Isolation of Y-chromosomal repetitive DNA sequences of Drosophila hydei via enrichment of chromosome-specific sequences by heterogeneous hybridization between female and male DNA

Journal of Biochemical and Biophysical Methods, 12 (1986) 37-50 Elsevier

37

BBM 00511

Isolation of Y-chromosomal repetitive D N A sequences of Drosophila hydei via enrichment of chromosome-specific sequences by heterogeneous hybridization between female and male D N A Alexander Awgulewitsch * and Hans Bianemann Institut fftr Genetik der Universiti~t, Universitgttsstr. 1, D - 4000 Dftsseldorf F.R.G.

(Received 22 May 1985) (Accepted 1 August 1985)

Summa~ Male or female DNA of Drosophila hydei was sheared by sonication, denatured, reannealed to different C0t-values and fractionated by hydroxyapatite. The highly repetitive, moderately repetitive and unique fractions of female DNA were denatured again and coupled via diazotization or cyanogen bromide to macroporous Sephacryl S-500. Enrichment of Y-chromosomal sequences was achieved by cycling each of the different fractions of male DNA under optimized hybridization conditions over a column with a manifold excess of immobilized female DNA of the corresponding complexity. Thereby, Y-chromosomal sequences of D. hydei could be enriched about 100-fold for highly and moderately repetitive DNA and about 10-fold for unique DNA. When a library of male D. hydei DNA was screened with Y-enriched highly repetitive DNA, more than 98% of all hybridizing phages contained inserts of repetitive Y-chromosomal DNA of at least four different sequence familieS. Key words: immobilized DNA; heterogeneous hybridization; Y-chromosome-specific repetitive DNA.

Introduction T h e m o l e c u l a r s t r u c t u r e o f t h e Y - c h r o m o s o m e o f D r o s o p h i l a h y d e i is o f p a r t i c u l a r i n t e r e s t f o r s e v e r a l r e a s o n s . I t r e p r e s e n t s a b o u t 10% o f t h e g e n o m i c D N A o f D. h y d e i

* Present address: Dept. of Biology, Yale University, New Haven, CT 06511, U.S.A. Abbreviations: BrCN, cyanogen bromide; DPTE, 2-diazophenylthioether; TEACI, tetraethylammonium chloride; HR-, MR- and U-DNA, highly repetitive, moderately repetitive and unique DNA; HRp-, MRpand Up-DNA, different samples of prehybridized DNA; HRy-, MRy- and Uy-DNA, different samples of Y-enriched DNA; SSC, standard saline citrate. 0165-022X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

38 males [1,2]. The entire chromosome is heterochromatic in somatic cells and obviously of no importance for the development of male flies, since X / O flies, lacking the entire Y-chromosome, are phenotypically normal but sterile males. Indeed, solely the development of fertile sperm depends on the function of several 'fertility genes' which are distributed along the Y-chromosome [3]. The activity of these genes is restricted to the premeiotic phase of spermatogenesis and can be correlated with the unfolding of several giant and characteristic lampbrush loops [4]. This clear correlation between visible structures and genetic functions initiated many efforts towards the isolation of specific transcripts or proteins. Surprisingly, none of them could be characterized definitely to date [5,6]. Therefore, only a less direct approach to the molecular analysis of the fertility genes was left, which is based on the assumption that these genes might be constructed of DNA sequences specific for the Y-chromosome. Heterochromatic regions of chromosomes are often correlated with simple repetitive DNA sequences which are easily detected as 'satellites' by buoyant density centrifugation, as dominant bands by slab gel electrophoresis of genomic DNA after digestion with suitable restriction enzymes or by their kinetic properties in the course of in situ hybridizations on metaphase chromosomes. Surprisingly, all these classical methods could not detect any repetitive sequence specific for the heterochromatic Y-chromosome [1,7,8]. Even molecular cloning gave only very limited success. Only some clones of Y-specific repetitive DNA could be isolated when several thousand plasrnids of a library of D. hydei male DNA were differentially screened with labeled genomic male and female DNA [9,24,25]. The poor outcome of this experiment induced us to ask whether the limited resolution of such screening procedures for the isolation of chromosome specific sequences could be improved substantially by an enrichment prior to the actual screening step. An enrichment of Y-specific sequences, for example, should be obtained as the result of an extensive hybridization reaction between an excess of immobilized female DNA (X/X-genotype) and male DNA (X/Y-genotype) in the mobile phase (heterogeneous hybridization). Under these conditions the Y-specific DNA sequences should become concentrated in the mobile phase, since they should not find homologous molecules among the immobilized female sequences. The critical parameters of heterogeneous hybridization reactions were known in detail from corresponding studies performed with sonicated E. coli DNA [10,11]. Nevertheless, it was uncertain whether these results could be extrapolated from the procaryotic to the 20-50-fold more complex eucaryotic genome of D. hydei. Therefore, we studied the enrichment procedure more systematically with respect to the complexity of the DNA sample. In the first step the DNA of D. hydei was fractionated by hydroxyapatite into fractions of highly repetitive, moderately repetitive and unique DNA. In the second and third step the particular enrichment factor of Y-specific DNA was determined in the course of separate heterogeneous hybridization experiments for each DNA fraction. Finally we tested one of the Y-enriched DNA probes as to whether the calculated enrichment factor could be verified by the result of a screening experiment with a library of D. hydei male D N A in EMBL3-phages.

39

Materials and Equipment

Preparation of D. hydei DNA Wild-type male and female flies were starved overnight in a flask with damp filter paper to prevent dehydration before they were used for DNA extraction (M. Schwochau, personal communication). About 1 g of loosely packed flies were transferred into liquid nitrogen and ground in a porcelain mortar to a fine powder, which was suspended at 60°C in 10 ml of buffer (0.05 M Tris-HC1, 0.1 M EDTA, 0.1 M NaC1, 0.03 M 2-mercaptoethanol, 1% (w/v) Sarkosyl, pH 7.9). 1 mg of proteinase K was added and the viscous solution was incubated at 63°C for 3-4 h. The solution was filled into Quick-Seal tubes, mixed with saturated solution of CsC1 in 0.1 x SSC and centrifuged in a type VTi50 rotor (8 = 1.70 g / c m 3, 41 000 rpm, 20°C, 20-40 h). The fractions containing polysaccharides and DNA were pooled and centrifuged again under the same conditions. Finally, the DNA-containing fractions were collected, dialyzed against 10 mM Tris-HC1, pH 8.0, concentrated by precipitation with ethanol and stored frozen as stock solution in 10 mM Tris-HC1, pH 8.0, for all further experiments.

Preparation of phage DNA Preparative amounts of phage DNA were isolated according to Thomas et al. [12]. For analytic purposes minilysates were prepared as described by Cameron et al. [13].

Library of D. hydei male DNA Genomic DNA of D. hydei males was partially digested with Sau3A and size fractionated essentially as described by Maniatis et al. [14]. Then 4.5 #g of DNA fragments, larger than 13 kilobases (kb), were treated with 0.1 unit of calf intestine phosphatase (Boehringer) and ligated with 15/xg EMBL3 vector DNA. The vector had been previously digested to completion with EcoRI and BamHI and the small EcoRI-BamHI linkers removed by precipitation with isopropanol [15]. 5 /xl of the ligation mixture (total 45 /xl) were packaged by an in vitro packaging kit of Amersham [16]. The packaged phages were diluted and plated on NM 528 bacteria [15]. A total of 1.5 × 105 plaques were obtained from 45 plates (q~ = 14.5 cm). From a collection of ten randomly chosen phages an average insert length of 14.2 kb was determined. The library, therefore, should represent more than 99% of the 2.2-4.6 x 108 base pairs (bp) comprising the genome of D. hydei [1,2].

Screening of the library The library was screened with 32p-labeled Y-enriched highly repetitive DNA as described by Benton and Davis [17].

Immobilization of DNA Samples of sonicated denatured DNA were coupled to Sephacryl S-500 (Pharmacia) via diazotization or cyanogen bromide, designated as standard procedures previously [10,11]. For reasons of optimal hybridization rates two different procedures were used: the cyanogen bromide method (BrCN) for unique-DNA and the

40 TABLE 1 COMPILATION OF DATA CHARACTERIZING THE PREHYBRIDIZATION REACTIONS BETWEEN MOBILE AND IMMOBILIZED FRACTIONS OF MALE DNA IN 2.4 M TEACI AT 45°C 1

2

3

4

5

6

Immob. DNA (/tg/xg wet S-500)

Mobile DNA (/~g)

Quantity of batches

Time of reaction (h)

Prehybrid. DNA (% of mobile)

Total amount of recovered prehybridized DNA (~g)

,:3 HR-DNA 40/0.5 (DPTE)

d HR-DNA 26

5

1

89

~, MR-DNA (1) cycle

~ MR-DNA 36

7

22

46

95/0.31 (DPTE) (2) cycle U-DNA 200/0.83 (BrCN)

J, 18 ~ U-DNA 125

HRp 116 116

74 10

8 72-96

87.5 50

MRp 63 Up 625

The meaning of the data in columns 1-6 is interpreted in detail for the prehybridization reactions of MR-DNA. In the course of the first reaction cycle 36 txg of male MR-DNA (isolated by hydroxyapatitechromatography according to the diagram in Fig. 1) were hybridized to 95 /~g of the same DNA immobilized by DPTE on 0.31 g of wet Sephacryl S-500. The reaction was stopped after 22 h when 46% of DNA in the mobile phase had formed hybrids with their immobilized counterpart on the support. At the end of the first cycle 116/~g of reannealed DNA could be collected from seven identical runs with the same column. A part of this DNA was further purified in a second hybridization cycle (see arrow from column 6 to 2). Finally, 63 #g of prehybridized DNA (MRp) were collected by a series of four identical reactions each performed with 18 ~tg of mobile DNA and stopped after 8 h.

2 - d i a z o p h e n y l t h i o e t h e r ( D P T E ) m e t h o d for highly a n d m o d e r a t e l y repetitive sequences. The quantities of stably fixed D N A (see below) were d e t e r m i n e d by nuclease digestion a n d are listed in c o l u m n 1 of Tables 1 a n d 2.

Renaturation and hybridization reactions All reactions i n the course of the e n r i c h m e n t procedure were performed with sonicated D N A of 0.3-0.5 kb length [10] in 0.12 M p h o s p h a t e buffer or 2.4 M t e t r a e t h y l a m m o n i u m chloride (TEAC1). Since the reaction rates at 65 or 45°C, respectively, were nearly identical for b o t h m e d i a the same C0t-values could be used for experiments i n b o t h of them. (a) Prefractionation. The p r e f r a c t i o n a t i o n of male or female D N A in highly repetitive (HR), m o d e r a t e l y repetitive (MR) a n d u n i q u e ( U ) - D N A sequences was achieved b y r e n a t u r a t i o n in 0.12 M p h o s p h a t e buffer at 6 5 ° C [18]. After r e n a t u r a tion to Cot = 5.6 i n the first a n d to Cot = 0.013 in the second step the samples were passed over hydroxyapatite. The r e n a t u r e d sequences were finally eluted b y 0.24 M p h o s p h a t e buffer. The resultant fractions of HR-, MR-, a n d U - D N A were dialyzed against 0.1 M NaC1, 10 m M Tris-HC1, p H 8.0, precipitated with 2 volumes of

41 TABLE 2 C O M P I L A T I O N OF D A T A C H A R A C T E R I Z I N G T H E E N R I C H M E N T H Y B R I D I Z A T I O N REACT I O N S B E T W E E N I M M O B I L I Z E D F E M A L E D N A A N D MOBILE M A L E D N A OF D. H Y D E 1 IN 2.4 M TEAC1 A T 45°C 1

2

3

lmmob. D N A ( / ~ g / x g S-500)

Mobile D N A (~g) (batches)

Time of Y-enriched = reaction mobile (h) DNA (% of input)

2 HR-DNA 61/0.5 (DPTE)

8 HRp-DNA 40 (2)

2

HRy-DNA 8.8 0.22

128

128

8 HRy-DNA 4.0

MR-DNA 170/0.4 (DPTE)

8 MRp-DNA 27 (2)

16

MRy-DNA 3.5 0.66

150

84

~ MRy-DNA 0.4

? U-DNA (1) cycle ~ Up-DNA 260/0.56 (BrCN) 163 (2)

4

107

5

32.2

6

7

Effective Enrichment factor release _ + rate release release (%/24 h)

1

4.4

3.6

Total a m o u n t of recovered Y-enriched D N A (/.tg)

105

T (2) cycle 250/0.56 (BrCN) 100 (1)

137

$ Uy-DNA 45.1

1

x, 3.1 --13.6

x 2.4 = 8.6

3 (Jy-DNA 50

The meaning of the data in columns 1 - 7 is interpreted in detail for the enrichment of U - D N A . In the course of the first reaction cycle 163 t~g of prehybridized (Up) male D N A were hybridized to 260 fig of prefractionated (U) female D N A immobilized by BrCN to 0.56 g of wet Sephacryl S-500. The reaction was stopped after 107 h when 32.2% of input male D N A was left in the supernatant, corresponding to an enrichment factor of 3.1. If the enrichment by hydroxyapatite-prefractionation is taken into consideration, the enrichment factor with respect to genomic D N A is changed to 4.4. In reality it is lowered to 3.6 due to the release rate of 1% per day of the immobilized female D N A [10,11]. To improve the poor results, 105/~g of Y-enriched male D N A recovered from the mobile phase of two hybridizations were reused for a second cycle of enrichment hybridization (see arrow from column 7 to 2). Finally 50 fig of U y - D N A were obtained 8.6-fold enriched for Y-chromosomal sequences.

ethanol, dried, resuspended in small volumes of 10 mM Tris-HC1, p H 8.0, and stored at - 2 0 ° C . Their renaturation profiles were measured in 2.4 M TEAC1 at 45°C. (b) Prehybridization. Part of each fraction of male D N A (HR, MR, and U) was coupled to Sephacryl S-500, as described above. The DNA-supports were pretreated under reaction conditions for several days to detach all loosely bound sequences. Then the particles with stably bound DNA were filled into a small temperature controlled column (0.5 x 10 cm) and hybridized in 2.4 M TEAC1 at 45°C with a similar amount of male DNA of the same kinetic complexity. The aqueous phase with the mobile DNA was pumped through the column continuously. Since the D N A solution passed a heated loop (80-90°C) of Teflon tubing at the entrance of the column, the D N A was kept permanently in the denatured form [10,11]. Taking into account a ten-fold retardation of heterogeneous hybridization reactions in comparison to the analogous homogeneous ones, each reaction could be stopped at a precalculated C0t-value. The mobile phase was replaced by fresh 2.4 M TEACI before the DNA sample was melted from the column by raising the temperature to

42 70°C. The different samples of prehybridized DNA (HRp, MRp and Up) were collected from the 2.4 M TEAC1 solution by dialysis against 10 mM Tris-HC1, pH 8.0, concentration by 2-butanol and precipitation with 2 volumes of ethanol. For renaturation profiles and enrichment hybridization they were resuspended in small volumes of 2.4 M TEAC1. (c) Enrichment hybridization. The enrichment hybridizations were performed between an excess of immobilized prefractionated female DNAs and corresponding fractions of HRp, MRp, and Up male DNA in the aqueous phase, essentially as described for the prehybridization above. Since the female DNAs were 32p-labeled, their release rates from the column and their total content in the circulating mobile phase could be controlled permanently in the course of the proceeding enrichment hybridizations. The reactions were normally stopped as soon as a steady state between hybridized and released DNA was reached, corresponding to maximal enrichment of Y-specific DNA in the circulation. The samples of Y-enriched DNA were recovered from the mobile phase by precipitation as described above. The pellets of Y-enriched highly repetitive (HRy), moderately repetitive (MRy), and unique (Uy) DNA were resuspended in 10 mM Tris-HC1, pH 8.0, for screening or cloning experiments.

Labeling of DNA by 'nick-translation' Nick translation was carried out according to Maniatis et al. [19].

Renaturation of DNA The renaturation profiles for samples from different fractionation steps were performed regularly in 2.4 M TEAC1 solution at 45°C in a Gilford model 2400 recording spectrophotometer. If necessary the optical pathlength of the 1-cm cuvette was decreased by glass stoppers to 0.1 or 0.2 cm.

Gel electrophoresis and blotting of DNA 1 /~g samples of AluI-digested DNAs were separated on 1.2% agarose gels and transferred to nitrocellulose filters by the method of Southern [22].

Methodology Preliminary remarks to the enrichment procedure Basically, the enrichment procedure consists of an optimized hybridization reaction between male DNA and a manifold excess of immobilized female DNA. As described in detail previously, the heterogeneous hybridization is performed in a small temperature-controlled column in 2.4 M TEAC1 at 45°C to guarantee a fast and uniform hybridization rate independent of base composition [10,11]. The completeness of the hybridization reaction is achieved by continuously pumping the male DNA through the column containing the immobilized female DNA. In addition, a heated loop (80-90°C) of Teflon tubing at the top of the column keeps the male DNA reactive throughout the experiment. Since most sequences of male

43 DNA (X/Y-genotype) in the mobile phase finally hybridize with homologous sequences of immobilized female DNA (X/X-genotype) on the support, Y-specific sequences are enriched in the mobile phase. However, it was known from studies of the essential parameters of heterogeneous hybridization reactions with sonicated immobilized E. coli-DNA that this type of reaction generally proceeds about ten times slower than the analogous reaction in solution [10,11]. These experiments also demonstrated that one cannot overcome this limitation simply by coupling a ten times larger amount of DNA. In our hands none of the methods and materials, commonly used for immobilization of DNA, produced materials with substantially more than about 1 mg of sonicated single stranded DNA bound per 1 ml of settled material. Unfortunately, the rate increase observed after addition of NaC1 or LiC1 to the 2.4 M TEAC1 solution [20] was useless for our experimental approach due to the permanent loss of DNA by unspecific adsorption to all surfaces inside the hybridization device under these conditions. Enhancement of the reaction by addition of dextransulfate or polyethyleneglycol also was unfavorable because of the high viscosity unacceptable for the permanent cycling of the medium in the course of the enrichment hybridization.

Definition of prefractionation, prehybridization, and enrichment hybridization Under these circumstances it was uncertain whether the enrichment procedure for Y-chromosomal sequences could be performed successfully with the complete unfractionated genomic DNA of D. hydei. An extrapolation of our results with E. coli to the 20-50-fold more complex DNA of Drosophila predicted a reaction time of about 1-2 weeks necessary for the hybridization of 95% of DNA in the mobile phase, equivalent to a 20-fold enrichment of Y-specific sequences in the mobile phase. For these reasons we decided to fractionate the genomic DNA of D. hydei by hydroxyapatite chromatography in highly repetitive, moderately repetitive, and unique DNA prior to the enrichment hybridization. In addition, this prefractionation would allow a precise correlation of the degree of enrichment for specific sequences with the complexity of the DNA sample. Furthermore, the fraction of higher kinetic complexity would remain free of contamination by highly repetitive sequences throughout the actual enrichment procedure. Therefore, the Y-enriched fractions could be tested directly in differential screenings of genomic libraries without severe impairment due to background signals from repetitive sequences. Further improvement in the resolution of the enrichment procedure should be achieved by prehybridization. Each fraction of male DNA, used in the course of the final enrichment hybridization against the corresponding fraction of immobilized female DNA, was hybridized at first to an identical sample of immobilized male DNA. Only that part of DNA which hybridized under the appropriate C0t-conditions, was recovered from the colunm at 70°C and pooled for further experiments. In this way the actual final enrichment hybridization could be performed with well characterized DNA allowing the evaluation of a separate enrichment factor for each of the three DNA fractions. The complete enrichment procedure is schematically shown in Fig. 1.

44

Results and Discussion

Prefractionation by hydroxyapatite chromatography The studies on renaturation of sheared genomic D N A of D. hydei in 0.12 M phosphate buffer by Hennig [21] several years ago, show the tripartite C0t-curve, typical for eucaryotic genomes. Our corresponding measurements in 0.12 M phosphate buffer and 2.4 M TEAC1 solution are in agreement with the published data and demonstrate that both media are practically interchangeable when their hybridization rates at 65 and 45°C, respectively, are compared. Therefore, it was possible to transfer the C0t-values, evaluated in the course of prefractionation in phosphate buffer, without any changes to the prehybridization and enrichment hybridization performed in 2.4 M TEAC1. From the overall profile of the renaturation reaction with total genomic DNA in Fig. 2A it was derived that renaturation to Cot = 5.6 (log Cot = 0.75) should separate the total DNA in fractions of approximately 10% HR, 20% MR, and 70% U sequences. When the corresponding fractionation was performed in the classical way by reannealing of denatured sonicated DNA in 0.12 M phosphate buffer and chromatography over hydroxyapatite [18], the results in Figs. 1 and 2A were obtained and are basically consistent with the prediction. However, in

sonicofed genomic DNA I Cot =5.G HAP-prefractionation I cot=0.013 ~10% ~19% 171°1o HR MR U prehybridization i ~ ~mob./ ~immob. HRp MRp Up ~ ~ enrichmenthybridization HRy WRy Uy ~mob./ ~ immob. library- screening Fig. 1. Schematic representation of the procedure for enrichment of Y-chromosomal DNA sequences by hydroxyapatite (HAP)-prefractionation, prehybridization and enrichment hybridization. HAP-prefractionation of sonicated samples of male or female DNA was realized by two succeeding cycles of HAP-chromatography in 0.12 M phosphate buffer. The first cycle at Cot = 5.6 (log Cot = 0.75) separated 29% of renatured DNA from 71% of single stranded unique sequences (U). The second cycle was performed with the DNA recovered from the HAP column by 0.24 M phosphate buffer and reannealed to Cot = 0.013 (log Cot = -1.9). The resultant fractions of renatured HR DNA and single stranded MR DNA represented 10 and 19% of genomic DNA, respectively. For prehybridization minor amounts of the three fractions of male DNA were immobilized separately on Sephacryl S-500 and reacted in several batches with the rest of the corresponding DNA fractions. All reactions were accomplished in 2.4 M TEACI at 45°C. Finally the different fractions of prehybridized male DNA (HRp, MRp and Up) were eluted from the prehybridization column by raising the temperature to 70°C. In the course of enrichment hybridization between the HRp, MRp and U p male DNA fractions and the corresponding immobilized fractions of prefractionated female DNA the Y-chromosomal sequences were left in the mobile phase of the column. At the end of hybridization the samples of Y-enriched DNAs (HRy, MRy and Uy) were recovered from the supernatant and used as Y-specific probes for the screening experiments.

45

¢ 20

~

!

o

"',

-- ,,

-/U

"6 ~0

~ 60

i

MR

~BO

A -1

-

0

-1.9

} "~ 60

¢,-,R\ /! ". N. ....

1

2

log Cot

0.75

.....--MRp

~

I;

[

I

-2

-1

0

"', \ "',. :,E.coL, \ \"

I

~,.

I

J

1

2 tog Cot

Fig. 2. Results of hydroxyapatite-prefractionation and prehybridization of D. hydei male DNA performed according to Fig. 1. (A) Renaturation profiles for the fractions of HR, MR and U-DNA at the end of HAP-prefractionation step. For comparison the corresponding profile of unfractionated genomic DNA is also shown. (B) Renaturation profiles of prehybridized DNA fractions (HRp, MRp and Up) recovered from the DNA columns at the end of the prehybridization reaction between immobilized and mobile samples of the same fractions of prefractionated DNA. IR represents the fraction of HR-DNA (about 1% of genomic DNA) which is prevented from hybridization to immobilized DNA by the very fast 'snap back' reaction between inverted repeats. All renaturation experiments were performed in 2.4 M TEACI at 45°C. Dotted lines at log Cot = -1.9 and 0.75 represent the limiting Cot-values between HR, MR and U-DNA used throughout all fractionation experiments.

contrast to the kinetically more u n i f o r m fractions of H R a n d U - D N A the fraction of M R - D N A was c o n t a m i n a t e d with a b o u t 50% of u n i q u e D N A which finally could be removed by the successive p r e h y b r i d i z a t i o n step.

Prehybridization between immobilized male and mobile male DNA of identical complexity I n the course of heterogeneous h y b r i d i z a t i o n experiments with E. coli we detected that a b o u t 5% of the d e n a t u r e d D N A in the circulation never hybridized to the excess of identical D N A i m m o b i l i z e d on the Sephacryl S-500 support. There the fraction of ' u n r e a c t i v e D N A ' could be reduced to less than 1% if the h y b r i d i z a t i o n reaction was repeated u n d e r identical c o n d i t i o n s with that part of D N A , which had b o u n d to the c o l u m n in a preceding h y b r i d i z a t i o n (prehybridization). D u r i n g the e n r i c h m e n t of Y-specific D N A of D. hydei we used the p r e h y b r i d i z a t i o n n o t only for e l i m i n a t i o n of unreactive D N A , b u t a d d i t i o n a l l y for a s u b s t a n t i a l i m p r o v e m e n t of the kinetic u n i f o r m i t y of the three different D N A samples. F o r this p u r p o s e we

46 measured the concentration of the immobilized male DNA and performed the prehybridization with a comparable concentration of the same DNA in the mobile phase. Under these conditions the heterogeneous hybridization proceeds as secondorder reaction comparable to normal renaturation in solution but about ten times slower [10,11 ]. Therefore, we could adapt the limiting C0t-values of prehybridization reactions to those of the corresponding prefractionation steps. Table 1 summarizes the reaction parameters chosen for the various prehybridizations. The advantage of prehybridization is demonstrated most clearly by the renaturation profile of MRpD N A in Fig. 2B in comparison to that of the M R - D N A in Fig. 2A. The MRp-sample renatures nearly quantitatively within the C0t-range (log Cot = - 1 . 9 to 0.75) selected originally for prefractionation. It is essentially free of unique sequences. This improvement of kinetic uniformity could be achieved by two cycles of prehybridization under different Cot-values (Table 1, column 4). On the other hand, the renaturation profiles of the kinetically more uniform HR- and U-DNA in Fig. 2A are not altered remarkably by the prehybridization as seen from HRp and Up in Fig. 2B. The IR-DNA in Fig. 2 represents inverted repeat DNA which was recovered as double stranded DNA from the hydroxyapatite column. It was left in the circulation during prehybridization since its very fast intramolecular snap-back reaction prevented hybridization to the immobilized D N A on the column.

Enrichment of Y-specific DNA sequences by enrichment hybridization The final 'enrichment hybridization' was performed between an excess of immobilized 3H-labeled, prefractionated female DNA and unlabeled prehybridized male DNA of the same kinetic complexity (Fig. 1). The reaction conditions are summarized in Table 2. Principally the enrichment hybridization is the most critical step of the complete enrichment procedure. The degree of enrichment (enrichment factor) depends mainly upon the chemical stability of the linkage between D N A and support. For both materials used throughout these experiments (DPTE S-500 and BrCN S-500), the release rate was about 1% per day under hybridization conditions (2.4 M TEAC1, 45°C) as measured previously [10]. There, the release rate was calculated from the total amount of labeled D N A released from the column, taking into account the rate of rehybridization back to the column. In contrast, the 'effective release rate' measured during enrichment hybridization, considers only the actual daily increase of labeled DNA in the circulation of the column. While the Y-specific sequences are enriched by hybridization they are simultaneously contaminated with released DNA from the column. Since the effective release rate essentially depends on the kinetic complexity of the immobilized D N A sample (column 5 of Table 2), its influence on the enrichment factor is negligible for short reaction times with DNA samples of low complexity (HR-DNA), but crucial for longer reaction times with more complex samples (MR- and U - D N A in column 6 of Table 2). The calculation of the enrichment factor in Table 2 generally takes into account the enrichment of Y-specific sequences in two different ways: an indirect enrichment by fractionation on hydroxyapatite (percentages in Fig. 1) and a direct enrichment by the enrichment hybridization. It represents the theoretical degree of enrichment of Y-specific sequences within the different DNA

47 f r a c t i o n s in c o m p a r i s o n to the o r i g i n a l s i t u a t i o n w i t h i n the g e n o m i c D N A . T h e r e is a c l e a r g r a d u a l d e c r e a s e o f e n r i c h m e n t f r o m 128 a n d 8 4 - f o l d for H R y - a n d M R y - D N A , r e s p e c t i v e l y , to o n l y 8.6-fold for U y - D N A ( c o l u m n 6, T a b l e 2), e v e n a f t e r a r e p e t i t i o n o f the e n r i c h m e n t h y b r i d i z a t i o n in the last case.

Fig. 3. Detection of recombinant clones with Y-chromosomal DNA inserts by differential screening of a genomic library of D. hydei male DNA in EMBL3 phages. Two pairs of identical replica filters, each with about 7000 different recombinant phages of a D. hydei male library, were differentially screened with 32p-labeled samples of male HR-DNA (a) and female HR-DNA (b) or male HRy-DNA (c) and female HR-DNA (d), respectively. While the filters a and b show only minor differences in the distribution of their hybridization signals (arrows) the contrary is true for c and d. The circles in c and d marking the positions of hybridization signals in d and c, respectively, clearly demonstrate that only the two strongest signals on both replicas are found at identical positions. Therefore about 98% of the positive clones on filter c should contain inserts of Y-chromosomal sequences.

48

Screening of the male DNA library with HRy-DNA The results in column 7 of Table 2 show that only tzg amounts of Y-enriched D N A could be obtained at the end of the enrichment procedure. Despite the preciousness of these DNA samples, which were to be preserved for cloning, we tried to prove the enrichment of Y-specific sequences by a single direct screening experiment with the HRy-DNA. For this purpose 0.1 ~g of H R y - D N A was renatured, 32p-labeled to a specific activity of 1.0 x 108 c p m / ~ g and hybridized to a nitrocellulose filter ( ~ = 14.5 cm) with about 7000 phage plaques from a library of D. hydei male D N A in phage vector EMBL3 (see Materials and Equipment). For comparison an identical replica filter was hybridized with female H R - D N A recovered from the prefractionation step. Both filters, represented in Fig. 3c, d, show about 200 hybridization signals. Only two of them are at identical positions and they correspond only to the strongest signals on the filter probed with female DNA. The difference was so striking that we performed a control experiment with a similar pair of filters and with male H R - D N A in comparison to female H R - D N A

o~ ~

YL I(3)

o~ ~

o~ .~

o~ ~

YL II(2)

YL III(7)

Ys I19)

o~ 9r M

Fig. 4. The clones with inserts of Y-chromosomal DNA represent four different families of repetitive DNA. Identical Southern filters of AluI digests of genomic male and female DNA (1 /xg) were hybridized separately with 32P-labeled DNA of single recombinant phages. Interestingly, only four different characteristic hybridization patterns were obtained, designated as YL I, YLII, YLIII and Ys I in the figure. The numbers, put in parentheses, define the number of individual phages for each family of repetitive Y-chromosomal sequences among a total of 22 isolated phages. The different families show no detectable cross-hybridization.

49

(both are homologous fractions from the prehybridization.step). The several hundred resulting hybridization signals are identical on both plates with only very few exceptions indicated by arrows in Fig. 3a, b. When the results of both experiments are compared, a 10-100-fold enrichment of Y-specific sequences within the HRyDNA sample can be estimated in agreement with the data calculated from the enrichment experiment. Isolation of Y-specific or Y-chromosomal repetitive DNA sequences For a preliminary characterization of the phages, which showed specific hybridization with HRy-DNA in Fig. 3c, d, about twenty of them were isolated (see Materials and Equipment). Their DNA was purified, 32p-labeled and used to produce final evidence for the Y-chromosomal origin of their fly DNA inserts (average size about 14.2 kb) by hybridization to Southern filters with AluI-digested genomic DNA of D. hydei males and females. All phages produced patterns of multiple bands with male DNA which could be grouped into four families, each of them clearly recognizable by a characteristic hybridization pattern designated as YLI-III and YsI in Fig. 4. The numbers, put in parentheses, define the number of individual phages for each family among a total of 22 isolated phages. Ys I and YLIII phages are clearly more frequently found than YLI and YLII ones. The hybridization experiments show that the repetitive sequences which belong to the families Ys I, YLII, and YLIII are located most likely exclusively on the Y-chromosome and can be designated as Y-specific sequences. In contrast, the sequences of the YLI family are predominantly found on the Y-chromosome but are present in smaller amounts elsewhere in the genome, too, as seen from the weak bands with female DNA in Fig. 4.

Simplified description of the method and its applications The isolation of several families of repetitive D N A specific for the Y-chromosome of D. hydei by the procedure of enrichment hybridization clearly demonstrates the applicability of this method for the isolation of chromosome-specific repetitive DNA-sequences from moderately complex eucaryotic genomes (e.g.D. hydei: 4.58×108 bp). The method basically consists of the production of a D N A sample enriched in chromosome-specific sequences. Naturally it depends on the available a m o u n t of the enriched D N A whether the sample can be used directly for differential screening of a genomic library or whether it has to be amplified by cloning first. Our screening experiments were performed with some percents of the H R y - D N A directly. The results in Fig. 3 illustrate the effectiveness of this enrichment procedure. Approximately 98% of about 200 hybridization signals on a replica filter with about 7000 different phages represent phages with inserts of Y-chromosomal DNA. In other words, the enrichment step practically renders the otherwise obligatory control hybridization with the homologous fraction of female D N A superfluous. Therefore the procedure can be recommended for improvement of the screening methods recently used for detection of restriction fragment length polymorphism on the h u m a n Y-chromosome [25]. Nevertheless, from a comparison of enrichment factors in Table 2 one can assume a similar good screening result with the M R y - D N A but not for the Uy-DNA. A 10-fold enrichment of Y-specific sequences is definitely not sufficient to obtain samples of unique D N A suitable for a successful screening. Therefore, to make this enrichment procedure for isolation of chromosome-specific unique sequences generally applicable, the rate of heterogeneous hybridization would have to be increased 10-100-fold. At present such conditions are not available. Under these limitations the novel method can only be recommended for the isolation of repetitive chromosome-specific sequences, e.g. from somatic cell hybrids

50 with extra chromosomes. In this case a crude prefractionation in repetitive and unique DNA should be sufficient before the repetitive DNA is immobilized and used for enrichment hybridization with the corresponding DNA sample of a cell line containing an extra chromosome. The value of somatic cell hybrids for the isolation of chromosome-specific sequences recently has been demonstrated by Wolfe et al. [26], who succeeded in the isolation of Y-specific repetitive sequences from a mouse cell line containing four human Y-chromosomes. Probably their screening procedure could be improved substantially by a DNA probe enriched in Y-chromosomal sequences. At present we are trying to isolate differentially expressed mRNAs by an analogous enrichment hybridization with complete cDNA libraries from two different cell clones. A similar experiment has been done recently by Scott et al. [27]. They performed an enrichment hybridization between a cDNA library of 3T3 cells immobilized on cellulose and a cDNA library of SV-40 transformed cells for the enrichment of genes specifically expressed in transformed cells.

Acknowledgements The authors thank Dr. M. Sch~fer for critical reading of the manuscript. The skillful technical assistance of Mrs. C. Gieseler is gratefully acknowledged. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Bu 342/2 and 342/3-2).

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