RESUMO
In a database search for homologs of acyl-coenzyme A oxidases (ACX) in Arabidopsis, we identified a partial genomic sequence encoding an apparently novel member of this gene family. Using this sequence information we then isolated the corresponding full-length cDNA from etiolated Arabidopsis cotyledons and have characterized the encoded recombinant protein. The polypeptide contains 675 amino acids. The 34 residues at the amino terminus have sequence similarity to the peroxisomal targeting signal 2 of glyoxysomal proteins, including the R-[I/Q/L]-X5-HL-XL-X15-22-C consensus sequence, suggesting a possible microsomal localization. Affinity purification of the encoded recombinant protein expressed in Escherichia coli followed by enzymatic assay, showed that this enzyme is active on C8:0- to C14:0-coenzyme A with maximal activity on C12:0-coenzyme A, indicating that it has medium-chain-specific activity. These data indicate that the protein reported here is different from previously characterized classes of ACX1, ACX2, and short-chain ACX (SACX), both in sequence and substrate chain-length specificity profile. We therefore, designate this new gene AtACX3. The temporal and spatial expression patterns of AtACX3 during development and in various tissues were similar to those of the AtSACX and other genes expressed in glyoxysomes. Currently available database information indicates that AtACX3 is present as a single copy gene.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/enzimologia , Oxirredutases/genética , Acil-CoA Oxidase , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA , DNA Complementar , Escherichia coli/genética , Dados de Sequência Molecular , Oxirredutases/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Muscle glycogen phosphorylase (MGP) transcript and protein levels increase during skeletal muscle development in tandem with the products of other muscle genes responsible for glucose and glycogen metabolism. Previous studies demonstrated that a 269 bp region 5' to exon 1 of MGP is sufficient for developmental regulation in the C2C12 myogenic cell line (Froman et al., 1994). This genomic region (-209 to +60) contains four consensus E box motifs, a CArG-like sequence, and a GC-rich domain. Native MGP transcripts were not detected in pluripotent CH310T1/2 fibroblasts, but low levels of MGP mRNA were measured in CH310T1/2 cells that were stably transfected with MyoD. Three of the E box motifs in the MGP proximal promoter interacted with C2C12 nuclear proteins. However, cotransfection of the MGP promoter with myogenic regulatory factors, including MyoD and myogenin, produced less than 2-fold activation compared with 20-fold activation of the desmin promoter. Mutational analyses of the MGP promoter demonstrated that increased expression in C2C12 myotubes did not require any of the E box motifs or the CArG-like element. A small region (-76 to -68) upstream of GC-rich domain (-64 to -51) significantly reduced promoter activities in both myoblasts and myotubes. The functional studies suggest that MGP is developmentally regulated during myogenesis by alternative pathways that utilize unidentified regulatory elements or ancillary factors.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Músculo Esquelético/citologia , Fosforilases/genética , Regiões Promotoras Genéticas , Animais , Diferenciação Celular , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Proteína MyoD/fisiologia , Proteínas Nucleares/metabolismo , Mutação Puntual , RNA Mensageiro/biossíntese , Sequências Reguladoras de Ácido Nucleico , Deleção de SequênciaRESUMO
The muscle isozyme of glycogen phosphorylase (MGP) catalyzes the hydrolysis hydrolysis of intracellular glycogen in mammalian tissues and is produced in skeletal muscle, brain and heart. The MGP gene is developmentally and neutrally regulated in skeletal muscle, but little is known about the gene's transcriptional regulation. We have isolated and characterized the 5' flanking region of rat MGP. Truncated portions of the MGP 5' flanking region were coupled to the bacterial cat reporter gene and used in transient transfection assays in the mouse muscle C2C12 cell line. The region between -211 and +62 contained the smallest regulatory domain capable of demonstrating developmentally regulated myogenic expression in C2C12 cells. This was in contrast with findings from another investigation that transfected this cell line with human MGP [Lockyer and McCracken, J. Biol. Chem. 266 (1991) 20262-20269]. A 172-nucleotide (nt) region between -839 and -666 functioned as a potent enhancer in C2C12 cells when coupled to its cognate promoter, but not when coupled to a simian virus 40 promoter. This rat MGP enhancer region is 78% identical to a comparable region of the human MGP 5' flanking region, but contains only one putative regulatory element that has been previously identified in other muscle genes. These data suggest that rat MGP transcription in C2C12 muscle cells is modulated by a potent enhancer that utilizes novel regulatory elements.
Assuntos
Elementos Facilitadores Genéticos , Regulação Enzimológica da Expressão Gênica , Isoenzimas/biossíntese , Músculos/enzimologia , Fosforilases/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/biossíntese , Feminino , Humanos , Fígado/enzimologia , Camundongos , Dados de Sequência Molecular , Desenvolvimento Muscular , Músculos/citologia , Regiões Promotoras Genéticas , Ratos , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Vírus 40 dos Símios/genética , Transcrição Gênica , Transfecção , Útero/enzimologiaRESUMO
RNA blot analysis of rat liver and adipose tissues detected two glycogen phosphorylase (GP)-encoding transcripts. The polymerase chain reaction was used to characterize the 3'-noncoding region of the gene (L-GP) encoding liver-GP (L-GP) from the lean Zucker rat (Fa/Fa). Three distinct classes of colinear cDNA clones were identified by nucleotide (nt) sequence analysis, demonstrating that the L-GP gene contains at least three functional polyadenylation sites. The predominant L-GP transcript was generated by polyadenylation 130 nt 3' from the end of the coding region. A previously uncharacterized L-GP transcript is generated by polyadenylation at 346 nt 3' of the first polyadenylation site. Polyadenylation site selection does not appear to be regulated in a tissue-specific fashion. The relative steady-state L-GP mRNA levels in the different types of adipose tissues were comparable to, or exceeded transcript levels in liver.
Assuntos
Fígado/enzimologia , Fosforilases/genética , Poli A/metabolismo , Tecido Adiposo/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Ratos Zucker , Mapeamento por RestriçãoRESUMO
Glycogen phosphorylase (alpha-1,4-glucan:orthophosphate D-glucosyltransferase, EC 2.4.1.1) is the rate-determining enzyme catalyzing glycogen degradation. Human brain has been demonstrated previously to express genes of both the liver and muscle isozymes of glycogen phosphorylase. In this report, a human fetal brain cDNA and genomic DNA corresponding to the brain isozyme of glycogen phosphorylase were isolated and characterized. Transcripts corresponding to this isozyme are present in human adult and fetal brain, and at low levels in other human fetal tissues. The predicted C-terminal sequence of the protein encoded by this cDNA and gene differ from that encoded by a phosphorylase cDNA isolated from a human astrocytoma cell line.
Assuntos
Encéfalo/enzimologia , DNA/isolamento & purificação , Isoenzimas/genética , Fosforilases/genética , Sequência de Aminoácidos , Astrocitoma , Sequência de Bases , Linhagem Celular , Humanos , Dados de Sequência Molecular , RNA Mensageiro/metabolismoRESUMO
The nucleotide sequence of the gene encoding the glycolytic enzyme phosphoglucose isomerase (PGI) from Escherichia coli is presented. The gene encodes a polypeptide of 549 amino acids. The transcriptional start point of the gene was determined and found to lie within a consensus promoter region. The amino acid sequence derived from the E. coli PGI gene can be aligned without insertions or deletions to the predicted amino acid sequence of a nuclear-encoded chloroplast isozyme of PGI from a higher plant, and the two sequences have a similarity of 87.6%. The amino acid sequence similarity between E. coli and that predicted from cDNA sequences for mouse and pig PGI is approximately 65%.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Glucose-6-Fosfato Isomerase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , DNA , Sondas de DNA , DNA Bacteriano , Eletroforese em Gel de Poliacrilamida , Endonucleases , Glucose-6-Fosfato Isomerase/biossíntese , Dados de Sequência Molecular , Plantas/genética , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Especificidade da EspécieRESUMO
Nuclear genes that appear to encode both cytosolic and plastid isozymes of phosphoglucose isomerase (PGI), an essential glycolytic enzyme, have been isolated from three diploid species of the annual wild flower genus Clarkia (Onagraceae). The genes do not contain introns and are expressed to varying degrees in Escherichia coli when cloned in either Charon 35 phage or pUC plasmid vectors. The PGI proteins synthesized in E. coli form dimers, are catalytically active, and their electrophoretic mobilities are similar to those of appropriate Clarkia PGIs. The nucleotide sequence of a gene encoding a plastid isozyme of C. unguiculata is described.
RESUMO
The 3' terminal region from the LEU2 gene of yeast has been sequenced. Two open reading frames (ORF) have been identified, one of which constitutes the 3' terminus of beta-isopropylmalate dehydrogenase, the product of the LEU2 gene. A noncoding spacer region located between the ORFs contains two consensus-type transcriptional terminators. The terminator-like sequences are oriented in opposing directions on opposite DNA strands. The noncoding spacer region may represent a single terminator for the LEU2 gene or two separate terminators involved in blocking convergent transcription from an as yet unidentified yeast gene.
Assuntos
Oxirredutases do Álcool/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , 3-Isopropilmalato Desidrogenase , Sequência de Aminoácidos , Sequência de Bases , Genes , Saccharomyces cerevisiae/enzimologia , Transcrição GênicaRESUMO
A new Type II restriction endonuclease XcyI, purified from Xanthomonas cyanopsidis 13D5, is an isoschizomer of SmaI and XmaI that cleaves at the nucleotide sequence 5'-C decreases CCGGG-3' of double-stranded DNA. The single restriction activity present in this strain permits rapid purification of 8000 units of cleavage activity from 10 g of freshly harvested cells. The resulting XcyI preparation is free of contaminating nuclease activities that interfere with in vitro manipulation of DNA.