Chromosoma (Berl.) 62, 155-174 (1977)

CHROMOSOMA 9 by Springer-Verlag 1977

Direct Correlation between a Chromosome Puff and the Synthesis of a Larval Saliva Protein in Drosophila melanogaster G/inter Korge Zoologisches Institut der UniversitS.t, Luisenstr. 14, 8000 Mtinchen 2, Federal Republic of Germany

Sgs-4 responsible for larval saliva protein 4 of Drosophila melanogaster was localized, with the help of Notch deficiencies, within the section between bands 3C10 and 3D1 of the X chromosome. In this chromosome section there is, very probably, only one fine band. In the third larval instar chromosome this section is transcriptionally active and forms a puff. When the ecdysone concentration increases, about 5 h before prepupa formation, it becomes inactive. - In section 3C of X chromosomes of third instar larvae of the stock Hikone-R no puff is formed. The saliva of these larvae lacks protein 4. However, female hybrids (H/B and H/O) from Hikone-R crossed with Berlin and Oregon respectively produce a Hikone-specific saliva protein 4h. The synthesis of protein 4h in the hybrids H/B and H/O is ascribed to an activation of the gene Sgs-4 in the Hikone chromosome. - In the saliva of heterozygotes (FM1/H) carrying one inversion chromosome In(1)FM1 and one X chromosome from Hikone, protein 4h could not be detected. In these inversion heterozygotes in 90% of all nuclei the homologues are not paired in 3C, and 3C is puffed only in the FAll chromosome. This suggests that a precondition for the activation of Hikone gene Sgs-4 in heterozygotes may be intimate homologue pairing. Intersexes with one of their X chromosomes from Hikone-R and the other from Berlin produce relatively more protein 4h than do diploid H/B females, indicating facilitated transcription as a result of dosage compensation.

Abstract. The structural gene

Introduction

As the larval development of Drosophila melanogaster nears completion the salivary glands attain high rates of protein synthesis. In about 20 h more than 30% of the total gland protein is synthesized to saliva protein, bound to mucopolysaccharides and finally discharged within 1 h into the gland lumen (Korge, in press). The saliva is composed of only a few proteins and is easy to extract in

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G. Korge

pure form. Synthesis of these proteins is induced towards the middle of the third larval instar by factors yet unknown. The sharp rise in the ecdysone concentration about 5 h before prepupa formation inhibits synthesis and induces extrusion of the saliva into the lumen (Poels, 1970, 1972). The ease of genetic manipulation of Drosophila melanogaster and the presence of polytene chromosomes make its larval salivary glands ideal for investigating the genomic control of protein synthesis. Preliminary investigations had already indicated correlations between the activities of two chromosome puffs and the synthesis of two saliva proteins (Korge, 1975). In the present paper an attempt is made to precisely localize Sgs-4, the structural gene responsible for saliva protein 4, in terms of the polytene chromosome map and to describe the behaviour of this chromosome site with respect to its transcriptional activity. The wild-type stock Hikone-R, which carries an apparent 0-mutation for protein 4, is employed to gain further insight into the possible mode of gene regulation on the transcriptional level. Material and Methods The following stocks of Drosophila melanogaster were used: (1) Berlin, wild-type. (2) OregonR(ORN), wild-type. (3) Hikone-R, wild-type. (4) Df(1)NS/dl-49, y Hw m2g 4. In the X chromosome carrying the deficiency Notch-8 the section from 3C1 through 3D6-E1 is lacking. The chromosome is kept balanced with the inversion chromosome [n(1)delta-49 marked with y HwmZg ~. (5) Df(1)N2~4-1~ y31dscSw"lz~B. The chromosome carrying N 264-1~ lacks the section from 3C6-7 through 3D2-3. The inversion chromosome FM1 is marked with y3ZescSw~Iz~B. (6) Df(1)bF69~9/ FMI, y31dscSw~lzSB. The X chromosome carrying N '69h9 lacks the section from 3C5 through 3C12. (7) Df(1)N54t9/dl-49, y Hw m2g *. The X chromosome carrying deficiency N 5419 lacks section from 3C6 through 3C10. In the following genotypes X marks an X chromosome and A marks a set of autosomes. The indices H and B mark their origin, Hikone-R and Berlin, respectively. (8) Triploids, 1XH/2XB; 1A"/2AB. (9)Metafemales, 1Xn/2XB; 1Aa/1AB. (10)Intersexes, 1Xn/1XB; 1An/2AB.-The X chromosomes from Berlin (X B) in (8), (9), and (10) were marked with yellow ~. Salivary gland squash preparations were made from late third instar larvae and from prepupae. In addition, autoradiographs were taken of salivary glands from third instar larvae after 10-15 min incubation in vitro with 100 ~tCi ~H-uridine(5) per ml Ringer's solution or, alternatively, after 5-15 min incubation in vivo with 0.2 or 0.4 gCi 3H-uridine(5). Stripping film AR i0 (Kodak) was used. The exposure time was 40 d. Saliva plugs were prepared frmn fixed salivary glands of larvae just about to form prepupae. Saliva proteins were reduced, alkylated and electrophoreticaliy separated in 7% acrylamide disc gels (Grossbach, 1969; Korge, in press). Further details on methods will be published elsewhere (Korge, in press).

Results

Localization of the Gene Sgs-4 The investigations which revealed a direct correspondence between the chromosome puff 3C in the X chromosome and saliva protein 4 were carried out on the heterogenous wild-type stock Oregon-R(ORN) (Korge, 1975). This stock carried the three codominant alleles Sgs-4 b, Sgs-4 c and Sgs-4 ~ coding for 1 For further information on the genetic markers and the chromosome mutations see Lindsley and Grell (1968)

Chromosome Puff and Saliva Protein Synthesis

157

Fig. 1. Electrophoretically separated reduced and alkylated saliva proteins from the Drosophila stocks Berlin and Oregon-R(ORN) and their hybrids. Under each gel is the chromosome constitution of the saliva donor. B, chromosome from Berlin; O, from Oregon-R(ORN); B/O, first, second or third chromosome (CHR. 1-3) heterozygous for B and O. P, parental generations; F 1, hybrids B x O, B e r l i n - ~ x Oregon-R(ORN)@c~; O • B, the reciprocal cross. For each gel 8 saliva plugs from four to five larvae were used. 7% acrylamide disc gels, 5 mm in diameter, la-5, saliva proteins. F, front

protein 4. A strain from this stock carrying only the allele 4c has since been isolated and bred. Fraction 4c is the one most distinct in its electrophoretic properties as compared with Berlin stock fraction 4a. The results of reciprocal crosses of the Berlin stock with the Oregon stock carrying S g s - 4 c are shown in Figures 1 and 2. The two stocks do not differ with respect to fraction 5. The conditions with respect to fraction 1 are not quite clear. Therefore, attention is paid only to fractions 3 and 4. Hybrid females from the reciprocal crosses between Berlin and Oregon present the protein types of both parents for all fractions. The males exhibit both parental proteins in the case of fraction 3 while for fraction 4 they show only the maternal protein type. This indicates autosomal inheritance of fraction 3 and X-chromosomal inheritance of fraction 4. An analysis of all possible combinations of Berlin and Oregon chromosomes substantiates this result. In animals

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G. Korge

Fig. 2. E1ectrophoretically separated reduced and alkylated saliva proteins from larvae with all possible combinations of Berlin (B) and Oregon-R(ORN) (O) first, second and third chromosomes (CHR. 1-3). For each gel 8 saliva plugs from four to five larvae were used. 7% acrylamide disc gels, 5 mm in diameter, la 5, saliva proteins. F, front

carrying X chromosomes derived from the Berlin stock, fraction 4 is invariably represented by the Berlin stock protein type 4a: with X chromosomes from our Oregon strain, only Oregon protein 4c is present (Fig. 2). As regards fraction 3, whenever the third chromosomes come from Berlin, fraction 3 is represented by protein 3a alone, if the third chromosomes are from Oregon, both proteins, 3 a and 3 b, occur (Fig. 2). Thus a gene on chromosome 3 lays down the type of protein 3b. In earlier investigations, analysis of the saliva of recombinants showed gene Sgs-4 to be located in X chromosome section 3C (Korge, 1975). By means of a preliminary cytological localization with the help of deficiencies Df(1)N8 and Df(I)N 26~- lO5 the site of gene Sgs-4 was identified in chromosome section 3C8-3D1 (Korge, 1975). The two other deficiencies Df(1)N '69h9 and Dffl)N 5419 were employed to narrow down this result as far as possible to a single chromosome band. The deficiencies employed are homo- and hemizygously lethal and can only be kept heterozygously. To prevent recombination the chromosomes with the deficiencies were kept balanced with chromosomes carrying inversions In(1)FM1 or In(1)dl-49. The saliva of all the heterozygous deficiency females-Df(1)N/ FM1 or Df(1)N/dl-49-contained only protein 4a in fraction 4. These females were crossed with Oregon males whose saliva contains fraction 4c. In the offspring of these crosses two distinct types of female occur. One

Chromosome Puff and Saliva Protein Synthesis

159

4c

4a

Fig. 3. Electrophoretically separated reduced and alkylated saliva proteins from heterozygous female larvae. FMJ, X chromosome carrying the inversion In(1)First Multiple (with Berlin-type fraction 4a-gene); Df(1)N, X chromosomes carrying the Notch deficiencies Df(1)N s or Df(1)N26#-1~ or Df(1)Nr69hg; O, X chromosomes from Oregon-R(ORN). 7% acrylamide disc gels, 5 mm in diameter. 4a and c, saliva proteins

FM1/Df(7)N O/Df(1)N FM 1/0

type has one X chromosome which carries inversions and one X chromosome from Oregon; the other type has one chromosome with a deficiency and one Oregon chromosome. The saliva of the inversion heterozygotes all contained both protein 4a and protein 4c (Fig. 3). The saliva of the heterozygotes with deficiencies Df(1)N s, Df(1)N 26~-1~ and Df(1)N r69h9 contained only protein 4c; protein 4a was missing (Fig. 3). Only the females of genotype Df(1)N5419/O presented both 4c and 4a. This means that the chromosomes with deficiencies N 8, N26r - los and j~69h9 lack gene Sgs-4 whereas the chromosome with deficiency N 5419 carries it. Absence of the gene Sgs-4 in the first three of the above deficiencies is expressed as a decrease of the relative protein yields in fraction 4. Comparing the values of fraction 3 and 4 obtained planimetrically from the densitometer curves of the gels, the quotient Q4/3 of the Berlin stock saliva was 0.71 as against 0.33 obtained for heterozygous Df(1)N/B (Fig. 4). Thus heterozygous deficiency females produce only half as much fraction 4 protein (in relation to fraction 3) as do normal Berlin females. The results of investigations on the four different Notch deficiencies permit localization of gene Sgs-4 as follows: The gene must lie to the right of 3C5, because the chromosome with deficiency N 264-105 possesses band 3C5 but lacks gene Sgs-4. The gene must lie to the left of 3D1, because the chromosome with deficiency N v69h9 possesses band 3D1 but lacks gene Sgs-4. The gene must lie within the chromosome section between 3C10 and 3D1, since the chromosome with deficiency N 5419 carries gene Sgs-4 and it possesses this chromosome section: this being the only point in which it differs from deficiencies N z64-1~ and N r69h9 (Fig. 5). Between 3C10 and 3D1 Bridges (1938) observed two faint bands, 3Cll

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G. Korge

O. Dh.m 59O 0,8[

Berlin - s163

Q4/3 = 0,71 0.6

y

0.4

0.2 0.8 0.6

F

Df(1)NS/B Q4/3 = 0 , 3 3

0.4 0.2L Fig. 4. The optical density curves of gels with electrophoretically separated reduced and alkylated saliva proteins of female larvae from the Berlin stock (above) and from the heterozygotes Df(1)NS/B (below), one X chromosome carrying the deficiency Df(1)N8, the other a normal X from Berlin (B). Q4/3, the quotient of the absorption units from proteins 4 and 3, ascertained planimetrically. For each gel four saliva plugs were used. 7% acrylamide disc gels, 3.4 mm in diameter.- The quotient Q4/3 of the gel from Df(1)N8/B heterozygotes is about half the value of normal Berlin females (dosage effect)

and 3C12. These bands are described by Becker (1959) and Ashburner (1969) as forming a puff which becomes visible already from the middle of the third larval instar on and which remains active until near the end of the third larval instar. With the rise in the ecdysone concentration about 5 h before prepupa formation the puff becomes i n a c t i v e . - The site from which the puff is formed coincides with the locus established for gene Sgs-4, and its phase of activity shows positive correlation with the phase of saliva synthesis (Korge, in press). Therefore this puff may be considered to be the morphological expression of the activity of gene Sgs-4 (Korge, 1975). This correlation is further corroborated by a cytological study of heterozygous deficient animals. In those instances where the deficiency includes the locus of gene Sgs-4 (Fig. 5), puffing in 3C is restricted to the non-deficient homologue (Fig. 6). Deficiency Df(1)N 54~9 does not include the locus of gene Sgs-4; i.e. fraction

Chromosome Puff and Saliva Protein Synthesis

; < ~ ~ , '~!'r

.........

161

~; f f l ......

ilii; )!i)ii llniulii ,

: ~ i'= ~ ~i~ii =:~ I.~[.,,~ ~ ' ~ ~ ~~~I || .,,............,..... _ , .

lilll[llllll

~',: i ............

I

A AI IA A/II IIAA A t o

23 ~67.91o..112..~6T2 3~ ~

SgS-4

Df(I)N 8

Df(I)N 264-105

Df(I)N regh9

Df(I)N54 L9

+

Fig. 5. The Notch deficiencies used in this study and their effect on the expression of gene Sgs-4. Gaps in the black horizontal bars: deficient chromosome sections. Cytological map of Bridges (1938) modified by Beermann (1972). Deficiency heterozygous females were investigated. The absence of the gene Sgs-4 ( - ) in the deficiency chromosomes or its presence in the chromosome N s*~9 ( + ) was concluded from the absence or presence of coding protein 4 in the deficiency chromosomes

4 protein is in this case produced (Fig. 5). As expected, the heterozygotes Df(1)NS4~9/+ exhibit puffing of 3C in both the structurally normal and the deficiency chromosome, most clearly seen when both homologues are unpaired (Fig. 6, bottom).

Chromosome Pairing and Gene Activation

The saliva of the two wild-type stocks Hikone-R and Kochi-R differs from that of other wild-type stocks in that it lacks fraction 4 completely. Yet, cytologically, there is no indication of a deficiency in chromosome section 3C. That the absence of protein 4 does not result from loss of gene Sgs-4 became evident from results obtained by crossing Berlin and Hikone animals. While, as expected, the saliva of male offspring from the reciprocal crosses shown only the maternal type of fraction 4, the analysis of saliva from female offspring gave surprising results. Saliva of the F I - ~ from both crosses contained Berlin stock protein 4a and an additional fine yet distinct band between fractions 3 and 4a (Fig. 7). This protein fraction, denominated 4h, is clearly different from any other saliva protein found in the wild-type stocks investigated. It is unique to hybrids carrying the Hikone X chromosome in heterozygous condition since it also occurs in the female offspring of crosses between Hikone and Oregon (Fig. 8). In acrylamide gel electrophoresis it moves slightly faster than protein 4c (Fig. 8). The occurrence of fraction 4h in the heterozygous H/B and H/O females

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+/+

Df(1)NS/+

Df(1)N264 - 105/+

Df(1)Ne69h9/--

Df(1)NS4t9/+

Fig. 6. Left end of X chromosomes of female larvae, 93 h after oviposition, fi'om the Berlin stock ( + / + ) and from heterozygotes with Notch-deficiencies and Berlin (+). The arrows indicate puffing in section 3C. In the heterozygotes Df(1)NS/+, Df(1)N~64-1~ and Df(1)Nr69h9/+ only the structurally normal Berlin chromosome shows puffing in 3C; in the heterozygote Df(1)N54~9/+ both chromosomes are puffed in 3C (bottom)

is evidence for the presence of an allele of gene Sgs-4 in the X chromosome of Hikone. The activation, in hybrids, of this allele which remains inactive in the Hikone stock itself, could have two principal causes. (1) Autosomes originating from the Berlin parents could exert an influence on the state of activity of the gene. The absence of protein 4h in the FI-d'~ from cross H-~?~ • B - ~ (Fig. 7) is evidence against this. The protein was also missing in the saliva of male and female larvae with X chromosomes of Hikone origin and autosomes from Berlin. (2) The gene in the Hikone chromosome could be activated under

Chromosome Puff and Saliva Protein Synthesis

163

Fig. 7. Electrophoretically separated reduced and alkylated saliva proteins from the stocks Hikone-R and Berlin and their hybrids. Underneath each gel chromosome constitution of the saliva donor. H, chromosome from Hikone-R; B, chromosome from Berlin; H/B, first, second or third chromosome (CHR. 1 3) heterozygous for H and B. P, parental generations; F1, hybrids B x H, Berlin@~ x Oregon-R(ORN)@o~ ; H x B, the reciprocal cross. For each gel 10 saliva plugs from five to six larvae were used. 7% acrylamide disc gels, 5 mm in diameter. The arrows indicate the Hikone specific protein 4h. 4a, saliva protein from Berlin

Fig. 8. Electrophoretically separated reduced and alkylated saliva proteins from the stocks Hikone-R (H/H) and Oregon-R(ORN) (O/O), and from the filial generation of Hikone-~ crossed with Berlin-SS. 4c, Oregon specific saliva protein; 4h, Hikone specific. Underneath each gel chromosome constitution of the saliva donor. H, chromosome from Hikone-R; O, chromosome from Oregon-R(ORN) ; H/O, first, second or third chromosome (CHR. 1-3) heterozygous for H and O. For each gel 10 saliva plugs were used. 7% acrylamide disc gels, 5 mm in diameter

the i n f l u e n c e o f the X c h r o m o s o m e s f r o m B e r l i n or O r e g o n . E v i d e n c e for this is the o c c u r r e n c e o f p r o t e i n 4h in F I - ~ f r o m b o t h crosses o f B e r l i n a n d O r e g o n w i t h H i k o n e a n d its a b s e n c e i n m a l e o f f s p r i n g . T h i s X - c h r o m o s o m a l i n f l u e n c e c o u l d either be exerted, i n d i r e c t l y , i.e. b y w a y o f the n u c l e a r sap a n d / o r the c y t o p l a s m , or b y i m m e d i a t e c o n t a c t b e t w e e n the h o m o l o g o u s c h r o m o s o m a l sites i n v o l v e d . T o c h e c k t h e l a t t e r p o s s i b i l i t y the H i k o n e X was c o m b i n e d with an X carrying an inversion (FM1) that interferes with homologous pairing in heterozygotes.

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G. Korge

Table 1. Relative amount of saliva protein 4h in different genotypes. It was determined by using the quotients (4h/4a and 4h/3) of the absorption units from proteins 4h, 4a and 3. These in turn were ascertained planimetrically from the optical density curves Genotype

n

n~

4h/4a

4h/4a per X B

4h/3

4h/3 per III

H/B-$~ 1Xn/1xB; 1AH/1As

29

270

0.148

0.148_+0.007 (100)

0.033 (100)

0.065_+0.004 !100)

Triploids 1xH/2xB; 1AH/2As

7

72

0.076

0.152 + 0.007 (103)

0.020 (67)

0.059 _+0.006 (91)

Metafemales 1XH/2xB; 1AH/1AB

4

29

0.090

0.179 _+0.030 (121)

0.034 (103)

0.068 _+0.006 (105)

Intersexes 1Xn/1XB; 1An/2A B

6

61

0.198

0.198_+0.011 (134)

0.059 (179)

0.178_+0.010 (274)

X H and A u, X chromosome and set of autosomes from Hikone-R; X B and A B, from Berlin. n, number of gels. ns, number of saliva plugs used. In the columns 5 and 7 the values of proteins 4a and 3 were related to the number of X B and chromosomes III, respectively. Values are given with their standard deviations. Percentages based on the H/B values in parentheses.

X chromosomes of Berlin (or Oregon) derivation almost always strictly pair with those of the Hikone stock in heterozygotes, and instances of nonpairing in section 3C are rare (Fig. 11 d). This is not so in the case of H/FM1 heterozygotes where pairing of the two X chromosomes in section 3C could only be observed in 26 of a total of 282 nuclei from five larvae. Protein 4h could not be detected in the saliva of these heterozygotes. This indicates but does not prove a causal relation between the synthesis of saliva protein 4h in the heterozygous H/B females and chromosome pairing in section 3C. Subsequently an attempt was made to activate gene Sgs-4 in the Hikone chromosome more strongly than was possible in the case of diploid H/B females. These experiments were based on the two following expectations. (1) More intense pairing of the X chromosomes of Hikone and Berlin than occurs in diploid H/B females might be achieved by combining one Hikone X chromosome with two Berlin X chromosomes in triploids and metafemales. (2) The Hikone chromosome when paired with a Berlin chromosome might become more strongly activated on a triploid autosomal background as in intersexes because of dosage compensation. To distinguish XH/XB from xB/x B intersexes Hikone males were crossed with triploid yellow females. Among the offspring, non-yellow intersexes, carrying one Hikone and one Berlin X chromosome, were examined. The activity of the gene Sgs-4 of Hikone in the larvae of the various genotypes was determined indirectly by comparing the quantity of synthesized protein 4h with that of the X-chromosomal protein fraction 4a or with that of the autosomal protein fraction 3. From the values obtained planimetrically on the optical density curves quotients were formed for 4h in relation to 4a and 3 respectively (Table 1 ; Fig. 9). Quotient 4h/4a in the diploid H/B females (0.148) is considerably higher than in triploids (0.076) though lower than in intersexes (0.198; Table 1). It

Chromosome Puff and Saliva Protein Synthesis

165

O,D. 590nm

3.0-

3 H/8-99

2.0-

1.0 - 1

4a

F

3.0-

2.0 F

A II

H/B-

Intersexes

I

Fig. 9. The optical density curves of the gels with electrophoretically separated reduced and alkylated saliva proteins from heterozygous diploid H/B larvae (above) and from intersexes with one X chromosome from Hikone-R (H) and one X chromosome from Berlin (B) (below). 1-5, saliva proteins, 4h, Hikone specific; P, possibly cytoplasmic protein; F, front. The quotient Q4/3 per autosome set (see Table 1) is for H / B - ~ (upper curve) 0.074, but for the H/B-intersexes (lower

curve) 0.169 must be remembered that triploids and metafemales both carry t w o Berlin X chromosomes. If the amount of protein 4h is viewed in relation to the amount of protein 4a produced per X B it can be seen that in diploid H/B females, triploids and metafemales roughly equal amounts of protein 4h were produced, though significantly less than in intersexes (Table 1; 4h/4a per X~; Fig. 10). This result becomes clearer when the amount of protein 4h is compared with the amount of autosomal fraction 3 in relation to one autosome III (4h/3 per III; Table 1; Fig. 10). Only the quotient of the intersexes (0.178_+0.010) is significantly higher than that of diploid H/B females (0.065 +0.004; Table 1). The saliva samples of triploids, metafemales and intersexes in most cases present an additional faint protein band between bands 3 and 4h (Fig. 9, P). This is presumably a cytoplasmic impurity-here more concentrated than in the saliva samples of diploid larvae as a result of the somewhat difficult preparation. To extract the saliva of triploids, metafemales and intersexes more larvae had to be used than larvae of diploids because the saliva in most cases formed flaky precipitates and often only very small saliva plugs were obtained from

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G. Korge

4 h / 4 a per X 8

4h/3 per A

0.1

iliiii

iliii

XH X B3

A

I 1 2

I 2 3

2 2

I 1 3

1 1 2

1 2 3

1 2 2

1 1 3

Fig. 10. The a m o u n t of protein 4 h in various genotypes, on the left related to the a m o u n t of protein 4a (quotient Q4h/4a) and on the right related to the a m o u n t of protein 3 (quotient Q4h/3). Under each column is the n u m b e r of X c h r o m o s o m e s from Hikone-R (X H) and Berlin (XB), and the number of autosome sets (A) of the saliva donor. Relative a m o u n t s are given with their standard deviations. For further information on the material used see Table 1

a larva. The genotype of the saliva donor was identified from chromosome preparations. With regard to the problem posed the result of these experiments is unmistakeable: (1) The pairing of just one X chromosome from Hikone-R with two X chromosomes from Berlin in triploids and metafemales does not bring about a significantly higher production of protein 4h than in diploid H/B females. (2) Intersexes, which carry an X chromosome from Hikone-R paired to an X chromosome from Berlin produce more protein 4h than do diploid H/B females. If the reason why Hikone stock larvae fail to produce one of the saliva proteins in fraction 4 is that the gene somehow cannot be activated, no puff should appear in chromosome section 3C10 through 3D1 during the saliva synthesis phase. In fact, at no time during the third larval instar of Hikone was a puff in this chromosome section observed (Fig. 11 a). Throughout the third larval instar, this section in the X chromosomes of Hikone always appears as a narrow, pale region like the one seen in Berlin chromosomes towards the beginning of prepupa formation when the puff in this section has already become inactive. Heterozygous females with one X chromosome from Berlin and one from Hikone would be expected to exhibit some kind of puffing heterozygosity in chromosome section 3C from the middle of the third larval instar on. However, in the paired state of the section, this structural heterozygosity was rarely distinct. In most of the nuclei examined, puff formation in the Berlin chromosome appeared to be inhibited whereas the Hikone chromosome did brighten and

Chromosome Puff and Saliva Protein Synthesis

167

Fig. lla-d. Left end of X chromosomes from female larvae of the Drosophilastock Hikone-R (a) and of heterozygotes with one X chromosome from Hikone-R and one X chromosome from Berlin (b-d), The arrows indicate the chromosome section 3Cll/12. ]n Hikone (a) 3Cll/12 is not puffed, in the heterozygotes Hikone/Berlin this section is asymmetrically puffed, if the chromosomes are strongly paired (b, c); in unpaired condition only one chromosome shows puffing in 3C (d) swell to some extent in this section (Fig. 11 b, c). The expected structural heterozygosity, however, was distinct in just a few nuclei in which section 3C was not paired (Fig. 11 d). This is particularly true in those heterozygous females which possess an X chromosome from Hikone and one carrying the FM1 inversions: a 3C puff is formed only in the FM1 c h r o m o s o m e (Fig. 12). Intersexes with one X c h r o m o s o m e from Hikone and one X chromosome from Berlin were expected to show more marked puffing in 3C of the Hikone chromosome than did dipoid H/B females, in view of dosage compensation and the resulting increased activity of gene Sgs-4 (Table 1; Fig. 10). In fact, both the Berlin and Hikone X showed distinct puffing in 3C but with almost no indication of heterozygosity in any of the chromosomes e x a m i n e d - a t any rate, much less so than in diploid H/B females. The puff is visually symmetric and just about as pronounced as in homozygous Berlin females (Fig. 13).

Transcription in 3C-D Salivary glands of third instar larvae were incubated in vivo and in vitro with 3H-uridine and autoradiographs taken of the squash preparations.

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G. Korge

Fig. 13. Left end of X chromosomes from a heterozygous intersex, one X chromosome from Hikone-R, one X chromosome from Berlin. The chromosome section 3C-D is symmetrically puffed

Fig. 12. X chromosomes from female larvae, one X chromosome from Hikone-R (/4), one X chromosome carrying the inversion F M 1. The chromosome pairing in 3C is disturbed by the inversion. The arrows indicate the chromosome section 3C1 !/12. Though the chromosomes seem to be paired normally (a) only one chromosome is puffed in 3C (upper chromosome). In unpaired condition only the FM1 chromosome shows puffing in 3C (b)

At low levels of labeling region 3CD of the Berlin X chromosome showed a concentration of label between 3C10 and 3D1, very close to band 3D1. A few silver grains were invariably found above 3D1-2 (Fig. 14a). With slightly elevated incorporation the presence of label at both sites became more distinct (Fig. 14b). However, the site between 3C10 and 3D1 was always more intensily labeled (Fig. 14c). In contrast to the situation in the Berlin stock the chromosomes of Hikone larvae appeared in most cases labeled only in section 3D, i.e. mainly over 3D1-2 (Fig. 14d, e). Thus, in X chromosomes of Hikone third instar larvae very little if any transcription appears to take place at the site between 3C10 and 3C1. In autoradiographs of heterozygous H/B larvae an asymmetric labeling in section 3CD was occasionally seen. However, the resolving power of the autora-

Chromosome Puff and Saliva Protein Synthesis

169

Fig. 14a-e. Autoradiographs from the left end of X chromosomes from female larvae of the stocks Berlin (a-e) and Hikone-R (d, e) after 10-15 min 3H-uridine incubation, in vivo. 0.2 gCi 3H-uridine (0.2 ~tl) were injected into larvae, about 90 h after oviposition. Exposition time: 40 d. Each upper figure: stained chromosomes in bright-field; each lower figure: phase contrast, a Weak labeling in 3C adjacent to 3D1 and 3D1-2; b and c strong labeling in 3C and 3D1-4; fl and e relatively weak labeling in 3D1-6

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G. Korge

Fig. 15a-e. Left ends of X chromosomes from prepupae of the Drosophila stocks Hikone-R (a-e) and Berlin (d, e); a 5 h after eversion of the spiracles ; b 8 h; e--e 9 h. The arrows indicate c h r o m o s o m e section 3D1-2. a no puffing within 3DI-2; b-e puffing starting slightly to the right of 3D1-2

diographic method is hardly sufficient to distinguish between the two closely adjacent active loci in 3C and 3D, especially in heterozygous condition.

Puffing in the Prepupa Prepupa formation marks the end of the synthesis of saliva proteins 1, 3 and 4 (Korge, in press). These proteins do not appear again in the course of prepupal development, so their structural genes should not become active again either. The puff in section 68C of the third chromosome, which probably contains the gene responsible for protein 3 (Korge, 1975), does not in fact appear again once it has been inactivated 5 h before prepupa formation (Becker, 1959; Ashburner, 1967a). However, in section 3C of the X chromosome Becker (1959) and Ashburner (1969) observed the formation of a puff in 6 and in 4 h old prepupae respectively, which disappears again after 5-6 h. If this were the same locus in 3C as the one active in the third instar larva and established as the site of the active gene Sgs-4, the absence of protein 4 in the salivary glands of prepupae would be surprising. There are two possible explanations for this apparent contradiction. Supposing gene Sgs-4 is reactivated in the prepupa, the interval between the puff's first appearance (four or six hours after prepupa

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formation) and the onset of autolysis (11-12h) might be so brief that too little protein 4 to be detected by electrophoresis is produced. A simpler explanation for the absence of protein 4 in the prepupa would be that not gene Sgs-4 but a very close neighbour of this gene is activated. If this is the case, formation of a puff is to be expected during the prepupal development of Hikone, too, since it is most unlikely that in Hikone yet another gene, adjacent to Sgs-4, would fail to be activated in the prepupa. In 5-h-old Hikone prepupae there is no puff in X chromosome section 3C (Fig. 15a). About 6 h after prepupa formation bands 3D1-2 grow diffuse and begin to d i s t e n d - a typical sign of activation in this region (Fig. 15b). In 8-h-old prepupae a puff is already formed. It seldom extends distally as far as band 3C10; proximally it includes in most cases bands 3D5-6 (Fig. 15c). This puff has the same properties as the puff in chromosomes of Berlin prepupae: it appears at the same time and is of the same morphological extent (Fig. 15 d, e). Therefore, from the morphological results, it can be concluded that in the X chromosomes of prepupae no puff originates from section 3C, as it does in the third instar larva.

Discussion

Gene Sgs-4, which determines the type of larval saliva protein 4, must be the structural gene of this protein. This can be concluded, above all, from the codominant behaviour of the alleles (Fig. 1), the absence of a second fraction of protein in heterozygotes of deficiencies including section 3C (Figs. 3, 5) and the dependence of the relative amount of the protein synthesized upon gene dosage (Fig. 4; Korge, 1975). Experiments on recombinants and preliminary cytological analysis had already shown gene Sgs-4 to be situated within a section limited by bands 3C8 and 3D1 of the X chromosome (Korge, 1975). With the help of further Notch deficiencies the site of the gene could be placed between bands 3C10 and 3D1, leaving, according to Bridges (1938), only two very fine, closely adjacent bands, 3Cll and 3C12. Actually, the presence of two bands at this site is doubtful. Own light-microscopic observations always showed at most just one very fine, diffuse band between 3C10 and 3D1 like the one drawn in Bridges' chromosome 1935 map. Berendes (1970), drawing upon electron optical results, also describes only one diffuse band in this section. The problems involved in proving the existence of so-called "double bands" are discussed in depth by Beermann (1962). To determine whether, in fact, one band or two bands are present in section 3C10 through 3D1, light microscopic observations alone will not suffice. It will be even harder to determine whether or not the band, or one of the bands, contains the structural gene Sgs-4. On the basis of the cytogenetic investigations and from the 1:1 ratio of the number of genes to the number of bands in very carefully examined chromosome sections, it has been deduced that the genes are located in the bands, only one gene in each band (Beermann, 1972). The interbands were not thought to contain structural genes, though this possibil-

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ity could not be excluded. The chromosome model conceived by Crick (1971) ascribes coding function only to the interbands and regulative function to the bands. Zhimulev and Belyaeva (1975a, b) go even further: from the results of their autoradiographic investigations on salivary gland chromosomes they conclude that all interbands are transcriptionally active sites. The bands, which also, in their opinion, can contain several genes, are presumed to contain the inactive genes which in the salivary gland cells are not needed neither for basal metabolism nor for specific synthetic activities. In contrast, Paul (1972) ascribes a regulative function to the bands and an informative function to the interbands. The molecular weight of protein 4 has been determined as being below 100• Daltons (Beckendorf and Kafatos, 1976; Korge, in press). This protein might be equivalent to a coding DNA section of at least 2100 base pairs-the total DNA of an average interband (Beermann, 1972). Supposing that the m-RNA for protein 4 derives from a larger precursor, participation of a band (3Cll or 12) in the transcription of gene Sgs-4 is highly probable. The results suggest that the synthesis of protein 4 is dependent upon the appearance of the puff in section 3Cll/12. The puff in 3Cll/12 is the only pronounced puff in the X chromosome to appear during the third larval instar before the actual puffing period (Becker, 1959; Ashburner, 1969; Korge, in press). There is a very clear positive correlation between the duration of activity of this puff and the phase of larval saliva synthesis. The puff is present at least five hours before the first appearance of protein 4 and becomes inactive at the start of the puffing period (Becker, 1959; Ashburner, 1969). This puff was said to reappear in the prepupa (Becker, 1959 ; Ashburner, 1969) though it is now clear that not this site, but chromosome site 3D1-2 very near by, becomes active (Fig. 15). The greatest dilation of the chromosome is visible proximal to 3D1 and probably coincides with the site of this puffs centre of activity (Fig. 15; also see Becker, 1959, Abb. 7; Ashburner, 1969, Fig. 3~f). The two puffs appearing in section 3C-D in the third larval instar and in the prepupa are clearly distinct and independent from each other as can be seen in the case of Hikone. In X chromosomes from Hikone there is no puff in section 3C-D during the third larval instar whereas in the prepupa a puff in 3D1-2 appears as it does in the case of Berlin (Fig. 15). Investigation of the Hikone stock led to a further interesting result. The homozygous Hikone larvae lack both the puff in 3C11/12 and protein 4, whereas the heterozygous females from crosses between Hikone and Berlin or Oregon have Hikone-specific protein (4h) in their saliva (Figs. 7, 8). The assumption that activation of the gene in the Hikone chromosomes takes place when there is strict chromosome pairing in section 3C is substantiated by the results from crossing FMl-animals with Hikone. In the inversion heterozygotes section 3C was unpaired in 90% of cases, and in these cases only the FM1 chromosomes presented a puff (Fig. 12). Protein 4h was not found in the saliva of these larvae. However, this lack of protein 4h in the inversion heterozygotes could be due to causes specific for FM1 other than the prevention of chromosome pairing in 3C. This question could be solved by using additional inversions which also impair homologue pairing in this region.

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An influence of homologue pairing on the expression of a phenotype has been observed in earlier investigations on pseudoalleles of the bithorax group (Lewis, 1954). Heterozygotes with pseudoalleles in trans-configuration show modification of the mutant phenotype, when chromosome rearrangements impaire homologue pairing. This "trans-vection effect" was explained by diffusion of a substance from one chromosome to the other in the paired condition (Lewis, 1954; 1963); diffusion was assumed to be suppressed if homologous bithorax regions were not paired. - T h e same way Ashburner (1967 b) interpreted his observation that in vg6/Oregon heterozygotes section 64C in the Oregon chromosome is puffed only if strongly paired with the active homologous section of vg6. He believes that section 64C in the Oregon chromosome is puffed either by taking over and storing transcription products (NP-Function, Beermann, 1965) of the vg6-puff or by activation of a gene in the Oregon chromosome, which is otherwise inactive. Only this second assumption is consistent with the activity of 3C in Hikone and in Berlin or Oregon. In heterozygous H/B or H/O, in paired chromosomes the gene inactive in the pure Hikone stock must be activated in the Hikone chromosome, in view of the fact that a specific translation product (protein 4tl) occurs. The much greater production of protein 4h in heterozygous intersexes is very clear evidence for an activation of gene Sgs-4 in the X chromosome of Hikone (Table 1; Fig. 10). This large yield must result from dosage compensation. The nearly symmetric form of the puff in 3C in these heterozygous intersexes could be the morphological expression of the gene's activity in the Hikone chromosome (Fig. 13). Synthesis of protein 4a is also intensified in the intersexes, though to a lesser extent than 4h (Table 1). The way in which gene Sgs-4 in the X chromosome of Hikone becomes activated cannot be explained by the regulation models of Britten and Davidson (1969) and Paul (1972) unless additional assumptions are made. It is possible that in the case of strict chromosome pairing, transcription in the Berlin chromosome brings about a change in the blocked receptor function in the pairing partner Hikone. Another possibility is that the Hikone chromosome, through the activation in the Berlin chromosome, is passively uncoiled and thus put into a suitable state for transcription. Investigation of recombinants within the operative unit of gene Sgs-4 of Hikone and Berlin and electron microscopical examination of the chromosome region 3C11/12 of Hikone, Berlin and heterozygotes may help to solve this problem. Acknowledgements. I should like to thank Drs. B. Rasmuson, H. Ursprung, and W.J. Welshons for providing me with wild-type and deficiency stocks. I am very grateful to Drs. H.J. Becker and W. Beermann for helpful advice and criticism in preparing the manuscript.

References Ashburner, M.: Patterns of puffing activity in the salivary gland chromosomes of Drosophila. I. Autosomal puffing patterns in a laboratory stock of Drosophila melanogaster. Chromosoma (Berl.) 21, 398 428 (1967a) Ashburner, M.: Gene activity dependent on chromosome synapsis in the polytene chromosomes of Drosophila melanogaster. Nature (Lond.) 214, 1159 1160 (1967b) Ashburner, M.: Patterns of puffing activity in the salivary gland chromosomes of Drosophila. II. The X-chromosome puffing patterns of D. melanogaster and D. simulans. Chromosoma (Berl.) 27, 47 63 (1969)

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Beckendorf, S.K., Kafatos, F.C. : Differentiation in the salivary glands of Drosophila melanogaster: characterization of the glue proteins and their developmental appearance. Cell 9, 365-373 (1976) Becker, H.J. : Die Puffs der Speicheldrfisenchromosomen von Drosophila melanogaster. I. Beobachtungen zum Verhalten des Puffmusters im Normalstamm und bei zwei Mutanten, giant und lethal-giant-larvae. Chromosoma (Berl.) 10, 654-678 (1959) Beermann, W.: Riesenchromosomen. Protoplasmatologia, VI D, pp. 1-161. Wien: Springer 1962 Beermann, W.: Operative Gliederung der Chromosomen. Naturwissenschaften 52, 365 384 (1965) Beermann, W.: Chromosomes and genes. In: Results and problems in cell differentiation (W. Beermann, ed.), Vol. 4, pp. 1-35. Berlin, Heidelberg, New York: Springer 1972 Berendes, H.D.: Polytene chromosome structure at the submicroscopic level. I. A map of region X, 1 4E of Drosophila melanogaster. Chromosoma (Berl.) 29, 118-130 (1970) Bridges, C.B.: Salivary chromosome maps with a key to the banding of the chromosomes of DrosophiIa melanogaster. J. Hered. 26, 60 64 (1935) Bridges, C.B.: A revised map of the salivary gland X-chromosome of Drosophila melanogaster. J. Hered. 29, 11-13 (1938) Britten, R.J., Davidson, E.H.: Gene regulation for higher cells: a theory. New facts regarding the organization of the genome provide clues to the nature of gene regulation. Science 165, 349-357 (1969) Crick, F.: General model for the chromosomes of higher organisms. Nature (Lond.) 234, 25-27 (1971) Grossbach, U. : Chromosomen-Aktivitht und biochemische Zelldifferenzierung in den Speicheldrfisen yon Camptochironomns. Chromosoma (Berl.) 28, 136 187 (1969) Korge, G. : Chromosome puff activity and protein synthesis in larval salivary glands of Drosophila melanogaster. Proc. nat. Acad. Sci. (Wash. 72, 4550-4554 (1975) Korge, G.: Larval saliva in Drosophila melanogaster: production, composition and relationship to chromosome puffs. Develop. Biol. 58, (in press, 1977) Lewis, E.B. : The theory and application of a new method of detecting chromosomal rearrangements in Drosophila melanogaster. Amer. Naturalist 88, 225-239 (1954) Lewis, E.B. : Genes and developmental pathways. Amer. Zoologist 3, 33-56 (1963) Lindsley, D.L., Grell, E.H. : Genetic variations of Drosophila melanogaster. Carnegie Inst, Wash. Publ. 627 (1968) Paul, J. : General theory of chromosome structure and gene activation in eukaryotes. Nature (Lond.) 238, 444-446 (1972) Poels, C.L.M. : Time sequence in the expression of various developmental characters induced by ecdysterone in Drosophila hydei. Develop. Biol. 23, 210~25 (1970) Poels, C.L.M. : Mucopolysaccharide secretion from Drosophila salivary gland cells as a consequence of hormone induced gene activity. Cell Diff. 1, 63 78 (1972) Zhimulev, I.F., Belyaeva, E.S. : 3H-uridine labeling patterns in the Drosophila melanogaster salivary gland chromosomes X, 2R and 3L. Chromosoma (Berl.) 49, 219--231 (1975a) Zhimulev, I.F., Belyaeva, E.S. : Proposal to the problem of structural and functional organization of polytene chromosomes. Theoret. appl. Genet. 45, 335-340 (1975 b)

Received February 17-March 11, 1977 / Accepted March 12, 1977 by W. Beermann Ready for press March 16, 1977