metallothionein and Albinism

Mice homozygous for chromosomal deletions at or around the albino locus on chromosome 7 express reduced levels of a group of liver genes, including tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), and generally die perinatally. Sequences within the deleted region are thought to encode a regulatory factor(s) that affects expression of these genes in trans. To facilitate study of the putative factors, we immortalized hepatocytes derived from newborn cch wild-type and c14CoS deletion homozygous mice as well as cch/c14CoS heterozygous mice using a SV40 temperature-sensitive A255 mutant virus. Three c14CoS deletion homozygous hepatocyte lines were characterized and compared with the homozygous wild-type and heterozygous lines. The SV40 tsA255 mutant-transformed hepatocyte lines were temperature-sensitive for maintenance of transformation and expressed many liver-specific genes. In agreement with in vivo studies, hepatocyte lines derived from mice homozygous for the deletion expressed reduced mRNA levels of a number of liver genes including TAT, PEPCK, X1, X2, and X7 in comparison with heterozygous and wild-type cell lines. Similar mRNA levels of transferrin and albumin, genes whose expression is unaffected by the mutation in vivo, were observed in all cell lines. The expression of two genes, X5 and metallothionein, reported to be reduced in newborn mutant mice, did not differ appreciably among cell lines. TAT and PEPCK have been shown to respond poorly to glucocorticoids and cAMP in newborn mutant mice. Interestingly, all affected liver genes tested were responsive to glucocorticoids and dibutyryl cAMP in deletion homozygous cell lines as well as in wild-type and heterozygote-derived cell lines. This may suggest that effects of the deletion on expression of liver-specific genes do not cause loss of responsiveness to glucocorticoids and cAMP. These immortalized hepatocyte lines, which express most, if not all, liver-specific genes, should provide a useful means for further investigation of the effects of the albino lethal deletion.

Topics: Albinism; Albumins; Animals; Blotting, Southern; Bucladesine; Cell Line, Transformed; Cells, Cultured; Chromosome Deletion; Dexamethasone; DNA; Gene Expression Regulation; Genes, Lethal; Heterozygote; Homozygote; Liver; Metallothionein; Mice; Nucleic Acid Hybridization; Radioimmunoassay; RNA, Messenger; Transferrin

Melanocyte cell lines, with characteristic dendritic morphology and melanosomes, were established from young mice of wild-type (C57BL/6) and of two albino (C57BL/6-c2J/c2J and BALB/c) inbred strains. The albino cells were cotransfected with two plasmids: pMTtyr1, containing the full-length tyr1 cDNA for tyrosinase encoded by the c locus, under the control of the inducible mouse metallothionein-I (MT-I) promoter; and pSVneo beta, allowing selection of transformants by G418 resistance. The intrinsic albino defect was corrected by the tyr1 cDNA in transfected cells, thereby validating the coding capability of tyr1 for tyrosinase. Black melanin was formed in the genetically black (B/B) C57BL/6-c2J/c2J cells and brown melanin in the genetically brown (b/b) BALB/c cells. Pigment was produced even without adding heavy metal (for induction of the MT-I promoter), thus obviating the need for adding it, but was formed more rapidly upon addition of ZnSO4 up to 100 microM. Stable transfected albino melanocyte lines with active tyrosinase and melanization were obtained. Addition of ZnSO4 at 200 microM was lethal to the cells. However, this toxicity--attributable at least in part to melanin precursors--was prevented if the cells sojourned at 100 microM ZnSO4 for two weeks before being exposed to the 200 microM level. Adaptation was lost when the cells were removed from 200 microM ZnSO4 for one week and then returned to it. Avoidance of toxicity under these conditions is thus the result of physiological detoxification mechanisms rather than selection for a genetic change.

Topics: Albinism; Animals; Cell Line; Cell Survival; Female; Gene Expression Regulation, Enzymologic; Melanins; Melanocytes; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Monophenol Monooxygenase; Promoter Regions, Genetic; Transfection; Zinc

Deletions in chromosome 7 of the mouse affect the expression of the metallothionein gene Mt-1, which maps on chromosome 8, and steady-state levels of Mt-1 mRNA are reduced to 15-40% of normal in livers of newborn mice homozygous for either the c3H or c14CoS deletion. Glucocorticoids fail to induce hepatic Mt-1 mRNA levels in deletion homozygotes in contrast to normal littermates. However, zinc chloride is effective in inducing Mt-1 mRNA levels in livers of deletion homozygotes as well as of their normal littermates. Other tissues (e.g., kidney and intestine) of deletion homozygotes express basal levels of Mt-1 mRNA higher than those of normal littermates. In the intestine these are furthermore inducible by both hormonal and metal agents. Thus, loss of inducibility of the Mt-1 gene in deletion homozygotes concerns glucocorticoids only and is furthermore restricted to specific cell types (i.e., hepatocytes). The trans-acting factor(s) normally encoded in the deleted region of chromosome 7 appears to be instrumental in conferring on the metallothionein gene in hepatocytes the essential competence to respond to hormonal inducing signals.

Topics: Albinism; Animals; Chlorides; Chromosome Deletion; Chromosome Mapping; Dexamethasone; Gene Expression Regulation; Homozygote; Intestinal Mucosa; Kidney; Liver; Metallothionein; Mice; Mice, Mutant Strains; RNA, Messenger; Zinc; Zinc Compounds