Leu-enkephalin purification from E. coli cells carrying the plasmid with fused synthetic leu-enkephalin gene.

Chemically synthesized leu-enkephalin gene was fused to a large Eco RI-Bam HI fragment of pBR322 along with a Eco RI fragment of Ch4A phage DNA carrying the promoter and most of the E.coli beta-galactosidase gene. The resulting recombinant DNA was used to transform E. coli cells. Transformants were screened for Tc-sensitivity, Am-resistance, and beta-galactosidase constitutional synthesis. Restriction endonuclease analysis combined with DNA sequencing of the plasmid DNAs revealed a complete nucleotide leu-enkephalin sequence and Eco RI lac-operon fragment in two possible orientations. Radioimmunoassay for leu-enkephalin activity in BrCN-treated bacterial extracts showed that in vivo leu-enkephalin is synthesized only in strains carrying plasmids with the proper lac-fragment orientation. About 5.10(4) molecules of the former are synthesized per single E. coli cell. One of the clones was used for leu-enkephalin purification. Using 100 g of cells it is possible to obtain about 2 mg of practically pure leu-enkephalin.Images

[Cloning of fragments of lambda phage DNA, containing red and gam genes]. / Klonipovanie fragmenta DNK faga lamba, soderzhashchego geny red i gam.

On the base of plasmid pCV20 (Apr, Tcr mol. weight 5.2 x 10(6) a recombinant plasmid pEH60 (Apr, mol. weight 17.0 x 10(6) with BamHI fragment of phage DNA, containing red+ and gam+ genes was constructed. Selection was found on the ability of phage red- and gam- to propagate in strain E. coli K12 recA-, which was transformed by recombinant plasmid with active red and gam genes. Influence of recombinant plasmid pEH60 on processes of repair and recombination of phage lambda DNA and bacterial DNA was studied. It was shown that red gene in plasmid pEH60 compensates deficiency of redA gene in these processes with phage lambda DNA; in the case of E. coli K12 AB2480 uvr- recA- (pEH60) the processes of multiple reactivation and decombination of phage red- were presented. In the case of bacterial cells, plasmid pEH60 did not compensate deficiency of recA function of bacteria, although it partly compensates deficiency of recBC function. Increase of survival after introduction of plasmid pEH60 in the cell was obtained only for recBC- strain, but not for wild type and recA- strains.

[Repair of recombinant plasmids]. / Reparatsiia rekombinantnykh prlazid.

Comparative analysis of UV-sensitivity was carried out on plasmids of various molecular weight. Recombinant plasmids containing fragments of prokaryotic DNA (E. coli, phage lambda) are repaired in E. coli cells more effectively than those containing eukaryotic DNA fragments. It was also shown that UV-sensitivity of recombinant plasmids is independent of their molecular weight provided that the active repair process in fact occurs. UV-sensitivity was strongly proporational to DNA size only when E. coli double mutant strain was used as host. The survival of plasmids strongly increased after M. luteus UV-endonuclease treatment when E. coli mutant strain uvr- was used as host, but remained practically constant in case of wild type strain. Agarose gel electrophoresis data provide evidence that DNA double-stranded breaks appear un UV-irradiated as well as in UV-endonuclease treated plasmids. One can suggest that UV-inactivation of plasmids results from DNA breaking as a consequence of repair gaps overlap when wild type strain is used as host. Mathematical analysis was carried out assuming this possibility. Experimental data are shown to fit theoretical values calculated assuming that the repair gap size in one DNA strand is equal to 2000-3000 bases.

[Phenomenon of w-reactivation in plasmids]. / Fenomen w-reaktivatsii na plazmide.

An analysis of the action of the bacterial repair system on the UV-irradiated pMB9 plasmid has been carried out. It has been shown that the UV-irradiated plasmid is repaired in the bacteria similarly to the DNA of bacteriophage (lambda): the effectiveness of the UVR-system is lowered two-three times, and the postreplicativing REC system acts only at low doses of the UV-light. The phenomenon of omega-reactivation is observed both with plasmid and phage DNAs.