Functional analysis of the porcine alpha-skeletal actin gene
Abstract
Three series of experiments were conducted to (1) clone and sequence the porcine $\alpha$-skeletal actin gene, and (2) define the cis-acting elements that regulate porcine $\alpha$-skeletal actin transcription in cell culture and in skeletal muscle. We cloned and sequenced a 5.2 kb Hind III DNA fragment which contained the coding region of porcine $\alpha$-skeletal actin plus 1.9 kb of $5\sp\prime$ and 0.5 kb of $3\sp\prime$ flanking sequences. The amino acid sequence was strictly conserved with respect to previously sequenced $\alpha$-skeletal actin genes. In addition, the proximal promoter region was 81.6% homologous to the human $\alpha$-skeletal actin promoter. The porcine $\alpha$-skeletal actin promoter (bp $-$1929 to +250) was capable of directing high-level, tissue-specific and developmental expression, relative to pCAT-Control. The porcine $\alpha$-skeletal actin promoter can be divided into three regions (1) $5\sp\prime$ distal (bp $-$1929 to $-$550), (2) proximal (bp $-$84 to +55), and (3) intron (bp +55 to +250). The $5\sp\prime$ distal region enhanced porcine $\alpha$-skeletal actin promoter activity in C2C12 myotubes and inhibited promoter activity in Hela cells. The proximal region was responsible for basal levels of transcriptional. The inter region was necessary for tissue-specific expression. In addition, intron 1 sequences enhanced porcine $\alpha$-skeletal actin promoter activity in C2C12 myoblasts. We also identified three regions of the porcine $\alpha$-skeletal actin gene with cis-acting elements (1) $5\sp\prime$ distal regulatory element (bp $-$1929 to $-$550), (2) internal regulatory element (bp $-$550 to $-$138 and bp +55 to +1200), and (3) $3\sp\prime$ distal regulatory element (bp +1973 to +2973). All three regions enhanced SV40 promoter activity in C2C12 myotubes and inhibited SV40 promoter activity in Hela cells. The porcine $\alpha$-skeletal actin promoter (bp $-$1929 to +55) was capable of directing high-level transgene expression in skeletal muscle. However, in contrast to our cell culture results the $5\sp\prime$ distal region did not enhance and intron 1 sequences inhibited porcine $\alpha$-skeletal actin promoter activity. A novel cis-acting element in the promoter region (bp $-$550 to $-$388) was identified by direct injection experiments. These results demonstrate that the transcriptional control of the porcine $\alpha$-skeletal actin is complex and differs between cell culture and skeletal muscle.
Degree
Ph.D.
Advisors
Grant, Purdue University.
Subject Area
Molecular biology|Cellular biology|Genetics
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