RESUMEN
Gene duplications play a key role in the emergence of novel traits and in adaptation. But despite their centrality to evolutionary processes, it is still largely unknown how new gene duplicates are initially fixed within populations and later maintained in genomes. Long-standing debates on the evolution of gene duplications could be settled by determining the relative importance of genetic drift vs. positive selection in the fixation of new gene duplicates. Using the Drosophila Global Diversity Lines (GDL), we have combined genome-wide SNP polymorphism data with a novel set of copy number variant calls and gene expression profiles to characterize the polymorphic phase of new genes. We found that approximately half of the roughly 500 new complete gene duplications segregating in the GDL lead to significant increases in the expression levels of the duplicated genes and that these duplications are more likely to be found at lower frequencies, suggesting a negative impact on fitness. However, we also found that six of the nine gene duplications that are fixed or close to fixation in at least one of the five populations in our study show signs of being under positive selection, and that these duplications are likely beneficial because of dosage effects, with a possible role for additional mutations in two duplications. Our work suggests that in Drosophila, theoretical models that posit that gene duplications are immediately beneficial and fixed by positive selection are most relevant to explain the long-term evolution of gene duplications in this species.
Asunto(s)
Drosophila melanogaster/genética , Transcriptoma , Animales , Variaciones en el Número de Copia de ADN , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Evolución Molecular , Duplicación de Gen , Frecuencia de los Genes , Masculino , Selección GenéticaRESUMEN
One of the persistent problems in biology is understanding how genetic variation contributes to phenotypic variation. Associations at many levels have been reported, and yet causal inference has remained elusive. We propose to rely on the knowledge of causal relationships established by molecular biology approaches. The existing molecular knowledge forms a firm backbone upon which hypotheses connecting genetic variation, transcriptional variation and phenotypic variation can be built. The sex determination pathway is a well-established molecular network, with the Yolk protein 1-3 (Yp) genes as the most downstream target. Our analyses reveal that genetic variation in expression for genes known to be upstream in the pathway explains variation in downstream targets. Relationships differ between the two sexes, and each Yp has a distinct transcriptional pattern. Yp expression is significantly negatively correlated with longevity, an important life history trait, for both males and females.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Variación Genética , Longevidad/genética , Modelos Moleculares , Biología de Sistemas , Animales , Análisis por Conglomerados , Compensación de Dosificación (Genética) , Drosophila melanogaster/fisiología , Proteínas del Huevo/genética , Femenino , Redes Reguladoras de Genes , Masculino , Procesos de Determinación del Sexo , Transcriptoma , Vitelogeninas/genéticaRESUMEN
Laboratory selection for resistance to starvation has been conducted under relatively controlled conditions to investigate direct and correlated responses to artificial selection. With regard to starvation resistance, there are three physiological routes by which the trait can evolve: resource accumulation, energy conservation and starvation tolerance. A majority of energetic compounds and macromolecules including triglycerides, trehalose and other sugars, and soluble protein increased in abundance as a result of selection. Movement was additionally investigated with selected males moving less than control males and selected females exhibiting a similar response to selection. Results obtained from this study supported two of the possible evolutionary mechanisms for adaptation to starvation: energy compound storage and conservation. If the response to selection is based on an evolutionarily conserved pattern of genetic correlations (elevated lipid, elevated sugars and reduced movement), then the response to selection is medically relevant and the genetic architecture should be investigated in depth.
Asunto(s)
Evolución Biológica , Drosophila melanogaster/metabolismo , Animales , Composición Corporal , Tamaño Corporal , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Femenino , Privación de Alimentos , Metabolismo de los Lípidos , MasculinoRESUMEN
Sperm storage organs are common and broadly distributed among animal taxa. However, little is known about how these organs function at the molecular level. Additionally, there is a paucity of knowledge about the evolution of genes expressed in these organs. This investigation is an evolutionary expressed sequence tag (EST) study of genes expressed in the seminal receptacle, one of the sperm storage organs in Drosophila. The incidence of positive selection is higher for the seminal receptacle genes than Drosophila reproductive genes as a whole, but lower than genes associated with the spermatheca, a second type of Drosophila sperm storage organ. By identifying overrepresented classes of proteins and classes for which sperm storage function is suggested by the nature of the proteins, candidate genes were discovered. These candidates belong to protein classes such as muscle contraction, odorant binding and odorant receptor, protease inhibitor and immunity.
Asunto(s)
Drosophila melanogaster/genética , Etiquetas de Secuencia Expresada , Genes de Insecto/genética , Genitales Femeninos/metabolismo , Animales , Secuencia de Bases , ADN Complementario/genética , Drosophila melanogaster/metabolismo , Evolución Molecular , Femenino , Funciones de Verosimilitud , Modelos Genéticos , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The occurrence of female sperm storage across taxa indicates the importance of this complex and dynamic process. Organs responsible for sperm storage (SSOs) and proteins expressed therein, are important in fundamental aspects of reproduction and could play a major role in evolutionary processes such as post-mating sexual selection. Given the essential role of SSOs, it is surprising that the process of sperm storage is so poorly understood. This study investigated the transcriptome of female Drosophila melanogaster SSOs (seminal receptacle and spermathecae). Spermathecae were enriched for proteases and metabolic enzymes while the seminal receptacle was enriched for genes involved in localization, signaling and ion transport. Differences in functional gene categories indicate that these organs play unique roles in sperm storage.
Asunto(s)
Drosophila melanogaster/metabolismo , Perfilación de la Expresión Génica , Genitales Femeninos/metabolismo , Animales , Análisis por Conglomerados , Sistema Enzimático del Citocromo P-450/metabolismo , Drosophila melanogaster/genética , Femenino , Regulación de la Expresión Génica/fisiología , Hormonas de Insectos/metabolismo , Masculino , Reproducción/fisiología , EspermatozoidesRESUMEN
The 458 amino acid sequence of a mature JHE protein from the cricket Gryllus assimilis was identified after isolating the partial cDNA sequence encoding this protein from a fat body and midgut cDNA library. This hemimetabolan JHE sequence shows over 40% amino acid similarity to the known JHE sequences of several holometabolous insects. It also includes previously determined peptide sequences for G. assimilis JHE as well as two other motifs associated with JHE enzymes in holometabolous insects. The predicted molecular weight of the protein agrees with that of the JHE previously purified from G. assimilis. Partial genomic sequence encoding the Jhe contains two large (1330 and 2918bp) introns. No coding DNA sequence variation was observed over a 1293bp region between selected lines differing six to eight-fold in hemolymph JHE activity. However, a 19bp indel was found in one of the introns; the insertion was strongly associated with elevated hemolymph activity, both in the selected lines and in the F(2) progeny of crosses between them. Phylogenetic analyses localised the G. assimilis JHE to a clade containing dipteran and coleopteran JHEs, with lepidopteran JHEs occurring in a separate clade.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Cuerpo Adiposo/enzimología , Gryllidae/enzimología , Hemolinfa/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Clonación Molecular , Tracto Gastrointestinal/enzimología , Biblioteca de Genes , Gryllidae/química , Gryllidae/genética , Intrones , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Técnicas de Sonda Molecular , Datos de Secuencia Molecular , Filogenia , Selección Genética , Análisis de Secuencia de ADN , Relación Estructura-ActividadRESUMEN
Identification of genes underlying complex traits is an important problem. Quantitative trait loci (QTL) are mapped using marker-trait co-segregation in large panels of recombinant genotypes. Most frequently, recombinant inbred lines derived from two isogenic parents are used. Segregation patterns are also studied in pedigrees from multiple families. Great advances have been made through creative use of these techniques, but narrow sampling and inadequate power represent strong limitations. Here, we propose an approach combining the strengths of both techniques. We established a mapping population from a sample of natural genotypes, and applied artificial selection for a complex character. Selection changed the frequencies of alleles in QTLs contributing to the selection response. We infer QTLs with dense genotyping microarrays by identifying blocks of linked markers undergoing selective changes in allele frequency. We demonstrated this approach with an experimental population composed from 20 isogenic strains. Selection for starvation survival was executed in three replicated populations with three control non-selected populations. Three individuals per population were genotyped using Affymetrix GeneChips. Two regions of the genome, one each on the left arms of the second and third chromosomes, showed significant divergence between control and selected populations. For the former region, we inferred allele frequencies in selected and control populations by pyrosequencing. We conclude that the allele frequency difference, averaging approximately 40% between selected and control lines, contributed to selection response. Our approach can contribute to the fine scale decomposition of the genetics of direct and indirect selection responses, and genotype by environment interactions.
Asunto(s)
Alelos , Mapeo Cromosómico , Drosophila melanogaster/genética , Frecuencia de los Genes/genética , Genoma de los Insectos/fisiología , Sitios de Carácter Cuantitativo/genética , Animales , Femenino , Genética de Población , Endogamia , Masculino , Selección GenéticaRESUMEN
The present study tests the hypothesis that reproduction is correlated with decreased oxidative stress resistance. In numerous species, it has been observed that longevity is negatively correlated with reproduction but the physiological basis of this cost is not well understood. In the present study, female egg production was stimulated by adding live yeast to the surface of Drosophila food. After females were held on yeast-supplemented and unmodified medium for 6-12 days, susceptibility to oxidative stress was measured by exposure to methyl viologen. Added yeast was associated with stress susceptibility of fertile females but not of sterile females. The results of the present study suggest that oxidative stress susceptibility is a physiological cost of reproduction.
Asunto(s)
Drosophila melanogaster/fisiología , Oogénesis/fisiología , Estrés Oxidativo , Envejecimiento/metabolismo , Animales , Femenino , Fertilidad/fisiología , Infertilidad Femenina/metabolismo , Longevidad/fisiología , Modelos Biológicos , Reproducción/fisiologíaRESUMEN
Little is known about physiological mechanisms that underlie the cost of reproduction. We tested the hypothesis that stress susceptibility is a cost of reproduction. In one test of our hypothesis, Drosophila melanogaster females were exposed to a juvenile hormone analog (methoprene) to stimulate egg production followed by stress assays. A sterile stock of D. melanogaster was employed as a control for reproduction. Exposure of fertile females to methoprene resulted in an increase in female reproduction and increased susceptibility to oxidative stress and starvation (compared to solvent controls). Sterile females did not exhibit a decrease in stress resistance. Mating also stimulated egg production. As a second test of our hypothesis, mated females were compared to virgin females. Mated fertile females were relatively susceptible to oxidative stress, but this relationship was not evident when mated and virgin sterile females were compared. The results of the present study support the hypothesis that stress susceptibility is a cost of reproduction.
Asunto(s)
Drosophila melanogaster/fisiología , Oocitos/fisiología , Estrés Oxidativo/fisiología , Animales , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Femenino , Radicales Libres/metabolismo , Herbicidas/farmacología , Masculino , Metopreno/farmacología , Mortalidad , Paraquat/farmacología , Reproducción , InaniciónRESUMEN
The Drosophila melanogaster gene chico encodes an insulin receptor substrate that functions in an insulin/insulin-like growth factor (IGF) signaling pathway. In the nematode Caenorhabditis elegans, insulin/IGF signaling regulates adult longevity. We found that mutation of chico extends fruit fly median life-span by up to 48% in homozygotes and 36% in heterozygotes. Extension of life-span was not a result of impaired oogenesis in chico females, nor was it consistently correlated with increased stress resistance. The dwarf phenotype of chico homozygotes was also unnecessary for extension of life-span. The role of insulin/IGF signaling in regulating animal aging is therefore evolutionarily conserved.
Asunto(s)
Envejecimiento/fisiología , Proteínas de Drosophila , Drosophila melanogaster/fisiología , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Longevidad/fisiología , Proteínas Tirosina Quinasas Receptoras , Receptor de Insulina/metabolismo , Alelos , Animales , Constitución Corporal , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cruzamientos Genéticos , Drosophila melanogaster/genética , Femenino , Fertilidad , Genes de Insecto , Heterocigoto , Calor , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina , Masculino , Mutación , Estrés Oxidativo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Reproducción , Transducción de Señal , Somatomedinas/metabolismo , Inanición , Superóxido DismutasaRESUMEN
Stress resistance is associated with longevity in Drosophila melanogaster and other model organisms used for genetic research. The present study tests for oxidative stress resistance in one set of lines selected for late-life reproduction and extended longevity. Both females and males from the selected lines were appreciably more resistant to oxidative stress than were flies from the control lines. A relative increase in oxidative stress resistance is a correlated response to selection in this laboratory selection experiment. Increased oxidative stress resistance appears to be a relatively robust correlated response to laboratory selection for late-life reproduction and extended longevity.
Asunto(s)
Longevidad , Estrés Oxidativo/fisiología , Selección Genética , Análisis de Varianza , Animales , Drosophila melanogaster , Femenino , Herbicidas/efectos adversos , Longevidad/efectos de los fármacos , Longevidad/genética , Masculino , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Paraquat/efectos adversos , Reproducción/genética , Tasa de Supervivencia , AguaRESUMEN
Vitellogenic and previtellogenic oocyte stages, as well as ovariole numbers, were characterized in the selected (O) lines of D. melanogaster that have been selected for late-life reproduction and extended longevity. On day 4 post-eclosion, O females had more ovarioles than control (B) females, but this difference diminished at older ages. From days 4 to 24, O females showed a marked increase in average vitellogenic oocyte stage, whereas this parameter did not increase in B females as they aged. The previtellogenic gradient of oocyte maturation declined in both B and O females. Specifically, terminal previtellogenic stages were lost with advancing age, and this loss occurred relatively gradually in O females regardless of food regime. Among the reproductive traits assessed, differential persistence of the previtellogenic gradient of maturation is most strongly associated with selected versus control line differences in longevity.
Asunto(s)
Drosophila melanogaster/fisiología , Longevidad/fisiología , Ovario/citología , Envejecimiento/fisiología , Animales , Dieta , Drosophila melanogaster/anatomía & histología , Femenino , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oogénesis , Ovario/fisiología , Reproducción/fisiología , VitelogénesisRESUMEN
Lines of Drosophila melanogaster selected for late-life female reproduction typically exhibit correlated responses of reduced early fecundity and increased longevity. This relationship suggests a tradeoff between reproductive effort and somatic maintenance, which in turn, underlies some evolutionary theories of senescence. The mechanistic basis of the apparent tradeoff between increased longevity and reduced early-age fecundity has remained obscure. The present manuscript addresses the issues of whether the reduced early-age fecundity in selected lines corresponds to reduced yolk-protein mRNA production, and whether long-lived flies exhibit somatic maintenance in terms of relatively reduced yolk-protein mRNA production in the fat body. Yolk protein is one of the most abundant proteins used for female reproduction. By comparing a set of lines selected for late life reproduction with the corresponding control lines, we show that that yolk-protein gene mRNA relative abundance during the first four days posteclosion did not correspond to reduced early-life fecundity in the selected lines. In D. melanogaster, yolk protein is produced in the fat body and ovarian follicle cells. On the fourth day posteclosion, relatively more yolk-protein gene mRNA was present in the fat body. On day 1 posteclosion, supplemental yeast did not alter relative yolk-protein gene mRNA abundance. However, on day 4 posteclosion, supplemental yeast stimulated yolk-protein gene mRNA production in the fat body, which suggests an underlying mechanism for the nutrition-based phenotypic plasticity of fecundity previously documented in these lines. On medium without supplemental yeast, the relatively low abundance of fat body yolk-protein gene mRNA in the selected lines on day 4 posteclosion corresponds to a prediction derived from the disposable soma theory.
Asunto(s)
Drosophila melanogaster/genética , Proteínas del Huevo/genética , Longevidad/genética , Envejecimiento/genética , Envejecimiento/fisiología , Animales , Drosophila melanogaster/fisiología , Cuerpo Adiposo/metabolismo , Femenino , Fertilidad/genética , Fertilidad/fisiología , Longevidad/fisiología , Ovario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Five independent populations (lines) of Drosophila melanogaster were selected for female starvation resistance. Females and males from the selected lines were relatively starvation resistant when compared to flies from five control lines. Moreover, flies from selected lines were resistant to other stresses: desiccation, acetone fumes, ethanol fumes, and paraquat (a source of oxygen radicals). Data from a variety of previous studies indicate an association between stress resistance and longevity. In this context, the present study addressed the question of whether flies from the stress-resistant lines were relatively long-lived. Replicate population cages from each selected and control line were used to assess longevity. Neither females nor males from the selected lines were relatively long-lived. In at least some cases, stress resistance may be necessary, but not sufficient, for longevity.
Asunto(s)
Drosophila melanogaster/fisiología , Longevidad/fisiología , Inanición/fisiopatología , Acetona , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Desecación , Etanol , Femenino , Herbicidas , Masculino , Estrés Oxidativo/fisiología , Paraquat , Solventes , Especificidad de la EspecieRESUMEN
There is a complete absence of information about the endocrinology of Drosophila melanogaster in relation to genetic-based differential longevity in this model species. In the present study, aspects of the endocrine system of D. melanogaster were investigated in selected and control lines characterized by relative differences in life span. By using extracts from whole bodies, steroid hormone (ecdysteroid) titers were determined by radioimmunoassay in all replicate selected and control lines on the first and fourth day of adult life. The results suggest that ecdysteroid titers were relatively reduced on the first day post-eclosion in females from the long-lived lines, but this difference was not present on the fourth day posteclosion. The reduction in early-age ecdysteroid titers in long-lived females might be related to the decrease in early-age fecundity in the selected lines. There was no difference between line types in male ecdysteroid titers on either day post-eclosion. Two classes of enzymes that act on juvenile hormone were also investigated in the present study. Esterase and epoxide hydrolase activity on juvenile hormone was assessed in females in all replicate selected and control lines at approximately 12 h or 4 days post-eclosion. There was no difference between selected and control lines in the specific activity of either class of enzymes that metabolize juvenile hormone.
Asunto(s)
Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Sistema Endocrino/fisiología , Longevidad/fisiología , Animales , Drosophila melanogaster/metabolismo , Ecdisteroides , Femenino , Hormonas Juveniles/metabolismo , Masculino , Especificidad de la Especie , Esteroides/metabolismoRESUMEN
A substantial number of Drosophila studies have investigated variation in desiccation and starvation resistance, providing an opportunity to test for consistent patterns of direct and correlated responses across studies and across the species and population levels. In general, responses to laboratory selection for these traits in D. melanogaster are rapid and indicate abundant genetic variation in populations. However, slower responses to selection for desiccation resistance occur in other species including D. simulans. Clines suggest adaptive divergence although specific selection pressures have not been documented empirically. Drosophila species differ markedly in desiccation and starvation resistance and there is also marked variation within species for desiccation resistance that may be linked to local climatic conditions. Laboratory selection experiments on starvation resistance in D. melanogaster suggest that changes in lipid content are largely responsible for resistance variation but this factor may be less important in explaining variation among species. For desiccation, lines with increased resistance show reduced rates of water loss but no changes in the minimum water content that flies can tolerate. Changes in life history traits are sometimes associated with altered levels of stress resistance. Increased starvation resistance is associated with longer development time and reduced early age reproduction in different studies. However, other associations are inconsistent between studies as in the case of stress resistance changing following selection for longevity. Multiple mechanisms may underlie genetic variation in stress resistance and future studies should address the evolutionary importance of the different mechanisms at the population and species levels.
Asunto(s)
Drosophila/genética , Variación Genética , Estadios del Ciclo de Vida , Animales , Deshidratación/genética , Deshidratación/metabolismo , Drosophila/metabolismo , Población , Inanición/genética , Inanición/metabolismoRESUMEN
Radioimmunoassay was used to determine ecdysteroid titers in mated or unmated Drosophila melanogaster females. Whole-body ecdysteroid titers increase after mating and this response is more pronounced after 12-24 hours than it is immediately after mating. In one experiment, females were mated to transgenic males deficient in accessory gland proteins to test whether these peptides mediate the observed increase in female whole-body ecdysteroid titers. Females mated to such transgenic males do not show a pronounced increase in whole-body ecdysteroid titers. The effect of mating on female hemolymph ecdysteroid titers was also investigated. Hemolymph ecdysteroid titers decrease after mating. The ecdysteroid titer change in the hemolymph may result from yolk protein uptake of ecdysteroids into developing vitellogenic oocytes as a consequence of male accessory gland protein stimulation of female oocyte maturation and yolk protein synthesis following mating.
RESUMEN
Studies on transcriptional control of gene expression play a pivotal role in many areas of biology. In non-Drosophilid insects, the cuticle, chorion, immune response, silk gland, storage proteins, and vitellogenin are foci for advances in basic research on promoter elements and transcription factors. Insects offer other advantages for gene regulation studies, including the availability of applied problems. In non-Drosophilid insects, the most serious problem for transcriptional control studies is the lack of homologous in vivo expression systems. Once this deficiency is addressed, the full impact of research on transcription control will be realized throughout the field of entomology.
Asunto(s)
Regulación de la Expresión Génica , Insectos/genética , Transcripción Genética , Animales , Hormonas de Insectos/fisiologíaRESUMEN
Genes that influence mating and/or fertilization success may be targets for strong natural selection. If females remate frequently relative to the duration of sperm storage and rate of sperm use, sperm displacement may be an important component of male reproductive success. Although it has long been known that mutant laboratory stocks of Drosophila differ in sperm displacement, the magnitude of the naturally occurring genetic variation in this character has not been systematically quantified. Here we report the results of a screen for variation in sperm displacement among 152 lines of Drosophilia melanogaster that were made homozygous for second and/or third chromosomes recovered from natural populations. Sperm displacement was assayed by scoring the progeny of cn;bw females that had been mated sequentially to cn;bw and tested males in either order. Highly significant differences were seen in both the ability to displace sperm that is resident in the female's reproductive tract and in the ability to resist displacement by subsequent sperm. Most lines exhibited nearly complete displacement, having nearly all progeny sired by the second male, but several lines had as few as half the progeny fathered by the second male. Lines that were identified in the screen for naturally occurring variation in sperm displacement were also characterized for single-strand conformation polymorphisms (SSCP) at seven accessory gland protein (Acp) genes, Glucose dehydrogenase (Gld), and Esterase-6 (Est-6). Acp genes encode proteins that are in some cases known to be transmitted to the female in the seminal fluid and are likely candidates for genes that might mediate the phenomenon of sperm displacement. Significant associations were found between particular Acp alleles at four different loci (Acp26Aa/Ab, Acp29B, Acp36DE and Acp53E) and the ability of males to resist displacement by subsequent sperm. There was no correlation between the ability to displace resident sperm and the ability to resist being displaced by subsequent sperm. This lack of correlation, and the association of Acp alleles with resisting subsequent sperm only, suggests that different mechanisms mediate the two components of sperm displacement.
Asunto(s)
Drosophila melanogaster/fisiología , Genes de Insecto/genética , Proteínas/genética , Espermatozoides/fisiología , Alelos , Análisis de Varianza , Animales , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Femenino , Fertilidad/genética , Fertilidad/fisiología , Variación Genética , Genética de Población , Haplotipos , Humanos , Desequilibrio de Ligamiento , MasculinoRESUMEN
The gene for juvenile hormone esterase (JHE) was cloned from Heliothis virescens (Lepidoptera: Noctuidae). A genomic library was constructed from embryonic DNA and screened with a homologous N-terminal probe from the JHE cDNA. Five genomic clones were isolated and analyzed by dot blot hybridization using regions of the JHE cDNA as probes. Clone C hybridized to both 5' and 3' probes from the JHE cDNA, suggesting that clone C contains both ends of JHE gene. This was verified by sequencing the ends of the JHE gene from clone C using primers from both the 5' and 3' ends of the JHE cDNA. Additional sequencing and restriction mapping were used to characterize the gene. The gene is c. 8 kb long and contains four introns with consensus intron-exon junctions. One of the introns is relatively large (4 kb) and is situated near the extreme 5' end of the gene. Genetic analysis of RFLP variation in interspecific and intraspecific crosses shows that the JHE locus is single-copy with no closely related paralogs and is autosomally encoded in Heliothis. Therefore the developmental pattern of expression of this gene and the previously documented sequence variation in cDNA clones is not explainable by reference to a JHE gene family with distinct structural loci for the different forms.