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Results for “sequencing libraries”

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PubMed2005

Characterization of a normalized cDNA library from bovine intestinal muscle and epithelial tissues.

Tissue-specific cDNA library sequences (expressed sequence tags, or EST) yield a detailed snapshot of gene expression and are useful in developing second-generation molecular resources (i.e., microarrays) for gene expression profiling. The objective of this study was to develop and characterize an intestine-specific cDNA library to examine the transcriptome of the bovine gut and identify expressed genes that influence ruminant nutrition and health. We describe BARC-8BOV, a normalized cDNA library developed from mRNA isolated from four distinct intestinal locations (duodenal, jejunal and ileal small intestine, colon) of Holstein dairy cattle resulting in 19,110 5'-EST deposited into the NCBI GenBank EST database. Assembly and clustering of these 19,110 clone sequences yielded 11,208 unique elements (3,419 contigs and 7,789 singletons) with an average length of 695 base pairs. Analysis strongly suggests normalization and tissue pooling were effective at increasing the discovery rate of new bovine sequence. A total of 1,123 sequence elements not previously identified in cattle, but with similarity to known genes in other animal species, were identified and shown to be involved in numerous critical biological processes. An additional 745 transcripts were not previously represented as EST in nucleotide or protein databases, and further analysis of these could lead to the identification of gut-specific transcript variants of known genes or potentially the discovery of novel bovine genes. Of the 11,208 assembled sequences, 11,034, or 98.4%, match sequences present in the bovine DNA trace archive at NCBI, and add to a bovine EST database previously lacking significant gut tissue representation. Ultimately, these data will also contribute in efforts to annotate the bovine genome.

Animals↗

PubMed1990

Production and use of bovine DNA libraries: DNA-sequencing.

An important part in the use of genomic DNA libraries is the sequencing of identified clones for detailed information. In this study, methods for DNA sequence analysis were elaborated and employed for the k-casein gene, a bovine milk protein. The results encourage further research.

Animals↗

PubMed1994

The cloning of zebrin II reveals its identity with aldolase C.

The sagittal organization of the mammalian cerebellum can be observed at the anatomical, physiological and biochemical level. Previous screening of monoclonal antibodies produced in our laboratory has identified two intracellular antigens, zebrin I and II, that occur exclusively in adult cerebellar Purkinje cells. As their name suggests, the zebrin antibody staining of the Purkinje cell population is not uniform. Rather, zebrin-positive Purkinje cells are organized in stripes or bands that run from anterior to posterior across most of the cerebellum; interposed between the zebrin-positive cells are bands of Purkinje cells that are zebrin-negative. Comparison of the position of the antigenic bands with the anatomy of afferent projections suggests that the bands are congruent with the basic developmental and functional 'compartments' of the cerebellum. We report the isolation of cDNA clones of the 36 x 10(3) M(r) antigen, zebrin II, by screening of a mouse cerebellum cDNA expression library. Sequence analysis reveals a 98% identity between our clone and the glycolytic isozyme, aldolase C. In order to more rigorously demonstrate the identity of the two proteins, we stained adult cerebellum with an independent monoclonal antibody raised against aldolase C. Anti-aldolase staining occurs in a previously unreported pattern of sagittal bands of Purkinje cells; the pattern is identical to that revealed by the zebrin II monoclonal. Further, in situ hybridization of antisense aldolase C riboprobe shows that the accumulation of zebrin II/aldolase C mRNA corresponds to the pattern of the zebrin antigen in Purkinje cells. Zebrin II/aldolase C gene expression is thus regulated at the level of transcription (or mRNA stability). In light of previous work that has demonstrated the cell-autonomous and developmentally regimented expression of zebrin II, further studies of the regulation of this gene may lead to insights about the determination of cerebellar compartmentation.

Amino Acid Sequence↗

PubMed1995

Cloning of a fibroblast growth factor receptor 1 splice variant from Xenopus embryos that lacks a protein kinase C site important for the regulation of receptor activity.

A cDNA clone, predicted to encode a variant form of the type 1 fibroblast growth factor receptor (FGFR1) containing a dipeptide Val-Thr (VT) deletion at amino acid positions 423 and 424 located within the juxtamembrane region, was isolated from a Xenopus embryo (stage 8 blastula) library. Sequence analysis of genomic DNA encoding a portion of the FGFR1 juxtamembrane region demonstrated that this variant form arises from use of an alternative 5' splice donor site. RNase protection analysis revealed that both VT- and VT+ forms of the FGFR1 were expressed throughout embryonic development, the VT+ being the major form. Amino acid position 424 is located within a consensus sequence for phosphorylation by a number of Ser/Thr kinases. We demonstrate that a VT+ peptide was specifically phosphorylated by protein kinase C (PKC) in vitro, but not by protein kinase A (PKA). A VT- peptide, on the other hand, was not a substrate for either enzyme. Phosphorylation levels of in vitro synthesized FGFR-VT+ protein by PKC were twice that of FGFR-VT- protein. In a functional assay, Xenopus oocytes expressing FGFR-VT- or FGFR-VT+ protein were equally able to mobilize intracellular Ca2+ in response to basic fibroblast growth factor (bFGF). However, pretreatment with phorbol 12-myristate 13-acetate significantly reduced this mobilization in oocytes expressing FGFR-VT+ while having little effect on oocytes expressing FGFR-VT-. These findings demonstrate that alternative splicing of Val423-Thr424 generates isoforms which differ in their ability to be regulated by phosphorylation and thus represents an important mechanism for regulating FGFR activity.

Alternative Splicing↗

PubMed2000

Identification and cDNA cloning of alveolin, an extracellular metalloproteinase, which induces chorion hardening of medaka (Oryzias latipes) eggs upon fertilization.

Chorion hardening is triggered by the contents of cortical alveoli that are released upon fertilization of medaka (Oryzias latipes) eggs. We purified the chorion hardening-inducing activity as a single protein from the exudate of cortical alveoli of medaka eggs. This activity was co-purified with proteolytic activity of the chorion protein ZI-1,2. Based on the amino acid sequence of purified protein, we cloned the cDNA of this protein from a medaka ovarian cDNA library. Sequence analyses revealed typical sequence features, a zinc-binding motif and a methionine turn motif, of the astacin metalloproteinase family. We termed this protein "alveolin." Alveolin has a molecular mass of 21.5 kDa deduced by the amino acid sequence and neutral optimal pH range. Alveolin hydrolyzes ZI-1,2. Alveolin activity was strongly inhibited by metal-chelating agents but not by various proteinase inhibitors. To our knowledge, this is the first description of the isolation and identification of the chorion hardening-inducing factor from cortical alveoli exudate of teleost eggs.

Amino Acid Sequence↗

PubMed1992

Cloning of the guinea pig 5-lipoxygenase gene and nucleotide sequence of its promoter.

The guinea pig 5-lipoxygenase (5-LO) gene and its promoter were cloned from a guinea pig genomic DNA library. Sequencing analysis of the guinea pig promoter revealed that expression of the 5-LO gene in this rodent is probably governed by cis acting nucleotide sequences quite similar to the human gene. Nucleotide sequences that bind factors like Sp-1, AP-2, NF-kB and c-Ha-ras were identified in the guinea pig 5-LO promoter region.

Amino Acid Sequence↗

PubMed1988

Detection of H-2K mRNA in mouse 8-cell embryo by cDNA cloning.

Mouse MHC class I gene expression in 8-cell embryo was examined by cDNA cloning. We constructed a cDNA library from 8-cell embryos of ICR mice and isolated a class I cDNA from 3.0 x 10(5) phage clones of the library. Sequencing analysis of this clone revealed it to include the cDNA fragment extending from the exon 6 of the cytoplasmic portion to 3' untranslated region 1 of H-2K gene. Qa, Tla or other embryonic class I cDNA have not been isolated in the library.

Animals↗

PubMed1995

Cloning and sequence of ftsZ and flanking regions from the cyanobacterium Anabaena PCC 7120.

Using degenerate oligodeoxyribonucleotide primers based on conserved regions of the cell-division protein FtsZ, a 220-bp fragment of DNA was amplified by the polymerase chain reaction from Anabaena PCC 7120 (Ana). This fragment, which showed significant homology with Escherichia coli ftsZ, was used as a probe to isolate a 15-kb genomic clone containing ftsZ from an Ana DNA library. Sequence analysis revealed an open reading frame (ORF) encoding a protein of 379 amino acids, with 49% identity with E. coli FtsZ. Upstream of Ana ftsZ is a small, unidentified ORF, transcribed in the same direction. An ORF lying downstream of the ftsZ coding region and transcribed in the opposite orientation, shows homology with bacterial glutathione synthetase-encoding genes. Single copies of ftsZ have been identified in Ana and two other cyanobacteria. Multiple transcripts hybridising to ftsZ were detected by Northern hybridisation.

Amino Acid Sequence↗

PubMed1996

An insecticidal N-acetylglucosamine-specific lectin gene from Griffonia simplicifolia (Leguminosae).

Griffonia simplicifolia II, an N-acetylglucosamine-specific legume lectin, has insecticidal activity when fed to the cowpea weevil, Callosobruchus maculatus (F.). A cDNA clone encoding G. simplicifolia II was isolated from a leaf cDNA library, sequenced, and expressed in a bacterial expression system. The recombinant protein exhibited N-acetylglucosamine-binding and insecticidal activity against cowpea weevil, indicating that glycosylation and multimeric structure are not required for these properties. These results support the hypothesis that genes of the legume lectin gene family encode proteins that function in plant defense against herbivores.

Acetylglucosamine↗

PubMed1995

Sequence analysis of the rat jun-D gene.

A rat jun-D genomic clone was isolated from a rat liver lambda EMBL3 library. Sequence analysis revealed a high sequence similarity with mouse jun-D, and relatively low similarity with human and chicken jun-D. In both the trans-activation and DNA-binding domains, however, the amino-acid sequences were well conserved among rat, mouse, human and chicken.

Amino Acid Sequence↗

PubMed1993

Molecular characterization of a leukotoxin gene from a Pasteurella haemolytica-like organism, encoding a new member of the RTX toxin family.

A Pasteurella haemolytica-like organism, a new species of bacterium isolated from piglets with diarrhea, secretes a leukotoxin into the culture media. Western blot (immunoblot) analysis indicated that this leukotoxin cross-reacted with antileukotoxin antibody derived from cattle immunized with P. haemolytica. Five overlapping recombinant bacteriophages carrying the gene for this 105-kDa polypeptide were identified with a DNA probe containing sequences from the P. haemolytica lktCA genes from a P. haemolytica-like organism strain 5943 genomic library. Sequence analysis of a region of the cloned DNA revealed two open reading frames encoding proteins with predicted masses of 19.4 and 101.6 kDa. These genes, which we designate pllktC (P. haemolytica-like organism leukotoxin C gene) and pllktA (A gene), respectively, are similar in sequence to the RTX (repeat of toxin) toxin family. The structure of the 101.6-kDa protein derived from the DNA sequence shows three transmembrane domains in the N-terminal part of the protein, 13 glycine-rich repeat domains in the second half of the protein, and a hydrophobic C-terminal part. pllktC and pllktA are strongly homologous to P. haemolytica lktC and lktA genes. However, this leukotoxin kills both BL-3 and pig leukocytes and is not hemolytic.

Amino Acid Sequence↗

PubMed2001

cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG.

Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis.

Acyltransferases↗

PubMed1993

Cloning and structural analysis of a human thymidylate synthase pseudogene splitted by several Alu sequences.

An unidentified genomic DNA fragment of 2.4kb that is weakly hybridizable with thymidylate synthase (TS) cDNA was cloned from a human genomic DNA library. Sequencing of the cloned DNA fragment and comparison of the sequence with that of the known human TS cDNA revealed that the DNA fragment contained a human TS processed pseudogene with unusual features. Based on the rate of nucleotide substitutions for neutral mutations in the 3'-untranslated regions between the gene and the pseudogene, it was estimated that the human TS pseudogene was formed about 16 million years ago.

Biological Evolution↗

PubMed1995

Studies of cloning, chromosomal mapping, and embryonic expression of the mouse Rab geranylgeranyl transferase beta subunit.

The mouse Rab geranylgeranyl transferase beta subunit has been cloned from a mouse E8.5 embryonic cDNA library. Sequence comparison reveals 97.4% sequence identity at the amino acid level to the rat clone isolated from an adult rat brain cDNA library. This gene, given a gene symbol of Rabggtb, is mapped in the distal region of mouse chromosome 3. It is ubiquitously expressed in adult animals but displays an interesting pattern of expression during a specific time of embryonic development. The expression of this gene can be detected in the whole embryos during early embryonic stages and is specifically concentrated in the developing brain, heart, and liver between gestation stages of E11.5 and E13.5. In addition, the expression of this gene is induced by retinoic acid in a mouse embryonal carcinoma cell line, P19.

Animals↗

PubMed2000

Identification of Hox Gene Sequences in the Sea Cucumber Holothuria glaberrima Selenka (Holothuroidea: Echinodermata).

The Echinodermata is a unique animal group forming an early branch in the deuterostomes phylogenetic tree. In echinoids and asteroids a single Hox cluster with nine cognates of the vertebrate Hox paralogous groups has been reported, but no data are available from other echinoderm classes. We report here nine Hox-type sequences from the sea cucumber Holothuria glaberrima, a member of the class Holothuroidea. Partial homeodomain sequences were amplified by polymerase chain reaction from genomic DNA and from a regenerating gastrointestinal tract complementary DNA library. Sequence analyses suggest that the holothuroid cluster has at least three genes of the anterior, one of the medial, and five of the posterior groups. This is the first evidence of five posterior sequences in echinoderms.

Journal Article↗

PubMed1995

The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5'-nucleotidase family.

The saliva of hematophagous insects contains a variety of pharmacologically active substances that counteract the normal hemostatic response to injury in vertebrate hosts. The yellow-fever mosquito, Aedes aegypti, secretes an apyrase that inhibits ADP-dependent platelet aggregation. Apyrase was purified as an active enzyme from adult female salivary glands and subjected to tryptic digestion, and the resulting peptides were sequenced. The amino acid sequences obtained match the conceptual translation product of a cDNA clone isolated from an adult female salivary gland library. Sequence comparisons indicate similarities with a ubiquitous family of 5'-nucleotidases. The mosquito protein differs from other members of the family by lacking a carboxyl-terminal hydrophobic domain. The apparent conversion of a gene encoding an enzyme involved in a common metabolic event at the cellular level to a gene involved in the antihemostatic response of mosquitoes illustrates one way this particular insect has adapted to the challenges of bloodfeeding.

5'-Nucleotidase↗

PubMed1994

Isolation and characterization of a novel gene encoding nuclear protein at a locus (D11S636) tightly linked to multiple endocrine neoplasia type 1 (MEN1).

To identify a gene responsible for multiple endocrine neoplasia type 1 (MEN1), we attempted to isolate potentially transcribable fragments from cosmid clones derived from a region on chromosome 11q13 where genetic linkage studies and analyses of loss of heterozygosity in MEN1-associated tumors have localized the MEN1 gene. By an exon-amplification method, we recovered three exon-like sequences from one of these clones, cCI11-367, and using these sequences as probes we were able to isolate new clones from cerebrum, cerebellum, and fetal-liver cDNA libraries. Sequence analysis of these cDNA clones revealed that the transcribed gene, designated ZFM1, encodes a novel 623-amino-acid protein containing domains with interesting structural properties including a nuclear transport domain, a metal binding motif, and glutamine- and proline-rich regions. Analysis by the reverse-transcriptase polymerase chain reaction (RT-PCR) indicated that this gene is expressed in various tissues including endocrine organs such as thyroid gland, pancreas, adrenal gland, and ovary. These data suggest that ZFM1 might be a candidate for mutations that cause MEN1.

Amino Acid Sequence↗

PubMed2005

Deconvolution of a complex target using DNA aptamers.

In vitro selection of single-stranded nucleic acid aptamers from large random sequence libraries is now a straightforward process particularly when screening with a single target molecule. These libraries contain considerable shape diversity as evident by the successful isolation of aptamers that bind with high affinity and specificity to chemically diverse targets. We propose that aptamer libraries contain sufficient shape diversity to allow deconvolution of a complex mixture of targets. Using unfractionated human plasma as our experimental model, we aim to develop methods to obtain aptamers against as many proteins as possible. To begin, it is critical that we understand how aptamer populations change with increasing rounds of in vitro selection when using complex mixtures. Our results show that sequence representation in the selected population changes dramatically with increasing rounds of selection. Certain aptamer families were apparent after only three selection rounds. Two additional cycles saw a decline in the relative abundance of these families and the emergence of yet another family that accounted for more than 60% of sequences in the pool. To overcome this population convergence, an aptamer-based target depletion method was developed, and the library screen was repeated. The previous dominant family effectively disappeared from the selected populations but was replaced by other aptamer families. Insights gained from these initial experiments are now being applied in the creation of second generation plasma protein screens and also to the analysis of other complex biological targets.

Aptamers, Nucleotide↗