Chapter 1 Nzlcleic Acid Hyhidixation to the D N A of Cytological Preparations MARY LOU PARDUE
AND
JOSEPH G. GALL
Department of Biology, Massachusetts Institute of Technology, Olmbridge, Massachusetts and Depamnent of Biology, Yale University, New Haven, Connecticut
Introduction . . . . . . . I. Procedure for in Situ Hybridization . . . A. Fixation and Squashing of Tissue . . . B. Removal of EndogenousRNA . . . C. Denaturation . . . . . . D. Hybrid Formation . . . . . E. Removal of NonspecificallyBound Nucleic Acid F. Autoradiography . . . . . . G. Staining Preparations after Autoradiography . 11. Equipment and Reagents . . . . . 111. Radioactive Nucleic Acids for Hybridization . . IV. Conditions of Reaction . . . . . V. General Comments . . . . . . References . . . . . . . .
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Introduction Techniques for annealing two molecules of single-stranded nucleic acid and characterizing the resulting hybrid molecule make it possible to compare nucleic acid sequences even though the actual order of nucleotides t Supported by research grants GB-35736 from the National Science Foundation and GM-12427 from the National Institute of General Medical Sciences.
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MARY LOU PARDUE AND JOSEPH G . GALL
in the sequences may not be known. When combined with cytological procedures that permit in situ detection of the cellular or chromosomal localization of the hybridized nucleic acid, these techniques can be used in several ways to study the organization and function ofthe genetic material in higher organisms. (1) In situ hybridization to condensed chromosomes can be used to map the sites of DNA sequences that have not been mapped by genetic breeding experiments. (2) Just as experiments with known nucleic acid sequences in the hybridizing solution can be used to identify chromosomal sites, hybridization to known chromosomal regions can be used in characterizing the nucleic acid sequences in the hybridizing solution. (3) Hybridization to cytological preparations gives information on the functional organization of particular sequences in the diffuse chromatin of interphase nuclei. This permits comparison of the organization in cells of different types. (4) Because cytological techniques permit analysis at the level of single cells, in situ hybridization can be used to obtain preliminary evidence on gene amplification or underreplication in single cell types in tissues composed of several cell types or cell stages. ( 5 ) The distribution of specific RNA species within a cell or in mixed cell populations can be studied by hybridization with DNA transcribed in vitro from that RNA by RNA-directed DNA poly merases. Several years ago we published a general review of techniques for in situ hydridization (Gall and Pardue, 1971). Since that time we have continued to use and study in situ hybridization and have made several changes in the procedures described in our earlier article. Because there are a number of reviews on nucleic acid hybridization in vitro (Wetmur and Davidson, 1968; Walker, 1969; McCarthy and Church, 1970; Birnstiel et al., 1972; Bishop, 1972; Britten et al., 1974), and on techniques for in situ hybridization (Jacob et al., 1971; Harrisonetal., 1973;Hennig, 1973;Jones, 1973;Wimber and Steffensen, 1973), this article describes only the procedures that have been used in our laboratories.
I. Procedure for in Situ Hybridization
A. Fixation and Squashing of Tissue Many conventional cytological procedures can be used to make slides that give good results in in situ hybridization experiments. However, fixatives containing formaldehyde should not be used, since they apparently interfere with denaturation of the DNA. The best preparations are those that are well spread and very flat.
1. NUCLEIC ACID HYBRIDIZATION TO DNA
3
The following procedures are the ones we routinely use to make slides for
in situ hybridization.
1. FORSMALL PIECES
OF
TISSUE
Fresh tissue is teased into very small pieces not more than 5 mm in their longest dimension. It is then fixed for 5-10 minutes in freshly mixed ethanolacetic acid (3 : I). Next a small drop of 45% acetic acid is placed on a siliconized 18-mm2 cover slip. A small piece (less than 1 mm’) is teased from the fixed tissue and transferred to this drop. Because addition of the fixative to 45% acetic acid causes violent mixing, the tissue is held for a few seconds in air to permit most of the fixative to evaporate before being placed in the acetic acid. However, the morphology may be ruined if the tissue becomes too dry in this step. The tissue is thoroughly minced in the drop of acetic acid, and any large pieces that remain are removed. Then a “subbed” slide is carefully lowered onto the cover slip, and the cells are squashed. A simple way to squash the cells is to place the slide on filter paper with the cover slip side down and apply firm thumb pressure on the back of the slide over the cover slip. Some tough tissues will give a better preparation if they are allowed to soften on a 45”C warming plate for 3-5 minutes before being squashed. The flattest squashes tend to have the best preservation of morphology during the hybridization steps, so it is wise to use small amounts of tissue and to remove any bits of material that might interfere. After squashing, the slides are placed on a flat surface of Dry Ice for a few minutes. When the preparation is completely frozen (5-10 minutes), the cover slip is flipped off with a razor blade and the slide is immediately placed in 95% ethanol. After the slide has been in ethanol for at least 5 minutes, it is air-dried and stored. Slides can be stored dry for very long periods of time, although the level of hybridization tends to drop after several months. The level of hybridization drops much more rapidly if the slides are stored in ethanol.
2. FORDROSOPHILA SALIVARY GLANDS The glands are dissected in insect Ringer’s solution and transferred to a small drop of 45% acetic acid on a siliconized 18-mmzcoverslip. After the glands are allowed to fix briefly in the 45% acetic acid, asubbed slide is lowered onto the cover slip m d the glands are squashed. There are many techniques for squashing salivary glands. The cover slip can be tapped with the point of a pencil, or with the eraser end, or it can be pressed with pointed forceps. The initial steps in squashing salivary glands are intended to spread out the chromosomes and should be checked in the phase micro-
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MARY LOU PARDUE AND JOSEPH G. GALL
scope. When a satisfactory degree of spreading is obtained, the slide is placed on filter paper with its cover slip side down and firm thumb pressure is applied to the back of the slide over the cover slip. The slide is then placed on a flat piece of Dry Ice until the preparation is frozen. Next the cover slip is flipped off with a razor blade, and the slide is immediately plunged into freshly made ethanol acetic acid (3: 1) for fixation. After 2 minutes fixation in 3: 1, the slide is transferred to 95% ethanol for at least 10 minutes and air-dried. Salivary gland slides may be stored dry as noted for tissues squashed by the procedure described in Section I,A,l. A slightly different technique for making squashes of Drosophilu salivary glands is given by Atherton and Gall (1972). 3. FORCELLSUSPENSIONS Tissue culture cells are gently spun out of the medium and resuspended in a hypotonic solution composed of 1 part medium and 3 parts distilled water. The amount of hypotonic solution added should be about 40 times the volume of the cell pellet. After being resuspended very gently in the hypotonic solution, the cells are left for 5 minutes at room temperature. The cells are gently spun down and the hypotonic solution is replaced with an equal volume of 50% acetic acid to fix the cells. The acetic acid is added without disturbing the pellet, and the cells are left on ice for 20 minutes. After the fixation the acetic acid is removed. A drop or two of fresh 50% acetic acid is added and the cells are gently resuspended. Squashes are made by putting a small drop of the fixed cell suspension on a siliconized cover slip and completing the squashing as described for small pieces of tissue in Section I,A,l .
B. Removal of Endogenous RNA Endogenous RNA, which might act as a competitor for the hybridizing RNA, is removed by treating the squashes with pancreatic RNase (EC 2.7.7.16). The RNase is dissolved in 2 x SSC at a concentration of 100puglml. Each slide is placed in a moist chamber prepared from a plastic petri plate (Fig. l), 200 p1 of the RNase solution is placed over the preparation, and the RNase solution is covered with a 22 x 40 mm cover slip. The moist chambers can be held at 37°C for 1 hour or at room temperature for 2 hours. After the RNase treatment the cover slips are gently removed by dipping the slide into a beaker of 2 x SSC to float off the cover slip. Slides are then washed three times in 2 x SSC, once in 70% ethanol, and once in 95% ethanol before air-drying.
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NUCLEIC ACID HYBRIDIZATION TO DNA
5
Solution of RNA-3H or DNA-3H
Coverslip
q
Microscope slide
-- PIas t ic petri dish
Hybridization buffer
/
\
Rubber washer or grommet
FIG 1. Moist chamber used in cytological hybridization procedure.
C. Denaturation All the agents, such as alkali, heat, and organic solvents, that denature purified DNA also denature the DNA of cytological preparations, as measured by in situ hybridization. Acid treatments also denature DNA on slides but give a reduced amount of in situ hybridization. Chromosome morphology is excellently preserved during acid denaturation, but the acid treatments that denature DNA in cytological preparations also produce some depurination (M. L. Pardue and H. C. Macgregor, unpublished). In our laboratories a1kali denaturation consistently gives the best results. Slides are placed in 0.07 NNaOH at room temperature for 2-3 minutes to denature the DNA of the preparation. This is followed by three changes of 70% ethanol over a period of about 10 minutes and two washes of 95% ethanol of about the same duration. The slides are again air-dried. Preparations that suffer loss of morphology during this step usually have not been squashed flat enough.
D. Hybrid Formation Our preliminary experiments to study the parameters of in situ hybridization have indicated that they should be similar to those in hybridizations with DNA fixed to nitrocellulose filters. Therefore conditions of ionic strength, nucleic acid concentration, temperature, and length of time for hybridization are chosen for each experiment as one would choose them for filter hybridization experiments. The conditions chosen determine the stringency of the hybridization conditions and set limits on the amount of sequence mismatching that can occur.
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MARY LOU PARDUE AND JOSEPH G . GALL
When performing hybridization at high temperature, we ordinarily use an SSC or an SNB buffer at a concentration determined by the amount of sodium ion desired. For example, reactions can be done in 2 x SNB at 65°C. Hybridizations can be done at lower temperatures by adding formamide to the reaction mixture. We frequently use 40% formamide (Matheson, Coleman, Bell; Norwood, Ohio) in 4 x SSC at 37°C. Addition of a large excess of nonradioactive, noncompeting nucleic acid helps to eliminate nonspecific binding of radioactive nucleic acid to the slide. The radioactive nucleic acid is placed over the preparation and covered with a cover slip. The slide is then placed in a moist chamber and incubated at the chosen temperature. Ordinarily we use 10-15 p1 of solution under an 18-mm2 cover slip. Much smaller volumes can be used with 9-mm2 cover slips if the moist chamber is sealed. We routinely do 10- to 15-hour incubations at 65°C without noticeable loss of liquid from under the cover slip. Although it is possible to seal the cover slip over the liquid, it is simpler to seal the moist chambers. Such a procedure eliminates the possibility that the hybridization medium will extract some components from the sealing mixture. Some brands of cover slips leach alkali into the incubation mix. It is useful to test a new brand of cover slip before using it. This can be done by doing a mock hybridization using the hybridization buffer plus phenol red under the cover slip. If this test shows that the cover slips are leaching alkali during the incubation, the cover slips can be boiled in 1 N HCl and rinsed well in pH 7 buffer before they are used.
E. Removal of Nonspecifically Bound Nucleic Acid 1. FORRNA-DNA HYBRIDS After the hybridization reaction the slides are removed from the oven. The cover slip and hybridization mix are immediately washed off by dipping the slide into a beaker of 2 x SSC. Slides are then washed in 2 x SSC at room temperature for 15 minutes before being incubated with pancreatic RNase (20 pg/ml in 2 x SSC) for 1 hour at 37°C. Finally, the slides are rinsed twice for 10 minutes in 2 x SSC and passed through 70 and 95% ethanol before being air-dried.
2. FORDNA-DNA HYBRIDS The cover slips are removed from the slides by dipping in 2 x SSC at a temperature 5" less than the temperature used for the hybridization reaction. Nonspecifically bound DNA is then removed by three additional 10-minute washes in 2 x SSC which is also 5" less than the hybridization temperature. Finally, the slides are washed in 70 and 95% ethanol and dried.
1. NUCLEIC ACID HYBRIDIZATION TO DNA
7
F. Autoradiography The dried slides are coated with autoradiographic emulsion by standard procedures. We use Kodak NTB-2 liquid emulsion diluted 1: 1 with distilled water. A thicker coating of emulsion on the slide would not increase the information gathered, since the beta particles emitted by 3H are not energetic enough to reach the upper part of the film. However, a thicker film would increase the number of background grains produced extraneously. Kodak NTB-2 is purchased in 4-oz bottles (1 12 ml) and is a solid at room temperature. It should be used only in absolute darkness or with a Kodak Wratten Series I1 or OA safelight. We heat each new bottle to 45°C for 30 minutes and then add it to 112 ml ofwarm, distilled water. The two solutions mix readily, and this step should be done carefully to avoid producing bubbles in the emulsion. Diluted emulsion is distributed into nylon scintillation vials in aliquots of about 10 ml. The vials are wrapped in aluminum foil and stored in a light-tight box in a refrigerator. Each vial contains enough emulsion for about 30 slides and is used only once. The emulsion will keep for many months. The caps used for the scintillation vials should not have cork inserts, since these can give off organic compounds which may produce background grains in the emulsion during storage. If one suspects that the emulsion may have picked up some background grains before use, it is wise to dip a test slide, dry it for 20 minutes, and then develop and check it before dipping the experimental slides. A good emulsion has 0.1-0.2 x grains per square micrometer. For use the emulsion is melted in a 45°Cwater bath for 10 minutes and then poured into a small plastic dipping chamber, taking care that no bubbles are formed. (A polyethylene two-place slide mailer available from most supply houses makes a good dipping chamber.) The dipping chamber is held at 45°C. Slides are dipped into the chamber, withdrawn slowly, drained, and placed vertically in a rack. Slides should dry in the dark for 2 hours. A nonsparking fan can be used to reduce the drying time to 1 hour. Dried slides are stored in light-tight plastic boxes with a small container of silica gel included to maintain dryness. The boxes are sealed with electrician’s tape and stored in lightproof containers in a refrigerator. Autoradiographic exposures for in sifu hybridization may range from a few hours to many days. It is wise to have several slides, so that test slides may be developed at intervals to determine the proper exposure time. After the appropriate exposure time, slides are developed for 2+ minutes in Kodak D-19, rinsed well in distilled water, and fixed for 2-5 minutes in
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MARY LOU PARDUE AND JOSEPH G. GALL
Kodak fixer. The slides are then rinsed through several changes of distilled water over a period of 30 minutes and stained before drying.
G. Staining Preparations after Autoradiography There are several ways of staining cytological preparations through autoradiographic emulsions. We use Giemsa stain. A stock solution of Giemsa blood stain is diluted approximately 1 :20 with 0.01 M phosphate buffer (pH 6.8) immediately before adding the slides. The staining time depends on the cytological preparation and on the batch of Giemsa stain. It is usually somewhere between 2 and 45 minutes. A metallic film forms rapidly over the surface of the Giemsa solution once it has been mixed with the buffer. Since this film will adhere to the slide if the slide is pulled out of the solution, the film is floated out of the staining dish with distilled water before removing the slides. The slides are rinsed with distilled water, airdried, and covered with a cover-glass mounted in Permount. Slides may also be left uncovered and observed under immersion oil. Petroleum ether is used to remove oil from unmounted slides.
11. Equipment and Reagents Subbed slides. Microscope slides are washed in either acid or detergent and thoroughly rinsed in distilled water. They are then immersed briefly in subbing solution and allowed to dry for several hours before being used. Subbing solution. This is an aqueous solution of 0.1% gelatin and 0.01% chrome alum [chromium potassium sulfate, CrK(SO,), 12H,O]. The gelatin is first dissolved in hot water, and the chrome alum is added after the solution cools. Siliconized cover slips. Cover slips are immersed briefly in a 1% solution of Siliclad (Clay-Adams; Parsippany, N.J.), rinsed thoroughly in distilled water, and allowed to dry overnight at room temperature. They are stored dry. Before using, the cover slips can be rinsed with 95% ethanol and dried. SSC. 0.15 M NaCl, 0.015 M sodium citrate; pH 7.0 SNE. 0.15 M NaCl, M tris; pH 7.0. Insect Ringer’s solution. 7.5 gm NaCl, 0.35 gm KCl, and 0.21 gm CaCl, in 1 liter of water. RNuse. Pancreatic RNase (EC 2.7.7.16) is dissolved at 1 mg/ml in 0.02 M sodium acetate (pH 5). This solution is placed in a boiling water bath for for 5 minutes, cooled, and stored at -20°C.
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NUCLEIC ACID HYBRIDIZATION TO DNA
9
FIG.2. Metaphase chromosomes hybridized with 18 and 28 S rRNA. The DNA sequences complementary to the rRNA are found at the secondary constrictions on the sex chromosomes in both karyotypes. The muntjac has an additional site of ribosomal sequences on the largest autosome. The specific activity of the rRNA-3H was 5 x lo6 dmplpg. It was hybridized in 2 x SNB at 65°C for 10 hours. (a) Bat, Olrolliaperspicillafa. Male karyotype. rDNA only on X chromosome. Exposure 37 days. x 1445. (b) Indian mutjac, Munriaarsmunrjak. Male karyotype. rDNA on X, Y,and one pair ofautosomes. Exposure, 75 days. x 1870. (FromT. C. Hsu, F. E. Arrighi, S. Spinto, and M. L. Pardue, unpublished.)
FIG. 3. Pachytene nucleus from the testis of the toad X la& hybridized with cRNA-’H transcribed in vitro from DNA coding for 18 and 28 S rRNA. In X.laevik the genes for 18 and 28 S rRNA are localized on one chromosome of the haploid complement, and thus the tightly paired homologous chromosomes of the pachytene nucleus show only one site of hybridization in this experiment. The cRNA-’H had a calculated specific activity of 10’ dpndpg. Hybridization was carried out in 2 x SSC for 10 hours at 65°C. Exposure, 41 days. ~ 2 2 0 0 . FIG. 4. Pachytene nucleus from the testis of the toad X. laevis hybridized with cRNA-’H transcribed in vitro from the DNA coding for 5 S rRNA. The ends of all of the chromosomes are oriented toward one side of the nucleus. The 5 S cRNA binds to DNA sequences located at the end of one arm of most, if not all, of the chromosomes. The cRNA-’H had a specific activity of 4 x lo7 dpmlpg. It was hybridized in 4 x SNB for 8 hours at 65°C. Exposure, 26 days. x 17%. (From Pardue er al., 1973.)
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MARY LOU PARDUE AND JOSEPH G. GALL
Moist chamber. (see Fig. 1). Moist chambers are made from 4 inch plastic petri plates. The bottom of the petri plate is lined with filter paper and contains 5-10 ml of the incubation buffer. The slide is supported above the liquid by two rubber grommets or a U-shaped glass rod. The solution in the bottom of the moist chamber must have the same salt concentration as the solution under the cover slip to prevent distillation and subsequent change in concentration. Glass petri plates do not make satisfactory moist chambers, because the moisture that condenses on the lid tends to coalesce into large drops which may fall onto the preparation. Moisture condensing on plastic lids remains in small droplets on the lid. Some types of plastic petri plates melt at temperatures used for hybridization reactions. Giemsa stain. We use commercially prepared stock solutions of Giemsa blood stain.
111. Radioactive Nucleic Acids for Hybridization The feasibility of detecting any particular DNA sequence by cytological hybridization depends on two factors, either of which may be the limiting one. The first factor is the sensitivity of detection. This depends on the amount of radioactivity in agiven region and the efficiency of the autoradiographic procedure. The second factor is the rate of hybridization, which depends on the concentrations of the sequences being studied. Thus in choosing a source of radioactive nucleic acid for in situ hybridization experiments, one should consider both the specific radioactivity of the nucleic acid and the amounts of nucleic acid that can be obtained for use in the hybridization medium. The minimum levels of radioactivity needed for a particular experiment are determined by the amount of nucleic acid bound at each chromosomal site. Repeated sequences clustered at a single chromosomal locus can be detected more easily than the same number of sequences scattered over sites on several chromosomes. In general, we find that lo6 d p d p g is the lower useful limit. There are several possible ways of obtaining nucleic acid with a specific activity greater than lo6 dprn/pg. 1. IN Vwo LABELED NUCLEIC ACID Cultured cell lines grown in a medium containing tritiated nucleic acid precursors can yield both DNA and RNA with sufficient tritium label for use in in situ hybridization (Gall and Pardue, 1971). If culture and labeling conditions are optimized for the particular nucleic acid fraction desired, both stable and rapidly turning over fractions can be obtained with specific
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NUCLEIC ACID HYBRIDIZATION TO DNA
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FIG. 5 . Spermatozoa from X laevis hybridized with cRNA-IH transcribed in vitro from DNA coding for 18 and 28 S rRNA. The rRNA genes are apparently packed into the sperm head in a tight cluster, but the position of this cluster is not constant from sperm to sperm. The conditions for hybridization are those given in Fig. 3. Exposure, 31 days. x 1400.
radioactivity of 106-107d p d p g . There are other biological systems that can be used to produce highly radioactive nucleic acids in vivo. For example, sea urchin embryos grown in sea water containing tritiated uridine during the early cleavage stages yield histone mRNA containing enough tritium label for in situ experiments (Pardue et al., 1972).
FIG. 6. Salivary gland chromosomes from Drosophila rnelanogaster hybridized in situ with 9 S mRNA-’H from the sea urchin Psmrnechinus rnilaris. These stretched chromosomal preparations show hybridization of the putative histone mRNA fractions to bands 39D and 39E, as well as the intervening interband region. The 9-S mRNA-IH was prepared from sea urchin blastulas grown in sea water containing uridine-’H. The RNA was hybridized in 4x SNB at 65°C for 10 hours. Exposure, 109 days. (a) x 1600; (b) ~ 2 6 0 0(From . M. L. Pardue, L. H. Kedes, E. H. Weinberg, and M. L. Birnstiel, unpublished.)
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MARY LOU PARDUE AND JOSEPH G. GALL
2. IN VITRO TRANSCRIPTION OF NUCLEIC ACID By far the most successful method for preparing extremely radioactive nucleic acid is by in vitro transcription. In cases in which a specific DNA fraction can be isolated, Escherichia coli RNA polymerase can be used in vitro to produce RNA copies of the DNA (Gall and Pardue, 1971). RNA fractions can be transcribed into DNA by RNA-directed DNA polymerases (Verma and Baltimore, 1974), or by DNA polymerase from E. coli (Loeb et al., 1973). The product of an in vitro transcription should be characterized carefully, since any of the polymerases may transcribe certain nucleic acid sequences preferentially. When a mixture of nucleic acids is transcribed, the product may not be equally transcribed from all fractions of the template. The specific radioactivity of nucleic acids obtained from in vitro transctiption is limited only by the specific activity of the nucleotide precursors. The highest specific activity precursors currently available yield a product with greater than lo* dpm/pg. OF PREFORMED NUCLEIC ACIDS 3. CHEMICAL LABELING
In cases in which the desired nucleic acid cannot be labeled either in vivo or by in vitro transcription, radioactive atoms can be added chemically. Although there are several methods for chemical addition of 3H to nucleic acids, none of the methods now in use yields the levels of radioactive labeling required for in situ hybridization. However, techniques have been developed for labeling nucleic acids with lz5I(Commerford, 1971;Prensky et al., 1973). The specific radioactivity obtained with lZ5Iis about equal to that produced by in vitro transcription with 'H precursors. Theoretically, *ZSI-labeled nucleic acids of even higher specific activity can be produced, but the characteristics of hybridization may be affected by the increased iodination (Commerford, 1971). The radiation emitted by lzSI is somewhat more energetic than that emitted by 3H. For this reason lz5I is more efficient than 3H for autoradiography, but gives less precise cytological localization.
IV. Conditions of Reaction We have assumed that in situ hybridization shares some of the kinetic properties of the nitrocellulose filter hybridization technique. Our experimental evidence is consistent with this assumption, and we use filter
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NUCLEIC ACID HYBRIDIZATION TO DNA
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FIG. 7. Salivary gland chromosomes from D. melanogaster hybridized in situ with cytoplasmic RNAJH. The RNA was prepared from the cytoplasmic fraction of cultured D. melanogaster cells grown in a medium with uridine-lH. These poly-A-containing sequences were isolated by oligo-dT chromatography and hybridized in 2x SNB at 65°C for 10 hours. This RNA binds to a reproducible series of chromosomal bands and to thep-heterochromatin of the chromocenter (c). Exposure, 35 days. (a) x 1100; (b) x 1400. (From A. Spradling, S. Penman, and M. L. Pardue, unpublished.)
hybridization experiments when necessary to optimize reaction conditions for in situ hybridization. In both in situ and filter hybridizations, one strand of the nucleic acid involved in the hybridization reaction is immobilized. We have not been able to detect either loss or renaturation of the DNA of the cytological preparation during the periods we have used for the annealing reaction, although there may be losses of DNA during the denaturation steps. Insitu hybridization does differ from filter hybridization in having extremely high local concentrations of DNA sequences and in having some of the protein components of chromatin still associated with the DNA. We have not yet
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MARY LOU PARDUE AND JOSEPH G. GALL
been able to assess the effect of either of these factors on the kinetics of hybridization in situ. Precise quantitation of in situ hybridization experiments has proved difficult for several technical reasons. One source of variation is the degee of compaction of the DNA in different nuclei. Whether the compaction affects the amount of denaturation of the DNA, the access of the hybridizing nucleic acid to chromosomal sites, or the autoradiographic efficiency, it makes quantitative comparisons between different types of nuclei questionable in some cases. In many studies it is preferable to use information from in situ experiments to design filter hybridization experiments for precise quantitation. Published in situ hybridization experiments have been calculated to have 5-1 5% efficiency of hybridization. Approximately the same efficiency is obtained whether the hybridization is to the diffuse amplified ribosomal genes in amphibian oocytes, to repeated sequences in heterochromatic regions of condensed chromosomes, or to repeated genes in euchromatic portions of chromosomes. The low efficiency of hybridization may be a reflection of the availability of the DNA in cytological preparations. However, it is also possible that none of the hybridizations were done with saturating concentrations of nucleic acid in solution. It is unlikely that the concentrations and times now used for in situ hybridization reactions would permit the detection of unique sequences in the DNA of diploid cells of higher organisms. However, unique sequences can easily be studied in polytene chromosomes in which the close alignment of multiple chromatids provides high local concentrations of the sequences. Each metaphase chromosome in the toad Xenopus luevis has about 1000 copies of the sequences coding for 5 S rRNA. These can be detected by in situ hybridization using in vivo labeled 5 S RNA (Pardue et ul., 1973). The lateral repetitions of a unique gene in the polytene chromosomes of Drosophilu salivary glands should be no more difficult to detect than the 5 S genes on a metaphase chromosome in X . luevis. A polytene band of lo3chromatids, each containing a single copy of agene coding for a typical protein (a DNA sequence of 105-106daltons, perhaps), should be a much better target for hybridization than the estimated 106-107daltons of DNA detected when a x . luevis metaphase chromosome is hybridized with 5 S RNA.
V. General Comments One of the first problems we encountered in our experiments on cytological hybridization was that of preserving nuclear morphology during
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NUCLEIC ACID HYBRIDIZATION TO DNA
15
the denaturation step. In our early experiments we dipped the slides into 0.5% agar just before the alkali denaturation. However, we have since found that, for preparations that are very flat and thoroughly dried to the slide, such protection is not necessary. Slides are dried several times during the hybridization procedure to ensure the adherence of the preparation. When we began our experiments we were concerned that basic proteins might either interfere with the hybridization reaction or cause nonspecific binding of the radioactive nucleic acid. However, a large fraction of the histones is extracted by the ethanol-acetic acid fixation and subsequent 45% acetic acid treatment (Dick and Johns, 1967), and this is sufficient to permit hybridization. Much protein, of course, remains in a cytological preparation and is a possible source of nonspecific binding of radioactive nucleic acid. In our earlier article on the technical details of in situ hybridization (Gall and Pardue, 1971), we recommended treating preparations with 0.2 N HCI to further remove basic protein before hybridization. Since that time we have found that treating a preparation with 0.2 N HCl significantly reduces the amount of in situ hybridization that can occur. We have not determined whether the HCl treatment simply depurinates the DNA or whether it also disposes the DNA to loss during the subsequent steps of the hybridization procedure. We now omit the 0.2 N HCl treatment entirely. Instead we add an excess of nonradioactive, noncompeting nucleic acid to the hybridization mixture to reduce nonspecific binding to chromosomal proteins. We originally avoided the use of RNase to remove endogenous RNA, because in filter hybridizations the enzyme binds to the filter and causes nonspecific attachment of radioactive RNA (Gillespie and Spiegelman, 1965). However, we found that treating Xenopus oocyte preparations with RNase before denaturation did not increase the background binding. Instead, it noticably improved the specific hybridization of ribosomal RNA, presumably by removing unlabeled ribosomal RNA from the tissue. We now routinely include the RNase step, although we have not investigated its effect on the hybridization of RNAs other than ribosomal RNA. The genotypes of several organisms contain chromosomal pairs that are so similar that they can not be distinguished simply on the basis of size and centromere position. Recently, staining techniques have been developed that produce characteristic banding patterns on each pair of chromosomes in some organisms (reviewed in Hsu, 1973).We have found that it is possible to use at least one of these powerful techniques for chromosome identification, quinacrine banding, in conjunction with in situ hybridization (Evans et al., 1974). In our experiments human chromosomes were stained with quinacrine, and the fluorescence banding patterns of selected metaphase plates were photographed by the techniques of Evans et al. (1971). After the chromo-
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MARY LOU PARDUE A N D JOSEPH G. GALL
somes had been photographed, the slides were washed for 5 or 6 hours with a gentle stream of running tap water to remove the quinacrine. The slides were dried and used for in situ hybridization experiments. By comparing the autoradiographs with the photographs of fluorescent-banded chromosomes, it was possible to identify unambiguously the chromosomes that had bound the radioactive RNA. In choosing a staining technique for the identification of chromosomes before in situ hybridization, it is important to note that some chromosome stains can cause exposure of autoradiographic emulsions by chemical reactions. If such stains are used and not completely washed out, autoradiography may show chromosome regions that have a high affinity for the stain, as well as regions that bind the radioactive RNA. REFERENCES Atherton, D., and Gall, J. G. (1972). Drosophilu Inform. Sen? 49, 131-133. Birnstiel, M. L., Sells, B. H., and Purdom, I. F. (1972). J. Mol. Biol. 63, 21-39. Bishop, J. 0. (1972). Biochem. J. 126. 171-185. Britten. R. J., Graham, D. E., and Neufeld, B. R. (1974). In “Nucleic Acids and Protein Synthesis” (L. Grossman and K. Moldave, eds.), Methods in Enzymology, Vol. 29, 363-418. Academic Press, New York. Commerford, S . L. (1971). Biochemistry 10, 1993-2000. Dick, C., and Johns, E. W. (1967). Biochem. J. 105,46P. Evans, H. J., Buckton, K. E., and Sumner, A. T. (1971). Qlromosomu 35, 310-325. Evans, H. J., Buckland, R. A., and Pardue, M. L. (1974). Qlromosoma 48,405-426. Gall, J. G., and Pardue, M. L. (1971). In “Nucleic Acids Part D ’(L. Grossman and K. Moldave, eds.), Methods in Enzymology, Vol. 21, 470-480. Academic Press, New York. Gillespie, D., and Spiegelman, S. (1965). J. Mol. Biol. 12, 829-842. Harrison, P. R., Cronkie, D., Paul, J., and Jones, K. (1973). FEBS (Fed. Eur. Biochem. Soc.) Lerr. 32, 109-112. Hennig, W. (1973). IN. Rev. c)ltol. 36, 1-44. Hsu, T. C. (1973). A m . Rev. Genet. 7 , 153-176. Jacob, J., Todd, K., Birnstiel, M. L., and Bird, A. (1971). Biochim. Biophys. Actu. 228, 761766. Jones, K. W. (1973). New Tech. Biophys. Cell Biol. 1, 29-66. Loeb, L., Tartof, K. D., and Travaglini, E. C. (1973). N u m e (London), New Biol. 242,66-69. McCarthy, B. J., and Church, R. B. (1970). A m . Rev. Biochem. 39, 131-150. Pardue, M. L., Kedes, L. H., Weinberg, E. H., and Bimstiel, M. L. (1972). J. Cell Biol. 55, 199a. Pardue, M. L., Brown, D. D., and Birnstiel, M. L. (1973). Uwomosomu 42, 191-203. Prensky, W., Steffensen, D. M., and Hughes, W. L. (1973). Proc. Nut. Amd. Sci. U.S. 70, 1860-1 864. Verma, I. M., and Baltimore, D. (1974). In “Nucleic Acids and Protein Synthesis” (L. Grossman and K. Moldave, eds.), Methods in Enzymology, Vol. 29, 125-130. Academic Press, New York. Walker, P. M. B. (1969). Progr. Nucl. Acid Res. Mol. Biol. 9, 301-326. Wetmur, J. G., and Davidson, N. (1968). J. Mol. Biol. 31, 349-370. Wimber, D. E., and Steffensen, D. M. (1973). Annu. Rev. Genet. 7,205-223.
AND
JOSEPH G. GALL
Department of Biology, Massachusetts Institute of Technology, Olmbridge, Massachusetts and Depamnent of Biology, Yale University, New Haven, Connecticut
Introduction . . . . . . . I. Procedure for in Situ Hybridization . . . A. Fixation and Squashing of Tissue . . . B. Removal of EndogenousRNA . . . C. Denaturation . . . . . . D. Hybrid Formation . . . . . E. Removal of NonspecificallyBound Nucleic Acid F. Autoradiography . . . . . . G. Staining Preparations after Autoradiography . 11. Equipment and Reagents . . . . . 111. Radioactive Nucleic Acids for Hybridization . . IV. Conditions of Reaction . . . . . V. General Comments . . . . . . References . . . . . . . .
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Introduction Techniques for annealing two molecules of single-stranded nucleic acid and characterizing the resulting hybrid molecule make it possible to compare nucleic acid sequences even though the actual order of nucleotides t Supported by research grants GB-35736 from the National Science Foundation and GM-12427 from the National Institute of General Medical Sciences.
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MARY LOU PARDUE AND JOSEPH G . GALL
in the sequences may not be known. When combined with cytological procedures that permit in situ detection of the cellular or chromosomal localization of the hybridized nucleic acid, these techniques can be used in several ways to study the organization and function ofthe genetic material in higher organisms. (1) In situ hybridization to condensed chromosomes can be used to map the sites of DNA sequences that have not been mapped by genetic breeding experiments. (2) Just as experiments with known nucleic acid sequences in the hybridizing solution can be used to identify chromosomal sites, hybridization to known chromosomal regions can be used in characterizing the nucleic acid sequences in the hybridizing solution. (3) Hybridization to cytological preparations gives information on the functional organization of particular sequences in the diffuse chromatin of interphase nuclei. This permits comparison of the organization in cells of different types. (4) Because cytological techniques permit analysis at the level of single cells, in situ hybridization can be used to obtain preliminary evidence on gene amplification or underreplication in single cell types in tissues composed of several cell types or cell stages. ( 5 ) The distribution of specific RNA species within a cell or in mixed cell populations can be studied by hybridization with DNA transcribed in vitro from that RNA by RNA-directed DNA poly merases. Several years ago we published a general review of techniques for in situ hydridization (Gall and Pardue, 1971). Since that time we have continued to use and study in situ hybridization and have made several changes in the procedures described in our earlier article. Because there are a number of reviews on nucleic acid hybridization in vitro (Wetmur and Davidson, 1968; Walker, 1969; McCarthy and Church, 1970; Birnstiel et al., 1972; Bishop, 1972; Britten et al., 1974), and on techniques for in situ hybridization (Jacob et al., 1971; Harrisonetal., 1973;Hennig, 1973;Jones, 1973;Wimber and Steffensen, 1973), this article describes only the procedures that have been used in our laboratories.
I. Procedure for in Situ Hybridization
A. Fixation and Squashing of Tissue Many conventional cytological procedures can be used to make slides that give good results in in situ hybridization experiments. However, fixatives containing formaldehyde should not be used, since they apparently interfere with denaturation of the DNA. The best preparations are those that are well spread and very flat.
1. NUCLEIC ACID HYBRIDIZATION TO DNA
3
The following procedures are the ones we routinely use to make slides for
in situ hybridization.
1. FORSMALL PIECES
OF
TISSUE
Fresh tissue is teased into very small pieces not more than 5 mm in their longest dimension. It is then fixed for 5-10 minutes in freshly mixed ethanolacetic acid (3 : I). Next a small drop of 45% acetic acid is placed on a siliconized 18-mm2 cover slip. A small piece (less than 1 mm’) is teased from the fixed tissue and transferred to this drop. Because addition of the fixative to 45% acetic acid causes violent mixing, the tissue is held for a few seconds in air to permit most of the fixative to evaporate before being placed in the acetic acid. However, the morphology may be ruined if the tissue becomes too dry in this step. The tissue is thoroughly minced in the drop of acetic acid, and any large pieces that remain are removed. Then a “subbed” slide is carefully lowered onto the cover slip, and the cells are squashed. A simple way to squash the cells is to place the slide on filter paper with the cover slip side down and apply firm thumb pressure on the back of the slide over the cover slip. Some tough tissues will give a better preparation if they are allowed to soften on a 45”C warming plate for 3-5 minutes before being squashed. The flattest squashes tend to have the best preservation of morphology during the hybridization steps, so it is wise to use small amounts of tissue and to remove any bits of material that might interfere. After squashing, the slides are placed on a flat surface of Dry Ice for a few minutes. When the preparation is completely frozen (5-10 minutes), the cover slip is flipped off with a razor blade and the slide is immediately placed in 95% ethanol. After the slide has been in ethanol for at least 5 minutes, it is air-dried and stored. Slides can be stored dry for very long periods of time, although the level of hybridization tends to drop after several months. The level of hybridization drops much more rapidly if the slides are stored in ethanol.
2. FORDROSOPHILA SALIVARY GLANDS The glands are dissected in insect Ringer’s solution and transferred to a small drop of 45% acetic acid on a siliconized 18-mmzcoverslip. After the glands are allowed to fix briefly in the 45% acetic acid, asubbed slide is lowered onto the cover slip m d the glands are squashed. There are many techniques for squashing salivary glands. The cover slip can be tapped with the point of a pencil, or with the eraser end, or it can be pressed with pointed forceps. The initial steps in squashing salivary glands are intended to spread out the chromosomes and should be checked in the phase micro-
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MARY LOU PARDUE AND JOSEPH G. GALL
scope. When a satisfactory degree of spreading is obtained, the slide is placed on filter paper with its cover slip side down and firm thumb pressure is applied to the back of the slide over the cover slip. The slide is then placed on a flat piece of Dry Ice until the preparation is frozen. Next the cover slip is flipped off with a razor blade, and the slide is immediately plunged into freshly made ethanol acetic acid (3: 1) for fixation. After 2 minutes fixation in 3: 1, the slide is transferred to 95% ethanol for at least 10 minutes and air-dried. Salivary gland slides may be stored dry as noted for tissues squashed by the procedure described in Section I,A,l. A slightly different technique for making squashes of Drosophilu salivary glands is given by Atherton and Gall (1972). 3. FORCELLSUSPENSIONS Tissue culture cells are gently spun out of the medium and resuspended in a hypotonic solution composed of 1 part medium and 3 parts distilled water. The amount of hypotonic solution added should be about 40 times the volume of the cell pellet. After being resuspended very gently in the hypotonic solution, the cells are left for 5 minutes at room temperature. The cells are gently spun down and the hypotonic solution is replaced with an equal volume of 50% acetic acid to fix the cells. The acetic acid is added without disturbing the pellet, and the cells are left on ice for 20 minutes. After the fixation the acetic acid is removed. A drop or two of fresh 50% acetic acid is added and the cells are gently resuspended. Squashes are made by putting a small drop of the fixed cell suspension on a siliconized cover slip and completing the squashing as described for small pieces of tissue in Section I,A,l .
B. Removal of Endogenous RNA Endogenous RNA, which might act as a competitor for the hybridizing RNA, is removed by treating the squashes with pancreatic RNase (EC 2.7.7.16). The RNase is dissolved in 2 x SSC at a concentration of 100puglml. Each slide is placed in a moist chamber prepared from a plastic petri plate (Fig. l), 200 p1 of the RNase solution is placed over the preparation, and the RNase solution is covered with a 22 x 40 mm cover slip. The moist chambers can be held at 37°C for 1 hour or at room temperature for 2 hours. After the RNase treatment the cover slips are gently removed by dipping the slide into a beaker of 2 x SSC to float off the cover slip. Slides are then washed three times in 2 x SSC, once in 70% ethanol, and once in 95% ethanol before air-drying.
1.
NUCLEIC ACID HYBRIDIZATION TO DNA
5
Solution of RNA-3H or DNA-3H
Coverslip
q
Microscope slide
-- PIas t ic petri dish
Hybridization buffer
/
\
Rubber washer or grommet
FIG 1. Moist chamber used in cytological hybridization procedure.
C. Denaturation All the agents, such as alkali, heat, and organic solvents, that denature purified DNA also denature the DNA of cytological preparations, as measured by in situ hybridization. Acid treatments also denature DNA on slides but give a reduced amount of in situ hybridization. Chromosome morphology is excellently preserved during acid denaturation, but the acid treatments that denature DNA in cytological preparations also produce some depurination (M. L. Pardue and H. C. Macgregor, unpublished). In our laboratories a1kali denaturation consistently gives the best results. Slides are placed in 0.07 NNaOH at room temperature for 2-3 minutes to denature the DNA of the preparation. This is followed by three changes of 70% ethanol over a period of about 10 minutes and two washes of 95% ethanol of about the same duration. The slides are again air-dried. Preparations that suffer loss of morphology during this step usually have not been squashed flat enough.
D. Hybrid Formation Our preliminary experiments to study the parameters of in situ hybridization have indicated that they should be similar to those in hybridizations with DNA fixed to nitrocellulose filters. Therefore conditions of ionic strength, nucleic acid concentration, temperature, and length of time for hybridization are chosen for each experiment as one would choose them for filter hybridization experiments. The conditions chosen determine the stringency of the hybridization conditions and set limits on the amount of sequence mismatching that can occur.
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MARY LOU PARDUE AND JOSEPH G . GALL
When performing hybridization at high temperature, we ordinarily use an SSC or an SNB buffer at a concentration determined by the amount of sodium ion desired. For example, reactions can be done in 2 x SNB at 65°C. Hybridizations can be done at lower temperatures by adding formamide to the reaction mixture. We frequently use 40% formamide (Matheson, Coleman, Bell; Norwood, Ohio) in 4 x SSC at 37°C. Addition of a large excess of nonradioactive, noncompeting nucleic acid helps to eliminate nonspecific binding of radioactive nucleic acid to the slide. The radioactive nucleic acid is placed over the preparation and covered with a cover slip. The slide is then placed in a moist chamber and incubated at the chosen temperature. Ordinarily we use 10-15 p1 of solution under an 18-mm2 cover slip. Much smaller volumes can be used with 9-mm2 cover slips if the moist chamber is sealed. We routinely do 10- to 15-hour incubations at 65°C without noticeable loss of liquid from under the cover slip. Although it is possible to seal the cover slip over the liquid, it is simpler to seal the moist chambers. Such a procedure eliminates the possibility that the hybridization medium will extract some components from the sealing mixture. Some brands of cover slips leach alkali into the incubation mix. It is useful to test a new brand of cover slip before using it. This can be done by doing a mock hybridization using the hybridization buffer plus phenol red under the cover slip. If this test shows that the cover slips are leaching alkali during the incubation, the cover slips can be boiled in 1 N HCl and rinsed well in pH 7 buffer before they are used.
E. Removal of Nonspecifically Bound Nucleic Acid 1. FORRNA-DNA HYBRIDS After the hybridization reaction the slides are removed from the oven. The cover slip and hybridization mix are immediately washed off by dipping the slide into a beaker of 2 x SSC. Slides are then washed in 2 x SSC at room temperature for 15 minutes before being incubated with pancreatic RNase (20 pg/ml in 2 x SSC) for 1 hour at 37°C. Finally, the slides are rinsed twice for 10 minutes in 2 x SSC and passed through 70 and 95% ethanol before being air-dried.
2. FORDNA-DNA HYBRIDS The cover slips are removed from the slides by dipping in 2 x SSC at a temperature 5" less than the temperature used for the hybridization reaction. Nonspecifically bound DNA is then removed by three additional 10-minute washes in 2 x SSC which is also 5" less than the hybridization temperature. Finally, the slides are washed in 70 and 95% ethanol and dried.
1. NUCLEIC ACID HYBRIDIZATION TO DNA
7
F. Autoradiography The dried slides are coated with autoradiographic emulsion by standard procedures. We use Kodak NTB-2 liquid emulsion diluted 1: 1 with distilled water. A thicker coating of emulsion on the slide would not increase the information gathered, since the beta particles emitted by 3H are not energetic enough to reach the upper part of the film. However, a thicker film would increase the number of background grains produced extraneously. Kodak NTB-2 is purchased in 4-oz bottles (1 12 ml) and is a solid at room temperature. It should be used only in absolute darkness or with a Kodak Wratten Series I1 or OA safelight. We heat each new bottle to 45°C for 30 minutes and then add it to 112 ml ofwarm, distilled water. The two solutions mix readily, and this step should be done carefully to avoid producing bubbles in the emulsion. Diluted emulsion is distributed into nylon scintillation vials in aliquots of about 10 ml. The vials are wrapped in aluminum foil and stored in a light-tight box in a refrigerator. Each vial contains enough emulsion for about 30 slides and is used only once. The emulsion will keep for many months. The caps used for the scintillation vials should not have cork inserts, since these can give off organic compounds which may produce background grains in the emulsion during storage. If one suspects that the emulsion may have picked up some background grains before use, it is wise to dip a test slide, dry it for 20 minutes, and then develop and check it before dipping the experimental slides. A good emulsion has 0.1-0.2 x grains per square micrometer. For use the emulsion is melted in a 45°Cwater bath for 10 minutes and then poured into a small plastic dipping chamber, taking care that no bubbles are formed. (A polyethylene two-place slide mailer available from most supply houses makes a good dipping chamber.) The dipping chamber is held at 45°C. Slides are dipped into the chamber, withdrawn slowly, drained, and placed vertically in a rack. Slides should dry in the dark for 2 hours. A nonsparking fan can be used to reduce the drying time to 1 hour. Dried slides are stored in light-tight plastic boxes with a small container of silica gel included to maintain dryness. The boxes are sealed with electrician’s tape and stored in lightproof containers in a refrigerator. Autoradiographic exposures for in sifu hybridization may range from a few hours to many days. It is wise to have several slides, so that test slides may be developed at intervals to determine the proper exposure time. After the appropriate exposure time, slides are developed for 2+ minutes in Kodak D-19, rinsed well in distilled water, and fixed for 2-5 minutes in
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MARY LOU PARDUE AND JOSEPH G. GALL
Kodak fixer. The slides are then rinsed through several changes of distilled water over a period of 30 minutes and stained before drying.
G. Staining Preparations after Autoradiography There are several ways of staining cytological preparations through autoradiographic emulsions. We use Giemsa stain. A stock solution of Giemsa blood stain is diluted approximately 1 :20 with 0.01 M phosphate buffer (pH 6.8) immediately before adding the slides. The staining time depends on the cytological preparation and on the batch of Giemsa stain. It is usually somewhere between 2 and 45 minutes. A metallic film forms rapidly over the surface of the Giemsa solution once it has been mixed with the buffer. Since this film will adhere to the slide if the slide is pulled out of the solution, the film is floated out of the staining dish with distilled water before removing the slides. The slides are rinsed with distilled water, airdried, and covered with a cover-glass mounted in Permount. Slides may also be left uncovered and observed under immersion oil. Petroleum ether is used to remove oil from unmounted slides.
11. Equipment and Reagents Subbed slides. Microscope slides are washed in either acid or detergent and thoroughly rinsed in distilled water. They are then immersed briefly in subbing solution and allowed to dry for several hours before being used. Subbing solution. This is an aqueous solution of 0.1% gelatin and 0.01% chrome alum [chromium potassium sulfate, CrK(SO,), 12H,O]. The gelatin is first dissolved in hot water, and the chrome alum is added after the solution cools. Siliconized cover slips. Cover slips are immersed briefly in a 1% solution of Siliclad (Clay-Adams; Parsippany, N.J.), rinsed thoroughly in distilled water, and allowed to dry overnight at room temperature. They are stored dry. Before using, the cover slips can be rinsed with 95% ethanol and dried. SSC. 0.15 M NaCl, 0.015 M sodium citrate; pH 7.0 SNE. 0.15 M NaCl, M tris; pH 7.0. Insect Ringer’s solution. 7.5 gm NaCl, 0.35 gm KCl, and 0.21 gm CaCl, in 1 liter of water. RNuse. Pancreatic RNase (EC 2.7.7.16) is dissolved at 1 mg/ml in 0.02 M sodium acetate (pH 5). This solution is placed in a boiling water bath for for 5 minutes, cooled, and stored at -20°C.
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1.
NUCLEIC ACID HYBRIDIZATION TO DNA
9
FIG.2. Metaphase chromosomes hybridized with 18 and 28 S rRNA. The DNA sequences complementary to the rRNA are found at the secondary constrictions on the sex chromosomes in both karyotypes. The muntjac has an additional site of ribosomal sequences on the largest autosome. The specific activity of the rRNA-3H was 5 x lo6 dmplpg. It was hybridized in 2 x SNB at 65°C for 10 hours. (a) Bat, Olrolliaperspicillafa. Male karyotype. rDNA only on X chromosome. Exposure 37 days. x 1445. (b) Indian mutjac, Munriaarsmunrjak. Male karyotype. rDNA on X, Y,and one pair ofautosomes. Exposure, 75 days. x 1870. (FromT. C. Hsu, F. E. Arrighi, S. Spinto, and M. L. Pardue, unpublished.)
FIG. 3. Pachytene nucleus from the testis of the toad X la& hybridized with cRNA-’H transcribed in vitro from DNA coding for 18 and 28 S rRNA. In X.laevik the genes for 18 and 28 S rRNA are localized on one chromosome of the haploid complement, and thus the tightly paired homologous chromosomes of the pachytene nucleus show only one site of hybridization in this experiment. The cRNA-’H had a calculated specific activity of 10’ dpndpg. Hybridization was carried out in 2 x SSC for 10 hours at 65°C. Exposure, 41 days. ~ 2 2 0 0 . FIG. 4. Pachytene nucleus from the testis of the toad X. laevis hybridized with cRNA-’H transcribed in vitro from the DNA coding for 5 S rRNA. The ends of all of the chromosomes are oriented toward one side of the nucleus. The 5 S cRNA binds to DNA sequences located at the end of one arm of most, if not all, of the chromosomes. The cRNA-’H had a specific activity of 4 x lo7 dpmlpg. It was hybridized in 4 x SNB for 8 hours at 65°C. Exposure, 26 days. x 17%. (From Pardue er al., 1973.)
10
MARY LOU PARDUE AND JOSEPH G. GALL
Moist chamber. (see Fig. 1). Moist chambers are made from 4 inch plastic petri plates. The bottom of the petri plate is lined with filter paper and contains 5-10 ml of the incubation buffer. The slide is supported above the liquid by two rubber grommets or a U-shaped glass rod. The solution in the bottom of the moist chamber must have the same salt concentration as the solution under the cover slip to prevent distillation and subsequent change in concentration. Glass petri plates do not make satisfactory moist chambers, because the moisture that condenses on the lid tends to coalesce into large drops which may fall onto the preparation. Moisture condensing on plastic lids remains in small droplets on the lid. Some types of plastic petri plates melt at temperatures used for hybridization reactions. Giemsa stain. We use commercially prepared stock solutions of Giemsa blood stain.
111. Radioactive Nucleic Acids for Hybridization The feasibility of detecting any particular DNA sequence by cytological hybridization depends on two factors, either of which may be the limiting one. The first factor is the sensitivity of detection. This depends on the amount of radioactivity in agiven region and the efficiency of the autoradiographic procedure. The second factor is the rate of hybridization, which depends on the concentrations of the sequences being studied. Thus in choosing a source of radioactive nucleic acid for in situ hybridization experiments, one should consider both the specific radioactivity of the nucleic acid and the amounts of nucleic acid that can be obtained for use in the hybridization medium. The minimum levels of radioactivity needed for a particular experiment are determined by the amount of nucleic acid bound at each chromosomal site. Repeated sequences clustered at a single chromosomal locus can be detected more easily than the same number of sequences scattered over sites on several chromosomes. In general, we find that lo6 d p d p g is the lower useful limit. There are several possible ways of obtaining nucleic acid with a specific activity greater than lo6 dprn/pg. 1. IN Vwo LABELED NUCLEIC ACID Cultured cell lines grown in a medium containing tritiated nucleic acid precursors can yield both DNA and RNA with sufficient tritium label for use in in situ hybridization (Gall and Pardue, 1971). If culture and labeling conditions are optimized for the particular nucleic acid fraction desired, both stable and rapidly turning over fractions can be obtained with specific
1.
NUCLEIC ACID HYBRIDIZATION TO DNA
11
FIG. 5 . Spermatozoa from X laevis hybridized with cRNA-IH transcribed in vitro from DNA coding for 18 and 28 S rRNA. The rRNA genes are apparently packed into the sperm head in a tight cluster, but the position of this cluster is not constant from sperm to sperm. The conditions for hybridization are those given in Fig. 3. Exposure, 31 days. x 1400.
radioactivity of 106-107d p d p g . There are other biological systems that can be used to produce highly radioactive nucleic acids in vivo. For example, sea urchin embryos grown in sea water containing tritiated uridine during the early cleavage stages yield histone mRNA containing enough tritium label for in situ experiments (Pardue et al., 1972).
FIG. 6. Salivary gland chromosomes from Drosophila rnelanogaster hybridized in situ with 9 S mRNA-’H from the sea urchin Psmrnechinus rnilaris. These stretched chromosomal preparations show hybridization of the putative histone mRNA fractions to bands 39D and 39E, as well as the intervening interband region. The 9-S mRNA-IH was prepared from sea urchin blastulas grown in sea water containing uridine-’H. The RNA was hybridized in 4x SNB at 65°C for 10 hours. Exposure, 109 days. (a) x 1600; (b) ~ 2 6 0 0(From . M. L. Pardue, L. H. Kedes, E. H. Weinberg, and M. L. Birnstiel, unpublished.)
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MARY LOU PARDUE AND JOSEPH G. GALL
2. IN VITRO TRANSCRIPTION OF NUCLEIC ACID By far the most successful method for preparing extremely radioactive nucleic acid is by in vitro transcription. In cases in which a specific DNA fraction can be isolated, Escherichia coli RNA polymerase can be used in vitro to produce RNA copies of the DNA (Gall and Pardue, 1971). RNA fractions can be transcribed into DNA by RNA-directed DNA polymerases (Verma and Baltimore, 1974), or by DNA polymerase from E. coli (Loeb et al., 1973). The product of an in vitro transcription should be characterized carefully, since any of the polymerases may transcribe certain nucleic acid sequences preferentially. When a mixture of nucleic acids is transcribed, the product may not be equally transcribed from all fractions of the template. The specific radioactivity of nucleic acids obtained from in vitro transctiption is limited only by the specific activity of the nucleotide precursors. The highest specific activity precursors currently available yield a product with greater than lo* dpm/pg. OF PREFORMED NUCLEIC ACIDS 3. CHEMICAL LABELING
In cases in which the desired nucleic acid cannot be labeled either in vivo or by in vitro transcription, radioactive atoms can be added chemically. Although there are several methods for chemical addition of 3H to nucleic acids, none of the methods now in use yields the levels of radioactive labeling required for in situ hybridization. However, techniques have been developed for labeling nucleic acids with lz5I(Commerford, 1971;Prensky et al., 1973). The specific radioactivity obtained with lZ5Iis about equal to that produced by in vitro transcription with 'H precursors. Theoretically, *ZSI-labeled nucleic acids of even higher specific activity can be produced, but the characteristics of hybridization may be affected by the increased iodination (Commerford, 1971). The radiation emitted by lzSI is somewhat more energetic than that emitted by 3H. For this reason lz5I is more efficient than 3H for autoradiography, but gives less precise cytological localization.
IV. Conditions of Reaction We have assumed that in situ hybridization shares some of the kinetic properties of the nitrocellulose filter hybridization technique. Our experimental evidence is consistent with this assumption, and we use filter
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NUCLEIC ACID HYBRIDIZATION TO DNA
13
FIG. 7. Salivary gland chromosomes from D. melanogaster hybridized in situ with cytoplasmic RNAJH. The RNA was prepared from the cytoplasmic fraction of cultured D. melanogaster cells grown in a medium with uridine-lH. These poly-A-containing sequences were isolated by oligo-dT chromatography and hybridized in 2x SNB at 65°C for 10 hours. This RNA binds to a reproducible series of chromosomal bands and to thep-heterochromatin of the chromocenter (c). Exposure, 35 days. (a) x 1100; (b) x 1400. (From A. Spradling, S. Penman, and M. L. Pardue, unpublished.)
hybridization experiments when necessary to optimize reaction conditions for in situ hybridization. In both in situ and filter hybridizations, one strand of the nucleic acid involved in the hybridization reaction is immobilized. We have not been able to detect either loss or renaturation of the DNA of the cytological preparation during the periods we have used for the annealing reaction, although there may be losses of DNA during the denaturation steps. Insitu hybridization does differ from filter hybridization in having extremely high local concentrations of DNA sequences and in having some of the protein components of chromatin still associated with the DNA. We have not yet
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MARY LOU PARDUE AND JOSEPH G. GALL
been able to assess the effect of either of these factors on the kinetics of hybridization in situ. Precise quantitation of in situ hybridization experiments has proved difficult for several technical reasons. One source of variation is the degee of compaction of the DNA in different nuclei. Whether the compaction affects the amount of denaturation of the DNA, the access of the hybridizing nucleic acid to chromosomal sites, or the autoradiographic efficiency, it makes quantitative comparisons between different types of nuclei questionable in some cases. In many studies it is preferable to use information from in situ experiments to design filter hybridization experiments for precise quantitation. Published in situ hybridization experiments have been calculated to have 5-1 5% efficiency of hybridization. Approximately the same efficiency is obtained whether the hybridization is to the diffuse amplified ribosomal genes in amphibian oocytes, to repeated sequences in heterochromatic regions of condensed chromosomes, or to repeated genes in euchromatic portions of chromosomes. The low efficiency of hybridization may be a reflection of the availability of the DNA in cytological preparations. However, it is also possible that none of the hybridizations were done with saturating concentrations of nucleic acid in solution. It is unlikely that the concentrations and times now used for in situ hybridization reactions would permit the detection of unique sequences in the DNA of diploid cells of higher organisms. However, unique sequences can easily be studied in polytene chromosomes in which the close alignment of multiple chromatids provides high local concentrations of the sequences. Each metaphase chromosome in the toad Xenopus luevis has about 1000 copies of the sequences coding for 5 S rRNA. These can be detected by in situ hybridization using in vivo labeled 5 S RNA (Pardue et ul., 1973). The lateral repetitions of a unique gene in the polytene chromosomes of Drosophilu salivary glands should be no more difficult to detect than the 5 S genes on a metaphase chromosome in X . luevis. A polytene band of lo3chromatids, each containing a single copy of agene coding for a typical protein (a DNA sequence of 105-106daltons, perhaps), should be a much better target for hybridization than the estimated 106-107daltons of DNA detected when a x . luevis metaphase chromosome is hybridized with 5 S RNA.
V. General Comments One of the first problems we encountered in our experiments on cytological hybridization was that of preserving nuclear morphology during
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NUCLEIC ACID HYBRIDIZATION TO DNA
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the denaturation step. In our early experiments we dipped the slides into 0.5% agar just before the alkali denaturation. However, we have since found that, for preparations that are very flat and thoroughly dried to the slide, such protection is not necessary. Slides are dried several times during the hybridization procedure to ensure the adherence of the preparation. When we began our experiments we were concerned that basic proteins might either interfere with the hybridization reaction or cause nonspecific binding of the radioactive nucleic acid. However, a large fraction of the histones is extracted by the ethanol-acetic acid fixation and subsequent 45% acetic acid treatment (Dick and Johns, 1967), and this is sufficient to permit hybridization. Much protein, of course, remains in a cytological preparation and is a possible source of nonspecific binding of radioactive nucleic acid. In our earlier article on the technical details of in situ hybridization (Gall and Pardue, 1971), we recommended treating preparations with 0.2 N HCI to further remove basic protein before hybridization. Since that time we have found that treating a preparation with 0.2 N HCl significantly reduces the amount of in situ hybridization that can occur. We have not determined whether the HCl treatment simply depurinates the DNA or whether it also disposes the DNA to loss during the subsequent steps of the hybridization procedure. We now omit the 0.2 N HCl treatment entirely. Instead we add an excess of nonradioactive, noncompeting nucleic acid to the hybridization mixture to reduce nonspecific binding to chromosomal proteins. We originally avoided the use of RNase to remove endogenous RNA, because in filter hybridizations the enzyme binds to the filter and causes nonspecific attachment of radioactive RNA (Gillespie and Spiegelman, 1965). However, we found that treating Xenopus oocyte preparations with RNase before denaturation did not increase the background binding. Instead, it noticably improved the specific hybridization of ribosomal RNA, presumably by removing unlabeled ribosomal RNA from the tissue. We now routinely include the RNase step, although we have not investigated its effect on the hybridization of RNAs other than ribosomal RNA. The genotypes of several organisms contain chromosomal pairs that are so similar that they can not be distinguished simply on the basis of size and centromere position. Recently, staining techniques have been developed that produce characteristic banding patterns on each pair of chromosomes in some organisms (reviewed in Hsu, 1973).We have found that it is possible to use at least one of these powerful techniques for chromosome identification, quinacrine banding, in conjunction with in situ hybridization (Evans et al., 1974). In our experiments human chromosomes were stained with quinacrine, and the fluorescence banding patterns of selected metaphase plates were photographed by the techniques of Evans et al. (1971). After the chromo-
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MARY LOU PARDUE A N D JOSEPH G. GALL
somes had been photographed, the slides were washed for 5 or 6 hours with a gentle stream of running tap water to remove the quinacrine. The slides were dried and used for in situ hybridization experiments. By comparing the autoradiographs with the photographs of fluorescent-banded chromosomes, it was possible to identify unambiguously the chromosomes that had bound the radioactive RNA. In choosing a staining technique for the identification of chromosomes before in situ hybridization, it is important to note that some chromosome stains can cause exposure of autoradiographic emulsions by chemical reactions. If such stains are used and not completely washed out, autoradiography may show chromosome regions that have a high affinity for the stain, as well as regions that bind the radioactive RNA. REFERENCES Atherton, D., and Gall, J. G. (1972). Drosophilu Inform. Sen? 49, 131-133. Birnstiel, M. L., Sells, B. H., and Purdom, I. F. (1972). J. Mol. Biol. 63, 21-39. Bishop, J. 0. (1972). Biochem. J. 126. 171-185. Britten. R. J., Graham, D. E., and Neufeld, B. R. (1974). In “Nucleic Acids and Protein Synthesis” (L. Grossman and K. Moldave, eds.), Methods in Enzymology, Vol. 29, 363-418. Academic Press, New York. Commerford, S . L. (1971). Biochemistry 10, 1993-2000. Dick, C., and Johns, E. W. (1967). Biochem. J. 105,46P. Evans, H. J., Buckton, K. E., and Sumner, A. T. (1971). Qlromosomu 35, 310-325. Evans, H. J., Buckland, R. A., and Pardue, M. L. (1974). Qlromosoma 48,405-426. Gall, J. G., and Pardue, M. L. (1971). In “Nucleic Acids Part D ’(L. Grossman and K. Moldave, eds.), Methods in Enzymology, Vol. 21, 470-480. Academic Press, New York. Gillespie, D., and Spiegelman, S. (1965). J. Mol. Biol. 12, 829-842. Harrison, P. R., Cronkie, D., Paul, J., and Jones, K. (1973). FEBS (Fed. Eur. Biochem. Soc.) Lerr. 32, 109-112. Hennig, W. (1973). IN. Rev. c)ltol. 36, 1-44. Hsu, T. C. (1973). A m . Rev. Genet. 7 , 153-176. Jacob, J., Todd, K., Birnstiel, M. L., and Bird, A. (1971). Biochim. Biophys. Actu. 228, 761766. Jones, K. W. (1973). New Tech. Biophys. Cell Biol. 1, 29-66. Loeb, L., Tartof, K. D., and Travaglini, E. C. (1973). N u m e (London), New Biol. 242,66-69. McCarthy, B. J., and Church, R. B. (1970). A m . Rev. Biochem. 39, 131-150. Pardue, M. L., Kedes, L. H., Weinberg, E. H., and Bimstiel, M. L. (1972). J. Cell Biol. 55, 199a. Pardue, M. L., Brown, D. D., and Birnstiel, M. L. (1973). Uwomosomu 42, 191-203. Prensky, W., Steffensen, D. M., and Hughes, W. L. (1973). Proc. Nut. Amd. Sci. U.S. 70, 1860-1 864. Verma, I. M., and Baltimore, D. (1974). In “Nucleic Acids and Protein Synthesis” (L. Grossman and K. Moldave, eds.), Methods in Enzymology, Vol. 29, 125-130. Academic Press, New York. Walker, P. M. B. (1969). Progr. Nucl. Acid Res. Mol. Biol. 9, 301-326. Wetmur, J. G., and Davidson, N. (1968). J. Mol. Biol. 31, 349-370. Wimber, D. E., and Steffensen, D. M. (1973). Annu. Rev. Genet. 7,205-223.
