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.

scholarly journals Structure and expression of B-myc, a new member of the myc gene family

1988 ◽  
Vol 8 (8) ◽  
pp. 3168-3174
Author(s):  
S Ingvarsson ◽  
C Asker ◽  
H Axelson ◽  
G Klein ◽  
J Sümegi
Keyword(s):  
Southern Blotting ◽  
Cdna Libraries ◽  
Rat Tissues ◽  
Coding Region ◽  
Specific Expression ◽  
Protein Coding ◽  
Myc Gene ◽  
The Brain ◽  
Mouse Somatic Cell ◽  
Specific Mrna

The myc family of genes contains five functional members. We describe the cloning of a new member of the myc family from rat genomic and cDNA libraries, designated B-myc. A fragment of cloned B-myc was used to map the corresponding rat locus by Southern blotting of DNA prepared from rat X mouse somatic cell hybrids. B-myc mapped to rat chromosome 3. We have previously mapped the c-myc to rat chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature [London] 306:497-498, 1983) and N-myc and L-myc to rat chromosomes 6 and 5, respectively (S. Ingvarsson, C. Asker, Z. Wirschubsky, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Somat. Cell Mol. Genet. 13:335-339, 1987). A partial sequence of B-myc had extensive sequence homology to the c-myc protein-coding region, and the detection of intron homology further indicated that these two genes are closely related. The DNA regions conserved among the myc family members, designated myc boxes, were highly conserved between c-myc and B-myc. A lower degree of homology was detected in other parts of the coding region in c-myc and B-myc not present in N-myc and L-myc. A 1.3-kilobase B-myc-specific mRNA was detected in most rat tissues, with the highest expression in the brain. This resembled the expression pattern of c-myc, although at different relative levels, and was in contrast to the more tissue-specific expression of N-myc and L-myc. B-myc was expressed at uniformly high levels in all fetal tissues and during subsequent postnatal development, in contrast to the stage-specific expression of c-myc.

scholarly journals Structure and expression of B-myc, a new member of the myc gene family.

1988 ◽  
Vol 8 (8) ◽  
pp. 3168-3174 ◽  
Author(s):  
S Ingvarsson ◽  
C Asker ◽  
H Axelson ◽  
G Klein ◽  
J Sümegi
Keyword(s):  
Southern Blotting ◽  
Cdna Libraries ◽  
Rat Tissues ◽  
Coding Region ◽  
Specific Expression ◽  
Protein Coding ◽  
Myc Gene ◽  
The Brain ◽  
Mouse Somatic Cell ◽  
Specific Mrna

The myc family of genes contains five functional members. We describe the cloning of a new member of the myc family from rat genomic and cDNA libraries, designated B-myc. A fragment of cloned B-myc was used to map the corresponding rat locus by Southern blotting of DNA prepared from rat X mouse somatic cell hybrids. B-myc mapped to rat chromosome 3. We have previously mapped the c-myc to rat chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature [London] 306:497-498, 1983) and N-myc and L-myc to rat chromosomes 6 and 5, respectively (S. Ingvarsson, C. Asker, Z. Wirschubsky, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Somat. Cell Mol. Genet. 13:335-339, 1987). A partial sequence of B-myc had extensive sequence homology to the c-myc protein-coding region, and the detection of intron homology further indicated that these two genes are closely related. The DNA regions conserved among the myc family members, designated myc boxes, were highly conserved between c-myc and B-myc. A lower degree of homology was detected in other parts of the coding region in c-myc and B-myc not present in N-myc and L-myc. A 1.3-kilobase B-myc-specific mRNA was detected in most rat tissues, with the highest expression in the brain. This resembled the expression pattern of c-myc, although at different relative levels, and was in contrast to the more tissue-specific expression of N-myc and L-myc. B-myc was expressed at uniformly high levels in all fetal tissues and during subsequent postnatal development, in contrast to the stage-specific expression of c-myc.

Selection on Rapidly Evolving Proteins in the Arabidopsis Genome

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 723-733 ◽  
Author(s):  
Marianne Barrier ◽  
Carlos D Bustamante ◽  
Jiaye Yu ◽  
Michael D Purugganan
Keyword(s):  
Expressed Sequence Tag ◽  
Amino Acid Replacement ◽  
Adaptive Divergence ◽  
Coding Region ◽  
Protein Coding ◽  
Hierarchical Bayesian Analysis ◽  
Brassicaceae Species ◽  
Replacement Site ◽  
Orthologous Loci ◽  
Selection Intensities

Abstract Genes that have undergone positive or diversifying selection are likely to be associated with adaptive divergence between species. One indicator of adaptive selection at the molecular level is an excess of amino acid replacement fixed differences per replacement site relative to the number of synonymous fixed differences per synonymous site (ω = Ka/Ks). We used an evolutionary expressed sequence tag (EST) approach to estimate the distribution of ω among 304 orthologous loci between Arabidopsis thaliana and A. lyrata to identify genes potentially involved in the adaptive divergence between these two Brassicaceae species. We find that 14 of 304 genes (∼5%) have an estimated ω > 1 and are candidates for genes with increased selection intensities. Molecular population genetic analyses of 6 of these rapidly evolving protein loci indicate that, despite their high levels of between-species nonsynonymous divergence, these genes do not have elevated levels of intraspecific replacement polymorphisms compared to previously studied genes. A hierarchical Bayesian analysis of protein-coding region evolution within and between species also indicates that the selection intensities of these genes are elevated compared to previously studied A. thaliana nuclear loci.

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.

scholarly journals Structure and tissue-specific expression of the Drosophila melanogaster organellar-type Ca2+-ATPase gene

1995 ◽  
Vol 310 (3) ◽  
pp. 757-763 ◽  
Author(s):  
A Magyar ◽  
E Bakos ◽  
A Váradi
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Drosophila Genome ◽  
Coding Region ◽  
Specific Expression ◽  
S1 Nuclease ◽  
Protein Coding ◽  
Atpase Gene ◽  
High Level

A 14 kb genomic clone covering the organellar-type Ca(2+)-ATPase gene of Drosophila melanogaster has been isolated and characterized. The sequence of a 7132 bp region extending from 1.1 kb 5′ upstream of the initiation ATG codon over the polyadenylation signal at the 3′ end has been determined. The gene consists of nine exons including one with an exceptional size of 2172 bp representing 72% of the protein coding region. Introns are relatively small (< 100 bp) except for the 3′ intron which has a size of 2239 bp, an exceptionally large size among Drosophila introns. Five of the introns are in the same positions in Drosophila, Artemia and rabbit SERCA1 Ca(2+)-ATPase genes. There is only one organellar-type Ca(2+)-ATPase gene in the Drosophila genome, as was shown by Southern-blot analysis [Váradi, Gilmore-Hebert and Benz (1989) FEBS Lett. 258, 203-207] and by chromosomal localization [Magyar and Váradi (1990) Biochem. Biophys. Res. Commun. 173, 872-877]. Primer extension and S1-nuclease assays revealed a potential transcription initiation site 876 bp upstream of the translation initiation ATG with a TATA-box 23 bp upstream of this site. Analysis of the 5′ region of the Drosophila organellar-type Ca(2+)-ATPase gene suggests the presence of potential recognition sequences of various muscle-specific transcription factors and shows a region with remarkable similarity to that in the rabbit SERCA2 gene. The tissue distribution of expression of the organellar-type Ca(2+)-ATPase gene has been studied by in situ RNA-RNA hybridization on microscopic sections. A low mRNA abundance can be detected in each tissue of adult flies, suggesting a housekeeping function for the gene. On the other hand a pronounced tissue specificity of expression has also been found as the organellar-type Ca(2+)-ATPase is expressed at a very high level in cell bodies of the central nervous system and in various muscles.

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 Discrimination of distinct proteinases at the four structural levels of rat liver mitochondria

1986 ◽  
Vol 233 (1) ◽  
pp. 283-286 ◽  
Author(s):  
M C Duque-Magalhães ◽  
P Régnier
Keyword(s):  
Outer Membrane ◽  
Rat Liver ◽  
Degradation Products ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Intermembrane Space ◽  
Peptidase Activity ◽  
Inner Membrane ◽  
Mitochondrial Fractions ◽  
The Matrix

Rat liver mitochondrial fractions corresponding to four morphological structures (matrix, inner membrane, intermembrane space and outer membrane) contain proteinases that cleave casein components at different rates. Proteinases of the intermembrane space preferentially cleave kappa-casein, whereas the proteinases of the outer membrane, inner membrane and matrix fractions degrade alpha S1-casein more rapidly. Electrophoretic separation of the degradation products of alpha S1-casein and kappa-casein in polyacrylamide gels shows that different polypeptides are produced when the substrate is degraded by the matrix, by both membranes and by the intermembrane-space fraction. Some of the degradation products resulting from incubation of the caseins with the mitochondrial fractions are probably the result of digestion by contaminating lysosomal proteinase(s). The matrix has a high peptidase activity, since glucagon, a small peptide, is very rapidly degraded by this fraction. These observations strongly suggest that distinct proteinases, with different specificities, are associated respectively with the intermembrane space and with both membrane fractions.

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.

scholarly journals The complete sequence of the mouse skeletal alpha-actin gene reveals several conserved and inverted repeat sequences outside of the protein-coding region.

1986 ◽  
Vol 6 (1) ◽  
pp. 15-25 ◽  
Author(s):  
M C Hu ◽  
S B Sharp ◽  
N Davidson
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Inverted Repeat ◽  
Actin Gene ◽  
Coding Region ◽  
Specific Expression ◽  
Protein Coding ◽  
Repeat Sequences ◽  
Actin Genes ◽  
Alpha Actin

The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.

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