Abstract
We have isolated an alternative transcript of the rat Gs alpha signal transduction protein gene, referred to as Gs alpha N1. Gs alpha N1 was isolated by differential hybridization screening of genes induced upon dexamethasone treatment of the neuronal-like CA77 rat thyroid C-cell line. The 1-kilobase Gs alpha N1 transcript is generated by alternative splicing and polyadenylation of a novel terminal exon. This exon lies 800 base pairs downstream of exon 3 in the Gs alpha gene. Dexamethasone differentially induced Gs alpha N1 severalfold relative to Gs alpha mRNA in the CA77 cells, similar to the bias seen with alternative processing of the calcitonin/calcitonin gene-related peptide transcript. In addition to the differential regulation by dexamethasone, the expression pattern of Gs alpha N1 in rat tissues differed markedly from Gs alpha. Gs alpha N1 mRNA was much more abundant in the brain, with intermediate levels in skeletal muscle and very low levels in other tissues. This was in contrast to the more ubiquitously expressed Gs alpha mRNA. Within the brain, Gs alpha N1 was particularly abundant in discrete regions of the brainstem and hypothalamus that modulate autonomic functions. Examination of rat embryos demonstrated that Gs alpha is expressed in both brain and nonneural tissue at least 1 day before Gs alpha N1 mRNA could be detected in the embryonic brain. Based on the regulated expression of the Gs alpha N1 transcript and previous studies on G alpha proteins, the predicted Gs alpha N1 protein may potentially modulate several heterotrimeric G protein functions in the nervous system.
Highlights
We have isolated an alternative transcripotf the rat sone can partly repress these properties to induce features
GsaN1 wasisolated by differential hybridiza- The morphological changes are an increased cell roundness tion screening of genes induced upon dexamethasone and neurite remodeling, seen as a partial retraction andthintreatment of the neuronal-likeCA77 rat thyroidC-cell ning with an increased number of varicosities
GsaN1 mRNA was much more abundant in the crest (Anderson, 1989)and suggests that common differentiabrain, with intermediate levels in skelemtaulscle and tion mechanisms maybe shared between the sympathoadvery low levels in other tissues
Summary
CA77 cells and screened withradiolabeled cDNA probes prepared from either control or dexamethasone-treatedcells. To screening a second CA77 cDNA library and by reverse tran- determine whether the GsaNsel quences were contributed by scription and PCR amplification (RT-PCR) of CA77 mRNA a single exon or multiple exons, the PCR amplification was (see "Experimental Procedures") (Fig. 1).These cDNA clones repeatedusing a GsaN1-specific primer located nearthe were designated GsaN1 to indicate that the mRNA containspolyadenylation site (D3) (Fig. 2 0 ). The productwas 1.3 kb, only the NH2-terminalcoding region of Gsa (Fig. 1).GsaNl which is the size predicted by the additionalcDNA sequences mRNA encodes the first 86 amino acids of Gsa, followed by between the splice junction andpolyadenylation primers. Following exon 3of the human Gsngene (Kozasa et al, 1988) Alternative splicing in this region of the Gsa transcript has Single bands of 4.0,4.4,and 9.8 kb were detected with genomic DNAdigested withHindIII, BglII, and BamHI restriction.
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