Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells

1987 ◽  
Vol 7 (7) ◽  
pp. 2435-2443
Author(s):  
I L Andrulis ◽  
J Chen ◽  
P N Ray
Keyword(s):  
Cell Lines ◽  
Untranslated Region ◽  
Asparagine Synthetase ◽  
Synthetase Activity ◽  
Coding Region ◽  
Protein Coding ◽  
Reading Frame ◽  
Amino Terminal ◽  
Multiple Copies ◽  
Sarcoma Cells

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.

scholarly journals Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

1987 ◽  
Vol 7 (7) ◽  
pp. 2435-2443 ◽  
Author(s):  
I L Andrulis ◽  
J Chen ◽  
P N Ray
Keyword(s):  
Cell Lines ◽  
Untranslated Region ◽  
Asparagine Synthetase ◽  
Synthetase Activity ◽  
Coding Region ◽  
Protein Coding ◽  
Reading Frame ◽  
Amino Terminal ◽  
Multiple Copies ◽  
Sarcoma Cells

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.

Structure and expression of canary myc family genes

1991 ◽  
Vol 11 (3) ◽  
pp. 1770-1776
Author(s):  
R G Collum ◽  
D F Clayton ◽  
F W Alt
Keyword(s):  
Untranslated Region ◽  
Untranslated Regions ◽  
Coding Region ◽  
Protein Coding ◽  
Coding Regions ◽  
Neuronal Precursors ◽  
Myc Gene ◽  
Mature Neurons

We found that the canary N-myc gene is highly related to mammalian N-myc genes in both the protein-coding region and the long 3' untranslated region. Examined coding regions of the canary c-myc gene were also highly related to their mammalian counterparts, but in contrast to N-myc, the canary and mammalian c-myc genes were quite divergent in their 3' untranslated regions. We readily detected N-myc and c-myc expression in the adult canary brain and found N-myc expression both at sites of proliferating neuronal precursors and in mature neurons.

DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells

1989 ◽  
Vol 9 (7) ◽  
pp. 2922-2927
Author(s):  
I L Andrulis ◽  
M T Barrett
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary Cells ◽  
Methylation Status ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Methylation Pattern ◽  
Asparagine Synthetase ◽  
Synthetase Activity ◽  
Dna Hypomethylation ◽  
Ovary Cells

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.

The destabilizing elements in the coding region of c-fos mRNA are recognized as RNA

1993 ◽  
Vol 13 (8) ◽  
pp. 5034-5042
Author(s):  
C L Wellington ◽  
M E Greenberg ◽  
J G Belasco
Keyword(s):  
Codon Usage ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Polypeptide Chain ◽  
Coding Region ◽  
Protein Coding ◽  
Present Evidence ◽  
Reading Frame ◽  
Nascent Polypeptide

The protein-coding region of the c-fos proto-oncogene transcript contains elements that direct the rapid deadenylation and decay of this mRNA in mammalian cells. The function of these coding region instability determinants requires movement of ribosomes across mRNAs containing them. Three types of mechanisms could account for this translational requirement. Two of these possibilities, (i) that rapid mRNA decay might be mediated by the nascent polypeptide chain and (ii) that it might result from an unusual codon usage, have experimental precedent. Here, we present evidence that the destabilizing elements in the c-fos coding region are not recognized in either of these two ways. Instead, the ability of the c-fos coding region to function as a potent mRNA destabilizer when translated in the +1 reading frame indicates that the signals for rapid deadenylation and decay reside in the sequence or structure of the RNA comprising this c-fos domain.

ACE2, an activator of yeast metallothionein expression which is homologous to SWI5

1991 ◽  
Vol 11 (1) ◽  
pp. 476-485
Author(s):  
G Butler ◽  
D J Thiele
Keyword(s):  
Steady State ◽  
Gene Product ◽  
Large Deletion ◽  
Yeast Genome ◽  
Coding Region ◽  
Reading Frame ◽  
Enhanced Expression ◽  
Multiple Copies ◽  
Steady State Levels ◽  
Cup1 Gene

Transcription of the Saccharomyces cerevisiae metallothionein gene CUP1 is induced in response to high environmental levels of copper. Induction requires the ACE1 gene product, which binds to specific sites in the promoter region of the CUP1 gene. In this study, we found that deleting the entire coding sequence of the ACE1 gene resulted in a decrease in basal-level transcription of CUP1 to low but detectable levels and conferred a copper-sensitive phenotype to the cells. We have isolated a gene, designated ACE2, which when present on a high-copy-number plasmid suppresses the copper-sensitive phenotype of an ace1-deletion strain. The presence of multiple copies of the ACE2 gene enhanced expression of an unlinked CUP1-lacZ fusion integrated in the yeast genome and resulted in an increase in the steady-state levels of CUP1 mRNA in an ace1-deletion background. A large deletion of the coding region of the genomic copy of ACE2 resulted in a decrease in steady-state levels of CUP1 mRNA, indicating that ACE2 plays a role in regulating basal-level expression of CUP1. The ACE2 open reading frame encodes a polypeptide of 770 amino acids, with putative zinc finger structures near the carboxyl terminus. This protein is 37% identical to the SWI5 gene product, an activator of HO gene transcription in S. cerevisiae, suggesting that ACE2 and SWI5 may have functional similarities.

Retroviral insertions in the murine His-1 locus activate the expression of a novel RNA that lacks an extensive open reading frame

1994 ◽  
Vol 14 (3) ◽  
pp. 1743-1751
Author(s):  
D S Askew ◽  
J Li ◽  
J N Ihle
Keyword(s):  
Cell Lines ◽  
Wild Mouse ◽  
Myeloid Cell ◽  
Single Copy ◽  
Open Reading Frame ◽  
Chromosome 2 ◽  
Coding Region ◽  
Reading Frame ◽  
Novel Gene ◽  
Myeloid Leukemias

The His-1 locus is a common site of viral insertion in murine myeloid leukemias induced by the wild mouse ecotropic retrovirus, CasBrM. In this report, we describe the cloning of a novel gene at the His-1 locus and show that His-1 expression is associated with the transformed phenotype. Northern (RNA) blot analysis identified His-1 transcripts in four transformed myeloid cell lines but in no normal tissues examined. Two of these cell lines were derived from retrovirus-induced myeloid leukemias that harbor integrated proviruses which drive His-1 gene expression by promoter insertion. The two other cell lines expressed a discrete 3-kb His-1 RNA that is derived from a novel gene consisting of three exons that span 6 kb on mouse chromosome 2. The His-1 gene is conserved as a single-copy sequence in multiple vertebrate species and is expressed as a spliced and polyadenylated RNA. A protein-coding region is not evident from analysis of the His-1 sequence because of the presence of multiple small open reading frames, none of which are greater than 219 bp. This lack of an extensive open reading frame is an unusual feature that is shared by other RNA molecules believed to function in the absence of translation.

A preprogalanin cDNA from the turtle pituitary and regulation of its gene expression

2007 ◽  
Vol 292 (4) ◽  
pp. R1649-R1656 ◽  
Author(s):  
John Yuh-Lin Yu ◽  
Chin-Hon Pon ◽  
Hui-Chen Ku ◽  
Chih-Ting Wang ◽  
Yung-Hsi Kao
Keyword(s):  
Mrna Expression ◽  
Untranslated Region ◽  
Signal Sequence ◽  
Biological Effects ◽  
In Vitro Study ◽  
The Body ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame

Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle ( Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5′-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3′-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH2 terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly1 and Thr29 substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48–81% and 76–96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17β-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.

l-Asparaginyl-Trna Synthetase and l-Asparagine Synthetase Activities of l-Asparaginase-Sensitive and -Resistant Forms of the Mouse Gardner Lymphoma 6C3HED

1979 ◽  
Vol 56 (6) ◽  
pp. 539-545 ◽  
Author(s):  
M. R. Davies ◽  
Lucy P. Lambert ◽  
R. D. Marshall
Keyword(s):  
Cell Lines ◽  
Tumour Cell ◽  
Trna Synthetase ◽  
Asparagine Synthetase ◽  
Ascites Fluid ◽  
Synthetase Activity ◽  
Early Passage ◽  
Tumour Cell Lines ◽  
Cells Cultured

1. The mouse Gardner lymphoma 6C3HED was grown in ascites fluid in a form sensitive to the action of l-asparaginase (line 1), in another form which was resistant to l-asparaginase (line 2) and in a third form with partial sensitivity to l-asparaginase (line 3). 2. The l-asparaginyl-tRNA synthetase activities of extracts of the tumour cells, cultured both in the mouse and in vitro, were determined. Two of the lines, 1 and 3, in early passage numbers, showed a derepression mechanism involving l-asparagine. Mutation occurred with these lines resulting in the l-asparaginyl-tRNA synthetase activity of all the tumour cell lines being the same. 3. Cells of line 1 had low l-asparagine synthetase activity, which was unchanged by altering the supply of l-asparagine in vitro. Cells of lines 2 and 3 exhibited l-asparagine synthetase activities, which changed with the supply of l-asparagine. 4. It is not certain that l-asparagine synthetase activity of l-asparaginase-sensitive cells is controlled by l-asparaginyl-tRNA acting as a corepressor.

scholarly journals Structure of transcripts from the homeotic Antennapedia gene of Drosophila melanogaster: two promoters control the major protein-coding region.

1986 ◽  
Vol 6 (12) ◽  
pp. 4676-4689 ◽  
Author(s):  
A Laughon ◽  
A M Boulet ◽  
J R Bermingham ◽  
R A Laymon ◽  
M P Scott
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Polyadenylation ◽  
Transcription Unit ◽  
Cdna Clones ◽  
Major Protein ◽  
Coding Region ◽  
Developmentally Regulated ◽  
Protein Coding ◽  
Reading Frame ◽  
C Terminus

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster regulates segmental identity in the thorax. Loss of Antp function results in altered development of the embryonic thoracic segments or can cause legs to be transformed into antennae. Certain combinations of Antp recessive lethal alleles complement to permit normal development. The structure of the Antp gene, analyzed by sequencing cDNA clones and exons and by transcript mapping, revealed some of the basis for its genetic complexity. It has two promoters governing two nested transcription units, one unit 36 and one 103 kilobase pairs (kb) long. Both units incorporated the same protein-coding exons, all of which are located in the 3'-most 13 kb of the gene. The two promoters resulted in the attachment of either of two long noncoding leader sequences (1.5 and 1.7 kb) to a 1.1-kb open reading frame. Both transcription units used the same pair of alternative polyadenylation sites 1.4 kb apart; the choice of sites was developmentally regulated. Some of the mutations that disrupt the larger transcription unit complemented a mutation affecting the smaller one. Dominant mutations that transform antennae into legs split the gene but left the coding exons intact. The encoded protein has unusually long runs of glutamine and a homeodomain near the C terminus.

scholarly journals Biochemical and genetic characterization of a murine class Kappa glutathione S-transferase

2003 ◽  
Vol 373 (2) ◽  
pp. 559-569 ◽  
Author(s):  
Ian R. JOWSEY ◽  
Rachel E. THOMSON ◽  
Terry C. ORTON ◽  
Clifford R. ELCOMBE ◽  
John D. HAYES
Keyword(s):  
Expressed Sequence Tag ◽  
Subcellular Fractionation ◽  
Liver Mitochondria ◽  
Rat Tissues ◽  
Coding Region ◽  
Specific Expression ◽  
Protein Coding ◽  
Reading Frame ◽  
Mitochondrial Fractions ◽  
The Matrix

The class Kappa family of glutathione S-transferases (GSTs) currently comprises a single rat subunit (rGSTK1), originally isolated from the matrix of liver mitochondria [Harris, Meyer, Coles and Ketterer (1991) Biochem. J. 278, 137–141; Pemble, Wardle and Taylor (1996) Biochem. J. 319, 749–754]. In the present study, an expressed sequence tag (EST) clone has been identified which encodes a mouse class Kappa GST (designated mGSTK1). The EST clone contains an open reading frame of 678 bp, encoding a protein composed of 226 amino acid residues with 86% sequence identity with the rGSTK1 polypeptide. The mGSTK1 and rGSTK1 proteins have been heterologously expressed in Escherichia coli and purified by affinity chromatography. Both mouse and rat transferases were found to exhibit GSH-conjugating and GSH-peroxidase activities towards model substrates. Analysis of expression levels in a range of mouse and rat tissues revealed that the mRNA encoding these enzymes is expressed predominantly in heart, kidney, liver and skeletal muscle. Although other soluble GST isoenzymes are believed to reside primarily within the cytosol, subcellular fractionation of mouse liver demonstrates that this novel murine class Kappa GST is associated with mitochondrial fractions. Through the use of bioinformatics, the genes encoding the mouse and rat class Kappa GSTs have been identified. Both genes comprise eight exons, the protein coding region of which spans approx. 4.3 kb and 4.1 kb of DNA for mGSTK1 and rGSTK1 respectively. This conservation in primary structure, catalytic properties, tissue-specific expression, subcellular localization and gene structure between mouse and rat class Kappa GSTs indicates that they perform similar physiological functions. Furthermore, the association of these enzymes with mitochondrial fractions is consistent with them performing a specific conserved biological role within this organelle.

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