Experimental manipulation of gene expression / edited by Masayori Inouye.

Date:
1983
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Experimental manipulation of gene expression / edited by Masayori Inouye. Source: Wellcome Collection.

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    12 a. Shatzman, y.-s. Ho, and m. Rosenberg inserted into this vehicle. Blunt-ended fragments can be inserted directly, where¬ as other restriction fragments must first be made blunt-ended. This is accom¬ plished by either removing the 5' and 3' overhanging ends with mung bean nuclease (as described previously) or by filling in the 5' overhanging ends with DNA polymerase. Of course, even this procedure still limits the use of pASl to those genes that contain appropriate restriction information near their 5' termini. In order to make the pASl system generally applicable to the expression of any gene, a procedure was developed that allows precise placement of a new restriction site at the second codon (or any other codon) of any gene. Creation of this site permits fusion of the gene in frame to the cIl initiation codon of pAS 1. The general procedure has been outlined by Maniatis et al. (1982) and described in detail by Rosenberg et al. (1982a). This procedure has been used to adapt several genes for insertion and expression into pAS 1. The small-t-antigen gene of SV40 does not contain an appropriate restriction site at its 5' end. Using BaB\ exonucleolytic digestion from an upstream re¬ striction site, the first base (G) of the second codon of the small-t gene (ATG GAT. . .) was fused to an upstream, filled-in Aval restriction site (. . .CCCGA) (C. Queen, personal communication). The fusion, (...CCCGAGAT...) recreates the Aval site precisely at the second codon of the small-t gene. Restriction of this vector with Aval followed by mung bean nuclease digestion produces a blunt end that was fused in frame to the blunt-ended ВатШ site of pAS 1. The resulting vector pASlt expresses authentic SV40 small-t antigen entirely from phage- regulatory signals. After only a 60-min induction period, small-t antigen repre¬ sented some 10% of the total cellular protein (Fig. 7, left). Moreover, ^^S-pulse- labeling experiments indicate that small t is the major product synthesized in these bacteria after temperature induction (Fig. 7, right). D. Expression of Functional Mammalian Metallothioneines Procedures similar to those just described have also been used to adapt, insert, and express both the mouse and monkey metallothioneine genes in the pASl vector system. The mouse gene (a gift from R. Palmiter) contains a unique Avail restriction site at its second codon (5' ATG GAC CCC 3'). Cleavage with Avail, followed by fill-in of the 3-base 5' overhanging end with DNA polymerase, creates a blunt end before the first base pair of the second codon. This blunt- ended fragment was inserted into the mung bean nuclease-treated pAS 1 vector. The resulting fusion places the second codon of metallothioneine in frame with the initiation codon of ell. This construction (pASl-MTLl) results in high levels of synthesis of the authentic metallothioneine gene product in E. coli (Fig. 8). The protein produced in bacteria has been partially purified and shown to have