Chromosoma (Berl.) 59, 23-42 (1976) CHROMOSOMA �9 by Springer-Verlag 1976

Chromosomal Localizations by in situ Hybridization of the Repetitious Human DNA Families and Evidence of Their Satellite DNA Equivalents

Kenneth A. Marx 1, James R. Allen 2 and John E. Hearst 3

Dept. of Chemistry, University of California, Berkeley, California 94720, U.S.A. ; present addresses: 1 Institute of Animal Genetics, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JN, U.K, ; 2 Dept. of ZooIogy, Oregon State University, Corvallis, Oregon, U.S.A. ; 3 To whom requests for reprints should be addressed

Abstract. Four of the five major repetitious human DNA families ,have been mapped by the in situ hybridization technique at their Tom r values. Two of the lighter density DNA families have autoradiographic grain pat- terns over heterochromatic chromosomal regions that resemble those of known satellite DNAs. The two heaviest density D N A families have autora- diographic grain patterns of middle repetitious DNAs, with all chromosomes showing labelling. Some evidence suggests that one of these DNA families is concentrated in certain chromosomal regions. Both DNA families exhibit biphasic TopT curves. The presence of two thermal stability classes of hybrids suggests sequence interspersion. By co-enrichment studies in Ag+-CszSO4 gradients, evidence suggests the origin of the three lightest density renaturated human DNA families to be satellites I, II and III.

Introduction

A number of reports have appeared which describe the locations of human satellite DNAs (Jones and Corneo, 1971; Saunders et al., 1972; Jones et al., 1973; Jones et al., 1974; Gosden et al., 1975) on human chromosomes by the in situ hybridization technique (John et al., 1969; Gall and Pardue, 1969). In this report we utilize the in situ hybridization technique to map the locations of four of the highly repetitious renatured human DNA families characterized in previous studies (Hearst et al., 1973; Marx et al., 1976) and present evidence which suggests the origin of three of these renatured D N A families to be human Satellites I, I! and III.

Materials and Methods

DATA Isolation and Fractionation

Human DNA was isolated from placental tissue by methods previously described (Hearst et al., 1973). This includes digestion with Pronase, RNase and c~-amylase, but avoids treatment with phenol in favor of repeated chloroform: iso-octanol (24:1) extractions.

24 K.A. Marx et al.

H u m a n total D N A was fractionated at a 0.20 Ag + : D N A phosphate ratio in Cs2SO4 gradients essentiaUy as described by Corneo et al. (1971). The gradients were established at 30,000 rpm for 4 d at 25~ in an 8 x 40 rotor in the MSE 65 Centrifuge. Fractionation was from the top by slowly pumping with a Sigmamotor. Refractive indices were taken immediately to calibrate the gradient. Optical densities of each fraction were measured at 260 nm in the Unicam 800. Pooled fractions were dialysed exhaustively against 5 M NaC1, 0.01 EDTA, 0.01 M Tris pH 7.0 to remove Ag + before dialysis into 0.12 M phosphate buffer pH 6.8.

Hydroxyapati te chromatography was performed as previously described in Marx et al. (1976). The individual renatured Cot 1.0 h u m a n D N A families, used as templates for the preparation of cRNA, were isolated by repeated preparative CsC1 gradient fractionation.

Analytical ultracentrifugation was performed in a Beckman Model E Ultracentrifuge using the A N F rotor at 44,000 rpm 2 5 ~ for 24 h. Equilibrium ultraviolet photographs were taken and traced on a Joyce-Loebl Recording Microdensitometer.

Preparation of 3H cRNA

In each case between 2 and 10 gg D N A in 20-50 gl 0.04 M Tris HC1, pH 7.9 was used to prime a single transcription reaction. The highly aggregated D N A samples were first denatured and allowed to renature for about 0.5 h.

The DNA, in the above buffer, was placed in an acid cleaned conical bot tom centrifuge tube in which 5-10 m g moles of 3H-ATP (20 Ci/mmole, C-8 labelled) had been evaporated nearly to dryness in a stream of N 2. Then 50gl of a 0 .025M MgCI2, 2 . 5x10 AM EDTA, 0 .03M fl- mercaptoethanol, and 0.2 M Tris-HC1 pH 7.9 solution and 15 ~tl of millipore filtered 3 M KC1 were added and mixed R N A polymerase (Burgess, 1969) was added and the reaction initiated by the addition of 20 m g moles each GTP, CTP and UTP. The solution was mixed gently and incubated at 37 ~ C for 60 min. The final reaction volume was 250 gl and contained 0.15 M KCI, 0.005 M MgClz, 7 x 10- 5 M EDTA, 0.006 M fi-mercaptoethanol and 0.04 M Tris, pH 7.9.

The reaction was terminated by addition of 20 gg of electrophoretically pure DNAse and incubating at 37 ~ C for 30 min. 200 gg of yeast R N A carrier was then added and the volume brought to 1.0-1.5 ml. SDS was added to 1% and this solution was extracted with an equal volume of Tris buffer saturated phenol. The phenol phase was itself extracted and the pooled aqueous phases were loaded o n t o a 30 cm (30 ml) G-50 fine Sephadex column. The column was eluted with vacuum de-gassed glass-distilled H20.

Two millilitre fractions were collected and small aliquots assayed for TCA precipitable counts. The fractions having more than one third acid precipitable activity were pooled and dialyzed vs. 10 x SSC, 0.05 M Na z E D T A pH 7.4 to remove nucleotides and then vs. 2 x SSC. The c R N A had a specific activity of 3.6 x 107 dpm/gg.

Determination of Toer

The Tof, T profile was determined somewhat differently than in Moar et al. (1975). Rather than use driver 3H c R N A in excess over filter bound DNA, the 3H c R N A was reacted as tracer in 2 x SSC solution with total h u m a n D N A in excess (> 100,000 : 1). Aliquots at the temperatures indicated were reacted to identical Cot values (corresponding to less than 40% reaction completion) before quenching on ice. Pancreatic RNase was added (10 gg/ml) for 30 rain at 25 ~ C. The aliquots were made 10% TCA and incubated at 0 ~ for 30 min before being vacuum filtered through Millipore filters and ethanol washed. Filters were counted in PPO-POPOP toluene based scintillation fluid.

Cytologieal Materials

Peripheral leukocytes were collected in sterile ampoules from healthy adults. They were transferred to TC chromosome culture kit (Difco Labs) blood separation vials and mixed by inversion. After incubation at 37~ for about 3 h, or until at least 4 ml of Plasma-Leukocyte suspension had

Localizations of Repetitious Human DNA Families 25

separated, each bottle of rehydrated chromosome medium was innoculated with 1.5-2.5 ml of Plasma-Leukocyte suspension. Each bottle was stored at 370 C for 3 or 4 d. Colcemid was added at the third or fourth day. Six hours later the cell suspension was hypotonically swollen and then fixed in (3:1) methanol:acetic acid (Moorhead etal., 1960) and resuspended in less than 1 ml of fixative.

Two or three drops of the freshly resuspended cell suspension were dropped from a height of 2 3 feet onto the freshly wetted surface of an acid cleaned slide. The slide was tilted a number of times to spread the cells and the excess water wiped quickly from the bottom of the slide. Passing through an alcohol lamp a few times ignited the excess fixative and promoted spreading of metaphase cells. The slides were never allowed to become warmer than skin temperature.

After air drying, slides of good quality were collected for use in in situ hybridization. The slides were digested with boiled RNAse (100 gg/ml) at 37 ~ C for 1 h, washed in several changes of 2 x SSC at 37 ~ C. deproteinized and the DNA denatured with 0.2 N HC1 at room temperature for 30 min, then rapidly dehydrated by passage through 2 changes each of 50%, 70%, 95% and 100% ethanol.

Hybridization in situ

3H cRNA (200,000 cpm) was applied to the surface of each slide in 150 gl of 2 x SSC. The RNA solution was covered with a coverslip. Slides were incubated at Topv for 16 h in thermal contact with a 2 x SSC reservoir and then washed in several changes of 2 x SSC at Top ~. Next, the slides were incubated in RNAse (10 20 Itg/ml) at 37 ~ C for 1 h to remove any nonspecifically adsorbed RNA. Again the slides were washed thoroughly in 2 • SSC and dehydrated in an ethanol series.

After air drying, the slides were dipped in 41-42 ~ C Kodak NTB-2 Nuclear Emulsion diluted 1:1 with glass distilled water. The emulsion was applied without any safelights and the slides were dried in a stream of warm air. Slides were stored well-sealed in a cold room at 4 ~ C for one week to one month.

Developing took 4 min in full strength D-19 developer at room temperature. The slides were rinsed in distilled water and fixed in full strength Kodak Acid Fixer 5 min. Staining quality depended crucially upon complete removal of fixer, so the slides were commonly rinsed twice in Kodak Hypo Clearing Agent for a total of 5 rain, then rinsed 5 times with distilled water before staining in 20:1 0.005 M Tris pH 7.4:Giemsa stain for 10 rain.

Results

The Repetitious Human DNA Families

T o t a l h u m a n D N A c o n t a i n s n o d e n s i t y sa te l l i t e s in CsC1 g r a d i e n t s ( M a r x et al. ,

1976). H o w e v e r , a n u m b e r o f sa t e l l i t e D N A f r a c t i o n s h a v e b e e n i s o l a t e d in

A g § o r H g + § 4 g r a d i e n t s a n d t h e i r p r o p e r t i e s , i n c l u d i n g in s i tu l oca l i z a -

t i o n s , d e s c r i b e d ( J o n e s a n d C o r n e o , 1971; S a u n d e r s et al. , 1972; J o n e s et al. ,

1973; J o n e s et al . , 1974; G o s d e n et al. , 1975). U s i n g a n a l t e r n a t e a n d c o m p l e m e n -

t a r y a p p r o a c h t o g e n o m e f r a c t i o n a t i o n t h e r e p e t i t i o u s h u m a n D N A k i n e t i c

c lass Co t 0 - 1 . 0 , i s o l a t e d o n h y d r o x y a p a t i t e , c a n b e s h o w n to b e c o m p r i s e d

o f f ive m o l e c u l a r f a m i l i e s (F ig . 9 a) w i t h d e f i n e d p r o p e r t i e s ( M a r x et al. , 1976).

E a c h o f t h e r e n a t u r e d h u m a n D N A fami l i e s , e x c e p t t h e 1.696 D N A , was i s o l a t e d

o n p r e p a r a t i v e CsC1 g r a d i e n t s in su f f i c i en t q u a n t i t y to ac t as t e m p l a t e f o r E.

coli R N A p o l y m e r a s e ; t h e c R N A s t r a n s c r i b e d we re in s i tu h y b r i d i z e d to m e t a - p h a s e c h r o m o s o m e s p r e a d s .

26 K.A. Marx et al.

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Fig. l a and b. Initial rate of c R N A : D N A hybrid formation (RNAse resistant) as a function of temperature. Total D N A in excess over aH c R N A (>105:1) in 2 x S S C solution was divided into aliquots and hybridized at varying temperatures to a Cot value where only about 30% of the cRNA had reacted, a 1.687 cRNA. b - o - 1.703 cRNA; - � 9 1.714 c R N A

1.687 DNA

This lightest density renatured DNA family has physical properties (Marx et al., 1976) which suggest its similarity to human Satellite I DNA (Corneo et al., 1971 ; Jones et al., 1974). When the 1.687 DNA cRNA (1.687 cRNA) Top T (Moar et al., 1975) was determined by DNA excess reassociation in 2 x SSC solution, the result in Figure 1 a was obtained. The To~T is about 47~ in 2 x SSC. In 4 x SSC solution the T o p T would be about 52 ~ C, which is close to the T o p T (56 ~ C) for Satellite I (Jones et al., 1974) in 4 x SSC, considering the accuracy of such determinations.

When 1.687 cRNA was hybridized in situ at Tom- to human metaphase chromosomes, grains were seen only at a restricted number of chromosomal loci. A typical result of one week exposure time is shown in Figure 2. The metaphase spread has been karyotyped, although in the absence of banding studies it is difficult to make certain individual chromosomal assignments (Harris et al., 1973). Realizing this limitation we have exercised caution in our claims of chromosomal identity.

A statistical analysis of the distribution of 1.687 cRNA autoradiographic grains within chromosomal groups from many karyotypes is presented in Fig- ure 3 a. For each chromosomal group the average grain percentage (of each metaphase spread) normalized to the absolute diploid DNA mass is presented with bar limits corresponding to one standard deviation about the normalized average. The solid line at 1.78 is the line describing a completely random grain distribution for any chromosomal group. Normalized averages of D and total G group chromosomes are above the random value. Especially the G group seems to be enriched in 1.687 cRNA hybridizable sequences. When the presump- tive Y chromosome is identified, its normalized average is quite high and even the remaining G group chromosomes appear to be slightly enriched.

Localizations of Repetitious Human DNA Families 27

Fig. 2. In situ hybridization of cRNA from the 1.687 DNA family to human leukocyte metaphase chromosomes at TopT in 2 x SSC solution. The slides were pretreated with 0.2 N HC1 for 30 min. Autoradiographic exposure time was 1 week. The bar indicates 10 gm

When slides are exposed for 2 months (Fig. 4), it can be seen that only one small chromosome (the presumptive Y) is intensely labelled (large arrow). All other G group chromosomes have only one or two grains. Thus, it would appear that a significant port ion of the 1.687 D N A sequences are concentrated on the Y chromosome, perhaps at the distal fluorescent port ion of the long arm, as is the case for Satellite I (Jones et al., 1974). Additional support for this conclusion comes f rom hybridization of 1.687 cRNA to female metaphase chromosome spreads (unpublished). After long exposure times no large grain concentration is seen over any small acrocentric chromosome. The minor loci, however, (Figs. 2 and 4) still stand out. These loci are most likely chromosomes 1, 4 or 5 and a C group chromosome, probably 9, (small arrow) containing concentrations of grains close to their centromeres. It is known that chromo- somes 1 and 9 contain pericentromeric heterochromatin adjacent to their centro- meric regions and Satellite I shows localization to these regions as well (Jones et al., 1974).

1.700 DNA

The 1.700 D N A has many physical properties (Marx et al., 1976) similar to human Satellite I I I (Corneo et al., 1971, 1972; Prosser, 1974). Due to a lack

28 K.A. Marx et al.

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Fig. 3 a-d. Statistical analyses of various cRNA autoradiographic grain distributions within chromo- somes or chromosomal groups, a 1.687 cRNA. b 1.700 cRNA. e 1.703 cRNA. d 1.714 cRNA. A number of metaphase spreads, hybridized with each cRNA, were photographed and karyotyped. Grains were counted over each chromosome (presumptive) or chromosomal group and the average grain count _+ a determined for the number of karyotypes. This average grain count and the _+ a values were normalized by dividing by the absolute diploid human DNA mass of the appropriate chromosome or chromosomal group. The horizontal line drawn at 1.78 represents the normalized average grain % for a random grain distribution

of sufficient 1.700 cRNA a Top T curve was not measured. However, in light of its similarity to human Satellite III (discussed later), a value of 60 ~ C in 2 x SSC, close to the TOpT for Satellite III cRNA, was adopted for use.

When 1.700 c R N A was hybridized in situ at 60 ~ C to male human metaphase chromosomes the autoradiographic grain distribution appeared as in Figure 5 after 1 month exposure time. The major location appears to be on 1 pair of C group chromosomes, most probably the pericentromeric heterochromatin of chromosomes 9. Smaller concentrations of grains appear at the pericentromeric heterochromatin in chromosomes 1, the centromere regions of chromosomes 2

Localizations of Repetitious H u m a n D N A Families 29

Fig. 4. In situ hybridization of 1.687 c R N A to human leukocyte metaphase chromosomes at Top T in 2 x SSC solution. The slides were pre-treated with 0.2 N HCI for 30 rain. Autoradiographic exposure time was 2 months. The bar indicates 10 gm. Large arrow indicates labelling of the presumptive Y chromosome. Small arrow indicates minor grain concentrations

and 4, the centromeric regions of most D group chromosomes, some E group chromosomes and G group chromosomes. The Y chromosome contains a size- able grain concentration as well and here, as in the case of the 1.687 cRNA hybridization mentioned, no grain localization was seen on the distal portion of any small acrocentric chromosome arms in female metaphase chromosomes. Thus, the major and minor loci of the 1.700 DNA are coincident with those of human satellite III DNA (Jones et al., 1973; Moar et al., 1975).

The high concentration of 1.700 DNA sequences in specific heterochromatic regions of human chromosomes is evident as well in interphase nuclei. Two prominent grain clusters appear over a number of the nuclei in Figure 6 (small arrows). These are most likely the locations of the pericentromeric heterochroma- tin of chromosomes 9, which form separate chromocenters in interphase nuclei. Minor grain concentrations correspond no doubt to the minor chromosomal loci discussed in relation to the karyotype in Figure 5. Occasionally, association of a number of the smaller grain clusters (Fig. 6, large arrow) into a loosely organized chromocenter can be seen. This is congistent with the tendency of human acrocentric chromosomes to form loose associations at metaphase and perhaps is related to nucleolar structure or function since the rDNA cistrons have been located at or close to these centromeric regions (Henderson et al., 1972). Similar hybridization patterns were noted for Satellite III cRNA to inter- phase nuclei (Jones et al., 1973).

30 K.A. Marx et al.

Fig. 5. In situ hybridization of 1.700 cRNA to human leukocyte metaphase chromosomes at 60 ~ C in 2 x SSC solution. The slides were we-treated with 0.2 N HC1 for 30 rain. Autoradiographic exposure time was 1 month. The bar indicates 10 gm

A statistical description of the distribution of 1.700 cRNA autoradiographic grains within chromosomal groups from many karyotypes of short exposure time is presented in Figure 3b. The low normalized grain percentages for A and B group chromosomes are a reflection of the relatively small percentages of 1.,700 DNA sequences in the minor centromeric or pericentromeric loci of these large chromosomes. Chromosomes of the C group are naturally enriched due to the very heavy labelling of chromosomes 9. This chromosome pair alone can be seen to have a normalized grain percentage some 6 to 7 fold higher than an average chromosome pair. Chromosomes of the total D and G group show enrichment and if the heavier labelling of the single chromosome thought to be the Y is examined, it shows a normalized grain percentage some 8 fold greater than an average chromosome pair.

1.703 DNA and 1.714 DNA

These DNA families together constitute a large portion of total human D N A - a t least 1 6 % - a n d have physical properties very different from satellite DNAs (Marx et al., 1976). Each is composed primarily of a family of highly diverged middle repetitious DNA in addition to a smaller fraction of more highly repeti- tious DNA sequences. Evidence suggests as well that the diverged middle repeti-

Localizations of Repetitious Human DNA Families 31

Fig. 6. In situ hybridization of 1.700 cRNA to human leukocyte interphase nuclei at 60~ in 2 x SSC solution. Two prominent grain clusters (smali sets of arrows) appear in most nuclei, An occasional association of a number of smaller grain clusters (large arrows) is seen. The slides were pre-treated with 0.2 N HCI for 30 rain. Autoradiographic exposure time was 1 month. The bar indicates 10 gm

tious D N A sequences of these two D N A families are related and can hybridize under non-stringent reassociation conditions.

When the 1.703 c R N A and 1.714 cRNA Tom r profiles were determined (Fig. 1 b) the results were found to be consistent with the hybridization of a large fraction of c R N A transcribed f rom families of highly diverged middle repetitious D N A sequences. The initial reassociation occurs over a wide range of temperatures due to the formation of hybrids of widely varying thermal stability. The profiles have broad maxima both at roughly Top T = 45 ~ C, although this opt imum temperature cannot be fixed with certainty.

At higher temperature, a second sharper set of maxima are seen in the Tor t profiles of both cRNAs. This peak in the 1.714 c R N A profile is more pronounced and has a Tom-=67 ~ C. Its sharpness suggests the reassociation of a family of D N A sequences diverged to a considerably lesser extent than the middle repetitious D N A - w i t h broad T o r t = 4 5 ~ C - p r o b a b l y the highly repetitious sequences (Marx et al., 1976) within each D N A family. Some unpub- lished reassociation data indicate that this assignment is indeed correct.

32 K.A. Marx et al.

Table 1. Intrachromosomal autoradiographic grain distribution of various cRNAs Chromosomal cRNA

region 1.687 1.703 1.714

centromeric 1.49 1.43 1.15 telomeric 1.23 1.26 1.10 arm 0.87 0.87 0.95

Analysis of the longitudinal distribution of autoradiographic grains of various cRNAs along the metacentric A, B and C group chromosomes (excluding chromosomes 1 and 9) of a number of karyotypes. The various cRNAs were in situ hybridized as previously described and a number of photographed metaphase spreads for each were karyotyped. Each metacentric chromosome was measured and divided into ten equivalent regions. Grains lying in the two terminal regions were designated telocentric; those lying in the single region spanning the centromere were designated centromeric; and grains lying in the remaining 7 interstitial regions were designated as arm. Each value in the table expresses the average grain percentage of the representative area normalized by the percentage linear length of that area (i.e. - 10%, 20%, 70%). A normalized value of unity is indicative of a completely random grain distribution and no intrachromosomal preferences.

When the 1.703 cRNA and 1.714 c R N A fractions were hybridized to meta- phase chromosome spreads at 60 ~ C the autoradiographic results (Fig. 7a and b) indicate a lack of major grain concentration over any chromosomal area. This remains so even at longer exposure times.

A statistical description of the distribution of both cRNA autoradiographic grains with chromosomal groups is presented in Figure 3c and d. With the exception of the slight B group enrichment and E group depletion in 1.703 D N A hybridizable sequences there appears to be a generally random grain distribution resulting from hybridization of these two cRNAs at 60 ~ C.

I

Although there are hints of grain clustering in certain chromosomes (arrows Fig. 7a), no consistent associations were noted. Of course, to do such a study properly would necessitate performing chromosomal banding studies before in situ hybridization. Even then, care must be exercised in interpretation (Sanchez and Yunis, 1974) due to the limitation of autoradiographic resolution and the great complexity of the cRNA probes.

A limited analysis of the longitudinal distribution of autoradiographic grains within A, B and C group metacentric chromosomes (excluding chromosomes 1 and 9) of a number of karyotypes is presented in Table 1. Each chromosome was measured and divided into centromeric, telomeric and arm regions; individu- al grains were assigned to each region and the final grain percentage normalized to the percentage linear distance, a value of unity representing a random distribu- tion. It can be seen that the 1.703 cRNA probe has hybridized to a greater extent to centromeric and telomeric regions than to arm regions. It is much more concentrated in these regions than the 1.714 cRNA, which has only a weak preference for centromeric or telomeric regions over arm regions. For the satellite like 1.687 c R N A localization, as expected, a similarly high concentra- tion of grains appears over centromeric regions, although this value would be much greater if chromosomes 1 and 9 had been included. In addition, telomere

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Fig, 8. Preparative equilibrium density gradient centrifugation of total high molecular weight human DNA in Ag+-CszSO4 (0.2 Ag+/DNA phosphate). The indicated positions of human Satellites I, II and III in the gradient are based on the known densities of these satellite DNAs in a Ag+-Cs2SO4 (0.2 Ag+/DNA phosphate) gradient (Corneo et al., 1971). The gradient was divided into the regions A, B and C as shown

regions also appear to be enriched over arm regions. This serves, as well, as a control illustrating the localized case versus the random case (almost ex- emplified by the 1.714 cRNA distribution).

Identification of Three Renatured DNA Families with Human Satellite DNAs I, H and III

A number of physical properties (Marx et al., 1976) as well as the in situ localization data described here strongly suggest that the three lightest density renatured DNA families are synonymous with human Satellite DNAs I, II and III (Jones and Corneo, 1971 ; Jones et al., 1973; Jones et al., 1974) respective- ly.

The following experiment was designed to test the above statement more directly. Total high molecular weight human DNA was banded (Fig. 8) in a preparative equilibrium Cs2SO4 gradient at a Ag+/DNA phosphate ratio of 0.2 as described by Corneo et al. (1971). This somewhat overloaded gradient may be compared to Figure 1 b in the above reference. The position of Satellite I is clearly evident and there are small bumps in the OD profile at the density positions corresponding to Satellite DNAs II and III. This gradient was divided into regions A, B and C as shown, the Ag § removed, the DNA sonicated

Localizations of Repetitious Human DNA Families 35

Fig. 9a-d. Analytical ultracentrifugation in CsCI of the equivalent Cot 1.0 DNA sequences isolated from: a total human DNA; b region A DNA; e region B DNA; d region C DNA. Each DNA sample was sonicated prior to hydroxyapatite isolation. The equivalent Cot values for fractions A, B and C were calculated correcting for the assumed complete enrichment of sequences within each region. Thus, the equivalent Cot 1.0 = Cot 1.0 x (enrichment factor)-1. Of course, the assumed enrichment factor is simply the inverse of a given regions fraction of the total DNA. Before banding in CsC1, each Cot 1.0 DNA isolate was stringent renatured (Marx et al., 1976) (>20Dz6o/ml at 67 ~ C in 0.12 M phosphate buffer pH 6.8 for 20 h) to form high molecular weight DNA networks

a

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~/~17 Z, 1.733 '-69t " I

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36 K.A. Marx et al.

and then prepared for hydroxyapatite chromatography. DNA was isolated from each sample on hydroxyapatite at Co t 1.0. Each sample's Cot value was deter- mined with a correction factor assuming complete DNA enrichment within that pooled region (Fig. 9, legend). Therefore, any DNA family present entirely within a single region will reassociate and be isolated on hydroxyapatite to roughly the same extent as in the unfractionated case, although its proportion of the total yield may vary if the distribution of DNA frequency classes in that region is significantly different than in the unfractionated genome. After hydroxyapatite chromatography the Cot 1.0 isolated DNA was further stringent renatured and then banded in CsC1 gradients in the analytical ultracentrifuge. The results are shown in Figure 9b-d and may be compared to the typical Cot 1.0 DNA profile (Fig. 9a) isolated from sonicated total human DNA in the normal fashion (Marx et al., 1976).

Region A of the Ag § gradient can be seen (Fig. 9b) to be consider- ably enriched in 1.687 DNA and 1.700 DNA. Some of the 1.714 DNA is present as well. Thus, the Satellite I and III DNA region yields an enrichment in 1.687 DNA and 1.700 DNA in the Cot 1.0 distribution. This fact, coupled with the expected lack of spillover of Satellite I DNA into region B (Fig. 9 c) and the complete absence of 1.687 DNA sequences in this region as well as region C (Fig. 9 d), quite clearly identifies the origin of the 1.687 DNA family as Satellite I DNA.

Consistent with the expected spillover of Satellite III DNA sequences into region B, some 1.700 DNA sequences are seen in the Cot 1.0 DNA of region B in Figure 9c. Some 1.700 DNA appears in region C Cot 1.0 DNA as well, although the considerably reduced 1.703 DNA peak makes this peak appear more prominent. Thus, the 1.700 DNA clearly correlates with the presence of Satellite III sequences and, no doubt, arises, at least in part, from its reassocia- tion.

Satellite II DNA, enriched in region C, is correlated with the presence of the 1.696 DNA, although not as convincingly as the previous two cases. The 1.696 DNA appears to be present in both regions B and C, but, as expected, not region A. Due to the poor resolution of the 1.696 DNA peak it is difficult to describe the enrichment in regions B and C. However, the clear implication is that Satellite II DNA sequences are the origin of the 1.696 DNA family.

It is of interest to note that the two heaviest density DNA families (Fig. 9 a), having physical properties (Marx et al., 1976) and in situ localizations suggesting middle repetitious DNA sequences, have different distributions in the three re- gions of the Ag+-Cs2SO4 gradient. The 1.703 DNA occurs almost exclusively in the middle B region, indicative of its main band DNA character and its relative homogeneity with respect to Ag § binding. The 1.714 DNA, on the other hand, appears in all regions of the Ag § -Cs2SO4 gradient, indicating consid- erable heterogeneity in either: a) sequence or b) sequence organization.

Discussion

1. Renatured DNA Families with Satellite Like Properties

The repetitious human DNA sequences have been shown to be composed of five major molecular families (Marx et al., 1976). The three lightest density

Localizations of Repetitious Human DNA Families

Table 2. Chromosomal localizations and identities of the renatured human DNA families

37

Renatured Top T(~ C) Chromosomal localizations Native DNA DNA family (2 x SSC) equivalent a

1,687 DNA 47 Y distal arm; 1, (9) secondary constrictions; Satellite I few C, D, G group centromeres; statistical preference for A, B, C group centromeres and telomeres

- (1, 16 secondary constrictions) b

-- 9 secondary constrictions; 1 secondary

1.696 DNA Satellite II

1,700 DNA Satellite III

1.703 DNA 45 (broad); 67

1.714 DNA 45 (broad); 67

constrictions; 2, 4 centromeres; C, D and G group centromeres; Y distal arm; two major loci in interphase nuclei

Some hints of clustering on chromosomes (Fig. 7a). All chromosomes labelled. Clear statistical preference for centromere, telomere regions (T=60 ~ C)

All chromosomes labelled; weak statistical preference for centromeres and telomeres (T=60 ~ C)

a A more thorough comparison of the human repetitious DNA-Satellite DNA literature has already appeared (Marx et al., 1976) b This is based upon an unpublished observation of the in situ hybridization of cRNA tran- scribed from a mixture of 1.696 DNA and 1.700 DNA

rena tu red D N A famil ies have satel l i te D N A like p roper t i e s which include sharp melts and low complex i ty m o n o p h a s i c r eassoc ia t ion kinetics.

In this c o m m u n i c a t i o n we have presen ted in situ hybr id i za t ion evidence o f the res t r ic ted loca t ions o f c R N A s f rom two of these D N A famil ies in hetero- c h r o m a t i c regions o f h u m a n me taphase c h r o m o s o m e s (Table 2: Summary) and evidence of thei r na t ive satel l i te D N A identi t ies.

The 1.687 c R N A shows a m a j o r gra in locus on the Y c h r o m o s o m e when hybr id i zed at TopT to male me taphase ch romosomes . Other loci showing consis- tent label l ing include c h r o m o s o m e s 1 and the p resumpt ive 9 at or near bo th cen t romeres and pe r i cen t romer ic he t e roch romat in . This is very s imi lar to the Satel l i te I c R N A in situ hyb r id i za t i on results o f bo th Jones et al. (1974) and G o s d e n et al. (1975). In add i t i on to shar ing s imi lar phys ica l p roper t i e s ( M a r x et al. , 1976) with Satel l i te I D N A , the 1.687 D N A was shown to be comple te ly enr iched in the Ag+-Cs2SO4 grad ien t reg ion con ta in ing Satel l i te I D N A , no d o u b t ind ica t ing its na t ive state to be Satel l i te I D N A .

The 1.700 c R N A shows m a j o r loci at the pe r icen t romer ic h e t e r o c h r o m a t i n of c h r o m o s o m e s 9 as well as m i n o r sites at o ther cen t romer ic regions and the dis ta l a r m of the Y c h r o m o s o m e . This d i s t r ibu t ion o f label led loci s t rongly resembles tha t of h u m a n Satel l i te I I I D N A (Jones et al., 1973). In add i t i on to s imilar i t ies in every phys ica l p r o p e r t y inves t iga ted ( M a r x et al., 1976) the 1.700 D N A fami ly was found to be enr iched in the Cot 1.0 D N A iso la ted f rom the Satel l i te I I I D N A rich region o f a A g § gradient , suggest ing its nat ive state to be Satel l i te I I I D N A .

38 K.A. Marx et al.

Heterochromatic regions of human metaphase chromosomes may be classified into five types on the basis of their differential staining behaviour (Pearson et al., 1972). This heterogeneity in staining behaviour is matched by the heterogeneity in type and occurrence of human satellite DNAs-renatured DNAs. The strongly quinacrine fluorescent distal arm of the Y chromosome is a primary location of the 1.687 DNA family - Satellite I DNA. The Giemsa 11 positive secondary constrictions of chromosomes 9 contain the major concentra- tions of the 1.700 DNA family- Satellite III DNA, Satellite IV DNA (Gosden et al., 1975) and Satellite C DNA (Saunders et al,, 1972). The secondary constric- tions of chromosomes 1 and 16, which are only C band positive, contain almost all (Gosden et al., 1975) of the Satellite II DNA (1.696 DNA) sequences (Jones and Corneo, 1971). Another class, the acrocentric chromosomes, known to be the sites ofrDNA localization (Henderson et al., 1972) and probably involved in nucleolus organization, contain characteristic concentrations of Satellite DNAs I-IV (Gosden et al., 1975: Table 1) as well as the 1.687 DNA and 1.700 DNA sequences. Notwithstanding the ability of in situ hybridization to reflect true DNA concentration it is tempting to infer that the differential stainability of each region is in some way a consequence of its unique concentra- tion distribution of satellite DNAs. This may be brought about by non-histone proteins (Comings et al., 1973) recognizing and interacting with specific satellite DNA sequences or geometries.

Recent results of Melli et al. (1975) demonstrate cross-hybridization of satel- lite DNAs II and III. This suggests a common origin for these two DNA families and the possibility of cross-hybridization in situ. The quantitative results (Gosden et al., 1975) of the satellite II and III cRNA grain distributions at single loci may be a reflection of this partial homology, when, in reality, the homologous Satellite II and III DNA sequences might share no common chro- mosomal loci. This homology may be responsible as well for the very small amount of 1.696 DNA-Satellite II DNA and the unexpectedly large amount of 1.700 DNA-Satellite III DNA seen in the Cot 1.0 DNA sequences of the Satellite II DNA rich region C of the Ag § gradient in Figure 9d.

The occurrence of common satellite DNA sequences amongst the higher primates (Jones et al., 1972; Prosser et al., 1973) is unusual since even closely related species often contain totally different types of satellite sequences. As Jones et al. (1972) have suggested, the common satellite DNAs of the higher primates may have been responsible, perhaps by Robertsonian fusion, for the evolutionary success of these primate families. As was seen here for the 1.700 DNA localization, associations of constitutive heterochromatin in inter- phase occur to varying degrees. It is clear in Mus musculus that different tissues show widely varying but characteristic numbers of chromocenters in interphase nuclei. Such transient situations could allow for the testing of genetic arrange- ments before commitment to a permanent fused condition (Jones et al., 1972).

The rainbow trout (Salmo irideus) exhibits epigenetic Robertsonian fusion, such that each tissue possesses a characteristic metaphase karyotype (Ohno et al., 1965). This unusual and striking form of tissue specific interaction has been shown by the first author to be at least partially associated with the presence of Ba(OH)2 C banding heterochromatin occurring in tissue specific numbers of chromocenters in interphase nuclei (unpublished).

Localizations of Repetitious Human DNA Families 39

Mazrimas and Hatch (1972) have demonstrated significant correlations be- tween the amount of satellite DNA in twelve closely related species of kangaroo rat (Dipodomys) and the degree of specialization of each species, the number of subspecies within each species and the fundamental number of chromosome arms. Thus, it seems that genetic changes in adaptation may be facilitated by the presence of satellite DNA. This could arise through phenomena such as the variegated position effect (Baker, 1968; McClintock, 1951).

2. Middle Repetitious Renatured DNA Families

The two heaviest density renatured human DNA families have previously been shown to have the physical properties of middle repetitious DNA sequences and to share, in common, DNA sequences of weak homology (Marx et al., 1976). It is not surprising then to find that these two DNA families have very similar bimodal Top T profiles and behave very similarly in in situ hybridiza- tion patterns, showing no obvious chromosomal localizations, although the 1.703 DNA shows a statistical preference for centromeres and telomeres and some clustering of autoradiographic grains.

Saunders et al. (1972) have noted a preference of cRNA from different thermal classes of total repetitious human DNA for centromeres and telomeres of certain human chromosomes. This is to be expected. However, the complex nature of their template and the consequent hybridization parameters makes their results difficult to interpret in light of the present study. A similar difficulty arises in the claim of Sanchez and Yunis (1975) to have demonstrated the association of chromosomal bands with repetitious DNA. In addition to the complex nature of the template, the low resolution of autoradiography would suggest a cautious interpretation.

The bimodal TorTs of these DNA families are consistent with their biphasic reassociation kinetics. Broad 45 ~ C ToPvS and sharper 67 ~ C ToPTS of both DNA families correspond respectively to a class of highly diverged and related middle repetitious DNA sequences and a class of more highly repetitious less diverged DNA sequences (unpublished). The presence of two DNA kinetic classes within each DNA family of 400 nucleotide fragment size suggests a distribution in the genome where these two kinetic classes of sequences would be, at least in part, interspersed rather than tandemly repetitious.

Schmid and Deininger (1975) have recently described such an organization in the human middle repetitious DNA sequences. An interspersed arrangement of middle repetitious sequences appears to be widespread in nature (Davidson et al., 1975) and has been demonstrated in many other systems in different ways (Cech and Hearst, 1975; Manning et al., 1974; Clarkson et al., 1973; Lewin, 1974 review). The fact that the 1.714 DNA was found in all regions of a Ag+-Cs2SO4 gradient argues for heterogeneity in any interspersion of the two DNA kinetic classes, producing all possible Ag § :DNA binding ratios. Both the restriction of 1.703 DNA sequences to the central region of the Ag § Cs2SO4 gradient and the pronounced statistical preference of 1.703 cRNA for telomere and centromere chromosomal areas suggests much less interspersion of this DNA family with other sequences and some chromosomal concentration

40 K.A. Marx et al.

of sequences, probably close to or in heterochromatic regions and perhaps associated with chromosomal G bands.

A similar suggestion of the preferential localization of a single middle repeti- tious DNA family in mouse heterochromatin was noted by Cech et al. (1973). Consistent with this is the observation that only a portion of mouse heterochro- matin has high concentrations of mouse satellite DNA (Rae and Franke, 1972). In addition, Cech and Hearst (1976) have described a repetitious mouse main band DNA family which is tandemly repeated.

Although the results presented here do not support a particular model of function (see Botchan et al., 1971; Kram et al., 1972; Paul, 1972; Sutton, 1972; Davidson and Britten, 1973; Lewin, 1975 for review) the suggestions of different localisations and sequence arrangements in the 1.703 DNA and 1.714 DNA, in spite of their weak sequence homology, and, therefore, probable common origin, imply that these DNAs have somewhat different biological functions. Melli et al. (1975) recently presented evidence for the clustering of repetitive DNA sequences complementary to repetitive nuclear RNA in HeLa cells. While this is only one possibility for a general function involving transcription of the different middle repetitious DNA families, it may begin to be tested by first looking directly at sequence organization in both middle repetitious DNA families.

With regard to sequence organization it is interesting to note that Gummer- son (1972) has found, amongst primate repetitious DNAs, that large fractions of diverged repetitious DNAs are held in common, without significant change. New, undiverged repetitious DNA, on the other hand, is changing much more rapidly. Therefore, an analysis of the sequence organization of these distinct but related middle repetitious DNA families in different primates may suggest ways in which genome organization evolves and thereby provide clues as to the function of satellite and repetitious DNA sequences.

Acknowledgements. The authors would like to thank Dr. W. Palmer, Chief Pathologist at Alta Bates Hospital, Berkeley, Calif., for his help in obtaining DNA samples and his interest in this work. Mrs. Judith Hughes cheerfully helped with the karyotype analyses. This work was supported by the United States Public Health Service Grant GMl1180. K.A.M. would like to acknowledge a Muscular Dystrophy Society of America Postdoctoral Fellowship under which a small portion of this work was performed.

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Received March 22-August 5, 1976 / Accepted April 22, 1976 by J.G. Gall Ready for press August 7, 1976