Insect juvenile hormone resistance gene homology with the bHLH-PAS family of transcriptional regulators

Abstract

Juvenile hormone analog (JHA) insecticides are relatively nontoxic to vertebrates and offer effective control of certain insect pests. Recent reports of resistance in whiteflies and mosquitoes demonstrate the need to identify and understand genes for resistance to this class of insect growth regulators. Mutants of the Methoprene-tolerant (Met) gene in Drosophila melanogaster show resistance to both JHAs and JH, and previous biochemical studies have demonstrated a mechanism of resistance involving an intracellular JH binding-protein that has reduced ligand affinity in Met flies. We cloned the Met+ gene by transposable P-element tagging and found reduced transcript level in several mutant alleles, showing that underproduction of the normal gene product can lead to insecticide resistance. Transformation of Met flies with a Met+ cDNA resulted in susceptibility to methoprene, indicating that the cDNA encodes a functional Met+ protein. MET shows homology to the basic helix-loop-helix (bHLH)-PAS family of transcriptional regulators, implicating MET in the action of JH at the gene level in insects. This family also includes the vertebrate dioxin receptor, a transcriptional regulator known to bind a variety of environmental toxicants. Because JHAs include a diverse array of chemicals with JH activity, a mechanism whereby they can exert effects in insects through a common pathway is suggested.

Figure 1

Met gene region. Genomic organization of an 8-kb region including the Met ORF (boxed). P-element insertional sites in the Met^A3 and Met^K17 alleles are shown at the downward facing arrows. The solid arrow represents the direction of transcription of Met. The locations of the transcripts as deduced from cDNA sequencing (3.3-kb transcript) and RT-PCR analysis (5.5-kb transcript) are noted below the map. The genomic transformation fragments are indicated below the transcriptional units; those that did not rescue the resistance phenotype are noted (−) and the fragment that produced methoprene susceptibility in transformant flies is noted (+). The fragments are designated by the restriction enzyme sites. D , HindIII; S, SalI; K, KpnI; St, StuI; B, BamHI; X, XhoI; H, HpaI.

Figure 2

Northern blot of total RNA isolated from wandering third-instar larvae homozygous for v or any of various Met alleles and probed with a ³²P-labeled riboprobe of the Met gene. Each lane was loaded with 40 μg of total RNA, subjected to denaturing gel electrophoresis in a formaldehyde-agarose gel, and blotted onto Hybond-N membrane. Control loading (Lower) was evaluated by stripping the blot and reprobing with a [³²P]dCTP random-primed cDNA of Rp49. Met, Met², and Met³ are ethyl methanesulfonate-induced alleles; Met^A3 and Met^K17 are P-element alleles, and the remaining alleles were gamma-ray induced from methoprene-susceptible v flies. Met¹²⁸ has consistently shown overproduction of transcript on Northern blots.

Figure 3

Developmental Northern blot analysis of total RNA isolated from the methoprene-susceptible Oregon-RC strain at various times in development and probed with a [³²P]UTP-labeled 331-bp riboprobe for Met. Each lane was loaded with 40 μg of total RNA, and the blot was probed with the Met gene riboprobe followed by a DNA probe for the Rp49 gene as described for Fig. 2. The decreased levels of Rp49 in pupae reflect decreased expression during this stage of development (22). Embryos were collected from overnight cultures and either frozen in liquid nitrogen or maintained at 25°C until the desired age. Larvae were staged from timed embryo collections; the indicated times are ±8 hr. Pupae were staged from the white prepupal stage, which lasts for about 1 hr. Adult males consistently show only the 5.5-kb transcript; females show both and, when fully gravid, show more of the 3.3-kb transcript than appears on this blot.

Figure 4

(A) Nucleotide sequence and predicted amino acid sequence of the Met ORF, derived from sequence determination of 3.3-kb cDNA clones. The bHLH domain is underlined, the PAS-A domain is boldly underlined, and the PAS-B domain is double underlined. Single-letter abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (B) Amino acid sequence alignment of the homologous regions for the bHLH, PAS-A, and PAS-B domains of MET with those of human AHR and ARNT. Number in parenthesis indicates the position of the initial amino acid of each domain in the respective protein. Identical sequences are boxed.