Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae

2001 ◽  
Vol 359 (2) ◽  
pp. 295-304 ◽  
Author(s):  
Hilary RANSON ◽  
Louise ROSSITER ◽  
Federica ORTELLI ◽  
Betty JENSEN ◽  
Xuelan WANG ◽  
...  
Keyword(s):  
Gene Family ◽  
Anopheles Gambiae ◽  
Malaria Vector ◽  
Sequence Similarity ◽  
Mrna Levels ◽  
Class Iii ◽  
Glutathione S Transferase ◽  
Close Proximity ◽  
Genes Encoding ◽  
Glutathione S Transferases

The sequence and cytological location of five Anopheles gambiae glutathione S-transferase (GST) genes are described. Three of these genes, aggst1-8, aggst1-9 and aggst1-10, belong to the insect class I family and are located on chromosome 2R, in close proximity to previously described members of this gene family. The remaining two genes, aggst3-1 and aggst3-2, have a low sequence similarity to either of the two previously recognized classes of insect GSTs and this prompted a re-evaluation of the classification of insect GST enzymes. We provide evidence for seven possible classes of insect protein with GST-like subunits. Four of these contain sequences with significant similarities to mammalian GSTs. The largest novel insect GST class, class III, contains functional GST enzymes including two of the A. gambiae GSTs described in this report and GSTs from Drosophila melanogaster, Musca domestica, Manduca sexta and Plutella xylostella. The genes encoding the class III GST of A. gambiae map to a region of the genome on chromosome 3R that contains a major DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] resistance gene, suggesting that this gene family is involved in GST-based resistance in this important malaria vector. In further support of their role in resistance, we show that the mRNA levels of aggst3-2 are approx. 5-fold higher in a DDT resistant strain than in the susceptible strain and demonstrate that recombinant AgGST3-2 has very high DDT dehydrochlorinase activity.

Cytosolic glutathione S-transferases of Oesophagostomum dentatum

Parasitology ◽  
2008 ◽  
Vol 135 (10) ◽  
pp. 1215-1223 ◽  
Author(s):  
A. JOACHIM ◽  
B. RUTTKOWSKI
Keyword(s):  
Amino Acids ◽  
Pcr Primers ◽  
Mrna Levels ◽  
Fourth Stage ◽  
Glutathione S Transferase ◽  
Cdna Sequences ◽  
Oesophagostomum Dentatum ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases

SUMMARYOesophagostomum dentatum stages were investigated for glutathione S-transferase (GST) expression at the protein and mRNA levels. GST activity was detected in all stages (infectious and parasitic stages including third- and fourth-stage larvae of different ages as well as males and females) and could be dose-dependently inhibited with sulfobromophthalein (SBP). Addition of SBP to in vitro larval cultures reversibly inhibited development from third- to fourth-stage larvae. Two glutathione-affinity purified proteins (23 and 25 kDa) were detected in lysates of exsheathed third-stage larvae by SDS-PAGE. PCR-primers were designed based on peptide sequences and conserved GST sequences of other nematodes for complete cDNA sequences (621 and 624 nt) of 2 isoforms, Od-GST1 and Od-GST2, with 72% nucleotide similarity and 75% for the deduced proteins. Genomic sequences consisted of 7 exons and 6 introns spanning 1296 bp for Od-GST1 and 1579 and 1606 bp for Od-GST2. Quantitative real-time-PCR revealed considerably elevated levels of Od-GST1 in the early parasitic stages and slightly reduced levels of Od-GST2 in male worms. Both Od-GSTs were most similar to GST of Ancylostoma caninum (nucleotides: 73 and 70%; amino acids: 80 and 73%). The first three exons (75 amino acids) corresponded to a synthetic prostaglandin D2 synthase (53% similarity). O. dentatum GSTs might be involved in intrinsic metabolic pathways which could play a role both in nematode physiology and in host-parasite interactions.

scholarly journals The effect of hepatic regeneration on the expression of the glutathione S-transferases

1993 ◽  
Vol 293 (1) ◽  
pp. 137-142 ◽  
Author(s):  
S J Lee ◽  
T D Boyer
Keyword(s):  
Transcriptional Activity ◽  
Enzymic Activity ◽  
Hepatic Regeneration ◽  
Mrna Levels ◽  
Glutathione S Transferase ◽  
Toxic Compounds ◽  
Protein Levels ◽  
Post Surgery ◽  
Glutathione S Transferases ◽  
Early Phases

The effect of hepatic regeneration on expression of four glutathione S-transferase (GST) subunits (Ya, Yc, Yb1, Yb2) was examined in rats following partial hepatectomy (PH). mRNA levels of the Ya and Yc subunits (Alpha class) decreased and were 13% and 42% of levels in sham-operated animals respectively 12 h after surgery. mRNA levels for the Yb1 subunit (Mu class) also decreased but were not maximally reduced until 24 h after PH (22% of sham-treated level). mRNA levels of the Yb2 subunit were affected little by PH. Changes in levels of mRNA appeared to reflect a decrease in both transcriptional activity and mRNA stability. The decrease in mRNA levels was associated with a fall in enzymic activity and in protein levels of Alpha-class GSTs. Within 48 h of surgery, levels of mRNA, protein enzymic activity and transcriptional activity had all fully recovered. GSH levels also decreased in the first 6 h after PH. However, 24 h after surgery GSH levels in animals having undergone PH exceeded those in sham-treated animals by 2-fold and this difference persisted for 72 h. These findings suggest that during the early phases of hepatic regeneration, because of decreased GST and GSH levels, the liver may be unusually susceptible to injury by toxic compounds. However, by the first round of cell division (36-48 h post-surgery) the liver has fully recovered its ability to metabolize toxic electrophiles.

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.

scholarly journals Glutathione S-transferases from the white-rot fungus, Phanerochaete chrysosporium

1997 ◽  
Vol 324 (1) ◽  
pp. 243-248 ◽  
Author(s):  
Caitriona A. DOWD ◽  
Catherine M. BUCKLEY ◽  
David SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Phanerochaete Chrysosporium ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Preliminary Evidence ◽  
White Rot ◽  
White Rot Fungus ◽  
Glutathione S Transferase ◽  
Glutathione S Transferases ◽  
A Subunit

A glutathione S-transferase (GST) was purified to homogeneity from the white-rot fungus, Phanerochaete chrysosporium, by affinity chromatography on glutathione–agarose followed by Mono-Q ion-exchange FPLC. This protein immunoblotted with antisera to rat Theta class GST 5-5 and also showed N-terminal sequence similarity to the Theta class, including the presence of a conserved serine residue that has been specifically implicated in catalysis in this class [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133–2143] and other residues conserved in plant sequences. Catalytic activity was found to be highly labile in the purified protein, although preliminary evidence for activity (approx. 120 m-units/mg) with 1,2-epoxy-3-(p-nitrophenoxy)propane was obtained in some preparations. The enzyme seems to be a dimer with a subunit molecular mass of 25 kDa by SDS/PAGE. The native molecular masses estimated by non-denaturing electrophoresis and by Superose-12 gel filtration were 58 and 45 kDa respectively. A second protein purified in this study also gave low level of activity with 1,2-epoxy-3-(p-nitrophenoxy)propane and had a subunit molecular mass of 28 kDa (native size 62–63 kDa), but did not immunoblot with any GST class and seemed to be N-terminally blocked.

scholarly journals Cloning and characterization of two glutathione S-transferases from a DDT-resistant strain of Anopheles gambiae

1997 ◽  
Vol 324 (1) ◽  
pp. 97-102 ◽  
Author(s):  
Hilary RANSON ◽  
La-aied PRAPANTHADARA ◽  
Janet HEMINGWAY
Keyword(s):  
Anopheles Gambiae ◽  
Resistant Strain ◽  
Sequence Similarity ◽  
Amino Acid Sequences ◽  
Class I ◽  
Kinetic Properties ◽  
Coding Region ◽  
High Sequence Similarity ◽  
Glutathione S Transferases ◽  
High Level

Two cDNA species, aggst1-5 and aggst1-6, comprising the entire coding region of two distinct glutathione S-transferases (GSTs) have been isolated from a 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) resistant strain (ZANDS) of Anopheles gambiae. The nucleotide sequences of these cDNA species share 80.2% identity and their derived amino acid sequences are 82.3% similar. They have been classified as insect class I GSTs on the basis of their high sequence similarity to class I GSTs from Drosophila melanogaster and Musca domestica and they are localized to a region of an An. gambiae chromosome known to contain further class I GSTs. The genes aggst1-5 and aggst1-6 were expressed at high levels in Escherichia coli and the recombinant GSTs were purified by affinity chromatography and characterized. Both agGST1-5 and agGST1-6 showed high activity with the substrates 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene but negligible activity with the mammalian theta class substrates, 1,2-epoxy-3-(4-nitrophenoxy)propane and p-nitrophenyl bromide. Despite their high level of sequence identity, agGST1-5 and agGST1-6 displayed different kinetic properties. Both enzymes were able to metabolize DDT and were localized to a subset of GSTs that, from earlier biochemical studies, are known to be involved in insecticide resistance in An. gambiae. This subset of enzymes is one of three in which the DDT metabolism levels are elevated in resistant insects.

scholarly journals Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae

2001 ◽  
Vol 359 (2) ◽  
pp. 295 ◽  
Author(s):  
Hilary RANSON ◽  
Louise ROSSITER ◽  
Federica ORTELLI ◽  
Betty JENSEN ◽  
Xuelan WANG ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Malaria Vector ◽  
Glutathione S Transferases

scholarly journals Pl-Bh, an anthocyanin regulatory gene of maize that leads to variegated pigmentation.

Genetics ◽  
1993 ◽  
Vol 135 (2) ◽  
pp. 575-588 ◽  
Author(s):  
S M Cocciolone ◽  
K C Cone
Keyword(s):  
Gene Family ◽  
Dna Sequences ◽  
Sequence Similarity ◽  
Regulatory Gene ◽  
Regulatory Genes ◽  
Anthocyanin Synthesis ◽  
Mrna Levels ◽  
Wild Type ◽  
Specific Expression ◽  
Organ Specific

Abstract Anthocyanins are purple pigments that can be produced in virtually all parts of the maize plant. The spatial distribution of anthocyanin synthesis is dictated by the organ-specific expression of a few regulatory genes that control the transcription of the structural genes. The regulatory genes are grouped into families based on functional identity and DNA sequence similarity. The C1/Pl gene family consists of C1, which controls pigmentation of the kernel, and Pl, which controls pigmentation of the vegetative and floral organs. We have determined the relationship of another gene, Blotched (Bh), to the C1 gene family. Bh was originally described as a gene that conditions blotches of pigmentation in kernels homozygous for recessive c1, suggesting that Bh could functionally replace C1 in the kernel. Our genetic and molecular analyses indicate that Bh is an allele of Pl, that we designate Pl-Bh. Pl-Bh differs from wild-type Pl alleles in two respects. In contrast to the uniform pigmentation observed in plants carrying Pl, the pattern of pigmentation in plants carrying Pl-Bh is variegated. Pl-Bh leads to variegated pigmentation in virtually all tissues of the plant, including the kernel, an organ not pigmented by other Pl alleles. To address the molecular basis for the unusual pattern of expression of Pl-Bh, we cloned and sequenced the gene. The nucleotide sequence of Pl-Bh showed only a single base-pair difference from that of Pl. However, genomic DNA sequences associated with Pl-Bh were found to be hypermethylated relative to the same sequences around the wild-type Pl allele. The methylation was inversely correlated with Pl mRNA levels in variegated plant tissues. Thus, we conclude that DNA methylation may play a role in regulating Pl-Bh expression.

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