Determination of the sequence requirements for the expression of a Xenopus borealis embryonic/larval skeletal actin gene

Abstract

In this study, we demonstrate that all sequences necessary and sufficient for the expression of a Xenopus borealisα3B embryonic/Iarval skeletal actin gene, reside in a 156‐nucleotide fragment of the promoter that spans nucleotides ‐197 to ‐42. This region of the promoter contains three imperfect repeats of the CC(A/T)6GG (CArG) box motif that have been demonstrated to be important in the expression of other sarcomeric actin genes. Deletion of the actin promoter, using Xenopus microinjection techniques as a transient assay system for promoter activity, shows that the most distal CArG box (CArG box3) is essential for the full expression of the gene. Under our assay conditions, the most proximal CArG box (CArG box1) exhibits two binding activities using band‐shift analysis. One of these binding activities contains components antigenically related to a serumresponse factor (transcription factor), whilst the second does not. In contrast, CArG box3 produces only a single retarded band using electrophoretic mobility‐shift analysis. Although the shifted complex coelectrophoreses with the CArG box1/serum‐response factor complex, the band produced by CArG box3 appears to be distinct from SRF. In addition to the CArG motifs, a further upstream regulatory element has been identified in the actin promoter between nucleotides ‐197 and ‐167. In the actin promoter, a downstream region can apparently fulfil this function.

Transposon-Mediated Transgenesis in the Short-Lived African KillifishNothobranchius furzeri, a Vertebrate Model for Aging

“…We generated a pCska-gfp Tol2 construct encoding enhanced GFP driven by the Xenopus cytoskeletal actin promoter, a promoter that has been shown to be active in Xenopus and zebrafish and allows ubiquitous expression (Figure 1) (Lakin et al 1993; Thermes et al 2002). The pCska-gfp Tol2 construct was injected into one- or two-cell stage embryos of the MZM-0403 strain of N. furzeri in the presence or absence of Tol2 transposase mRNA.…”

Liver hyperplasia after tamoxifen induction of Myc in a transgenic medaka model

“…For ubiquitous expression of the construct, the cytoskeletal actin promoter of Xenopus borealis was utilized (Lakin et al, 1993; Thermes et al, 2002). Stable integration of the construct into the medaka genome resulted in two independent transgenic lines.…”

“…Additionally, the myc17 sequence was subsequently replaced by the full-length cloned myc20 sequence. The myc17ER construct was then subcloned into pI-SceI containing the cytoskeletal-actin promoter of Xenopus borealis for ubiquitous expression in the entire embryo (Lakin et al, 1993) and an SV40-poly A and named myc17ER I-SceI plasmid.…”

Characterizations of Hirudo medicinalis DNA promoters for targeted gene expression

“…The promoter regions of most vertebrate ␤-actin genes contain three CArG boxes and at least one CCAAT box (Danilition et al, 1991). Deletion studies of the vertebrate CArG box, which is thought to interact with soluble serum response factors, leads to significant loss of promoter activity (Lakin et al, 1993). Among invertebrates however, the picture is less clear.…”