Section 3 — Molecular and Microbial Genetics 



introduced transforming marker, or whether 

 the original mutation had been replaced by the 

 introduced marker. In inter-specific transfor- 

 mations involving two low-level and one high- 

 level pneumococcal mutations, one low-level 

 and one high-level streptococcal mutations, 

 the results demonstrate that mutations affecting 

 a similar function (streptomycin-resistance) 

 but arising in different bacterial species are 

 genetically linked. 



3.34. Biochemical and Genetic Studies of Integra- 

 tion and Recombination in the B. subtilis 

 Transformation. Walter F. Bodmer and A. T. 

 Ganesan (Palo Alto, U.S.A.). 



DNA was isolated from a multiple marked 

 strain of B. subtilis grown on N 15 H 2 medium 

 with added H 3 thymidine. Competent cells 

 were prepared in an N 14 H 1 medium containing 



p32 



Using the labelled DNA as donor, transforma- 

 tion was terminated at 30 min with deoxyribonu- 

 clease. The DNA purified from the donor- 

 recipient complex was fractionated pycnogra- 

 phically. Donor and recipient DNA differed 

 in buoyant density and were traceable by differ- 

 ential counting of P 32 and H 3 . Donor atoms were 

 found in density strata corresponding to native 

 donor but also in native recipient, hybrid, and 

 "denatured" donor DNA. These DNA fractions 

 were assayed for their genotypic content by 

 transformation. Donor and recombinant geno- 

 types were found in all of these strata but the 

 last, which is biologically inactive. 



The association of donor atoms (i.e. H 3 ) with 

 a recipient stratum persisted on refractionation 

 of the DNA, and was also found when transfor- 

 mation was terminated after only 10 min. The 

 number of donor atoms found in the other strata 

 decreases when the cells were allowed to grow 

 after transformation. However, the H 3 count 

 in the recipient DNA stratum remains approxi- 

 mately constant. The continuous distribution of 

 H 33 label over strata of varying densities implies 

 the integration of donor DNA segments, which 

 were considerably smaller than the average size 

 molecule present in our preparations of trans- 

 forming DNA. 



3. 35. DNA - mediated Transformations to Improved 

 Enzymatic Activities. Stephen Zamenhof and 

 Lilo H. Heldenmuth (New York, U.S.A.). 



A mutation resulting in an improvement in 



enzymatic activity (and, sometimes, in survival 

 or in adaptive value) over and above that of a 

 wild strain, may be caused by a change in regu- 

 latory mechanisms or an "improvement" in the 

 structural gene determining the enzyme. At 

 least the latter should be a change in DNA 

 and might be transferable by DNA-mediated 

 transformation. 



In the present study several spontaneous 

 mutants of Bacillus subtilis were isolated having 

 various enzymatic activities improved over 

 those of the wild strain. The DNA of some of 

 these mutants was able to transform the wild 

 strains in a single step to strains with improved 

 enzymatic activities. The transformable im- 

 provements studied to date were: (1) In utiliza- 

 tion of citrate as the only carbon source; (2) In 

 production of paraminobenzoic acid (PABA); 

 (3) In production of phenylalanine. Other im- 

 provements will also be discussed. Sulfanilamide- 

 resistant strains of B. subtilis, first isolated by 

 S. Greer in this laboratory, did not require sul- 

 fanilamide for growth but were better PABA- 

 producers than the wild-type; both features are 

 transformed concomitantly, in both directions. 

 Some of the (3-thienylalanine-resistant mutants 

 were better phenylalanine-producers; both fea- 

 tures are also transformed concomitantly. The 

 improvements that could not be transferred by a 

 single step transformation were those which 

 accumulated in different strains as a result 

 of more than one mutation, e.g. slime produc- 

 tion, or utilization of pyruvate as the only car- 

 bon source. 



3.36. Participation of RNA in the Transfer of 

 Genetic Information of a Virus of the DNA- 

 type. S. Gershenson (Kiev, U.S.S.R.). 



Injection into healthy larvae or pupae of 

 Bombyx mori of RNA isolated from those in- 

 fected with nuclear polyhedrosis virus causes a 

 high frequency of the disease, whereas RNA 

 from healthy B. mori is non-infectious. The 

 technique used for isolating the infectious 

 RNA (extraction with phenol, high speed cen- 

 trifugation) makes it improbable that the infec- 

 tivity of the preparations is due to an accidental 

 admixture of the virus. This possibility is further 

 disproved by the fact that these preparations 

 are infectious for larvae of Pier is brassicae wich 

 are completely immune to the virus itself. The 

 virus developing in P. brassicae infected with 

 RNA from virus-diseased B.mori is infectious 

 for B.mori but not for P. brassicae. Infectivity 

 of the RNA preparations from virus-diseased 

 B.mori is destroyed by RN-ase but not by DN- 



30 



