CLONING OF THE XENOPUS-LAEVIS ALDOLASE-C GENE AND ANALYSIS OF ITS PROMOTER FUNCTION IN DEVELOPING XENOPUS EMBRYOS AND A6 CELLS

A Xenopus aldolase C gene (XAC lambda 3-1), much longer (9.6 kb) than human and rat genes (3.7-3.6 kb), was isolated and characterized, and expression studies were performed using Xenopus embryos and A6 cells, a kidney cell line constitutively expressing aldolase C gene. The Xeno pus gene contained nine exons, and in its proximal 5'-upstream region a GC box and a 16 bp long aldolase C-specific element (ACSE), and in a ddition, a CCAAT box and a TATA-like element, both missing in mammalia n genes. The lacZ gene connected to the 5'-upstream region (1.6 kb) of the aldolase gene containing many potentially regulative sequence ele ments was expressed in embryos temporally and spatially like the endog enous aldolase C gene. Deletion experiments using embryos and A6 cells suggested that this 5'-upstream DNA contained in its distal part a re gion which negatively affected on its expression in embryos, but not i n A6 cells. The proximal-most region contained a basal promoter (68 bp ) essential for expression in both embryos and A6 cells. Deletion expe riments using A6 cells failed to detect such regulative regions within the first intron (spanning ca. 4 kb). Analyses with mutated promoters in A6 cells revealed that the GC box was the crucial element in the b asal promoter, although the TATA-like element appeared to have a sligh tly stimulative effect on the GC box functioning. Gel retardation and foot-printing assays revealed the occurrence in A6 cells of a nuclear factor(s) that binds specifically to the GC box. Since Xenopus aldolas e C gene has several unique structural features, we expect that it wil l provide an interesting material for studying the evolution and devel opmental control of the aldolase C gene. (C) 1998 Elsevier Science B.V . All rights reserved.