Isozyme differentiation among three pathotypes of the entomogenous fungus Nomuraea rileyi.

The zymogram technique has been applied to three pathotypes of the entomopathogenic fungus Nomuraea rileyi. Isozyme profiles of isolates from Heliothis zea, Pseudoplusia includens, and Anticarsia gemmatalis were compared for 17 enzymes of known metabolic function. Electrophoretic data supported the taxonomic differences inferred for the three pathotypes based on host specificity. The isolate from A. gemmatalis was found consistently to be the most distinct. This study demonstrates that isozyme analysis may be used to distinguish closely related fungal isolates of N. rileyi.

Gene expression patterns in the black blowfly (Phormia regina) as revealed by two-dimensional electrophoresis of proteins. I. Developmental stage-specific and sex-specific differences.

The black blowfly, Phormia regina, has been implicated in human myiasis and as a contact vector of viral and bacterial diseases present in carrion to which female flies are attracted for egg deposition. Inbred strains of P. regina are an excellent model system for studying gene expression in the developmental stages of such holometabolous dipteran parasites. However, information regarding gene and protein expression patterns in P. regina is limited. We used ISO-DALT high-resolution, two-dimensional electrophoresis with silver staining to establish fundamental protein maps for examination of the stage-specific gene expression patterns in the 615 most abundant proteins of the eggs, first- and third-instar larvae, pupae, and male and female adults. We also used a differential extraction technique to identify the major cuticular proteins of the adults. The results show 48 clearly identifiable stage-specific and sex-specific proteins. Thus, approximately 8% of the most abundant proteins exhibit developmental changes. These analyses serve as an initial data base for further studies of ontogenetic regulation, organellar origin, and physiologic function of the stage-specific proteins in the life cycle of these opportunistically parasitic dipterans.

Mitochondrial DNA of the blowfly Phormia regina: restriction analysis and gene localization.

A study of an invertebrate mitochondrial genome, that of the blowfly Phormia regina, has been initiated to compare its structural and functional relatedness to other metazoan mitochondrial genomes. A restriction map of mitochondrial DNA (mtDNA) isolated from sucrose gradient-purified mitochondria has been established using a combination of single and double restriction endonuclease digestions and hybridizations with isolated mtDNA fragments, revealing a genome size of 17.5 kilobases (kb). A number of mitochondrial genes including those encoding the 12 S and 16 S ribosomal RNA, the cytochrome c oxidase I subunit (COI) and an unidentified open reading frame (URF2) have been located on the Phormia mtDNA by Southern blot analysis using as probes both isolated mtDNA fragments and oligonucleotides derived from the sequences of previously characterized genes from rat and Drosophila yakuba mtDNAs. These data indicate that for those regions examined, the mitochondrial genome organization of blowfly mtDNA is the same as that of Drosophila yakuba, the order being COI-URF2-12 S-16 S. These data also report the presence of an A + T-rich region, located as a 2.5-kb region between the URF2 and the 12 S rRNA genes, and its amplification by the polymerase chain reaction is described.

Brown larva: an allele of the green larva mutation in the malaria mosquito, Anopheles stephensi.

We isolated a new larval color mutant, brown larva (b), from the Bangalore, India strain of Anopheles stephensi Liston. The gene b is an autosomal recessive with uniform expression and complete penetrance. We conducted extensive crosses to establish allelism between brown larva (b) and green larva (g) reported previously in An. stephensi from our laboratory. The wild-type is dominant to green larva, which, in turn, is dominant to brown larva. These larval color mutants belong to an allelic series.

Reciprocal translocations and partial correlation of chromosomes in the stable fly.

An initial investigation of the genetics of the stable fly, Stomoxys calcitrans (L.), is described. Two recessive, autosomal mutants, carmine eye (ca) and rolled down wing (rd), were assigned to chromosomes 2 and 4, respectively. Sex is apparently determined by a locus on chromosome 1. Crossing over is restricted to females. Six reciprocal translocations were induced with gamma radiation and used to assign ca and rd to their respective chromosomes.

Activation of immediate-early gene expression by peroxisome proliferators in vitro.

The hepatocarcinogenicity of peroxisome proliferators (PPs) in rodents has been attributed both to oxidative DNA damage resulting from excessive leakage of peroxisomal H2O2 and to increased hepatocellular replication that may be independent of peroxisome proliferation. Because of the growing association between tumor promotion and alterations in growth-regulatory signal transduction pathways, we investigated whether PPs can modulate these pathways in a mouse liver epithelial cell line, BNL-CL.2. We tested two PPs that differ markedly in rodent tumorigenicity for their ability to activate immediate-early proto-oncogene expression. 4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy-14643), a highly tumorigenic PP, was an exceptionally strong inducer of c-fos expression. Wy-14643 was also stronger than DEHP in stimulating c-jun expression, whereas both PPs were fairly strong inducers of jun-B and jun-D. The induction of fos and jun expression by Wy-14643 was specifically inhibited by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H-7). DEHP-induced gene expression was strongly inhibited by H-7, but was also partially inhibited by an inhibitor of protein kinase A. The activation of fos and jun gene expression by PPs was independent of peroxisome proliferation since it was an immediately-early response not requiring protein synthesis and since the cell lines used in this study do not undergo peroxisome proliferation. Our r results raise the possibility that the carcinogenicity of PPs may be due, in part, to epigenetic modulation of growth-regulatory signal transduction pathways.

Dose-related changes in the profile of ras mutations in chemically induced CD-1 mouse liver tumors.

We investigated the role of dosing regimen on ras mutations in chemically induced CD-1 mouse liver tumors. The spectra of ras gene mutations in liver tumors that were induced by 15 daily i.p. injections of 7,12-dimethylbenz[a]anthracene (DMBA), 4-aminoazobenzene (AAB), N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or N-nitrosodiethylamine (DEN) were compared to those previously obtained for tumors induced by a single but higher dose of each carcinogen. The principal assay used was a direct tumor analysis involving sequencing of polymerase chain reaction (PCR)-amplified tumor DNA; additional mutations that were present in only a small fraction of tumor cells were detected using a transfection assay or a PCR-engineered restriction fragment length polymorphism method. Spontaneous liver tumors had a relatively low frequency of ras mutations, all found in Ha-ras codon 61, and most of these mutations were present in only a small fraction of tumor cells. With the exception of multiple-dose DEN, each group of single- and multiple-dose carcinogen-induced tumors exhibited a higher frequency of ras mutations compared with spontaneous tumors. For AAB, N-OH-AAF and DEN, the dosing regimen was found to affect significantly the profile of ras mutations. For each of these carcinogens, the multiple-dose tumor group (versus single-dose group) had fewer Ki-ras and N-ras mutations and more tumors in which the Ha-ras codon 61 (C-->A) mutation was present in a large fraction of cells. Our results demonstrate that the dosing procedure can materially affect the pattern of ras gene mutation in mouse liver tumors.

Induction of minisatellite DNA rearrangements by genotoxic carcinogens in mouse liver tumors.

We investigated whether somatic rearrangements in minisatellite DNA are more frequent in chemically induced mouse liver tumors than they are in spontaneous tumors. CD-1 mouse liver tumors were induced by either a single dose or 15 consecutive daily doses of 7,12-dimethylbenz[alpha]anthracene, 4-aminoazobenzene, N-hydroxy-2-acetyl-aminofluorene or diethylnitrosoamine (DEN). Using DNA fingerprinting analysis, we found that the single- and multiple-dose carcinogen treatments caused a 2- to 5-fold higher frequency of minisatellite DNA rearrangements compared with that found in spontaneous tumors--with the exception of single-dose DEN tumors, which showed no increase in rearrangements. Our results suggest that DNA fingerprinting may be a valuable assay for differentiating certain chemically induced tumors from spontaneous tumors.

Activation of the Ki-ras gene in spontaneous and chemically induced lung tumors in CD-1 mice.

As part of an evaluation of the effectiveness of using ras mutation analysis for distinguishing carcinogen-induced from spontaneous tumors, we examined the profile of ras gene point mutations in spontaneous, 7,12-dimethylbenz[a]anthracene (DMBA)-induced, and N-nitrosodiethylamine (DEN)-induced lung tumors from Crl:CD-1(ICR)BR (CD-1) mice. Although all of the lung tumors were assayed for mutations in the Ha-ras, Ki-ras, and N-ras genes (codons 12, 13, and 61), only Ki-ras mutations were found, which is consistent with other studies that have noted a strong preference for Ki-ras gene activation in mouse, rat, and human lung tumors. We found that spontaneous CD-1 mouse lung tumors had a very high frequency of Ki-ras gene activation (17 of 20 tumors; 85%), distributed among codons 12 (5 of 20), 13 (1 of 20), and 61 (11 of 20). DMBA-induced lung tumors had a slightly higher frequency of Ki-ras gene mutations (16 of 16; 100%), again distributed among codons 12 (5 of 16), 13 (2 of 16), and 61 (9 of 16). However, seven of the DMBA tumors had mutations qualitatively different from those found in spontaneous tumors. In contrast to DMBA-induced tumors, DEN-induced tumors had a lower frequency of Ki-ras mutations (36%) when compared with spontaneous lung tumors, suggesting that DEN primarily induces lung carcinogenesis by a mechanism other than ras gene activation. Thus, although spontaneous and induced CD-1 mouse lung tumors have a strong tissue-specific preference for carrying an activated Ki-ras gene, the nature of the initiating carcinogen can influence the frequency or profile of Ki-ras mutations.