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PubMed1989

Identification of a repetitive DNA sequence specific to Mycobacterium paratuberculosis.

A 0.2-kb DNA sequence specific to Mycobacterium paratuberculosis, the causative organism of Johne's disease, was isolated from a partial genomic library. The sequence was part of a larger repetitive DNA element and was present in strains of M. paratuberculosis from cattle, sheep, goat, deer and also a woman with Crohn's disease but not in M. paratuberculosis strain 18. The sequence was not present in strains of 19 other mycobacterial species including 31 reference serotype strains of the M. avium-M. intracellular-M. scrofulaceum (MAIS) complex, some strains of which are closely related to M. paratuberculosis. The sequence may be useful for developing a diagnostic test for Johne's disease.

Animals↗

PubMed2004

Aspergillus flavus expressed sequence tags for identification of genes with putative roles in aflatoxin contamination of crops.

Aflatoxins, produced primarily by Aspergillus flavus and A. parasiticus, are among the most toxic and carcinogenic naturally occurring compounds. In an attempt to identify genes potentially involved in aflatoxin contamination of crops, and to better understand the biology of A. flavus, a large scale sequencing of A. flavus expressed sequence tags (EST) was conducted. The 5' ends of 26,110 cDNA clones from a normalized cDNA expression library were sequenced. After annotation, a total of 7218 unique ESTs in A. flavus were assembled into 3749 tentative concensus sequences and 3469 singleton sequences. The functional classifications of the genes or Gene Ontology (GO) terms were assigned to these ESTs. Genes potentially involved in the aflatoxin contamination process were identified in the ESTs sequenced. These include the aflatoxin biosynthetic pathway, signal transduction, global regulation, pathogenicity of the fungus, and stress response.

Aflatoxins↗

PubMed1997

Dog mast cell alpha-chymase activates progelatinase B by cleaving the Phe88-Gln89 and Phe91-Glu92 bonds of the catalytic domain.

In prior work we showed that a metallogelatinase is secreted from dog mastocytoma cells and directly activated by exocytosed mast cell alpha-chymase. The current work identifies the protease as a canine homologue of progelatinase B (92-kDa gelatinase, MMP-9), determines the sites cleaved by alpha-chymase, and explores the regulation of gelatinase expression in mastocytoma cells. To obtain a cDNA encoding the complete sequence of mastocytoma gelatinase B, a 2. 3-kilobase clone encoding progelatinase was isolated from a BR mastocytoma library. The sequenced cDNA predicts a 704-amino acid protein 80% identical to human progelatinase B. Regions thought to be critical for active site latency, such as the Cys-containing propeptide sequence, PRCGVPD, and the catalytic domain sequence, HEFGHALGLDHSS, are entirely conserved. Cleavage of progelatinase B by purified dog alpha-chymase yielded an approximately 84-kDa product that contained two NH2-terminal amino acid sequences, QTFEGDLKXH and EGDLKXHHND, which correspond to residues 89-98 and 92-101 of the cDNA predicted sequence, respectively. Thus, alpha-chymase cleaves the catalytic domain of gelatinase B at the Phe88-Gln89 and Phe91-Glu92 bonds. Like BR cells, the C2 line of dog mastocytoma cells constitutively secrete progelatinase B which is activated by alpha-chymase. By contrast, non-chymase-producing C1 cells secrete a gelatinase B (which remains in its proform) only in response to 12-O-tetradecanoylphorbol-13-acetate. Whereas 12-O-tetradecanoylphorbol-13-acetate stimulation of BR cells produced a approximately 15-fold increase in gelatinase B mRNA expression, dexamethasone down-regulated its expression by approximately 5-fold. Thus, extracellular stimuli may regulate the amount of mast cell progelatinase B expressed by mast cells. These data further support a role for mast cell alpha-chymase in tissue remodeling involving gelatinase B-mediated degradation of matrix proteins.

Amino Acid Sequence↗

PubMed1997

Characterization and evolution of a gene encoding a Trimeresurus flavoviridis serum protein that inhibits basic phospholipase A2 isozymes in the snake's venom.

The proteins that bind phospholipase A2 (PLA2) isozymes of Trimeresurus flavoviridis (habu snake, crotalinae) venom were fractionated from sera on four columns, each conjugated with one of four PLA2 isozymes. Five proteins, termed PLA2 inhibitors (PLI) I-V, were obtained as the binding components. The combinations of the binding components differed depending on the PLA2 isozymes. PLI-IV and PLI-V correspond to PLI-A and PLI-B, respectively, which were known to bind to a major [Asp49]PLA2, PLA2, and contained a segment similar to the carbohydrate-recognition domain of C-type lectins. PLI-I, which is a major component of inhibitory proteins against three basic PLA2 isozymes, PLA-B (a basic [Asp49]PLA2) and basic proteins I and II (both [Lys49]PLA2s), has been isolated, and its partial amino acid sequence has been determined. A cDNA encoding PLI-I was isolated from a T. flavoviridis liver cDNA library and sequenced. PLI-I cDNA encoded 200 amino acid residues, including a signal peptide of 19 amino acid residues. One sugar chain was predicted to occur at position 157. A gene coding for PLI-I was isolated. It is 9.6-kb long and consists of five exons and four introns. Comparison of the exon-intron structure of the PLI-I gene with those of genes encoding urokinase-type-plasminogen-activator receptor (uPAR), Ly-6, CD59 and neurotoxins showed that they have characteristic unit encoding approximately 90 amino acid residues, which is divided over two exons. This strongly suggests that the PLI-I gene belongs to the uPAR, Ly-6, CD59 and neurotoxin gene family. There are two types of structurally different inhibitors against PLA2 isozymes in T. flavoviridis serum with different evolutionary origins.

Amino Acid Sequence↗

PubMed1992

The sequence and tissue expression of ovine renin.

The primary structure of the sheep renin precursor has been determined from its cDNA sequence. A library of cDNA clones was constructed from adrenalectomized sheep kidney poly(A)+ RNA and screened for sheep renin sequences with a cloned mouse renin cDNA probe. Of the 300,000 clones generated, 24 were hybridization positive and the nucleotide sequences of two of the longest clones were determined. These clones coded for the mature sheep renin protein and the 3'-untranslated sequence but did not extend to the amino-terminal region of preprorenin. Clones corresponding to the 5' region of renin mRNA were generated by the polymerase chain reaction and their nucleotide sequences determined. The sheep renin precursor consists of 400 amino acids with a putative leader sequence of 14 amino acids and a putative 45 or 53 amino acid prosegment. The mature sheep renin protein has a 73% sequence identity with human renin. Northern analysis demonstrated the presence of renin mRNA in the kidney but not in other tissues in the sheep. While sodium depletion of sheep caused a rise in renin mRNA in the kidney, adrenalectomy also led to a large increase in renal renin mRNA. Southern analysis of genomic DNA suggests that there is only one gene coding for renin in the sheep.

Amino Acid Sequence↗

PubMed2000

Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication.

Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNA(Pro)molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian retro-viruses have revealed evidence of molecular adapt-ation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites. While most of the selected primer binding sites are complementary to the 3'-end of tRNA((Pro)), we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNA(Arg(CCU)), tRNA(Phe(GAA))and a hitherto unknown human tRNA(Ser(CGA)).

3T3 Cells↗

PubMed2004

Characterisation of Mal d 1-related genes in Malus.

It has been suggested that there are at least 15 Mal d 1-related (PR10) genes in one genotype of apple (Malus x domestica Borkh). We sequenced cDNA libraries of cultivar 'Royal Gala' and identified 12 members of the Mal d 1 family, including the previously reported Mal d 1b and Mal d 1d, an allelic variant of the previously reported Mal d 1a. Eight Mal d 1 gene products were expressed in tree-ripened fruit, in either the cortex or the skin, and most of these were also expressed in leaves in response to challenge with Venturia inaequalis -a fungal disease of apple. Mal d 1 gene products were identified from a large number of different tissues. Degree of ripeness as measured by standard parameters was shown not to predict either the amount of protein able to bind to a specific monoclonal antibody 5H8, previously shown to bind to an allergenic epitope in Mal d 1b and a/d, or the amount of Mal d 1 mRNA present. Mal d 1d and Mal d 1b were the most highly expressed isoforms in 'Royal Gala', particularly in the skin of fruit, and these isoforms were also predominant in other cultivars and species of apple. Genotypes, however, differed in relative predominance of Mal d 1b and Mal d 1d. The predominantly expressed Mal d 1 genes in ripe apple fruit were translated in vivo into proteins and proteins binding to the antibody were found in all cultivars and species examined. New Mal d 1 proteins were identified that bound to the 5H8 antibody. At least two new subfamilies have been identified, and while some structural differences are predicted between groups of isoforms, the P-loop motif is identical in all except two isoforms. A role in intracellular signalling in plants is suggested and in vitro expression of the isoforms should help in assessing their relative roles in disease, allergic responses, senescence and nucleotide-, cytokinin- and brassinosteroid-binding.

Allergens↗

PubMed1994

Sequence of ornithine decarboxylase from Lactobacillus sp. strain 30a.

A gene encoding biodegradative ornithine decarboxylase from Lactobacillus sp. strain 30a was isolated from a genomic DNA library and sequenced. Primer extension analysis revealed two transcription initiation sites. The deduced amino acid sequence is compared with the amino acid sequences of five previously reported bacterial decarboxylases, and conserved pyridoxal phosphate motif residues are identified.

Amino Acid Sequence↗

PubMed2003

Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia.

We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

Animals↗

PubMed2004

cDNA array analysis of Japanese quail lines divergently selected for four-week body weight.

Decades of divergent selection for 4-wk BW has produced 3 lines of growth-selected Japanese quail. P line quail have been selected for >110 generations for 4-wk posthatch BW and are nearly 3-fold larger than the randomly bred control C line. The H line has been selected for high 4-wk BW for 52 generations and the L line has been selected for low 4-wk BW for 54 generations. To identify differentially expressed genes that may play a role in defining the differences in these lines, a DNA array containing 4,704 random anonymous cDNA clones from 8-d C line embryos was screened using isotopically-labeled cDNA from the different quail lines. Array analysis yielded 3 differentially expressed cDNA clones that were confirmed by Northern blot analysis. The 35-kD quail EB1 protein, previously unidentified, was shown to have elevated transcripts in the L line and decreased transcripts in the H and P lines, compared with the C line. It was also widely expressed in embryonic and adult tissues by BLASTN analysis of chicken expressed sequence tag (EST) libraries. Two other cDNA clones are novel sequences expressed at higher levels in the L line and at lower levels in the H and P lines, one of which was more selectively expressed in embryos and adult tissues by BLASTN analysis. These limited findings suggest that anonymous cDNA array analysis is a productive means to identify differentially expressed genes in growth-selected poultry.

Animals↗

PubMed1996

Uroguanylin gene expression in the alimentary tract and extra-gastrointestinal tissues.

Uroguanylin, a member of the guanylin peptide family, is a novel peptide regulator for intestinal salt and water transport. A cDNA encoding a precursor for rat uroguanylin was cloned from a rat jejunum cDNA library and sequenced. The precursor was 106 amino acids long and included a 21 residue putative signal peptide at the N-terminus. Rat uroguanylin consisted of 15 amino acids similar to, but distinct from human uroguanylin; the C-terminal leucine residue was deleted and 3 residues were substituted compared to those in the human peptide. Synthetic rat uroguanylin-15 dose-dependently increased the cyclic GMP level in cultured T84 cells. RNA blot analysis showed that rat uroguanylin mRNA is expressed not only in the gastrointestinal tract but also in the lung, pancreas and kidney. Evidence for uroguanylin expression in extra-gastrointestinal tissues indicates the possible existence of a novel system for water and electrolyte homeostasis, and a more global effect of uroguanylin on epithelial cell function.

Amino Acid Sequence↗

PubMed2000

Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency.

We describe a general approach for identifying components of subcellular structures in a multicellular organism by exploiting the ability to generate thousands of independent transformants in Arabidopsis thaliana. A library of Arabidopsis cDNAs was constructed so that the cDNAs were inserted at the 3' end of the green fluorescent protein (GFP) coding sequence. The library was introduced en masse into Arabidopsis by Agrobacterium-mediated transformation. Fluorescence imaging of 5,700 transgenic plants indicated that approximately 2% of lines expressed a fusion protein with a different subcellular distribution than that of soluble GFP. About half of the markers identified were targeted to peroxisomes or other subcellular destinations by non-native coding sequence (i.e., out-of-frame cDNAs). This observation suggests that some targeting signals are of sufficiently low information content that they can be generated frequently by chance. The potential of the approach for identifying markers with unique dynamic processes is demonstrated by the identification of a GFP fusion protein that displays a cell-cycle regulated change in subcellular distribution. Our results indicate that screening GFP-fusion protein libraries is a useful approach for identifying and visualizing components of subcellular structures and their associated dynamics in higher plant cells.

Arabidopsis↗

PubMed1996

Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori.

A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori. The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced. Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases. Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody. Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms. Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge. An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line. These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.

Acute-Phase Proteins↗

PubMed2006

Molecular basis of plant gene expression during aphid invasion: wheat Pto- and Pti-like sequences are involved in interactions between wheat and Russian wheat aphid (Homoptera: Aphididae).

The Russian wheat aphid, Diuraphis noxia (Mordvilko) (Homoptera: Aphididae), is a major pest of bread wheat, Triticum aestivum L. (em Thell), in most wheat-growing areas worldwide. Aphid-resistant cultivars are used to combat this pest, but very little is known about the molecular basis of resistance. In this study, differential gene expression in D. noxia biotype 1-resistant wheat plants containing the Dnx gene and D. noxia biotype 1 feeding on Dnx plants was investigated using suppressive subtraction hybridization. The derived subtracted cDNA library includes sequences similar to Pto and Pti1, genes involved in gene-for-gene recognition of and resistance to bacterial speck disease in tomato, Lycopersicon esculentum (L.). Pto- and Pti1-like sequences contain an activation domain with conserved amino acid residues crucial for avr protein recognition and binding by Pto, and avr-Pto phosphorylation of Pti1. Wheat defense signaling is represented by sequences putatively involved in producing sterols, jasmonates, Ca2+, and abscisic and gibberellic acids. We suggest that reductions in populations of D. noxia fed Dnx plants are related to the expression of sequences involved in defensive chemical production, cellular transport, and exocytosis. Dnx plant tolerance of D. noxia feeding is proposed to be based on the expression of sequences putatively involved in self-defense against reactive oxygen species and toxins, and proteolysis; DNA, RNA, and protein synthesis; chloroplast and mitochondrial function; carbohydrate metabolism; and maintenance of cell homeostasis. D. noxia unsuccessfully counter Dnx by expressing sequences putatively involved in detoxification; proteolysis; DNA, RNA, protein, and lipid synthesis; carbohydrate metabolism; and mitochondrial function.

Amino Acid Sequence↗

PubMed2006

Culture isolation and culture-independent clone libraries reveal new marine Synechococcus ecotypes with distinctive light and N physiologies.

Marine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacterium Synechococcus can be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of a Synechococcus community from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of seven Synechococcus phylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed among Synechococcus strains than previously known. Thus, as in its sister genus Prochlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marine Synechococcus lineage.

Bacterial Typing Techniques↗

PubMed1992

cDNA sequence for rat dermatan sulfate proteoglycan-II (decorin).

A cDNA clone for dermatan sulfate proteoglycan-II, or decorin, has been isolated from a rat uterus library and sequenced. The cDNA and deduced amino acid sequences are 79 and 77% identical to the previously reported human and bovine sequences, respectively. The rat protein contains potential attachment sites for two glycosaminoglycan chains and four N-linked oligosaccharides, six conserved cysteine residues and multiple repeats of a leucine-rich sequence, LXXLXLXXNXL/I. Overlapping the C-end of one of these repeats is an NKISK sequence, which has been implicated in binding to fibronectin.

Amino Acid Sequence↗

PubMed1995

Cloning and sequencing a putative pyrophosphate-dependent phosphofructokinase gene from Entamoeba histolytica.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) gene from Entamoeba histolytica was cloned from its genomic library and sequenced. The open reading frame has 1149 bp and codes for a protein of 41.5 kDa. The deduced amino acid sequence of E. histolytica PPi-PFK has 25 to 28% identity to the PPi-PFKs from Propionibacterium freudenreichii, Naegleria fowleri and potato. The amino acid residues known to contribute to the active site of PPi-PFK from P. freudenreichii are conserved.

Amino Acid Sequence↗

PubMed1989

Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity.

Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. We have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46,XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development.

Alleles↗