Genomic sequencing and in vivo footprinting

176,201-208

ANALYTICALBIOCHEMISTRY

(1989)

REVIEW Genomic

Sequencing

and in Viva Footprinting

H. P. Saluz and J. P. Jost Friedrich

Miescher

Institut,

P. 0. Box 2543, CH 4002 Basel, Switzerland

Information about base modifications in DNA and protein-DNA interactions in viuo is permanently lost during cloning in procaryotic hosts. In addition, naturally occurring mutations and genetic polymorphisms could, until recently, be studied only by cloning each mutation prior to sequencing, a labor intensive approach. Indeed, the cloning procedures themselves may introduce artifacts, such as deletions or duplications, into the sequence of interest. It was necessary, therefore, to develop new techniques to overcome these limitations. So far, base modifications, such as cytosine methylation, have usually been studied with methylation-sensitive restriction endonucleases. Clearly this approach will reveal only modifications occurring within recognition sequences of these specific enzymes. Moreover, the existence of hemimethylated DNA, inferred from in uitro enzymatic methylation experiments (1,2), would not be detected by such restriction enzymes. Genomic sequencing, first described by Church and Gilbert (3), overcomes the shortcomings of restriction enzymes revealing the methylation of any cytosine on either strand of the DNA. The existence of hemimethylated DNA in the upstream region of the avian vitellogenin gene was successfully demonstrated using the genomic sequencing technique (4). Genomic sequencing is a relatively complex procedure consisting of many steps (Fig. 1). First, purified genomic DNA is digested to completion with a suitable restriction enzyme. After purification of the reaction products by repeated phenol-chloroform extractions and ethanol precipitation, the restricted DNA is subjected to the base-specific chemical reactions described by Maxam and Gilbert (5), Rubin and Schmid (6), or Fritzsche et al. (7). Conditions are chosen that give a partial reaction of about one cleavage event every 500-700 bases. The resulting fragments are separated by high resolution gel electrophoresis (5,8) and subsequently transferred to a nylon membrane by electroblotting. They are immobilized on the membrane by uv irradiation or a combination of irradiation with heat treatment at reduced pressure. The sequence of interest is visualized by hy0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form

Inc. reserved.

bridizing a labeled single-stranded DNA or RNA probe to one end of the appropriate restriction fragment, as depicted in Fig. 2, followed by autoradiography. It is important that a unique sequence be chosen for this indirect end-labeling. Furthermore, the subfragments (wavy lanes in Fig. 2) not contributing to the sequence may also hybridize to the probe and should be kept to a minimum as they increase the background within a sequencing ladder. In viva footprinting allows the interaction between nuclear proteins and DNA to be studied with precision in intact cells (9-18). This procedure differs from genomic sequencing in that intact living cells are treated with either dimethyl sulfate, to modify guanosine residues not protected by DNA-binding proteins, or with uv light, which will react with pyrimidine residues in contact with the proteins. Otherwise, the technique is fundamentally the same as genomic sequencing, described in detail by Saluz and Jost (19). Footprinting techniques using isolated nuclei fall into a category of in vitro experiments and are thus not discussed here. COMPARISON OF GENOMIC

OF THE DIFFERENT SEQUENCING

SYSTEMS

A major disadvantage of genomic sequencing is the inverse relationship between the strength of hybridization signals and the complexity of a genome. Consequently, for single copy genes in higher eucaryotes, it is necessary to optimize the amount of genomic DNA to be used. Another approach is dideoxynucleotide chain termination sequencing applied directly to genomic DNA templates by annealing a radiolabeled oligonucleotide primer to a unique site in the total DNA and extending with avian myeloblastosis virus reverse transcriptase. Although this approach has been successful with Saccharomyces cereuisiae (20), attempts to apply it to the chicken genome, which is about 150 times more complex than yeast (Table l), have failed (Saluz, unpublished results). The availability of Taq DNA polymerase, which functions at an optimum temperature of about 8O”C, in201

202

SALUZ genomic

DNA

cloned

AND

JOST

DNA

cleavage

with resfricendonuclease

bon Cl

mC2

C3

Cl

-UPPER STRAND

c2

c3 1

Cl

c2

I partial chemical

3

C or mC reactions

Cl I

'i=:c4

ljii?-Q~

FIG. 1. Genomic sequencing scheme. Genomic sequencing involves the isolation of intact native DNA directly from a tissue or cells in culture. The genomic DNA is digested to completion by using a restriction endonuclease. The restriction fragments are subjected to the appropriate chemical sequencing reaction, separated by size on a sequencing gel, electrophoretically transferred to a nylon membrane, and covalently bound by means of uv irradiation. In an independent experiment, a short stretch of DNA flanking the area of interest is cloned in Ml3 or another single-stranded vector in order to produce a radioactively labeled single-stranded probe which is used for indirect endlabeling (hybridization) to either the lower or upper strand of the filter bound genomic DNA. Highly diluted cloned DNA is used as described above for control reactions.

traduces the possibility (21,22 and references therein) of first amplifying a limited sequence within the DNA under stringent conditions and then using, for example, the dideoxynucleotide chain termination method. This would have the advantage of eliminating the need to transfer DNA to a membrane. COMPARISON I’i’ivo

OF

THE

DIFFERENT

SYSTEMS

OF

TABLE

Organism E. coli Yeast Drosophila Chicken Mammal Mais Lily

1

of Various

Number of base pairs 4X 1.35 x 1.65 X 2x 5 x 1.5 x 3 x

LOWER STRAND

Cells Relative complexity

lo6 lo7 10’ lo9

1 3.3 41 500

lo9

1,250

1O’O 10”

3,750 75,000

c5- c5

1

8

FIG. 2. Indirect end-labeling. For a given restriction fragment (RF) there are four possible probes (boxes numbered l-4). The diagram shows examples of the products of the C-specific sequencing reaction. By using the probes l-4 the fragments designated by S will hybridize. The DNA fragments created by more than one cut and represented by the wavy lanes will not hybridize with the labeled probe (1 and 3, open boxes; 2 and 4, filled boxes).

tissue was treated with either dimethyl sulfate or uv light. After the chemical reaction is quenched, DNA is isolated and further processed in vitro. When the dimethyl sulfate reaction is used it is essential to optimize the incubation conditions for each cell system, i.e., concentration of dimethyl sulfate, temperature, and time of the reaction. This procedure has been used in studies with both procaryotes and eucaryotes (9-18) and the results show that a protein or protein complex that is very tightly bound to DNA leads to little if any reaction of guanosine residues with dimethyl sulfate. This lack of reaction provides a protected area or “window” within the DNA sequence. By contrast, uv light, which has been used only with procaryotes and lower eucaryotes, shows a strong reaction at the pyrimidines that are protected by the protein (9,14,15,23). ANALYTICAL

In all the in. viva footprinting procedures published so far, a cell suspension from tissue culture or directly from

Content

c3 2 'RF 4

in

FOOTPRINTING

DNA

c5

I--

Cl -

c3n

CL

DNA

PROBLEMS

Isolation

Since restriction endonucleases are used to cleave total genomic DNA, it is important to begin by preparing highly purified DNA. Any contamination with carbohydrates, proteins, or phenol may inhibit the restriction endonuclease (for review see Ref. (30)). Degraded genomic DNA will increase the background in the sequencing tracks and should be avoided. Excellent results have been obtained by first isolating nuclei from tissues or cells and then digesting the nuclei with proteinase K in the presence of 0.5% sodium dodecyl sulfate. To prevent the formation of clumps of undigested material, nuclei should be resuspended in sucrose buffer prior to the addition of the SDS buffer. To avoid degradation of DNA by nucleases during the isolation of nuclei, spermidine

GENOMIC

SEQUENCING

AND

and spermine (19) were added to the buffers. The addition of low concentrations of detergents (Triton X-100, 0.05%; or NP40, 0.5%) to the sucrose buffer facilitates proteinase K treatment by allowing the soluble nucleoplasmic proteins to leak out during the preparation of the nuclei. Clearly, it would be inadvisable to add detergents if the nuclei were required for in vitro footprinting. Restriction

203

FOOTPRINTING

C

GATC

H i-i-i?i

R

Digest of Genomic DNA

In order to obtain a subset of identical DNA fragments containing the target sequence, it is essential that the genomic DNA is digested. Incomplete digests will drastically impair the resolution of a genomic sequencing ladder. The best results were obtained when genomic DNA was digested overnight at the temperature recommended by the supplier and with a 3- or IO-fold excess (units/ pg DNA) of restriction enzyme (6-bp cutters and 4-bp cutters, respectively). The amount of DNA to be digested for genomic sequencing depends on the size of the genome and the copy number of the gene to be studied. For example, to arrive at a strong hybridization signal, a single-copy gene from a genome of 2 X lo6 kb (avian, see Table 1) will require approximately 25 pg of genomic DNA for each lane of the sequencing gel. The most suitable enzymes are those which yield DNA restriction fragments of approximately 1000 bp. Shorter or longer fragments can also be used providing that the genomic fragment (G,) is equal to or greater than P, + E,, where P, is the length of the probe in nucleotides and En is the size exclusion of the gel in nucleotides. If shorter DNA fragments are used, a second superimposed sequencing ladder will be created. On the other hand, we have successfully tested fragments of over 2000 bp in length. In ideal genomic sequencing experiments the sites of interest should be situated at least 50 bp and at the most 270 bp from one end of the restriction fragment (Fig. 3). A more detailed explanation of the theory of genomic sequencing is provided elsewhere (19). Chemical Reactions

600-w” :‘E

630-

640-

pL x-c

FIG. 3. Example of a genomic sequencing result. The left panel shows four control reactions (G,A+G,T+C,C; upper strand) performed using cloned vitellogenin DNA from the estrogen-receptor interacting area of the chicken vitellogenin II gene. The right panel shows the methylation state of three CpGs (arrowheads) in C-tracks (C) of different genomic DNA of hen (H) and rooster (R): 0, oviduct; L, liver; E, erythrocytes (4).

in Vitro and in Vivo

The three principal steps in all chemical sequencing procedures are essentially the same: modification of a base, removal of this base from its sugar, and the piperidine-induced cleavage of the sugar-phosphate backbone at this position. The first steps are base specific, random, and limited. The p-elimination cleavage step is quantitative (5). The thymidine-specific reaction (6) is necessary only when a strand-specific methylation pattern is studied. 5Methylcytosine can be distinguished from cytosine by its lack of reaction with hydrazine resulting in the absence of a band in the C-specific sequencing lane (3). Thymidine-specific sequencing reactions have to be performed to ascertain that the absence of a band in the

sequence represents 5-methylcytosine and not a thymidine resulting from the deamination of 5-methylcytosine. Methylated cytosines, however, can be detected directly using the permanganate reaction described by Fritzsche et al. (7). One of the major differences between the Maxam and Gilbert sequencing procedure and genomic sequencing is the amount of DNA used in each chemical reaction. In genomic sequencing reactions, a much higher concentration of unlabeled DNA is required to guarantee a strong hybridization signal and to reduce the number of cuts per unit length of DNA. The number of cuts per target DNA molecule directly affects the number of bases that can be read on the sequencing ladder. Ideally, there

204

SALUZ HEN /

U CTAG

AND

JOST

By incubating uv-irradiated DNA with piperidine at 90°C the DNA backbone is cleaved quantitatively at the so-called PydC photoproduct (9). These photoproducts occur at T-C, C-C, and, to a lesser extent, T-T dinucleotide residues. Since these photoproducts contain a pyrimidine saturated at its 5,6 bond, they can be detected by NaBH, acidic aniline technique (9). This method has been used successfully to detect the in uiuo interaction of the lac repressor and the lac operator in Escherichia coli (9,lO) and the interaction of the GAL 4 protein at sites within upstream activating sequences in yeast (13-15).

12345G

Gel Electrophoresis

d= ‘_ I 6

FIG. 4. Example of an in viva DMS footprint. The figure represents the upper strand (Ul of the chicken vitellogenin II gene promoter area. The four different controls are labeled C, T, A, and G. A decreasing concentration of DMS (lanes 1-5: 0.5, 0.05, 0.005, 0.0005, and 0.00005%, respectively) was used to reveal the details of the footprint made with hen liver cells in suspension. The stars indicate the positions of two CpGs. B2 represents a DNase I hypersensitivity site (24).

should be about one cut per genomic restriction fragment or, more precisely: 1 cut/E, + P,,. A greater number of cuts may result in additional bands that fail to contribute to the sequence and would lead to great difficulty in interpretation (19). Most investigators have used a fixed concentration of 0.5% dimethyl sulfate for in uivo footprinting (10-12,1618). In our hands, the concentration of dimethyl sulfate proved to be critical if a fine resolution footprint was to be obtained and we routinely use several concentrations of dimethyl sulfate for each trial (see Fig. 4 and Ref. (24)). With 0.5% dimethyl sulfate almost all bands corresponding to guanosine residues were visible on the Xray film and only very few were protected, presumably by very stable complexes with binding proteins. Lower concentrations of dimethyl sulfate, however, led to major differences between active and inactive genes (24). Furthermore, we found that upper and lower DNA strands had different sensitivities to the same high concentration of dimethyl sulfate (0.5%).

Millions of different fragments of genomic DNA produced by the restriction digest and the chemical sequencing reactions are separated by size on polyacrylamide gels. Nonspecific nicks within the target sequence increase the background and reduce the band definition. Gels of up to 1 m long improve resolution by increasing the distance between the bands; the background due to nonspecific degradation products or hybridization mismatches is thereby diluted. Excellent resolution, up to 300 bases, was obtained with l-m-long 8% sequencing gels with a ratio of 29:l of acrylamide:bisacrylamide (25). Electrotransfer

to Nylon Membranes

Many factors influence the transfer of DNA to nylon membranes: electrical resistance, strength of the electrical field, diffusion, Joule heating, and stability of the buffer. Most of the transfer systems described in the literature (26-29) are not suitable for genomic sequencing. A high resolution electrotransfer of the sequencing ladder requires an absolutely tight contact between the gel and the nylon membrane. Trapped gas bubbles in the transfer system disturb the electrical field and result in the distortion or loss of DNA bands in the blot. The electrical field, therefore, must be sufficiently low to avoid buffer electrolysis, and sufficiently high to ensure quantitative, diffusion-free transfer of DNA. The buffer capacity has to be chosen such that the pH is constant throughout the transfer. Excellent results were obtained with the system shown in Fig. 5. The distance between the two electrodes is only about 3 cm so that a current of 1.2 to 2 A requires only 32 to 35 V for quantitative transfer of the DNA within 30-35 min at room temperature in 1X Tris-borate-EDTA buffer (19). Immobilization

of DNA

on Nylon Membranes

Two types of membranes suitable for nucleic acid hybridization are commercially available: nylon and nitrocellulose filters. The nylon membranes are generally preferred because of their greater physical strength. Ge-

GENOMIC

SEQUENCING

AND

205

FOOTPRINTING

a

transfer system as modified from VaesFIG. 5. The electrophoretic sen et&. (1981). Place the lower sieve plate electrode (cathode) on four Teflon cubes in a plastic box. Then add a layer of Whatman 17 paper, the sequencing gel, the immobilizing membrane matrix, a second layer of Whatman 17 paper, and the upper sieve plate electrode (anode). A weight placed on the top of the anode ensures a tight contact between the gel and the membrane (15,25).

-

b

/ X AiB -

~-L--l

R

nomic sequencing requires nylon membranes of highly homogeneous composition. Unfortunately, the quality of commercially available membranes varies from one batch to another. Such variation seems to be less important for Southern or Northern blotting. However, where the limits of detection need to go as low as a few femtograms of nucleic acid, as in the case of genomic sequencing, even slight changes in local binding capacity of the filter will result in irregularity in the strength of the hybridization signal. We have tested three different types of membranes for their ability to bind DNA stably: Gene Screen, Zeta-Probe, and Millipore membranes. Since Gene Screen membranes gave the lowest background (25) on autoradiography for a given hybridization signal, it has been used throughout our work (4,19,24,25). DNA is usually fixed to the nylon by uv irradiation. Figure 6 shows the relationship between the amount of filter-bound DNA and the strength of the hybridization signal; above a certain level of covalent binding, the hybridization signal decreases (25). The Hybridization

Probe

Choosing the length of the hybridization probe is important as this governs the number of bases that can be

;;!

10

z

DNA

30 BOUND

50 TO

FILTER

70

90 %

MEMBRANE

FIG. 6. Relationship between the amount of filter-bound DNA and the strength of the hybridization signal. The experiment was carried out with Gene Screen membranes and the labeled DNA was crosslinked to the membrane by means of a combination of uv light and heat treatment. A-D represent different buffer conditions, for details see the original work (25).

C

GEL

*

’ -

loo0 R

FIG. 7. Choice of the hybridization probe when there is only one cut (X) per target restriction fragment. In example (a) Fragment A, created by the sequencing reaction, is separated on a gel, transferred to a filter membrane, and after hybridization with the probe (+++) appears as a band in the sequencing ladder. Fragment B can also be hybridized, but because of its size it will stay on top of the gel. In (b) the indirect end-labeling is not correct: The probe (e++) hybridizes only with fragment B which remains on top of the gel and does not contribute to the sequence. Fragment A cannot hybridize with the probe. Example (c) shows that for a restriction fragment R of 1000 bp, a size exclusion of the gel of 300 bp (0 - 0 - 0) and one cut per target DNA fragment (hypothetical case), the labeled probe (l-*) should not exceed 700 nucleotides.

read from the sequence ladder (Fig. 7). The maximal length of the probe depends on the exclusion size of the sequencing gel, on the size of the restriction fragment to be studied, and on the number of chemical cleavages within the target sequence (19). In the theoretical case of only one cut per genomic restriction fragment, no background bands will be generated as long as P, < G, - E,. Only fragments smaller than or equal to E, are resolved by the gel. The use of a longer probe (P, > G, - E,) results in a second superimposed sequencing ladder. Since more than one cut per unit length of DNA may occur in practice, probes of only about 100-180 nucleotides should be used to guarantee an optimal length of readable sequence. In genomic sequencing and footprinting, singlestranded DNA or RNA probes have been used (3,4,919,25,26). A review on the different methods of labeling hybridization probes has recently become available (30). Regardless of which procedure is used, it is important that the probe has both high specific radioactivity and sequence homogeneity (19,31). According to Anderson and Young (32), an increase in the concentration of sin-

206

SALUZ

gle-stranded nucleic acid probes in solution results in an increase in both the rate of hybridization and the amount of hybrid formation. They suggest that 100 ng of 32P-labeled probe/ml of hybridization mixture is the upper limit for the concentration of the labeled probe. At higher concentrations, the probe DNA will bind irreversibly to the membrane. We have routinely used up to 32 ng of highly purified labeled probe per milliliter of hybridization buffer. To obtain the same mole equivalent of pure probe by using a nonfractionated, labeled Ml3 vectors probe one would have to exceed the limit of 100 ng 32P-labeled material/ml of hybridization mixture. Hybridization The optimal temperature of hybridization membranes was determined both empirically the equation of Meinkoth and Wahl(33), T, = 16.6(log M) + 0.41(%G

+ C) + 815°C

on nylon and from

- 500/n,

where M is the molarity of the monovalent cations and (%G+C) is the percentage of guanosine and cytosine residues in the hybridization probe. This equation can be used for duplexes of a minimal length of 50 nucleotides in which n is the length of the shortest chain in the duplex. Usually n is between 50 and 60 nucleotides for genomic experiments. The term 500/n is, therefore, equal to between 8 and 10. Knowing that the hybridization rate increases up to a maximal level of about 25°C below the T, of DNA-DNA annealing (34), an approximate hybridization temperature (HT) for use in genomic sequencing experiments is given by the following formula (19), provided that the original buffer described by Church and Gilbert (3) is used: HT = 44.4”C + 0.41(%G (tested for sequences

+ C);

with

(G + C) content

We have observed that 58°C was sufficient iments. Processing

of the Hybridization

of 30-47%). for our exper-

Filters

The two-buffer system described by Church and Gilbert (3) is ideal for washing filters under stringent conditions, in which the sodium ion concentration is kept at 74 mM in the more stringent buffer. The optimal washing temperature was found to be approximately 20°C below the calculated T,. This allowed the determination of a general formula for the processing temperature (WT) of genomic sequencing membranes upon hybridization with DNA probes:

AND

JOST

WT = 33°C + O.Ol(%G

+ C).

We have observed that a WT of 48°C was sufficient for most experiments; all sequences tested at this temperature had a G + C content of 30 to 47%. Additional details have been provided (19). Autoradiography It is tempting to use a very sensitive X-ray film, e.g., XAR-5 from Kodak, when weak signals are encountered, without considering that the large size and low density of the silver halide crystals in such a film will reduce the resolution of the autoradiograms. For this reason, X-ray films of an intermediate sensitivity are recommended so as to attain very good resolution (X-OMAT S from Kodak or equivalent). It is interesting that, without an intensifying screen, the Kodak direct exposure film DEF5 that is used for X-ray diffraction studies is two to three times more sensitive than X-OMAT AR films or four to six times more sensitive than X-OMAT S film from Kodak. Owing to its very thin layer and high density of silver halide crystals DEF film has a power of resolution comparable to that of X-OMAT S film. Sensitivity of the film can be increased by preflashing and using intensifying screens (exposure at -40 to -90°C) (35). The sensitivity can be enhanced another two- to fourfold by baking the film at 65°C in an 8% “forming” gas environment (8% Hz + 92% N,) as suggested by Philips et al. (36) and Smith et al. (37,38). Since heating and preflashing act on the film in different ways their effects should be additive. FUTURE

DEVELOPMENTS

It is clear that at present genomic sequencing is a rather complex procedure and that new, less time-consuming techniques will be developed. Two major problems can account for the present difficulties in obtaining high quality genomic sequences and in viuo footprints in higher eucaryotes: the high dilution of the single copy genes and the unreliable quality of the nylon membranes. One way to circumvent the first problem is to increase the copy number of the gene of interest. The second problem can be solved by avoiding the use of membranes. With the availability of the thermostable enzyme Taq DNA polymerase it is now possible to increase the copy number of a gene by many orders of magnitude in uitro and then use, for example, the strategy outlined by Saiki et al. (21). This approach could be very useful for studying point mutations of any given gene without cloning. We also think that with appropriate modifications the same approach could be used for the study of DNA methylation and protein-DNA interactions in vivo. In all cases, these new techniques would circumvent the difficulties described.

GENOMIC

SEQUENCING

Novel in viuo footprinting procedures can be expected. For example, it is conceivable that the in vitro hydroxyl radical footprinting described by Tullius and Dombroski (39) could also be used in vivo. Other techniques using cis- and trczns-diaminedichloroplatinum (II) (40) or formaldehyde (41) to crosslink chromosomal proteins with DNA in viuo offer interesting alternatives for study of protein-DNA interactions. It is likely that there will be additional modifications to in uivo footprinting with uv light, although, as described above, uv light can be used only to detect proteins bound tightly to DNA (9), whereas less tightly associated proteins might go undetected. Becker and Wang (9) have developed an alternative photochemical technique which may be more sensitive for proteins binding less tightly to DNA. One of the techniques involved a triplet sensitizer, P-dimethylaminopropiophenone (P-DAP).’ When excited by 315-nm light in vivo, @-DAP can transfer its excitation energy to DNA, causing the DNA to assume an excited triplet state (9,42). This triplet state produces many of the same photoproducts formed by irradiation of DNA with uv light and allows detection of proteins that are weakly bound to DNA. Another small organic molecule that can be used to footprint DNA photochemically is the intercalator psoralen (9). When excited by light at 350 nm, psoralen becomes covalently linked to DNA (9,43,44). Because covalent binding of psoralen saturates the 5,6 double bonds of pyrimidine (9,45), the chemical labilization procedure used to detect, uv- and sensitizer-induced photoproducts can also be used to detect. psoralen binding (9). The main problem for the use of such techniques in uivo is the permeability of cells to the chemicals. In addition, the chemicals should not alter the native state of the chromatin or kill the cells. Meanwhile, we should remain open to any alternative techniques to study the specific interactions of proteins with DNA in vivo. APPLICATIONS

Direct genomic sequencing is the only technique presently available that allows the methylation state of any given cytosine in DNA to be studied. It also offers a powerful means of studying specific protein-DNA interactions in uiuo and permits the identification of mutations without prior cloning of the mutant gene in a bacterial host. Thus, this technique could be used for the diagnosis of genetic disorders, the detection of pathogens, and the analysis of allelic sequence variations. With the newly introduced method of gene amplification using Taq thermostable DNA polymerase (22 and references therein) it will be possible to overcome many difficulties presented by the original procedure of Church and Gil-

AND

207

FOOTPRINTING

bert (3) and open a new road for a quick early prenatal diagnosis of genetic disorders. ACKNOWLEDGMENTS We are grateful to I. McEwan, I. M. Feavers, M. Hughes, and H. Moser for critically reading the manuscript.

REFERENCES 1. Ward, C. A., Bolden, A. H., Nalin, J. Biol. Chem. 262,11057-11063.

used: fi-DAP,

fi-dimethylaminopropiophenone.

C. M., and Weissbach,

2. Bolden, A. H., Nalin, C. M., Ward, Weissbach, A. (1986) Mol. Cell. Biol. 3. Church,

G. M., and Gilbert,

A. (1987)

C. A., Poonian. 6, 1135-1140.

W. (1984)

Proc.

Nat/.

M.

Acad.

S., and Sci. USA

81,1991-1995. 4. Saluz, H. P., Jiricny, Sci. USA 83,.7167-7171.

,J., and Jost,

5. Maxam, A. M., and Gilbert, (Grossman, L., and Moldave, demic Press, New York. C. M., and Schmid, 6. Rubin. 4613-4619.

d. P. (1986)

Proc.

Natl.

Acad.

W. (1980) in Methods in Enzymology K.. Eds.), Vol. 65, pp. 499-560, AcaC. W. (1980)

Nucleic

Acids

Res. 8,

I. Fritzsche, E.. Hayatsu, H., Igloi, G. L., Iida, (1987) Nucleic Acids Res. 15, 5517-5528. ,J.. and Vassilenko. S. (1978) Nature 8. Stanley, 89.

S.. and Koessel, (London)

274,

9. Becker, 687.

(London)

309,682-

10. Nick,

M. M., and Wang, H., and Gilbert,

11. Ephrussi, A., Church, Science 227,134-140.

J. C. (1984)

W. (1985)

Nature

Nature

S., and Gilbert,

12. Becker, P. B., Gloss, B.. Schmid, W., Straehle, G. (1986) Nature (London) 324, 686m688. 13. Giniger, E., and Ptashne. 85,382-386. 14. Selleck, 177. 15. Selleck,

S. B.. and Majors,

16. Gimble,

d. M., and Max,

S. B., and Majors,

M.

(1988)

Proc.

J. (1987)

Natuw

J. (1987)

Mol.

E. E. (1987)

87-

W. (1985)

U., and Schuetz,

Natl.

Acad.

(London)

Cell. Biol.

Mol.

H.

313,795-798.

(London)

G. M., Tonegawa,

Cell. Biol.

Sci. 325,

USA 173-

7, 3260-3267. 7, 15-25.

17. Pauli, U., Chrysogelos, S., Stein. G., Stein. J., and Nick, H. i 1987) Science 236,1308-1311. 18. Becker, P. B., Ruppert, S., and Schuetz. G. (1987) Cell 51, 435443. 19 Saluz, H. P., andJost, J. P. (1987) A Laboratory Guide to Genomic Sequencing, Birkhaeuser Verlag, Basel/Boston. 20 Huibregtse, J. M., and Engelke, D. R. (1986) Gene 44, 151-158. 21. Saiki, R. K.. Scharf, S., Faloona, F., Mullis. K. B., Horn, G. T., Erlich, H. A.. and Amheim, N. (1985) Science 230,1350-1354. 22. Erlich, H. A., Gelfand, D. H., and Saiki, R. K. (1988) Nature (London)33 1,461-462. 23 Cullis. P. M., -Jones, G. D. D., Symons, M. C. R., and Lea. J. S. (1987) Nature (London) 330,773-774. 24. Saluz, H. P., Feavers, I. M., Jiricny, J., and Jost, J. P. (1988) Proc. N&l. Acad. Sci. USA 85,6697-6700. 25. Saluz, H. P., and Jost. J. P. (1986) &ne 42, 151-157. 26. Towbin, H., Staehelin, Sci. USA 76,4350-4354.

’ Abbreviation

J. Jiricny,

27. Gibson,

W. (1981)

Anal.

T., and Gordon, Biochem.

d. (1979)

118,1-3.

Proc.

Natl.

Acad.

208

SALUZ

28. McCellan, T., and Ramshaw, J. A. M. (1981) Biochem. Genet. 19, 647-654. 29. Shuttleworth, A. D. (1984) Electrophoresis 3, 178-179. 30. Mattews, J. A., and Kricka, L. J. (1988) Anal. Biochem. 169, l25. 31. Jackson, P. D., and Felsenfeld, G. (1987) in Methods in Enzymology (Wu, R., and Grossman, L., Eds.), Vol. 152, pp. 735-755. Academic Press, Orlando, FL. 32. Anderson, M. L. M., and Young, B. D. (1985) Quantitative Filter Hybridization: Nucleic Acid Hybridization (Hames, B. D., and Higgins, S. J., Eds.), IRL Press, Oxford/Washington, DC. 33. Meinkoth, J., and Wahl, G. (1984) Anal. Biochem. 138,267-284. 34. Marmur, J. G., and Doty, P. (1961) J. Mol. Biol. 3,584-594. 35. Laskey, R. A., and Mills, A. D. (1977) FEBS Lett. 82,314-316. 36. Phillips, C. A., Smith, A. G., and Hahn, E. J. (1986) Proceedings from the Second International Symposium on the Synthesis and Application of Isotopically Labeled Compounds (Muccino, R. R., Ed.), pp. 189-194, Elsevier, New York.

AND JOST 37. Smith,

A. G., Phillips,

C. A., and Hahn,

E. J. (1985)

J. Zmag.

Z’ech-

nol. 11,27-32. 38. Smith, A. G., Phillips, C. A., Hahn, AAS Photo-Bull. 39,8-14.

39. Tullius, USA

T. D., and Dombroski, 83,5469-5473.

E. J., and Leacock,

B. A. (1986)

40. Banjar, Z. M., Hnilica, L. S., Briggs, (1984) Biochemistry 23,1921-1926. 41. Salomon, M. J., and Varshavsky, USA 82,6470-6474. 42. Meistrich, Photobiol. 43. Musajo, 27-35.

M.,

Lamola,

R. C., Stein, A. (1985)

A. A., and Gabbay,

Proc.

Natl.

Acad.

Sci.

J., and Stein, N&l.

E. (1970)

Acad.

G. Sci.

Photochem.

11, 169-178. L., and Rodighieno,

44. Cole, R. S. (1970) 45. Musajo, Photobiol.

Proc.

R. J. (1985)

Biochim.

L., Bondin, 6,927-931.

G. (1970) Biophys.

F., and

Photochem. Acta

Bevilacqua,

Photobiol.

217,30-39. R. (1967)

Photo&em.

11,