scholarly journals Structural and functional analysis of a glutathione S-transferase from Ascaris suum

1997 ◽  
Vol 324 (2) ◽  
pp. 659-666 ◽  
Author(s):  
Eva LIEBAU ◽  
Volker H. O. ECKELT ◽  
Gabriele WILDENBURG ◽  
Paul TEESDALE-SPITTLE ◽  
Peter M. BROPHY ◽  
...  
Keyword(s):  
Specific Activity ◽  
Ascaris Suum ◽  
Homology Modelling ◽  
Single Copy ◽  
Regulatory Elements ◽  
Polyclonal Antiserum ◽  
Nucleotide Sequence Analysis ◽  
Flanking Sequence ◽  
Glutathione S Transferase ◽  
Secondary Products

A recombinant glutathione S-transferase (GST) (EC 2.5.1.18) from the parasitic nematode Ascaris suum(AsGST1) displays specific activity with a variety of model substrates and secondary products of lipid peroxidation. The AsGST1 interacts with a range of model inhibitors, haematin-related compounds, bile acids and anthelminthics. The reported variations in biochemical activity correlate with structural differences observed by homology modelling. Here, differences in the topography of the proposed substrate binding site between the AsGST1 and the host GSTs were identified. A rabbit polyclonal antiserum was raised against the glutathione-binding proteins ofA. suum and specific antibodies against AsGST1 were affinity-purified using the recombinant protein. These antibodies were used to localize the AsGST1 in adult worms by immunohistochemical staining. The strongest immunostaining for AsGST1 was localized in the intestine in all worms examined. This suggests that the enzyme may be responsible for the metabolism of materials that are incorporated from the environment, as well as for molecules that are excreted or secreted from the parasite to the environment. It also demonstrates the accessibility of the enzyme to an inhibitor or blocking antibody. In addition, the structure and sequence of the gene encoding AsGST1 have been determined. Southern-blot analyses of the AsGST1 gene suggests that it is a single-copy gene. The nucleotide sequence analysis revealed that the gene is composed of four exons and three introns, and potential regulatory elements were identified in the 5′ flanking sequence.

scholarly journals Cell-specific activity of cis-acting regulatory elements in the promoter of the mouse multidrug resistance gene mdr1.

1990 ◽  
Vol 10 (11) ◽  
pp. 6036-6040 ◽  
Author(s):  
M Raymond ◽  
P Gros
Keyword(s):  
Multidrug Resistance ◽  
Resistance Gene ◽  
Reporter Gene ◽  
Specific Activity ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Mouse Cell ◽  
Nucleotide Sequence Analysis ◽  
Multidrug Resistance Gene ◽  
Cis Acting

To define cis-acting elements implicated in transcriptional regulation of the mouse multidrug resistance gene mdr1, we have cloned and characterized the 5' end of the gene. Nucleotide sequence analysis identified TATA, GGGCGG, and CCAAT consensus sequence elements at positions -27, -47, and -83, respectively. The transcriptional activities of 5' deletion fragments from the promoter linked to a reporter gene were tested in mouse cell lines of different tissue origins shown to express different levels of endogenous mdr1 RNA. Sequences located between nucleotides -93 and +84 were able to confer basal promoter activity and cell specificity to the reporter gene. The addition to the basal promoter of sequences upstream of position -141 was found to up or down regulate the basal level of expression of the reporter gene in a cell-specific manner.

Cell-specific activity of cis-acting regulatory elements in the promoter of the mouse multidrug resistance gene mdr1

1990 ◽  
Vol 10 (11) ◽  
pp. 6036-6040
Author(s):  
M Raymond ◽  
P Gros
Keyword(s):  
Multidrug Resistance ◽  
Resistance Gene ◽  
Reporter Gene ◽  
Specific Activity ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Mouse Cell ◽  
Nucleotide Sequence Analysis ◽  
Multidrug Resistance Gene ◽  
Cis Acting

To define cis-acting elements implicated in transcriptional regulation of the mouse multidrug resistance gene mdr1, we have cloned and characterized the 5' end of the gene. Nucleotide sequence analysis identified TATA, GGGCGG, and CCAAT consensus sequence elements at positions -27, -47, and -83, respectively. The transcriptional activities of 5' deletion fragments from the promoter linked to a reporter gene were tested in mouse cell lines of different tissue origins shown to express different levels of endogenous mdr1 RNA. Sequences located between nucleotides -93 and +84 were able to confer basal promoter activity and cell specificity to the reporter gene. The addition to the basal promoter of sequences upstream of position -141 was found to up or down regulate the basal level of expression of the reporter gene in a cell-specific manner.

scholarly journals Evidence for Positive and Negative Regulatory Elements in the 5′-Flanking Sequence of the Mouse Sparc (osteonectin) Gene

1989 ◽  
Vol 264 (21) ◽  
pp. 12201-12207 ◽  
Author(s):  
S Nomura ◽  
S Hashmi ◽  
J H McVey ◽  
J Ham ◽  
M Parker ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Flanking Sequence

scholarly journals Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

1991 ◽  
Vol 11 (3) ◽  
pp. 1488-1499 ◽  
Author(s):  
H J Roth ◽  
G C Das ◽  
J Piatigorsky
Keyword(s):  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Regulatory Elements ◽  
Dnase I ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

Overlapping positive and negative regulatory elements determine lens-specific activity of the delta 1-crystallin enhancer

1993 ◽  
Vol 13 (9) ◽  
pp. 5206-5215 ◽  
Author(s):  
Y Kamachi ◽  
H Kondoh
Keyword(s):  
Mutational Analysis ◽  
Specific Activity ◽  
Regulatory Elements ◽  
Positive Element ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Negative Element ◽  
Positive Elements ◽  
Lens Cells ◽  
Specific Regulation

Lens-specific expression of the delta 1-crystallin gene is governed by an enhancer in the third intron, and the 30-bp-long DC5 fragment was found to be responsible for eliciting the lens-specific activity. Mutational analysis of the DC5 fragment identified two contiguous, interdependent positive elements and a negative element which overlaps the 3'-located positive element. Previously identified ubiquitous factors delta EF1 bound to the negative element and repressed the enhancer activity in nonlens cells. Mutation and cotransfection analyses indicated the existence of an activator which counteracts the action of delta EF1 in lens cells, probably through binding site competition. We also found a group of nuclear factors, collectively called delta EF2, which bound to the 5'-located positive element. delta EF2a and -b were the major species in lens cells, whereas delta EF2c and -d predominated in nonlens cells. These delta EF2 proteins probably cooperate with factors bound to the 3'-located element in activation in lens cells and repression in nonlens cells. delta EF2 proteins also bound to a promoter sequence of the gamma F-crystallin gene, suggesting that delta EF2 proteins are involved in lens-specific regulation of various crystallin classes.

Fine-structure mapping of the three mouse alpha-fetoprotein gene enhancers

1988 ◽  
Vol 8 (3) ◽  
pp. 1169-1178
Author(s):  
R Godbout ◽  
R S Ingram ◽  
S M Tilghman
Keyword(s):  
Nucleotide Sequence ◽  
Specific Activity ◽  
Biological Activities ◽  
Transcriptional Start Site ◽  
Alpha Fetoprotein ◽  
Nucleotide Sequence Analysis ◽  
Structure Mapping ◽  
Transient Expression Assay ◽  
Enhancer Activity ◽  
Positive Elements

Multiple cellular enhancers have been identified previously in the 5'-flanking region of the mouse alpha-fetoprotein gene by transient expression assay. In this report the enhancers have been localized to three regions 200 to 300 base pairs in length at 2.5, 5.0, and 6.5 kilobases of DNA upstream of the transcriptional start site. Nucleotide sequence analysis of the three enhancers revealed areas of homology among them, the most significant of which were two regions of 10 and 18 nucleotides in length. Two of the enhancers were analyzed in detail and shown to be composed of multiple nonidentical domains, none of which was sufficient for full enhancer activity; rather, they acted in an additive fashion in generating the full activity of the enhancer. The tissue-specific activity of the enhancer at -2.5 kilobases was assessed by comparing the activities of subdomains in liver- and non-liver-derived cell lines and was found to be the result of both positive elements within the enhancer and at least one negative element to its 5' end. In contrast, the tissue specificity of the enhancer at -5.0 kilobases was maintained when the minimal essential region was tested alone. The nucleotide sequence similarities, as well as the differences among the enhancers, may explain their differing biological activities both in tissue culture and in vivo.

scholarly journals X-linked sideroblastic anemia: identification of the mutation in the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) in the original family described by Cooley

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3915-3924 ◽  
Author(s):  
PD Cotter ◽  
DL Rucknagel ◽  
DF Bishop
Keyword(s):  
Specific Activity ◽  
Restriction Analysis ◽  
Affinity Purification ◽  
Large Family ◽  
Flanking Sequence ◽  
Sideroblastic Anemia ◽  
Catalytic Core ◽  
Aminolevulinate Synthase ◽  
Original Family

In 1945, Thomas Cooley described the first cases of X-linked sideroblastic anemia (XLSA) in two brothers from a large family in which the inheritance of the disease was documented through six generations. Almost 40 years later the enzymatic defect in XLSA was identified as the deficient activity of the erythroid-specific form of delta-aminolevulinate synthase (ALAS2), the first enzyme in the heme biosynthetic pathway. To determine the nature of the mutation in the ALAS2 gene causing XLSA in Cooley's original family, genomic DNAs were isolated from two affected hemizygotes, and each ALAS2 exon was PCR amplified and sequenced. A single transversion (A to C) was identified in exon 5. The mutation predicted the substitution of leucine for phenylalanine at residue 165 (F165L) in the first highly conserved domain of the ALAS2 catalytic core shared by all species. No other nucleotide changes were found by sequencing each of the 11 exons, including intron/exon boundaries, 1 kb of 52-flanking and 350 nucleotides of 32-flanking sequence. The mutation introduced an Mse I site and restriction analysis of PCR-amplified genomic DNA confirmed the presence of the lesion in the two affected brothers and in three obligate heterozygotes from three generations of this family. Carrier diagnosis of additional family members identified the mutation in one of the proband's sisters. After prokaryotic expression and affinity purification of both mutant and normal ALAS2 fusion proteins, the specific activity of the F165L mutant enzyme was about 26% of normal. The cofactor, pyridoxal 52-phosphate, activated and/or stabilized the purified mutant recombinant enzyme in vitro, consistent with the pyridoxine-responsive anemia in affected hemizygotes from this family.

Chromosomal location and expression of a herbicide safener-regulated glutathione S-transferase gene in Triticum aestivum and linkage relations in Hordeum vulgare

Genome ◽  
1998 ◽  
Vol 41 (3) ◽  
pp. 368-372 ◽  
Author(s):  
Dean E Riechers ◽  
Andris Kleinhofs ◽  
Gerard P Irzyk ◽  
Stephen S Jones
Keyword(s):  
Triticum Aestivum ◽  
Hordeum Vulgare ◽  
Chromosomal Location ◽  
Single Copy ◽  
Rflp Marker ◽  
Glutathione S Transferase ◽  
Triticum Tauschii ◽  
Herbicide Safener ◽  
Mrna Transcripts ◽  
Copy Gene

The chomosomal location of a glutathione S-transferase (GST) gene was determined in both hexaploid wheat (Triticum aestivum) and barley (Hordeum vulgare). The GST cDNA used to map the gene was cloned from the diploid wheat Triticum tauschii. GST loci were located on the short arms of chromosomes 6A, 6B, and 6D in T. aestivum and also on the short arm of chromosome 6H in H. vulgare. The GST locus in barley was absolutely linked to the RFLP marker E148A and was located 0.8 cM proximal to the RFLP marker ABC169B on barley chromosome 6H. At least two copies of the GST gene were present in each of the T. aestivum A, B, and D genomes, and a homologous GST gene was present as a single-copy gene in the barley genome. GST mRNA transcripts were not detected in RNA isolated from shoots of control (unsafened) seedlings of T. tauschii or T. aestivum. It was determined that the expression of the GST gene was regulated by herbicide safener treatment in T. tauschii and T. aestivum by detecting safener-increased GST mRNA transcript levels.Key words: Triticum aestivum, Triticum tauschii, Hordeum vulgare, herbicide safener, glutathione S-transferase, genetic mapping.

scholarly journals Characterization of glutathione S-transferases in rat kidney. Alteration of composition by cis-platinum

1988 ◽  
Vol 252 (1) ◽  
pp. 127-136 ◽  
Author(s):  
G M Trakshel ◽  
M D Maines
Keyword(s):  
Specific Activity ◽  
Rat Kidney ◽  
Male Rats ◽  
Glutathione S Transferase ◽  
Aberrant Form ◽  
Glutathione S Transferases ◽  
Exchange Matrix ◽  
Rat Kidney Cytosol

We have developed chromatographic and mathematical protocols that allowed the high resolution of glutathione S-transferase (GST) subunits, and the identification of a previously unresolved GST monomer in rat kidney cytosol; the monomer was identified tentatively as subunit 6. Also, an aberrant form of GST 7-7 dimer appeared to be present in the kidney. This development was utilized to illustrate the response of rat kidney GST following cis-platinum treatment in vivo. Rat kidney cytosol was separated into three ‘affinity families’ of GST activity after elution from a GSH-agarose matrix. The affinity peaks were characterized by quantitative differences in their subunit and dimeric compositions as determined by subsequent chromatography on a cation-exchange matrix and specific activity towards substrates. By use of these criteria, the major GST dimers of affinity peaks were tentatively identified. The major GST dimers in peak I were GST 1-1 and 1-2, in affinity peak II it was GST 2-2, and in peak III they were GST 3-3 and 7-7. GST 3-6 and/or 4-6, which have not been previously resolved in kidney cytosol, were also present in peak II. Alterations in the kidney cytosolic GST composition of male rats were detected subsequent to the administration of cis-platinum (7.0 mg/kg subcutaneously, 6 days). This treatment caused a pronounced alteration in the GST profile, and the pattern of alteration was markedly different from that reported for other chemicals in the kidney or in the liver. In general, the cellular contents of the GSTs of the Alpha and the Mu classes decreased and increased respectively. It is postulated that the decrease in the Alpha class of GSTs by cis-platinum treatment may be related to renal cortical damage and the loss of GSTs in the urine. The increase in the Mu class of GSTs could potentially stem from a lowered serum concentration of testosterone; the latter is a known effect of cis-platinum treatment.

scholarly journals Cloning and expression of a chick liver glutathione S-transferase CL 3 subunit with the use of a baculovirus expression system

1992 ◽  
Vol 281 (2) ◽  
pp. 545-551 ◽  
Author(s):  
L H Chang ◽  
J Y Fan ◽  
L F Liu ◽  
S P Tsai ◽  
M F Tam
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Relative Activity ◽  
Cloning And Expression ◽  
Glutathione S Transferase ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
A Subunit

Glutathione S-transferase CL 3 subunits purified from 1-day-old-chick livers were digested with Achromobacter proteinase I and the resulting fragments were isolated for amino acid sequence analysis. An oligonucleotide probe was constructed accordingly for cDNA library screening. A cDNA clone of 1342 bases, pGCL301, encoding a protein of 26209 Da was isolated and sequenced. Including conservative substitutions, this protein has 75-79% sequence similarity to other Alpha family glutathione S-transferases. The coding sequence of pGCL301 was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The expressed protein has a high relative activity with ethacrynic acid (47% of the specific activity with 1-chloro-2,4-dinitrobenzene). The enzyme has a subunit molecular mass of 25.2 +/- 1.2 kDa (by SDS/PAGE), a pI of 9.45 and an absorption coefficient A1%1cm of 13.0 +/- 0.5 at 280 nm.

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