DNA of mammalian and avian heterochromatin

Experimental Cell Research 71 (1972) 113-131

DNA OF MAMMALIAN

AND AVIAN

HETEROCHROMATIN

D. E. COMINGS and E. MATTOCCIA Department of Medical Genetics, City of Hope National Medical Center, Duarte, Calif. 91 010, USA

SUMMARY The buoyant density, satellite composition, and in some cases renaturation kinetics of the DNA of whole nuclei, euchromatin, heterochromatin + nucleoli and pure nucleoli, were determined in the mouse, guinea pig, horse, dog, Chinese hamster, Micro&s agrestis, chicken and Japanese quail. These and other studies suggest there are several different types of DNA which may be localized to the constitutive heterochromatin. These include the following: I. Repetitious satellite DNA: (a) AT-rich, (b) GC-rich, (c) same density as main band DNA. II. Repetitious main band DNA. III. Non-repetitious DNA: (a) GC-rich heavy shoulder DNA, (b) AT-rich main band DNA.

Recent studies utilizing in situ hybridization [21, 27, 31, 32, 36, 42-44, 461 or nuclear subfractionation [35, 37, 40, 49, 56, 57, 601have shown that satellite DNA tends to be localized to the constitutive heterochromatin of eukaryotes. A related question of whether there are other types of non-repetitious DNA which are also localized to heterochromatin, remains to be answered. To investigate this the intranuclear distribution of the various classes of DNA has been determined in a number of mammals andibirds. MATERIALS AND METHODS Nuclear isolation The animals used were fasted overnight. In the studies of the chicken and quail the liver was first perfused with normal saline to remove the nucleated red blood cells. The liver was cut into small pieces and placed in0.25 M sucrose containing 3.3 mM calciumchloride at 4°C. Ten volumes of 2.4 M sucrose, 3.3 mM calcium chloride was then added, mixed well, and homogenized with 4 to 6 strokes of a Teflon homogenizer until most of the cells were broken, as confirmed by phase contrast microscopy. The solution was filtered through one layer of cheesecloth, adjusted to a density reading of 12”C, 15 min, with an Abbe refractometer, placed in 8 - 721803

Spinco SW 41 Ti centrifuge tubes and spun at 16 000 rpm for 1 h at 4°C. The nuclear pellet was resuspended in 0.34 M sucrose without calcium chloride and subsequently washed in this three times by centrifugation for 5 min at 1 000 g [37, 561.

Nuclear subfractionation The nuclear pellet was resuspended in 1.5-2.0 ml of 0.34 M sucrose and exposed to three 5-set bursts of sonication at setting No. 4 using a Branson sonicator (Model S-75) with a microtip. The sonication was carefully controlled by phase contrast microscopy until more than 90 % of the nuclei had been ruptured. The suspension was gently spun at 400 g for 3 min to pellet the unbroken nuclei. If necessary, this nuclear pellet was son&ted once more and the supernatant combined with the first supernatant. The nuclear sonicate was then spun at 4000 g for 20 min in a Sorvall swinging bucket rotor and the pellet was taken as the heterochromatin + nucleoli fraction [371. In some preparations, to further remove contaminating euchromatin, it was resuspended in 0.34 M sucrose and spun once again at 4 000 g for 20 min. Usually this supematant was taken as euchromatin, but in some preparations an intermediate fraction was pelleted from the euchromatin by centrifugation at 20 000 g for 20 min [56]. In all samples the heterochromatin+nucleoli fraction contained from 12 to 20 % of the total DNA.

Isolation of nucleoli The heterochromatin + nucleoli pellet was resuspended in 0.4 M sucrose and 1% Tween 20 was added to bring it to a final concentration of 0.1% Tween. This Exptl Cell Res 71

114 D. E. Comings & E. Mattoccia suspension was then sonicated at setting No. 4 for 20 & and an aliquot of the sonicate examined after adding a drop of 0.05 % solution of Azure-B. This stained the nucleoli intensely and allowed visualization of the degree to which the chromatin had been removed from the nucleoli. The sonicate was layered over 0.88 M sucrose 1411and spun at 1 000 g for 10 min. The pellet cont&ed nuileoli with a variable amount of chromatin contamination. The procedure was repeated until a pure nucleolar preparation was obtained. In the mouse the nucleolar fraction contained 2.5 % of the total DNA. Further extraneous DNA was removed by treatment with 2 M NaCl [37, 491 or with DNase 2.0 pug/ml for 7 min at room temperature.

Electron microscopy of nuclear subfractions Aliquots were taken of all nuclear subfractions and fixed in 3 % glutaralderhyde at 4°C for 1 h. They were then washed in a balanced salt solution and subsequently stained with 1 % osmic acid for 1 h. They were washed again and sequentially placed in increasing concentrations of 25-100 % ethanol, then in propylene oxide, propylene oxide plus EponAraldite, and finally embedded in Epon-Araldite. The embedded soecimens were sectioned with LKB ultratome, stained with uranyl acetate and lead citrate and examined in the Hitachi HS-8-1 electron microscope.

DNA Isolation Aliouots of whole nuclei or the nuclear subfractions were placed in 1 % sodium dodecyl sulfate, 0.15 M sodium chloride. 0.01 M EDTA. OH 7.2 and intermittently shaken at room temper&e for 1 h. 5.0 M sodium chloride was then added in sufficient volume to bring the final molarity to 0.5 M sodium chloride and the solutions were piaced on ice for 30 min to precipitate the SDS. The SDS was then pelleted at 10 000 g for 20 min. The supernatants were repeatedly extracted with chloroform iso-amyl alcohol (24 : 1) until no protein interface remained. The DNA was then dialvsed against SSC (0.15 M sodium chloride. 0.015 M sodium citrate, pfi 7) for overnight at 4°C: These samples were used for analytical uitracentrifugation in cesium chloride. In occasional specimens, especially when isolating DNA from nucleoli, the DNA was further purified by exposure to RNase-A (Sigma) 25 pug/ml and RNaseTl (CalBiochem) 25 units/ml at 37°C for 1 h. Both RNases had previously ‘been heated to 90°C for 5 min to destroy residual DNase. Following RNase treatment they were extracted once again with iso-amyl alcohol and dialysed overnight against SSC.

Analytical ultracentrifugation For analytical ultracentrifugation, 5 to 10 pg of DNA were combined with 1 to 2 pg of Micrococcus lysodeikticus DNA and the volume brought to 0.7 ml with SSC. Dry, optical grade cesium chloride (Schwarz) was then added to bring the volume to 1 ml and the density adjusted to 1.70 g/cm* with an Exprl Cell Res 71

Abbe refractometer. The sample was then spun to equilibrium with a Beckman- Model E analytical ultracentrifuge at 42 040 rpm at 20°C using a standard cell with a Kel-F centerpiece and an AN-D two cell rotor. The photographs were scanned with a JoyceLoebel microdensitometer utilizing an arm ratio of 1:75 and the buoyant densities of the peaks determined [38]. The photographs were scanned once again at a considerably greater enlargement utilizing an arm ratio of 1:20 and an appropriate wedge. This gave curves which filled one or two 8 x 11 inch sheets of graph paper. These enlarged curves were then analysed with a DuPont 310 curve resolver. This instrument allows the superimposition of electronically generated Poisson distributions on top of the densitometer tracings and allows determination of the minimal number of sub-components which are present in a given tracing. The percentage of each sub-component can then be determined. The results were quite reproducible if the sub-components were relatively distinct (as in the mouse, horse, chicken, quail and others) but in some cases where the subcomponents were close together several curve resolviIw solutions were possible (as in the renatured DNA OFMicrotus). In these cases no solutions were presented. Most of the samples were centrifuged twice, some as many as five times.

Centrifugation of partially

renatured DNA

To detect the presence of hidden peaks of highly repetitious satellite DNA, samples were denatured at 100°C in SSC for 5 min, then renatured at 60°C for 5 h. Marker DNA was then added and the sample centrifuged as described above. The concentration of DNA in the re-annealing mixture ranged from 5-12 pg/ml, and 5 h of re-annealing resulted in an average Cot of 0.3 moles nucleotides x set x L-l.

Preparative ultracentrifugation To study individual components, some of the samples of whole nuclear DNA were subfractionated by preparative ultracentrifugation. From 75 to 200 pg of DNA were centrifuged in CsCl in a Beckman 50 Ti fixed angle rotor [24] at 31 000 rpm for 65 h. Eight drop fractions were collected, diluted with 0.5 ml of -SSC, the OD determined,’ and the appropriate fractions pooled. In some cases the fractions were further purified by a second centrifugation.

DNA replication studies To investigate the variation in base composition of DNA replicated during different portions of the S period, synchronized Chinese hamster cells were utilized. The Don cells were grown in McCoy medium containing 10 % fetal calf serum and 1 %- essential and non-essential amino acids. The cells were synchronized by the mitotic selection technique [52]. This produced a suspension containing between 94 and 98 % cells in mitosis. These cells were then resuspended in McCoy solution containing amethopterin 2 mM and adenosine 10 mM [47]. After incubation for 4 h, DNA replicating during the first 2 h of the S period

DNA of mammalian and avian heterochromatin was labeled by the addition of fresh media containing sH-thymidine 25 @X/ml (spec. act. 16.9 Ci/mM, New England Nuclear). In other preparations these cells were released by the addition of 20 .u.aof cold thvmidine for t h and then washed twice and re-incubated in McCoy medium. After further incubation for 4 h, medium containing *H-thymidine ulus ametbonterin and adenosine was added for an additional- 2 h. Previous studies [14] have shown that this technique produces a highly synchronous population of cells. Autoradiography demonstrated that heterochromatin was replicating in the 4 to 6-h specimens. Unsynchronized cells labeled with W-thvmidine (Schwarz. spec. act. 40 mCi/mM, 0.5 &i/nil for 15‘h), were utilized for controls. In some experiments the synchronized cells were labeled for the first 2 h of S period with W-thymidine. The mouse L cells were synchronized by double excess thymidine block, as described previously [14]. The initial part of the DNA extraction procedure was as described above. ineluding the BNase treatments. The DNA was dialysed against 0.12 M uhosohate. DH 6.8, and further uurified bi placing it on a hydroxyapatite column. The contaminants were eluted with 0.12 M phosphate buffer and the DNA eluted with 0.3 M phosphate buffer. This DNA was then dialysed back to 0.12 M phosphate buffer and sheared by passing twice through a specially designed French Press at 25 000 psi. This results in DNA fragments with a mean molecular weight of 400000 as determined by velocity sedimentation [19].

DNA Tm by hydroxyapatite chromatography

column

The hydroxyapatite was prepared and the columns run by the procedure of Miyazawa & Thomas [39]. The samples containing SH-labeled DNA derived from specific portions -of the S period and l*Cthvmidine-labeled DNA from unsvnchronized cells were both placed together on a hydroxyapatite column and the temperature increased at increments of 5°C. After each temperature increment the denatured DNA was washed from the column with 0.12 M phosphate buffer. The eluted DNA was precipitated with 10% TCA at 4°C and placed on filters. The radioactivity was counted in a Beckman ambient temperature scintillation counter and the counts corrected for background and cross spill-over between the two channels. The percentage of 8Hand ‘“C DNA denatured at a given temperature was calculated to give a DNA Tm curve.

COT Curve The renaturation kinetics of euchromatin and heterochromatin DNA from the Chinese hamster were determined on unlabeled DNA by hydroxyapatite column chromatograohv. The DNA was uurified and sheared as d&&bed above for labeled DNA. 250 ~a of DNA in 0.12 M nhosnhate buffer was then den&-ed by boiling. It was then incubated at 60°C and at various time intervals aliquots were removed and the ratio of renatured to denatured DNA was determined by elution on hydroxyapatite. The

115

concentration of DNA was determined by optical density with no correction for the hyperchromisity of the denatured DNA. The renaturation kinetics of the horse main band and heavy satellite were determine in a recording spectrophotometer.

Heterochromatin staining Chromosome preparations of all the species studied were stained by the technique of Arrighi & Hsu [l] for constitutive heterochromatin. An estimate of the amount of such heterochromatin was made by measuring the length of the stained and unstained segments.

RESULTS Electron microscopy of nuclear subfractions

The purity of all subfractions was demonstrated by electron microscopy. The whole nuclei were clean with no cytoplasmic tags. The euchromatin fraction washomogenous in appearance with no nucleoli or masses of condensed chromatin. The heterochromatin + nucleoli fractions contained dense masses of chromatin that were still adherent to long sections of the nuclear membrane, plus fibrillar and granular portions of nucleoli. The purified nucleoli showed only fibrillar and granular portions of the nucleoli. Illustrations of thesefractions have been presented elsewhere [37]. Analytical ultracentrifugation Mouse (Mus musculus)

The buoyant density and satellite content of nuclear subfractions of the mouse have been presented previously [37]. These studies demonstrated that the pure nucleoli and nucleoli treated with 2 M sodium chloride contained the greatest concentration of satellite DNA. They also demonstrated that the buoyant density of the main band DNA underwent a progressive shift from 1.7010 g/cm3 in whole nuclei, to 1.7007g/cm3 in DNA from heterochromatin + nucleoli, to 1.6999 g/cm3 in pure nucleoli, to 1.6991 g/cm3 in nucleoli treated with 2 M NaCl. Since the later fractions Exptl Cell Res 71

116 D. E. Comings & E. Mattoccia purified nucleoli, 40 % in nucleoli treated with DNase, and 47 % in nucleoli treated with 2 M sodium chloride. This indicated that the more the nucleoli were purified the higher became their content of satellite DNA. Small sub-components or ‘minor shoulders’ ranging between 3 and 5 % of the total DNA were seenin the heterochromatin, EUCHROMATIN heterochromatin + nucleoli, and nucleoli + DNase fractions, but were not seen in the other samples. These minor shoulders were WHOLE NUCLEI probably the result of excessivefragmentation of the DNA with the result that short HETEROCHROMATIN stretches of relatively CC-rich DNA (heavy minor shoulders) or AT-rich DNA (light minor shoulders) could be separated from the main band. This interpretation is consisHETEROCHROMATIN tent with the following. (a) The minor shoul8 NUCLEOLI ders could be seen on both the light and heavy side of the main band; (b) they were most frequently observed in DNA from NUCLEOLI fractions that were the most heavily sonicated; (c) they were sometimes absent from the euchromatin DNA probably as a result of the fact that euchromatin DNA was so Fig. 1. Analytical ultracentrifugation of DNA isolated from mouse nuclear subfractions. The DNA in SSC heavily sheared that the curves were broad has been denatured at 100°C for 5 min, renatured at tending to obscure non-gaussian sub-com60°C for 5 h, and centrifuged. The DNA from whole nuclei was freshly prepared, that of the other fracponents; (d) the minor shoulders were espetions had been frozen for 1 year (see text). cially prominent in DNase-treated material, but rarely seen in DNA from whole nuclei probably represent progressively purer hetero- that were neither sonicated nor DNasechromatin, these observations were com- treated. The DNA from the mouse nuclear subpatible with the replication studies which have shown that early replicating presumably fractions was utilized to determine if centrieuchromatic DNA is relatively GC-rich, and fugation of the DNA after denaturation and late replicating presumably heterochromatic partial renaturation (see Methods) was a DNA is relatively AT-rich [4-6, 9, 23, 541. valid technique for detecting the presence of The euchromatin appeared to contain 100% highly repetitious satellite DNA. This techmain band DNA. The whole nuclei showed nique has previously been used in studies of 10 % satellite and this progressively increased mouse, guinea pig, and calf and human DNA to 12% in heterochromatin (the supernatant [17, 181. The results, shown in fig. 1, allow the capabilities of this technique to be from the heterochromatin + nucleoli fraction after removal of most of the nucleoli), 16 % scrutinized. The buoyant density of minimally in the heterochromatin+ nucleoli, 25 % in renatured main band, euchromatin DNA was

L

Exptl Cell Res 71

DNA of mammalian and avian heterochromatin

1.716 g/cm3.In the heterochromatin + nucleoli and nucleoli fractions it shifted to 1.714 g/cm3 consistent with a similar slight shift to AT richness seen in the main band when the native DNA was centrifuged [37]. The buoyant density of the satellite peak in most of the samples was 1.695 g/cm3 compared with 1.691 g/cm3 when native DNA was centrifuged. This technique demonstrated that there was still some satellite DNA in the euchromatin fraction. It progressively increased in the samples containing more heterochromatin. The percentage of satellite DNA obtained was consistently greater than with centrifugation of native DNA. This might be explained in the following manner. Flamm et al. [25] have shown that some base sequencessimilar to those found in satellite DNA are present in the main peak but presumably do not band separately because they are not clustered. After denaturation and renaturation some of these sequences, along with their associated non-repetitious DNA, may anneal with the satellite sequences and travel with the satellite peak, thus increasing its size. The shift in density of the main band and satellite peak in the DNA from whole nuclei deserves comment. All of the other samples had been stored frozen for approx. 1 year before this denaturation-renaturation experiment was done. The whole nuclei DNA, however, was made fresh. Since the length of the DNA molecules is an important factor in determining the rate of renaturation [7], it is possible that the stored sampleshave suffered slow breakage to smaller molecular weights and thus underwent more rapid renaturation than the freshly prepared sample. This underscores the fact that additional variables are present in the denaturation-renaturation studies that are not involved in the centrifugation of native DNA. One additional feature of the denaturation-

117

Fig. 2. Analytical ultracentrifugation of DNA isolated from nuclear subfractions of the chicken.

renaturation experiments is of importance. It has been suggested that all constitutive heterochromatin is enriched in rapidly or moderately repetitious DNA [60]. Centrifugation of native mouse DNA showed that the buoyant density of the main band was less in the DNA from pure nucleoli fractions than in the DNA from the whole nuclei or euchromatin fraction. This was consistent with the AT richness found in late replicating presumably heterochromatic DNA and indicated that this nonsatellite DNA was not just the result of contaminating euchromatin. This indicated that a significant portion of heterochromatic DNA was not satellite DNA but it did not rule out the possibility that this might be moderately or even highly repetitious DNA with a base composition near that of the main band. The denaturationrenaturation experiments, however, tend to rule this out, since the shift toward the density of native DNA (a measure of rates of renatuExptl Cell Res 71

118 D. E. Comings & E. Mattoccia

EUCHROMATIN,

chicken DNA demonstrated the presenceof a rapidly renaturing satellite fraction constituting 5 % of the total in DNA from whole nuclei and increasing to 9 % in DNA from the heterochromatin + nucleoli and pure nucleoli fractions (fig. 3). To determine whether this satellite was hidden within the main band

D-R

NUCLEI,D-R

HETEROCHROMATIN,

D-R

A : : % *\ 1

y

NUCLEOLI,

MAIN

BAND

MAIN BAND,

D-R BAND,

Fig. 3. Analytical ultracentrifugation of denatured and renatured DNA from nuclear subfractions of the chicken. ration) was no greater than expected on the basis of its slight AT richness.

Chicken (Gallus domesticus)

Analytical ultracentrifugation of DNA from nuclear subfractions of the chicken was unremarkable. There was no satellite present and the buoyant density of the DNA of the different fractions did not change appreciably (fig. 2). Chicken DNA, however, proved to be of great interest in that there was a significant heavy shoulder component constituting 30 % of the total DNA in all nuclear subfractions. Detailed studies of heavy shoulder DNA are reported elsewhere [lo]. Centrifugation of denatured and renatured Exptl Cell Res 71

DENATURED

D-R

Jj-L HEAVY

SHOULDER

SHOULDER-

DENATURED

Fig. 4. Analytical ultracentrifugation of isolated chicken main band and heavy shoulder DNA. Denatured, denatured in SSC at 100°C for 5 min, quickly cooled in ice and centrifuged. D-R, denatured in SSC at 100°C for 5 min, incubated at 60°C for 5 h, cooled and centrifuged. The main band and heavy shoulder component were isolated by double passage through preparative ultracentrifugation.

DNA of mammalian and avian heterochromatin

or heavy shoulder DNA, these fractions were separated by double passage through preparative ultracentrifugation in CsCl. The results are shown in fig. 4. Main band DNA presented as a perfect gaussian curve with a mean buoyant density of 1.700 g/cm3. When this was denatured and centrifuged without renaturation it showed a main peak (96 “/) with a density of 1.716 g/cm3 and a minor light component (4 %) of questionable significance. When centrifuged after renaturation for 5 h the minor component, banding at a mean density of 1.705 g/cm3, was now clearly significant (14%). The enriched heavy shoulder DNA was bimodal containing 60 %

119

EUCHROMATIN,D-R

NUCLEI, D- R

.HETEROCHROMATIN El NUCLEOLI, D-R

EUCHROMATIN NUCLEOLI,D-R

:r: rrNUCLEI

HETEROCHROMAlIN 8 NUCLEOLI

Fig. 5. Analytical ultracentrifugation of DNA isolated from guinea pig nuclear subfractions. There is a progressive increase in the heavy satellite peak in the heterochromatin + nucleoli and nucleoli fractions.

I ;;i 2

Fig. 6. Analytical ultracentrifugation of denatured and renatured DNA isolated from nuclear subfractions of the guinea pig.

heavy shoulder DNA with a density of 1.706 g/cm3 and 40% main band DNA. When denatured it showed an apparently single but broad curve. After renaturation for 5 h there was only a tiny (4 %) light component. These results indicate that the rapidly renaturing satellite was hidden within the main band DNA. The observation that upon renaturation the shift in buoyant density of the main band and heavy shoulder component were approximately the same, agrees with the Cot studies [lo] which indicated that the heavy shoulder did not contain a disproportionate amount of repetitious DNA. Exptl Cell Res 71

120 D. E. Comings &YE. Mattoccia

heavy shoulder component (55 and 60%) and a moderate increase in the satellite DNA (15 and 20 %) [ 151.The renaturation kinetics of these isolated components indicated that EUCHROMATIN the heavy shoulder did not contain a disproportionate amount of rapidly renaturing DNA. These observations show that in the WHOLE NUCLEI quail the major fraction to be increased in the heterochromatin was a non-repetitious HETEROCHROMATlN t NUCLEOLI DNA component. Since this component was increased in the heterochromatin of the quail but not of the chicken and since the microNUCLEOLI chromosomes are heterochromatic in the quail but not in the chicken, this suggests that in birds heavy shoulder DNA may be localized to the microchromosomes [ 151. Extensive centrifugation studies of the Fig. 7. Analytical ultracentrifugation of DNA isolated renaturation of quail DNA were done. The from nuclear subfractions of the horse. There is a slight ,decreasein the buoyant density of the main resulting curves, however, were complex, and band in nucleolar DNA. have provided no increased understanding of problems investigated in the present study. Japanese quail (Coturnix

coturnix, Japonica)

Previous studies [13] have shown that the Japanese quail possessesa heavy GC-rich satellite and that the DNA of this satellite renatures more rapidly than main band DNA, indicating it is enriched in repetitious sequences.Purified satellite DNA centrifuged under denaturing conditions (pH 13) separated into three bands demonstrating that at least in a portion of the satellite the bases were non-randomly distributed between the two half helices. These studies also indicated that the microchromosomes in the quail were late replicating and nucleolus organizing. Curve resolving shows that the DNA of the quail consists of three sub-components, a main band constituting 70 %, a satellite constituting IO%, and a heavy shoulder on the main band constituting 20 % [15]. These ratios remained approximately the same in DNA from euchromatin but in the heterochromatin + nucleoli and pure nucleoli fractions there was a striking increase in the Exptl Cell Res 71

Guinea pig (Cavia porcellus)

Analytical ultracentrifugation of native guinea pig DNA (fig. 5) shows a main band at 1.700 g/cm3 and a small secondary peak close to the main band with a density of 1.706 g/cm3 [17, 18, 261. Centrifugation in Ag+-Cs,SO, demonstrates the presence of three satellite bands [18]. Once separated these bands can be dialysed back into SSC and when recentrifuged the native molecules show the following densities: satellite I [18] or t( [26, 511 (1.706 g/cm3); satellite II or /? (1.704 g/cm”) and satellite III [18] (1.704 g/cm”). They consti-

tute 5.5, 2.5 and 2.5 % of the total DNA respectively [18]. When centrifuged in alkaline CsCl the tc satellite separates into two widely

separate bands [18, 26, 291. The

base sequence of this satellite has been shown by Southern [51] to consist of a basic repeat of 5’-CCTAA-3’ light strand 3’-GGGATT-5’

heavy strand.

DNA of mammalian and avain heterochromatin

Previous studies of nuclear subfractions of the guinea pig utilizing preparative ultracentrifugation in Ag+-C@O, have shown an enrichment of the satellites in the heterochromatin + nucleoli fraction [60]. The present results from analytical ultracentrifugation of native and denatured-renatured DNA from nuclear subfractions are show in figs 5, 6. Centrifugation of native DNA from whole nuclei showed two peaks with up to 32% of the total DNA contributing to the second component. This is greater than the total amount of satellite DNA in this region (10.5 %) and probably represents the additional presenceof someheavy shoulder DNA. The second peak at 1.706 g/cm3 is no longer apparent in DNA from euchromatin, but this peak progressively increases in DNA flom heterochromatin + nucleoli and pure nucleoli (fig. 10) until it accounts for 50% of the total DNA in the nucleoli fraction. In this respect it resembles the shift in main band density with increasing purity of nucleoli seen in the quail [15]. The significance of the extra heavy component increasing from 3 % in whole nuclei to 15 % in nucleoli is unknown. Centrifugation of denatured-renatured DNA allowed an evaluation of the degree to which there was an alteration in rapidly renaturing DNA in these fractions (fig. 6). After 5 h of renaturation the DNA from whole nuclei showed a main band density of 1.715 g/cm3 and a peak of rapidly renaturing DNA at 1.711 g/cm” constituting approx. 6% of the total. This was reduced to 3 % in the euchromatin fraction, and increased to 12 % in the heterochromatin +nucleoli and 28 % in the pure nucleoli fraction. On the photographs this was a very sharp band comparable to that seen with the satellite DNA in the nucleoli fraction of mice. Since studies of the isolated light and heavy strands of satellites I, II and III [17, 181show the satellites renature to a

121

EUCHROMATIN

WHOLE

NUCLEI

HETEROCHROMATIN

NUCLEOLI

resolvingof the DNA from nuclear subfractionsof the horse. In the DNA of whole nuclei the heavy sateIlite constitutes 28 % of the Fig. 8. A curve

total. It progressively increases in heterochromatin and nucleoli fractions.

density of 1.711 g/cm3, this band probably represents the renaturation of all three satellites. Thus in the guinea pig there is a significant increase in the concentration of Exptl Cell Res 71

122 D. E. Comings & E. Mattoccia

centrifugation of denatured and renatured DNA were surprising (fig. 9). Instead of demonstrating a large rapidly renaturing satellite component they showed essentially EUCHROMATIN a unimodal distribution with no rapidly renaturing band in any nuclear subfraction. To clarify this observation, main band and heavy satellite DNA were separated by NUCLEI ultracentrifugation and their renaturation HETEROCHROMATIN kinetics followed in a recording spectropho8 NUCLEOLI tometer. This demonstrated (fig. 10) that NUCLEOLI about 38 % of the heavy satellite fraction was composed of rapidly renaturing DNA, compared to 15% in the main band. When these Fig. 9. Analytical ultracentrifugation of denaturedfractions were examined by analytical ultrarenatured DNA isolated from nuclear subfractions of the horse. In these samples there was no evidence centrifugation, additional interesting features for any rapidly renaturing satellite peaksand the were observed (fig. 11). The mean density main band showed a unimodal distribution. of the main band was 1.699 g/cm3, and that satellite DNA in the heterochromatin frac- of the heavy satellite was 1.712 g/cm3. When tions, but there is also a large amount (66 %) the main band was denatured and renatured, of non-satellite DNA which does not renature a rapidly renaturing satellite constituting any more efficiently than DNA from whole about 3% of the DNA and banding at a density of 1.694 g/cm3, became apparent. nuclei or euchromatin. However, when the heavy satellite, or whole Horse (Equus caballus) nuclear DNA, was denatured and renatured Arrighi et al. [3] reported a prominent heavy no such rapidly renaturing component was seen. This suggested that the presence of satellite constituting 28 % of the total DNA heavy satellite DNA during renaturation of the horse. It was thus of interest to investigate the intranuclear distribution of such a prevented the appearance of the rapidly large satellite. The results are shown in renaturing peak in the main band. To test figs 7, 8. They confirm the observation that this, isolated main band and heavy satellite in DNA from whole nuclei there is a promi- DNA were mixed together, denatured, renanent satellite with a density of 1.712 g/cm” tured and centrifuged. This resulted in the constituting 28 % of the DNA. This was disappearance of the rapidly renaturing peak. decreased to 12% in euchromatin but pro- If the main band and heavy satellite compogressively increased to 35 and 38 % in the nents were sonicated (Branson sonicator, heterochromatin + nucleoli and pure nucleoli setting no. 6, 20 set), there was a moderate fractions. A heavy shoulder component of increase in the amount of satellite in the intermediate density was also present that main band, but still none was seen in the did not change significantly in concentration heavy satellite. The most likely explanation for these obin the different fractions. There was a downward shift in the density of the main band servations is that there are some highly from 1.699 g/cm3in the euchromatin to 1.698 repetitious but non-clustered sequencesin the g/cm3 in the pure nucleoli. The results of heavy satellite, and some similar sequences Exptl Cell Res 71

DNA of mammalian and avian heterochromatin

123

Fig. 10. Abscissa: cot (mole x set/l); ordinate: % renaturation. Renaturation kinetics of isolated main band and heavy satellite DNA from the horse. DNA in SSC was sonicated and placed in a recording spectrophotometer. The temperature was increased to 100°C to denature the DNA and then rapidly brought down to 60°C and the incubation continued for up to 48 h. The main band contained approx. 15 % rapidly renaturing DNA, while this component constituted over 35 % in the heavy satellite DNA. The second point from the left on each curve is the first reading at which a temperature of 60°C was obtained. The rapid fall in OD in the heavy satellite can be due either to a very highly repetitious fraction of DNA, or to partial renaturation of single stranded DNA [8]. The curves are the result of two different runs with a concentration of main band DNA of 58 and 27 pg/ml and a concentration of heavy satellite DNA of 30 and 18 pg/ml.

only a single component in DNA from whole nuclei, and only small artifactual heavy and light components (3 % or less) in the DNA from the other fractions that had been exposed to sonication. There were, however, significant differences in the profiles of denatured and renatured DNA from the different fractions (fig. 13). The renatured main band of euchromatin and heterochromatin DNA showed a mean density of 1.717 g/cm3 and a small rapidly renaturing satellite European field vole (Microtus agrestis) constituting 3 and 5 % respectively. This Microtus agrestis was of particular interest rapidly renaturing component did not into investigate becauseof the large amount of crease significantly in the heterochromatin + constitutive heterochromatin localized to the nucleoli fraction and increased only slightly X and Y chromosomes [55]. No satellite to 7 % in the pure nucleoli fraction. The bands were observed and the buoyant density most striking difference was in the main was the same for the DNA of all the nuclear peak. In the heterochromatin + nucleoli and subfractions (fig. 12). Curve resolving showed pure nucleoli fractions this showed a down-

which can come to equilibrium within the main band, at a density of about 1.694 or less, because they are clustered together and thus form a minor satellite. When the total DNA is denatured and renatured, the minor satellite DNA re-anneals to the greater number of non-clustered sequences in the heavy satellite. If the heavy satellite is missing, they re-anneal to themselves to form the rapidly renaturing peak.

Exptl Cell Res 71

D. E. Comings & E. Mattoccia

MPlN BAND

HEAVY

MAIN

SATELLITE

ward shift in density to 1,712 g/cm3. This peak was difficult to reproducibly analyse with the curve resolver so only the rapidly renaturing component is shown. However, as an approximate estimate, the main band DNA from the nucleolar fraction had a component at 1.712 g/cm3 constituting 60 % and a heavy component at 1.717 g/cm3 constituting 30 % of the total DNA. These results suggestthat there is a speciesof DNA in the heterochromatin of Microtus whichis of intermediate repetitiousness.

EAND.DR.

Dog (Canus familaris)

HEAVY

WHOLE

SATELL1TE.D.R.

NUCLEI&R.

The main band of DNA from the various subnuclear fractions of the dog showed no significant variation in density (fig. 14). However, in all fractions there was a heavy shoulder component, a portion of which became so marked in the heterochromatin+ nucleoli and pure nucleoli fractions that it almost appeared as a distinct band. In DNA

MAIN BAND 8 HEAVY SATELLITE,D.R.

MAIN BAND. S0NICATED.D

WHOLE

R

NUCLEI

EUCHROMATIN

HEAVY SATELLITE SONCATED.0 R

HETERDCHROMATIN + NUCLEOLI

Fig. II. Analytical ultracentrifugation of isolated main band and heavy satellite DNA from the horse. D-R, denatured in SSC at 100°C for 5 min and renatured at 60°C for 5 h. The rapidly renaturing satellite peak banding at 1.694 g/cm8 was present only when isolated main band DNA was denaturedrenatured and-centrifuged.

Exptl Cell Res 71

Ljl

NUCLEOLI

Fig. 12. Analytical ultracentrifugation of DNA isolated from nuclear subfractions of Microtus agrestis.

DNA of mammalian and avian heterochromatin 125 from whole nuclei, heavy shoulder components constituted about 10% of the total. In heterochromatin + nucleoli they increased to 12 % and in nucleoli they constituted 22 % of the DNA. Centrifugation of denatured and renatured DNA failed to show a satellite component. The heavy shoulder component was not isolated to determine if it was composed of an excessof repetitious sequences. Chinesehamster (Cricetulus griseus) The centrifugation of native DNA from the various nuclear subfractions of the Chinese hamster was unremarkable. There was no shift in the density of the main band (fig. 15).

EUCHROMATIN

NUCLEI

HETEROCHROMATIN 8 NUCLEOLI

NUCLEOLI

Fig. 14. Analytical ultracentrifugation of DNA isolated from nuclear subfractions of the dog. There was a progressive increase in the amount of heavy shoulder components in heterochromatin + nucleoli and pure nucleoli DNA.

Curve resolving showeda small heavy shoulder component which increased slightly from 4 % in the whole nuclei to 8 % in nucleoli. Centrifugation of denatured and renatured DNA was unremarkable and showed no rapidly renaturing peak except in DNA from purified nucleoli where there was a small component constituting 3 % of the DNA (fig. 16). Fig. 13. Analytical ultracentrifugation of denatured and renatured DNA isolated from nuclear subfractions of Microtus aarestis. All fractions showed a small rapidly renatu&sg satellite peak and in addition, the main band DNA from the heterochromatin+ nucleoli and purified nucleoli showed a greater shift or more rapid rate of renaturation than main band DNA from whole nuclei or euchromatin.

Rem&ration kinetics of euchromatic and heterochromatic DNA

To further investigate the possibility that the heterochromatin in the Chinese hamster might contain a disproportionate amount of Exptl Cell Res 71

126 D. E. Comings & E. Mattoccia

Whole

Nuclei

Euchramotin

4 I

Heterochromatin + Nucleoli

I

labeled at various times during the S period. The DNA Tm of various samples was compared utilizing hydroxyapatite column chromatography. This technique allowed the DNA Tm of two different samples to be determined simultaneously and thus any differences could be accurately evaluated. In one pair, Chinese hamster DNA labeled from O-2 h of S had a DNA Tm of 88.9”C while that labeled at 4-6 h had a Tm of 87.7”C. In a second pair, mouse L cells labeled from O-2 h of S had a DNA Tm of 88.5”C, and that labeled 4-6 h of S had a Tm of 87.7”C. These results indicate that the early replicating DNA of the Chinese hamster and mouse is relatively GCrich compared with late replicating DNA. Heterochromatin staining

Nucleoli

The percentage of constitutive heterochromatin, stained by the technique of Arrighi & Hsu [l], was roughly quantitated with the Fig. 15. Analytical ultracentrifugationof DNA isolatedfrom nuclear subfractionsof the Chinese following results: mouse 12-l 6 %, horse hamster. lo-12 %, Chinese hamster 18-20 %, Microtus agrestis 18-24 %, human 10 %, quail 9-15 %, rapidly renaturing DNA the renaturation and guinea pig 18-20 %. The results of the kinetics of euchromatic and heterochromatic staining in the horse and the quail are shown DNA was studied by hydroxyapatite column in fig. 18. The quail was of particular interest chromatography. The renaturation of the since someheterochromatin wasfound around two was similar over a Cot [lo] of 1O-2to 1 the centromeres of most of the microchro(fig. 17), indicating that most of the heterochromatic DNA of the Chinese hamster did not contain a disproportionate amount of rapidly renaturing DNA. This is in agreement with the similar results based on studies of early and late replicating DNA [14]. This technique could easily miss a small amount (l-3 %) of highly repetitious DNA NUCLEOLI, D-R localized to constitutive heterochromatin and detectable by in situ hybridization. DNA Tm of early and late replicating

DNA

Chinese hamster and mouse L cells synchronized as described previously [14] were Exptl CeN Res 71

Fig. Id. Analytical ultracentrifugationof denatured and renaturedCliinesehamsterDNA from isolated nucleoli.Thereis a smallrapidly renaturingfraction whichis not presentin DNA from any of theother nuclearsubcomponents.

DNA of mammalian and avian heterochromatin

127

0.0 Fig. 17. Abscissfz: Cot (mole x set/l); ordinate: fraction absorbed on hydroxyapatite. 0, heterochromatin DNA; 0, euchromatin DNA. Renaturation kinetics of DNA isolated from purified heterochromatin and euchromatin from the Chinese hamster. The kinetics of the two DNAs were similar.

0.1

0.2

0.3 1 10-s

162

16'

100

quail, and to a slight extent in the Chinese hamster. Further evidence for the relationship between satellite DNA and heterochromatin comesfrom in situ hybridization experiments. DISCUSSION In the mouse [31, 431, Drosophila [27, 29, 32, On the basis of these and other observations 44, 461, Rynchoschiarra [21], Plethodon cineit is possible to suggest that several different reus [36], Xenopus [43], and Micro&s agrestis types of DNA may be localized to the con- [2], RNA synthesized from satellite or repetistitutive heterochromatin of eukaryotes. They tious DNA tends to hybridize to constitutive heterochromatin localized at the centromere may be classified as follows: I. Repetitious satellite DNA: (a) AT-rich, and other regions. Both nuclear subfractionation and in situ (b) GC-rich, (c) same density as main band hybridization studies have also shown that DNA. repetitious DNA seems to localize to the II. Repetitious main band DNA. III. Non-repetitious DNA: (a’) GC-rich chromatin associated with the nucleoli. In heavy shoulder DNA, (b) AT-rich main our studies on the mouse in which nucleoli were separated from the rapidly sedimenting band DNA. fraction, and extraneous DNA further reRepetitious satellite DNA moved by brief treatment with DNase or 2 Schildkraut & Maio [49] demonstrated a 2-3 M NaCI, there was a progressive increase in fold increase in satellite DNA in purified content of satellite DNA reaching a maximum mouse nucleoli and Yasmineh & Yunis [56] or 47 % [37]. This is probably a reflection of showed that the fraction from sonicated the fact that heterochromatin is intimately mouse nuclei that was rich in heterochromatin associated with the nucleolus [30] and these and nucleoli showed a significant increase in various treatments result in progressively satellite DNA. A similar enrichment has been greater and greater purification of that found in the guinea pig [60], calf [571, rat heterochromatin. kangaroo [40] and African Green Monkey The repetitious satellite DNA that has [35]. In the present studies an increase in the been localized to the heterochromatin may be amount of repetitious satellite DNA was either AT-rich [21, 27, 31, 37, 42, 56, 57, 601, found in the heterochromatin fractions of GC-rich [15, 36, 40, 48, 57, 691 or have mouse, guinea pig, horse, chicken, Japanese approximately the same buoyant density as

mosomes, while other parts of the microchromosomes were unstained. The results with the chicken were not adequate for quantitation.

Exptl Cell Res 71

128 D. E. Comings & E. Mattoccia

Fig. 18. Heterochromatin staining of horse chromosomes (a) and of quail chromosomes (b). It can be seen in the quail that the densely staining centromeric heterochromatin also occurs on the microchromosomes but that all the microchromosomes are not densely staiiied. a, x 3 280; b, x 5 000.

[7]. In some species a portion of these dispersed sequencesseem to be homologous to the clustered sequencesof satellite DNA [21, 251. If all main band repetitious DNA is evenly dispersed among non-repetitious sequences, one would not expect that it would be free to become localized in constitutive heterochromatin. In some species this appears to be the case. For example, in the Chinese hamster early replicating DNA contains essentially the same amount of repetitious Repetitious main band DNA DNA as late replicating DNA [14]. HowIn addition to satellite DNA a significant ever, there are some interesting exceptions. amount of repetitious DNA is dispersed In Drosophila, Gall et al. [27] found that the throughout the genome [7]. It can be detected chromocenter of the salivary gland chromoif it is freed from the non-repetitious fraction somes contained repetitious sequencesfrom by shearing the DNA into short pieces, dena- both the satellites and from the main band turing it, and separating the rapidly renaturing DNA. A second example is seen in Microtus material on hydroxyapatite. On the basis of agrestis. Yasmineh & Yunis [58] have shown its rate of renaturation it can be separated that in this species about 8 % of the DNA occurs as minor highly repetitive compointo highly and moderately repetitious DNA

main band DNA [I 11.In the latter caseit can be detected by centrifugation in Hg+ or AG+-CsSO, [16, 171, by centrifugation of rapidly renaturing DNA separated by hydroxyapatite [34], or by centrifugation of denatured and renatured DNA [16, 171. The latter technique was utilized extensively in the present study and showed the presence of hidden satellites in the chicken, Microtus agrestis, and a tiny satellite in nucleolar DNA of the Chinese hamster.

Exptl CeN Res 71

DNA of mammalian and avian heterochromatin

nents, and there is a main band component of intermediate repetitiousness. Arrighi et al. [2] demonstrated that the large blocks of constitutive heterochromatin on the sex chromosomesand at the centromeres are enriched in repetitious DNA. These observations are in agreement with the present findings of a rapidly renaturing peak constituting about 5 %, plus a larger main band component renaturing at an intermediate rate, which is markedly enriched in the heterochromatin and nucleoli fractions. These studies suggest the presenceof a moderately repetitious main band component enriched in constitutive heterochromatin. GC-rich, non-repetitious, heavy shoulder DNA

When the DNA from a number of organisms is examined by analytical ultracentrifugation it presents a non-gaussian curve which is skewed to the heavy side. This has been termed heavy shoulder DNA [lo]. It is particularly prominent in the chicken where it makes up 30 % of the total DNA. Renaturation studies indicate it does not contain an increased amount of repetitious DNA [lo, 151. In the chicken, where the microchromosomes are not heterochromatic, the heavy shoulder component is not enriched in the heterochromatic fractions. In the Japanese quail, which possessesheterochromatic microchromosomes [ 131there is a marked enrichment of heavy shoulder DNA from 20 % in whole nuclei to 60 % in the DNA from purified nucleoli [15]. A similar but less striking enrichment of heavy shoulder DNA in the heterochromatin and nucleolar fractions was noted in the present studies in the guinea pig and dog. These findings indicate that in some organisms the heterochromatic DNA is enriched in non-repetitious GC-rich DNA. 9-

721803

129

AT-rich, non-repetitious, main band DNA

Numerous studies, utilizing preparative ultracentrifugation in CsCl, have shown that early replicating main band DNA tends to be relatively GC-rich, and late replicating DNA tends to be AT-rich [4-6, 9, 23, 541. These conclusions, based on buoyant density of pulse labeled DNA from synchronized cultures have been confirmed by direct analysis of the GC content of BUdR labeled DNA [54]. Similar results were obtained in the present study by determining the DNA Tm of early and late replicating Chinese hamster and mouse DNA. There are two potential explanations for this observation. The first is that the heterochromatic DNA in these species tends to be AT-rich and the observed change represents a shift to heterochromatin replication late in the S phase [9]. An alternative explanation is that there is something about GC-rich replicons that predisposes them to replicate early and AT-rich ones to replicate late [5, 61. In favor of the idea that heterochromatin is relatively ATrich is the observation that nuclear subfractions in the mouse were progressively enriched in heterochromatin, and showed a progressive decrease in the buoyant density of the main band DNA [37]. A similar shift was seen in the horse (fig. 7). In the Chinese hamster, chicken, Microtus agrestis, and dog, a shift was not seen. The ease with which the cell synchrony-pulse labeling technique is able to demonstrate this shift, and the relative difficulty with which it is demonstrated in nuclear subfraction experiments is probably a reflection of two factors. (1) In cell synchronization studies the S period is from 8 to 10 h long, the duration of the pulse is 30 to 60 set, the degree of synchrony (at least with the mitotic selection technique in Chinese hamster cells) is excellent, and quantitation of the labeled DNA shows a distinct late S peak [33] probably representing Exptl Cell Res 71

130 D. E. Comings & E. Mattoccia predominantly heterochromatin replication. Thus with this technique it is quite likely that much more precise separation of euchromatic and heterochromatic DNA can be obtained, than is possible with the sonication of nuclei and differential centrifugation of the debris. (2) It is quite likely that the centromeric heterochromatin containing highly repetitious satellite DNA is more densely condensed [27] and thus relatively easy to isolate by nuclear fragmentation, while the heterochromatin that is enriched in AT-rich DNA is probably non-centromeric [II] and probably less compacted and more difficult to separate from euchromatin. Other evidence that the shift to AT-richness late in S is due to heterochromatin replication is presented in ref. [9]. Evidence that the AT-rich heterochromatic DNA is not unduly enriched in repetitious DNA comes from three observations. (1) Cot studies show that late replicating ATrich Chinese hamster DNA shows essentially the same renaturation kinetics as the early replicating GC-rich DNA [14]. (2) Centrifugation of denatured and partially renatured Chinese hamster DNA pulse labeled at various times throughout the S period gave no evidence for more extensive renaturation in late replicating than early replicating DNA [9]. (3) Following analytical ultracentrifugation of denatured and partially renatured DNA from the heterochromatin rich subfractions of the mouse there was no evidence of rapid renaturation of the AT-rich main band DNA (fig. 1). Relation between repetitious DNA and C-band staining

Pardue & Gall [43] noted that after chromosomeswere prepared for in situ hybridization the regions of centromeric hetetochromatin stained heavily with Giemsa. Arrighi & Hsu [l] have capitalized on this observation Exptl Cell Res 71

and developed a staining technique which preferentially stains the same regions which tend to show localization of repetitious DNA by in situ hybridization, This technique which usually stained predominantly the centromerit heterochromatin has been called the C-banding technique [22]. Modifications of this procedure (G-banding techniques [22]) show numerous additional bands in the arms of the chromosomes [20, 45, 50, 53, 591 which, in human chromosomes, with few exceptions correspond to the bands seen by quinacrine fluorescence [22]. In human cells these bands also tend to correlate well with regions of late replication [28]. There are thus numerous chromosomal segments that appear to possessconstitutive heterochromatin on the basis of late replication and by G and Q banding which do not stain by C-banding. It seems reasonable to suggest that there may be two general types of constitutive heterochromatin [ 1I] : centromeric heterochromatin which is enriched in repetitious satellite DNA, and intercalary [l l] heterochromatin which may be more enriched in the types of DNA discussed above which are not unusually repetitious. This has been discussedelsewhere [I 1, 121. Although there is a good correlation between the amount of satellite DNA and the amount of C-band staining material in mouse, human and Microtus agrestis cells, this does not hold as well in the Chinese hamster. We could find evidence for only a tiny amount of satellite DNA in the renatured nucleolar DNA of the Chinese hamster. Yet despite the apparent presence of less than 1 % satellite DNA, more than 10 % of the genome seemsresponsive to C-band staining. There are several possible explanations. (1) The stain may be sufficiently sensitive that it gives a positive result even if only a relatively small amount of highly repetitious DNA is present. (2) It may be detecting other

DNA of mammalian and avian heterochromatin

131

characteristics of this type of heterochro- 28. Ganner, E & Evans, H J. Submitted for publication. matin, or (3) it may be detecting a relatively 29. Hennig, W, Hennig, I & Stein, H, Chromosoma low level of repetition such as might be seen 3. 32 (1970) 31. Hsu, T C, Cooper, J E K, Mace, M L & Brinkley, with a few random repeats. B R, Chromosoma 34 (1971) 73. This work was supnorted by NIH grant no. GM-_ 15886.

REFERENCES 1. Arrighi, F E & Hsu, T C, Cytogenetics 10 (1971) 81. 2. Arrighi, F E, Hsu, T C, Saunders, P & Saunders, G F, Chromosoma 32 (1970) 224. 3. Arrighi, F E, Mandel, ‘M, Bergendahl, J & Hsu, C, Biochem genet 4 (1970) 367. 4. Bostock, C J & Prescott, D M, Exptl cell res 64 (1971) 267. 1 Exptl cell res 64 (1971) 481. 2. - J mol biol 60 (1971) 151. 7: Britten, R J & K&n< D E, Science 161 (1968) 529. 8. B&ten, R J & Smith, J, Carnegie inst Wash yearbook 68 (1969) 378. Comings. D E. Exntl cell res 71 (1972) 106. 1;: - Submitted for publication. 11. - Exptl cell res 67 (1971) 441. 12. - Advances in human genetics (ed H Harris & K Hirschhom) 3 (1972). In mess. E; Chromosoma 30 13. Comings, D E & M&c&a, (1970) 202. 14. - Proc natl acad sci US 67 (1970) 448. 15. - Exptl cell res 70 (1972) 256. 16. Comeo, G, Ginelli, E & Polli, E, J mol biol 33 (1968) 331. 17. - Biochemistrv 9 (1970) 1565. 18. Comeo, G, Ginelli; E, Soave, C & Bemardi, G, Biochemistrv 7 (1968) 4373. 19. Doty, P, McGill, B B & Rice, S A, Proc natl acad sci US 44 (1958) 432. 20. Drets, M E & Shaw, M W, Proc natl acad sci US 68 (1971) 2073. 21. Eckhardt, R A & Gall, J G, Chromosoma 32 (1971) 407. 22. IVth intemat conf on standardization in human cytogenetics, Paris, Sept 1971 (1971). 23. Flamm, W G, Bemheim, N J & Brubaker, P E, Exptl cell res 64 (1971) 97. 24. Flamm, W G, Bond, H E & Burr, H E, Biochim biophys acta 129 (1966) 310. 25. Flamm, W G, Walker, P M B & McCallum, M, J mol biol40 (1969) 423. 26. - J mol bio142 (1969) 441. 27. Gall, J G, Cohen, E H & Polan, M L, Chromosoma 33 (1971) 319.

31. Jones, K W, Nature 225 (1970) 912. 32. Jones, K W & Robertson, F W, Chromosoma 31 (1970) 331. Klevecz, R R, Science 166 (1969) 1536. 34. Laird, C D, Chromosoma 32 (1971) 378. 35. Maio, J J, J mol biol 56 (1971) 579. 36. Mcgregor, H C & Kezer, J, Chromosoma 33 (1971) 167. 37. Mattoccia, E & Comings, D E, Nature new biol 229 (1971) 175. 38. Mandel, M, Schildkraut, C L & Marmur, J, Methods in enzymology. Nucleic acids, part B, 12 (1968) 184. 39. Miyazawa, Y & Thomas, C A, J mol biol 11 (1965) 223. 40. Mazrimas, J A & Hatch, F T, Exptl cell res 63 (1970) 462. 41. Maramatsu, M, Smetana, K & Busch, H, Cant res 23 (1963) 510. 42. Pardue, M L & Gall, J G, Proc natl acad sci US 64 (1969) 600. - Science 168 (1970) 1356. Pardue, M L, Gerbi, S A, Eckhardt, R A & Gall, J G, Chromosoma 29 (1970) 268. 45. Patil, S R, Merrick, S & Lubs, H A, Science 173 (1971) 821. 46. Rae, P M, Proc natl acad sci US 67 (1970) 1018. 47. Rueckert, R R & Mueller, G C, Cant res 20 (1960) 1548. 48. Saunders, G F, Hsu, T C, Getz, M J, Simes, E L & Arrighi F E. Submitted. 49. Schildkraut, C L & Maio, J J, Biochim biophys acta 161 (1968) 76. 50. Schnedl, W, Chromosoma 34 (1971) 448. 51. Southern, E M, Nature 227 (1970) 794. 52. Stubblefield, E & Klevecz, R R, Exptl cell res 40 (1966) 660. 53. Summer, A T, Evans, H J & Buckland, R A, Nature new bio1232 (1971) 31. 54. Tobia, A M, Schildkraut, C L & Maio, J J, J mol biol 54 (1970) 449. 55. Wolf, U, Flinspach, G, Bohm, R & Ohno, S, Chromosoma 16 (1965) 609. 56. Yasmineh, W G & Yunis, J J, Exptl cell res 59 (1970) 69. 57. - Exptl cell res 64 (1971) 41. 58. - Biochem biophys res commun 43 (1971) 580. 59. Yunis, J J, Roldan, L & Yasmineh, W G, Nature new biol 231 (1971) 532. 60. Yunis, J J & Yasmineh, W G, Science 168 (1970) 263.

2

Received June 14, 1971 Revised version received December 6, 1971

Exptl Cell Res 71