Genomic organization of alpha1 and beta1 subunits of the mammalian soluble guanylyl cyclase genes

Abstract

The structures of the genes encoding the alpha(1) and beta(1) subunits of murine soluble guanylyl cyclase (sGC) were determined. Full-length cDNAs isolated from mouse lungs encoding the alpha(1) (2.5 kb) and beta(1) (3.3 kb) subunits are presented in this report. The alpha(1) sGC gene is approximately 26.4 kb and contains nine exons, whereas the beta(1) sGC gene spans 22 kb and consists of 14 exons. The positions of exon/intron boundaries and the sizes of introns for both genes are described. Comparison of mouse genomic organization with the Human Genome Database predicted the exon/intron boundaries of the human genes and revealed that human and mouse alpha1 and beta1 sGC genes have similar structures. Both mouse genes are localized on the third chromosome, band 3E3-F1, and are separated by a fragment that is 2% of the chromosomal length. The 5' untranscribed regions of alpha(1) and beta(1) subunit genes were subcloned into luciferase reporter constructs, and the functional analysis of promoter activity was performed in murine neuroblastoma N1E-115 cells. Our results indicate that the 5' untranscribed regions for both genes possess independent promoter activities and, together with the data on chromosomal localization, suggest independent regulation of both genes.

Figure 1

Genomic structure and size of introns of murine α₁ and β₁ sGC. (A) Exon-intron distribution of murine α₁ and β₁ sGC genes (drawn to scale). Exons are represented by black boxes. (B) Estimation of the intron sizes by PCR using primers located in cDNA sequences flanking the introns (see Materials and Methods). The amplified DNA products were separated on 1% agarose gel and stained with ethidium bromide. The molecular mass markers (mixture of Lambda DNA–HindIII digest and φX174 DNA–HaeIII digest, New England Biolabs) are shown at the left of each panel.

Figure 2

Chromosomal localization of murine α₁ and β₁ sGC genes. DNA from BAC clones containing genomic regions of α₁ and β₁ sGC genes was labeled with digoxigenin dUTP and hybridized to normal metaphase chromosomes derived from mouse embryo fibroblast cells (for details see Materials and Methods). Cohybridization of α₁ sGC and β₁ sGC probe with a probe specific for telomeric region on chromosome 3 is shown on A and B, respectively. Arrows indicate the sites of specific hybridizations.

Figure 3

Promoter activity of 5′ flanking regions of α₁ and β₁ sGC in N1E-115 cells. Summary of luciferase expression assays of α₁ and β₁ sGC putative promoter regions in murine neuroblastoma N1E-115 cells. Luciferase activity was assayed as described in Materials and Methods. Values were normalized by using a β-gal construct (cytomegalovirus-β-gal) cotransfections and the total protein concentration of each group. The values represent the normalized means ± SD of three different experiments.