to any known protein sequence. This domain may be involved in PTP functions unique to plants.
Plant Mol Biol 1999 Feb
PMID:Higher plant tyrosine-specific protein phosphatases (PTPs) contain novel amino-terminal domains: expression during embryogenesis. 1009 85

Telomere repeat binding factor 2 (TERF2) is one of two recently cloned mammalian telomere binding protein genes. TERF2 binds as a dimer with high affinity to the double-stranded TTAGGG telomeric repeat through an evolutionarily conserved myb-type DNA binding domain. TERF2 prevents telomere end-to-end fusion and may be important in maintaining genomic stability. We localized the transcribed TERF2 gene to human chromosome 16q22.1, tightly linked to the EST HUM000S343. The mouse Terf2 gene is situated by itself in a newly defined "bin" on chromosome 8 one crossover distal to Psm10 and Sntb2. Human TERF2 and mouse Terf2 are therefore part of a large evolutionarily conserved linkage group comprised of at least 25 known paralogous genes between human chromosome 16q and mouse chromosome 8.
Somat Cell Mol Genet 1998 May
PMID:Chromosomal sublocalization of the transcribed human telomere repeat binding factor 2 gene and comparative mapping in the mouse. 1022 53

Human mitochondrial phenylalanyl-tRNA synthetase (mtPheRS) has been identified from the human EST database. Using consensus sequences derived from conserved regions of the alpha and beta-subunits from bacterial PheRS, two partially sequenced cDNA clones were identified. Unexpectedly, sequence analysis indicated that one of these clones was a truncated form of the other. Detailed analysis indicates that unlike the (alphabeta)2 structure of the prokaryotic and eukaryotic cytoplasmic forms of PheRS, the human mtPheRS consists of a single polypeptide chain. This protein has been cloned and expressed in Escherichia coli. Gel filtration and analytical velocity sedimentation centrifugation indicate that the human mtPheRS is active in a monomeric form. The N-terminal 314 amino acid residues appear to be analogous to the alpha-subunit of the prokaryotic PheRS, while the C-terminal 100 amino acid residues correspond to a region of the beta-subunit known to interact with the anticodon of tRNAPhe. Comparisons with the sequences of PheRS from yeast and Drosophila mitochondria indicate they are 42 % and 51 % identical with the human mtPheRS, respectively. Sequence analysis confirms the presence of motifs characteristic of class II aminoacyl-tRNA synthetases. KM and kcat values for ATP:PPi exchange and for the aminoacylation reaction carried out by human mtPheRS have been determined. Evolutionary origins of this small monomeric human mtPheRS are unknown, however, implications are that this enzyme is a result of the simplification of the more complex (alphabeta)2 bacterial PheRS in which specific functional regions were retained.
J Mol Biol 1999 May 14
PMID:Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase. 1032 63

Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.
Plant Mol Biol 1999 Mar
PMID:Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase. 1034 1

The arabinose-sensitive ara1-1 mutant of Arabidopsis is deficient in arabinose kinase activity. A candidate for the ARA1 gene. ISA1, has been previously identified through the Arabidopsis genome sequencing initiative. Here we demonstrate that (1) the ARA1 gene coincides with ISA1 in a positional cloning strategy; (2) there are mutations in the ISA1 gene in both the ara1-1 mutant and an intragenic suppressor mutant; and (3) the ara1-1 and suppressor mutant phenotypes can be complemented by the expression of the ISA1 cDNA in transgenic plants. Together these observations confirm that ISA1 is the ARA1 gene. ARA1 is a member of the galactose kinase family of genes and represents a new substrate specificity among this and other families of sugar kinases. A second gene with similarities to members of the galactose kinase gene family has been identified in the EST database. A 1.8 kb cDNA contained an open reading-frame predicted to encode a 496 amino acid polypeptide. The GAL1 cDNA was expressed in a galK mutant of Escherichia coli and in vitro assays of extracts of the strain expressing GAL1 confirmed that the cDNA encodes a galactose kinase activity. Both GAL1 and ARA1 cross-hybridise at low stringency to other sequences suggesting the presence of additional members of the galactose kinase gene family.
Plant Mol Biol 1999 Mar
PMID:The arabinose kinase, ARA1, gene of Arabidopsis is a novel member of the galactose kinase gene family. 1034 5

Homologs of the eyes absent (eya) gene in animals function at multiple stages in the development of organs. Their functional roles in the genetic network that regulates eye development in Drosophila have recently been extensively analyzed. A rice homolog of eya was identified from a cDNA library made from embryo RNA. The corresponding gene (OSEya1) encodes a conserved ED1 domain and a short N-terminal peptide. The ED1 domain of OSEya1 shows 25% identity and 36% similarity to the product of Drosophila eya. Mammalian and squid eya homologs show about 35% similarity to OSEya1. Homologous sequences were also found in the alfalfa EST database (53% identity and 65% similarity to OSEya1) and in the Arabidopsis genome sequence (63% identity). Therefore, eya homologs are present in both monocots and dicots. Three regions in the ED1 domain are well conserved in animals and plants. Plant eya products deduced from the nucleotide sequences also have short N-terminal peptides. The OSEya1 gene is located between the wx gene and the telomere on the short arm of chromosome 6. OSEya1 is expressed in the embryo, shoot apex, and caryopsis in rice. Expression in the embryo increases during embryogenesis until 7 days after pollination, with preferential localization in leaf primordia and the shoot apical meristem. Expression in the influorescence was observed in floral meristems. The functions of OSEya1 in higher plants are discussed and compared with those of their animal homologs. OSEya1 might regulate the morphogenesis of lateral organs as a subunit of a transcription factor.
Mol Gen Genet 1999 Aug
PMID:Homologs of animal eyes absent (eya) genes are found in higher plants. 1050 44

ESTs constitute rapid and informative tools with which to study gene-expression profiles of the diverse stages of the schistosome life cycle. Following a comprehensive EST study of adult worms, analysis has now targeted the cercaria, the parasite larval form responsible for infection of the vertebrate host. Two Schistosoma mansoni cercarial cDNA libraries were examined and partial sequence obtained from 957 randomly selected clones. On the basis of database searches, 551 (57.6%) ESTs generated had no homologs in the public databases whilst 308 (32.2%) were putatively identified, totaling 859 informative ESTs. The remaining 98 (10.2%) were uninformative ESTs (ribosomal RNA and non-coding mitochondrial sequences). By clustering analysis we have identified 453 different genes. The most common sequences in both libraries represented Sm8 calcium binding protein (8% of ESTs), fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, cytochrome oxidase subunit 1, ATP guanidine kinase and triose phosphate isomerase. One hundred and nineteen identified genes were sorted into 11 functional categories, with genes associated with energy metabolism being the most abundant (13%) and diverse. The diversity and abundance of genes associated with the transcription/translation machinery and with regulatory/signaling functions were also marked. A paramyosin transcript was identified, indicating that this gene is not exclusively expressed in adult worms and sporocysts (as had been suggested previously). The possible physiological relevance to cercariae of the presence of transcripts with homology to calcium binding proteins of the EF-hand superfamily, Gq-coupled rhodopsin photoreceptor, rod phosphodiesterase 8 subunit and peripheral-type benzodiazepine receptor is discussed.
Mol Biochem Parasitol 1999 Sep 20
PMID:Analysis of the gene expression profile of Schistosoma mansoni cercariae using the expressed sequence tag approach. 1051 83


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