Chromosoma (Berl.) 32, 378-406 (1971) 9 by Springer-Verlag 1971

Chromatid Structure : Relationship between DNA Content and Nucleotide Sequence Diversity CHARLES D. LAIRD Department of Zoology, University of Texas, Austin, Texas Received September 29, 1970 / Accepted October 8, 1970

Abstract. Models of chromatid structure are based on inferences made from genetic, cytological, and cytochemical observations. An alternative approach can provide limits as to the number of identical subunits present in chromatids. This method is based on the demonstration that nuc]eotide sequence diversity may be estimated from the kinetics of renaturation of denatured DNA. Measurements of DNA content and renaturation rate constants are given for several eukaryotic DNAs. Control experiments involved measurements of renaturation kinetics of DNAs from bacteria and bacteriophage. These estimates show that most of the nucleotide sequences in mouse, Drosophila, and Ciona DNA are present only once per sperm. Since the reduction of DNA content during meiosis indicates that mouse sperm contain a haploid set of chromatids, it follows that a set of mouse meiotic chromatids contains a single copy of most sequences. Models of chromatid structure which postulate multiple subunits with identical nucleotide sequences are therefore not tenable for mouse meiotic ehromatids. This method of analyzing nucleotide sequence diversity may be of general use in designing models of chromatid structure in other organisms. Introduction A n u n d e r s t a n d i n g of chromosome s t r u c t u r e d e p e n d s in p a r t on a resolution to t h e p r o b l e m of c h r o m a t i d strandedness. The unit chromarid model, b a s e d on s t r u c t u r a l d a t a , implies t h a t c h r o m a t i d s c o n t a i n each gene only once (see D u P r a w , 1965). The u n i t c h r o m a t i d is therefore n o t subdivisible into equal p a r t s , either in a genetic, i n f o r m a t i o n a l , or s t r u c t u r a l sense. W h i l e this m o d e l is consistent w i t h m u c h genetic segregation, m u t a t i o n a l , a n d cytological d a t a , bineme or m u l t i n e m e models of c h r o m a t i d s t r u c t u r e have been s u p p o r t e d b y a v a r i e t y of cytological a n d biochemical d a t a . F o r example, light m i c r o s c o p y of corn meiotic chromosomes (Maguire, 1968) a n d c o m p a r a t i v e D N A values of r e l a t e d species of Vicia (Wolfe a n d Martin, 1968) or a n e m o n e s (Rothfels et al., 1966) are consistent w i t h b i n e m e a n d m u l t i n e m e chromatids. S u p p o r t for m u l t i p l e s u b c h r o m a t i d s has also come from d a t a on D N A r e p l i c a t i o n in Chlamydomonas reinhardii (Sueoka et al., 1967). Cytological observations of s u b c h r o m a t i d s are n o t l i m i t e d to plants. S t o c k e r t (1969), for example, has r e p o r t e d t h a t h u m a n m i t o t i c ehro-

DNA Content and Nucleotide Sequence Diversity

379

matids often appear to contain subunits (see Wolff, 1969, and Prescott 1970, for a review of these and other data). The purpose of this communication is to present an alternative approach to the analysis of chromatid structure. This approach is based on measurements of renaturation M~etics of denatured DNA. Such rates depend in part on the numbers of different nueleotide sequences present in a DNA preparation (Marmur and Dory, 1961; Britten and Kohne, 1967; Wetmur and Davidson, 1968; Gillis et al., 1970). Although analysis of sequence diversity is relatively direct for DNAs from mitoehondria, virus, and bacteria (Britten and Kohne, 1968; Wells and Birnstiel, 1969; Kingsbury, 1969; tIollenberg, Borst, and van Bruggen, 1970; Gillis et al., 1970), DNAs from m a n y eukaryotic organisms do not follow the expected second-order renaturation kinetics. DNA which renatures more rapidly than expected m a y represent" repeated nucleotide sequences", i.e., m a n y copies of very similar sequences (Britten and Kohne, 1968). Attention will be focused here on the non-repeated sequences, i.e., those molecules in a population of DNA fragments which are slowest to renature and which follow second-order renaturation kinetics. Reassoeiation rates of such fragments provide information about the number of different nucleotide sequences in DNAs of eukaryotes. These data m a y then be related to the amount of DNA contained in a haploid set of chromatids. The relationship between these two values places limits on the extent to which identical subunits m a y exist in ehromatids. Results and D i s c u s s i o n 1

1. Unit Genome: A De/inition A unit genome m a y be defined as the chromosomal DNA of a cell such t h a t the least frequent nuc]eotide sequences are present only once, and such t h a t all nucleotide sequences are represented in their original proportions (Bultmann, personal communication). For example, Escherichia coli is thought to have a unit genome consisting of a circular molecule of DNA about 2.6 • 109 daltons in size, or about 4 • 106 nucleotide pMrs (Cairns, 1963). This amount of DNA is sufficient for perhaps 4000 cistrons. Implicit is the assumption that nueleotide sequences of most of these cistrons are different from all others in the E. coli chromosome. If this assumption is correct, then a unit genome and a haploid genome are equivalent in E. coll. To make the concept of unit genome more biologically meaningful, it is useful to designate an arbitrary nueleotide sequence length. This avoids the ease of short polynucleotides (trinucleotides, for example) which are present m a n y times in a haploid cell. Fragments of about 1 Materials and Methods see Appendix, p. 396-404.

380

C.D. Laird:

400 nucleotides were used in the experiments reported here (see Materials and Methods), since a homogeneous population of small fragments is necessary for precise interpretation of kinetic data (Wetmur and Davidson, 1968) and to reduce viscosity problems. Such pieces contain sufficient information to specify a polypeptide of about 130 amino acids. For these reasons the term "nucleotide sequences" as used here will refer to sequences of about 400 nucleotides. This arbitrary usage is not intended to imply a precise relationship between gene, a term with functional connotations, and nucleotide sequences in fragments of DNA. It is instead art attempt to consider, independently of DNA function, the quantitative arrangement of nucleotide sequences in chromatids. Returning to the question of ploidy among prokaryotic organisms, one can say with some certainty that for the bacteriophages 2, T2, and T4, the unit and haploid genomes are equivalent. The amount of DNA in a single T4 phage, for example, represents about 2 • 10 a base pairs, or perhaps 200 cistrons (Tomizawa and Anraku, 1965; Thomas, 1966). Physical studies on the ability of fragments of T4 DNA to form circles (see Thomas, 1966) and analysis of polynucleotides of deletion mutants of T 4 (Mazaitis and Bautz, 1967) provide strong evidence that the T4 equivalent of DNA (130 • 106 daltons) represents a collection of nucleotide sequences which are represented but once in the T4 DNA molecule. (Terminally redundant sequences, which comprise 1-3 % of the T4 genome, are exceptions to this generalization). 2. D N A Content

Precise information is required concerning the amount of DNA per cell in different organisms. This information may then be related to nucleotide sequence diversity to determine the degree of ploidy at the informational level. This section summarizes published data and presents some confirmatory experiments on the DNA content of cells of several prokaryotie and eukaryotie organisms. a) Prokaryotes DNA contents of bacteria and bacteriophage have been estimated using a variety of methods. Contour lengths of DNA molecules as visualized in electron micrographs are in agreement with the amount of DNA per cell when phage or bacterial "chromosomes" are single molecules of DNA (see Thomas, 1966). These data, summarized in the Table, indicate that T4, Bacillus subtilis, and E. coli contain 1.3 • l0 s daltons DNA/phage (Tomizawa and Anraku, 1965; Thomas, 1966); 2.1 X 109 daltons DNA/nuelear body (see Gillis et al., 1970) ; and 2.6 • 109 daltons DNA/circular chromosome (Cairns, 1963).

DNA Content and Nucleotide Sequence Diversity

381

b) Eukaryotes The DNA content of Ciona intestinalis (sea squirt) was estimated, by comparative microphotometry of Feulgen-stained cells, to be 6% of h u m a n cells (Atkin and Ohno, 1967). Although Ciona epithelial cells were measured, I will assume the DNA content of Ciona sperm to be 9 x 10~~ daltons, i.e., one-half t h a t of epithelial cells. Microphotometric estimates b y Kurniek and Herskowitz (1952) of diploid Drosophila melanogaster cells suggest that sperm of D. melanogaster contain between 5 and l0 x 10 l~ daltons of DNA. These measurements have been repeated and extended to sperm using more sensitive methods (Rasch, Burr, and Rasch, 1970). Rasch et al. conclude that D. melanogaster sperm contain 11.3 X 10 l~ daltons of DNA, assuming that chicken erythrocytes, their standard reference cells, contain 2.5 pg DNA. This value for chicken diploid cells is about 20% higher than that reported by Atkia et al. (1965), who found that chicken erythrocytes had 30-34% as much DNA as mouse erythrocytes. I will use this latter value of 2.0 pg DNA for chicken erythrocytes to arrive at an estimate of 9.0 x 10 l~ daltons (0.14 pg) DNA for Drosophila melanogaster sperm based on the comparative data of l~asch et al. (1970). An accurate estimate of the DNA content of mouse meiotic cells is especially crucial for the experiments reported here. Mammaliau sperm contain about 1.8 X l01~ daltons (3 pg) DNA (see Sober, 1968). Sperm from mouse strain C57 Black/6 J, the source of DNA used in the experiments reported here, also contain 3.0 pg DNA, as measured by the modified diphenylamine assay for deoxyribose (Burton, 1956) using deoxyadenosine and salmon sperm DNA as standards. The DNA content of other mouse meiotic cells is equally important to the argument. From the reduction of chromatid number per cell during meiosis, from 4N to 157 (see Wilson, ]934; Griffen, 1966), it was anticipated that DNA content was also reduced by four (from 4C to 1 C) in sperm relative to primary spermatocytes. This was verified by Swift (1950), who measured relative DNA content of mouse cells. Because of the importance of relating the relative DNA content of mouse cells to a standard with known DNA content, an experiment comparable to that of Swift (1950) was carried out (Fig. 1). Mierophotometric measurements of Feulgen-stained mouse meiotic cells confirm the 4-fold reduction in DNA during formation of sperm from primary spermatoeytes. Two additional controls were used to relate these relative Feulgen values to absolute amount of DNA per cell. The Feulgen measurements for diploid erythroeytes from the house sparrow (Passer domesticus) erythrocytes are about 70% that observed for mouse sperm cells (Fig. 1), in agreement with DNA content of sparrow erythrocytes of 1.9 pg DNA (Vendreley, 1952). The ratio of DNA content of mouse sperm (3.0 pg) to sparrow erythrocytes (1.9 pg)

C.D. Laird:

382

Mean 60C E m

50C

9

Pg DNA

650

12,2

300

5.6

O

>, 40C o

O 30G

O 20C D_

160

5.O mouse sperm

105

1.9 sparrow

Z s

o ]OC

t

t

]C

2C

t 4C

erythrocyle

Fig. 1. Relative D2VA content o/mouse meiotic cells. Cells from homogenized mouse testes were stained with Feulgen reagent for dual-wave-length microspectrophotometric measurements, as described in the text. Values for different cell types clustered about means of 160 (sperm and spermatids, corresponding to 3.0 pg DNA) ; 300 (spermatogonial and secondary spermatocytes), and 650 units (primary spermatocytes). No meiotic cells with absorption values significantly greater than 4 times that in spermatids were observed. As an additional control, a sparrow blood sample was processed in parallel with the mouse tissue. A mean Feulgen value of 105 was observed for the diploid erythrocytes, as expected for cells with 1.9 pg DNA

is 1.6, compared to the ratio of 1.7 observed with Feulgen staining (Fig. 1). This close correspondence b e t w e e n relative values obtained b y Feulgen staining and the a m o u n t of D N A per cell, as determined b y other chemical methods, lends confidence to the conclusion that D N A content has been reduced by four during the formation of mouse sperm from primary spermatoeytes. The observation that ehromatid number in mouse meiotic cells is also reduced by four, during formation of sperm from primary spermatocytes, implies that no substantial D N A synthesis has occurred during the mouse meiotic divisions. The chromatids as seen in the tetrad of meiosis I are therefore comparable in D N A content to the chromatids being distributed to spermatids at anaphase II. It follows, from the relative D N A measurements, that a haploid set of ehromatids present in mouse spermatids ( n = 2 0 ) contains a sperm equivalent of D N A , n a m e l y 1.8 • 1012 daltons. This conclusion is neither surprising, from genetic segregation data, nor original and unique for mouse meiosis. I emphasize it here to point out that if it can be s h o w a that a mouse

DNA Content and Nueleotide Sequence Diversity

383

sperm contains a unit genome, then it must follow that a haploid set of mouse meiotic ehromatids also contains a unit genome. The evidence establishing this point is presented in the next section.

3. Renaturation Kinetics o / D N A Rates at which the separated strands of DNA reassociate are influenced by salt concentration, base composition, size of DNA fragments, reaction temperatures, as well as the intrinsic nucleotide sequence diversity and concentration of the DNA (Murmur and Dory, 1961; Wetmur and Davidson, 1968). Data presented in Materials and Methods section of this paper illustrate some of these relationships for the experimental approach described here. To minimize the effects of parameters not related to sequence diversity, rates were measured b y mixing denatured DNAs from unrelated organisms and analyzing, b y hydroxylapatite column chromatography, the extent of renaturation of each DNA as a function of time. Renaturation rates of differentially radio-labeled DNAs of similar base composition and size, renaturing in a common solvent, are sufficiently reproducible to distinguish between chromatid uninemy or binemy, which depends on detecting a two fold difference in renaturatioa rate. (This "internal s t a n d a r d " method has also been used b y Britten and Kohne, 1967, and Davidson and Hough, 1969, for renaturation of DNA). a) Control DNAs The purpose of this section is to validate the nse of bacterial or phage DNAs as internal standards for studying solution renaturation of DNAs from eukaryotes. This depends on showing firstly that the renaturation of bacterial DNA is not affected by a high concentration of heterologous DNA, and secondly that the internal standard method gives consistent results when applied to other bacterial or phage DNAs. Renaturation rates of DNAs from T4 and Bacillus subtilis (Fig. 2a), and Bacillus subtilis and Escherichia coli (Fig. 2b) indicate t h a t Cot1/2 values are proportional to the amount of DNA per single phage particle or nuclear body. (Cotl/9, is the product of the initial concentration of denatured DNA expressed as molarity of nueleotides, and time in seconds at 50% renaturation, Britten and Kohne, 1968). These data, corrected for the effect of base composition on renaturation rate (see p. 404, Fig. 11) are summarized in the Table (p. 386). They confirm the expected inverse proportionality between rate of renaturation and amount of DNA per phage or nuclear body (Murmur and Dory, 1961; Wetmur and Davidson, 1968; Britten and Kohne, 1968). This relationship is predictable since renaturation of DNA depends on the concentration of

384

C.D. L a i r d :

[

100

'

' ~ IIIIII

' ~IIllll

'

r ~illll

' ~lJllll

~ . ~ ---0~ ~.0.~

<~ Z

a

'

\4

80

~B.

~

\

subtH/s

\ T4\ E (1) X~

\

\o \

\

\

\

b

X

\

~\

m 20 i

r J I llll[

,

r

Illllll

'

,

~ I llllll

'

tllllll

'

J IIIIII

IIII

,

10~

10-1 '

\xo

101 '

~

J

Illl

IIIlll

100 o

<[

~

8o

2

6O

"~'N.

o

o A

zZ

\

--B. sub/ills 0 c-

E co//' ~ \

22J

x2

"%

\

~d 4O o

~ 20 b , , i iiIrJi , p iiil11[ , 10-1 10~ '

IIIlllJ

'

~Jll[ll

r l iitlll ld '

r

illllJ

, I ITII '

'

Jillll

tO0 ~g/ml

<~

;57

-

2

6O

~f-

40

0 c n3

G)

a_ 20 -C ~ i I IIrli]

10-1

, r i llllll

, ~

10~ Cot(mote sec/Uter)

r[

101

Renaturation kinetics o] bacterial and bacteriophage D N A s a8 measured by hydroxylapatite chromatography, a H 3 - 1 a b e l e d Bacillus subtilis D h T A ( 3 0 0 c p m / Fig. 2a--c.

Fg) a n d C 1 4 - ] a b e l e d T 4 D N A ( 8 2 0 c p m / F g ) w e r e m i x e d i n 0 . 1 2 M P B t o g i v e f i n a l DNA concentrations of 7 0 t z g / m l a n d 6 t~g/ml, r e s p e c t i v e l y . Each DNA had been

DNA Content and Nucleotide Sequence Diversity

385

c o m p l e m e n t a r y sequences. F o r example, suppose t h a t fragments of T 4 a n d B. subtilis DNAs were present at equal c o n c e n t r a t i o n i n terms of nueleotides per u n i t volume. T h e n each different T 4 nucleotide sequence would be present at a higher relative c o n c e n t r a t i o n t h a n would be each B. 8ubtilis sequence, since B. subtilis has 16 times more D N A per cell t h a n does T4. As expected, DNAs from B. subtilis a n d E . coli r e n a t u r e d 14 a n d 20 times more slowly, respectively, t h a n D N A from phage T 4 (Fig. 2 a a n d b, a n d the Table). Of especial i m p o r t a n c e is the use of DNAs, from u n r e l a t e d organisms, m i x e d together during the r e n a t u r a t i o n procedure. A t t e m p e r a t u r e s 25 ~ below the Tm of the DNAs (midpoint of the t h e r m a l dissociation) each c o m p o n e n t r e n a t u r e d i n d e p e n d e n t l y . For example, H~-thymidine-labeled D N A from B.subtilis was half-renatured when Cotl/~ = 1.7 :~ 0.1 (mole-see/ liter J: 2a, normalized to 50% g u a n i n e + c y t o s i n e , as described in the appendix). This value was also o b t a i n e d when B. 8ubtilis D N A was r e n a t u r e d at concentrations ranging from 5-70 ~g/ml i n the presence of u n l a b e l e d heterologous DNAs from mouse (1500 ~g/ml), bee (1500 ~g/ ml), cockroach (900 ~g/ml), or T 4 (6 ~tg/ml) (Fig. 2c). Such i n d e p e n d e n t

sheared previously by passage through a pressure cell at 12000 p.s.i. The mixture was heat-denatured at 100~ for 10 min, and incubated at 60~ C. At various times, 0.1 ml aliquots were diluted into 0.9 ml of 0.12M PB and frozen for subsequent hydroxylapatite chromatography. Cot values were calculated (Britten and Kohne, 1968) on the basis of either the concentration of T4 DNA (molarity of nucleotides and time in seconds) or on the basis of the concentration of B. subtilis DNA. The dashed lines are theoretical second-order curves, C/C o = 1/(1 + k2Cot) where Co is the initial concentration of denatured DNA. Values of ks used to place the curve are 0.52 (M sec)-1 for 17. subtilis DNA (0) and 4.5 (3/[ sec)-1 for T4 DNA (A). b HMabeled E. coli DNA (4600 cpm/~g) and C14-1abeled Bacillus subtilis DNA (820 epm/tzg) were mixed in 0.12 M PB at concentrations of 60 and 20 ~g/ml, respectively; denatured by heating to 105~ C for 10 rain; and renatured at 63~ C. Aliquots were removed at various times and the percent denatured DNA was determined by hydroxylapatite chromatography, as described in Materials and Methods. The rate constant for the second-order rate curve is 0.58 (3/[see)-~. c Renaturation of Bacillus subtilis DNA at different concentrations and in the presence of heterologous DNAs. Results of 4 separate experiments are superimposed in this figure: 9 Ha-labeled B. subtilis DNA (21000 epm/~g, 5 tzg/ml) plus mouse DNA (1540 ~g/ml). 9 Cl~-labeled B. subtilis DNA (820 epm/~g, 7 tzg/ml) plus 1500~g/ml bee (Apis mellifica) DNA. zx C14-1abeled B. subtilis DNA (820epm/~g, 24 ~xg/ml) plus 902 ~g/ml cockroach (Byrsotria ]umigata) DNA. o Ha-labeled B. subtilis DNA (300 epm/vg, 70 tzg/ml) plus T4 DNA (6 ~zg/ml). All DNAs were sheared at 12000 p.s.i. Each mixture was heat denatured and allowed to renature at 60~ C. Samples were assayed on hydroxylapatite columns. Only the B. subtilis DNA renaturation kinetics are shown in Fig. 2 c to illustrate the reproducibility of these measurements in the presence of varying concentrations of homologous and heterologous DNAs

60

2/1us musculus (40)

(3)

1733 ~ 94

(3)

65 • 13

(1)

90

(4)

1.8 • 0.4

1.7 ~= 0.1 (16)

(5)

0.17 • 0.05

1282 ~ 69

59 =J_ 12

63

1.8 • 0.4

1.4 ~= 0.08

0.10 ~ 0.03

712

32.8

35.0

1.0

0.78

0.055

E. coli

relative to

1.85 X l 0 t2

8.5 X 10 l~

9.1 x l01~

2.6 x 109

2.0 x l09

1.43 • l 0 s

unit genome (kinetic in da]tons) c

1.8 • l012

9.0 x 10 l~

9.0 x 10 l~

2.6 x 109

2.1 X 109

1.3 x l 0 s

daltons

2.7 X 109

1.4 x l 0 s

1.4 x l 0 s

4.0 • 106

3.2 X 106

2.0 x 10 s

base pairs

haploid genome ( a m o u n t of D N A ) a

E s t i m a t e of m i n i m u m g e n o m e size

a Cotv ~ is e x p r e s s e d in ( m o l e - s e e / l i t e r ) • 2a. T h e figure in p a r e n t h e s e s r e p r e s e n t s t h e n u m b e r of i n d e p e n d e n t d e t e r m i n a t i o n s of r e n a t u r a t i o n r a t e s as m e a s u r e d b y h y d r o x y l a p a t i t e c h r o m a t o g r a p h y . b See Fig. 11. c T h i s e s t i m a t e is b a s e d o n size of t h e E. cell g e n o m e , 2.6 • 10 ~ d a l t o n s (Cairns, 1963). d R e f e r e n c e s for t h e s e v a l u e s a r e g i v e n in t h e t e x t .

85

Drosophila melanogaster (43)

> 98

Escherichia coli (50)

70

> 98

Bacillus subtilis (43.5)

Ciona iutestinalis (38)

> 98

corrected for r e p e a t e d sequences

unique

c o r r e c t e d to 50 % G + C base eomposition ~

C9tl/2 (mole-see/liter) a

%

T 4 (34)

D N A (% G + C )

Table. Comparison o/the kinetic complexity o / D N A and amount o / D N A per cell (haploid)

~

9:

Oo

DNA Content and Nueleotide Sequence Diversity |

I

_

387

10 o

b:

G

c o O9

=

,o 3

o

10-~ g

9

z K3

z

~s s

O o

@

10~

10-2~s

._~

9

~z

0

a

o

10~--

1@-3~ C3

o

g

9

9

c) <1

r

2

I

Illll

5

T

10

2

r

r

cd

[

5

10

Cob/2(: l/k2) of Baci//us sub////s DNA renaturing in the presence of h e i e r o l o g o u s DNA

Fig. 3. Bacillus subtilis D N A renatures independently o~ heterologous D N A s /rom unrelated organisms. Experiments similar to those described in Fig. 2 are summarized here. Three different 17. subtilis DNAs, eight different DNAs from eukaryotes, one bacterial DNA (E. coli), and one bacteriophage DNA (T4) were used. I n P a r t A, the reciprocal of the second-order rate constant for Bacillus subtilis DNA renat u r a t i o n is plotted versus the concentration of heterologous DNA. All values of 1/k 2 fall between 1.5 and 2.0 mole-sec/liter indicating little effect of heterologous DNA on renaturation of B. subtilis DNA, under these conditions of salt concentration, temperature, and polynucleotide fragment size. These data are expressed in P a r t B as a function of the fraction of B. subtilis DNA in the total mixture

renaturation of B. 8ubtilis DNA is expected, since the heterologous DNAs are from organisms so distantly related that no cross-reaction should be observed under restrictive salt and temperature cortditions. (Under these conditions, large fragments of DNA with less than about 60 to 70% base sequence homology do not form stable duplexes and would therefore not reassociate, see Laird et al. 1969.) However, it was not clear at what point high concentrations of DNA would affect renaturation due to viscosity and ionic changes. Data presented in Fig. 3 indicate that with the fragments of DNA used in these experiments, and at the salt and temperature conditions employed [0.12 M sodium phosphate buffer (PB), p i t 6.8, and 62 ~ C], B. subtilis DNA renatures

388

C.D. Laird: '

100

' --

*lll[ll

'

'

: Jllll[

'

: al/lll[

'

= I IsflJI

'

,

IJig

~-.

~-1--.~

9 __ ~ ' ~

<~

"~ 2nd order kmehcs ~ assuming 7 0 % unique

o 80-0

40

n 20

\ \ O

\ \

,

I

ill[Ill

,

~llillll

10"1 '

100 ~

+L+JIIJ

,

II;llll

,"?,

10~ '

'

=+llllj

IIII

illllll

101 '

~

Z '~ ~ o 80

,

'

IlJllll

102 '

'

JJlllll

'

J

illl

" ~ .

~

o\..

85 % unique

"\

"13

--

60

\

melanogaster ~

"\\

40 n 20

% \

b ,

~ rllilll

,

10~

;

itlil[1

,

,

%-...

LII]III

10~ 101 Cot(mole sec/titer)

~.~..

, Illllll

,

t IIIlll

102

Fig. 4 a - d . Renaturation #inetics o/ D N A s ~tom eu#aryotes, a Renaturation o/tunicat D N A . Ha-labeled E. coli I)NA (3300 cpm/b~g, 5 [,g/ml) was mixed with unlabeled DNA (470 Fg/m]) from the sea squirt, Ciona intestinalis. The mixture was denatured a t 100 ~ C, and allowed to renature at 61 ~ C. Aliquots containing 50 ~g of Cioua DNA were assayed on hydroxylapatite columns. The dashed line represents the theoretical second-order kinetic curve which best fits the Ciona data, assuming 70% non-repeated sequences. The rate constant for the second-order curve is 0.016 (M see) -1. b Renaturation #inetics o/ D N A ~tom Drosophila melanogaster. Unlabeled D N A /rom Drosophila melanogaster pupae and Ha-labeled DNA B. subtilis (21000 cpm/b~g ) were mixed in 0.12 M P B a t final DNA concentrations of 600 ~zg/ml and 7.7 Fg/ml, respectively. 0.1 aliquots were analyzed by hydroxylapatite chromatography. The dashed curves are theoretical second-order rates (/cz = 0.63 M sec) -1 for B. subtilis; the v a l u e / ~ = 0.014 (M sec) -1 for the Drosophila DNA assumes 85 % nonrepeated sequences), c Renaturation lcinetics o~ mouse D N A . Unlabeled I)NA from mouse spleen was mixed with Cl~-labeled T4 DNA (870 cpm/Fg) a n d Ha-labeled B. 8ubtilis DNA (21000 cpm/Fg ) in 0.12 5/[ P B a t final concentrations of 1540 Fg/ml, 8.9 Fg/ml a n d 5 Fg/ml, respectively. Aliquots (50 bd)

DNA Content and R~ucleotide Sequence Diversity ,

,

i

llllll

,

,

i

tl~lll

'

'

'

'llllH

' htlllll

'

""~''~'~.

\

389

' '~11111

'

~ JlJl/J

6 0 % unique mouse ~" \ DNA

k k \

mouse

\

A

\ X

\

\

\

NT4

k

O \ B. subli/is \

N c t

I I IIll

10-1 IJ]IH

I IIIIIII

,""7zZlllIIzZA,

10~ '

t IIII1[[

101

~ IIl[ll

'

,

,

,IJll]l

'lalllll

' ,llllll

' 'llilli

(total)

c,

I IIIII

\

A"~

'X

-'X \,,:

Boc/llus \

'~

\

M ou s e ~ " ' - ~

\

IIIII

Mouse " [ 3 \ (unique)

9 \

I

103

~

\

,

10 2

_

sublil/~;

,,% O

d J IIIII

10"I

,

t IlIIIll

,

lo o

~ Itl[lll

I

lO1

i illllll 102

, i ~llllll , 103

i

r I[l[ll

Cot (mote sec/titer) were analyzed by hydroxylapatite chromatography. Dashed lines are theoretical second-order reaction curves (k 2 ~ 6.2 (M sec) -I for T4; 0.5 (IV[ see) -I for B. subtilis; and 0.0005 (Msec) -I for mouse, assuming mouse DNA contains 60% nonrepeated sequences, d Renaturation o/ non-repeated sequences o/ mouse JDNA. HS-labeled DNA (210000 cpm/~g) from mouse L-cells was ffactionated to remove repeated sequences by renaturation to a Cot value of 250 mole-sec/liter, followed b y hydroxylapatite chromatography at 60~ in 0.12M PB. The unrenatured sequences which passed through the columnn a t 0.12 M P B were recovered, and added to a mixture of unlabeled DNA from mouse testes, and Cla-labeled B. subtilis (880 cpm/Fg ). Final DNA concentrations for the renaturation experiment summarized in Fig. 4 d were 1.3 Fg/ml (Ha-labeled mouse non-repeated DNA); 1735 ~g/ml (unlabeled mouse DNA); and 15.2 Fg/ml (B. subtilis). The mixture was heat denatured, and allowed to renature at 60 ~ C. Aliquots (0.05 ml) were analyzed b y hydroxylapatite chromatography. Dashed lines represent theoretical second-order reaction curves, where ]c2 is 0.67 (M sec) -1 for B. subtilis DNA and 0.00056 (M sec) -~ for unique mouse DNA

390

C.D. Laird:

independently of heterologous DS~As present at concentrations up to approximately 3 mg/ml. Cot values for B. subtilis DNA at half renaturation (=l/k2) ranged from 1.5 to 2.0 mole-see/liter when heterologous DNA concentrations varied from 5 to 2800 ~g/ml (Fig. 3a). These same data, plotted in a different manner, show that comparable renaturation rates were observed for B. subtilis DNA when heterologous DNA was present at levels ranging from 1-99.7% of the total DNA (Fig. 3b). Translation of these data into unit genome estimates requires the assumption that for one test organism, the unit and haploid genomes are equivalent. T4 has been used as this standard, on the assumption, justified above, that a T4 phage equivalent of 2 X 105 base pairs (1.3 X l0 s daltous) of DNA contains but once each different sequence 400 nucleotides in length. Based on this assumption for T4, data for B. subtilis and E. coli DNAs suggest that these organisms have unit genomes of 2.9 X 106 and 3.9 X 106 base pairs, respectively (Table, p. 386). Gillis et al. (1970), using an optical assay of renaturation rates, also estimate that the B. subtilis genome is somewhat smaller than that of E. coli. These estimates are within 15% of the published DNA values, suggesting t h a t B. subtilis and E. coli nuclear bodies contain a single copy of each different nucleotide sequence or gene. These estimates support the use of internal DNA standards for measurements of renaturation kinetics. b) DNAs from Eukaryotes Multicellular eukaryotes have a DNA content in gametes which is, in general, several hundred fold greater than that present in bacteria. Two interesting exceptions are tunieates (Atkin and Ohno, 1967) and Drosophila (Kurniek and tterskowitz, 1952). Ciona intestinalis and Drosophila melanogaster have about 30 to 35 times as much DNA per sperm as is present in the E. coli haploid genome. In. keeping with this greater DNA content per cell, DNAs from Drosophila and Ciona renature 33 and 35 times more slowly than does DNA from E. coli (Fig. 4a and b, Table). Cot1~2 values for non-repeated sequences indicate that the unit genome sizes for Drosophila melanogaster and Ciona intestinalis are 8.5 • l01~ daltons and 9.1 • 10 I~ daltons, respectively. This value for Drosophila compares well with the estimate of 7 • 10 l~ daltons obtained b y optical renaturation measurements (Laird and McCarthy, 1969). I t appears that for Drosophila and Ciona, unit genomes and haploid genomes are equivalent as is the case for T4, B. subtilis, and E. coli. I n the experiments just described for Drosophila and Ciona DNAs, data for non-repeated nucleotide sequences have been used to calculate unit genome sizes. This is necessary because the basis of the inference that repeated sequences are present in eukaryotic DNA was that such

DNA Content and Nueleotide Sequence Diversity

391

DNAs renature more rapidly than expected (Britten and Kohne, 1968). This fast-renaturing fraction represents about 15% of the Drosophila DNA and about 30% of the Ciona DNA when renaturation rates of 400 nucleotide fragments are measured by hydroxylapatite chromatography using reassoeiation criteria of 62 ~ C and 0.12 M PB (Fig. 4a and b, Table, p. 386). The dashed lines in Fig. 4 are second-order rate curves, where C/Co • 100 (percentage denatured DNA, where Co represents the concentration of denatured DNA at time = 0) is plotted vs. Cot. A rate constant, /~2, is estimated for the non-repeated sequences by a curve fitting procedure described previously (Laird and McCarthy, 1969). The equation, C/Co=I/(1 4-keCot), describes the dashed line. If data for un/ractionated DNA are used to estimate ~2, then this value represents the rate eonstant which would be obtained if all the sequences in the population of molecules were non-repeated. Although there are other equivalent methods to determine these values, 1 have chosen this method to include repeated sequences in the estimate of unit genome size. I n the data already presented, then, unit genomes are equivalent to haploid genomes. The crucial question, as far as mouse DNA is concerned, is whether the mouse sperm equivalent of DNA, 1.8 • 101~ daltons, represents a unit mouse genome. As discussed in the previous section, mouse sperm contain about 700 times more DNA than an E. coli unit genome. If the DNA in a mouse sperm represents a unit mouse genome, then mouse DNA (again, using rate estimates of reassociation of the non-repeated sequences) should renature 700 times more slowly than E. coli DNA, and 14000 times more slowly than T4 DNA. On the other hand, if a mouse sperm contains two of each nueleotide sequence, then mouse DNA should renature 350 times more slowly than DNA from

E. coll. The data support the former alternative, showing that mouse DNA renatures about 900 times more slowly than DNA from B. subtilis and 9000 times more slowly than DNA from T4 (Fig. 4c and d). When these data are corrected for the effect of base composition, the rate constant for mouse DNA is 712 times less than that for E. coli DNA (Table, p. 386). This value clearly supports the conclusion that mouse sperm contain a unit genome, i.e., each non-repeated sequence is present once per sperm. The experiment shown in Fig. 4 e utilized u~raetionated mouse DNA. The dashed line represents second-order renaturation kinetics for the final 60 % renaturation of the mouse DNA. The observed Cot,!2 of 1900 for the second-order curve represents the value which would be obtained if all the mouse sequences were non-repeated. To verify that this value is correct, non-repeated mouse sequences were prepared by incubating H a27

C h r o m o s o m a (tlerl.) ]34. 32

392

C.D. Laird:

labeled sheared and denatured DNA from mouse L-cells to a Cot of 250 mole-see/liter. About 40% of the labeled DNA bound to an hydroxylapatite column under these conditions. This represents the fast renaturing DNA thought to contain repeated sequences (Britten and Kohne, 1968). The urtrenatured DNA, recovered in the column eluent at 0.12 M PB, was mixed and renatured with a hundred-fold excess of sheared, denatured DNA from mouse testes (1100 ag/ml). A low concentration of C~
393

D N A Content and Nucleotide Sequence Diversity 1 0 I~

~

' , Iir1111

i J ,l/ill

'

I

[lllll/prirnary'epe~mat'ocyte'(4C)

mouse
spermatocyte

o '

' l Jllll

(2C) o /

--

sperm t i C ) / E c o

._c

~Ciono

/?test/no/is Drosophila me/onogoster

i011

E

1=

o

10~c

F

u Ld Q_ <

Bacillussubt/l/~.~7"Escherich/a col/

iO 9

g I0 s

i~rlllr

IO i

, ,Jpllllr

10 0

, ,rllllll

~ r rlrFIII

10~ l / k z (= Cotm, J

i0 a

, ,lllllll

, ,~llll/ 10 3

Fig. 5. Correlation between #inetic and analytical complexity o / D N A . Duta in the Table (p. 386) indicate that the reciprocal of the second-order rate constant for non-repeated D N A is proportional to the experimentally determined amount of D N A per cell. D N A contents of T 4 bacteriophages (Tomizawa and Anraku, 1965), E. coli (Cairns, 1963), and B. subtilis (see Gillis et al., 1970) are thought to represent haploid and unit genome values. These values fix the slope and placement of the straight line (see text). The estimate of D N A content of Ciona intestinalis is one-half that measured in epithelial cells (Atkin and Ohno, 1967) which are thought to be 2C. The vulue of 9.0 • 101~ daltons for Drosophila melanogaster is taken from the I C estimate of Rasch, Burr, and Rasch (1970). For mouse cells, the D N A contents of sperm and other meiotic cells (see Fig. 1) are plotted for comparison. Rate constants for D N A from mouse liver, spleen, and testes have been averaged. Horizontal lines about each point indicate 95% confidence limits ( ~ 2a) for rate constant determinations. All rate constants have been corrected for effects of base composition on renaturution kinetics (Table, p. 386, and Fig. 11). The observation that 1C D N A values for mouse, Drosophila, and Ciona sperm full on the line indicates that these cells are of the sume informational nemy as bacteria and bucteriophage

27*

394

C.D. Laird:

As discussed above, the DNA contents of a mouse sperm and of a haploid set of mouse ehromatids are identical. Since a mouse sperm contains a unit mouse genome, it therefore follows that mouse meiotic ehromatids are unineme in nit informational sense. There is not stlffieient DNA in these ehromatids for each non-repeated sequence to be present more than once. Potential semantic problems should not obscure the above conclusion. To say that unique nueleotide sequences are present but once in a haploid cell is not strictly a matter of definition. "Uniqueness" is a relative term when compared to the repeated sequences which are present in eukaryotie DNAs. From the data presented here, unique sequences m a y be thought of in an absolute sense in being present as single copies in mouse sperm. I t should also be emphasized that the fraction of DNA which displays "uniqueness" m a y be a function of experimental conditions. For example, high temperatures m a y be used to prevent less specific reassoeiation of polynueleotides of only very distant homology. I n principle, renaturation at temperatures several degrees below the T~ of native DNA would prevent reassoeiation of m a n y repeated sequences which have only partial base sequence homology. I n this ease, most of the DNA should reassoeiate with the second-order kinetics expected for non-repeated sequences. Since intrinsic renaturation rates are also a function of temperature (Wetmur and Davidson, 1968), it is necessary to carry out renaturation of standard polydeoxyribonueleotides, for instance T4 DNA, under these more restrictive temperature eonditions. Britten and Kohne (1968) have concluded t h a t bovine DNA contains about 60% unique sequences under reassoeiation conditions of 0.12 M PB and 60 ~ C. Their experiment involved renaturing labeled E. coli DNA together with high concentrations (8.6 mg/ml) of calf thymus DNA. Cot values at half renaturation, as measured by hydroxylapatite chromatography, were 6 and 4000 for E. coli DNA and non-repeated bovine DNA, respectively. When corrected for the effect of base composition, bovine DNA renatured 530 times more slowly (3200/6) than did the E. coli DNA. This value is slightly less than the ratio of 690 which would be expected if the unit and haploid genomes were equivalent in bovine DNA, although it is considerably greater than a ratio of 345 which would be expected if these sperm contained two of each bovine nueleotide sequence. A similar experiment involving renaturation of Xenopus laevis nonrepeated DNA and E. coli DNA has been referred to by Davidson and Hough (1969). Their unpublished data indicate that Xenopus DNA renatures 700 times more slowly t h a n the E. coli DNA included as an internal standard. These data, corrected for base composition, suggest

DNA Content and Nucleotide Sequence Diversity

395

t h a t the haploid genome of Xenopus contains about 2.6 • 101~ daltons of DNA. This agrees with a published 1C value of 2.5 x 101~ daltons (Bristow and Deucher, 1964). These data pertain to the structure of amphibian lamprush chromosomes. If it can be shown that the diploid complement of lampbrush chromosomes in germinal vesicles of Xenopus laevis contains a 4C amount of DNA, then it would follow that each ehromatid in lampbrush chromosomes is unineme informationally, as well as structurally (Gall, 1963). Since the unit genome and haploid genome are equivalent for Xenopus laevis, a 4C amount of DNA in the lampbrush chromosomes would imply that each lampbrush chromatid contained a sperm equivalent of DNA. Conversely, a value larger than 4C for chromosomal DNA (i.e., excluding the amplified ribosomal cistrons) would support the concept of a ehromatid structure which is informatiortally polyneme. The available data (Brown and Dawid, 1968) suggest t h a t chromosomal DNA in Xenopus germinal vesicles is about 5 times the 4C amount. This estimate for chromosomal DNA was made after separation of the amplified ribosomal cistrons by CsC1 centrifugation. Unfortunately, these authors could not rule out contamination of chromosomal DNA by mitochondrial DNA, and the resolution to the problem of the structure of lampbrush chromosomes will await further experimentation.

4. Concluding Remarks I n this communication I discuss the relationship between kinetics of strand reassoeiation of DNA and classical cytochemieal measurements of the DNA content of meiotic cells. Careful analysis of both of these parameters permits the conclusion t h a t a haploid set of mouse ehromatids are unineme in the sense t h a t they contain one copy of each non-repeated nucleotide sequence. An alternative statement of these results is that mouse meiotic chromatids do not contain sufficient DNA to support the concept of subchromatid units with identical nucleotide sequence information. I t is possible that mouse chromatids are also unineme in the strict sense for repeated sequences. Since most members of a family of such sequences are thought to be similar but not identical (Britten and Kohne, 1968), each individual member of a group m a y also be present only once in a meiotic chromatid. This generalization is not intended to obscure important exceptions to the unit chromatid conclusion. A small number of precisely repeated nucleotide sequences m a y be present in chromatids, and these would not be detected by measurements on unfractionated DNA. The arrangement of nucleotide sequences in unineme chromatids m a y be exceedingly complex. Electron microscopy of dispersed chromosomes

396

C.D. Laird:

(see Prescott, 1970) indicates the existence of complex matrices of fibers. I n the case of mouse meiotic chromatids, however, it is clear that parallel longitudinal fibers must represent packing of different nueleotide sequences, and not lateral repeating elements. Thus the bipartite ehromatid as seen by light microscopy (see Maguire, 1968; Wolff, 1969) must be species specific and perhaps cell specific. Occasional endoreduplication in mammalian meiotic cells m a y represent an abnormal situation leading to binemy. The measurements reported here and b y Swift (1950) on mouse meiotic cells suggest, however, that this is not a general condition of mouse meiotic chromatids. The demonstration of truly unique genes in animal tissue (see also Britten and Kohne, 1968; Laird and McCarthy, 1969; Davidson and Hough, 1969) has important implications for questions of gene function as well as chromosome structure. The observation that 60% of mouse DNA is non-repeated as defined under these experimental conditions implies that mouse tissues have the equivalent of about 2 million structurally di//erent genes (where a gene is defined rather arbitrarily as 1000 nueleotides of DNA). Whether this structural diversity results in a corresponding functional diversity is presently under investigation. Evidence for a functional role in the form of RNA transcription will be presented elsewhere (Hahn and Laird, in preparation). I n the ease of Drosophila melanogaster, the estimated sequence diversity is equivalent to about 120000 different genes. Their arrangement in polytene chromosomes is an intriguing problem. I t is possible that only a small proportion of the total sequences are endoreduphcated during formation of the polytene chromosome to give rise to the several thousand chromosome bands visible with the light and electron microscopes. I n this case, DNA from Drosophila polytene chromosomes would be expected to renature more rapidly than non-polytene DNA. On the other hand, if most of the nucleotide sequence diversity is retained during polytenization, then DNA from these chromosomes will renature with the same kinetics as non-polytene DNA. Results of these experiments support the latter possibility, suggesting that individual bands in D. hydei polytene chromosomes contain sufficient sequence diversity to specify 50-100 di]/erent cistrons (Dickson, Boyd, and Laird, in preparation).

Appendix Materials and Methods

a) Cytology Mouse (Mus musculus, strain C57 Black/6J) testes and sparrow (Passer domesticus) blood samples were gently homogenized in 0.25 M sucrose. Aliquots were spread on Mbumin-eoated slides and air dried. In some experiments, tissues were

DNA Conten~ and Nueleotide Sequence Diversity

397

homogenized and fixed in ethanol-acetic acid (3:1) for 10 min, washed 3 times with fixative, and spread on slides to air-dry. Feulgen staining was carried out as follows: slides were dehydrated in absolute methanol, dried, and rinsed in cold, saturated picrie acid (2,4,6-trinitrophenol) for 1 rain. Depurination was carried out in saturated pieric acid at 60 ~ C for 20 rain. For staining, slides were transferred to pieric acid-Schiff's reagent (0.1 M picrie acid-0.1 M K~S205-1% basic fuchsin) for 2 h at 25 ~ C. This was followed by three five minute rinses in 2 mM pierie acid-0.02 M K2S~05; two rinses in water; and two rinses in 70% ethanol. Slides were left 15-20 h in 70% ethanol at 4 ~ C, and then dehydrated serially in absolute alcohol and xylene. Cover slips were mounted over immersion oil with a refractive index comparable to that of the cells on the slide. Microphotometric readings were made with a Canaleo-Zeiss mierospeetrophotometer, following the dual wavelength method described by Patau (1952).

b) Preparation o] DNAs Techniques described previously were modified for purification of DNA from mouse and Drosophila tissue (Laird and McCarthy, 1968). Mouse tissues or Drosophila pupae were homogenized in 0.05 M tris-0.025 M KC1-0.005 M magnesium acetate-0.35 M sucrose (pH 7.6). The homogenate was filtered through gauze before eentrifugation at 1000 • g for ten minutes. The pellet, containing cells and nuclei, was resuspended in 0.15 M NaC1-0.1 M E D T A - p H 8.0 or SSC (0.15 M NaC1-0.015 M trisodium citrate) and lysed with sodium lauryl sulfate (1% final concentration). After lysis, NaClO 4 was added to a concentration of 0.8 M. Deproteinization was effeeted by shaking the lysate with distilled phenol (water-saturated and neutralized). DNA was precipitated with ethanol from the aqueous phase and resuspended in 0.1 • SSC. After traces of phenol were removed by ether-extraction or dialysis, polysaecharides and R N A were hydrolyzed by digestion with alpha amylase (100~g/ml), pancreatic ribonuelease (5 ~g/ml), and T 1 ribonuclease (5 units/ml). Subsequent phenol deproteinization and ethanol precipitations were carried out until DNAs of greater than 90 percent purity were obtained. Routine analyses of DNA concentrations involved thermal denaturation (see Mandel and Marmur, 1968), diphenylamine assays (see Burton, 1968), and analytical CsC1 eentrifugation (see Mandel et al., 1968). Bacterial DNAs (Bacillus subtilis strain W23, and E. coli strain K12) were purified by the method of Marmur (1961). DNA from bacteriophage T4 (grown on Escherichia coli B) was prepared by the NaCtO 4 extraction method (see Freifelder, 1968). Ciona intestinalis DNA was purified from sperm as described by Lambert and Laird (in preparation). DNAs were radiolabeled with thymidine-2C 14 or thymidine-methyl-H ~ (New England Nuclear Corp. 156 and 027X).

c) Fragmentation o] DNA Sheared DNAs were prepared by passage of DNA solutions through a pressure cell at approximately 1 ml/min. Since the size of the orifice and the velocity of passage affect the resulting fragment size, it was desirable to determine an empirical relationship between pressure and molecular weight of the resulting fragments. The data presented in Fig. 6 represent single and double-stranded molecular weights measured by band velocity sedimentation, using corrections and coefficients determined by Studier (1965). Sedimentation was at 42040 rpm in a Spinco AnD rotor, using the band-forming centerpiece Spineo No. 331346. Size homogeneity increased after shear, as expected for fragmentation forces of this kind (Fig. 7). For example, the single strand size of a preparation of DNA from mouse testes had a weight average of 11.5 • 106 daltons. This same DNA, after

398

C.D. Laird: I

J

i

I

i

io5 h

e3 S IO'

v E E

i,

• 0

lO3

~

~ Double-stranded I

"S

(D

Single -s~randed 1o:

X

I 4

I

I

I

8 12 16 Sheer Force (p.s,i. X 10-3)

I

20

Fig, 6. Molecular weights o/ mouse D N A s subjected to various shear ]orces in a pressure cell. DNAs from mouse spleen (filled symbols) and mouse testes (open symbols) were subjected to increasing shear forces. Samples were centrifuged under neutral (1 M NaC1) and alkaline (0.9 M NaC1-0.1 M Na0H) salt to determine double and single strand molecular weights. A bandforming centerpiece (Spinco 331346) was used for velocity sedimentation measurements as described by Studier (1965). Additional experimental details are given in the section on Materials and Methods

passage through a pressure cell at 12000 p.s.i., was relatively homogeneous and had a single strand molecular weight of 1.2 • l05 daltons, which corresponds to about 400 nucleotides. The data presented in Fig. 4 c result from an experiment in which high molecular weight DNAs from mouse liver, B. subtilis, and T 4 were mixed before shearing and denaturation. All other renaturation experiments involving a mixture of DNAs were done with individually-sheared preparations. A ratio of about ten was observed for the rate constants of the T4 and B. subtilis components (Table, p. 386) regardless of the shearing procedure. d) Fractionation o/Denatured and Renatured D N A Hydroxylapatite chromatography permits ffactionation of denatured from renatured DNA (see Bernardi, 1969). In 0 . 1 2 M P B , renatured DNA binds to hydroxylapatite crystals, while denatured DNA passes through the column. Renatured DNA may then be eluted by sodium phosphate concentrations above 0.27 M (0.5 M was used in the experiments presented here). Bernardi has suggested that this binding is a result of complexing between the calcium in the hydroxylapatite and the phosphate groups in nucleic acids. This suggestion is based on elution patterns with higher phosphate concentrations and the decreased binding of DNA in the presence of E D T A and citrate, presumably due to

DNA Content and Nueleotide Sequence Diversity

399

4

8

UNSHEARED

SHEARED

Fig. 7. Band sedimentation o/ mouse testes D N A in alkali be]ore (left) and after (right) shearing at 15000 p.s.i. Densitometer tracings are shown of ultraviolet absorption photographs during sedimentation in 0.9 ~ NaC~0.1 M N a 0 I t (left to right) at 42040 rpm (Spinco AnD rotor). Twenty-second exposures were t a k e n at times (in minutes) indicated

chelation of calcium. Since commercial sources of hydroxylapatite are variable, each b a t c h m u s t be checked for capacity of binding of nucleic acids as well as for salt elution specificity, ttydroxylapatite batches which have poor capacity or specificity sometimes m a y be regenerated by following the three-step boiling procedure of Miyazawa and Thomas (1965). This has proved successful for preparations of hydroxylapatite from Clarkson Chemical Company (used in the experiments reported here). Less t h a n 100 ~g DNA per cm a of packed H A was used in fraetionating renatured and denatured DNA. For hydroxylapatite chromatography, samples were diluted into 0.12 M PB, heated to 60 ~ C, and passed through a 1 cm 3 column of hydroxylapatite equilibrated at 60 ~ C, in 0.12 M PB. Four 2 ml washes of 0.12 M P B were collected to wash through unrenatured DNA. Similar washes with 0.5 M P B were used to elute renatured DNA. For scintillation spectrometry of radiolabeled DNAs, 0.8 ml of the 0.12 M P B washes was added to 10 ml toluene containing P P 0 (4 gm/1), P O P O P (50 rag/l), and 10% Bio-solv B B S I I I (Beckman Instruments). The 0.5 M P B fractions were diluted four fold with water to reduce the salt concentration; 0.8 ml of this dilution was counted as above. Correction for counting of dual isotopes was based on appropriate H a and C14 standards. A Beckman LS250 scintillation spectrometer was used; counting efficiencies were about 25 % for H a and 75 % for C14 under the quench a n d channel discrimination conditions of these experiments. No corrections have been made for background binding of denatured DNA to hydroxylapatite. To verify t h a t the hydroxylapatite procedures used in these experiments do fractionate DNA into denatured and renatured classes, samples of Drosophila melanogaster DNA, t a k e n after various extents of renaturation, were separated

C.D. Laird:

400

Cot DENATURED

RENATURED

(Msec

880

1.727

1.69) 1.704 DENSlTY(gm/cm 3)

Percent Satellite Renol"ured Total

16

0.42

~0

0.19

57

0.14

~2

0.08

1.704

CsCl pyenography o/ DNA /rom Drosophila melanogaster pupae/raetionated by hydroxylapatite chromatography after various extents o/ renaturation. Sheared, denatured Drosophila DNA (1,8 mg/ml) was renatured at 60~ in 0.12 M PB. Samples containing 50 ~g DNA were removed at times indicated by the Cot

Fig. 8.

value a n d passed through hydroxylapatite columns. " D e n a t u r e d " column indicates CsC1 pyenography of DNA eluted at 0.12M PB. T h e ' " r e n a t u r e d " column indicates pycnography of DNA retained a t 0.12 M P B b u t eluted with 0.5 M PB. Sample sizes were adjusted to give 1-5 Fg DNA in each CsC1 gradient. The peak in each picture at 1.727 gm cm -3 represents DNA (from Myxoeoceus xanthus) used as a density marker. The lines a t 1.704 and 1.691 gm cm -~ represent densiLies of native Dro.sophila melanogaster DNA (not shown) which were verified for this DNA preparation (see Laird and McCarthy, 1968, 1969). The " p e r c e n t r e n a t u r e d " column is an expression of the relative amounts of D N A eluted from H A with 0.12 M P B and 0.5 M P B for each sample. The ratio of satellite DNA (1.691 gm/cm 3) to total DNA in the renatured fractions was determined from areas under the appropriate peaks in the densitometer tracings, a n d should be considered approximate

401

DNA Conten~ and Nucleotide Sequence Diversity Renaturation kinetics of mouse DNA '

'

'

' illlll

i

i

lll[lJ

'

~

i

I

Illl

'

,

i

lllllJ

100

,

i

J Jlltl

,

,

lllll

'

'

,tillln

,

,

,~11

n: I000

Hyperchromici|y

o

< 8O z n -(3

~ 6O 5

Hydroxylopafife

~ 4O 20 ,

ill[lll

,

,

IIIIII

10~ +

,

,

,l

IIII

101

iJgllll

+

,

llillll

102

JlllllJ

' +PlltlJ

103 9

+

'+141[I

100 n = 5oo

7< 8O cl "73

P 6O

Hydroxylopotite

0 r-

~ ~ , .

c~ 4o 20 t

iIIIll[

,

10~

,

r]FIIIl

,

,

,,~qJ[J

101 102 Cot (mo[e sec/[iter)

,

,

,~lnlll

103

Fig. 9a and b. Optical and hydroxylapatite analyses o/ renaturation kinetics o/ mouse D~JA. D~TA from mouse tissue (pooled livers and spleens) was sheared at 5000 and 15000 p.s.i., resulting in fragments of approximately 1000 and 300 nucleotides (single-strand) size, respectively. These DNAs were denatured at 100 ~ C, and allowed to renature at 60~ in 0.12 ~ PB at concentrations of 1.8 and 1.5 mg/ml, respectively. Duplicate samples were removed from each reaction mixture at various times; one sample from each reaction was passed through hydroxylapatite, while the other was subjected to thermal denaturation in 0.12 M PB. Optical densities were monitored continuously using a Gilford 2400 spectrophotometer. The percent denatured D:NA in each sample analyzed by thermal denaturation was calculated from the relative percent hyperchromicity as compared with the analogous valves for native, sheared DNA, i.e., 34% and 35% for the 1000 and 300 nucleotide samples, respectively. With the smaller pieces of D~NA (Fig. 9b), the close correspondence between hyperchromicity and hydroxylapatite measurements is interpreted as indicating re-establishment of extensive basepairing during renaturation. The disparity between these two measurements for the longer fragments (Fig. 9a) is consistent with physical linkage between the slowly-renaturing non-repeated sequences and the more rapidly reassociating repeated polynucleotides. Such an interspersion has been suggested by Thomas et al. (1970) who used other techniques for analysis of eukaryotic D:NAs

402

C.D. Laird:

on hydroxylapatite into denatured (eluted at 0.12 M PB) and renatured fractions (eluted at 0.5 M PB). Aliquots were mixed with 1 ~g Myxococcus xanthus DNA as a density marker, and the buoyant densities were determined by equilibrium sedimentation in CsC1 (Fig. 8). As expected, DNA from 0.12 M PB fractions had a buoyant density of 1.721 g e m -s, characteristic of denatured Drosophila DNA (Laird and McCarthy, 1969). The density homogeneity of each band of renatured Drosophila DNA verifies earlier reports of limited diversity among Drosophila "repeated sequences" (Laird and McCarthy, 1968, 1969). In addition, the density satellite DNA (1.691 gm cm -3) renatures more rapidly than the main band component (compare ratios of satellite to main band in last column of Fig. 8). Experiments are in progress to determine if this density satellite in Drosophila DNA is anMogous to the density satellite of mouse DNA in its nuclear location and high sequence repetition. In addition, investigators in several laboratories report that denatured Drosophila DNA hybridizes to chromocenter DNA in polytene chromosomes (F. Robertson; J. Gall; personal communications), suggesting a localization similar to that for mouse satellite DNA of rapidly renaturing Drosophila DNA in cen~ric heterochromatin (Jones, 1970; Pardue and Gall, 1970). From the standpoint of hydroxylapatite fractionation, however, it is clear that DNA not retained at 0.12 M PB at 60 ~ C has the density of denatured DNA, while DNA retained under these conditions, and eluted at 0.5 M PB, has a density comparable to that of native DNA. Moreover, this discrimination is maintained even with DNA renatured for many hours (Fig. 8). Of the molecules which had renatured, base pairing was re-established over a very large proportion of the total length, as evidenced by the essentially complete restoration of native buoyant density in CsC1. Although the data are not yet extensive, the pycnographic patterns in Fig. 8 suggest a slightly greater return to native density with longer incubation. This is consistent with concatenation of fragments, permitting single-strand ends of partially renatured duplexes to renature with other molecules. These analytical CsC1 analyses verify the fractionation of renatured and denatured DNA by hydroxylapatite chromotography.

e) E]]ect o/Size o] DNA Fragments on Extent o/Renaturation Since DNAs of most multicellular eukaryotes contain a mixture of repeated and non-repeated sequences, physical linkage of these two kinds of polynucleotides should result in partial renaturation, after short incubations, when fragment size is large relative to the repeat unit. Fig. 9 illustrates how such physical association may be detected. Aliquots of mouse DNA taken at various stages of renaturation were passed through hydroxylapatite, or subjected to thermal denaturation to determine the percent hyperchromicity. This latter measurement is an index of the extent to which complementary hydrogen bonding had been re-established during renaturation (Marmur and Doty, 1961). These data show that when fragments are large (1000 nucleotides), only about half the base pairs are re-formed during renaturation of the repeated sequences (excluding the density satellite) of mouse DNA (Fig. 9a). When fragments are smaller (300 nucleotides), base pairing is established over a greater proportion of the reassociated DNA, as indicated by the correspondence in kinetics measured by hydroxylapatite fractionation and thermal denaturation.

D Kinetics o/DNA Renaturation as Measured by Hydroxylapatite Chromatography The data presented in Fig. 2c show that Bacillus subtilis DNA renatures with second-order kinetics over concentrations ranging from 5 to 70 ~g/ml. These

DNA Content and Nucleotide Sequence Diversity I

I

I

l

I

403

I zx

Co/(

5-

2

l

lO 20 T I M E (hours)

50

Fig. 10. Second-order renaturation kinetics o/ B. subtilis DNA, as measured by hydroxylapatite chromatography. B. subtilis DNA was denatured at 100~ and allowed to renatnre a t 60 ~ C. Samples were assayed for the fraction renatured DNA by hydroxylapatite chromatography (the reciprocal of this fraction is shown in the figure). The data are from four different reactions at DNA concentrations of 5 ~g/ml (V); 7 Ixg/ml ( . ) ; 24 ~xg/ml (A) and 70 ~g/ml (9 These data, t a k e n from the central parts of the curves shown in Fig. 2c, indicate an approximate linear concentration-dependence between Co/C and time, as expected for a second-order reaction

same data are presented in Fig. 10 in the more conventional form of the reciprocal of the fraction of denatured DNA versus time. As expected for second-order reactions, a linear relationship was observed for each concentration of DNA. I n addition, the second-order rate constant (determined at 50% renaturation) is independent of the presence of heterologous DNA at concentrations of up to 2.7 mg/ml (see Fig. 3). Intrinsic rate constants apparently vary with the guanine plus cytosine content of DNA (Wetmur and Davidson, 1968). This relatively minor effect was measured b y comparing the expected renaturation rate constants of DNAs of known genome sizes with observed rate constants determined b y optical measurements of hypoehromieity. :For example, N1 bacteriophage DNA (66% G-t-C) renatures a b o u t twice as rapidly as T4 DNA (34% G + C ) after correcting for genome size. These data of W e t m u r and Davidson (1968), summarized in Fig. 11, have been used to correct for base composition effects on DNAs listed in the Table (p. 386). I t should be noted t h a t the principal DNAs used in the experiments reported here, from B. subtilis, Ciona intestinalis, D. melanogaster and mouse, all have base compositions between 3 8 4 4 % G - - C . Variation of base composition within this range would affect intrinsic renaturation rates b y less t h a n 20 percent.

404

C.D. Laird : I

I

i

i

i

I

2.0

o n-- 1.6

1 N]

o

1.2

0~ o

9$ 0.8

.E

7 4 0 1 1 -

0.4

r~

I 50

I

I

40

I

~

50

T 60

Base Composition (%G+C)

:Fig. l l. E//ect o/ base composition on the intrinsic renaturation rate o] DNA. Data from Wctmur and Davidson (1968) indicate that DNAs with high G - - C contents renature somewhat more rapidly than do those with low G-~ C contents, when the data are corrected for the relative sequence diversity. This minor correction has been used to normalize data in the Table (p. 386) to 50% G + C . Data for DNAs from T4, T7, N ] , and E. coli (all open circles) are compared with theoretically calculated G + C dependence (closed circles) (see Wctmur and Davidson, 1968)

Acknowledgements. I thank my many colleagues for their valuable contributions. David Bloch, Maryna Barnard, and Elizabeth Dickson provided especially critical assistance.--Financial support was provided by research grants from the National Science Foundation (GU-1598) and Public Itealth Service (GM-16982, GM-35558, and FR-07091). References

Atkin, N . B . , Mattinson, G., Becak, W., Ohno, S.: The comparative DNA content of 19 species of placental mammals, reptiles, and birds. Chromosoma (Berl.) 17, 1-10 (1965). - - Ohno, S.: DNA values of four primitive chordates. Chromosoma (Berl.) 23, 10-13 (1967). Bcrnardi, G.: Chromatography of nucleic acids on hydroxylapatite. Biochim. biophys. Acta (Amst.) 174, 423448 (1969). Bristow, D. A., Deuchar, E. M. : Changes in nucleic acid concentration during the development of Xenopus laevis embryos. Exp. Cell Res. 35, 580-589 (1964). Britten, R. J., Kohne, D. E. : Nucleotide sequence repetition in DNA. Carnegie Inst. Wash. Year Book 66, 73-108 (1967). - - - - Repeated sequences in DNA. Science 161, 529-540 (1968). Brown, D., Dawid, I. B. : Specific gene amplification in oocytes. Science 160, 272-280 (1968).

DNA Content and Nucleotide Sequence Diversity

405

Burton, K. : A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Bioehem. J. 62, 315 (1956). - - Determination of DNA concentration with diphenylamine. In: Methods in enzymology, X I I B, 163-166. New York: Academic Press 1968. Cairns, J.: The chromosome of E. coll. Cold Spring Harb. Symp. quant. Biol. 28, 43-46 (1963). Davidson, E. H., Hough, B. R. : High sequence diversity in the RNA synthesized at the lampbrush stage of oSgenesis. Proc. nat. Acad. SoL (Wash.) 611, 342-349 (1969). DuPraw, E. J. : Macromolecular organization of nuclei and chromosomes: a folded fibre model based on whole-mount electron microscopy. Nature (Lond.) 206, 338-343 (1965). :~reifelder, D. : The use of NaC104 to isolate bacteriophage nucleic acids. In: Methods in enzymology, X I I A, 550-554. New York: Academic Press 1968. Gall, J. G. : Kinetics of deoxyribonuclease action on chromosomes. Nature (Loud.) 198, 36-38 (1963). Gillis, M., De Ley, J., De Cleene, M.: The determination of molecular weight of bacterial genome DNA from renaturation rates. Europ. J. Biochem. 12, 143-153 (1970). Griffen, A. B. : Nuclear cytology. In: Biology of the laboratory mouse (E. L. Green, ed.), 2nd edit. New York: McGraw-Hill 1966. Hollenberg, C.P., Borst, P., Bruggen, E . F . J . van: Mitochondrial DNA. V. A 25 ~z closed circular duplex DNA molecule in wild-type yeast mitochondria. Structure and genetic complexity. Biochim. biophys. Acta (Amst.) 209, 1-15 (1970). Jones, K . W . : Chromosomal and nuclear location of mouse satellite DNA in individual cells. Nature (Loud.) 225, 912-915 (I970). Kingsbury, D . T . : Estimate of the genome size of various microorganisms. J. Bact. 98, 1400-1401 (1969). Kohne, D. E. : Isolation and characterization of bacterial R N A cistrons. Biophys. J. 8, 1104-1118 (1968). Kurnick, N. B., Herskowitz, I. H. : The estimation of polyteny in Drosophila salivary gland nuclei based on determination of DNA content. J. cell comp. Physiol. 119, 281-299 (1952). Laird, C.D., McCarthy, B . J . : Magnitude of interspecific nucleotide sequence variability in Drosophila. Genetics 60, 303-322 (1968). - - - - Molecular characterization of the Drosophila genome. Genetics 63, 865-882 (1969). - - McConaughy, B. L., McCarthy, B. J. : Rate of fixation of nucleotide substitutions in evolution. Nature (Lond.) 224, 149-154 (1969). Maguire, M. P. : Nomarski interference contrast resolution of subchromatid structure. Proc. nat. Acad. Sci. (Wash.) 60, 533-536 (1968). Mandel, M., Marmur, J.: Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA. In: Methods in enzymology, X I I B, 195-206. New York: Academic Press 1968. - - Schildkraut, C. L., Marmur, J.: Use of CsC1 gradient analysis for determining the guanine plus cytosine content of DNA. In: Methods in enzymology, X I I B, 184-195. New York: Academic Press ]968. Marmur, J.: A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. molec. Biol. 11, 208-218 (1961). - - Dory, P. : Thermal renaturation of deoxyribonucleic acids. J. molec. Biol. 11, 585-594 (1961).

406

C.D. Laird: DNA Content and Nucleotide Sequence Diversity

Mazaitis, A . J . , Bautz, E. K. F.: Partial isolation of an r l I B segment of T4 DNA by hybridization with homologous RNA. Proc. nat. Acad. Sci. (Wash.) 57, 1633-1637 (1967). Miyazawa, Y., Thomas, C.A., Jr.: lXlucleotide composition of short segments of DNA molecules. J. molec. Biol. 11, 223-237 (1965). Pardue, M.L., Gall, J . G . : Chromosomal localization of mouse satellite DNA. Science 168, 1356 1358 (1970). Patau, K. : Absorption microphotometry of irregular-shaped objects. Chromosoma (Berl.) 5, 341-362 (1952). Prescott, D. M. : The structure and replication of eukaryotic chromosomes. In: Advanc. Cell Biol. 1, 57-117 (1970). Rasch, E.M., Barr, H.J., l%asch, R.W. : The DNA content of sperm of Drosophila melanogaster. Chromosoma (Berl.) 33 (in press, 1971). Rothfels, K., Sexsmith, E., Heimburger, M., Kranse, M. O. : Chromosome size and DNA content of species of Anemone L. and related genera (Ranunculaceae). Chromosoma (Berl.) 20, 54-74 (1966). Sober, H.A. (editor): Handbook of biochemistry. Chemical Rubber Co. 1968. Stockert, J. C.: Half-chromatids in the human chromosomes from leucocyte cultures. Cytologia (Tokyo) 34, 160-162 (1969). Studier, 1~. W.: Sedimentation studies of the size and shape of DNA. J. molec. Biol. 11, 373-390 (1965). Sueoka, lq., Chiang, K., Kates, J. : DNA replication in Chlamydomonas reinhardi. I. Isotopic transfer experiments with an eight-zoospore-producing strain. J. molec. Biol. 25, 47 (1967). Swift, H. H. : The desoxyribose nucleic acid content of animal nuclei. Physiol. Zool. 23, 169-198 (1950). Thomas, C.A., Jr. : The arrangement of information in DNA molecules. J. gen. Physiol. 49, 143-168 (1966). - - Hamkalo, D. A., Misra, D. N., Lee, C. S. : The cyclization of eucaryotic DNA fragments. J. molec. Biol. 51, 621-632 (1970). Tomizawa, J., Anraku, 1~.: Molecular mechanisms of genetic recombination in bacteriophage. IV. Absence of polynucleotide interruption in DNA of T4 and ~ phage particles, with special reference to heterozygosis. J. molec. Biol. 11, 509 (1965). Vendreley, C.: L'acide d6oxyribonucl6ique du noyau des cellules animales. Son r61e possible dans la biochimie de l'h6r6dit6. Bull. Biol. France et Belg. 86, 1-87 (1952). Wells, R., Birnstiel, M. : Kinetic complexity of chloroplastal deoxyribonucleic acid and mitochondrial deoxyribonucleic acid from higher plants. Biochem. J. ll~, 777-786 (1969). Wetmur, J. G., Davidson, l~. : Kinetics of renaturation of DNA. J. molec. Biol. 31, 349-370 (1968). Wilson, E . B . : The cell in development and heredity, 3rd edit. New York: Macmillan 1934. Wolfe, S. L., Martin, P. G. : The ultrastructure of chromosomes from two species of Vicia. Exp. Cell Res. 50, 140-150 (1968). Wolff, S.: Strandedness of chromosomes. Intern. Rev. Cytol. 25, 279-296 (1969). Dr. Ch. D. Laird Department of Zoology University of Texas Austin, Texas 78712 U.S.A.