The 150 independently isolatedthy−mutants ofE. coliK12 Hfr 3.OSO were studied genetically and phenotypically. Variants were found among the mutants in respect to the lag period of thymineless death, and temperature sensitivity. The latter correlates with mutations located at a specific site on the genetical map.Thethylocus is located between thecysandser/glygenes, and is a linear structure where 134thymutants are distributed over more than 17 sites. The site distribution of the mutants is not regular: about a half of them (62) are localized within one site and all these are temperature-sensitive.Two further genes involving utilization of thymine—tlrandtd—were found. Mutations oftlrlead to a reduced thymine requirement (0·5 μg./ml.instead of 20 μg./ml.). A mutation oftdresults in thymidine sensitivity.This latter character is expressed when thetd-sallele is transferred intoE. coliK12, prototrophic for thymine, by conjugation. Thymidine inhibition can be reversed by the addition of any riboside to the growth medium. Both genes map at the proximal end of the Hfr 3.OSO chromosome and are linked with thethrgene. The most probable gene order is:tlr-td-thr.The following results have been obtained from14C-thymine incorporation experiments with wild-type cells, as well as withthy−tlr+andthy−tlr−cells: (1) Wild-type cells incorporate exogenous thymine extremely poorly, but incorporate thymidine better. (2) Thethy−tlr+mutants are able to incorporate thymine only when high concentration are used, but can utilize a low concentration of thymidine. (3) Thethy−mutants are able to incorporate exogenous thymine as well as thymidine at low concentration. (4) Thetlrmutation is a thymine-specific one.

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We used the hybrid plasmid pAS8 in order to conduct the genetic analysis of RP4 plasmid. The presence of two replicons in the hybrid plasmid permitted to expand the spectrum of deletion mutants of RP4 isolated, which are capable to autonomous replication. The shortening of the hybrid plasmid was achieved by P22 transduction, by induction of deletion mutants using mitomycin C, as well as by seletion of Tra- mutants on the basis of resistance of cells to P-specific phages. These techniques have lead to isolation of clones possessing different combinations of plasmid resistance determinants. Comparison of phenotypic characteristics of deletion plasmids pAS9, pAS10, pAS11, pAS12 and pAS10-2 permitted to propose the map for pAS8 plasmid with the following sequence of markers: tra--kam--ColE1--amp--tet... Heteroduplex analysis of deletion mutants of pAS8 permitted to construct a physical map and to elaborate in greater detail the functional map of RP4 plasmid. The correlation between the ability of mutants to replicate in polA(TS) strain at nonpermissive conditions and the length of the deleted segment permitted to map rep genes of RP4 on a region with coordinates 9.8--17.3 kb. A relationship between the manifestation of incompatibility of mutants with Inc P-1 plasmids and the length of deletions points out that inc genes are located on DNA region with coordinates 2.1--9.8 kb. The analysis of replication of deletion mutants and the manifestation of incompatibility just as of the data about the size of appropriate deletions permitted to make the conclusion about the functional and genetic independence of the replication control and incompatibility control in RP4 plasmid.