Mitchell Stuart Gross

[81] Screening of colonies by RNA-DNA hybridization with mRNA from induced and uninduced cells

Publisher Summary Two classes of positive clones are detected by the screening procedure. One class, which was comprised of 2-3% of the total, hybridized very strongly to the probe and appeared as intense signals on the autoradiogram. The second class, which consisted of 10% of the total, was significantly less intense than those of the first class and varied in intensity over a wide range. The positive colonies were examined further for the presence of interferon-specific cDNA sequences by an mRNA hybridization translation assay. Intense colonies are shown to have no interferon-specific sequences, but two interferon cDNA clones-were identified among 59 of the less intense positive colonies. Partially purified interferon mRNA from induced cells are used for synthesis of cDNA for transformation of Escherichia coli χ1776.

Expression Using Vectors with Phage λ Regulatory Sequences

In the expression system described here, plasmids (pSKF) utilize regulatory signals‐such as the powerful promoter pL ‐from the bacteriophage λ. Transcription from pL can be fully repressed and plasmids containing it are thus stabilized by the λ repressor, cI. The repressor is supplied by an E. coli host which contains a integrated copy of a portion of the λ genome. This so‐called defective lysogen supplies the λ regulatory proteins cI and N but does not provide the lytic components that would normally lead to cell lysis. Thus, cells carrying these plasmids can be grown initially to high density without expression of the cloned gene and subsequently induced to synthesize the product upon inactivation of the repressor. This system also ensures that pL‐directed transcription efficiently traverses any gene insert, which is accomplished by providing the phage λ antitermination function, N, to the cell and by including on the pL transcription unit a site necessary for N utilization (Nut site). The N protein interacts with and modifies the RNA polymerase at the Nut site so as to block transcription termination at distal sites in the transcription unit. In order to express the coding sequence, efficient ribosome‐recognition and translation‐initiation sites have been engineered into the pL transcription unit. Expression occurs after temperature or chemical induction inactivates the repressor (see first and second basic protocols). Restriction endonuclease sites for insertion of the desired gene have been introduced both upstream and downstream from an ATG initiation codon. Thus, the system allows either direct expression or indirect expression (via protein fusion) of any coding sequence, thereby potentially allowing expression of any gene insert. Protocols describe direct expression of “authentic“ gene products, as well as heterologous genes fused to highly expressed gene partners generates chimeric proteins that differ from the native form. In the latter case, the fusion partner can be removed to obtain an unfused version of the gene product.

UNIT 16.3 Expression Using Vectors with Phage λ Regulatory Sequences

In the expression system described here, plasmids (pSKF) utilize regulatory signals‐such as the powerful promoter pL ‐from the bacteriophage λ. Transcription from pL can be fully repressed and plasmids containing it are thus stabilized by the λ repressor, cI. The repressor is supplied by an E. coli host which contains a integrated copy of a portion of the λ genome. This so‐called defective lysogen supplies the λ regulatory proteins cI and N but does not provide the lytic components that would normally lead to cell lysis. Thus, cells carrying these plasmids can be grown initially to high density without expression of the cloned gene and subsequently induced to synthesize the product upon inactivation of the repressor. This system also ensures that pL‐directed transcription efficiently traverses any gene insert, which is accomplished by providing the phage λ antitermination function, N, to the cell and by including on the pL transcription unit a site necessary for N utilization (Nut site). The N protein interacts with and modifies the RNA polymerase at the Nut site so as to block transcription termination at distal sites in the transcription unit. In order to express the coding sequence, efficient ribosome‐recognition and translation‐initiation sites have been engineered into the pL transcription unit. Expression occurs after temperature or chemical induction inactivates the repressor (see first and second basic protocols). Restriction endonuclease sites for insertion of the desired gene have been introduced both upstream and downstream from an ATG initiation codon. Thus, the system allows either direct expression or indirect expression (via protein fusion) of any coding sequence, thereby potentially allowing expression of any gene insert. Protocols describe direct expression of “authentic“ gene products, as well as heterologous genes fused to highly expressed gene partners generates chimeric proteins that differ from the native form. In the latter case, the fusion partner can be removed to obtain an unfused version of the gene product.