Recombinant bHLH-PAS/JHR polypeptide and its use to screen potential insecticides
a polypeptide and insecticide technology, applied in the field of recombinant bhlhpas/jhr polypeptide and its use to screen potential insecticides, can solve the problems of high cost of discovery, negative public opinion, and high cost of discovery
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[0233] To isolate the Met JHR gene, genomic libraries were constructed from flies carrying either of two P-element alleles of Met, MetA3 and MetK17. These alleles were recovered in separate screens from methoprene-resistant flies. Wilson et al. Molecular Mech. of Insecticide Resistance (Am. Chem. Soc. Symp.) 505:99 (1992). Each allele conferred resistance to both the toxic and morphogenetic effects of JH and methoprene, and susceptible revertants could be recovered by standard genetic means. A 50 kilobase region surrounding the P-element insertion site was cloned from each library, as described by Turner and Wilson, Arch. Insect Biochem. Biophys. 30:133 (1995).
[0234] DNA sequencing and analysis of the genomic region located within one kilobase of the insertion sites revealed an open reading frame (ORF) located 273 base pairs from the insertion site in the MetA3 allele and 424 base pairs in the MetK17 allele. See FIG. 1. Transcription of this ORF occurs away from the P-element, sugg...
example 2
[0240] A DNA probe to the ORF described above failed to identify any transcript(s) on a Northern blot of RNA from a methoprene-susceptible Oregon-RC late third-instar larvae, but a more sensitive RNA probe recognized a transcript of approximately 5.5 kilobases. Total RNA was isolated with TriReagent (Molecular Research Center, Inc., Ohio) from staged animals. Each lane was loaded with 40 mg of total RNA, subjected to denaturing gel electrophoresis on a formaldehyde-agarose gel, and blotted onto Hybond-N membrane. Following cross-linking, membranes were prehybridized in a solution containing 5×SSPE, 5× Denhardt's, 0.5% SDS, 50% formamide, and 100 μg / ml yeast tRNA for about 5 to about 7 hours at about 65° C.
[0241] Membranes were then hybridized in the same solution at about 68° C. for about 15 to about 17 hours with a [32p]-UTP labeled riboprobe (Promega Co., Wis.) synthesized from a fragment of the Met-JHR gene. This fragment extended from nucleotide 771 through 1102 of the open rea...
example 3
[0246] cDNA molecules corresponding to the region containing the Met-JHR ORF, as well as to the smaller (3.3 kb) transcript, were isolated as apparent full-length cDNAs from a Drosophila wild-type Canton-S ovary cDNA library and were sequenced to establish a relationship of the transcript with the genomic nucleotide sequence. The probable transcription start site for this transcript begins 220 bp upstream from the start codon and the probably transcript ends 912 bp from the stop codon.
[0247] A comparison of the cDNA to the genomic sequence showed that the genomic ORF is 2.22 Kb and the cDNA ORF is 2.151 Kb. The difference between the two sequences is a 69 nucleotide intron, which corresponds to 23 codons, and does not change the open reading frame of the genomic and cDNA. The presence of the intron provides evidence for the possibility of alternatively spliced variants of Met-JHR and hence multiple isoform proteins of Met-JHR.
[0248] The longest single open reading frame in the cDN...
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