Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

1. The activity of an enzyme catalysing the loss of O6-methylguanine from methylated DNA was increasing during liver regeneration after partial hepatectomy. Activity was increased 3-fold by 24h and was maximal (6-fold increase) over the period 48-72h after operation. 2. This activity could also be induced by chronic treatment with dimethylnitrosamine, but the maximal response amounted to a 2-3-fold change (with the greater effect in male rats) after 4-6 weeks of exposure to daily doses of 2 mg of dimethylnitrosamine/kg. 3. Neither partial hepatectomy nor treatment with dimethylnitrosamine increased the activities of two other enzymes repairing alkylated DNA, DNA (7-methylguanine-)glycosylase and DNA (3-methyladenine-)glycosylase. 4. These results therefore indicate that there is a selective induction of the O6-methylguanine removal system during hepatocyte proliferation. Since this product is known to lead to mutations and its persistence in DNA throughout cell replication has been implicated in tumour initiation, this induction may play a role in resistance to carcinogenesis by alkylating agents.

An enzymatic activity present in rat liver extracts catalyzes the transfer of methyl groups from O6-methylguanine in DNA to protein. This activity was stimulated by treatment of rats with thioacetamide, carbon tetrachloride, 1,2-dimethylhydrazine, diethylnitrosamine, dimethylnitrosamine and by partial hepatectomy but not by treatment with N-methyl-N-nitrosourea or streptozotocin. These results suggest that an enhancement of this activity accompanies the increase in cell division brought about by these agents and is not necessarily a specific response to the presence of alkylated bases in DNA.

No abstract

No abstract