SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Two distinct cDNAs encoding protein synthesis initiation factor 4A (eIF-4A) were isolated from an Arabidopsis thaliana cDNA library and sequenced. The deduced amino acid sequences from the two cDNAs were compared to eIF-4A from tobacco, mouse and Saccharomyces cerevisiae. The putative ATP-binding sites and RNA helicase motifs were identified.
Antibodies against an inositol phospholipid-specific phospholipase C purified from bovine brain were used to screen rat brain lambda gt11 expression cDNA libraries. Complete sequences of three cDNA inserts yielded a cumulative sequence of 5106 base pairs. The deduced protein had 1289 amino acids with a calculated molecular weight of 148,431. The determination of an open reading frame was aided by the amino acid sequences of 21 tryptic peptides isolated from bovine brain phospholipase C. Only 9 residues of a total of 140 amino acid residues determined for the bovine enzyme were different from those deduced from the rat cDNA. Two regions of phospholipase C (amino acid residues 555-598 and 668-705) exhibited significant amino acid similarities to the products of various tyrosine kinase-related oncogenes (yes, src, fgr, abl, fps, fes, and tck). The homologous domain was located in the region that is not essential for the protein-tyrosine kinase activity but is likely to be involved in an interaction with cellular components that modulate kinase function. Therefore, this unexpected similarity raises the possibility that the 148-kDa phospholipase C and cytoplasmic tyrosine kinases are modulated by common cellular component(s).
A full-length cDNA coding for a novel human serine hydrolase has been cloned from a breast carcinoma cDNA library. Nucleotide sequence analysis has shown that the isolated cDNA contains an open reading frame coding for a polypeptide of 274 amino acids and a complete Alu repetitive sequence within its 3'-untranslated region. The predicted amino acid sequence contains the Gly-X-Ser-X-Gly motif characteristic of serine hydrolases and displays extensive similarity to several prokaryotic hydrolases involved in the degradation of aromatic compounds. The highest degree of identities was detected with four serine hydrolases encoded by the bphD genes of different strains of Pseudomonas with the ability to degrade biphenyl derivatives. On the basis of these sequence similarities, this novel human enzyme has been tentatively called Biphenyl hydrolase-related protein (Bph-rp). The Bph-rp cDNA was expressed in Escherichia coli, and after purification, the recombinant protein was able to degrade p-nitrophenylbutyrate, a water-soluble substrate commonly used for assaying serine hydrolases. This hydrolytic activity was abolished by diisopropyl fluorophosphate, a covalent inhibitor of serine hydrolases, providing additional evidence that the isolated cDNA encodes a member of this protein superfamily. Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues revealed that Bph-rp is mainly expressed in liver and kidney, which was also confirmed at the protein level by Western blot analysis with antibodies raised against purified recombinant Bph-rp. According to structural characteristics, hydrolytic activity and tissue distribution of Bph-rp, a potential role of this enzyme in detoxification processes is proposed.
The manB gene, coding for a unique beta-mannosidase (MANB) of Aspergillus aculeatus, was cloned from genomic and cDNA libraries, and sequenced. The gene consists of 2,811 bp encoding a polypeptide of 937 amino acid residues with a molecular mass of 104,214 Da. The A. aculeatus MANB shared amino acid sequence identity with MANB of human (24%), goat (24%), bovine (24%), and Caenorhabditis elegans (22%). When the A. aculeatus MANB was compared with other related enzymes, a Glu residue corresponding to the active site identified by the Escherichia coli beta-galactosidase and the human beta-guclonidase was conserved. This is the first fungal gene that encodes MANB.
The rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of the 34-kDa catalytic subunits (PRS I and II) and other 39- and 41-kDa proteins (Kita, K., Otsuki, T., Ishizuka, T., and Tatibana, M. (1989) J. Biochem. (Tokyo) 105, 736-741), which are termed here PRPP synthetase-associated proteins (PAPs). We have cloned the cDNA for the major one of 39 kDa (PAP39) from a rat liver cDNA library. Nucleotide sequencing showed that the clone encoded 356 amino acids containing sequences of all five peptides derived from PAP39. Surprisingly, the deduced amino acid sequence is markedly similar to those of the 34-kDa catalytic subunits. Excluding two regions (about 45 residues in total), PAP39 has a 48% identity with PRS I. Northern analysis detected a major 1.9-kilobase transcript in all 16 rat tissues examined, and the relative amounts of PAP39 mRNA to PRS I mRNA varied with tissues. Covalent cross-linking experiments gave definitive evidence for molecular interaction of PAP39 with the catalytic subunits. Immunoprecipitation experiments revealed that all the catalytic subunits existed as complexes containing PAP39. When PAPs were eliminated from the rat liver enzyme complex by gel filtration in the presence of 1 m MgCl2, a lyotrope, or by mild tryptic treatment, the enzyme activity of the remaining catalytic subunits increased. Based on these results, we propose that PAP39, the major component of PAPs, plays a negative regulatory role in PRPP synthesis and hence is an important factor controlling nucleotide syntheses in general.
Hair growth depends on maintenance of signalling between the dermal papilla and the germinative epithelium (GE), from which the differentiated layers of the hair fibre originate. Because no molecular studies have been reported which concentrate specifically on GE cells either in vivo or in vitro, we prepared a cDNA library enriched for messages which were highly expressed in GE cells to identify genes that may be involved in hair growth control. Of 35 subtracted library clones sequenced, 23 shared extensive homology with previously determined cDNA sequences, including LEF-1 and id4. Hair follicle organ culture models are often used to investigate the molecular basis of hair growth, although hair growth arrest occurs relatively rapidly in vitro. As an indicator of their role in follicle activities, we compared the expression of GE-specific clones in different regions of freshly isolated vibrissa follicles, with the corresponding regions of growth arrested, cultured follicles. Changes in the expression of some of these clones indicates that they could be related to fundamental cellular activities in the follicle. A library enriched for GE-specific clones therefore provides a useful source of candidate molecules for studies of follicular epithelial cell behaviour, both in vivo and in vitro.
Natural evolution has guided the development of 'molecular breeding' processes used in the laboratory for the rapid modification of subgenomic sequences including single genes. The most significant recent development has been the in vitro permutation of natural diversity. Homologous recombination of multiple related sequences produced high-quality libraries of chimeric sequences encoding proteins with functions that differ dramatically from any of the parents. Increasingly powerful screening methods are also being developed, allowing these libraries to be screened for novel biocatalysts.
Complementary DNA (cDNA) clones containing the entire coding sequence for Diazepam Binding Inhibitor (DBI) peptide, a 10-kDa precursor of putative natural ligands of benzodiazepine recognition sites, were isolated from rat, human and cow libraries. The sequence of all these clones is highly conserved; however, the N-terminal sequence predicted by the human DBI clone differed from that of the other two clones. DBI cDNA, utilized as hybridization probe in Southern blot analysis, revealed that DBI of both human and rat might be encoded by a multiple family of 4-6 genes. Furthermore, we have used in situ chromosomes hybridization to map human DBI genes. The results indicate that a human DBI gene is localized on chromosome 2 and that three of the four hybridization signals detected by the human DBI probe are located on three other chromosomes. These findings raise a question as whether multiple DBI genes encode for different molecular forms of DBI. In the attempt to test this hypothesis, cow cDNA and human genomic libraries were screened with DBI cDNA. In this paper I report the isolation of clones from these libraries which, although hybridizing well to DBI cDNA, possess a low percentage of homology (46.7%), randomly distributed within the coding region of DBI cDNA. Whether or not these clones encode for peptides sharing the same physiological role as DBI is under investigation.
The establishment and mapping of gene-specific DNA sequences greatly complement the ongoing efforts to map and sequence all human chromosomes. To facilitate our studies of human chromosome 7, we have generated and analyzed 2006 expressed-sequence tags (ESTs) derived from a collection of direct selection cDNA libraries that are highly enriched for human chromosome 7 gene sequences. Similarity searches indicate that approximately two-thirds of the ESTs are not represented by sequences in the public databases, including those in dbEST. In addition, a large fraction (68%) of the ESTs do not have redundant or overlapping sequences within our collection. Human DNA-specific sequence-tagged sites (STSs) have been developed from 190 of the ESTs. Remarkably, 180 (96%) of these STSs map to chromosome 7, demonstrating the robustness of chromosome enrichment in constructing the direct selection cDNA libraries. Thus far, 140 of these EST-specific STSs have been assigned unequivocally to YAC contigs that are distributed across the chromosome. Together, these studies provide > 2000 ESTs highly enriched for chromosome 7 gene sequences, 180 new chromosome 7 STSs corresponding to ESTs, and a definitive demonstration of the ability to enrich for chromosome-specific cDNAs by direct selection. Furthermore, the libraries, sequence data, and mapping information will contribute to the construction of a chromosome 7 transcript map.
For the Xanthomonas campestris pathovar campestris wild-type strain B100 a plasmid-based clone library was constructed. The plasmids carried chromosomal fragments of 3-4 kb in size that were tagged in vitro with the artificial transposon KAN-2. More than 3000 of the transposon target sites were characterized by DNA sequencing. The sequences obtained were compared to the recently published genome of Xanthomonas campestris pathovar campestris strain ATCC 33913. Most of the sequenced clones derived from strain B100 matched the chromosomal sequence of strain ATCC 33913. An alignment to the circular map of this chromosome revealed that the similarities were statistically distributed over the entire genome of strain ATCC 33913. The similarity was obvious for protein coding sequences, as well as for mobile genetic elements. However, four regions in the genome of Xanthomonas campestris pathovar campestris strain ATCC 33913, ranging in size from 11 to 37 kb, were not represented in the sequenced clone library of Xanthomonas campestris pathovar campestris strain B100. On the other hand, 1.2% of the sequenced clones originating from Xanthomonas campestris pathovar campestris strain B100 showed no or insignificant similarities to the genome of strain ATCC 33913.
The prokaryotic diversity of aerobic and anaerobic bacterial isolates and of bacterial and archaeal 16S rDNA clones was determined for a microbial mat sample from the moated region of Lake Fryxell, McMurdo Dry Valleys, Antarctica. Among the anaerobic bacteria, members of Clostridium estertheticum and some other psychrotolerant strains dominated whereas methanogens and other Archaea were lacking. Isolates highly related to Flavobacterium hibernum, Janthiniobacterium lividum, and Arthrobacter flavus were among the aerobic bacteria most frequently isolated. Assessment of more than 350 partial 16S rDNA clone sequences of libraries generated by Bacteria- and Archaea-specific PCR primers revealed a rich spectrum of bacterial diversity but only two different archaeal clone sequences. Among the Bacteria, representative sequences belonged to the class Proteobacteria, order Verrucomicrobiales, class Actinobacteria, Clostridium/Bacillus subphylum of Gram-positives, and the Cytophaga-Flavobacterium-Bacteroides phylum. The clones formed about 70 higher taxonomy groups (<98% sequence similarity) and 133 potential species, i.e., groups of clones sharing greater than 98% similarity. Only rarely were clone sequences found to be highly related to Lake Fryxell isolates and to strains of described species. Subsequent analysis of ten sequencing batches of 36 individual clones indicated that the diversity might be still higher than had been assessed.
Two overlapping cloned cDNAs encoding the entire amino acid sequences of the subunits of acylamino acid-releasing enzyme (AARE) [EC 3.4.19.1] have been isolated from porcine liver cDNA lambda gt10 cDNA libraries and sequenced. Sequence analyses of the cDNA and several Achromobacter protease I-digested peptides of the purified protein revealed that porcine liver AARE consists of four identical subunits, and each comprising a single chain of 732 amino acids with acetylmethionine at the N-terminus.
Based on sequence information from tryptic peptides an almost full-size cDNA coding for the human vascular anticoagulant was isolated from a placental cDNA library and sequenced. The coding region was cloned into an Escherichia coli expression vector and the protein expressed at high levels. The recombinant protein was purified and found to be indistinguishable from its natural counterpart in several biological assays.
BACKGROUND: The enormous amount of genome sequence data asks for user-oriented databases to manage sequences and annotations. Queries must include search tools permitting function identification through exploration of related objects. METHODS: The GenoList package for collecting and mining microbial genome databases has been rewritten using MySQL as the database management system. Functions that were not available in MySQL, such as nested subquery, have been implemented. RESULTS: Inductive reasoning in the study of genomes starts from "islands of knowledge", centered around genes with some known background. With this concept of "neighborhood" in mind, a modified version of the GenoList structure has been used for organizing sequence data from prokaryotic genomes of particular interest in China. GenoChore http://bioinfo.hku.hk/genochore.html, a set of 17 specialized end-user-oriented microbial databases (including one instance of Microsporidia, Encephalitozoon cuniculi, a member of Eukarya) has been made publicly available. These databases allow the user to browse genome sequence and annotation data using standard queries. In addition they provide a weekly update of searches against the world-wide protein sequences data libraries, allowing one to monitor annotation updates on genes of interest. Finally, they allow users to search for patterns in DNA or protein sequences, taking into account a clustering of genes into formal operons, as well as providing extra facilities to query sequences using predefined sequence patterns. CONCLUSION: This growing set of specialized microbial databases organize data created by the first Chinese bacterial genome programs (ThermaList, Thermoanaerobacter tencongensis, LeptoList, with two different genomes of Leptospira interrogans and SepiList, Staphylococcus epidermidis) associated to related organisms for comparison.
Antisecretory factor (AF) is a protein known to inhibit intestinal fluid secretion induced by cholera toxin. cDNA clones, expressing immunoreactivity to AF were isolated from a human pituitary gland library and sequenced. The sequence contained 1309 base pairs plus a poly(A) tail; Northern blot analysis of pituitary RNA confirmed this size. One large open reading frame was found to code for 382 amino acids. The protein was expressed in pGEX-lambda 1T/Escherichia coli and purified. The recombinant AF was extremely potent, 9 ng (2.10(-13) mol), giving a significant antisecretory activity against cholera toxin-induced fluid secretion in rat. Antiserum against recombinant AF was used in immunohistochemical and Western blot analysis. Sections from human pituitary glands manifested specific intracellular staining in cells exclusively located in the anterior part. Both recombinant AF and AF extracted from pituitary gland appeared in SDS-polyacrylamide to have a molecular mass of 60 kDa, although the renal value was 41 kDa. The protein sequence manifested homology (29% identity) with one protein, a putative Saccharomyces cerevisiae 30-kDa protein of unknown function.
Several cDNA clones encoding the human N-methyl-D-aspartate receptor modulatory subunit hNR2A, were isolated from human hippocampus and fetal brain libraries. DNA sequence analysis revealed overlapping clones permitting the reconstruction of full-length hNR2A cDNA. The hNR2A cDNA demonstrated an 88-89% nucleotide (nt) identity with the corresponding rodent cDNAs. The nt sequence of hNR2A would encode a 1464-aa protein that has a 95.2% identity with the rodent NR2A subunits.
Two full-length cDNA clones encoding rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were isolated from a lambda gt10 rabbit spleen cDNA library and sequenced. As predicted from the open reading frame (ORF) in vitro translation of a sense orientation GAPDH cDNA clone yielded a protein product with a molecular mass of 37 kDa. Rabbit GAPDH exhibits a high degree of homology to the mouse, rat, hamster, chicken and human GAPDH on both the nucleotide (nt) and amino acid (aa) levels.
A murine interferon gene (MuIFN alpha) has been isolated from a cosmid library. The sequence of a 1.2-kb HindIII-PstI fragment revealed a new MuIFN alpha gene which has not yet been described and which was termed MuIFN alpha 7. The coding sequence produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter. A comparison of the MuIFN alpha 7 gene with the known interferon genes in their coding and flanking sequences shows homologies between enhancer elements found in the 5' upstream region of the coding gene. The core element common to all known viral enhancers, GTGG(AAA/TTT)G is repeated four times in the MuIFN alpha 7 5'-flanking region, as in all known MuIFN alpha genes.