Two PCR products were obtained with type IV primers (Fig. 3) corresponding to the published sequence for rat type IV and a newly identified splice variant of type IV containing 24 nucleotides with two possible splice sites. It is possible that the splice site is either between nucleotides 2600 and 2601 or between nucleotides 2607 and 2608 of the published rat sequence. These two splice insertion sites arise due to a seven-nucleotide repeat (TGGAGAA) that could be the first or last seven nucleotides of the insert (Fig. 4a). Either splice insertion site results in an identical predicted amino acid insertion of eight amino acids (LTRLLLEN) between amino acids 833 and 834 of the initially described sequence of type IV adenylyl cyclase. Both splice variants of adenylyl cyclase type IV were coexpressed in uterine smooth muscle cells (Fig. 3). This region of the newly identified splice variant for type IV has a higher putative amino acid homology to types II and VII adenylyl cyclase than the previously published type IV sequence (Fig. 4b). flovent inhaler
These results obtained in fresh myometrial cells relied on total RNA extracted from freshly dissected uteri. Although this tissue consisted of primarily myometrial cells, it also contained multiple other cell types of vascular, stromal, and hematopoietic origin. Because of the high sensitivity of RT-PCR, it was possible that some or all of the PCR products obtained were amplified from RNA from non-myometrial cells.
FIG. 4. a) Nucleotide and amino acid sequence of the larger adenylyl cyclase type IV splice variant within the C2a region. Removal of either of the 24 nucleotides indicated by lines above or below the nucleotide sequence yields the adenylyl cyclase type IV sequence previously published. Either of these two splicing alternatives yields the same putative eight-amino acid addition (indicated by italics). Nucleotide 2591 and amino acid 828 refer to previously described adenylyl cyclase type IV sequence. b) Putative amino acid alignment of the 5′ end of the C2a region of related adenylyl cyclase types II, IV, and VII. Identical residues among sequences are indicated by —. Numbers in parentheses are references to previously published sequences. The newly identified adenylyl cyclase type IV splice variant contains eight additional amino acids that retain higher homology to adenylyl cyclase types II and VII than the previously published shorter version of adenylyl cyclase type IV.