Chromosome analysis by two-dimensional fingerprinting

Gene, 49 (1986) 93-102

93

Elsevier GEN 01830

Chromosome analysis by two-dimensional (Recombinant

DNA;

genome

fingerprinting

size; electrophoresis;

agarose

gels; restriction

endonucleases;

mycoplasma;

Escherichia coli)

Saibal K. Poddar and Jack Maniloff * Department of Microbiology and Immunology, Universityof Rochester, Medical Center Box 672, Rochester, NY 14642 (U.S.A.) Tel. (716)275-3413 (Received

August

(Accepted

September

4th, 1986) 9th, 1986)

SUMMARY

A two-dimensional fingerprinting technique has been developed that allows large, cell genome-size DNA’s to be analyzed by restriction endonuclease cleavage and separation of DNA fragments by agarose gel electrophoresis. Equations have been derived to determine the genome size and number of cleavage sites from analysis of the distribution of fragment lengths. Genome sizes of Escherkhia coli strain JM 101 and two strains of the mycoplasma Acholeplasma laidlawii measured by this method are in agreement with published values. Other uses of two-dimensional fingerprinting for studies of prokaryotic and eukaryotic genome structure and organization are described.

INTRODUCTION

Virus and plasmid DNA’s, < 50 kb in size, have been analyzed by restriction endonuclease digestion and separation of the resulting DNA fragments by agarose gel electrophoresis. This approach cannot be extended to larger, cell-genome-size DNAs, because so many fragments are produced by restriction endonuclease digestion that there is an inseparable number of DNA bands. To get around this problem, * To whom correspondence

and reprint

requests

should

be

addressed. Abbreviations: kilobase buffer, and

bp, base pair(s); or 1000 bp;

SDS,

EtdBr,

ethidium

bromide;

sodium

dodecyl

sulfate;

see section e of MATERIALS TES

METHODS.

buffers,

see

section b

AND of

METHODS;

MATERIALS

kb, TBE TE AND

several workers have tried two-dimensional electrophoretic separations. In these methods, chromosomal DNA is digested with a restriction endonuclease and the fragments are analyzed by agarose gel electrophoresis. Various methods are then used to treat the fragments with a second restriction endonuclease, and they are separated by a second agarose gel electrophoresis, with the electric field perpendicular to the original electrophoresis direction. This generates a two-dimensional fingerprint of the chromosome. The problem with two-dimensional methods has been in carrying out the second digestion. Two methods have been used. First, some workers have excised or eluted fragments from the first gel into a number of fractions (e.g., 50 to 90). The DNA in each fraction was purified, treated with a second nuclease, loaded onto an agarose gel, and separated

0378-l 119/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

94

by electrophoresis. analyze bacterial

This method

(Potter and Newbold,

et al., 1977; Chen (Hamer 1981)

and Thomas,

and Thomas, mouse

and human the number

1976; Potter

1983), Drosophila

1975; Smith

and Thomas,

(cited in Smith and Thomas,

DNAs (Guerin

the procedure

has been used to

is tedious of fractions

and Lucotte,

and resolution

1981),

5 g Bacto-Yeast

Extract,

and 5 g

NaCl per 1) at 37’ C on a rotary shaker with vigorous aeration. A. laidlawiistrains JA 1 and K2 were grown in tryptose medium at 37°C (Haberer et al., 1979; Liss and Maniloff,

1973).

1985), but is limited by

into which the first gel can

be divided. The second method is to treat the entire first-dimension gel with a second nuclease, then seal the treated

Bacto-Tryptone,

gel on top of a second gel and carry out

electrophoresis with the electric field perpendicular to the first gel. To give the second nuclease access to the DNA fragments, either the gel can be soaked in a nuclease solution, or the second nuclease can be incorporated directly into the first gel. This method has been applied to bacteriophage (Rosenvold and Honigman, 1977), bacterial (Yee and Inouye, 1982; 1984; Komano et al., 1985) and rat DNAs (Peacock et al., 1985). Although the second method resolves the problems associated with the first method, it introduces several new problems which limit its utility. These are practical difficulties in exposing DNA in the agarose to high-specific-activity nucleases, non-uniform exposure of DNA in the gel to nucleases, and inhibition of many nucleases by contaminants in agarose preparations, thereby limiting the choice of nucleases which can be used for the second digestion. We examined possible modifications of twodimensional fingerprinting to resolve the problems associated with the second digestion. In this paper,

(h) DNA isolation E. coli JMlOl

cells from a stationary

phase culture

(about 100 ml) were harvested by centrifugation for 5 min at 5000 rev./min in a Beckman (Palo Alto, CA) JA-20 rotor, and resuspended

in 2 ml of 25%

sucrose in 50 mM Tris . HCl buffer (pH 8.0). Lysozyme (Sigma, St. Louis, MO) was added to a final concentration of 3.4 mg per ml. After incubation at 37°C for 30 min, 0.8 ml of 0.25 M EDTA and 0.32 ml of 10% SDS were added and the mixture was swirled gently. After the mixture cleared, 40 ~1 of a 10 mg per ml solutio of proteinase K (Bethesda Research Labortories, Gaithersburg, MD) in 25 mM Tris . HCl buffer (pH 8.0) was added and the mixture was incubated at 37°C for 1 h. The lysate was then diluted with 4 ml of 50 mM Tris. HCl buffer (pH 8.0) and used for DNA extraction. A. luidluwiicells from an overnight culture (about 100 ml) were harvested by centrifugation as described above. The cell pellet was resuspended in 4 ml TES buffer (10 mM Tris * HCl, 1 mM EDTA, and 100 mM NaCl, pH 8.0) and used for DNA extraction. Each cell preparation was extracted twice with phenol and three times with chloroform-isoamyl

we describe a method involving transfer of the entire first-dimension DNA fragment distribution from the

alcohol (24 : 1, v/v). Nucleic acids were precipitated with ethanol, resuspended in TE buffer (10 mM Tris . HCl and 1 mM EDTA, pH 8.0) containing

gel to a DEAE cellulose membrane. This facilitates the second digestion, since all fragments are uniformly exposed to high-specific-activity nuclease, and agarose contaminants are absent, so that all nucleases can be used. The geometry of the DNA on the membrane surface also improves final gel resolution. We have used the method for rapid determination of bacterial genome sizes.

ribonuclease (Sigma) per ml, and incubated at 37 ‘C for 1 h. The mixture was then extracted once with chloroform-isoamyl alcohol and DNA was precipitated with ethanol. The DNA precipitate was redissolved in TE buffer.

MATERIALS

AND METHODS

(a) Cells and growth conditions E. coli strain JMlOl, a K-12 derivative (YanischPerron et al., 1981) was grown in YT medium (8 g

20 pg DNase-free

(Maniatis

et al., 1982) pancreatic

(c) Restriction endonucleases The DNA was digested with nucleases BstEII, HindIII, and BumHI using reaction conditions recommended by the supplier (Bethesda). One unit of enzyme activity is defined as the amount giving complete digestion of 1 pg of bacteriophage il DNA in 1 h, under the appropriate reaction conditions.

95

(d) First restriction endonuclease digestion

The DNA (30-40 pg) was digested with 30-40 u of restriction endonuclease, in a total volume of 60 ~1 of reaction buffer, for at least 3 h at the appropriate temperature. Following digestion, 5 ~1 of gel loading solution (25 y0 glycerol, 60 mM EDTA, 0.1 y0 bromphenol blue) was added to the mixture. (e) First-dimension

electrophoresis

A 0.7% agarose gel (Bethesda) in 2 x TBE buffer was cast on a glass plate. TBE buffer is 89 mM Tris . borate and 2 mM EDTA (pH 8.0). The gel was 15 cm wide, 19 cm long, and 2.5 mm thick, with sample wells 8.5 mm wide and 3 mm long. The gel was placed in a horizontal gel tank and each well was loaded with a chromosomal digestion mixture. In addition, one well was loaded with 1 DNA digested with HindIII, which was used as a size marker. Electrophoresis was carried out at 1.5 V/cm for 16-18 h in 2 x TBE buffer. (f) DNA transfer to DEAE cellulose membranes

Following electrophoresis, two longitudinal cuts were made in each gel lane, one just inside each side of the lane. This allowed a strip of agarose containing the central 4.5-5.0 mm of each lane to be removed. The remaining gel was stained with EtdBr (as described below) and DNA fragments in the trimmed edges were visualized with UV illumination, to determine the quality of the first-dimension separation. Removing the band edges also improved subsequent electrophoretic separation, because band edges tend to curve upward and decrease resolution in the second-dimension separation. From the positions of the fragments of HindIIIdigested ;1 DNA in the EtdBr-stained gel, the lower portion of each unstained gel strip, corresponding to DNA fragments ~2.0 kb, was removed. These small fragments were not needed in the subsequent analysis, and reducing the gel strip length made it less fragile. DEAE-cellulose membranes (grade NA45 from Schleicher and Schuell, Keene, NH) were marked on one side using permanent ink and cut into strips 12-16 cm long and 2.5-3.0 mm wide. To maximize ion exchange capacity, membranes were soaked in

10 mM EDTA (pH 7.6) for lo-15 min, in 0.5 M NaOH for 5-10 min, and washed several times in distilled water. Membranes can be stored for several weeks in water at 4°C. The DNA fragments in each gel strip were electroblotted onto a DEAE-cellulose membrane as follows. Agarose gel strips were placed side-by-side on a glass plate, with adjacent gels separated from each other by a DEAE-cellulose membrane strip. The membrane strips were the same size as the side of the gel strips, and arranged with the unmarked side of each membrane against the gel to be blotted onto it. The glass plate was then placed in a horizontal gel box so that electrophoresis would be perpendicular to the long axis of the strips. In this arrangement, for every gel-membrane pair, the gel was closer to the negative electrode and the unmarked membrane surface faced the gel and negative electrode. The gels were covered with 2 x TBE buffer and, to insure good contact between each gel and membrane, sponges were laid on the glass plate against the first and last strips to tightly sandwich all strips. Electrophoresis was carried out at 3.5 V/cm for 90 min. This was sufficient to transfer all DNA bands from each gel to the adjacent unmarked membrane surface. Longer electroblotting times did not affect subsequent steps, but were not necessary. Each membrane was removed, and gently washed three times with buffer appropriate for the second nuclease digestion. (g) Second restriction endonuclease digestion

A membrane strip containing bound DNA fragments was inserted into a tube, made by removing the ends of a lo-ml plastic disposable pipet. The tube was approximately half-tilled (3-4 ml was sufficient) with an appropriate reaction buffer containing 10 u of restriction endonuclease and 100 pg bovine serum albumin per ml, and the ends were tightly sealed with Parafilm. Tubes were sealed in a plastic bag, placed horizontally in a water bath at the appropriate temperature for the nuclease reaction, and incubated about 4 h with gentle shaking. (h) Second-dimension

electrophoresis

Following the second nuclease digestion, each membrane strip was removed from its tube and

96

placed against a comb. The comb was positioned near the top of a glass plate (so that the direction of electrophoresis would be perpendicular to the comb length) and a slab gel, 15 cm wide and 19 cm long, of 0.7% agarose in 2 x TBE bufIer was cast. The arrangement of the membrane was such that the unmarked surface (containing the DNA fragments) faced the positive electrode. The comb was then removed and a strip of agarose (5 mm wide and a few mm longer than the membrane), containing the membrane, was cut and removed from the slab gel. The agarose strip was placed in a solution of 2 x TBE buffer containing 1.5 M NaCl and 0.2% SDS and incubated at 60°C for 2 h, to break the ionic bonds between DNA and DEAE while holding the DNA fragment distribution in place along the membrane. The agarose strip was replaced in the agarose slab gel and sealed in place with melted agarose. Lambda DNA digested with Hind111 was loaded into a well at one end of the gel, to serve as size markers. Electrophoresis was carried out, in 2 x TBE buffer, at 3 V/cm for 30 min and then at 1.5 V/cm for 16 to 18 h. The gel was then soaked in 1 @g EtdBr per ml for 2 h to stain DNA fragments and destained in water with occasional shaking. Bands of DNA were visualized using UV illumination and recorded on Polaroid Type 55 P/N film.

M MO=-------= El + %

M,M,

(1)

M, + M,

The probability, p, , of a cleavage following a base after digestion by nuclease 1 is the number of cleavages divided by the number of base pairs: pi = n,/M = l/M,

(2)

and the probability of no cleavage following a base is (1 - p,). The number dist~bution of fragments of molecular size L after digestion with nuclease 1, N,(L), is the number of fragments (n,) times the probability, P,(L), of a fragment of size L:

N,(L) = M,(L)

(3)

P,(L) is the probability of no cleavage for L - 1 bases times the probability of one cleavage:

P,(L) = (1 - P,)=-‘P,= [1 - tWJL-’

(l/M,) (4)

L is of the order of lo3 to lo4 bp. Hence, expanding the first factor in a binomial series, we find [l - (l/M,)]=-’

m

(i) Genome size calculation

and

The genome size of an organism can be determined from an analysis of the fragment distribution in the two-dimensions DNA fingerprint. Consider a circular DNA molecule of M base pairs (bp) digested by one nuclease, followed by digestion with a second nuclease. Assume cleavage sites for both nucleases are distributed randomly along the molecule, and both cleavages are made independently of each other. There are supporting data for the first assumption (Smith and Thomas, 198 1) and the second is reasonable. Nuciease 1 alone cleaves the molecule at n, sites producing n, fragments, with number average ‘molecular size M, where M, = M/n,. Similarly, nuclease 2 alone cleaves the molecule at n2 sites producing n, fragments, with number average molecular size M, where M, = M/n,. If the DNA is digested with both nucleases, producing (n, + nJ fragments, the number average molecular size of the fragments, M,, is

N,(L) = Me-L’M1/M:

eeLiMI

(5)

(6)

This equation also has been derived from theoretical analyses of polymer de~adation (Kuhn, 1930; Montroll and Simha, 1940). If the fragments are now digested with nuclease 2, the probability, pZ, of a cleavage after any base is p2 = n,/M = l/M,

(7)

After the second digestion and fragment separation by two-dimensional gel electrophoresis, the number distribution of fragments migrating on the diagonal, M&L), is equal to the number of fragments of size L after the first digestion times the probabi~ty of no cleavages in a fragment of size L by nuclease 2:

N,(L) = N,tLXl -

~2

= N,(J-JIl- tl/WIL,

= MeWLjMI[1 - (l/M,)JL/Mt

(8)

91

Expanding the factor in the brackets, for L >>1: N,(L) = Me - WVMI)+ (l/Mz)l/~f

= Mu-

Since M > 10’ bp, and L and M, are of the order of lo3 bp, this becomes

“MO/M:

(9)

iELe-i/Mo

=

M,~-L/Mo

The number distribution of all fragments after digestion with nucleases 1 and 2, N,(L), is the total number of fragments (n, + nz) times the probability a fragment is of size L after the double digestion, P,,,(L). Hence,

and

N,(L) = (n, + 4&,&)

S,,(O) = MM,[U/M;) -

= M4.#)/M,

(10)

As before, the probability of a cleavage following any base after double digestion, ~i,~, is the number of cuts (n, + n,) divided by the number of base pairs: ~1.2

=

(n,

+

d/M

=

l/M,

(11)

and the probability of no cleavage following any base is (1 - p&. Hence, the probability of a fragment of size L after double digestion is

p1,m = (1 - Pl,d=- h,2

under the

= 5 J%(i) i=L

M[l/Ms)]

- (l/M:)]

5 eeiiMo

(15)

i=L

The summation is a geometrical progression:

igLe-i/Mo

=

e-L,Mo

eCcMmL+1YMo _ e-l/Ma

_

1

1

U/M:)1

(19)

and

S,.,( > L) = S,,(0)e-L/Mo

(20)

or m si,,(aL)

= ln si,@)

- (L/M,)

(21)

S,,,(aL)

(14)

The number of fragments of size >L diagonal, S, ,J > L), is

=

At L = 0,

= MM,[(l/Mg) - (l/M$)] e-L/Mo = = S,,,(O)e- L/Mo (22)

where

&,,KO = MW[(l/M:) emLIMO

(l/M:)1 ebLjMo (18)

(13)

N”(L) = Km - N,(L) = M[ l/M;) - (l/M:)]

-

(12)

Following double digestion and two-dimensional gel electrophoresis, the number distribution of fragments under the diagonal, N,(L), is

S,,,(aL)

= MM,[(l/Mz)

A plot of In S, ,J > L) as a function of L will be linear, with intercept In S,,JO) and slope -l/M,. If the digests are done in the reverse order, similar equations can be derived, where the total number of fragments of size 2 L under the diagonal, S,,,( > L), is

Using the steps in going from (3) to (6) N,(L) = Me - L/Mo/M$

s,.,(>L)

(17)

(16)

(l/M;)1

(23)

and ln &,,(>L)

= ln S,,,(O) - (L/M,).

(24)

In a plot of In S,,, (2 L) as a function of L, the intercept will be ln S,,,(O) and the slope -l/M,,. After plotting the data according to Eqns. 21 and 24 to determine S,,JO), S,,,(O), and M,, there are three equations (Eqns. 1, 19 and 23) with three unknowns (M, M,, and M2). Hence, Eqn. 19 can be solved for M, as a function of S,,(O), M,, and M; and Eqn. 23 for M, as a function of S,,(O), M,, and M. Putting these into Eqn. 1 M is obtained as a function of experimental values:

98

M =

(%/3)~~,,2(O) + S,,,(O) + 2KWV + %,*(o)* - &,*w,.,w1”‘~

+ (25)

With M, Me, S,,*(O), and S,,,(O), we calculate M, and M, from Eqns. 19 and 23:

M, = M,

M M - Wf%,&O

M, = M,

M M - M,S,,UN

112 1 112 1

(26)

(27)

Eqns. 21 and 24-27 were stated by Yee and Inouye (1982), based on an unpublished derivation in which molecular size was treated as a continuous variable.

RESULTS

AND DISCUSSION

(a) Experimental design The rationale of our two-dimensional DNA fingerprinting procedure for large DNAs is as follows. Genomic DNA, carefully isolated to reduce random breakage, is digested with a restriction endonuclease. The resulting fragments are separated by agarose gel electrophoresis, yielding a gel with an inseparable number of DNA fragments, appearing as almost a continuum of DNA bands. The entire fragment distribution is transferred by electroblotting from the gel to the surface of a DEAE cellulose membrane, which is positively charged and binds anions such as DNA. This transfer facilitates subsequent nuclease digestion (since all fragments are uniformly exposed to added nuclease, and agarose contaminants that might interfere with digestion are not present) and improves final gel resolution (because of the fragment geometry on the membrane surface). DNA fragments on the membrane are digested with a second restriction endonuclease, which cleaves those fragments containing recognition sites for that nuclease. The membrane then is embedded in a strip of agarose and soaked in high-salt btier, to break ionic bonds between DNA and DEAE, while holding the fragment ~st~bution is place along the paper. Finally, the strip is sealed on top of an agarose gel and electrophoresis is carried out perpendicular to the

direction of the paper. DNA fragments on the paper which are not cleaved by the second nuclease have the same mobility in the second electrophoresis as they had in the first, and migrate to form a diagonal in the second gel. However, those fragments on the membrane which are cleaved by the second nuclease produce smaller fragments, which have faster mobility in the second electrophoresis than the original fragment had in the first and form bands under the diagonal in the second gel. (b) Experimental cwsiderations To confirm the two-dimensional fingerprinting procedure, expe~ments were done to investigate whether electroblotting affects fragment mobility, and whether restriction endonuclease digestion of DNA bound to DEAE cellulose membranes produces the same products as digestion of DNA in solution. DNA mobility in the two-dimensional tingerprinting procedure was studied as follows. A Hind111 digest of phage ;1DNA was separated by agarose gel electrophoresis. The DNA fragments were transferred to a DEAE cellulose membrane by electroblotting and subjected to a second dimension electrophoresis. The second gel also contained a well loaded with HindIII-digested phage J DNA. The DNA fragments that had been blotted onto and eluted from the membrane were found to migrate along the diagonal in the second dimension, with each fragment corn&rating with the corresponding fragment in the lane of HindIII-digested phage 1 DNA (Fig. la). Hence, use of DEAE cellulose membranes for transferring DNA molecules between first and second dimension gels does not affect DNA electrophoretic mobility. Activity of restriction endonucleases on membrane-bound DNA was studied as follows. Lambda DNA was loaded into wells in a 0.2% agarose gel and a DEAE cellulose membrane was inserted into each lane, ahead of the DNA and perpendicular to the direction of electrophoresis. A brief electrophoresis transferred the DNA onto each membrane surface. The membranes were removed, washed, and digested with either IIindIII or EcaRI. The DNA digestion products on each membr~e were analyzed by agarose gel electrophoresis (Fig. lb). Both enzymes generated the expected number and size frag-

99

7.4 5.8 5.6 4.9 3.5

Fig. 1. Experiments designed to verify the two-dimensional fingerprinting method. (a) Two-dimensional electrophoresis of phage i DNA digested with HindHI, in a 0.7% agarose gel aslo containing a well (on the right) loaded with HindHI-digested I DNA. The sizes of the 1 DNA fragments are marked in kb. The DNA fragments were not heated before electrophoretic separation, so the 23.1-kb and 4.4-kb fragments annealed at their cohesive ends and migrated as a 27.5-kb fragment. The first electrophoresis was from left to right and the second from top to bottom. The bright strip across the top is the DEAE cehuiose membrane. (b) Phage 1 DNA ele~troblotted onto DEAE cellulose membranes was digested with either Hind111 (left lane) or EcoRI (right lane), and the fragments were separated by 0.7% agarose gel electrophoresis. The bright band at the top of each lane is the DEAE cellulose membrane. As expected from the known sequence of 2, DNA, Hind111 produced 23.1,9.4,6.7,4.4, 2.3 and 2.0-kb fragments, and EcoRI produced 21.2, 7.4, 5.8,5.6,4.9 and 3.5kb fragments. The sizes of the EcoRI fragments are marked in kb in the right margin.

ments for phage Z DNA. Hence, restriction endonucleases produce the same products for DNA bound to DEAE cellulose membranes as for DNA in solution. In addition, this procedure avoids the problem of ~ont~inants in agarose which inhibit restriction endonucleases Hind111 (Yee and Inouye, 1982) and EcoRI (Peacock et al., 1985). (c) Analysis of microbial genome size Two dimensional DNA fingerprints were used to determine genome sizes of E. coli and two strains of the mycoplasma A. faidlawii, using the equations derived in section i of MATERIALS AND METHODS. A typical electrophoretic pattern for E. coli strain JMlOl is shown in Fig. 2. For each fingerprint, ;1 DNA fragments were used as markers and the number of cell DNA fragments under the diagonal larger than or equal to each il fragment was counted and plotted as a function of fragment length. Each experi-

ment was repeated using the same nucleases, but in reverse order, and the data plotted in the same way. From Eqns. 21 and 24, the slopes and intercepts of these curves were used to calculate the genome size (Eqn. 25) and other parameters of the nuclease digestions. These are listed in Table I. We estimate the standard deviation of the genome size determined by this method from the variance of the residuals in the least squares analysis of the data fitted to Eqns. 21 and 24. Therefore, the standard deviations in these fingerprinting experiments are about + 12% for E. coli, f 9% for A. laidlawii strain JAl, and 5 17% for A. laidlawii strain KS.2 The range of genome sizes reported for 26 E. coli strains, measured by DNA renaturation kinetics, is 3333-4500 kb (references in Herdman, 1985), with a mean for K-12 strains of 3788 kb (references in Yee and Inouye, 1982). The size of 3369 kb determined in these studies for E. coli JMlOl (a K-12 derivative) is in agreement with the reported sizes and the value

23. I

9.4 6.7

4.4

2.3 2.0

Fig. 2. Two-dimensional agarose

fingerprint

gel electrophoresis,

separated

by electrophoresis

TABLE

The bright

gel also containing

are marked

strip across

DNA. The DNA was digested

a DEAE-cellulose

membrane,

a well (on the right) loaded

in kb in the left margin. The first electrophoresis

the top is the DEAE-cellulose

with BstEII,

and digested

and fragments

with BamHI.

with HindIII-digested

These

separated

fragments

by were

phage I DNA. The

was from left to right and the second from

membrane.

I

Parameters

Nuclease

of microbial

genomes

1

Number

of fragments

Average

molecular

Nuclease

size

2

Number

of fragments

Average

molecular

Double

onto

in an agarose

sizes of the I DNA fragments top to bottom.

of E. coli strain JMlOl

electroblotted

size

from two-dimensional

fingerprinting”

E. coli

A. laidlawiiJAI

A. laidlawii K2

BstEII

Hind111

Hind111

537

395

361

6.3 kb

4.3 kb

4.1 kb

BamHI

BstEII

BstEII

115

180

485

4.3 kb

9.4 kb

3.1 kb

1312

575

846

digest

Number

of fragments

Number

under

diagonal

Nucleases

l/2

1092

306

692

Nucleases

2/l

854

518

568

Average

molecular

size

2.6 kb

3.0 kb

1.8 kb

Genome

molecular

size

3369 kb

1705 kb

1483 kb

a Parameters fingerprints

of the nuclease using equations

digestions

were calculated

from plots of the distribution

[21], [24], [26], and [27], and genome

sizes from equation

of fragment [25].

sizes in two-dimensional

DNA

101

of 3520 kb measured for a K-12 strain in the twodimensional electrophoretic studies of Yee and Inouye (1982). A. Za~~a~iigenome sizes, determined by DNA renaturation kinetics, have been reported in the range of 1470-1879 kb, with a mean of 1682 kb (Bak et al., 1969; Saglio et al., 1973). The values found in these studies, 1705 kb for strain JAl and 1483 kb for strain K2, are in agreement with reported sizes. However, the fingerprinting method shows an interesting difference between these strains. Both have essentially the same number of Hind111 sites, but strain K2 has about 2.5 times more BstEII sites than strain JAl. (d) Conclusions In the past, cell genome sizes have been determined by DNA renaturation kinetics. The twodimensional fingerprinting method described here allows measurement of genome size, using less DNA than required for renaturation kinetics and providing genome information in addition to size. It should be noted that genome size measurement by this method does not require DNA fragment size dete~ination by agarose gel elec~ophoresis, since the sizes of the marker fragments that are used are obtained from sequence data and it is only necessary to count the number of genomic DNA fragments that are equal to or larger than each marker fragment. The two-dimensional procedure can be used to investigate particular aspects of genome structure. Methylation of specific sequences during cellular processes can be probed by choosing isoschizomers as restriction endonucleases, such that the first nuclease does not cleave if the cleavage site contains a methylated base and the second nuclease cleaves whether or not the site contains a methylated base (Yee and Inouye, 1982). Genome rearrangements can be studied by denaturing and renaturing the DNA fragments after the first restriction endonuclease digestion and using Sl nuclease (which cleaves single-stranded but not double-stranded DNA) for the second digestion (Yee and Inouye, 1984). In addition, two-dimensional fingerprinting is a high-resolution method for detecting restriction fragment length polymo~hisms and genetic patterns in populations. Hence, the method described here promises to be a significant experimental tool for a

variety of studies of prokaryotic and eukaryotic genome structure and organization.

ACKNOWLEDGEMENTS

We thank Dr. Thomas Yee for discussions about the derivation of his equations for calculating genome size. These studies were supported by grant GM32442 from the National Institutes of Health.

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