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in 5-bromouracil. Because of their origin and their measurable reversion fre- quencies, we shall refer to them as point mutants. These mutations (1-1-21 and.
ADDITIONAL EVIDENCE FOR TWO KINDS OF HETEROZYGOTES IN PHAGE T4* BY CHANNA SHALITINt AND FRANKLIN W. STAHL INSTITUTE OF MOLECULAR BIOLOGY AND DEPARTMENT OF BIOLOGY, UNIVERSITY OF OREGON, EUGENE

Communicated by A. D. Hershey, September 16, 1965

The work of Nomura and Benzerl suggested that there are two structurally distinct kinds of heterozygotes (HETS) in T4, and that genetic deletions can participate in the formation of only one of them. In the preceding paper2 it was shown that the frequency of point-mutant HETS, but not that of deletion HETS, is increased by fluorodeoxyuridine (FUDR) which reduces the amount of DNA duplication without diminishing the rate of genetic recombination. This observation confirms the suggestion of Nomura and Benzer. It furthermore lends support to the reasonable idea that point mutants can form heteroduplex ("internal") heterozygotes, whereas deletions are forbidden to do so. Deletion HETS presumably signal terminal redundancies in chromosomes whose genetic sequences are circular permutations of each other. In this paper we present further evidence of the existence of two kinds of HETS. Our observations again relate the two kinds of heterozygosity to genetic recombination. In the progeny of crosses between two rII mutants in the same cistron, an appreciable fraction of the particles that can grow in K(X) (i.e., the wild-type recombinants) are heterozygous at one or the other of the two loci involved. These particles have been termed "recombinant HETS" by Edgar.1 In the experiments described below, four mutants in the B cistron of the rII region were used. Two of these (r196 and r187) are rather small deletions which were isolated, mapped, and kindly provided by Dr. S. Benzer. The deletions lie near opposite ends of the B cistron of the rII region. The other two mutants were isolated by Stahl and Dr. M. Meselson from a wild-type stock of T4B after growth in 5-bromouracil. Because of their origin and their measurable reversion frequencies, we shall refer to them as point mutants. These mutations (1-1-21 and 2-1-4) also lie near opposite ends of the B cistron at approximately the same positions as r196 and r187, respectively. The following crosses were performed in the presence and absence of FUDR: (1) point mutant by point mutant, (2) deletion by deletion, and (3) point mutant by deletion. In the first two crosses, the relevant datum is the fraction of recombinant HETS among particles that can grow in K(X). This fraction is measured as originally described by Edgar.3 The lysate of the cross is adsorbed at low multiplicity (10-3) to Escherichia coli strain K12-112(Xh), in which the leaking rate for the mutants employed is about 10-4. Unadsorbed phage is inactivated by antiserum and further eliminated by centrifugation. The infected K(X) cells are distributed before lysis over plates seeded with E. coli S/6. On these plates wild-type, r, and mottled plaques are distinguishable. The fraction of HETS among wild-type recombinants is defined as the number of mottled plaques/the number of mottled plus wild-type plaques. Since a negligible number of r plaques is seen under the conditions em1340

VOL. 54, 1965

GENETICS: SHALITIN AND STAHL

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ployed, the denominator is essentially equal to the total number of plaques examined. In the third cross, the pertinent datum is the relative frequency of heterozygosis at point and deletion mutant sites. Progeny of the cross were plated as described above, and 200 mottled plaques were sampled from both the FUDR and the control lysates. Each sample was replated, and three of the resulting r plaques were spottested to determine genotype.4 As long as the original mottled plaques were picked from uncrowded plates (< 40 plaques/plate), the r's from any one mottler were invariably of the same genotype, either point mutant or deletion. Thus, any given mottled plaque could be classified as containing either the point mutant r or the deletion r in addition to both r+ alleles. The results of the three crosses are summarized in the table. All three show that FUDR exalts the fraction of recombinants that are heterozygous for a point mutant but not those heterozygous for a deletion mutant. TABLE 1 DIFFERENTIAL EFFECT OF FUDR ON THE TWO CLASSES OF RECOMBINANT HETS Type of cross

Condition

Point mutant X point mutant No FUDR FUDR NoFUDR Deletion X deletion FUDR NoFUDR Point mutant X deletion FUDR

Frequency of HETS among wild-type recombinants (%)

16 63 5.8 5.8 12.5 34

Fraction containing the deletion

Fraction containing the point mutant

-

-

28/200 5/200

172/200 195/200

The general procedure has been described.2 At 3 min after infection (at 370) the culture was divided and FUDR was added to one part at a final concentration of 4 X 10-' M. At 9 min after infection, 125 pg of chloramphenicol per ml were added to both parts. At 90 min after infection the chloramphenicol was removed, aeration was resumed for 90 min longer, and the bacteria were lysed with chloroform. FUDR-treated cultures yielded 2-14 phage particles per bacterium. Control cultures yielded 40-100. The resulting lysates were examined as described in the text.

We conclude that recombinant HETS, arising in crosses between rII markers and selected by passage through strain K(X), are of two types. These types appear to correspond precisely with the HETS arising in one-factor crosses and detected solely on the basis of their ability to form mottled plaques.2 One quantitative aspect of the results requires additional comment. The high frequency of HETS (16%) among wild-type recombinants arising in crosses between point mutants is an expectation of the model described in the preceding paper.2 On the other hand, the high frequency (5.8%) of HETS among wild-type recombinants arising in crosses between deletion mutants is not so obviously predictable from that model. It indicates a rather strong correlation between crossing over and terminal redundancy and implies either that ends of T4 chromosomes engage in crossing over at a higher rate than do other regions, or that crossing over determines the location of the end. * Supported by a research grant to F. W. Stahl from the National Science Foundation (GB-294). t Supported by a fellowship from the U.S. Public Health Service (FF-415). Present address: The Weizmann Institute, Rehovoth, Israel. 1 Nomura, M., and S. Benzer, J. Mol. Biol, 3, 684 (1961). 2 S6chaud, J., G. Streisinger, J. Emrich, J. Newton, H. Lanford, J. Reinhold, and M. M. Stahl, these PROCEEDINGS, 54, 1333 (1965). 3 Edgar, R. S., Genetics, 43, 235 (1958). 4 Benzer, S., these PROCEEDINGS, 47, 403 (1961).