Recently, we described a new biological function of p53 in inhibiting recombination processes when encountering mismatches in heteroduplexes (DudenhoÈ er et al., 1998). Here, we characterized protein domains of p53 participating in this process by in vitro analysis of mutated p53 proteins, and by applying our SV40-based assay system on monkey cells, which express di erent p53 variants. We present evidence that both binding of arti®cial recombination intermediates and p53-dependent recombination control require an intact p53 core and the oligomerization domain, strongly suggesting that the recognition of DNA undergoing recombination represents an essential step of this genomic surveillance mechanism. Further analyses indicated a role of the C-terminus in negatively regulating recombination control, an e ect which can be neutralized by concurrent mismatch recognition. p53 lacking the oligomerization domain totally lost its ability to suppress homologous recombination. The cancer-related mutant p53(273H) was also signi®cantly defective in this function, although we observed only twofold reductions in the corresponding transactivation activities on p53-response elements in episomal constructs. HDM2, an inhibitor of p53's transcriptional and growth regulatory activities, interfered with the inhibition of DNA exchange processes by p53 only weakly. Thus, functions of p53 in recombination control can be structurally dissociated from p53-dependent transcriptional transactivation.

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The serine/threonine-specific casein kinase I delta (CKIdelta) is ubiquitously expressed in all tissues, is p53 dependently induced in stress situations and plays an important role in various cellular processes. Our immunohistochemical analysis of the human placenta revealed strongest expression of CKIdelta in extravillous trophoblast cells and in choriocarcinomas. Investigation of the functional role of CKIdelta in an extravillous trophoblast hybrid cell line revealed that CKIdelta was constitutively localized at the centrosomes and the mitotic spindle. Inhibition of CKIdelta with the CKI-specific inhibitor IC261 led to structural alterations of the centrosomes, the formation of multipolar spindles, the inhibition of mitosis and, in contrast to other cell lines, the induction of apoptosis. Our findings indicate that CKIdelta plays an important role in the mitotic progression and in the survival of cells of trophoblast origin. Therefore, IC261 could provide a new tool in treating choriocarcinomas.

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Using CaCl, mediated transfection with Lambda DNA fragments, in vitro joining by ligase and in vivo recombination with helper phage DNA are effective systems for generating artificial recombinants. Recombination efficiencies are 20-30% in the i n vitro and i n vivo recombination systems.At 30 t o 37 "C T4 ligase mainly joins natural cohesive 1 ends, while at 12 "C the EcoRI-generated termini are preferentially ligated to form biologically active molecules, if the cloning vector 1401 is used, which has only one EcoRI target. The ligation products were characterized by gel electrophoresis and CaCI, transfection.For i n vivo recombination a new CaCl, transfection system was developed, termed postinfectiondependent CaCI, transfection system, which is based on the infection of recipient cells with helper phages after transfection. I n marker rescue experiments using this method not only single but also double recombination occurred between two independent 1 DNA fragments and the helper phage DNA.A direct and facile method for in vitro production of hybrid DNA molecules is ligase joining that of cohesive termini generated by restriction endonuclease digestion. It is known these artificial recombinants can be propagated using a vector DNA molecule capable of replication in a suitable host cell. The DNA of bacteriophage Lambda has become a useful vector for cloning experiments since constructed derivatives of 1 DNA with only one EcoRI restriction target per chromosome have been available (MURRAY and MURRAY 1974, MURRAY et al. 1977).The experiments described here were carried out in order t o determine T, ligase reaction conditions under which the majority of the reaction products are biologically active linear 1 DNA molecules. We investigated different T, ligase reaction conditions to analyse the influence of temperature, DNA concentration and incubation time on the character of ligation products of A wild type DNA and of the cloning vector A 401. We used agarose gel electrophoresis of ligated A wild type and CaCl, transfection of 1 401 DNA fragments to characterize the joining products.Furthermore a method for generating artificial recombinants in vivo was developed: the postinfection-dependent CaC1, transfeetion system, a modification of the standard CaCl, transfection system of MANDEL and HIGA (1970), based on the infection of recipient cells with heteroimmune helper phages after transfection. In this system recombination of A DNA fragments with the helper phage DNA takes place in the recipient cells.

This paper presents further parameters influencing the competence, the process of DNA uptake and the efficiency of plating of CaC12-treated E. coli D12 strains. We have found that the process of DNA uptake depends not only on the treatment of bacteria with a certain CaCl2-concentration but is also influenced considerably by a shift-down of the CaCl2-concentration in the reaction mixture. The pH of the growth media and of the reaction mixture plays an important role in maintaining of optimal transfection. The efficiency of plating is influenced by the thickness of the top layer and the concentration of bacteria on the plate. Without genetic variation of the strains, by only varying the mentioned factors we could improve the efficiency of CaCl2 transfection at about two orders of magnitude to a maximum of 6 X 105 pfu/microgram DNA.

This paper presents further parameters influencing the competence, the process of DNA uptake and the efficiency of plating of CaCI,-treated E. coli K12 strains. We have found that the process of DNA uptake depends not only on the treatment of bacteria with a certain CaClponcentrotion but is also influenced considerably by a shift-down of the CaC1,-concentration in the reaction mixture. The pH of the growth media and of the reaction mixture plays an important role in maintaining of optimal transfection. The efficiency of plating is influenced by the thickness of the top layer and the concentration of bacteria on the plate.Without genetic variation of the strains, by only varying the mentioned factors we could improve the efficiency of CaCl, transfection a t about two orders of magnitude to a maximum of 6 x lo6 pfu/pg DNA. 520-525. YAMAMOTO, K. R., ALBERTS, B. A., BENZINGER, R., LAWHORNE, L. and TREIBER, G., 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large scale virus purification. Virology, 40, 734-744.