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PubMed1992

Analysis of the chicken fast myosin heavy chain family. Localization of isoform-specific antibody epitopes and regions of divergence.

cDNAs encoding the rod region of four different fast myosin heavy chains (MYCHs) in the chicken were identified, using anti-MYCH monoclonal antibodies, in two expression libraries prepared from 19-day embryonic and adult chicken muscle. These clones were used to determine the amino acid sequences that encompass the epitopes of five anti-MYHC monoclonal antibodies. Additionally, the amino acid sequences were compared to each other and to a full length embryonic MYHC. Although there is extensive homology in the chicken fast myosin rods, sequences within the hinge, within the central portion of the light meromyosin fragment, and at the carboxy terminus exhibit the largest number of amino acid substitutions. We propose that divergence within these subdomains may contribute to isoform-specific properties associated with skeletal myosin rods.

Amino Acid Sequence↗

PubMed1990

Identification of a series of differentiation-associated gene sequences from GM-CSF stimulated bone marrow.

The production of terminally differentiated granulocytes and monocytes occurs by a program of orderly and sequential gene expression. This genetic program can be studied in vitro utilizing granulocyte-monocyte colony stimulating factor (GM-CSF) to stimulate proliferation and myeloid cell maturation. We have identified a series of genes expressed during early myeloid differentiation by differential screening of a cDNA library prepared from GM-CSF stimulated murine progenitor cells. From 72 potential early myeloid specific clones, three (B9, C9, C15) were characterized further. The time course of RNA accumulation during GM-CSF stimulated maturation demonstrated a unique pattern for each clone. B9 was expressed predominantly on day 3, and followed a pattern of accumulation similar to that for myeloperoxidase. (Jaffe et al. (1988), Oncogene, 2, 167-174). C9 expression increased gradually to a maximum level on day 3 and then plateaued; it appeared to be equally expressed in granulocytes and monocytes. C15 was expressed at a consistently high level in unstimulated cells and at 1-3 days post GM-CSF stimulation. It then decreased to undetectable levels. Hybridization of these clones to a panel of murine tissue RNAs demonstrated restricted expression of B9 to bone marrow, while C9 was present in most murine tissues, including thymus. C15 expression was relatively restricted to ovary/uterus, liver and adrenal, in addition to bone marrow. Partial DNA sequence analysis suggested that B9 was a novel sequence not previously identified. C9 was identified as thymosin beta-4, and C15 showed extensive homology to lactotransferrin. Thus, screening a bone marrow cDNA library by differential hybridization has successfully yielded a series of DNA sequences regulated during murine myelopoiesis. These include novel sequences (B9), genes previously known to be regulated during myelopoiesis (C15), as well as sequences not recognized previously as being associated with myeloid differentiation (C9).

Animals↗

PubMed2003

A cDNA clone for 3-carene synthase from Salvia stenophylla.

The essential oil of Salvia stenophylla contains (+)-3-carene as the principal monoterpene component. Using an enriched cDNA library prepared from mRNA isolated from S. stenophylla peltate glandular trichomes, and a homology-based cloning strategy, a full-length cDNA was isolated that encoded a preprotein of 69.7 kDa which resembled a monoterpene synthase in sequence. Heterologous expression of the gene in Escherichia coli provided a soluble recombinant enzyme capable of catalyzing the divalent metal ion-dependent conversion of geranyl diphosphate to (+)-3-carene and to lesser amounts of limonene, myrcene, 4-carene and beta-phellandrene. This multiple-product synthase is responsible for the production of all of the essential oil monoterpenes of S. stenophylla.

Alkyl and Aryl Transferases↗

PubMed1993

Species-specific recombinant DNA probes for Mycoplasma meleagridis.

Two recombinant DNA probes (pMM-2 and pMM-13) were isolated from a Mycoplasma meleagridis strain 17529 genomic library prepared in plasmid pUC8, and Escherichia coli strain JM83. In dot blot assays, 32P-labeled pMM-13 with a DNA insert of 3.5 kbp, hybridized with 18 isolates of M. meleagridis but not with 16 other known species of avian mycoplasmas. Except for weaker signals on hybridization with the M. meleagridis cultures, pMM-2 with an DNA insert of 0.85 kbp, showed a similar reaction pattern. The minimal concentration of M. meleagridis strain 17529 chromosomal DNA that pMM-13 and pMM-2 detected were 1 and 8 ng, respectively. Neither probe hybridized with chromosomal DNA of M. gallisepticum strain S6, M. synoviae strain WVU-1853, or M. iowae strain I-695 at concentration of 256 ng.

Animals↗

PubMed1993

Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function.

A defined synthetic medium was used to determine the amino acid requirements of Enterococcus faecalis OG1 and to demonstrate the absence of a requirement for exogenous purines or pyrimidines. Genomic libraries prepared from strain OG1 were transduced into Escherichia coli auxotrophic mutants, and cloned DNAs which complemented pyrC, pyrD, purF, purL, and guaAB mutations were identified. These and other cloned DNAs with known functions were localized on a restriction map of OG1 which was generated with SfiI (5 fragments), AscI (9 fragments), and NotI (15 fragments); the size of the OG1 chromosome was revised from a previous estimate of approximately 2,750 kb to 2,825 kb. The synthetic medium and the restriction map should be useful for studying enterococcal metabolic functions and the relationships between chromosomally encoded genes.

Amino Acids↗

PubMed1995

Structure of two cDNAs encoding cholecystokinin precursors from the brain of Xenopus laevis.

The skin secretions of many frogs, including Xenopus laevis, contain caerulein, a peptide related to mammalian cholecystokinin. We have screened a cDNA library prepared from the brain of this frog using a cloned cDNA encoding one of the caerulein precursors as a probe. Two clones were isolated which contained inserts encoding cholecystokinin precursors. It was found that the predicted precursor polypeptides resembled their mammalian counterparts rather than the caerulein precursors from the same species. The corresponding mRNAs of different size encoding the Xenopus cholecystokinin precursors are expressed in brain and in the gastrointestinal tract, but not in skin. The smaller mRNA was also detected in lung. These data demonstrate that a polypeptide homologous to mammalian cholecystokinin precursors was present early in the evolution of vertebrates. The possible evolution of the genes encoding the more complex caerulein precursors is discussed.

Amino Acid Sequence↗

PubMed1996

Isolation and characterization of two distinct growth hormone cDNAs from the goldfish, Carassius auratus.

As a first step toward the development of a ribonuclease protection assay for studying the regulation of growth hormone (GH) gene expression in pituitary cells of the goldfish, Carassius auratus, we report the isolation of two cDNA clones encoding goldfish GH from a cDNA library prepared from pituitary poly(A)+ RNA. The complete nucleotide sequences of these two GH cDNA clones have been determined and both of them were predicted to encode a polypeptide of 210 amino acids (aa) including a putative signal peptide of 22 aa. One of the GH cDNAs encodes a polypeptide (gfGHI) with five cysteine residues (similar to other carp Ms), whereas another encodes a polypeptide (gfGHII) with four cysteine residues (similar to most teleostean GHs). Because these two GH cDNAs have distinct nucleotide sequences at their coding and 3' untranslated regions, they are likely to be encoded by two different genes.

Amino Acid Sequence↗

PubMed1990

Cloning of specific diagnostic antigens of Onchocerca volvulus.

Specific, serological diagnosis is one of the main goals in onchocerciasis research. To date this objective has been hampered by (a) scarcity of parasite material, and (b) antigenic cross-reaction between Onchocerca volvulus and other nematode species. In order to obtain specific antigens, and in amounts suitable for study, molecular biological techniques have been adopted. A lambda gt11 cDNA expression library prepared from O. volvulus adult female worms was screened using infected human sera from onchocerciasis patients and rabbit hyperimmune sera raised against Onchocerca and genus-specific Onchocerca antigen extracts. Five clones were selected and their inserts expressed as beta-galactosidase fusion proteins. The fusion proteins were examined using individual sera from patients with O. volvulus or Wuchereria bancrofti infections. Three of the fusion proteins were recognised by more than 80% of O. volvulus sera and exhibited weak reactivity with a few W. bancrofti sera. One of these three clones was recognised to a significantly greater degree by sera from sowda than from generalised onchocerciasis patients.

Animals↗

PubMed1995

Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene.

The mutagen-sensitive CHO line irs1SF was previously isolated on the basis of hypersensitivity to ionizing radiation and was found to be chromosomally unstable as well as cross-sensitive to diverse kinds of DNA-damaging agents. The analysis of somatic cell hybrids formed between irs1SF and human lymphocytes implicated a human gene (defined as XRCC3; x-ray repair cross-complementing), which partially restored mitomycin C resistance to the mutant. A functional cDNA that confers mitomycin C resistance was transferred to irs1SF cells by transforming them with an expression cDNA library and obtaining primary and secondary transformants. Functional cDNA clones were recovered from a cosmid library prepared from a secondary transformant. Transformants also showed partial correction of sensitivity to cisplatin and gamma-rays, efficient correction of chromosomal instability, and substantially improved plating efficiency and growth rate. The XRCC3 cDNA insert is approximately 2.5 kb and detects an approximately 3.0-kb mRNA on Northern blots. The cDNA was mapped by fluorescence in situ hybridization to human chromosome 14q32.3, which was consistent with the chromosome concordance data of two independent hybrid clone panels.

Animals↗

PubMed1993

Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells.

Cloning of a ligand for the murine flt3/flk-2 tyrosine kinase receptor was undertaken using a soluble form of the receptor to identify a source of ligand. A murine T cell line, P7B-0.3A4, was identified that appeared to express a cell surface ligand for this receptor. A cDNA clone was isolated from an expression library prepared from these cells that was capable, when transfected into cells, of conferring binding to a soluble form of the flt3/flk-2 receptor. The cDNA for this ligand encodes a type I transmembrane protein that stimulates the proliferation of cells transfected with the flt3/flk-2 receptor. A soluble form of the ligand stimulates the proliferation of defined subpopulations of murine bone marrow and fetal liver cells as well as human bone marrow cells that are highly enriched for hematopoietic stem cells and primitive uncommitted progenitor cells.

Amino Acid Sequence↗

PubMedDate not supplied

Yeast artificial chromosomes: an alternative approach to the molecular analysis of mouse developmental mutations.

Mammalian genetics now allows a molecular study of genomic regions previously analysed by genetic and embryological techniques. To simplify such an analysis, we have established a number of libraries of mouse DNA in Yeast Artificial Chromosome (YAC) vectors, constructed either by partial digestion with EcoRI, or by complete digestion with enzymes which cut rarely in the mammalian genome. In this paper we report the construction of complete digest libraries prepared from mouse genomic DNA using the rare cutter enzymes NotI and BssHII, and the detection of gene loci from the H-2 complex, the t-complex, and other loci from the mouse genome. Due to their large insert size, YAC clones simplify the cloning of extended regions of the mouse genome surrounding known developmental mutations and should, after introduction into the germ line, offer a high probability of correct expression of the genes contained within the cloned region. We hope that this will allow the use of YAC clones to scan regions of interest such as the t-complex for specific genes by testing DNA introduced into transgenic mice for the ability to complement mutations localised to this region.

Animals↗

PubMed2003

Nylon filter arrays reveal differential gene expression in proteoid roots of white lupin in response to phosphorus deficiency.

White lupin (Lupinus albus) adapts to phosphorus deficiency (-P) by the development of short, densely clustered lateral roots called proteoid (or cluster) roots. In an effort to better understand the molecular events mediating these adaptive responses, we have isolated and sequenced 2,102 expressed sequence tags (ESTs) from cDNA libraries prepared with RNA isolated at different stages of proteoid root development. Determination of overlapping regions revealed 322 contigs (redundant copy transcripts) and 1,126 singletons (single-copy transcripts) that compile to a total of 1,448 unique genes (unigenes). Nylon filter arrays with these 2,102 ESTs from proteoid roots were performed to evaluate global aspects of gene expression in response to -P stress. ESTs differentially expressed in P-deficient proteoid roots compared with +P and -P normal roots include genes involved in carbon metabolism, secondary metabolism, P scavenging and remobilization, plant hormone metabolism, and signal transduction.

Algorithms↗

PubMed2004

Marker-based cloning of the region containing the UhAvr1 avirulence gene from the basidiomycete barley pathogen Ustilago hordei.

Race-cultivar specialization during the interaction of the basidiomycete smut pathogen Ustilago hordei with its barley host was described in the 1940s. Subsequent genetic analyses revealed the presence of dominant avirulence genes in the pathogen that conform to the gene-for-gene theory. This pathosystem therefore presents an opportunity for the molecular genetic characterization of fungal genes controlling avirulence. We performed a cross between U. hordei strains to obtain 54 progeny segregating for three dominant avirulence genes on three differential barley cultivars. Bulked segregant analysis was used to identify RAPD and AFLP markers tightly linked to the avirulence gene UhAvr1. The UhAvr1 gene is located in an area containing repetitive DNA and this region is undetectable in cosmid libraries prepared from the avirulent parental strain. PCR and hybridization probes developed from the linked markers were therefore used to identify cosmid clones from the virulent (Uhavr1) parent. By walking on Uhavr1-linked cosmid clones, a nonrepetitive, nearby probe was found that recognized five overlapping BAC clones spanning 170 kb from the UhAvr1 parent. A contig of the clones in the UhAvr1 region was constructed and selected probes were used for RFLP analysis of the segregating population. This approach genetically defined an approximately 80-kb region that carries the UhAvr1 gene and provided cloned sequences for subsequent genetic analysis. UhAvr1 represents the first avirulence gene cloned from a basidiomycete plant pathogen.

Base Sequence↗

PubMed1995

The complete cDNA and deduced amino acid sequence of equine IgE.

The cDNA from a transcript encoding the complete heavy chain of the equine immunoglobulin IgE has been cloned and sequenced. A fragment of the equine epsilon gene was amplified from cDNA using PCR and this fragment was then used to probe a horse cDNA library prepared from peripheral blood lymphocytes. A recombinant clone containing the cDNA encoding the complete horse epsilon chain and its associated V-D-J and leader, was subsequently isolated and sequenced. Comparison of the deduced amino acid sequence of equine IgE with the C epsilon heavy chains of other species indicates it to be most similar to human C epsilon (54%), followed by sheep C epsilon (52%); the greatest sequence similarity was found in the C epsilon 3 and C epsilon 4 domains among the species compared. The associated V-region had greatest similarity in FR1 to those reported for sheep and the cow (> 80%) but less than 60% to the VH sequences of other species in FR3.

Amino Acid Sequence↗

PubMed2005

Molecular cloning of cDNA for SPase, a monkey cathepsin L orthologue.

SPase is a cysteine protease isolated from an African green monkey kidney cell line, CV-1, and has selective cleavage activity toward transcription factor SP-1 and retinoblastoma susceptibility gene product RB. In this study, a cDNA encoding SPase was cloned from a cDNA library prepared from CV-1 cells. The cDNA clone encodes 333 amino acids and is 96.5% identical to human cathepsin L at the nucleotide and amino acid sequence levels. SPase appears to be translated as a preproenzyme based on the comparison between the deduced amino acid sequence and the N-terminal sequence of the purified enzyme. Northern blot analysis exhibited the considerably higher expression of SPase in CV-1 cells compared with COS-1 cells, showing a good correlation with enzymatic activity in these cell lines. Bacterially expressed SPase protein exhibited proteolytic activity toward SP-1 and RB proteins. These observations suggest that SPase is a monkey cathepsin L orthologue.

Amino Acid Sequence↗

PubMed2006

Upregulation of retinal transglutaminase during the axonal elongation stage of goldfish optic nerve regeneration.

Fish CNS neurons can repair their axons following nerve injury, whereas mammalian CNS neurons cannot regenerate, and become apoptotic within 1-2 weeks after the nerve lesion. One explanation for these differences is that one, or several molecules are upregulated in fish CNS neurons during nerve regeneration, and this same molecule is downregulated in mammalian CNS neurons before the development of apoptosis caused by nerve injury. A molecule satisfying these criteria might successfully rescue and repair the mammalian CNS neurons. In this study, we looked for such a candidate molecule from goldfish retinas. Transglutaminase derived from goldfish retina (TG(R)) was characterized as a regenerating molecule after optic nerve injury. A full-length cDNA for TG(R) was isolated from the goldfish retinal cDNA library prepared from axotomized retinas. Levels of TG(R) mRNA and protein increased only in the retinal ganglion cells (RGCs) between 10 and 40 days after optic nerve transection. Recombinant TG(R) protein enhanced neurite outgrowth from adult fish RGCs in culture. Specific interference RNA and antibodies for TG(R) inhibited neurite outgrowth both in vitro and in vivo. In contrast, the level of TG(R) protein decreased in rat RGCs within 1-3 days after nerve injury. Furthermore, the addition of recombinant TG(R) to retinal cultures induced striking neurite outgrowth from adult rat RGCs. These molecular and cellular data strongly suggest that TG(R) promotes axonal elongation at the surface of injured RGCs after optic nerve injury.

Animals↗

PubMed1993

Mapping of the 70 kDa, 34 kDa, and 11 kDa subunit genes of the human multimeric single-stranded DNA binding protein (hSSB/RPA) to chromosome bands 17p13, 1p35-p36.1, and 7p21-p22.

Human single-stranded DNA binding protein (hSSB/RPA) is a multimeric single-stranded DNA binding protein consisting of three subunits of 70 kDa, 34 kDa, and 11 kDa. Human SSB was isolated from HeLa cells as an essential factor for the in vitro replication of simian virus 40 DNA. We and others have isolated and sequenced cDNAs for each subunit of the SSB. The chromosome on which each gene is located was determined through the analysis of a panel of human/hamster somatic cell hybrids using the polymerase chain reaction with pairs of synthetic oligonucleotide primers from the 3'-untranslated sequences of the genes. Genomic clones for each gene were isolated from a genomic cosmid library prepared from human lymphoblastoid cells. Using those clones as probes, we have carried out fluorescence in situ hybridization to human metaphase chromosomes and have mapped the 70 kDa subunit gene to 17p13, the 34 kDa subunit gene to 1p35-p36.1, and the 11 kDa subunit gene to 7p21-p22. Since hSSB participates in replication, recombination and repair of DNA, the physical mapping of hSSB genes may aid in the identification of human hereditary diseases associated with aberrant DNA reactions caused by genetic alterations of the hSSB.

Animals↗

PubMed1989

cDNA cloning of human calpastatin: sequence homology among human, pig, and rabbit calpastatins.

cDNA of human calpastatin, an inhibitor protein specific for calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase) was isolated by screening of a library prepared from human liver mRNA with pig calpastatin cDNA fragment as a probe. The primary structure of human calpastatin was deduced from the nucleotide sequence of the cDNA and compared with that of pig and rabbit calpastatins already reported. Human calpastatin consisted of 673 amino acid residues and had 78% and 77% identity to pig or rabbit calpastatins, respectively. Human calpastatin had a domain structure with four internally repetitive sequences and one N-terminal non-homologous sequence like the other calpastatins. Human calpastatin had two deletions, 22 and 13 residues long in domain L and domain 1, respectively, compared to pig or rabbit calpastatins.

Amino Acid Sequence↗