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PubMed2003

A genomics approach to crop pest and disease research.

Genome-wide analyses of gene function and gene expression are beginning to yield valuable information in many areas of biological research, and these genomic tools are now being applied to crop pest and disease research. DNA sequencing of cDNA libraries to generate sets of expressed sequence tags (ESTs) are allowing gene compendiums for crop diseases to be compiled. Annotation of such data collections is also providing a wealth of functional information about gene products through similarities to proteins with known function. The next phase of the functional genomics era will be to employ large-scale techniques to knock out or silence genes in order to synthesize gene-specific mutants for phenotypic analysis and to use micro-array methodology to analyze global gene expression, protein turnover and protein processing during the processes of parasitism and colonization. Application of these technologies promises to accelerate the pace that biological information relevant to crop protection accrues. The ability of researchers to assimilate this information into complex models and workable hypotheses is, thus, set to revolutionize the way we study pests and diseases of crop plants.

Crops, Agricultural↗
PubMed1996

Baculovirus expression and purification of human and rat cytochrome P450 2E1.

High-level expression of human and rat cytochrome P450 2E1 (CYP2E1) was achieved using a baculovirus expression system. A full length cDNA encoding human CYP2E1 was cloned from a human liver cDNA library and sequenced using the dideoxy sequencing method. Insect cells were infected with the homologous recombinant baculoviruses containing the human and rat CYP2E1 cDNAs, respectively. The infected cells were harvested at a time when 450-nm peak intensities were at a maximal level and there was no 420-nm peak observed in the reduced CO difference spectrum. Both human and rat CYP2E1 were then purified to electrophoretic homogeneity by a relatively rapid and efficient procedure. The specific contents of the purified human and rat CYP2E1 were 13.8 and 17.0 nmol/mg protein, respectively. The lambda(max) of the reduced CO difference spectra of both purified rat and human CYP2E1 was found to be 451.5 nm. When the purified rat and human CYP2E1 were reconstituted with purified rat NADPH-P450 reductase and human cytochrome b5, they were able to metabolize several known CYP2E1 substrates: chlorzoxazone, p-nitrophenol, acetaminophen, and carbon tetrachloride. Interestingly, cytochrome b5 markedly stimulated the CYP2E1-mediated two-electron oxidation of the first three substrates, while it had almost no effect on the presumed one-electron reduction of carbon tetrachloride.

Animals↗
PubMed1998

Cloning and characterization of Syrian hamster testosterone 7 alpha-hydroxylase, CYP2A9.

We report here the cloning of a cDNA encoding testosterone 7 alpha-hydroxylase in the Syrian hamster, designated CYP2A9. Overlapping clones encoding Syrian hamster CYP2A9 were isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on the cDNA library. The sequence of the CYP2A9 cDNA contains an open reading frame of 1482 nucleotides encoding a polypeptide of 493 amino acids with a calculated molecular mass of 56,295 Da. The sequence is flanked by a 5'-untranslated region of 10 bp and a 3'-untranslated region of 178 bp including the poly(A) tail. The deduced amino acid sequence shares significant homology with members of CYP2A subfamily, notably with CYP2A1 and CYP2A12 which have testosterone 7 alpha-hydroxylase activity. We characterized the catalytic activity of CYP2A9 using microsomes obtained by transient expression of its cDNA in transfected COS-7 cells. CYP2A9 was found to hydroxylate testosterone at position 7 alpha. In Syrian hamster livers, a higher level of testosterone 7 alpha-hydroxylase activity as well as the mRNA of CYP2A9 in male than in female was obtained. The testosterone 7 alpha-hydroxylase activity and the mRNA level in liver were both decreased moderately by administration of 3-methylcholanthrene and slightly by administration of phenobarbital. In contrast, in kidney, both 3-methylcholanthrene and phenobarbital significantly decreased the mRNA level. These facts indicate that the regulation of the hamster testosterone 7 alpha-hydroxylase (CYP2A9) expression is different from that of the rat (CYP2A1) and hamster reported previously by other workers.

Amino Acid Sequence↗
PubMed1999

Structure and expression of the rat CYP3A1 gene: isolation of the gene (P450/6betaB) and characterization of the recombinant protein.

A P450 gene (P450/6betaB) of the CYP3A subfamily was isolated from a rat genomic library. Nucleotide sequencing of the exons revealed a high similarity with P450PCN1 cDNA (Gonzalez et al. (1985), J. Biol. Chem. 260, 7345-7441), but differed in 41 nucleotides, resulting in 11 changes and 2 deletions of amino acid residues. The P450/6betaB spanned about 30 kbp and consisted of 13 exons, and was in exon number and size identical with CYP3A2 gene except in the 6th exon, which was shorter than that of CYP3A2. 6beta-B mRNA, which may be transcribed from P450/6betaB, was detected on Northern blotting and by reverse transcription-polymerase chain reaction (RT-PCR). Profiles of the developmental change and induction by a treatment with several chemicals were very similar to those of P450PCN1 mRNA reported previously. P450PCN1 mRNA and gene, however, were not detected by PCR in rats. To determine whether P450/6betaB encodes an active protein, a cDNA was isolated and expressed. Expression of 6beta-B cDNA in COS-1 cells was carried out and revealed that the recombinant protein comigrated with purified P4506beta-4 previously identified as CYP3A1. The recombinant 6beta-B protein showed similar turnover rate and regioselectivity for testosterone with purified P4506beta-4 by the simultaneous addition of NADPH-cytochrome P450 reductase and cytochrome b5. These data suggest that P450/6betaB encodes an active P450 form corresponding to CYP3A1 and P450PCN1 reported previously does not exist in rats.

Amino Acid Sequence↗
PubMed1994

5'-flanking region surrounding a human cytosolic phospholipase A2 gene.

The 5'-flanking region and the first four exons of a gene encoding human cytosolic phospholipase A2 (cPLA2) were isolated from a human lambda EMBL3 genomic library and sequenced. The 5'-flanking region is characterized by CA repeats, one of microsatellites. The analysis of the 5'-flanking region with transcription factor database suggests the existence of the transcription factor binding sites such as NF-kappa B, NF-IL6, AP-1, AP-2, and PEA3. These factors are well known to be induced or activated by the reagents (tumor necrosis factor alpha, interleukin-1, epidermal growth factor, and phorbol myristate acetate) reported as the inducers of a cPLA2 gene.

Base Sequence↗
PubMed1994

Molecular cloning of two small GTP-binding proteins from human skeletal muscle.

Two distinct cDNAs encoding small GTP-binding proteins were isolated from a human fetal skeletal muscle cDNA library. Nucleotide sequence analysis revealed that one clone encodes a protein of 218 amino acids, which shares highest amino acid identities (over 90%) with the members of YPT3 GTP-binding proteins subfamily and was termed H-YPT3; and the second clone encodes a protein of 201 amino acids, which exhibits strongest homology to rab1A, rab1B, and YPT1 at its N-terminus but diverges notably from these rab family members at the C-terminus. The second cDNA may represents a novel member of the small GTP-binding protein family and was designated H-ray. Northern blot analysis shows that both genes are ubiquitously expressed. Using a bacterial expression vector, each clone was overexpressed in Escherichia coli. The two bacterially produced proteins possess GTP-binding activity.

Amino Acid Sequence↗
PubMed1995

Cloning and characterisation of a bovine P2Y receptor.

Using a chick P2Y1 receptor cDNA probe we have isolated a mammalian P2Y receptor clone from a bovine aortic endothelial cell library. The sequence has a high degree of similarity to the chick P2Y1 clone. When transfected into the Jurkat cell line, the cDNA conferred sensitivity to purinoceptor agonists. Using fura-2 loaded cells the potency order at the receptor was found to be 2-methylthioadenosine 5' triphosphate = adenosine 5' diphosphate > adenosine 5' triphosphate >> alpha,beta-methyleneadenosine 5' triphosphate and uridine 5' triphosphate. This corresponds to the agonist potency order expected for the bovine aortic endothelial cell P2Y receptor.

Adenosine Diphosphate↗
PubMed1996

Cloning and chromosomal localization of the human P2Y1 purinoceptor.

We have isolated two types of human P2Y1 cDNA clones from the human erythro leukemia cell cDNA library. The sequence of both clones codes for the same 373 amino acid polypeptide and these clones differ only in the length of the 3' untranslated region. The long form of the cDNA has 1165 nt 3' untranslated region while the 3' untranslated region in the short form is only 258 nt. Both forms are, however, polyadenylated. A multiple human tissue northern blot indicated two transcripts of approximately 4.4 kb and 7.0 kb. The 4.4 kb mRNA is present in all the eight tissues, while the approximately 7.0 kb transcript is expressed only in placenta, skeletal muscle, and pancreas. Using oligonucleotide primers specific for the human P2Y1 purinergic receptor to amplify a region from genomic DNA from a panel of mouse/human somatic cell hybrid cell lines, we have localized the P2Y1 gene to human chromosome 3.

Amino Acid Sequence↗
PubMed1997

Differential expression of PPAR gamma1 and gamma2 isoforms in human adipose tissue.

One of the essential factors for adipogenesis, peroxisome proliferator activated receptor gamma (PPAR gamma), is classified into two isoforms, gamma1 and gamma2 encoded by a single PPAR gamma gene. To examine the mode of expressions of both the PPAR gamma1 and gamma2 isoforms in human adipose tissue, we cloned a partial cDNA of the human PPAR gamma2 (hPPAR gamma2) from a human adipose tissue cDNA library. The sequence encoded an additional 28 amino acids amino-terminal to the first ATG codon of hPPAR gamma1. The simultaneous quantitation of hPPAR gamma1 and gamma2 mRNA in subcutaneous or visceral (mesenteric) fat tissue from 10 different individuals was performed by an RNase protection assay and revealed a relatively higher expression of gamma1 than gamma2 in all specimens examined.

Adipose Tissue↗
PubMed1998

Analysis of a human cDNA containing a tissue-specific alternatively spliced LIM domain.

A unique clone, isolated from a human pancreatic cDNA library, was sequenced and characterized. Northern blot analysis showed that the gene is active in a number of fetal and adult tissues, and immunoblots showed expression in nuclear and cytosolic cell fractions. The gene corresponding to the clone was localized to chromosome 13 by human/rodent somatic cell hybrid panels. The largest open reading frame contains a LIM domain, and the deduced peptide from the open reading frame appears to have the characteristics of a LIM-only protein, designated LMO7. RT-PCR and genomic sequence analyses indicate that expression of this gene product is subject to tissue-specific modulation by elimination of the LIM domain by alternative splicing in neural tissues.

Adult↗
PubMed1999

Molecular cloning of chick UCH-6 which shares high similarity with human UCH-L3: its unusual substrate specificity and tissue distribution.

A full-length cDNA encoding ubiquitin C-terminal hydrolase-6 (UCH-6) was isolated from the chick skeletal muscle cDNA library. The sequence of two peptides generated from purified UCH-6 matched perfectly with the predicted amino acid sequence. Nucleotide sequence analysis of the cDNA containing an open reading frame of 690 base pairs revealed that the protease consists of 230 residues with a calculated molecular mass of 26,315 Da. UCH-6 belonged to members of the UCH family containing highly conserved Cys, His, and Asp domains and showed 86% amino acid identity to human UCH-L3. Interestingly, most tissues examined contained significant amounts of UCH-6 mRNA, while human UCH-L3 is expressed only in the brain, lungs, and red cells. Moreover, UCH-6, unlike other UCH family enzymes including UCH-L3, could release free ubiquitin from ubiquitin-beta-galactosidase fusion proteins both in vivo and in vitro. The ubiquitous expression pattern and unusual substrate specificity of UCH-6 suggest that the enzyme may represent a distinct subfamily of UCH-L3.

Amino Acid Sequence↗
PubMed1993

Transient, localized accumulation of alpha-spectrin during sea urchin morphogenesis.

The mRNA and protein of alpha-spectrin in the sea urchin embryo is shown here to be transiently overexpressed in cells initiating certain morphogenetic changes. This expression was detected by use of a 4.5-kb cDNA clone that encodes alpha-spectrin isolated from a Lytechinus variegatus cDNA library. DNA sequence analysis demonstrated 80% similarity of this cDNA to human nonerythroid alpha-spectrin. Sea urchin alpha-spectrin RNA accumulated to a uniform basal level in all cells of the embryo with a marked, transient increased accumulation in the primary mesenchyme cells of the vegetal plate just prior to gastrulation. Cells of the endoderm also express increased levels of spectrin during gastrulation and these levels remained high in the myoepithelial cells of the sphincter constrictions that separates the gut into foregut, midgut, and hindgut domains. Antibodies made to recombinant sea urchin spectrin synthesized in Escherichia coli were used to localize the protein in situ. These experiments showed that all cells of the embryo accumulated alpha-spectrin protein confined to the cortical regions of cell-cell contact. In epithelial cells, this localization showed a distinct honeycomb pattern. Primary mesenchyme cells and the myoepithelial cells of the gut sphincters showed significantly enhanced spectrin signal corresponding to the pronounced RNA localization pattern seen by in situ RNA hybridization. These data demonstrated that alpha-spectrin from the sea urchin is a highly conserved member of the spectrin superfamily of proteins and that its elevated expression in specific cell types anticipates overt morphogenesis.

Animals↗
PubMed1995

Coexpression and promoter function in two muscle actin gene complexes of different structural organization in the ascidian Halocynthia roretzi.

During embryogenesis of the ascidian Halocynthia roretzi, 42 unicellular striated muscle cells are formed in the tail of the tadpole larva. Isolation of cDNA clones demonstrated that multiple genes for larval muscle actin are expressed in this process. Among them, at least five muscle actin genes (HrMA2, HrMA4a, HrMA4b, HrMA5, and HrMA6) form a cluster (HrMA2/4 cluster) within about 30 kb of the genome. The 5' flanking sequences of the five actin genes resemble each other. When constructs in which 184 bp of the 5' flanking region of each of these genes fused with lacZ were introduced into fertilized eggs, the reporter gene was expressed in muscle cells of the tailbud embryo, suggesting that the 5' flanking region of each cluster gene has promoter activity. In addition, a pair of muscle actin genes, HrMA1a and HrMA1b (HrMA1 pair), was isolated from a genomic region different from that of the HrMA2/4 cluster. The HrMA1a and HrMA1b are linked in a head-to-head arrangement on opposite strands and share a 340-bp 5' flanking sequence containing two symmetrically located TATA boxes. HrMA1a showed basically the same expression pattern as that of HrMA4a. When constructs in which the shared upstream region of HrMA1 pair fused with lacZ in either direction were microinjected into eggs, the reporter gene was expressed in muscle cells of the larval tail, suggesting a bidirectional promoter that regulates muscle-specific transcription of the HrMA1 pair. The tandem cluster of HrMA2/4 genes and the bidirectional promoter of the HrMA1 pair could expedite utilization of muscle-specific trans-acting factors. The organization of genes in the genome may play an important role in the synthesis of a large amount of actins during the process of rapid differentiation.

Actins↗
PubMed1997

SpHmx, a sea urchin homeobox gene expressed in embryonic pigment cells.

We describe the first complete sea urchin member of the H6 subfamily of homeobox genes. A cDNA encoding the complete protein was recovered from a Strongylocentrotus purpuratus library, and sequence comparisons demonstrate that this gene belongs to the same family as the Nkx-5.1, Nkx-5.2, and H6 genes of mammals, and the GH6 and SOHo-1 genes of chicken. In accord with current nomenclature this gene is named SpHmx. The gene is present in a single copy per haploid genome. Single-strand probe excess hybridization demonstrates that a rare maternal SpHmx mRNA is present in unfertilized eggs. Strong zygotic expression begins during the blastula stage, and the transcript is present at moderately high levels until late in development. Whole mount in situ hybridization reveals a remarkable pattern of expression: In midgastrula-stage embryos SpHmx is expressed throughout the archenteron, but particularly strongly in delaminating secondary mesenchyme cells. This more general expression resolves to a dramatic pigment cell-specific pattern in prism and pluteus stages. SpHmx apparently encodes a cell-type-specific transcription factor of embryonic pigment cells.

Amino Acid Sequence↗
PubMed1995

The murine nucleolin protein is an inducible DNA and ATP binding protein which is readily detected in nuclear extracts of lipopolysaccharide-treated splenocytes.

A 100-kDa DNA binding protein was found to be dramatically up-regulated upon the mitogenic stimulation of murine splenocytes with bacterial lipopolysaccharide (LPS). The induced DNA binding protein was also found to exhibit moderate binding specificity for the immunoglobulin isotype switch DNA repeats. Furthermore, the induction of the 100-kDa protein by LPS was found to be mediated by both an increase in the protein's stability and an increase in the synthesis of the protein. In vitro phosphorylation experiments revealed that the 100-kDa DNA binding protein was one of the most heavily phosphorylated proteins in both lymphoid and nonlymphoid nuclear extracts. Although this in vitro phosphorylation initially appeared to be mediated by a potent nuclear kinase activity, it was later determined that a significant part of the detected labeling was due to the direct binding of ATP by the 100-kDa protein. Antibodies raised to the 100-kDa DNA binding protein were used to isolate cDNA clones from a lymphocyte cDNA lambda gt11 expression library. Nucleotide sequence analysis revealed that the cloned cDNAs were identical to the mouse nucleolin gene. The beta-galactosidase fusion proteins (encoded by exons 3-14 of nucleolin) and a more severely truncated 45-kDa protein (encoded by exons 5-14 of nucleolin) were both found to bind strongly to DNA and ATP. Furthermore, the strength of DNA binding was found to be highly dependent on the overall dG content of the DNA probes. Our experiments also revealed that apart from binding ATP and G-rich DNA, nucleolin directly bound GTP, dATP, and dGTP, but not dCTP, dTTP, or dUTP. Computer analysis revealed that the putative ATP binding domains appear to fall within two of the phylogenetically conserved RNA binding domains of nucleolin.

Adenosine Triphosphate↗
PubMed1996

Two cardiac natriuretic peptide genes (atrial natriuretic peptide and brain natriuretic peptide) are organized in tandem in the mouse and human genomes.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which act as cardiac hormones, are produced mainly by the atrium and ventricle, respectively, and are involved in body fluid homeostasis and blood pressure control. The ANP and BNP gene expressions are markedly augmented in ventricles of patients with a wide variety of cardiovascular diseases. It has been demonstrated that the ANP and BNP genes are tightly linked on mouse chromosome 4 and on the distal short arm of human chromosome 1. However, the precise physical map of the ANP and BNP genes has never been elucidated. In the present study, we characterized the genomic DNA fragment containing the ANP and BNP genes in mice and humans. Three genomic DNA clones harboring the entire mouse BNP gene were isolated from a 129/Sv mouse genomic DNA library. Nucleotide sequence analysis revealed that a phage clone (lambda mBNP #3) contains at its 3'-end the 5'-flanking region and the first 209-bp sequence of the first exon of the mouse ANP gene. In mice, the BNP gene was located about 12 kb upstream of the ANP gene. By polymerase chain reaction, we isolated an approximately 11-kb human genomic DNA fragment containing the third exon of the BNP gene and the first and second exons of the ANP gene. In humans, the BNP gene was located upstream of the ANP gene, approximately 8 kb apart. The present study provides the direct evidence that the ANP and BNP genes are organized in tandem in the mouse and human genomes.

Animals↗
PubMed1998

Regulation of cardiac alpha-myosin heavy chain gene transcription by a contractile-responsive E-box binding protein.

The hemodynamic workload imposed on the heart modulates the expression of the cardiac-specific alpha-myosin heavy chain (MHC) gene. A hemodynamic responsive element (HME) has been mapped to an E box motif (CACGTG) located at position -47 of the promoter. The present studies showed that the HME is sufficient to confer contractile responsiveness to a heterologous promoter, the simian virus thymidine kinase gene, when expressed in cultured neonatal rat ventricular myocytes. Proximity of the HME to the TATA box of the alpha-MHC promoter appear necessary for high levels of basal transcription and for the four-fold induction in response to the contractile stimulus. An HME binding protein, approximately 43 kDa, was isolated from a neonatal rat ventricular myocyte cDNA library with sequence homology to the human upstream stimulatory factor-1 (hUSF1). Electrophoretic mobility shift assay showed that the in vitro translation product of the rat USF1 cDNA bound to the alpha-MHC HME motif and was recognized by an antibody to hUSF1. Overexpression of recombinant rat USF1 in spontaneously contracting cultured cardiomyocytes significantly increased activity of a cotransfected alpha-MHC promoter/luciferase reporter plasmid containing the HME motif plus core promoter elements (-40/+32), suggesting a role of rat USF1 in the contractile-mediated activation of the gene.

Amino Acid Sequence↗