Human argininosuccinate synthetase minigenes are subject to arginine-mediated repression but not to trans induction.
AUTOR(ES)
Boyce, F M
RESUMO
The human argininosuccinate synthetase locus is subject to metabolite-mediated repression by arginine in some cultured cell lines. To gain insight into the mechanism underlying this regulation, chloramphenicol acetyltransferase (CAT) minigenes under the transcriptional control of the human argininosuccinate synthetase promoter were constructed and tested for regulation. When the minigenes were introduced into RPMI 2650 cells, a human cell line that shows sixfold regulation of the argininosuccinate synthetase gene, CAT expression was repressed three- to fivefold when arginine was present in the culture medium. A minigene containing only 149 base pairs of 5'-flanking sequence was expressed at similar levels and regulated to the same degree as one having approximately 3 kilobases of 5'-flanking sequence. Therefore, the cis-acting sequences required for the arginine-mediated repression are likely to be located within the region of the transcription initiation site. The arginine-mediated repression of the CAT minigenes was not observed in canavanine-resistant variants of RPMI 2650 cells, and therefore they showed the appropriate cell-type specificity. Cultured cells having 200-fold-increased levels of argininosuccinate synthetase can be selected by growth in medium containing the arginine analog canavanine. It was previously demonstrated that the increased expression of argininosuccinate synthetase in canavanine-resistant human lymphoblasts was due to a trans-acting mechanism. To gain further support for a trans-acting mechanism, we tested our CAT minigenes for the trans induction in canavanine-resistant variants of RPMI 2650 cells. Transfection of the CAT minigenes into RPMI 2650 cells and canavanine-resistant variants of this cell line yielded no difference in transient CAT expression. Furthermore, cloned canavanine-resistant variant cells having integrated copies of the CAT minigenes expressed CAT at similar levels as compared to the parental cell lines. Since these cell lines do exhibit arginine-mediated repression of CAT but not trans induction, these data indicate that the argine-mediated repression is a regulatory event that occurs independently of the trans induction.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=367636Documentos Relacionados
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