Isolation and analysis of a rat genomic clone containing a long terminal repeat with high similarity to the oncomodulin mRNA leader sequence

The structure of a novel long terminal repeat (LTR) from an intracisternal A particle (IAP) DNA element in the rat (Sprague-Dawley) genome was determined. This LTR has a total length of 313 base pairs (bp). Several structural features typical for retroviral LTR promoters were identified, including a "CCAAT" box, a "TATA" box, a polyadenylation signal, and a polyadenylation site. The LTR is flanked by 3-bp inverted repeats, and it consists of the three typical LTR regions, U3, R, and U5. U3 contains 213 bp, R 46 bp, and U5 54 bp, which is within the usual size range of IAP LTRs. A sequence of 60 bp in the U3 region reveals considerable similarity to a murine IAP LTR U3 element, which is known to interact with nuclear proteins. A sequence of 69 bp in the U5 and R regions has 83 and 93% similarities to an endogenous retroviral LTR from Syrian hamster and to the cDNA leader sequence of (Buffalo) rat oncomodulin, respectively. Oncomodulin is an "EF-hand" Ca2+-binding protein and appears in many human and rodent tumors and in cells with tumor-like properties but not in normal tissues. We postulate that in the rat the tumor-specific expression of oncomodulin is controlled by a retroviral LTR promoter.     

Site-specific mutants of oncomodulin. 1H NMR and optical stopped-flow studies of the effect on the metal binding properties of an Asp59----Glu59 substitution in the calcium-specific site

High resolution 1H nuclear magnetic resonance spectroscopy and optical stopped-flow techniques have been used to study the metal binding properties of a site-specific mutant of bacterial recombinant oncomodulin in which glutamate has replaced a liganding aspartate at position 59 in the CD calcium-binding site. In particular we have followed the replacement of calcium by lutetium in bacterial recombinant oncomodulin and D59E oncomodulin to provide a measure of the protein's preferences for metal ions of different ionic radii. The result of the Asp----Glu substitution is to make the mutant oncomodulin more similar to rat parvalbumin in terms of its relative CD- and EF-domain affinities for lutetium(III), that is to increase its affinity for metal ions with smaller ionic radii. This finding supports the original hypothesis that the presence of Asp at sequence position 59 is an important factor in the reduced preference of the CD site of oncomodulin for smaller metals such as magnesium (Williams, T. C., Corson, D. C., Sykes, B. D., and MacManus, J. P. (1987) J. Biol. Chem. 262, 6248-6256). However, our studies show that both the CD and the EF sites are affected by this single residue substitution suggesting that many factors play a role in the metal binding affinity and interaction between the two sites.     

5' LTR complete nucleotide sequence of Chinese hamster intracisternal A-particle.

Retrovirus like sequences homologous to mouse IAP are present in Chinese hamster genome (Lueders K.K. and Kuff, E.L., 1981, 1983, Servenay et al., 1990). Murine IAP long terminal repeats (LTRs) can function as effective promoters in different cell types (Horowitz M. et al., 1984, Howe, C.C. et al., 1986). Thus CHO IAP sequences could act as retrotransposons in the cellular genome, and in this way affect the expression of other genes at the target sites. We had sequenced previously a Chinese hamster IAP genomic region corresponding mainly to the gag gene and including 57 nucleotides of U5 5' LTR (Servenay et al., 1988). In this paper, we present the 5' LTR complete nucleotide sequence of the Chinese hamster IAP element and its comparison with those of mouse and Syrian hamster.