Skip to main content
SpringerLink
Log in

Genetic analysis of factors controlling high-level expression of cytochrome P450, CYP6D1, cytochrome b5, P450 reductase, and monooxygenase activities in LPR house flies,Musca domestica

  • Published:
Biochemical Genetics Aims and scope Submit manuscript

Abstract

To understand better the biochemical genetics of cytochrome P450 monooxygenase-mediated insecticide resistance, we examined the microsomal monooxygenases in insecticide-susceptible (aabys) and pyrethroid-resistant (LPR) house fly strains, as well as 15 house fly lines derived from crosses of LPR and aabys. In comparison to the aabys strain, LPR had higher levels of total cytochromes P450, cytochrome b5, P450 reductase, CYP6D1, and three P450 monooxygenase activities: 7-ethoxycoumarinO-deethylase (ECOD), methoxyresorufinO-demethylase (MROD), and aromatic hydrocarbon hydroxylase (AHH). The elevated levels of cytochrome b5 were linked to factors on autosomes 1 and 2. This is similar to previous reports on monooxygenase-mediated resistance and is consistent with the idea that elevated cytochrome b5 levels are involved in monooxygenase-mediated resistance in the LPR strain. Linkage of the elevated P450 reductase is different from that of monooxygenase-mediated resistance. Strains having high levels of CYP6D1 (i.e., like LPR) had high levels of P450 reductase, while strains having intermediate levels of CYP6D1 also had high levels of reductase. Therefore, there is no clear evidence that the elevated P450 reductase in the LPR strain is required for the increased monooxygenase activity. Overexpression of total cytochromes P450, CYP6D1 (mRNA and protein), and CYP6D1-mediated monooxygenase activities (MROD and AHH) in LPR microsomes was linked to a combination of factors on autosomes 1 and 2. This demonstrates that increased expression of CYP6D1 in the LPR strain is both cis regulated by a factor(s) on autosome 1 and trans regulated by a factor(s) on autosome 2. The correlation between the overexpression of CYP6D1 mRNA and protein suggests that CYP6D1 expression is regulated transcriptionally. Monooxygenase-mediated resistance in LPR is controlled by factors on autosomes 1 and 2, which supports previous claims that CYP6D1 is responsible for monooxygenase-mediated resistance in the LPR strain.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72248.

    Article  CAS  PubMed  Google Scholar 

  • Burke, M. D., and Mayer, R. T. (1974). Ethoxyresorufin: Direct fluorimetric assay of a microsomalO-dealkylation which is preferentially inducible by 3-methylcholanthrene.Drug Metab. Dispos. 2583.

    CAS  PubMed  Google Scholar 

  • Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 162169.

    Article  Google Scholar 

  • Denison, M. S., Murray, M., and Wilkinson, C. F. (1983). Microsomal aryl hydrocarbon hydroxylase: comparison of the direct, indirect and radiometric assays.Anal. Lett. 1631.

    Google Scholar 

  • DeVries, D. H. and Georghiou, G. P. (1981). Absence of enhanced detoxication of permethrin in pyrethroid-resistant house flies.Pest. Biochem. Physiol. 15242.

    CAS  Google Scholar 

  • Estabrook, R. W., and Werringloer, J. (1978). The measurement of difference spectra: Application to the cytochromes of microsomes. In Fleischer, S., and Packer L. (eds.),Methods in Enzymology, Vol. 52 Academic Press, New York, pp. 212–220.

    Google Scholar 

  • Feyereisen, R., and Vincent, D. R. (1984). Characterization of antibodies to house fly NADPH-cytochromeP-450 reductase.Insect Biochem. 14163.

    CAS  Google Scholar 

  • Golly, I., Hlavica, P., and Schartau, W. (1988). The functional role of cytochrome b5 reincorporated into hepatic microsomal fractions.Arch. Biochem. Biophys. 260232.

    Article  CAS  PubMed  Google Scholar 

  • Lee, S. S. T., and Scott, J. G. (1989a). An improved method for preparation, stabilization, and storage of house fly (Diptera: Muscidae) microsomes.J. Econ. Entomol. 821559.

    CAS  PubMed  Google Scholar 

  • Lee, S. S. T., and Scott, J. G. (1989b). Microsomal cytochrome P450 monooxygenase in the house fly (Musca domestica L.): Biochemical changes associated with pyrethroid resistance and phenobarbital induction.Pest. Biochem. Physiol. 351.

    Article  CAS  Google Scholar 

  • Liu, N., and Scott, J. G. (1995). Genetics of resistance to pyrethroid insecticides in the house fly,Musca domestica.Pest. Biochem. Physiol. 52116.

    Google Scholar 

  • Liu, N., Tomita, T., and Scott, J. G. (1995). Allele-specific PCR reveals thatCYP6D1 is on chromosome 1 in the house fly,Musca domestica.Experientia 51164.

    Article  CAS  PubMed  Google Scholar 

  • Lubet, R. A., Mayer, R. T., Cameron, J. W., Nims, R. W., Burke, M. D., Wolff, T., and Guengerich, F. P. (1985). Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat.Arch. Biochem. Biophys. 23843.

    Article  CAS  PubMed  Google Scholar 

  • Mayer, R. T., Jermyn, J. W., Burke, M. D., and Prough, R. A. (1977). Methoxyresorufin as a substrate for the fluorometric assay of insect microsomalO-dealkylase.Pest. Biochem. Physiol. 7349.

    Article  CAS  Google Scholar 

  • Omura, T., and Sato, R. (1964). The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature.J. Biol. Chem. 2392370.

    CAS  PubMed  Google Scholar 

  • Oppenoorth, F. J. (1985). Biochemistry and genetics of insecticide resistance. In Kerkut, G. A., and Gilbert, L. I. (eds.),Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 12 Pergamon Press, New York, pp. 731–774.

    Google Scholar 

  • Peterson, J. A., and Prough, R. A. (1986). Cytochrome P-450 reductase and cytochrome b5 in cytochrome P-450 catalysis. In Ortiz de Montellano, P. R. (ed.),Cytochrome P-450 Structure, Mechanism, and Biochemistry Plenum, New York, pp. 89–117.

    Google Scholar 

  • Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). InMolecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  • Schenkman, J. B. (1993). Protein-protein interactions. In Schenkman, J. B., and Greim, H. (eds.),Cytochrome P450 Springer-Verlag, New York, pp. 527–546.

    Google Scholar 

  • Schonbrod, R. D., and Terriere, L. C. (1972). Inhibition of housefly microsomal epoxidase by the eye pigment, xanthommatin.Pest. Biochem. Physiol. 1409.

    Google Scholar 

  • Scott, J. G. (1991). Insecticide resistance in insects. In Pimentel, D. (ed.),Handbook of Pest Management, Vol. 2 CRC Press, Boca Raton, FL, pp. 663–677.

    Google Scholar 

  • Scott, J. G., and Georghiou, G. P. (1985). Rapid development of high-level permethrin resistance in a field-collected strain of the house fly (Diptera: Muscidae) under laboratory selection.J. Econ. Entomol. 78316.

    CAS  PubMed  Google Scholar 

  • Scott, J. G., and Georghiou, G. P. (1986a). Mechanisms responsible for high levels of permethrin resistance in the house fly.Pest. Sci. 17195.

    CAS  Google Scholar 

  • Scott, J. G., and Georghiou, G. P. (1986b). The biochemical genetics of permethrin resistance in the Learn-PyR strain of house fly.Biochem. Genet. 2425.

    Article  CAS  PubMed  Google Scholar 

  • Scott, J. G., and Lee, S. S. T. (1993). Purification and characterization of a cytochrome P-450 from insecticide susceptible and resistant strains of house fly,Musca domestica L., before and after phenobarbital exposure.Arch. Insect Biochem. Physiol. 241.

    Article  CAS  PubMed  Google Scholar 

  • Scott, J. G., Lee, S. S. T., and Shono, T. (1990). Biochemical changes in the cytochrome P450 monooxygenases of seven insecticide-resistant house fly (Musca domestica L.) strains.Pest. Biochem. Physiol. 36127.

    Article  CAS  Google Scholar 

  • Steel, R. G. D., and Torrie, J. H. (1980).Principles and Procedures of Statistics 2nd ed., McGraw-Hill, New York.

    Google Scholar 

  • Tomita, T., and Scott, J. G. (1995). cDNA and deduced protein sequence of CYP6D1: The putative gene for a cytochrome P450 responsible for pyrethroid resistance in house fly.Insect Biochem.Mol. Biol. 25275.

    CAS  Google Scholar 

  • Tomita, T., Liu, N., Smith, F. F., Sridhar, P., and Scott, J. G. (1995). Molecular mechanisms involved in increased expression of a cytochrome P450 responsible for pyrethroid resistance in the housefly,Musca domestica.Insect Mol. Biol. 4135.

    CAS  PubMed  Google Scholar 

  • Tsukamoto, M. (1983). Methods of genetic analysis of insecticide resistance. In Georghiou, G. P., and Saito, T. (eds.),Pest Resistance to Pesticides Plenum Press, New York and London, pp. 71–98.

    Google Scholar 

  • Ullrich, V., and Weber, P. (1972). TheO-dealkylation of 7-ethoxycoumarin by liver microsomes: A direct fluorometric test.Hoppe-Seyler Z. Physiol. Chem. 3531171.

    CAS  PubMed  Google Scholar 

  • Wheelock, G. D., and Scott, J. G. (1989). Simultaneous purification of a cytochrome P-450 and cytochrome b5 from the house fly,Musca domestica L.Insect Biochem. 19481.

    CAS  Google Scholar 

  • Wheelock, G. D., and Scott, J. G. (1990). Immunological detection of cytochrome P450 from insecticide resistant and susceptible house flies.Pest. Biochem. Physiol. 3130.

    Google Scholar 

  • Wheelock, G. D., and Scott, J. G. (1992a). Anti-P450lpr antiserum inhibits specific monooxygenase activities in LPR house fly microsomes.J. Exp. Zool. 264153.

    Article  CAS  PubMed  Google Scholar 

  • Wheelock, G. D., and Scott, J. G. (1992b). The role of cytochrome P450lpr in deltamethrin metabolism by pyrethroid-resistant and susceptible strains of house flies.Pest. Biochem. Physiol. 4367.

    Article  CAS  Google Scholar 

  • Wheelock, G. D., and Scott, J. G. (1994). Detection of cytochrome b5 from the house-fly,Musca domestica: Comparison of immunological and spectrophotometric methods.Comp. Biochem. Physiol. 108B209.

    CAS  Google Scholar 

  • Wilkinson, C. F. (1983). Role of mixed-function oxidases in insecticide resistance. In Georghiou, G. P., and Saito, T. (eds.),Pest Resistance to Pesticides Plenum Press, New York and London, pp. 175–206.

    Google Scholar 

  • Williams, C. H., and Kamin, H. (1962). Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver.J. Biol. Chem. 237587.

    CAS  PubMed  Google Scholar 

  • Zhang, M., and Scott, J. G. (1994). Cytochrome b5 involvement in cytochrome P450 monooxygenase activities in house fly microsomes.Arch. Insect Biochem. Physiol. 27205.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Entomology, Comstock Hall, Cornell University, 14853-0901, Ithaca, New York

    Nannan Liu & Jeffrey G. Scott

Authors
  1. Nannan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeffrey G. Scott
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, N., Scott, J.G. Genetic analysis of factors controlling high-level expression of cytochrome P450, CYP6D1, cytochrome b5, P450 reductase, and monooxygenase activities in LPR house flies,Musca domestica . Biochem Genet 34, 133–148 (1996). https://doi.org/10.1007/BF02396246

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02396246

Key words

Search

Navigation