Organization and expression of mouse nm23-M1 gene. Comparison with nm23-M2 expression

Abstract

Nm23 is a gene family encoding different isoforms of the nucleotide diphosphate kinase (NDPK), an enzyme involved in the synthesis of nucleoside triphosphates.

In the present study, the organization and expression of the nm23-M1 gene encoding the mouse NDPKA isoform are described. This gene is about 10 kb long and composed of five exons. The organization and the exon–intron boundaries are strictly conserved as compared to the human and rat related genes. The gene promoter region did not exhibit any consensus TATA box, SP1 binding element or Inr sequence. By contrast, TCF-1/LEF-1 binding elements and Pit-1 consensus sequence were present.

Northern blotting and in situ hybridization methods were carried out in adult and 18.5 days post-coitum (dpc) mouse embryo, respectively. They showed tissue-specific expression of nm23-M1 transcripts, despite housekeeping gene promoter features. The strongest signals were detected in the nervous system, sensory organs and embryonic thymus. In contrast nm23-M2 mRNA was shown to be more widely expressed.

The relationship between nm23-M1 gene tissue-specific expression and the putative binding element of the promoter region is discussed.

Introduction

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme which retains a ‘housekeeping’ activity as a major component of the pathway for the synthesis of triphosphate nucleotides except ATP (Parks and Agarwal, 1973). However, while the high conservation of the protein primary structure from higher to lower organisms (Fukuchi et al., 1993, Hama et al., 1991, Kimura et al., 1990, Lacombe et al., 1990, Munoz-Dorado et al., 1990, Nomura et al., 1992) strongly supports this first assumption about the basic function of this enzyme in the nucleotide metabolism of the cell, more recent studies provide evidence for new functional aspects in living organisms. For instance, in the fruit fly Drosophila melanogaster, Awd protein, the product of the abnormal wing discs (awd) gene, was found to display an NDPK activity (Biggs et al., 1990). In this species, lack of Awd protein leads to failure of normal development of several imaginal discs, and the mutant larvae cannot grow to adult size and finally die (Biggs et al., 1990). In mammals, it has been established that the nm23 gene family encodes the different NDPK isoforms. Until now, five isotypes of NDPK protein have been described in human (Milon et al., 1997, Munier et al., 1998, Rosengard et al., 1989, Stahl et al., 1991, Venturelli et al., 1995), but only two in rat (Kimura et al., 1990, Shimada et al., 1993) and in mouse (Urano et al., 1992). All the studies published to date suggest that although these different isoforms structurally resemble each other and exhibit the same phosphotransferase activity, they may display additional multifunctional and regulatory effects during cell differentiation or animal development. For instance, NDPKA was found to affect proliferation and differentiation into adrenergic neurones of murine PC12 cells in response to nerve growth factor in culture conditions (Gervasi et al., 1996). In the rat, it has been reported that the genes encoding NDPKβ and NDPKα proteins, respectively homologous to human nm23-H1 and nm23-H2 genes, are regulated independently (Ishikawa et al., 1992, Kimura et al., 1990). In human, the product of the nm23-H2 gene has been described as a possible transcription factor for c-myc proto-oncogene (Postel et al., 1993, Postel et al., 1996). Finally, human nm23-H1 and its mouse counterpart nm23-M1 both operate as a tumor metastasis suppressor gene for some cancer types (Freije et al., 1996, Lacombe and Jakobs, 1992).

Most of these additional functions of nm23 gene products seem to be unrelated to the NDPK enzyme activity of the proteins (Macdonald et al., 1993, Okabe-Kado et al., 1995, Postel and Ferrone, 1994), but their precise mechanisms of action are not fully understood. Moreover, the antimetastatic effect of nm23-H1 gene products has been demonstrated in different forms of human or mouse cancer, but not in all. Further studies on the functional role of the nm23 gene family require more detailed investigations of nm23 gene organization and expression. Thus, this study aims to characterize the nm23-M1 gene and determine its normal expression pattern in mouse as compared to that of the nm23-M2 gene.