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Cytochrome P450 Ipr is a developmentally regulated P450 responsible for monooxygenase-mediated pyrethroid resistance in the LPR strain of housefly. CYP6D1, the gene coding for P450 Ipr, has recently been sequenced. We investigated the molecular basis for CYP6D1-mediated pyrethroid resistance by comparison of mRNA levels, gene sequences, and gene copy number between LPR and pyrethroid susceptible strains of housefly. CYP6D1 mRNA levels were elevated in the LPR strain to a similar level as P450 Ipr protein, suggesting that over-expression of this important P450 in the resistant strain is probably due to increased transcription. Southern blots of susceptible and LPR strain DNA suggest that gene amplification is probably not involved in the increased expression of CYP6D1 protein. Five alleles of CYP6D1 were discovered and their possible role in resistance is discussed.

CYP6D1 is a cytochrome P450 found in the house fly, Musca domestica. Expression is greater in pyrethroid-resistant vs. -susceptible strains and can be induced by phenobarbital in adult susceptible flies. CYP6D1 is expressed only in adult flies. To gain information about possible regulatory elements involved in CYP6D1 expression, and to confirm the gene sequence that was previously determined by polymerase chain reaction (PCR), we screened a house-fly library prepared with genomic DNA from the pyrethroid-resistant LPR strain. A CYP6D1v1 clone was isolated and sequenced. This clone contained 887 nucleotides 5′ to the open reading frame and a previously unknown 2.4-kb intron. Using polymerase chain reaction with primers based on the CYP6D1v1 allele, the sequences 5′ to the ORF were obtained from five pyrethroid susceptible strains. The transcription initiation site (TIS) was identified at the same position in LPR and two susceptible strains (86 nucleotides upstream from the translation start site). A comparison of the 5′ flanking sequences revealed a high degree of similarity for most regions, although differences in the sequences were identified. The possible roles of these sequence differences in regulation of CYP6D1 expression are discussed.

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfidelinked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis. © 1992 Wiley-Liss, Inc.

We have used two radiolabeled oligonucleotide probes (TH2B and Hlt), Northern blotting, two-dimensional gel electrophoresis, and autoradiography to study the temporal expres- sion of TH2B and Hlt testis-specific histone genes during the development of rat spermatogenic cells in vitro. These studies were carried out to deter- mine whether meiotic prophase spermatocytes, known to synthesize in vivo THBB and Hlt his- tones among other histones, are capable of ex- pressing these testis-specific genes in vitro during an extended period of time. We have found abun- dant TH2B and Hlt mRNA steady state levels as well as newly-synthesized TH2B and Hlt histones after 5 days of coculture. Northern blots reprobed with Hlt-specific oligonucleotide showed that Hlt mRNA remained prominent when TH2B mRNA started to decline after 8-12 days of coculture. Phase-contrast and transmission electron micros- copy studies carried out throughout the course of the experiments demonstrated that the number of viable spermatogonia and meiotic prophase sper- matocytes was relatively constant during 12 days of coculture. Spermatocytes, in a clone-like ar- rangement, remained attached to Sertoli cell surfaces and displayed subcellular features con- sistent with those observed in the intact seminif- erous epithelium. Spermatogonia formed long, branching chains of interconnected cells. Results of this study indicate that spermatogenic cells in coculture with Sertoli cells express testis-specific histone genes for an extended period of time. Tes- tis-specific histone gene expression in vitro should facilitate further studies for understanding the role of these histones in chromatin structure, tran- scription, and genetic recombination during male meiotic prophase.