Cloning and Localization of a Glutathione S-transferase Class I Gene from Anopheles gambiae

Abstract

1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) resistance in both adults and larvae of Anopheles gambiae is mediated by stage-specific glutathione S-transferases (GSTs). On the basis of their biochemical characteristics the larval resistance-associated GSTs are likely to be insect class I GSTs. Aggst1-2, a class I GST gene, which is expressed in larvae, has been cloned from the malaria vector A. gambiae. The gene was inserted into a bacterial expression system, and the detection of 1-chloro-2,4-dinitrobenzene (CDNB) conjugating activity in Eschericia coli expressing the recombinant enzyme confirmed that aggst1-2 encodes a catalytically active GST. The gene encodes a 209 amino acid protein with 46% sequence similarity to a Drosophila melanogaster class I GST (GST-D1), 44% similarity with a Musca domestica class I GST (MdGST-1), but only low levels of homology with class II insect GSTs, including the adult specific AgGST2-1 from A. gambiae. Southern analysis of genomic DNA indicated that A. gambiae has multiple class I GSTs. In situ hybridization of class I genomic and cDNA clones to polytene chromosomes identified a single region of complementarity on chromosome 2R division 18B, suggesting that these class I GSTs in A. gambiae are arranged sequentially in the genome. Three positive overlapping recombinant clones were identified from an A. gambiae genomic library. Mapping and partial sequencing of these clones suggests that there are several GSTs and truncated GST pseudogenes within the 30kb of DNA that these clones span.

The role of alternative mRNA splicing in generating heterogeneity within the Anopheles gambiae class I glutathione S- transferase family

“…The sequences obtained matched the published 5Ј untranslated region sequence of the partial cDNA aggst1-1, which was obtained by 5Ј RACE (14). Comparison of the cDNA sequences with the genomic sequence upstream of aggst1␣ revealed the presence of a 146-nt intron 24 nt upstream from the initiator ATG and identified an additional noncoding exon, exon 1, within aggst1␣ (Fig.…”

“…The recombinant bacteriophage, Ag_B1 was isolated from a Suakoko 2La genomic library by screening with a partial A. gambiae class I GST cDNA, aggst1-1, as described (14). A contiguous sequence of the entire 15.3-kb insert of Ag_B1 was obtained by manual sequencing and automated sequencing using the ALF automatic sequencer (Pharmacia).…”

“…mRNA was extracted from ZAN͞U larvae, pupae, and adults and was used to synthesize cDNA as described (14). Genomic DNA was extracted from individual mosquitoes by using the method of Collins et al (22).…”

“…We previously have described a single, intronless class I GST gene, aggst1-2, from the A. gambiae genomic clone, Ag_B1 (14). Restriction mapping of Ag_B1 and two additional, overlapping genomic clones identified multiple fragments that hybridized to a class I GST probe.…”

“…In D. melanogaster, eight divergent intronless genes are found within a 14-kb DNA segment (12), and at least five different class I GST genes are present in M. domestica (13). We previously have shown that A. gambiae also contains multiple class I GSTs, which are arranged sequentially in the genome (14).…”