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BOTH NUCLEOLAR ORGANIZERS ARE REPLICATED IN DIPTERAN POLYPLOID TISSUES: A STUDY AT THE LEVEL OF INDIVIDUAL NUCLEI

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B O T H NUCLEOLAR ORGANIZERS ARE REPLICATED IN

DIPTERAN POLYPLOID TISSUES: A STUDY A T T H E

LEVEL

OF INDIVIDUAL NUCLEI

ESTHER J. BELIKOFF AND KATHY BECKINGHAM

Department of Biochemistry, Rice University, P.O. Box 1892, Houston, Texas 7 7 2 5 1

Manuscript received March 1 1, 1985 Revised copy accepted May 25, 1985

ABSTRACT

Working with the Dipteran Calliphora erythrocephala, we have tested the hypothesis that only one nucleolar organizer region (NO) is replicated during polyploidization. NO replication was examined in two very different highly polyploid nuclear types: salivary gland nuclei and nurse cell nuclei. Two strains of the organism containing NO regions with highly diagnostic nontranscribed spacer (NTS) polymorphisms were prepared and reciprocal single pair-matings between members of the strains were performed. The representation of the two distinguishable NOS in diploid and polyploid DNAs of individual F, prog- eny from each cross was then examined. DNA from a total polyploid nuclear DNA preparation and from individual polyploid nuclei of both tissue types was analyzed. Our results show conclusively that both genomic NOS are replicated in individual polyploid nuclei of both types. Further, evidence for variation in the relative replication of cistrons from the two NOS by individual nuclei was obtained. The cistron types present in the NOS of both strains showed differ- ential replication upon polyploidization. In general, the patterns of differential cistron replication seen in salivary gland and nurse cell nuclei were similar.

T is well established that, in Dipteran polyploid/polytene tissues, the genes

I

for ribosomal RNA (rDNA) are independently replicated to a level that differs from, and is usually lower than, that of the euchromatic regions of the genome (HENNIG and MEER 1971; SPEAR and GALL 1973; SPEAR 1974; REN-

KAWITZ and KUNZ 1975). T h e possibility that this differential replication is the result of replication of only one of the genomic NOS has been under consid- eration ever since this phenomenon was first elucidated (HENNIG and MEER

1971). T h e demonstration that the amount of rDNA in polytene DNA is independent of the number of NOS present in the diploid genome (SPEAR and

GALL 1973; SPEAR 1974) is often cited as evidence in favor of this hypothesis

(MACGREGOR 1973; ENDOW 1980; GLOVER 1981). However, as noted by SPEAR (1974), this theory is not adequate to explain the very similar rDNA levels achieved upon polyploidization of NOS with very different initial rDNA con- tents (SPEAR and GALL 1973; SPEAR 1974) and thus, as a hypothesis, provides little insight into the mechanisms regulating the polyploidization of the rDNA. Recently, crosses between strains of Drosophila melanogaster possessing rDNA

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326 E. J. BELIKOFF AND K. BECKINGHAM

gene sets of markedly different structure have been used to demonstrate that, when t w o nonidentical NOS are present in the same nucleus, cistrons from one N O may be polytenized to the virtual exclusion of cistrons from the other

(ENDOW and GLOVER 1979; ENDOW 1980, 1982a, b, 1983). It has been re- peatedly extrapolated that these experiments provide evidence in support of the idea that one N O is inactivated during polytenization (ENDOW and GLOVER

1979; GLOVER 1981; ENDOW 1982a, b, 1983). However, in crosses where two very different NOS are present in the same nucleus, an alternative explanation may be evoked to explain differences in their polytenization; that is, structural differences between the two NO types may result in their possessing very different intrinsic capacities to interact with, or compete for, the components of the replication machinery. Thus, in order to use differing N O regions to investigate definitively the possibility of N O inactivation, pairs of NOS must be obtained that appear to be replicated equally well at the level of the total hybrid polyploid tissue DNA, rather than showing very different degrees of replication. This prerequisite will establish that the two NOS in a particular cross show very similar capacity to interact with the replication machinery, and then, an examination of the rDNA present in individual polyploid/polytene nuclei will permit a critical test of the hypothesis of N O inactivation. If the hypothesis is correct, individual nuclei will be of two types, showing replication of cistrons from one or other NO-but never both- as a result of the inac- tivation of one or other N O in each cell; if incorrect, all nuclei will replicate cistrons diagnostic for both NO regions.

Working with the Dipteran Calliphora erythrocephala, we have used this ap- proach to examine the possibility of N O inactivation in two highly polyploid nuclear types: salivary gland and nurse cell nuclei. Our results show conclu- sively that, in both types of nuclei, both of the genomic NOS are replicated during the polyploidization process. Our experiments also revealed an unex- pected finding: the extent of replication of the two parental NOS during polyploidization can very considerably from cell to cell in a given animal.

MATERIALS AND METHODS

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parents are reported here. The behavior of the Cam NO type was identical in all Cam NO/Karla NO heterozygotes examined and therefore independent of whether the Cam NO was derived from a Cam strain or Cai strain parent.

DNA preparations from whole tissues and whole ovary nurse cell nuclear preparations: Brains and salivary glands from third instar larvae were dissected in insect saline (BECKINGHAM and THOMPSON

1982). The ring gland was removed from each brain, and the fat body and imaginal cells were removed from each pair of salivary glands. Adult females with batches of ovarian follicles at the stage of maximal nurse cell development were prepared as described previously (BECKINGHAM and

THOMPSON 1982). The brain of each female was dissected in insect saline, and nurse cell nuclei were prepared from both ovaries, using the procedure described earlier (BECKINGHAM and THOMP-

SON 1982), but with omission of the initial elastase treatment. DNA was extracted from these preparations and from early embryos (see Figures 2 and 3), using the method of BECKINGHAM and

THOMPSON (1982), with the following modifications. The pronase concentration for overnight digestions was increased to 1 mg/ml, and DNA samples were treated with boiled RNase A (25 rg/ml for 30 min at 37") before quantitation. DNA concentrations were estimated by ethidium bromide fluorescence (MANIATIS, FRITSCH and SAMBROOK 1982). Digestions with restriction en- donuclease RsaI were performed using conditions specified by the supplier (Boehringer-Mannheim) and an excess enzyme to DNA ratio (20 units enzyme/pg DNA). Horizontal gel electrophoresis, using 1 % agarose gels, was performed as previously described (BECKINGHAM and THOMPSON 1982).

Preparation and digestion of DNA from individual polyploid nuclei Individual salivary gland iiuclei were isolated from glands that had been disrupted by swelling in hypotonic saline (10 mM NaCl/ 10 mM Tris-HCI (pH 7.5)/0.15 mM spermine/0.5 mM spermidine) for 10 min at room temper- ature, followed by shearing through a 27-gauge hypodermic needle. Individual nurse cell nuclei were isolated from ovarian follicles, which were gently teased apart in insect saline with watch- makers' forceps. The largest (oocyte proximal) nuclei were always chosen for analysis. Silanized micropipettes were used to transfer single nuclei to individual wells of multiwell, silanized glass slides. Adherent saline was removed, and 1.35 pl of 10 mM Tris-HCI/l mM EDTA (pH 8.0) were added to each well. Each sample was immediately covered with light paraffin oil and was subjected to all further treatment under this medium. Addition of 0.15 pl of 1 mg/ml pronase was followed by a 19-24-hr incubation at 37". Subsequently, 0.5 pl of 0.1 mg/ml boiled RNase A was added, and incubation at 37" continued for a further 2 hr. Digestion of samples with RsaI was performed in a second overnight incubation at 37", using 7.5 units enzyme per sample in a total volume of 3 el adjusted to the buffer conditions specified by Boehringer-Mannheim. Samples were removed with silanized micropipettes for gel electrophoresis. In order to increase detection of the very small quantities of DNA present on these samples, vertical 1% agarose gels (3 mm thick) with slots 6 mm wide were used to produce very narrow sample loading zones and, consequently, very sharp bands of hybridization.

Assuming a genome size of 0.76 pg (BIER and MULLER 1969) and a ploidy level of 2048C for the four largest nurse cell nuclei (MAHOWALD and KAMBYSELLIS 1980), and knowing that the intron- rDNA cistrons are proportionately replicated in nurse cell nuclei (BECKINGHAM and THOMPSON 1982), it can be calculated that the total amount of rDNA in each nurse cell nucleus would be approximately 19 pg. Based on hybridization intensity relative to a standard DNA used as a molecular size marker (see Figure 3), it can be estimated that the individual salivary gland nuclei contain approximately four to five times less rDNA.

Southern transfers and filter hybridization: DNA was transferred to Gene-Screen filters (New England Nuclear) and prehybridized for 2-12 hr at 30" in 50% formamide/2 X SET [0.3 M

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328

H P A A A A A A

I

; I 1 1

l k b

FIGURE 1.-Restriction enzyme site map for the rDNA Hind111 fragment present in clone pBW1. Solid bars indicate regions that are parts of the 18s and 28s rRNA coding regions. Sites for AluI (A), Hind111 (H), PuuII (P), RsaI (R) and XbaI (X) are shown. Only the AluI and XbaI sites within RsaI fragment A are shown. T h e Alul sites in the right-hand region of fragment A are within the 350 bp repeating units present in this clone (SCHAFER, WYMAN and WHITE 1981). Although other cloned spacer regions with four repeating units contain an AluI site within each repeat (SCHAFER, WYMAN and WHITE 1981), the existence of an Alul site within the left-most unit of this particular clone was not confirmed. This site is therefore indicated by a dashed line.

at 30" and, finally, 1 X 10 min in 2 X SET at room temperature. Autoradiography was performed at -70" with Kodak XAR-5 film and intensifying screens.

Preparation and restriction enzyme site mapping of pBWl and PBW.2: Plasmid DNA was prepared by the procedure of BIRNBOIM (1 983). T h e structure and origin of plasmids pBW 1 and pBW2 are described by SCHAFER, WYMAN and WHITE (1981). In order to position RsaI fragment A of pBWl relative to the spacer repeating units present in this clone, the sites for Rsal and A h 1 within the largest HzndlII/PuuII fragment of pBWl (see Figure 1) were mapped by the procedure of SMITH and BIRNSTIEL ( 1 976). This HindIII/PuuII fragment was "P-labeled at its single Hind111 terminus, as described previously (SMITH and BECKINGHAM 1984), in the presence of the other Hind1111

PuuIl digestion products of pBW1. T h e largest '*P-labeled fragment was then isolated and sub- jected to RsaI and AluI digestion, as described by SCHAFER, WYMAN and WHITE (1981).

RESULTS

Strain-spec@ NTS-region polymorphisms used in this study: We have previously demonstrated that different length classes of NTS are present within the rDNA of two different laboratory strains of C. erythrocephala (BECKINGHAM 1982). Within one strain, the different length classes of NTS were shown to result from variation in the copy number of a 350 base pair (bp) repeating unit present in the central NTS region @CHAFER, WYMAN and WHITE 1981). We wished to obtain further strain-specific N T S polymorphisms, and from studies with two cloned rDNA spacers we established that the cleavage sites for the enzyme RsaI within the NTS region are particularly well positioned for iden- tification of variants containing different numbers of the repeating unit.

T h e cleavage sites for RsaI within the rDNA fragment of clone pBW1-a Hind111 fragment containing an N T S region with four repeats (SCHAFER, WY-

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329

taining the repeating units. T h e RsaI fragment pattern obtained from clone pBW2-which contains an equivalent Hind111 fragment spanning an NTS re- gion with five repeats-demonstrated that RsaI sites are identically positioned within this clone in such a way that fragment A is replaced by a fragment 350 bp, or one repeat unit, larger. Thus, it could be predicted that hybridization of pBWl DNA to RsaI cleaved genomic DNA from various strains of C. erythrocephala would yield a common pattern of small bands of hybridization [sizes 1.6 kb or less (see Figure I)] and variants of the 2.3-kb fragment de- pendent on the classes of NTS region present within the rDNA. DNA from individuals of a series of laboratory strains was therefore examined to identify strain-specific variants of this fragment that could be used as diagnostic markers for the replication of a given NO type. Two NO types, initially identified in fly stocks from the laboratories of Karlson and those of the Zoology Depart- ment in Cambridge, England, were selected for study, and strains homozygous for both of these NO types were generated by one to three rounds of single pair-matings as required (see MATERIALS AND METHODS). T h e Karla strain was homozygous for the Karlson N O type (Karla NO), and both the Cam and Cai strains (see MATERIALS AND METHODS) were homozygous for the Cambridge NO (Cam NO) type. These two NO types were selected because each contained very few length classes of NTS region, with most of the rDNA cistrons yielding only one diagnostic length variant of fragment A. Thus, the band characteristic of the NO is the major band of hybridization among this class of fragments and, therefore, is a very prominent marker for the NO.

In the Karla NO, the diagnostic RsaI fragment (Karla band) is -350 bp shorter than fragment A, and it is probable that this fragment results from spacers containing three repeating units. T h e diagnostic fragment of the Cai/ Cam strains (Cam band) is -1.5 kb larger than fragment A and therefore, could be explained by the presence of four additional repeat units within most of the spacers of this NO type. However, in Cai/Cam strain DNAs, a very low level of hybridization is seen at the position of the fragment B that lies adjacent to fragment A within the spacer (see Figures 1 and

2).

This suggests that cistrons yielding the diagnostic Cam fragment do not also produce fragment B. Thus, a more likely explanation for this variant fragment is that it is derived from spacers lacking the RsaI site that separates fragment A from fragment B. T h e size of fragment B (1.6 kb) is appropriate for this interpretation.

In both the Karla and Cai/Cam strain NOS, the only other spacers present, in addition to those yielding diagnostic fragments, are ones that produce frag- ment A (see Figure 2) and, therefore, probably contain four repeats.

Replication of the Cam and Karla NOS in polyploid tissues: In previous studies aimed at investigating the replication of two different NOS in polytene nuclei, differential replication of the various cistron types within each NO during the polytenization process has complicated interpretation of the findings (ENDOW

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330 E. J. BELIKOFF AND K. BECKINCHAM

1

2

1

2

M B S B

S

B

N B N M

A-

K-

-

- A

-K

A-

K-

B-

-A

-K

-B

FIGURE 2.-The diagnostic N T S polymorphisms of the two NO types used in this study and

their polyploidization in salivary gland and nurse cell nuclear DNA. T h e patterns of hybridization of pBW 1 DNA to blots of RsuI digested diploid and polyploid DNA from individual animals of the Karla and Cai strains are shown. In this figure and in Figure 3, most of the common, small bands of hybridization below -1.4 kb in size are not shown (see text). a and c. DNA from the brains (B) and salivary glands (S) of two individual third instar larvae of the Karla and Cai strains, respectively. 0.05 pg samples of brain DNAs and 0.2 pg samples of salivary gland DNAs were used to compensate for underreplication of the rDNA in salivary glands. b and d, DNA from the brains (B) and whole ovary nurse cell nuclear preparations (N) of two individual adult females of the Karla and Cai strains, respectively. Samples of both DNA types were 0.2 pg. A and B, Bands of hybridization to fragments equivalent to RsuI fragments A and B of pBWl (see Figure 1). C and K, T h e diagnostic Cam and Karla bands (see text). M (marker DNA), Rsul digest of early embryo DNA from a mixed fly population, which contains fragments A and B, and the diagnostic Cam and Karla fragments. Marker lanes in the different autoradiograms contain different amounts of DNA as follows: a. 0.05 pg; b, 0.1 pg; c, 0.4 pg; d, 0.2 pg.

ual third instar larvae, and from the brains and nurse cell nuclei of at least four individual females of the Karla and Cai strains, was examined.

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33

identical in all individuals, and the changes seen in whole tissue polyploid DNA of both types varied little in different animals (see Figure

2).

Hybridization of pBWl to DNA from individual salivary gland and nurse cell nuclei of animals from both strains was also attempted to investigate the possibility of different rDNA cistron polyploidization patterns in individual nuclei. Technically, it proved more difficult to obtain successful examples of hybridization to individual salivary gland nuclei for two reasons. First, the rDNA is underreplicated in the salivary glands (HENNIG and MEER 1971;

SPEAR and GALL 1973; (E. J. BELIKOFF and K. BECKINCHAM, unpublished observations), whereas it is almost proportionately replicated in nurse cells (BECKINGHAM and THOMPSON 1982). Thus, each salivary gland nucleus con- tains a much smaller quantity of rDNA, making detection more difficult. Sec- ond, endogenous DNase proved a problem with salivary gland nuclei. In gen- eral, therefore, fewer hybridization patterns for individual salivary gland nuclei were obtained.

For the Karla strain, the extreme underreplication of the fragment A-bear- ing cistrons seen at the level of the total polyploid tissue DNA (see Figure

2)

suggests that if, in some individual cells, these cistrons were extensively poly- ploidized, such cells would constitute an extremely minor fraction of the total population. Thus, although the hybridization patterns obtained for all of the individual polyploid nuclei examined (three salivary gland and 20 nurse cell) were identical to those seen for the total polyploid tissue DNA samples (that is, showing no detectable fragment A hybridization), the number of nuclei examined is probably insufficient for a definitive answer on this point.

For the Cam NO, detectable polyploidization of fragment A-bearing cistrons is seen at the level of the total polyploid tissue DNA (see Figure

2),

and therefore, it might be predicted that, if this NO region were variably poly- ploidized in individual cells, nuclei showing extensive replication of fragment A-type cistrons would be more frequent within the total nuclear population. However, of the

17

individual polyploid nuclei examined (three salivary gland and 14 nurse cell), none showed strong hybridization to the position of frag- ment A. Rather, hybridization to this band appeared to be close to the detec- tion limit of our system for all nuclei, being absent or barely detectable in all samples. A strong band of hybridization was seen at the diagnostic Cam frag- ment position for all 17 nuclei. Thus, although not conclusive, this finding suggests that no gross differences in polyploidization of the two cistron types of the Cam N O occur within individual nuclei.

Replication of Karla and Cam NOS in interstrain crosses: Single pair-matings between individuals of strains carrying the Cam and Karla NOS were per- formed, and the representation of the two parental NOS was examined in salivary gland and nurse cell nuclear DNA from individual F1 progeny. Table

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332 E. J. BELIKOFF AND K. BECKINGHAM

TABLE 1

The single pair strain crosses used in this study and the details of the individual F , progeny examined

Parental strain origins No. of individual F,

progeny examined No. of individual nuclei examined"

No. of

Third salivary No. of No. of No. of Female instar Adult gland original nurse cell original Cross no. parent Male parent larvae* females' nuclei larvae nuclei females

CaK7 Camd Karla 1 4 7 1

KCa 1 Karla Cam 1 4 1 1

CKI 1 Caid Karla 4 5 1 1 22 5

CK8 Cai Karla 8 2

CK9 Cai Karla 8 2

"In all crosses, except for CK8 and CK9, individual nuclei were derived from larvae or adult females for which analysis of whole salivary gland or whole ovary nurse cell nuclear DNA had been performed.

'For all larvae, DNA from both the whole brain and whole salivary glands was examined. 'For all females, DNA from both the whole brain and a whole ovary nurse cell nuclear prep-

dBoth the Cam and Cai strains are homozygous for the Cam N O (see MATERIALS AND METHODS).

aration was analyzed.

Diploid DNA from all individuals, both larvae and adult females, showed the expected hybrid pattern containing the Cam, Karla and fragment A bands of hybridization (see Figure 3). For both salivary gland and nurse cell DNA samples, the F, hybridization pattern was also a composite of the hybridization patterns seen for these tissue DNAs from the two parental strains; that is, strong and approximately equal representation of the Cam and Karla bands was seen in all individuals with very little detectable hybridization at the posi- tion of fragment A (see Figure 3). Thus, as judged at the level of the total polyploid nuclear DNA, despite major differences in rDNA cistron composi- tion, the Cam and Karla NOS appear to compete equally well for replication during polyploidization in both nuclear types examined and to retain their characteristic patterns of replication during this process.

In total, nine examples of hybridization to DNA from individual salivary gland nuclei originating from three different Fl larvae were obtained from these crosses. By contrast, a similar number of attempts yielded 38 successful examples of hybridization to individual nurse cell nuclei from nine different

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333

CAM x KARLA

B

S-B

S

M-B

S

M 1 2 3 4 5

1

2

3

SGSN

-7

- a

-7

1

i'

$5 I

4 6'

- -

L-& -ta ."irslls:

1

2

3

NCSN

B

N B N B

JU.

M_

M

I

1

FIGURE 3.-Representation of the Cam and Karla NOS in diploid and polyploid nuclei of individual FI progeny from Cam X Karla crows. T h e hybridization patterns of pBW 1 DNA to blots of RsaI DNA digests are shown. a, Brain (B) and salivary gland (S) DNA from three individual FI larvae of the C K l l cross (see Table 1); the DNA amounts are the same as for Figure 2. b, DNA from five individual salivary gland nuclei [salivary gland single nuclei (SGSN)] derived from one FI larva of the CaK7 cross (see Table 1). T h e diagnostic Cam and Karla bands are well represented in each nuclear pattern. c, DNA from brains (B) and whole ovary nurse cell nuclear preparations (N) from three adult females of the CKl 1 cross; DNA amounts are the same as for Figure 2. d. DNA from five individual nurse cell nuclei [nurse cell single nuclei (NCSN)] from a single adult female of the CKI 1 cross. Note that the Cam and Karla bands are present in each nuclear pattern, but their relative intensities differ in the five nuclei. The unmarked lane is a nuclear pattern from a different female. C, K, A and B, Band designations, as in Figure 2. M, marker DNA, as in Figure 2. DNA amounts in marker lanes of different autoradiograms: a, 0.05

rg; b, 0.85 ng; c, 0.2 rg; d, 3.4 ng.

ing polyploidization, the relative replication of the two NOS of a given geno- type is not a fixed genetic property but, rather, a more random process allow- ing one or other set of rRNA genes to undergo greater replication in any given cell.

DISCUSSION

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334 E. J. BELIKOFF AND K. BECKINCHAM

studies aimed at demonstrating the replication of a single NO during poly- ploidization were performed with strains of the species D. melanogaster (ENDOW and GLOVER 1979; ENDOW 1980, 1982a,b, 1983). It could be argued, there- fore, that different mechanisms of regulating the polyploidization of the rDNA may exist in different Diptera. It is certainly true that the chromosomal loca- tion and rDNA cistron composition of the two NO regions present in the diploid genomes of C. erythrocephala and D. melanogaster are very different (BECKINCHAM 1982). However, as pointed out earlier, the analyses performed to date for D. melanogaster do not provide a critical test of the hypothesis of NO inactivation in this species because an alternative explanation for the dif- ferences in replication seen between the NOS studied can be applied. Further- more, close examination of the data reveals that many of the NO pairs analyzed for

D.

melanogaster do not show complete replicative inactivity of one of the

NOS, but rather show much-reduced replication of one NO relative to the other (ENDOW and GLOVER 1979; Endow 1980, 1982a,b, 1983). We have also seen disproportionate polyploidization of this type for one NO relative to another with certain NO pairs in C. erythrocephala (BELIKOFF and BECKINCHAM

1985). Most importantly, several of the pairs of NOS examined in D. melano- gaster show equal replication at the level of the total polytene tissue DNA (ENDOW 1982, 1983). This is also true in

D.

hydei where a pair of strains with different NTS regions have been used to demonstrate replication of both NOS at the level of the total salivary gland polytene DNA (FRANZ, KUNZ and GRIMM 1983). T h e existence of pairs of NOS showing equal replicative behavior in these other Diptera suggests strongly that the replication of the N O regions during polyploidization will be similar in these organisms to that seen in C. erythrocephala. These NO pairs certainly provide the experimental material necessary for a critical test of this possibility in both D. melanogaster and D. hydei.

D@erential replication of rDNA cistron classes in salivary gland and nurse cell nuclei: both the NO types studied here show differential replication of their cistron types during polyploidization-a phenomenon noted previously (EN-

DOW and GLOVER 1979; ENDOW 1980; BECKINCHAM and THOMPSON 1982),

indicating that discrete regions of the N O must be selected for polyploidiza- tion. For both NOS studied here, the differential replication seen in nurse cell and salivary gland nuclei was very similar. Some of this similarity in tissue replication patterns may reflect the common property shown by these two nuclear types of underreplication of the intron+ rDNA cistrons (ENDOW and GLOVER 1979; BECKINCHAM and THOMPSON 1982). T h e intron+ rDNA cistrons of the Clever strain of C. erythrocephala were shown previously to contain NTS regions with four or five repeating units (BECKINGHAM 1981), and therefore, underreplication of cistrons generating fragment A, as seen for both these two NOS in both nuclear types, may in part reflect this phenomenon.

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335

the limited data obtained for individual salivary gland nuclei from these crosses prevent a conclusion on this point for this tissue. We have examined the replication of other N O pairs in individual salivary gland and nurse cell nuclei in more detail, however (BELIKOFF and BECKINCHAM 1985), and obtained evi- dence for variable replication of the two NOS present in individual salivary gland cells of these hybrids. The significance of this stochastic property of N O polyploidization at the level of individual cells is discussed in our paper de- scribing these additional hybrid studies (BELIKOFF and BECKINGHAM 1 9 8 5 ) .

Variable polyploidization

of

d@erent cistrons within a given NO in different nu- clei? Our discovery that the two NOS present in a given diploid genome are variably polyploidized in different individual nuclei raises the possibility that this might be true of different cistrons within a single NO. Individual polyploid nuclei from the Cai and Karla strain animals were examined, and our data for individual nuclei of the Cai strain perhaps provide some evidence in support of a constant, rather than variable, polyploidization pattern for a given N O in different individual cells. In order to address this question effectively, however, it would be necessary to study N O regions with at least two major diagnostic cistron types that show strong polyploidization at the level of the total DNA.

We are grateful to J. KOOLMAN (Marburg, West Germany), M. JAMRICH (New Haven, Con- necticut), J. LESTER (Cambridge, England), L. LEVENBOOK (Bethesda, Maryland), D. RIBBERT (Munster, West Germany) and E. THOMSEN (Copenhagen, Denmark) for gifts of laboratory stocks of Calliphora erythrocephala. We thank MOHIT NANDA for his initial screening of strain DNAs, MICHAEL O’REILLY for his help in maintaining our fly stocks, and MARIE MONROE and ANN VALVERDE for careful preparation of the manuscript. This work was supported by grant HD 17688 from the National Institutes of Health and grant C-848 from the Robert A. Welch Foundation.

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Figure

FIGURE 1.-Restriction enzyme site map for the rDNA Hind111 fragment present in clone pBW1
FIGURE 2.-The and K, The diagnostic Cam and Karla bands (see text). of of hybridization embryo Cam and Karla fragments
FIGURE 3.-Representation individual FI progeny from Cam

References

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