6. Results6.1. RhoA Gene Organisation Has Been Highly Conserved throughout Evolution and the Putative Promoter Contains Regulatory Elements Involved in Early Heart Development and OrganogenesisWe have previously shown that RhoA is click here necessary for normal heart formation in the developing chick [1]. In order to further investigate the regulation of RhoA expression in the early heart, the structure and organisation of the chick, mouse and human RhoA genes were obtained by genomic PCR analyses or comparative analysis of the known cDNA sequences against database genomic sequences. Subsequently, the putative promoter region of the mouse gene was deduced to permit the identification of cis-acting elements that might be involved in the regulation of expression of this gene in heart muscle.

We first determined the genomic organisation of the chick RhoA gene by comparing the known cDNA sequence with sequence obtained from genomic PCR and sequence analysis (data not shown). Based on these analyses, the chick RhoA gene spans at least 7kb and contains at least 4 introns. The first intron (designated Intron5��UTR1) is situated in the 5��UTR region at nucleotide position ?3/?2 (i.e., upstream of the ATG translation start codon, where the ��A�� of the ATG is nt +1). This is presumably a very long intron (as is the case with both the mouse and human RhoA genes; see Figure 1); however, its length could not be determined by genomic PCR because the sequence of the 5�� UTR of chick mRNA is not known.

In addition to this 5��UTR1 intron, three introns interrupt the chick RhoA open reading frame (ORF): Intron1 (2554bp), situated between nucleotides (nts) 156 and 157; Intron2 (1549bp), between nts 276 and 277; and Intron3 (750bp), between nts 408 and 409 (see Figure 1). Introns1 and 2 display consensus splice site sequences consistent with the GT/AG rule [7], but the Intron3 5��-splice donor sequence is GC. Based on these results for the chick RhoA gene, the genomic organisation of the mouse and human RhoA genes was deduced by comparative analysis of the known cDNA sequences against sequences in the respective genomic sequence databases. Alignment of the deduced chick RhoA gene organisation to that of mouse and human RhoA genes reveals a high degree of conservation for both coding sequence (alignment of the chick and human RhoA cDNA sequences is shown in our earlier paper [1]) and gene organisation (is shown in Figure 1). The mouse and human RhoA genes contain introns situated at the same positions relative to the ORF as the four Carfilzomib introns of the chick gene. The mouse RhoA gene contains an additional intron upstream of Intron5��UTR1, designated Intron5��UTR2. Figure 1Comparative organisation of the chick, mouse, and human RhoA genes.