Terminal Report to the Natural Sciences Division Rockefeller Feundaticn July 1, 1951. Froject: Genetics of Becteria Mey 1, 1948 to Avrfl 20, 1951. Submitted by: Joshua Lederverg, Associate ?rofessor of (enetics University of Wisecnsin Medison, Wis. 1. Wormel genetics end life cyele. Qur central problem has to do with the sexuel cycle of commen becteria, especially the colon bacillus, Escherichia golf. The methods used to i vosti~ nate this cycle have been indirect, and based upon the finding that genetic recombination takes place when cells of different genetic o-netitution -re alloved to grow in close preximity to esch other. Selective methous hve been used to detect the occurrence of these recombinetions of genes. 4 good devl of time has been spent on elucidating the so-called formal genetic structure of this orgenism by means of breeding tests involvin- = erast many different oharccteristics, mostly of a biochemical nature, tht “ve under eontrel of the genes. Ac a result, we can affirm that the st-nderd Life evcle of this bacterium is r«ther simil«r to thet of Neurospore or cer tain yeasts. The normal vegetative phase is haploid, i.e., the nucleus carries 2 single 36+ of genes, although there 19 both cytological and eenetic evidence that individual celle may carry two, four or eight aimiler nuclei which sort out in successive fissiocns. On rere occasions, the haploid nuclei from differ ant cells fuse to give a trensfient dirloi¢ zygote. Usually thie undergoes frmediste reduetion-division to restore the hea vloic ccendition, and in the course of this reduction, crossing-over with genetic recombination may take place. Certain exceptional strains heve verified this picture, These ere in~ stances vhere the reduction division ia delayed, or where there is = sec ndary re-fusion of products of reduction, giving rise to cultureswhich persist in the diploid condition. These cultures are heterozygous for most or 711 of the feeters that distinculshed the parents, end therefore make it possible t test for genetic dominance. ‘Ye find thet bicchemical capacities are in general, dosineant tt: defects, es is typiesl] of other orgeniame .1lso. Host interesting, suscertibility to deleterious azents such as bacterflophece or satrent mycin he. ge far seen douwinant to realstanee, so thet resistant mitations mey be nese 4n the heterozygous condition. The heterozyzotes heve provided the best proof, so far, of the reality of bacterial hybridization. Single cell faolations by M,R. Yelle end J. Lederberg showed unequivocally that the genetic qualities of two r-rantal celis coexist in stngle bectericl cells, subject tc later separati-n end recesortment. To date, we have been unsble to dem-natrste directly the norcho- legical basis of reeembinetion in Ee coll, mostly because of the infre uency with which 1% oceurs ~ one cell per million - in the experimental stocks of tiis organism, ‘Ye can infer, however, © sexual fusion probably invol- ine intact cells or elements of aimiler size, So far, we heve found no evidence of sexuel differentiation, 4.6., stocks whien might be lebelled meals =nd femsle vos;ectively, so thet enjugetion e p-rently cecurs st rand-m between ¢ lis Of wre or olxed cultures, but can be detected only in terms of reessortuent between cenetically differing celis. The diploid cultures have, however, been subjected to a cytological study in com rison with haploids, This stud; is still in progresa, but thera is no question of conaistent differences in nuclesr structure, the dirloids show ine lareer numbers of -ranular elements (chromosomes?) in eech nuclear arere ste. Studies are #iso in progress of the effects of radiationea on the cytozenctics of divloids., These show thet persistent effects can often be detected in some descendents of » treated call lones after cthers a pear tre have recovered com yletely. The main result so far fe thet the effects are ac comlex 2s t- require thet extreme caution be exercised in interpreting details of ex-eri-~ nenta on setinn of mutagente acents! the cell is an orgenized arrrezste of lex than a chenicel reaction between a mutagen moleaule with a localized gene. 2, Physiological genetics. The groundwork deseribed in the oreviouns section hea made it possible to cerry out work on mechaniems of gene section with some assurance thet these findings with bacterise will be vertinent to the general problems of physto-~ logical genetics. The character that has been subjected to the most intensive study hes been the fermentation of lectose, for the following reasons: a) it ie easily char acterized in single colonies of the becterle with the helr of indiector medla, b) 4t involves a simple enzyme whese acticn is limited to a single, directly measursble step, and which ie readily purified: «) a lerge number of utente affecting this character were readily obtained, end ad), it represents on important diagnostic charecteristic in taxonomy so that mutations effecting it may bear on the problem of bacterial speciation. On the other hand, although enzymes «re likely to be more or less direct products of gene ection, our leck of any real knowledge of the mechenisme of specific protein synthesis lergely precludes our learning very much at this time sbout the crucial rele leyed by genes in such ecyntheses,” This work was initiated vrimarily to test the "one-to-one theory" which sggested that the primary action of 2 given gene was to eontrol the a ecificity of a gingle enzyme, and conversely, that » given enzyme wae directly related to a single gene. With reapect to lactase, our findings sugeest thet et le-st seven different genes may affect the form=ticn of one enzyme, and elec thet at least tw: of these genes effect enzymes In addition to lactase. All of the mut-tions that could be analyzed proved to involve the conditions or extent of formation of lactase, rather than ite quelitative properties, so thet it = ow is possible thet none of these genes is directly related to lactase, If this voint-of-view fs insisted upon however, the one-to-one theory becomes immme to experimental disproof, and to thet extent of limited scientific value. It is expected thet different strains of B, soli may produce lacteses of sufficiently different specificities thet 1% may become possible tc investi- gate the details of gene control of enz;me quality, rather than solely its formation. Tt has been suggested that phyaiologioc] gramiles, if our present sup- positions are verified, is rather slong the lince of organized "gametes", worse inviabllity 49 a result cof limitations of the usual bactericlogical medic, Yn edditicn to their relevance to specifically genetic problems, the Ieforms mey play « hitherto poorly erprecisted role in many eapects of theoretical end ayplied microbiclogy. 4, Genes and viruses. In Fe gold K-12, all of the many characters ro far tested have been shown to denend on factors in the organized genotype, presumebdly in the ~ Fe nucleus.