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PubMed1992

Directed isolation of human genes that escape X inactivation.

Existing methodologies have been combined to produce a directed approach to the isolation of human genes that escape X inactivation. A mouse-human somatic cell hybrid line was established that has an inactive X as its only human chromosome, and nuclear RNA from this cell line was used to construct a cDNA library. Transcribed human sequences were isolated by screening the library with labeled human DNA. The corresponding genomic sequences were isolated in phage or cosmid clones, and exons were identified by detection of transcripts on northern blots. By these means three human loci have been identified that contain genes expressed from an inactive X chromosome. Fluorescence in situ hybridization has been used to map these genes to Xp21.1-22.1, Xp22.1-22.2, and terminal Xp/Yp. One of the three genes (XE45) corresponds to the ZFX gene, while the other two genes (XE7 and XE59) represent novel cloned sequences. Physical and genetic evidence indicate that XE7 is a newly identified pseudoautosomal gene.

Animals↗

PubMed2004

[Screening and identification of mimic epitopes of monoclonal antibodies against hantaan virus using phage display technique].

AIM: To obtain mimic epitopes of monoclonal antibody (mAb) against Hantaan virus, and identify preliminarily their immunological characteristics. METHODS: Group-specific mAbs F3 and B11 were used as selective molecules for biopanning. After biopanning, the positive phages were identified by ELISA and DNA sequencing. Among the positive phages, F3-8, B11-18, B11-20 and B11-24 were selected to immunize BALB/c mice three times, respectively. The antiserum was then titered and confirmed by sandwich ELISA and competition ELISA. RESULTS: After 3 to 4 rounds of effective screening, the majority of the selected clones were found able to react to mAb F3 or B11 in a dose-dependent manner, but not to BSA, rHBsAg or other unrelated mAbs. The amino acid sequences of the clones binding to mAb F3 contained an identical sequence MHGPTKNQMWHT, which had higher homology to 750-759 amino acids between a pair of cysteines within glycoprotein G2 of HTNV/SEOV, while those of clones binding to mAb B11 had no evident homologous regions within HTNV/SEOV proteins. The specific antibodies increased significantly following immunization with phage peptides, which indicated that the phage-displayed peptides had not only good antigenicity, but also strong immunoreactivity. CONCLUSION: The phage-displayed peptides could mimic the epitope of HFRSV antigen, which would provide the potential for preparing more effective epitope-based vaccines and specific diagnostic reagents.

Amino Acid Sequence↗

PubMed1988

Molecular cloning of the alpha subunit of human and guinea pig leukocyte adhesion glycoprotein Mo1: chromosomal localization and homology to the alpha subunits of integrins.

The cell-surface glycoprotein Mo1 is a member of the family of leukocyte cell adhesion molecules (Leu-CAMs) that includes lymphocyte function-associated antigen 1 (LFA-1) and p150,95. Each Leu-CAM is a heterodimer with a distinct alpha subunit noncovalently associated with a common beta subunit. Leu-CAMs play crucial roles in cell-cell and cell-matrix interactions. We describe the isolation and analysis of two partial cDNA clones encoding the alpha subunit of the Leu-CAM Mo1 in humans and guinea pigs. A monoclonal antibody directed against an epitope in the carboxyl-terminal portion of the guinea pig alpha chain was used for immunoscreening a lambda gt11 expression library. The sequence of a 378-base-pair insert from one immunoreactive clone revealed a single continuous open reading frame encoding 126 amino acids including a 26-amino acid tryptic peptide isolated from the purified guinea pig alpha subunit. A cDNA clone of identical size was isolated from a human monocyte/lymphocyte cDNA library by using the guinea pig clone as a probe. The human clone also encoded a 126-amino acid peptide including the sequence of an additional tryptic peptide present in purified human Mo1 alpha chain. RNA gel blots revealed that mature Mo1 alpha chain mRNA is approximately 5 kilobases in guinea pigs and humans. Southern analysis of DNA from hamster-human hybrids localized the human Mo1 alpha chain to chromosome 16, which has been shown to contain the gene for the alpha chain of lymphocyte function-associated antigen 1. A comparison of the Mo1 alpha chain coding region revealed significant homologies with carboxyl-terminal portions of the alpha subunits of fibronectin, vitronectin, and platelet IIb/IIIa receptors. These data suggest that the alpha subunits of Leu-CAMs evolved by gene duplication from a common ancestral gene and strengthen the hypothesis that the alpha subunits of these heterodimeric cell adhesion molecules on myeloid and lymphoid cells, platelets, and fibroblasts are evolutionary related.

Amino Acid Sequence↗

PubMed1992

[The construction of rich genomic library of bovine Y chromosome].

According to the fundamentals of DNA renatured kinetics, we made a study in which genomic DNA from male cattle was completely digested with Sau 3A, while female DNA was sheared to an average size of 500 bp fragments. The Y derived DNA sequence rich library of bovine has been contracted with pUC19 plasmid as vector and JM109 as host bacteria by reassociation of male and female DNA. Male and female DNA probes, were used. The result of colony hybridization showed that out of 1000 recombinants analysed, 6 were found to be stronger signal with male probe. Analysis of Pvu' I digested DNA fragment revealed that most of the inserts were in the 90-300 bp.

Animals↗

PubMed1985

Molecular cloning of the human cholecystokinin gene by use of a synthetic probe containing deoxyinosine.

A synthetic DNA based on the known amino acid sequence of the brain/gut peptide cholecystokinin (CCK) was synthesized. This DNA contained deoxyinosines at ambiguous codon positions and was used as a probe to isolate the CCK gene directly from a human genomic library. Nucleotide sequence analysis of the isolated gene revealed that human preprocholecystokinin consists of 115 amino acid residues, with 11 amino acids in common with the human gastrin precursor, another member of the gastrin-CCK family, and that the coding region is separated by a single, long intron. CCK appears to be encoded by a single-copy gene in the haploid human genome, as revealed by genomic Southern hybridization analysis, suggesting that the same gene is expressed both in gut and brain.

Amino Acid Sequence↗

PubMedDate not supplied

RFLP mapping of expressed sequence tags in the chicken.

Chicken cDNA probes were used to map 10 loci by restriction fragment length polymorphism (RFLP) analysis in progeny of the Jungle Fowl x White Leghorn chicken backcross gene mapping reference population. The cDNA probes were from a subset of a T-cell library whose sequence tags showed significant homology to other avian or mammalian sequences in the EMBL database. Placement of loci generated by Compton cDNA clones on the East Lansing linkage map contributes to development of a consensus from the two linkage maps and will help provide a framework to evaluate the phenotypic effects of these genes.

Animals↗

PubMed1988

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence.

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3'- and 5'-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34,462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases was found as a highly conserved sequence.

Amino Acid Sequence↗

PubMed2002

Analysis of a library of macaque nuclear mitochondrial sequences confirms macaque origin of divergent sequences from old oral polio vaccine samples.

Nuclear mtDNA sequences (numts) are a widespread family of paralogs evolving as pseudogenes in chromosomal DNA [Zhang, D. E. & Hewitt, G. M. (1996) TREE 11, 247-251 and Bensasson, D., Zhang, D., Hartl, D. L. & Hewitt, G. M. (2001) TREE 16, 314-321]. When trying to identify the species origin of an unknown DNA sample by way of an mtDNA locus, PCR may amplify both mtDNA and numts. Indeed, occasionally numts dominate confounding attempts at species identification [Bensasson, D., Zhang, D. X. & Hewitt, G. M. (2000) Mol. Biol. Evol. 17, 406-415; Wallace, D. C., et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14900-14905]. Rhesus and cynomolgus macaque mtDNA haplotypes were identified in a study of oral polio vaccine samples dating from the late 1950s [Blancou, P., et al. (2001) Nature (London) 410, 1045-1046]. They were accompanied by a number of putative numts. To confirm that these putative numts were of macaque origin, a library of numts corresponding to a small segment of 12S rDNA locus has been made by using DNA from a Chinese rhesus macaque. A broad distribution was found with up to 30% sequence variation. Phylogenetic analysis showed that the evolutionary trajectories of numts and bona fide mtDNA haplotypes do not overlap with the signal exception of the host species; mtDNA fragments are continually crossing over into the germ line. In the case of divergent mtDNA sequences from old oral polio vaccine samples [Blancou, P., et al. (2001) Nature (London) 410, 1045-1046], all were closely related to numts in the Chinese macaque library.

Animals↗

PubMed1993

Genomic structure of NKG5, a human NK and T cell-specific activation gene.

We reported previously the isolation of a cDNA clone, designated NKG5, encoding a secreted protein that is expressed only in natural killer and T cells and is strongly upregulated upon cell activation. In this report we have isolated the NKG5 gene from a human placental genomic library and sequenced the gene and two kilobases of 5'-flanking DNA. Comparison with the cDNA sequence reveals that the NKG5 gene consists of five exons and four introns. Intron 1 contains a DNA segment that was reported to occur as an exon in 519, a closely related cDNA clone that was isolated from a T-cell library. This result indicates that NKG5 and 519 are alternative splicing products of a single gene. The 5'-flanking region of the NKG5 gene was analyzed for homology with the promoter regions of cytokines and other activation-induced genes showing lymphocyte-specific expression. Several segments displaying sequence similarity were identified. We also identified numerous sequence elements that have strong similarity to known binding sites for transcriptional regulatory proteins including T cell-specific and activation-specific regulatory factors. These findings are consistent with the cell-specific expression and the tight regulatory control that is observed for the NKG5 gene.

Alternative Splicing↗

PubMed1985

Characterization of a rat tRNA gene cluster: comparison of nucleotide sequences of the gene for tRNALeu newly found in repeating units.

In the rat, DNA carrying a cluster of the genes for tRNAAsp, tRNAGly, and tRNAGlu, aligned in that order, is repeated about 10 times. Seven DNA clones corresponding to the independent repeating units were isolated from a rat gene library. Nucleotide sequence analysis of these clones revealed the presence of a fourth tRNA gene, the gene for tRNALeu, in the cluster. The tRNALeu gene is located about 600 base pairs (bp) upstream from the tRNAAsp gene and its polarity differs from those of the other three tRNA genes. Among the repeating units, the nucleotide sequence of tRNALeu is conserved to a relatively high degree.

Animals↗

PubMed2005

Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis.

Isoflavonoids are ecophysiologically active secondary metabolites of the Leguminosae and known for health-promoting phytoestrogenic functions. Isoflavones are synthesized by 1,2-elimination of water from 2-hydroxyisoflavanones, the first intermediate with the isoflavonoid skeleton, but details of this dehydration have been unclear. We screened the extracts of repeatedly fractionated Escherichia coli expressing a Glycyrrhiza echinata cDNA library for the activity to convert a radiolabeled precursor into formononetin (7-hydroxy-4'-methoxyisoflavone), and a clone of 2-hydroxyisoflavanone dehydratase (HID) was isolated. Another HID cDNA was cloned from soybean (Glycine max), based on the sequence information in its expressed sequence tag library. Kinetic studies revealed that G. echinata HID is specific to 2,7-dihydroxy-4'-methoxyisoflavanone, while soybean HID has broader specificity to both 4'-hydroxylated and 4'-methoxylated 2-hydroxyisoflavanones, reflecting the structures of isoflavones contained in each plant species. Strikingly, HID proteins were members of a large carboxylesterase family, of which plant proteins form a monophyletic group and some are assigned defensive functions with no intrinsic catalytic activities identified. Site-directed mutagenesis with soybean HID protein suggested that the characteristic oxyanion hole and catalytic triad are essential for the dehydratase as well as the faint esterase activities. The findings, to our knowledge, represent a new example of recruitment of enzymes of primary metabolism during the molecular evolution of plant secondary metabolism.

Amino Acid Sequence↗

PubMed2027

Workflow for Long-Read Amplicon Sequencing of Chikungunya Virus Using Oxford Nanopore Technology.

This protocol provides a comprehensive, step-by-step workflow for whole-genome sequencing of Chikungunya virus (CHIKV) using an amplicon-based strategy optimized for Oxford Nanopore Technologies (ONT) platforms. The procedure includes detailed instructions for sample handling, viral RNA extraction, quality control, cDNA synthesis, multiplex PCR amplification, library preparation, sequencing, and primary bioinformatic processing. The protocol is designed to maximize reproducibility across laboratories and is suitable for genomic surveillance applications, including outbreak investigation and molecular epidemiology, even when working with low-to-moderate viral loads.

Chikungunya virus↗

PubMed2006

Automated bacterial genome analysis and annotation.

More than 300 bacterial genome sequences are publicly available, and many more are scheduled to be completed and released in the near future. Converting this raw sequence information into a better understanding of the biology of bacteria involves the identification and annotation of genes, proteins and pathways. This processing is typically done using sequence annotation pipelines comprised of a variety of software modules and, in some cases, human experts. The reference databases, computational methods and knowledge that form the basis of these pipelines are constantly evolving, and thus there is a need to reprocess genome annotations on a regular basis. The combined challenge of revising existing annotations and extracting useful information from the flood of new genome sequences will necessitate more reliance on completely automated systems.

Bacterial Proteins↗

PubMed2006

A comprehensive SAGE database for the analysis of gammadelta T cells.

Gammadelta T cells have been conserved since the adaptive immune system arose, yet their importance is still unclear. In an attempt to compensate for the lack of a broad knowledge base of gammadelta T cells across species, global analyses of gammadelta T cell transcriptomes have been performed using serial analysis of gene expression (SAGE). Twelve new SAGE libraries were generated from the following bovine lymphocyte populations: magnetic bead-sorted blood gammadelta T cells, spleen gammadelta T cells and enriched spleen alphabeta T cells from a single calf, both rested and Con A/IL2 stimulated, and flow cytometry-sorted blood gammadelta and alphabeta T cells each either rested, Con A/IL2, or phorbol 12 myristate 13-acetate/ionomycin stimulated. These new libraries complement two earlier SAGE libraries of circulating gammadelta T cell subsets. These databases were analyzed using new web-based bioinformatic tools, which allow the user to rapidly compare gene expression patterns within these and other SAGE and standard expressed sequence tag libraries generated from different cell types and different species. These analyses revealed striking differences between blood and spleen gammadelta T cells and how these cells respond to mitogenic stimulation. These analyses also confirm previous studies that suggested that global gene expression in gammadelta and alphabeta T cells is quite similar; however, a 5-fold increase in gammadelta T cell-specific transcripts could be induced by Con A/IL2 stimulation. These new public databases provide additional resources for the annotation/analysis of global gene expression in gammadelta T cells, which will facilitate studies of the biology of this enigmatic lymphoid cell.

Animals↗

PubMed1988

Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid).

Osteonectin is a prominent noncollagenous protein of developing bone. A 2150-base-pair cDNA coding for osteonectin, isolated from a bovine bone cell lambda gt11 expression library, was sequenced and identified by comparison with protein sequence data. The nucleotide sequence predicts that osteonectin contains 304 amino acids, including a 17-residue signal peptide. Analysis of the deduced protein sequence suggests that the secreted protein contains at least four distinct structural domains. An acidic region at the amino terminus of the protein appears to be a potential hydroxyapatite-binding site. This is followed by a second domain, rich in cysteine, that shows sequence homology with cysteine-rich domains in turkey ovomucoid and other serine proteinase inhibitors. Two sequences homologous with central calcium-binding loops of "EF hands" and thus having potential to be high-affinity calcium-binding sites are located in two other domains within the carboxyl-terminal half of the protein. Finally, the osteonectin sequence shows near identity (greater than 90%) with another protein, SPARC (secreted protein, acidic and rich in cysteine), secreted by mouse parietal endoderm. These data suggest that osteonectin, a protein present in bone and other selected tissues, is a multifunctional protein.

Amino Acid Sequence↗

PubMed1999

Diversity of human endogenous retrovirus class II-like sequences.

Class II human endogenous retroviruses (HERVs), often referred to as mouse mammary tumour virus (MMTV)-like or HERV-K elements, have similarities to several animal infectious retroviruses. Single clones from each of nine class II HERV groups (NMWV 1 to NMWV 9), isolated from a human breast cancer cell genomic library, were sequenced over a 244 bp stretch of the conserved reverse transcriptase region. These sequences were aligned to related exogenous and endogenous retroviruses and a phylogenetic tree was constructed. Sequences with more than 80% identity were considered as members of one group and we report here that the class II HERV family consists of at least ten groups. Three of the sequenced clones, from groups NMWV 3, 7 and 9, could not be related to any other previously identified elements and constituted their own groups. NMWV 8 had no similarity to any retroviral sequences in the sequenced region and is so far considered to be non-retroviral.

Amino Acid Sequence↗

PubMed1990

Random peptide libraries: a source of specific protein binding molecules.

Libraries of random peptide sequences were constructed and screened to identify peptides that specifically bind to proteins. In one of these about 2 X 10(7) different 15-residue peptide sequences were expressed on the surface of the coliphage M13. Each phage encoded a single random sequence and expressed it as a fusion complex with pIII, a minor coat protein present at five molecules per phage. Phage encoding nine different streptavidin-binding peptide sequences were isolated from this library. The core consensus sequence was His-Pro-Gln and binding of these phage to streptavidin was inhibited by biotin. This type of library makes it possible to identify peptides that bind to proteins (or other macromolecules) that have no previously known affinity for peptides.

Adsorption↗

PubMed1993

Generation of expressed sequence tags of random root cDNA clones of Brassica napus by single-run partial sequencing.

Two hundred thirty-seven expressed sequence tags (ESTs) of Brassica napus were generated by single-run partial sequencing of 197 random root cDNA clones. A computer search of these root ESTs revealed that 21 ESTs show significant similarity to the protein-coding sequences in the existing data bases, including five stress- or defense-related genes and four clones related to the genes from other kingdoms. Northern blot analysis of the 10 data base-matched cDNA clones revealed that many of the clones are expressed most abundantly in root but less abundantly in other organs. However, two clones were highly root specific. The results show that generation of the root ESTs by partial sequencing of random cDNA clones along with the expression analysis is an efficient approach to isolate genes that are functional in plant root in a large scale. We also discuss the results of the examination of cDNA libraries and sequencing methods suitable for this approach.

Amino Acid Sequence↗