Cloning of a factor required for activity of the Ah (dioxin) receptor.

The aryl hydrocarbon (Ah) receptor binds various environmental pollutants, such as polycyclic aromatic hydrocarbons, heterocyclic amines, and polychlorinated aromatic compounds (dioxins, dibenzofurans, and biphenyls), and mediates the carcinogenic effects of these agents. The complementary DNA and part of the gene for an 87-kilodalton human protein that is necessary for Ah receptor function have been cloned. The protein is not the ligand-binding subunit of the receptor but is a factor that is required for the ligand-binding subunit to translocate from the cytosol to the nucleus after binding ligand. The requirement for this factor distinguishes the Ah receptor from the glucocorticoid receptor, to which the Ah receptor has been presumed to be similar. Two portions of the 87-kilodalton protein share sequence similarities with two Drosophila proteins, Per and Sim. Another segment of the protein shows conformity to the consensus sequence for the basic helix-loop-helix motif found in proteins that bind DNA as homodimers or heterodimers.

Genetic and molecular analysis of the Ah receptor and of Cyp1a1 gene expression.

The Ah receptor is a soluble protein complex that mediates carcinogenesis by a wide range of environmental pollutants, including polycyclic aromatic hydrocarbons, heterocyclic amines, and polychlorinated aromatic compounds. The best understood activity of the receptor concerns its role in the induction of cytochrome P450IA1. We undertook a somatic cell genetic analysis of P450IA1 induction using the mouse hepatoma cell line, Hepa-1. Clones of Hepa-1 were isolated that are defective in induction of P450IA1. Evidence was obtained that the clones are mutational in origin. Cell fusion experiments demonstrated that a few of the mutants are dominant, while the majority are recessive. The dominant mutants were shown to synthesize a repressor of P450IA1 transcription. The recessive mutants were assigned to 4 complementation groups (probably corresponding to 4 different genes). Complementation group A corresponds to the P450IA1 structural gene. Mutations in the B, C and D genes all affect functioning of the Ah receptor. A 'reverse selection procedure', whereby cells that express P450IA1 inducibility can be selected from a majority population of cells lacking inducibility, was developed. The reverse selection procedure was used to isolate transfectants of representative recessive mutants in which the mutational defects are complemented by exogenously applied genomic DNA. A human DNA-derived transfectant of a C- mutant was used to clone the human C gene. The C gene is not the ligand-binding subunit of the Ah receptor but is a protein that is required for translocation of Ah receptor-ligand complexes from cytoplasm to nucleus. In analogous experiments the dominant gene from one of the dominant mutants was transfected into wild-type Hepa-1 cells. Success in transfecting the dominant gene should provide the means for cloning it.

Fetuses of lean and obese swine in late gestation: body composition, plasma hormones and muscle development.

The development of obesity in porcine fetuses was investigated using a lean and obese strain of pigs at 80, 90, 100 and 110 d of gestation. In absolute terms, fetuses of obese gilts (FO) generally had lower carcass weight and contained less total protein, dry matter and ash than fetuses of lean gilts (FL). In relative terms (percentage of wet carcass weight) FO, compared with FL, generally had decreased percentages of water and increased percentages of protein and lipid. Comparisons based on absolute terms revealed body composition of the strains to be different at 90 d, indicating that factors responsible for obese-type growth were active before that time. Both body composition and hormone concentration differences were most pronounced at later gestation ages. Depressed growth hormone, elevated cortisol, and a tendency toward elevated insulin concentrations in fetal plasma were apparent in late gestation for FO compared with FL. These hormonal patterns are consistent with onset of obesity in FO in late gestation. Greater weights of semitendinosus and longissimus muscles were observed in FL vs FO at 90, 100 and 110 d of gestation (P less than .05). These greater muscle weights were generally accompanied by greater contents of RNA, DNA and protein in FL muscles at these same ages. However, at 80 d, FL had greater absolute DNA content in semitendinosus muscle whereas muscle weight was similar between the strains. This suggests that greater muscle weights for FL than FO were caused by more nuclei in muscle of FL. In general, indices of hypertrophy (protein/DNA) and protein synthetic capacity (RNA/DNA) of muscle were usually similar for both strains at all gestation ages. It is concluded that decreased muscle growth in late gestation of FO compared with FL is more related to fewer total nuclei and perhaps fewer myofibers than to an impaired cellular capacity for protein synthesis.