Proc. Nat. Acad. Sci. USAVol. 72, No. 11, pp. 4550-4554, November 1975Genetics

Chromosome puff activity and protein synthesis in larval salivaryglands of Drosophila melanogaster

(secretion protein analysis/gene localization/puffing/dosage compensation)

GUNTER KORGEZoologisches Institut der Universitat, 8 Munchen 2, Luisenstrasse 14, Federal Republic of Germany

Communicated by Wolfgang Beermann, September 2,1975

ABSTRACT Secretion proteins from larval salivarglands of Drosophila melanogaster were analyzed withacrylamide gel electrophoresis. Four fractions were found;three showed electrophoretic variants in different wild-typestocks. Crossbreeding and cytogenetic techniques were usedto localize the genes responsible for the two main fractions:The gene for fraction 3 was found to lie within a segment ofthe third chromosome which includes section 68C; the genefo'r fraction 4, Sgs-4, was found to lie within section 3C8-3D1of the X chromosome (1 - 3-5). The puffs within these sec-tions of the giant chromosomes are active before and duringsecretion synthesis and become inactive as secretion synthe-sis ceases. Larvae of one wild-type stock which lack proteinfraction 4 do not exhibit any puffing in 3C. The relativeamount of protein 4 in the salivary secretion shows a markeddependence on the dosage of the Sgs-4 gene in both duplica-tion and deficiency genotypes. The active site within puff3C11-12 apparently contains the structural gene for protein4.

A direct correlation between chromosomal sites which aretranscriptionally active and activities in the cytoplasm hasbeen established for puffs of the Balbiani ring type in thegiant chromosomes of the salivary glands of Chironomus(1-3) and Acricotopus (4). In Drosophila melanogaster untilnow only indirect evidence suggesting the existence of sucha relationship has been available (5-7). In the work on Dro-sophila melanogaster to be reported here we have now beenable to obtain direct evidence of a cytogenetic nature indi-cating the location of the genes responsible for certain secre-tion proteins in individual salivary gland chromosome puffs.Furthermore, the effect of changes in dosage of one of thegenes involved has been investigated.

MATERIAL AND METHODS

Drosophila stocks used: (1) Berlin, wild-type. (2) Oregon-R(ORN), wild-type. (3) Hikone-R, wild-type. (4) y v f and (5)y w spl cv f, X chromosome markers which were used for re-combination analysis. The salivary gland chromosome locusof w is at 3C2; that of spl, at 3C7; and that of cv, between4F1-2 - 5D1-2. (6) Df(1)N8/dl-49, y Hw m2g4. In the Xchromosome carrying Notch-S the section from 3C1through 3D6-E1 is lacking. The chromosome is kept bal-anced with the inversion chromosome In(l )delta-49,marked with y HW m2g4. (7) Df(l )N264-105/FM1, y3id_sctswalzsB. The X chromosome carrying Notch264 105 lacksthe section from 3C6-7 through 3D2-3. The inversion chro-mosome In(l )First Multiple is marked with y3ldSc8WalZsB.(8) C(1)RM, yf/Y. The females carry y f attached X chro-mosomes and a free Y. (9) C(1)RM, yf/Y & Dp(lf)z9;Df(1)scJ4R. All females carry attached X chromosomes andin addition they can carry the free duplication Dp(l;f)z9 ofthe X chromosome, i.e., section lAl through 3E7-F1;19-20.The males of this stock have an X chromosome with the de-

ficiency Df(1 )SCJ4R, i.e., the section lB to 3A3-C2. The defi-ciency is compensated by the duplication Dp(lf)z9. (10)Cy/Pm;CxD/Sb. Both second chromosomes carry inver-sions; one is marked with Cy and one with Pm. One thirdchromosome carries an inversion and is marked with CxD;the other third chromosome has the normal structure and ismarked with Sb.

For more information on the genetic markers and theirrespective salivary chromosome sections see ref. 8. Linkagedata for some X chromosome markers are given in Fig. 4.

Preparation of the Salivary Glands and Their Chromo-somes. The glands of late third instar larvae and of youngprepupae were dissected in Ringer's solution, washed, andsubsequently fixed in ethanol/glacial acetic acid (3:1). Thegland secretion, which became hard and opaque duringfixation (Fig. 1) was dissected out with needles. Secretionmasses were placed in small incubation tubes, 10 per tube,then vacuum dried and stored at -20°. The technique usedfor preparing chromosome slides and autoradiographs hasbeen described earlier (9).

Acrylamide-Gel-Electrophoresis. Gland and secretionproteins were reduced for 60 min at room temperature with0.01 M dithiothreitol (DTT; Cleland's Reagent; Sigma) atpH 8.0 and subsequently were alkylated with ethylenimine[2.5% (vol/vol); Schuchardt] for 30 min at room tempera-ture. The reduced and alkylated proteins were separatedelectrophoretically in 7% acrylamide disc gels, 5 mm in di-ameter. A modification of Grossbach's method (2) was usedfor preparing the gels. After electrophoresis the gels werestained with 0.25% Coomassie Brilliant Blue (R250; Serva) in7% acetic acid and destained in 10% methanol + 7% aceticacid. The destained gels were measured at 590 nm with theGilford spectrophotometer 240.

RESULTS

The main function of the larval salivary glands in Drosophi-la melanogaster appears to be the synthesis and extrusion ofthe mucopolysaccharide-containing secretion, which as ithardens, attaches the pupa case to a substrate. Ultrastructur-al (10, 11) and electrophoretic investigations have shownthat saliva begins to be formed about the middle of the thirdlarval instar. The extrusion of the secretion into the glandlumen begins about three hours before puparium formation.After puparium formation only a few secretion granules re-main in the cytoplasm of the salivary gland cells (12).The secretion of the wild-type stock Berlin contains at

least five protein components which account for most of thetotal gland protein (Fig. 1). Electrophoretic analysis of secre-tion proteins and proteins from other organs, e.g., epidermis,as well as gut and haemolymph of larvae and imagines allshowed the presence of fraction 2. This fraction seems to be

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I

12

-~~3

2a - a a2 -

la_ - : lb

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_ __ -- 4c

4a .40 M_ III* ^ i- 4b<_ _~iiia- 4d

5

5

G+S G G+S G

4.I 5

FS

FIG. 1. Salivary gland intact (G+S), salivary gland with secre-

tion mass removed (G), secretion mass alone (S), and their pro-

teins after acrylamide gel electrophoresis. Protein fractions 1-5 ofthe secretion and the front (F) are labeled. For each gel 10 speci-mens were used.

an artifact, possibly originating from microvilli torn off dur-ing preparation.

Variants of secretion proteinsDifferent wild-type stocks of Drosophila melanogaster were

tested for secretional variants. From the stock Oregon-R(ORN) one strain was isolated whose fractions 1, 3, and 4showed electrophoretic properties deviating from those ofthe Berlin wild-type stock (Fig. 2). The Berlin stock showedonly fraction la while Oregon-R (ORN) showed this frac-tion plus lb and an additional third faint fraction betweenla and b (This latter fraction is too faint to be recorded inFig. 2). Fraction 3 is represented in the Berlin stock by onlyone sub-fraction (Fig. 2, 3a), in Oregon by two sub-fractions(Fig. 2, 3a and b). Fraction 4a of the Berlin stock is missingin Oregon. In that position Oregon shows three fractions(Fig. 2, 4b, c and d) with different electrophoretic proper-

ties.The genes responsible for protein fractions 3 and 4 were

localized by means of conventional genetic techniques.

HybridsWhen Berlin and Oregon flies were crossed reciprocally thehybrid males from both crosses showed identical hybrid pro-

tein patterns in the case of fractions 1 and 3, while in thecase of fraction 4 only the maternal protein type appeared.Hybrid females from the two reciprocal crosses were foundto contain the protein types of both parents for all fractions.These results demonstrate autosomal inheritance of fractions1 and 3, and sex-linked inheritance of fraction 4.

Combination of Berlin and Oregon chromosomesWith the aid of the multichromosomal inversion stock Cy/Pm;CxD/Sb, which is used to prevent recombination, allpossible combinations of intact Berlin and Oregon chromo-somes were assembled. The small fourth chromosome was

disregarded. Animals of the inversion stock show the Berlinsecretion pattern. Fig. 3 shows the result of protein electro-phoreses using animals with various combinations of Berlinand Oregon chromosomes. (a) Whenever both third chro-mosomes come from the Berlin stock, fraction 3 shows only

CHR.1 e | d e1 B B B/OR OR B/OR OR

2 B B/OR B/OR B/OR B/OR OR

3 B B/OR B/OR B/OR B/ORp F1

BxOR ORxB

ORp

OR

OR

ORp

FIG. 2. Electrophoretically separated secretion proteins fromthe stocks Berlin and Oregon-R(ORN) and their hybrids. Undereach gel is the chromosome constitution of the secretion donor. Bchromosome from the Berlin stock; OR from the Oregon stock;B/OR first, second or third chromosome (CHR. 1-3) heterozygousfor B and OR. P, parental generation; F1, filial. B X OR B-9crossed with OR-8; OR X B the reciprocal cross. For each gel 10 se-

cretions from five to seven larvae were used.

one band;. if this chromosome comes from the Oregon stock,there are always two bands. (b) When the Berlin X chromo-some is present fraction 4 has only one band, 4a; when theOregon X chromosome is present there are three bands 4b, c

and d; i.e., the bands are those characteristic for the wild-type stocks that were the source of X chromosomes (Fig. 3).Neither type of chromosome 2 has an influence on protein

la -l--B--I -- --;-=-=fb

2 2.-2

3a _- m-m_:3a

d h- _ b_ 4c

_M _ft dM Adb 4b

4a ____4d

Ao

CHR.

mp..04s

oB B B B OR OR OR OR

2 B OR B OR B OR

3 B B OR OR OR B

B OR

B OR

FIG. 3. Electrophoretically separated secretion proteins fromlarvae with all possible combinations of Berlin (B) and Oregon(OR) first, second, and third chromosomes (CHR. 1-3). For eachgel 10 secretions from five to seven larvae were used.

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Proc. Nat. Acad. Sci. USA 72 (1975)

Linkage 0.0 1.5 3.0 13.7 33.0 56.7 Type ofGenotype W s CV V f protein 4y wspl cv f - - B

Y OR

Y WSPl 8B

YW SP/CV B

f == __ 1OR

v f 10 OR

cv f = 200R/1B

spicv f = - B

wspicv f - BFIG. 4. X chromosome maps of recombinant chromosomes

used for localizing the gene for protein 4. Black horizontal bars: Xchromosome or parts of it from the y w spi cv f or y v f stock.White horizontal bars: chromosome sections originating from theOregon stock. Hatched bars: Sections within which recombinationoccurred, i.e., consisting of one part from one of the marker stocks,the remainder from the Oregon stock. Left column: Genotype ofrecombinants. Right column: B, only protein 4a appeared; OR,only protein 4b or 4c or 4d appeared; in the decisive recombinantgroups numbers indicate numbers of recombinants with the re-spective protein type. The distances between markers are from thecytological map. For each gel 10 secretions from five to seven lar-vae were used.

fractions 3 and 4. Thus, chromosome 3 determines the typeof protein fraction 3, and the X chromosome determines thetype of protein fraction 4.The Oregon stock apparently has a mixture of autosomes,

some carrying the gene for protein la, and some carryingthe gene for lb. Therefore, it has not yet been possible to as-sign the genes responsible for these proteins to specific chro-mosomes.

Recombination analysisThe gene responsible for protein 3 lies on the third chromo-some. With the aid of a stock carrying eight markers ("ru cuca") an attempt was made to localize the gene within thechromosome by recombination analysis. Preliminary resultsindicate that the gene lies within a chromosome section be-tween hairy (salivary gland chromosome locus between66D2 and 66E1) and thread (between 72A2 and 72E5).

In order to localize the gene for protein 4 within the Xchromosome, y v f females and y wo spi cv f females whichshow the Berlin secretion type were crossed to Oregonmales. The F1-females were back-crossed to Berlin males.From the offspring of this second cross those males were se-lected that resulted from a recombination between one ofthe marked chromosomes and the Oregon chromosome.These recombinant males were mated singly to C(1)RM, yf/Y females in order to establish stocks representing individ-ual recombinant X chromosomes. Male larvae from each ofthese stocks were tested for the type of their salivary proteinfraction 4. As shown in Fig. 4 animals of the two markerstocks show the Berlin secretion pattern. Fraction 4 of y andy to recombinants was of the Oregon type. Eight recombi-nants derived from events between spi and cv (having thegenotypes y w spi) resulted in animals with the Berlin typeof fraction 4. All recombinations to the right of cv (havingthe genotypes y to spl cv and y v) also resulted in animals

The complementary recombination types all showed thecomplementary secretion type. Here the results of recombi-nations between spl and cv are of interest. Among 21 ani-mals with the genotype cv f 20 showed the secretion type ofOregon and only one that of Berlin.

These results demonstrate that the gene responsible forprotein fraction 4 lies in the X chromosome section betweenspl and cv. The gene obviously is close to spl, since amongthe 29 recombinants between spl and cv (8 y spl and 21 cvf) in 28 cases the recombination event took place betweenthe protein 4 gene and cv, and in only one case between theprotein 4 gene and spl. The protein 4 gene most probablylies even closer to spl than is suggested by the recombinationdata, since a comparison between the genetic and the cyto-logical map of Chromosome X (8) shows that recombina-tional events are cytologically more abundant immediatelyto the right of spl than immediately to the left of cv.

The investigation of the recombinants yielded another re-

sult. Each recombinant which showed the Oregon type offraction 4 had only one of the three Oregon fractions 4b, 4c,or 4d, never two or all three simultaneously. This indicatesthat the Oregon stock had been heterogeneous with respectto the gene for protein fraction 4, i.e., that it contained atleast three different alleles, one each for the protein types4b, 4c, and 4d. Investigations of single Oregon males con-

firmed this interpretation. Each male possessed only one ofthe three fractions. This heterogeneity of the stock was not a

disadvantage when it came to localizing the gene. With themethod applied all three alleles responsible for fraction 4were localized in one step. The heterogeneity of the Oregonstock explains the different intensities of fractions 4b, c, andd that were observed in different tests with the hybrids (Fig.2). There was a more or less random fluctuation in the distri-bution of the three alleles within the population.

Cytological localization of the gene for protein 4Secretion from Df(l)N8/dl-49, y Hw m2 g4 females con-

tains protein fraction 4a. Such females were crossed to Ore-gon males. The Fl-females were tested for their types offraction 4. Females with a dl-49 and an Oregon X chromo-some showed both Berlin and Oregon types of fraction 4.Females with a Df(1)N8 and an Oregon X chromosome(Fig. 5d) had protein fractions 4b or 4c or 4d of the Oregonstock, i.e., the Berlin type 4a was missing. Therefore, the Xchromosome with the deficiency Df(l)N8 covering the sec-

tion 3C1 through 3D6-E1 (Fig. Sd) lacks the gene for pro-tein 4.

For a more precise localization of the protein 4 gene thedeficiency Df(1 )N264-105 covering the section 3C6-7through 3D2-3 was used. It was found that this deficientchromosome also lacks the gene for protein 4. Consideringthat the gene lies to the right of spl (Fig. 4) it must be con-cluded that it is located within the chromosome section 3C8through 3D1 which contains six chromosome bands.

Correlation between chromosome puffs and secretionproteinsThe aim of the work reported here was to find correlationsbetween chromosome puffing and the synthesis of secretionproteins. The question to be answered was: Is there a chro-mosome puff at the gene locus which shows a positive corre-

lation with the synthesis of a specific secretion protein?Only one puff in the X chromosome specifically meets

these requirements. This puff lies within chromosome sec-

with the Berlin type of fraction 4.

4552 Genetics: Korge

tion 3C (Fig. 5c), which is also the site for the protein 4

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Proc. Nat. Acad. Sci. USA 72 (1975) 4553

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dFIG. 5. Distal part of the X chromosomes in females. (a) and

(b) Autoradiographs after 10 min [3H]uridine incubation; (a) lowlabeled X chromosomes showing one active site within 3C; (b) highlabeled X chromosomes with 3C in a well puffed stage; (c) X chro-mosomes of a wild-type female; (d) X chromosomes of a heterozy-gous deficiency female Df(1)N8/+. The upper deficient chromo-some lacks the puffed site within 3C; in the lower normal chromo-some the site can be seen.

gene. As soon as the salivary gland chromosomes in theyoung larva have reached the degree of polyteny that allowsspecific chromosome sites to be identified, this particular siteis already active. This means that it must be active 5-6 hrbefore the appearance of the secretion proteins. The puffbecomes inactive when the ecdysone-inducible puffs in sec-

tions 2B, 74EF and 75B appear (13, 14) and secretion syn-thesis decreases. The puff in SC is the only one covered bythe deficiencies Df(l )N8 and Df(l )N264-105. It contains onlyone active site (Fig. 5a and b); it originates from chromo-some band SC11 or 3C12. This active chromosome site ap-parently contains the active gene for protein 4.

Another puff which shows a positive correlation of activi-ty with secretion synthesis lies within section 68C of thethird chromosome (13), i.e., between 66D2 (left limit of thehairy locus) and 72E5 (right limit of the thread locus). Thissection apparently contains the gene for protein 3.

The effect of different gene doses of section 3C on thesynthesis of secretion protein 4

Since the gene for protein 4 is located on the X chromosome,normal females have two doses of this gene, whereas normalmales have only one dose. As a rule X chromosome genes inmales and females of Drosophila give almost the same phe-notype in spite of the two different doses (15, 16). This phe-nomenon was named dosage compensation by Muller (15).A test was made in order to find out whether the two sexes

synthesized different amounts of protein 4. The Berlin stockwas used, and the relative amount of protein 4 was deter-mined by using the quotient (Q4/3) of the absorption unitsfor proteins 4 and 3. These in turn were ascertained plani-metrically from the curves of absorbance. This quotient was

0.38 I 0.04 (n = 26 electrophoreses) for wild-type malesand was 0.44 0.08 (n = 49) for females, i.e., the gene dose

2C

0.0

0E

Gene * * m m*dosage aof3 -Sex 9 d: 9 d

FIG. 6. Dosage compensation for and dosage effect on synthe-sis of protein 4. Relative amount of protein 4 (see text) producedby normal females (gene dosage 2) is equated with 1. Relativeamounts of protein 4 produced by the other genotypes are com-pared with those of normal females. Relative amounts are givenwith their standard deviations. For each gel 10 secretions from fiveto seven larvae were used.

for protein 4 within section SC shows dosage compensationin males. The quotient Q4/3 for males with one dose wasabout 86% that of the female value, i.e., about 14% less thanmight be expected.

Further tests were made on animals with abnormal dosesof section SC (Fig. 6). When the dose in females was de-creased from two to one by means of the deficiencyDf(l)N8 (Fig. 5d), the quotient Q4/3 was 0.24 b 0.02, thatis, about 55% of the normal value. Thus there was practical-ly no dosage compensation in deficiency females. When thedose in females was increased from two to three the quotientQ4/3 was 0.60 i 0.07 (n = 6), about 136% of the normalvalue, i.e., about 14% less than might be expected.When the dose in males was increased from one to two by

means of the duplication Dp(l;f)z9 the quotient was 0.62 +0.08 (n = 19), that is 163% of the normal value, about 37%less than might be expected.

If the premise is correct that the protein of fraction 3 issynthesized in equal amounts in both the wild-type and mu-tant animals we have used, then the results can be summa-rized as follows: (a) Wild-type males show dosage compen-sation for the synthesis of protein 4 (Fig. 6). (b) Femaleswith only one gene dose of section SC show very little if anydosage compensation for this character. (c) There is amarked increase in protein 4 synthesis when the gene dosageis increased from two to three in duplication females andfrom one to two in duplication males.

DISCUSSIONIn this paper evidence is presented for a direct correspon-dence between the presence of certain chromosome puffsand the presence of specific secretion proteins in the larvalsalivary glands of Drosophila melanogaster. The nature ofthis relationship must be discussed in the light of the fol-lowing main findings: (1) Since the genetic localization dataare concerned with electrophoretic variants, we must bedealing with the structural genes for the protein fractionsstudied. The observations, in the case of protein 4, on dosageeffects and dosage compensation corroborate this conclusion.(2) The genetic and, in the case of protein 4, the cytogeneticlocalization data place the structural genes for protein frac-tions 3 and 4 into the polytene chromosome sections 68C

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and SC, respectively, both of which include a conspicuouspuff site at the time of secretion synthesis. In fact, the puffsare active at least 5 hr before the first appearance of secre-tion proteins. They become inactivated before the end of thethird larval instar as the synthesis of secretion protein ceases.In section SC a puff reappears during prepupal develop-ment (13), but no protein 4 was found at that stage. This ap-parent inconsistency will be discussed elsewhere.

While the presumed correlation between puff 68C and se-cretion fraction 3, though plausible, must remain tentativefor lack of more precise localization data and of other sup-porting evidence, a much better case can be made for thepostulated relation between puff SC and fraction 4. First ofall, the localization in this case has been almost narroweddown to the puff site (3C11/12) itself. In addition, a wild-type stock (Hikone-R) has recently been discovered whichlacks protein 4 and which does not exhibit any puffing in re-gion SC.

As pointed out above, the relative amount of protein frac-tion 4 present in the salivary secretion shows a marked de-pendence on gene dosage (Fig. 6). This is most clearly seenin females heterozygously deficient for section SC, wherethe relative content of fraction 4 is reduced to 55% of thewild-type level, and in duplication females with three 3Csections where the level of protein 4 is raised to 136% of thewild type. In normal males, as would be expected in the caseof X chromosome genes, there is dosage compensation. Thiscompensation is not complete. In duplication males carryingtwo SC sections the protein 4 level is increased to 163% ofthe wild type. The deviations of the dosage effects found induplication females and males-136 from the expected150% and 163 from the expected 200-might be explainedas follows: (1) The dosage effect may be incomplete at thetranscriptional level. This was indicated in earlier studiesusing other duplicated chromosome sections (9). (2) Theremight be a lack of substrate since a duplication requires arelatively high amount of protein 4 synthesis, particularly inmales, where an increase of 100% was expected. (3) The ab-sorbance of protein fraction 3 and 4 was often higher than1.5. Coomassie Blue gives valid results only up to 1.5 absorb-ance units (Beer's Law, ref. 19). Because of the increased op-tical density of fraction 4 in the duplication individuals, aquantitative determination of protein 4 is not completely re-

liable. (4) Finally, the possibility of a partial dosage compen-sation, which would decrease the dosage effect, should beconsidered.The structural gene for protein 4 will be named Sgs.4

(salivary gland secretion protein 4; 1 - 3-5, salivary glandchromosome locus 3C8-3D1). The gene lies apparently with-in the only active site of the puffed locus SC, which origi-nates from band SCil or 3C12. Further experiments mightallow us to narrow down the Sgs-4 locus to possibly a singleband.

I am very grateful to Drs. G. L. Becker and H. J. Becker for help-ful advice and criticism in preparing the manuscript. This work hasbeen supported by the Deutsche Forschungsgemeinschaft.

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38,619-627.4. Baudisch, W. & Panitz, R. (1968) Exp. Cell Res. 49, 470-476.5. Tissieres, A., Mitchell, H. K. & Tracy, U. M. (1974) J. Mol.

Biol. 84,389-398.6. McKenzie, S. L., Henikoff, S. & Meselson, M. (1975) Proc.

Nat. Acad. Sci. USA 72, 1117-1121.7. Spradling, A., Penman, S. & Pardue, M. L. (1975) Cell 4,

395-404.8. Lindsley, D. L. & Grell, E. H. (1968) Genetic Variations of

Drosophila melanogaster (Carnegie Inst. Wash. Publ. No.627).

9. Korge, G. (1970) Chromosoma 30,430-464.10. Gaudecker, B.v. (1972) Z. Zellforsch. Mikrosk. Anat. 127,

50-86.11. Lane, N. J., Carter, Y. R. & Ashburner, M. (1972) Wilhelm

Roux Arch. Entwicklungsmech. Org. 169,216-238.12. Gaudecker, B.v. & Schmale, E.-M. (1974) Cell Tiss. Res. 155,

75-89.13. Becker, H. J. (1959) Chromosoma 10, 654-678.14. Ashburner, M. (1967) Chromosoma 21,398-428.15. Muller, H. J. (1932) Proc. 6th Int. Congr. Hum. Genet. (Itha-

ca, N.Y., 1932) Vol. 1, pp. 213-255.16. Stern, C. (1960) Can. J. Genet. Cytol. 2, 105-118.17. Mukherjee, A. S. & Beermann, W. (1965) Nature 207, 785-

786.18. Holmquist, G. (1972) Chromosoma 36,413-452.19. Fenner, C., Traut, R. R., Mason, D. T. & Wikman-Coffelt, J.

(1975) Anal. Biochem. 63, 595-602.

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