examples of this pattern of investigation could be cited from developmental studies.

VOL. 49, 1963 GENETICS: B. WALLACE 801 4Taylor,J.H.,Genetics,43, 515(1958).

6Taylor, J. H.,Proc.10thIntern.Congr. Genet., 1, 63 (Montreal, 1958).

6Freese, E., in Exchange of Genetic Material; Mechanisms and Consequences, Cold Spring Harbor Symposia on Quantitative Biology, vol. 23 (1958), p. 13.

7LaCour,L. F.,and S. R.Pelc, Nature, 182, 506 (1958). 8Woods,P.S., and M. V. Schairer, Nature, 183,303 (1959).

9Kaufmann, B. P., H. Gay, and M. McDonald, Intern. Rev. Cytol., 9, 77 (1960); Ris, H., Canad.Journ. Genet. and Cytol., 3,95 (1961); Steffensen, D., Intern. Rev. Cytol., 12, 163(1961).

10Wilson,G.B., A. H. Sparrow, and V. Pond, Amer. Journ. Bot., 46, 309 (1959); Peacock, W. J., Nature, 191, 832 (1961).

I Howard, A.,and S. R. Pele, Exptl. Cell. Res., 2,178(1951).

12Neary, G. J., H. J. Evans, and S. M. Tonkinson, Journ.Genet., 56,363 (1959).

13Wimber,D.E.,thesePROCEEDINGS,45,839(1959).

14Forro, F.,andS. A. Wertheimer, Biochim. etBiophys.Acta, 40,9 (1960). 15Wimber,D.E., Amer. Journ. Bot., 47,828 (1960).

I' Taylor, J. H.,Intern. Rev. Cytol., 13,39 (1962).

17Lima-de-Faria,A., Progress in Biophysics and Biophysical Chemistry, 12, 282(1962).

Refer-ence tounpublisheddata of T.Nordquist,p. 292.

A COMPARISON OF THE VIABILITY EFFECTS OF CHROMOSOMES

IN HETEROZYGOUS AND HOMOZYGOUS CONDITION*

BYBRUCEWALLACE

CORNELL UNIVERSITY

CommunicatedbyTheodosiusDobzhansky, April 24,1963

Geneticiststendtothink ofmutantgenes in termsoftheireffectson

homozygous

carriers. In part thistendency exists because themajorityof genes are recessive;

for most practical purposes heterozygotes appear to be normal. Furthermore, homozygousstocks ofdiploid organismsare easier to maintain than heterozygous

ones; consequently, the homozygotesareavailableforexperimentation. Atypical studyof geneaction, for example, involvesacomparisonof normalindividualsand mutant homozygotes. A comparative study of heterozygotes, to determine the effects of the mutation in single

dose,

is made secondarily, if at all. Numerous

examples

ofthis pattern of

investigation

couldbecited from

developmental

studies.

In populationgenetics, work of Dobzhansky et

al.,'

of Hiraizumi andCrow,2 and

ofWallaceandDobzhansky3 serve asillustrations; theroutineprocedure hasbeen toinventory thegeneticvariation inapopulationbytestsofhomozygotesand then

toassaytheroleofthis variation in

populations by studying heterozygotes.

Inthe studyof

populations,

there is good reason to reverse the usualprocedure

for investigating gene effects. Suppose a previously nonexistent mutant allele arises within apopulationofcrossbreedingindividuals. Neglectingchance events thisnewallelewillbeeliminatedfromorestablishedinthepopulationaccordingto its effect on heterozygous individuals (Parsons and Bodmer;4 Wallace5). The

adaptedness of individuals homozygous for the new mutation becomes important only after the mutant allele has reached fairly high frequenciesin thepopulation.

The homozygotes do not even affect the elimination-establishment alternative; they merelyhelp todetermine final gene

frequencies.

In a sense, then, what occurs in natural populations is precisely the reverse of whathappensinexperiments; experimenters aregenerallyunaware thattheyhave amutationuntil theyhaveobtained ahomozygote; naturalpopulationscontain few or no homozygotesuntilheterozygotes havebeensubjectedforseveral generations to testsforsurvivalandhave provedthemselves superior in fitness to the old popula-tion average.

In a recent publication, Wallace and Dobzhansky3 were able to give a much

clearer pictureofthe relationbetween the viability effectsofchromosomes expressed

in homozygotes and heterozygotes than was previously available. In earlier analyses, chromosomeswerearranged inorder accordingtotheascendingviabilities of homozygotes indicated by ratios of contrasting types of flies in appropriately devised test cultures. These homozygotes were then grouped into a convenient

number of classes, and anaverageviability was computed for individuals carrying thechromosomes of each class inheterozygouscondition.

Thus,

oneobtained for groups of chromosomes seemingly comparable values expressing their average

via-bilityeffects inhomozygousandheterozygousindividuals.

Thevalues obtained bytheseprocedures were,however, notstrictly comparable. The "ordered" viabilities of homozygotes were fixed by the original observations

themselves, while the calculated viabilities of heterozygotes were averages based on a number of independent cultures. The latter are alwaysverysimilar to one

another; furthermore, theyarealwayssubstantiallylower than the viabilities of the

highest "ordered" homozygotes. As a rule, theregression of theviabilityof hetero-zygotes onthatofhomozygotes ispositive; this regression canbe interpretedas a measureofdominanceofdeleteriousmutations. Sincethe viabilities ofhomozygotes

and heterozygotes are in fact not comparable, estimates of dominance based on the slopeofthis

regression

aremeaningless.

The error inherent in the above procedure is eliminated by comparing the

via-bility of heterozygotes, notwiththatof"ordered" homozygotes, butwith the

via-bilities ofthese same chromosomes observed inreplicate cultures of homozygotes. Whereas the viability of heterozygotes generally increases steadily with that of

orderedhomozygotes (atleast in the upper portion of theviability range),the viabil-ity of individuals homozygous for these very same chromosomes does not increase, ordoes soat a ratelessthan that ofheterozygotes. Consequently,wefindthat the curverepresenting therelationshipbetweenthe viabilitiesofhomozygotes (as

meas-ured in replicate cultures) and heterozygotes is composed of a horizontal segment

andasharply upturned

(perhaps

vertical) terminal segment. In the caseofboth Drosophila melanogaster and D. pseudoobscura, theviability ofheterozygotes rep-resented by the horizontal segment is greater than that observed among replicate

culturesofthehighest ordered homozygotes.

The analysisdescribed below utilizes replicate cultures in evaluatingtheviability effects ofchromosomes inhomozygous and heterozygouscondition. Inthe present

analysis, however, the initial ordering is that of heterozygotes. The question we pose is, then, "What kinds of homozygotes are obtained from wild-type chromo-somesthat giveheterozygotesofsuccessively higherviabilities?" The experimental

technique presentsuswith ananswertoanother question aswell. There are two classes of heterozygous individuals in each culture, either one of

which

can be

"ordered";

thus,

we can also ask, "Whatkinds-of heterozygotes of one sortarise

VOL. 49, 1963 GENETICS: B. WALLACE 803

from chromosomes that give successively greater viabilities in heterozygotes of a different sort?"

The material used in this study is that ofDobzhansky, Krimbas, andKrimbas.1 It consists of about 1,000 second and third chromosomes of D. pseudoobscura from Texas andCalifornia tested generally in three replicate cultures at each of two tem-peratures,

160

and

250. The

statisticalanalysis wascarried outentirely within a temperature, butwith all other variables combined so that the results would be as general as possible.

The test of viability used by Dobzhansky et al. is that involving two laboratory chromosomesmarked withdominant genes,

D,

and D2, giving four classesof flies in each testculture:

D,/D2,

D,/+,

D2/+,

and

+/+.

(Theactual mutations used were Bare, Lobe, Blade

Soute,

and Delta.) The viability of the various classes

offlies ineach culture aremeasured relative to that of D1/D2whichisarbitrarily assigned the valueofunity.

To analyze thismaterial we have sorted cultures initially into ten classes on the

basis ofthe viability exhibited by one ofthe heterozygous classes

(D,/+,

for

ex-ample),and then, under that "ordered" classification, wehave noted the viabilities

ofD1/+ ("same"heterozygote), D2/+ ("other" heterozygote),and

+/+

(homozy-gote) ofthe two replicate cultures. Each ofthe hetero7ygous classes

(D,/+

and D2/+) served in turn as the basis for the initial, "ordered" classification (with

"same" and "other" heterozygotes reversed); furthermore, each of the replicate cultureswasused in turn asthebasis for the"ordered" classification.

The results of this analysis are

given

in

Table

1. In the leftmost column are listed

the

classes intowhichthe heterozygotes wereordered. To the right of- this

column arethree othersthatgivethe average viabiities observed inreplicate cul-turesfor heterozygotes thatwereofthesamegenotype asthose ordered

("same"),

heterozygoteg that were of the other genotype ("other"), and wild-type

homozy-gotes. The rightmost column gives the numberofentries uponwhich eachvalue isbased; the sum ofthese,of course,is roughlytwice theactual number ofcultures,

since inmostcaseseach ordered culturecalled for

the

entryof data from two

repli-catecultures.

The first pointtoemerge fromTable 1 (see Fig. 1) isthe linear relationship be-tweentheviabilityofthe "ordered"

heterozygote

andthe "same" heterozygotein

replicate cultures.

This

linearity is precisely what was not observed in a

com-parableanalysis-ofhomozygotes; inthelattercasethecurvedeviatesquicklyfrom

linearityand becomes horizontal (WallaceandDobzhansky3).

TABLE 1

COMPARISON OFTHE VIABILITIESOFORDEREDHEThROZYGOTES WITH VIABILITIES OFTHESAME HETEROZYGOTES,OTHERHETEROZYGOTES,ANDWILD-TYPEHOMOZYGOTESINREPLICATE CIYLTITRES

Ordered Same Other Homo. n

<0.69 1.026 1.075 0.734 536 0.70-0.89 1.043 1.067 0.748 1857 0.90-1.09 1.090 1.102 0.810 3197 1.10-1.29 1.130 1.120 0.806 2399 1.30-1.49 1.205 1.182 0.848 1286 1.50-1.69 1.267 1.254 0.889 642 1.70-1.89 1.321 1.246 0.946 289 1.90-2.09 1.379 1.320 0.990 175 2.10-2.29 1.444 1.402 0.824 82 2.30+ 1.615 1.517 1.015 113

Since strict

comparabil-...

/ ity does not exist between

./..

theordered viabilities

(left-most column) and the

1.2

others,

but does exist

be-tween the viabilities of heterozygotes and

homozy-.l 1i. Iis 1.40 1.66 1.36 2.66 2.26 2.40 gotes observed in replicate

61lIED*1T11t2YTE1 cultures,

viabilities

of the

FIG.1.-The relation between the viability of identical types "same" heterozygotes in

of heterozygous flies in (1) cultures grouped into 10classes

accordingtoincreasingviabilities of theseflies,and(2) replicate replicate cultures have

cultures of thoseinitially grouped. (The rather

sharp.

upturn been used as the horizontal

of the curve atits rightmostend indicates that 2.40islower

than the trueaverage viabilityofflies in thisclass,2.30+.) axis in depicting the

rela-tionships between the vari-ous groupslisted in Table 1 (see Fig. 2). In Figure 2we have drawna line with

slope 1 through thepoint (1,1); this linerepresents the viability of "same"

heter-ozygotesplotted onbothaxes; ineffect, this isthesame lineasthat ofFigure 1.

Sincethe horizontalaxis ofFigure 2 has beenconverted to values obtained from

replicate cultures, the regression slope has changedfrom 0.272, avalue of doubtful

significance, to 1.000, theobviouslycorrect value against which to make further comparisons.

Asecondinterestingrelationship canbeseeninFigure2. Here wefind evidence that the viability of heterozygotesisdetermined inpart by factorsspecific for each heterozygous combination itself. Thus, for the lowest viabilities of the "same" heterozygotes, thatof "other" heterozygotesissomewhathigher; atslightly higher viabilities of the "same" heterozygotes, that ofthe "other" heterozygotesis

some-what lower. The curve for "other" heterozygotes

then appears tobecome parallelto the line of slope

.

~

/5 1 representingthe "same"heterozygotes.

Thus,

in the upperend of theviability distribution the aver-age increase in

viability

of heterozygotes caused by

-/. one batch of

wild-type

chromosomesrelative to an-otherbatch is the same for different types of

hetero-.

A - -

zygotes.

Both batches of

wild-type

chromosomes

confer somewhat higher

viabilities

on those hetero-*

-l{ zygotesoriginallyscored

("ordered")

than

they

doon the second classof heterozygotes.

Another point of interest is the average viability

.S. of individuals

homozygous

for

wild-type

chromo-somes that

give

heterozygotes

of various viabilities.

1*

2.12

1.4 1 At the lower end of the viability scale there is a good

SAME

11HERI*U61S

correlation between the mean

viability

of

homozy--FIG. 2.-The relation between gotes and of

heterozygotes.

The observed correla-the viability of heterozygoxs tion does not extend into the higher viability ranges.

flies ("same" heterozygotes) im

replicate cultures and that of This last

point

appears in Table 1

only

by

virtue of "other" heterozygotes and the final two values in the column labeled "Homo."

homozYgotes also developing in

VOL.49, 1963 GENETICS: B. WALLACE 805

"ordered" viabilitieshasbeen made; it isquite apparentthatanorderly increase in

tije

viability of homozygotes ceases when that of the "same" heterozygotes is about 1.35-1.40. Within the range where the viabilities of homozygotes and

"same" heterozygotes arecorrelated, the slope of the regression ofhomozygotes on heterozygotes is considerably less than 1.000 (b = 0.683, Sb = 0.037). Succes-sive batches of chromosomes characterized by increasing viabilities of their hetero-zygous carriers do not make corresponding contributions to the viabilitiesof their

homozygous carriers.

TABLE 2

ADETAILED ANALYSISOFORDEREDCULTURESWITHVIABILITIES EXCEEDING 1.70

Ordered Same Other Homo. n

1.70-1.79 1.297 1.205 0.933 155 1.80-1.89 1.350 1.294 0.961 134 1.90-1.99 1.288 1.243 0.900 83 2.00-2.09 1.461 1.388 1.071 92 2.10-2.19 1.549 1.543 0.804 41 2.20-2.29 1.339 1.262 0.843 41 2.30-2.39 1.495 1.465 1.008 39 2.40+ 1.679 1.545 1.018 74

The wild-type chromosomes of thisstudycan bedivided arbitrarily into lethals (viability less than 0.20) and nonlethals (viability 0.20 or more). In Table 3 is listed thefrequency oflethalsamongchromosomes giving ordered heterozygotes of various viabilities; thereis asuggestioninthis material that lethalsareassociated

with heterozygotes of low viability (slope of theregression of lethal frequency on viability of"same" heterozygotes, -0.100, issignificantatthe

10%

level). Table 4gives the meanviability of flies homozygousfor thenonlethal chromosomes found

in each of the viability classes of ordered heterozygotes; the regression of these viabilities on those of the "same"heterozygotesis 0.380.

TABLE 3 TABLE 4

FREQUENCY OF LETHALS AND NEAR-LETHAL MEAN VIABILITY OFNONLETHAL WILD-TYPE WILD-TYPE CHROMOSOMES FOUND AMONG CHROMOSOMES WHICH GIVE HETEROZYGOTES

THOSE GIVING HETEROZYGOTES OF OFDIFFERENTVIABILITIES

DIFFERENT VIABILITIES Ordered Nonlethal

Viability of Percent heterozygotes homozygotes

orderedheterozygotes lethals <0.69 0.886 h0.016

<0.69 17.2 0.70-0.89 0.93340.009 0.70-0.89 20.1 0.90-1.09 0.96740.007 0.90-1.09 16.8 1.10-1.29 0.973 4 0.008 1.10-1.29 17.6 1.30-1.49 1.00440.012 1.30-1.49 16.2 1.50-1.69 1.0354 0.016 1.50-1.69 14.3 1.70-1.89 1.058 40.026 1.70-1.89 11.4 1.90-2.09 1.093 + 0.033 1.90-2.09 10.3 2.10-2.29 1.001 4 0.050 2.10-2.29 17.1 2.30+ 1.1654 0.045 2.30+ 13.3

DiscussionandSummary.-Thepurposeof this report has beentoapplyavariant ofatechniquedescribed earlier

by

Wallace and

Dobzhansky3

to

analyze

the effects of chromosomesontheviabilities of their carriers. The

technique

utilizes

replicate

cultures. Earlier

analyses

classified chromosomes

according

to their effect onthe

viability of homozygous individuals and then determined the

viability

effects of thesesamechromosomes in

heterozygous

individuals. The presentstudy reverses

viability ofheterozygouscarners; theviabilityeffectsof these samechromosomes

in

homozygotes

hasthen beendetermined.

Perhaps the main results of the analysis are the simple relationships revealed by the experimentaldata. Chromosomesthat confer high viability in one hetero-zygous combination do so in another as well. In the higher viability range the difference incontributionto viabilitymade by two batches of chromosomes is the same for differentheterozygouscombinations. At lowerviabilitiesit appears that chromosomes that are deleterious in one heterozygous combination are not quite as

deleteriousin asecond combination; lowviabilityresults, in part at least, from spe-cificchromosomal combinations. Finally, itappearsthatbatchesof chromosomes

thatgive successively higher viabilities in heterozygous combinations do not give comparable increases inviability totheirhomozygous carriers. Thus, incontrast totherelationbetween "same"and "other" heterozygotes, theslopeof the regres-sionofhomozygotes on "same" heterozygotes is muchless than 1.000; indeed, in the upperviabilityrangethe correlationbetweenhomozygotes and "same" hetero-zygotesdisappearscompletely.

*Contribution No.437, Department of Plant Breeding, Cornell University. This paper was prepared while the author heldcontract No.AT-(30-1)-2139, U.S. Atomic Energy Commission. Theexperimentaldata arethoseofDobzhansky, Krimbas,andKrimbas;' thepermissionofthese

authorsto usetheirdata isgratefullyacknowledged.

1Dobzhansky, Th., C. Krimbas, andM.G.Krimbas, Genetics, 45, 741-753(1960). 2Hiraizumi, Y., andJ. F. Crow, Genetics,45, 1071-1083(1960).

3Wallace, B.,and Th. Dobzhansky, Genetics, 47, 1027-1042 (1962). 4Parsons, P.A., and W.F.Bodmer, Nature, 190,7-12(1961).

Wallace, B., J. Genet., 54,280-293(1956).

SEQUENTIAL

REPLICATION OF THE BACILLUS SUBTILIS

CHROMOSOME,

II.

ISOTOPIC

TRANSFER

EXPERIMENTS*,t

BYHIROSHI YOSHIKAWA AND NOBORIJStJEOKA

DEPARTMENT OFBIOLOGY, PRINCETON UNIVERSITY

ComnunicatedbyPaulDoty, April25, 1963

Inthe previous report,' we presented evidence for the sequential replication of the chromosome in Bacillus subtilis. The experiments were based on the

com-parison of marker frequencies in DNA preparations from the exponential and stationary growth phases. The results indicated that the chromosome replicates sequentially from one end (the origin) to the other (the terminus), and that the

adenineless (ade) markeroccupiesthe chromosome region closeto theoriginwhile the methionineless (met) and isoleucineless (ileu) markers occupythe region close totheterminus.

This paper will report results of a different experimental approach designed to test thevalidity of the above-mentioned replication model of the B. subtilis chro-mosome. A part ofthis work has been

reported

briefly.2 The experiments were