Progress 01/01/05 to 12/31/05
Outputs
The hemolymph juvenile hormone binding protein (hJHBP) gene of Manduca sexta is a key target of its specific ligand, juvenile hormone (JH). While the cDNA for hJHBP has been partially characterized, little is known about the hJHBP gene structure or its promoter(s) and enhancers(s). Previous studies have demonstrated that JH stimulates a rapid accumulation of hJHBP mRNA in the fat body. To better understand the underlying molecular events affecting regulation, we sequenced the M. sexta hJHBP gene and its mRNA transcript, characterized its genomic organization, and determined the spatial and temporal expression patterns of the hJHBP gene. The gene is composed of 5 exons spanning 6.7 kb. Southern blot analysis indicates that the gene is present as a single copy. The earliest expression of hJHBP occurs 24 to 48 hours after fertilization. Distribution studies indicate that fat body is the only site for hJHBP expression. Elements displaying similarity with sequences of
other lepidopteran genes were discovered outside the open reading frame and may represent mobile insertion elements. Peripheral distribution of the insect juvenile hormones (JHs) requires hemolymph transport proteins. A comparison of three strains of the tobacco hornworm, Manduca sexta, indicates that hemolymph JH binding protein (hJHBP) levels in the Madison wild-type (Mwt) strain are significantly higher than in the black larval mutant (bl) and Seattle wild-type (Swt) strains. Interestingly, the structure of the hJHBP locus is slightly modified in different strains (GenBank Accession Number AF527636, AF527635; Young et al., 2003). To correlate differences in hJHBP levels between strain phenotypes with the hJHBP locus, we sequenced 8.4 kb of the hJHBP gene locus from each strain. Snb, an allele found in the Swt and bl strains, contains a 408 bp repetitive nuclear element flanked by 15 bp direct repeats. Mating studies coupled with molecular genotyping demonstrate that the presence of
Snb correlates with a 2-fold lower hJHBP level relative to the allele found in the Mwt strain. Despite the lower hJHBP levels in individuals carrying the Snb gene, hemolymph levels of JH do not appear to be significantly affected. As we have successfully completed the objectives of the current Hatch proposal, we have turned our attention during the last year to a parallel problem that involves modification of hJHBP levels. Parasitism of M. sexta by the wasp Cotesia congragata leads to a dramatic decline in hJHBP titers, so much so that hJHBP is non-existent in the hemolymph after parasitoid attack. Yet, the JH titers are very high. This has led us to look at the role of stress in controlling the levels of hJHBP and has open the possibility that hJHBP levels are controlled by a number of factors. These factors in turn, reduce the binding protein levels in the hemolymph to allow more unbound JH to reach the cellular target.
Impacts
From a fundamental standpoint, this provides the tools with which to better understand how this important insect hormone regulates development and potentially reproduction. From a practical standpoint, the JH analogs are used in pest insect control programs where the more toxic pesticides cannot be employed. Understanding how they act at the molecular level may offer insight into the development of new, safer insect control strategies. Because they are used in medically sensitive areas, it become imperative to understand how these hormones act on the nontarget organisms, particularly humans.
Publications
- Orth, A.P., Doll, S., Goodman, W.G. (2003) Structure and expression of the hemolymph juvenile hormone binding protein from Manduca sexta. Insect Biochemistry and Molecular Biology. 33:93-103.
- Mohamed, M.A., Hogg, D.B., Park, Y.C., Goodman, W.G. (2003) Dot-blot immunoassay detection of Microtonus aethiopoides (Braconidae, Euphorinae) an adult endoparasitoid in Alfalfa Weevil Hypera postica. Biocontrol Sci. Technol. 13:631-640.
- Orth, A.P., Tauchman, S.J., Doll, S.C., Goodman, W.G. (2003) Embryonic expression of juvenile hormone binding protein and its relationship to the toxic effects of juvenile hormone in Manduca sexta. Insect Biochemistry and Molecular Biology. 33:1275-1284
- Young, J.K., Orth, A.P., and Goodman, W.G. (2003) Allelic variation in the hemolymph juvenile hormone binding protein gene of Manduca sexta. Molecular and Cellular Endocrinology. 208:41-50.
- Goodman, W.G. and Granger, N.A. (2004) The Juvenile Hormones. In Comprehensive Molecular Insect Science (ed. by L.I. Gilbert, K. Iatrou, S.Gill). Vol. 3 311-408. Elsevier/Pergamon Press, Oxford.
|
Progress 10/01/01 to 09/30/05
Outputs
The hemolymph juvenile hormone binding protein (hJHBP) gene of Manduca sexta is a key target of its specific ligand, juvenile hormone (JH). While the cDNA for hJHBP has been partially characterized, little is known about the hJHBP gene structure or its promoter(s) and enhancers(s). Previous studies have demonstrated that JH stimulates a rapid accumulation of hJHBP mRNA in the fat body. To better understand the underlying molecular events affecting regulation, we sequenced the M. sexta hJHBP gene and its mRNA transcript, characterized its genomic organization, and determined the spatial and temporal expression patterns of the hJHBP gene. The gene is composed of 5 exons spanning 6.7 kb. Southern blot analysis indicates that the gene is present as a single copy. The earliest expression of hJHBP occurs 24 to 48 hours after fertilization. Distribution studies indicate that fat body is the only site for hJHBP expression. Elements displaying similarity with sequences of other
lepidopteran genes were discovered outside the open reading frame and may represent mobile insertion elements. Peripheral distribution of the insect juvenile hormones (JHs) requires hemolymph transport proteins. A comparison of three strains of the tobacco hornworm, Manduca sexta, indicates that hemolymph JH binding protein (hJHBP) levels in the Madison wild-type (Mwt) strain are significantly higher than in the black larval mutant (bl) and Seattle wild-type (Swt) strains. Interestingly, the structure of the hJHBP locus is slightly modified in different strains (GenBank Accession Number AF527636, AF527635; Young et al., 2003). To correlate differences in hJHBP levels between strain phenotypes with the hJHBP locus, we sequenced 8.4 kb of the hJHBP gene locus from each strain. Snb, an allele found in the Swt and bl strains, contains a 408 bp repetitive nuclear element flanked by 15 bp direct repeats. Mating studies coupled with molecular genotyping demonstrate that the presence of Snb
correlates with a 2-fold lower hJHBP level relative to the allele found in the Mwt strain. Despite the lower hJHBP levels in individuals carrying the Snb gene, hemolymph levels of JH do not appear to be significantly affected. As we have successfully completed the objectives of the current Hatch proposal, we have turned our attention during the last year to a parallel problem that involves modification of hJHBP levels. Parasitism of M. sexta by the wasp Cotesia congragata leads to a dramatic decline in hJHBP titers, so much so that hJHBP is non-existent in the hemolymph after parasitoid attack. Yet, the JH titers are very high. This has led us to look at the role of stress in controlling the levels of hJHBP and has open the possibility that hJHBP levels are controlled by a number of factors. These factors in turn, reduce the binding protein levels in the hemolymph to allow more unbound JH to reach the cellular target.
Impacts
From a fundamental standpoint, this provides the tools with which to better understand how this important insect hormone regulates development and potentially reproduction. From a practical standpoint, the JH analogs are used in pest insect control programs where the more toxic pesticides cannot be employed. Understanding how they act at the molecular level may offer insight into the development of new, safer insect control strategies. Because they are used in medically sensitive areas, it become imperative to understand how these hormones act on the nontarget organisms, particularly humans.
Publications
- Orth, A.P., Doll, S., Goodman, W.G. (2003) Structure and expression of the hemolymph juvenile hormone binding protein from Manduca sexta. Insect Biochemistry and Molecular Biology. 33:93-103.
- Mohamed, M.A., Hogg, D.B., Park, Y.C., Goodman, W.G. (2003) Dot-blot immunoassay detection of Microtonus aethiopoides (Braconidae, Euphorinae) an adult endoparasitoid in Alfalfa Weevil Hypera postica. Biocontrol Sci. Technol. 13:631-640.
- Orth, A.P., Tauchman, S.J., Doll, S.C., Goodman, W.G. (2003) Embryonic expression of juvenile hormone binding protein and its relationship to the toxic effects of juvenile hormone in Manduca sexta. Insect Biochemistry and Molecular Biology. 33:1275-1284
- Young, J.K., Orth, A.P., and Goodman, W.G. (2003) Allelic variation in the hemolymph juvenile hormone binding protein gene of Manduca sexta. Molecular and Cellular Endocrinology. 208:41-50.
- Goodman, W.G. and Granger, N.A. (2004) The Juvenile Hormones. In Comprehensive Molecular Insect Science (ed. by L.I. Gilbert, K. Iatrou, S.Gill). Vol. 3 311-408. Elsevier/Pergamon Press, Oxford.
|
Progress 01/01/04 to 12/31/04
Outputs
The hemolymph juvenile hormone binding protein (hJHBP) gene of Manduca sexta is a key target of its specific ligand, juvenile hormone (JH). Previous studies have demonstrated that JH stimulates a rapid accumulation of hJHBP mRNA in the fat body. The gene is composed of 5 exons spanning 6.7 kb. Southern blot analysis indicates that the gene is present as a single copy. Distribution studies indicate that fat body is the only site for hJHBP expression. Elements displaying similarity with sequences of other lepidopteran genes were discovered outside the open reading frame and may represent mobile insertion elements. We demonstrate that the preponderance of egg JHBP is maternally sequestered. However, 18 hours after fertilization, expression of the JHBP gene begins in both embryonic and extra-embryonic tissues, a time long before emergence of a functional circulatory system. Since JH regulates hJHBP expression during the larval stages, it may have a similar effect during
embryogenesis. Topical application of low JH doses to early embryos resulted in larval abnormalities while high-doses of the hormone lead to embryonic toxicity. Interestingly, toxicity rates are tightly correlated with the concentration of unbound hormone. This aspect plays into our current work (see below). Peripheral distribution of the insect juvenile hormones (JHs) requires hemolymph transport proteins. A comparison of three strains of the tobacco hornworm, Manduca sexta, indicates that hemolymph JH binding protein (hJHBP) levels in the Madison wild-type (Mwt) strain are significantly higher than in the black larval mutant (bl) and Seattle wild-type (Swt) strains. To correlate differences in hJHBP levels between strain phenotypes with the hJHBP locus, we sequenced 8.4 kb of the hJHBP gene locus from each strain. Snb, an allele found in the Swt and bl strains, contains a 408 bp repetitive nuclear element flanked by 15 bp direct repeats. Mating studies coupled with molecular
genotyping demonstrate that the presence of Snb correlates with a 2-fold lower hJHBP level relative to the allele found in the Mwt strain. Despite the lower hJHBP levels in individuals carrying the Snb gene, hemolymph levels of JH do not appear to be significantly affected. As we have successfully completed the objectives of the current Hatch proposal, we have turned our attention during the last year to a parallel problem that involves modification of hJHBP levels. Parasitism of M. sexta by the wasp Cotesia congragata leads to a dramatic decline in hJHBP titers, so much so that hJHBP is non-existent in the hemolymph after parasitoid attack. Yet, the JH titers are very high. This has led us to look at the role of stress in controlling the levels of hJHBP and has opened the possibility that hJHBP levels are controlled by a number of factors. These factors in turn, reduce the binding protein levels in the hemolymph to allow more unbound JH to reach the cellular target. This work is
still in progress.
Impacts
The present study indicates that genetic regulation of the expression of hJHBP can occur at several levels. Insertion of SINES of a particular sequence can have a significant impact on the levels of hJHBP.
Publications
- Goodman, W.G. and Granger, N.A. The Juvenile Hormones (2004) In Comprehensive Molecular Insect Science (ed. by L.I. Gilbert, K. Iatrou, S. Gill). Vol 3 pp. 319-408. Elsevier, Amsterdam.
|
Progress 01/01/03 to 12/31/03
Outputs
The hemolymph juvenile hormone binding protein (hJHBP) gene of Manduca sexta is a key target of its specific ligand, juvenile hormone (JH). While the cDNA for hJHBP has been partially characterized, little is known about the hJHBP gene structure or its promoter(s) and enhancers(s). Previous studies have demonstrated that JH stimulates a rapid accumulation of hJHBP mRNA in the fat body. To better understand the underlying molecular events affecting regulation, we sequenced the M. sexta hJHBP gene and its mRNA transcript, characterized its genomic organization, and determined the spatial and temporal expression patterns of the hJHBP gene. The gene is composed of 5 exons spanning 6.7 kb. Southern blot analysis indicates that the gene is present as a single copy. The earliest expression of hJHBP occurs 24 to 48 hours after fertilization. Distribution studies indicate that fat body is the only site for hJHBP expression. Elements displaying similarity with sequences of
other lepidopteran genes were discovered outside the open reading frame and may represent mobile insertion elements. A comparison of three strains of the tobacco hornworm, Manduca sexta, indicates that hJHBP levels in the Madison wild-type (Mwt) strain are significantly higher than in the black larval mutant (bl) and Seattle wild-type (Swt) strains. To correlate differences in hJHBP levels between strain phenotypes with the hJHBP locus, we sequenced 8.4 kb of the hJHBP gene locus from each strain. Snb, an allele found in the Swt and bl strains, contains a 408 bp repetitive nuclear element flanked by 15 bp direct repeats. Mating studies coupled with molecular genotyping demonstrate that the presence of Snb correlates with a 2-fold lower hJHBP level relative to the allele found in the Mwt strain. Despite the lower hJHBP levels in individuals carrying the Snb gene, hemolymph levels of JH do not appear to be significantly affected. In the larva of the tobacco hornworm, Manduca sexta, low
doses of JH can immediately increase JHBP gene expression. Less explored are the effects of JH on embryological development, where early hormonal treatment has been shown to either inhibit normal embryonic development or prevent pupation. This study examines JHBP protein and gene expression throughout embryonic development of M. sexta and the phenotypic effect JH treatment has on embryos and on JHBP expression. We here demonstrate that the preponderance of egg JHBP is maternally sequestered. However, 18 hours after fertilization, expression of the JHBP gene begins in both embryonic and extra-embryonic tissues, a time long before emergence of a functional circulatory system. Since JH regulates hJHBP expression during the larval stages, it may have a similar effect during embryogenesis. Topical application of JH in low doses resulted in latent larval effects while high-doses of the hormone lead to embryonic toxicity. These morphological effects are not mediated through regulation of the
JHBP gene, since embryonic expression appears invariant in response to JH challenge. Interestingly, toxicity rates are tightly correlated with the concentration of unbound hormone.
Impacts
The present study indicates that genetic regulation of the expression of hJHBP can occur at several levels. Insertion of SINES of a particular sequence can have a significant impact on the levels of hJHBP.
Publications
- Orth, A.P., Doll, S., Goodman, W.G. (2003) Structure and expression of the hemolymph juvenile hormone binding protein from Manduca sexta. Insect Biochemistry and Molecular Biology. 33:93-103.
- Mohamed, M.A., Hogg, D.B., Park, Y.C., Goodman, W.G. (2003) Dot-blot immunoassay detection of Microtonus aethiopoides (Braconidae, Euphorinae) an adult endoparasitoid in Alfalfa Weevil Hypera postica. Biocontrol Science and Technology.
- Orth, A.P., Tauchman, S.J., Doll, S.C., Goodman, W.G. (2003) Embryonic expression of juvenile hormone binding protein and its relationship to the toxic effects of juvenile hormone in Manduca sexta. Insect Biochemistry and Molecular Biology. 33:1275-1284
- Young, J.K., Orth, A.P., and Goodman, W.G. (2003) Allelic variation in the hemolymph juvenile hormone binding protein gene of Manduca sexta. Molecular and Cellular Endocrinology. 208:41-50.
|
Progress 01/01/02 to 12/31/02
Outputs
The juvenile hormones (JHs) are a class of acyclic sesquiterpenoids central to insect development and reproduction. Peripheral distribution of these lipophilic hormones requires hemolymph transport proteins. While the cDNA for hJHBP has been partially characterized, little is known about the hJHBP gene structure or its promoter(s) and enhancers(s). Previous studies have demonstrated that JH stimulates a rapid accumulation of hJHBP mRNA in the fat body. To better understand the underlying molecular events affecting regulation, we sequenced the M. sexta hJHBP gene and its mRNA transcript, characterized its genomic organization, and determined the spatial and temporal expression patterns of the hJHBP gene. The gene is composed of 5 exons spanning 6.7 kb. Southern blot analysis indicates that the gene is present as a single copy. The earliest expression of hJHBP occurs 24 to 48 hours after fertilization. Distribution studies indicate that fat body is the only site for
hJHBP expression. Elements displaying similarity with sequences of other lepidopteran genes were discovered outside the open reading frame and may represent mobile insertion elements. The juvenile hormones (JHs) are a class of acyclic sesquiterpenoids central to insect development and reproduction. Peripheral distribution of these lipophilic hormones requires hemolymph transport proteins. A comparison of hemolymph JH binding protein (hJHBP) levels in three strains of the tobacco hornworm, Manduca sexta, indicated that the Madison wild-type (Mwt) strain is significantly higher than the black larval mutant (bl) and Seattle wild-type (Swt) strains. To better understand the phenotypic differences among the strains, we sequenced 8.4 kb of the hJHBP gene locus from each strain. One hJHBP allele, Snb, found predominantly in Swt and bl strains, contained the insertion of a 407 bp repetitive nuclear element flanked by 15 bp direct repeats. With the exception of an inversion of the poly A/T
rich region from the 3' end to the 5', the element exhibits all the characteristics of a short integrated nuclear element. Snb also contains multiple deletions and sequence changes including a 217 bp deletion in intron 2 and several amino acid substitutions. Mating studies coupled with molecular genotyping techniques demonstrated that Snb correlates with decreased hJHBP production relative to the Mwt hJHBP allele, Md. These studies conclude that hJHBP gene is inherited in a non-linked Mendelian fashion. Despite the 2-fold lower hJHBP levels in individuals carrying the Snb gene, hemolymph levels of JH did not appear to be significantly affected.
Impacts
This is the first time that strain differences in the hJHBP gene have been reported and characterized. From a fundamental standpoint, this provides the tools with which to better understand how this important insect hormone regulates development and potentially reproduction. From a practical standpoint, the JH analogs are used in pest insect control programs where the more toxic pesticides cannot be employed. Understanding how they act at the molecular level may offer insight into the development of new, safer insect control strategies. Because they are used in medically sensitive areas, it becomes imperative to understand how these hormones act on the nontarget organisms, particularly humans.
Publications
- Anthony P. Orth, Sharon C. Doll and Walter G. Goodman (2003) Sequence, Structure and Expression of the Hemolymph Juvenile Hormone Binding Protein Gene in the Tobacco Hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology. 33:93-102.
|
Progress 01/01/01 to 12/31/01
Outputs
Maintenance and dispersal of Juvenile Hormone (JH) requires coordination of three activities: JH synthesis, transport and degradation. This proposal focused on one aspect of this important triad, that of transport by a specific hemolymph protein, hemolymph juvenile hormone binding protein (hJHBP). During a previous granting period, we discovered that larvae of the black strain of the tobacco hornworm, Manduca sexta, treated with small doses of JH I can regulate the titers of hJHBP. We now know that the hJHBP open reading frame (0.8 kb) is composed of 5 exons that are scattered over about 6 kb of genomic DNA. Several short interspersed nuclear elements (SINES), a type of mobile genetic element having no known function were found. However, in the black larval mutant, a region of intron 2 has a 470 nt insert followed by a 230 nt deletion near the beginning of exon 3. The black larval mutant appears to be heterozygous for this site with one normal allele and one mutated
allele. In examining the parental strain from which this mutant arose (Seattle wild-type), it was determined that the parents are homozygous for the insert/deletion. Since the black larval mutants have lower levels of hJHBP than the Madison wild-types, we hypothesized that the insert/deletion may be responsible for this lowered titer. The parental Seattle wild-type strain was tested for hJHBP levels and found to be significantly lower than either the black strain or the Madison wild-type strain. JH titers were tested in all three strains and found to be statistically similar. This region of the intron may contain potential promoters or enhancers that are necessary for JH action. Genetic crosses between Seattle wild-types and Madison wild-types are now being performed.
Impacts
This is the first time that a mutant of the hJHBP gene has been reported and characterized. From a fundamental standpoint, this provides the tools with which to better understand how this important insect hormone regulates development and potentially reproduction. From a practical standpoint, the JH analogs are used in pest insect control programs where the more toxic pesticides cannot be employed. Understanding how they act at the molecular level may offer insight into the development of new, safer insect control strategies. Because they are used in medically sensitive areas, it become imperative to understand how these hormones act on the nontarget organisms, particularly humans.
Publications
- No publications reported this period
|
|