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In situ hybridization—Application to gene localization and RNA detection
In Situ Hybridization Application to Gene Localization and RNA Detection Mary E. Harper and Lisa M. Marselle
ABSTRACT: In situ hybridization offers a direct approach for localization and quantitation of nucleic acid sequences in cellular preparations. Recent improvements in technology and methodology make possible the detection of DNA and RNA of relatively low copy number. For example, development of in situ hybridization methods for detection of single copy DNA sequences on mitotic chromosomes has led to general use of this technique for gene mapping of the human genome. More recently, improvements in methodology for detection of low abundancy RNA make possible a facilitated analysis of gene expression, both from cellular genes and exogenous sequences, such as viral genomes. In situ hybridization is now a powerful method for studying nucleic acid organization and function in normal cells, as well as in malignant cells, which should contribute to better understanding of the cell transformation process.
INTRODUCTION
In situ hybridization allows the visualization of nucleic acid sequences directly on cytologic preparations. Initially described in 1969 for chromosomal localization of DNA sequences of relatively high copy n u m b e r [1, 2], in situ hybridization n o w can be used to detect chromosomal sequences present in only one or two copies per cell [3, 4] or RNA present at less than 20 transcripts per cell [5] after short autoradiographic exposure. High sensitivity is attained by hybridization of cloned nucleic acid probes radiolabeled with 3H or 35S using high specific activity nucleotide triphosphates. Slide preparations are coated with nuclear track emulsion, exposed for several days to several weeks, developed, and visualized by light microscopy. In situ hybridization offers several advantages as a technique for detecting and/ or localizing nucleic acid sequences in eukaryotic cells. In situ hybridization is a direct method, is relatively rapid, and requires small tissue samples. Low abundancy sequences can be detected readily and, u n d e r similar conditions of probe hybridization and exposure, the n u m b e r of sequences can be reasonably quantitated. In situ hybridization as a gene m a p p i n g technique results in an immediate regional or band sublocalization, directly visualizes sequence relocation as a result of chromosomal rearrangement, and can detect breakpoints w i t h i n defined sequences.
From the Laboratoryof Tumor Cell Biology,NationalCancer Institute, Bethesda, MD. Address requests for reprints to Dr. Mary E. Harper, Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, MD 20205. Received August 5, 1985; accepted August 19, 1985.
73 ©1986ElsevierScience PublishingCo., Inc.
52VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet19:73-80 (1986) 0165-4608/86/$03.50
M. E. Harper and L. M. Marselle
74 CHROMOSOMAL
MAPPING
OF SINGLE
COPY SEQUENCES
Several years ago, improvements were introduced into the in situ hybridization methodology for mapping genes on chromosomes in order to allow higher sensitivity and detection of single copy sequences on mitotic chromosomes [3, 41. According to this method, cloned DNA probes are 3H-labeled by nick translation and denatured, generating randomly cleaved, single-stranded fragments. Hybridization to high quality chromosome preparations, previously ribonuclease-treated and denatured, is carried out in the presence of 10% dextran sulfate, which promotes the formation of DNA networks among the partially complementary fragments [6]. Careful control of temperature and pH results in efficient hybridization and preservation of chromosome morphology. Following autoradiography, chromosome preparations are G-banded directly by several series of staining-destaining-restaining with Wright’s stain [7]. Labeled G-banded chromosomes are visualized directly in the microscope and grain locations are recorded and analyzed. Probes are generally hybridized at a concentration resulting in visualization of 1-5 grains per cell (typically lo-200 ngiml). Hybridization at increasing DNA concentrations results in concordant nonspecific binding, manifested as increasingly frequent chromosomal grains and/or grain clusters. Hybridized preparations are analyzed by compilation of grains, or labeled sites consisting of multiple grains, on all chromosomes within multiple mitotic spreads. Assigned localizations should be based on a significant site or sites of labeling throughout the entire karyotype. MAPPING
ONCOGENES
BY IN SITU
HYBRIDIZATION
Oncogenes represent particular DNA sequences believed to affect normal cellular events and exert a direct role in cell transformation. Such sequences include cellular equivalents of viral one genes (called c-one genes), which may correspond to genes coding for certain growth factors and cell surface receptors, as well as genes defined by their ability to induce transformation of nonneoplastic cells upon DNA transfection [for review, see ref. 81. Recently, several oncogenes have been found to be associated with the consistent, nonrandom chromosomal aberrations observed in specific human neoplasms and believed to play an important role in the genesis or progression of malignancy. For example, the consistent translocations t(8;14), t(2;8), or t(8;22) observed in Burkitt’s lymphoma place the c-myc gene into close proximity with one of the immunoglobulin gene loci (heavy, kappa or lambda chain genes) [9, lo]. Also, in all Ph-positive chronic myelogenous leukemia (CML) cases studied to date, the c-abl gene is translocated from chromosome #9 to chromosome #22 [ll], which results in altered transcription of the gene [12, 131. In order to further investigate relationships between oncogenes and nonrandom chromosomal rearrangement, a direct approach is to determine oncogene positions within the normal genome, as compared with transformed genomes. In the case of the c-myb gene, in situ hybridization was carried out to sublocalize the gene, previously assigned to chromosome #6 [l4]. Hybridization of the ‘H probe resulted in significant label on 6q, at a position slightly distal to the midportion of the arm (Fig. 1). Of 163 cells analyzed, 50 (30.7%) exhibited grains on region q2 of one or both chromosomes #S. These sites represented 7.4% (511685)of all labeled sites throughout the 163 cells. Compilation of data from a large number of labeled chromosomes #6 (88) confirmed the hybridization to 6q2 and sublocalized c-myb to 6q22-24 (Fig. 2) [15]. These results suggested that c-myb may be associated with a nonrandom chromosomal abnormality consistently observed in certain human malignancies. Deletion or translocation of the distal half of 6q has been observed in a significant num-
75
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Figure 1 Representative human mitotic cell hybridized with the 3H-labeled c-myb-specific probe, demonstrating typical labeling of the long arm of chromosome #6 (arrow).
re 2 Distribution of labeled sites on 88 lachromosomes #6 following hybridization the c-myb probe, indicating sublocalization c-myb gene to 6q22-24.
6
76
M.E. Harper and L. M. Marselle ber of cases of acute lymphocytic leukemia (ALL) [16, 17], ovarian carcinoma [18], and melanoma [19-21]. In addition, elevated levels of c-myb RNA have been detected in primary ALL cells, as well as in two cell lines (Molt-4 and CEM) established from ALL patients [22]. Interestingly, Molt-4 cells exhibit a deletion/translocation of the q21-qter segment of one chromosome #6 [23]. Localization studies on c-myb in transformed cells containing the 6 q - chromosome, in conjunction with analyses of quantitative and qualitative changes in c-myb expression, are needed to determine the relationship of this oncogene to the chromosomal rearrangements. In situ hybridization has been utilized to sublocalize a variety of oncogenes within the human genome (Table 1). As these studies indicate, it has become increasingly apparent that other cellular oncogenes may be associated with specific chromosomal rearrangements observed in particular human neoplasms. The in situ hybridization technique allows direct examination of the position of any given cellular sequence in normal and transformed cells and also may reveal translocations not detected by standard kayotypic analysis.
DETECTION OF CELLULAR RNA BY IN SITU HYBRIDIZATION
Investigation of the expression of particular genes is important in determining the function of such genes in normal development, as well as their role in disease states such as malignancy. In situ hybridization offers several advantages as a method for studying the level and distribution of RNA transcription (see INTRODUCTION).In addition, in situ hybridization can be used to assay a wide variety of tissues for specific transcripts, and gives information regarding the type(s), as well as distribution, of cells expressing the gene within a particular tissue. In our laboratory, use of previously described methods for RNA in situ hybridization [24, 25] did not detect low abundancy RNA after short autoradiographic exposure (several days). Therefore, modifications were introduced in order to detect less than 100 copies of RNA per cell after 2-days exposure. This method makes use of 3~S-RNA probes with high specific activity (10 9 cpm/~g) synthesized from specific DNA sequences inserted into transcription vectors such as pSP64. Several pretreatment steps, stringent washing, and ribonuclease treatment following hybridization result in minimal background labeling [5]. DETECTION OF HTLV R N A BY IN SITU HYBRIDIZATION
Human T-lymphotropic virus (HTLV) represents a family of human retroviruses that share an increasing number of striking biological and biochemical properties [26]. Recent serologic, viral isolation, and nucleic acid studies have shown that HTLV-III is the etiologic agent of the acquired immunodeficiency syndrome (AIDS] [reviewed in ref. 26]. Using the improved in situ hybridization method, primary mononnclear cell preparations from peripheral blood obtained from AIDS and AIDS-related complex (ARC) patients were examined for the presence of HTLV-III RNA. Hybridization was carried out with 106 dpm 35S-RNA probe specific for HTLV-III; slides were autoradiographed and exposed for 2 days. Analysis of hybridized slides showed labeling of a very low percentage of cells (typically less than 0.01%) in the samples studied. The HTLV-III-infected cells exhibited morphologic characteristics consistent with that of lymphocytes, and contained approximately 20-100 grains per cell (Fig. 3). Mononuclear cell preparations from the same patients hybridized with a lambda-specific control probe were consistently negative. Similarly, mononuclear cells from normal individuals were negative when hybridized with the HTLV-III probe [5]. Specificity of hybridization was further shown by use of the T-cell line H9, a
77 Table 1
Oncogenes sublocalized in the h u m a n genome by in situ hybridization
Oncogene
Location
Ref.
ABL BLYM ERBA1
9q34 lp32 17q21-q22 17q11-q12 11q23-q25 11q23 15q25-q26 15q26.1 14q21-q31 11p14.1 11p15 11p13 12p12.1,12q24.2 8q22 6q22-q24 8q24 2p23-p24 1cen-p21 lp11-p13 lp22 3p25 22q13.1 22q12.3-q13.1 lp36.3,20q13.3
[11, 29] [30] [31] [32] [33] [34] [15] [29] [35] [36] [37] [38] [36] [39, 40] [15] [10, 39, 40] [41] [42] [43] [44] [45] [29] [46] [47]
ETS FES FOS HRAS1
KRAS2 MOS MYB MYC NMYC NRAS
RAF1 SIS SRC
Figure 3 Mononuclear cell preparation from uncultured peripheral blood of ARC patient hybridized with HTLV-III probe, demonstrating presence of rare infected lymphocyte expressing viral RNA.
78
M . E . Harper and L. M. Marselle neoplastic a n e u p l o i d line significantly resistant to the cytopathic effects of HTLVIII [27]. W h e n h y b r i d i z e d with the HTLV-III probe, uninfected H9 cells exhibited no grains, whereas, a clone of H9 cells infected with HTLV-III was highly labeled (average of 150 grains per cell). Comparison of n u m b e r of grains observed over the infected H9 cells with Northern blot analysis of cells from the same culture indicated a p p r o x i m a t e equivalency b e t w e e n grain n u m b e r and RNA c o p y n u m b e r when slides were exposed for 2 days [5]. The in situ h y b r i d i z a t i o n m e t h o d is n o w being used to detect the presence of HTLV-III viral sequences in a w i d e variety of tissues from infected individuals to determine the extent of cells and tissues infected. For example, hybridization to frozen sections detected HTLV-III RNA in brain tissue from AIDS patients with encephalopathy. However, the infected cells d i d not exhibit the cellular morphology of l y m p h o c y t e s [28]. Further use of in situ hybridization, in conjunction with i m m u n o c y t o c h e m i s t r y for detection of specific cellular antigens, should help to further define and characterize infected cells in HTLV-III disease.
APPLICATIONS OF IN SITU HYBRIDIZATION In situ h y b r i d i z a t i o n is a powerful technique for studying nucleic acid sequence organization and function in a w i d e variety of cell types and tissues. In addition to chromosomal m a p p i n g of DNA sequences, this m e t h o d has the capability to directly detect repositioning of sequences w i t h i n the karyotype as a result of chromosomal rearrangements. Also, small rearrangements not detectable by standard karyotypic analysis m a y be uncovered by direct h y b r i d i z a t i o n of relevant probes to chromosome spreads. A n o t h e r a p p l i c a t i o n is detection and characterization of breakpoints using probes for defined sequences; for example, the breakpoint on chromosome #22 in the variant t(8;22) in Burkitt's l y m p h o m a interrupts the l a m b d a chain variable region genes [48], and the m e t a l l o t h i o n e i n gene cluster is split by chromosome #16 rearrangements in m y e l o m o n o c y t i c l e u k e m i a [49]. Use of in situ hybridization for detection of cellular RNA should yield useful information regarding cellular specificity, quantity, and quality of expression of a w i d e variety of gene sequences. Furthermore, c o m b i n e d use of the two types of methods should contribute greatly to u n d e r s t a n d i n g the m o d e of action of oncogenes as well as other genes important in h u m a n malignancies.
REFERENCES 1. Gall JG, Pardue ML (1969): Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci USA 63:378-383. 2. John H, Birnstiel M, Jones K (1969): RNA-DNA hybrids at the cytological level. Nature 223:582-587. 3. Harper ME, Saunders GF (1981): Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization. Chromosoma 83:431-439. 4. Harper ME, Ullrich A, Saunders GF (1981): Localization of the human insulin gene to the distal end of the short arm of chromosome 11. Proc Natl Acad Sci USA 78:4458-4460. 5. Harper ME, Marselle LM, Gallo RC, Wong-Staal F (1985): Detection of HTLV-IlI-infected lymphocytes in lymph nodes peripheral blood from AIDS patients by in situ hybridization. Proc Natl Acad Sci USA (in press) 6. Wahl GM, Stern M, Stark GR (1979): Efficient transfer of large DNA fragments from agarose gels to diazobenzloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683-3687. 7. Chandler ME, Yunis JJ (1978): A high resolution in sitn hybridization technique for the
In Situe H y b r i d i z a t i o n
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direct visualization of labeled G-banded early metaphase and prophase chromosomes. Cytogenet Cell Genet 22:352-356. 8, Bishop JM (1983): Cellular oncogenes and retroviruses. A n n Rev Biochem 52:301-352. 9. Dalla-Favera R, Bregni M, Erikson J, Patterson D, Gallo RC, Croce CM (1982): Human cmyc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc N a t / A c a d Sci USA 79:7824-7827. 10. Taub R, Kirsch I, Morton C, Lenoir G, Swan D, Tronick S, Aaronson S, Leder P (1982): Translocation of the c - m y c gene into the immunoglobulin heavy chain locus in h u m a n Burkitt lymphoma and murine plasmacytoma cells. Proc Natl Acad Sci USA 79:78377841. 11. Bartram CR, de Klein A, Hagemeijer A, van Agthoven T, van Kessel AG, Bootsma D, Grosveld G, Ferguson-Smith MA, Davies T, Stone M, Heisterkamp N, Stephenson JR, Groffen J (1983): Translocation of c-abl oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukaemia. Nature 306:277-280. 12. Collins SJ, Kubonishi I, Miyoshi I, Groudine MT (1984): Altered transcription of the c-abl oncogene in K-562 and other chronic myelogenous leukemia cells. Science 225:72-74. 13. Gale RP, Canaani E (1984): An 8-kilobase abl RNA transcript in chronic myelogenous leukemia. Proc Natl Acad Sci USA 81:5648-5652. 14. Dalla-Favera R, Franchini G, Martinotti S, Wong-Staal F, Gallo RC, Croce CM (1982): Chromosomal assignment of the h u m a n homologues of feline sarcoma virus and avian myeloblastosis virus onc genes. Proc Natl Acad Sci USA 79:4714-4717, 15. Harper ME, Franchini G, Love J, Simon MI, Gallo RC, Wong-Staal F (1983): Chromosomal sublocalization of h u m a n c-myb and c-fes cellular onc genes. Nature 304:169-171. 16. Third International Workshop on Chromosomes in Leukemia (1983): Chromosomal abnormalities and their clinical significance in acute lymphoblastic leukemia. Cancer Res 43:868-873. 17. Kowalczyk JR, Grossi M, Sandberg AA (1985): Cytogenetic findings in childhood acute lymphoblastic leukemia. Cancer Genet Cytogenet 15:47-64. 18. Trent JM, Salmon SE (1981): Karyotypic analysis of h u m a n ovarian carcinoma cells cloned in short term agar culture. Cancer Genet Cytogenet 3:279-291. 19. Becher R, Gibas Z, Sandberg AA (1983): Chromosome 6 in malignant melanoma. Cancer Genet Cytogenet 9:173-175. 20. Trent JM, Rosenfeld SB, Meyskens FL (1983): Chromosome 6q involvement in h u m a n malignant melanoma. Cancer Genet Cytogenet 9:177-180. 21. Mitelman F (1985): Calalogue of Chromosome Aberrations in Cancer, 2nd Ed. Alan R. Liss, NY, pp.139-167. 22. Westin EH, Gallo RC, Ayra SK, Eva A, Souza LM, Baluda MA, Aaronson SA, Wong-Staal F (1982): Differential expression of the a m y gene in h u m a n hematopoietic cells. Proc Natl Acad Sci USA 79:2194-2198. 23. Hayaya I, Oshimura M, Minowada J, Sandberg AA (1975): Chromosomal banding of cultured T and B lymphacytes. In Vitro 11:361-368. 24. Brahic M, Haase AT (1978): Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc Natl Acad Sci USA 75:6125-6129. 25. Cox KH, DeLeon DV, Angerer LM, Angerer RC (1984): Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Devel Biol 101:485-502. 26. Wong-Staal F, Gallo RC (1985): Human T-lymphotropic viruses. Nature (in press) 27. Popovic M, Sarngadharan MG, Read E, Gallo RC (1984): Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and preAIDS. Science 224:497-500. 28. Shaw GM, Harper ME, Hahn BH, Epstein LG, Gajdusek DC, Price RW, Navia BA, Petito CK, O'Hara CJ, Cho ES, Oleske JM, Wong-Staal F, Gallo RC (1985): HTLV-III infection in brains of children and adults with AIDS encephalopathy. Science 227:177-182. 29. Jhanwar SC, Neel BG, Hayward WS, Chaganti RSK (1984): Localization of the cellular oncogenes ABL, SIS, and FES on h u m a n germ-line chromosomes. Cytogenet Cell Genet 38:73-75. 30. Morton CC, Taub R, Diamond A, Lane MA, Cooper GM, Leder P (1984): Mapping of the h u m a n Blym-1 transforming gene activated in Burkitt lymphomas to chromosome 1. Science 223:173-175.
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31. Dayton AI, Selden JR, Laws G, Dorney DJ, Finan J, Tripputi P, Emanuel BS, Rovera G, Nowell PC, Croce CM (1984): A h u m a n c-erbA oncogene homologue is closely proximal to the chromosome 17 breakpoint in acute promyelocytic leukemia. Proc Natl Acad Sci USA 81:4495~t499. 32. Le Beau MM, Westbrook CA, Diaz MO, Rowley JD, Oren M (1985): Translocation of the p53 gene in t(15;17) in acute promyelocytic leukaemia. Nature 316:826-828. 33. de Taisne C, Gegonne A, Stehelin D, Bernheim A, Berger R (1984): Chromosomal localization of the h u m a n proto-oncogene c-ets. Nature 310:581-583. 34. Diaz MO, Le Beau MM, Pitha P, Rowley JD (1985): Interferon and c-ets-1 genes in the t(9;11)(p22;q23) in h u m a n acute monocytic leukemia. Science (in press). 35. Barker PE, Rabin M, Watson M, Breg WR, Ruddle FH, Verma IM (1984): Human c-fos oncogene mapped within chromosomal region 14q21-q31. Proc Natl Acad Sci USA 81:5826-5830. 36. Jhanwar SC, Neel BG, Hayward WS, Chaganti RSK (1983): Localization of c-ras oncogene family on h u m a n germ-line chromosomes. Proc Natl Acad Sci USA 8 0 : 4 7 9 4 4 7 9 7 . 37. Zabel BU, Naylor SL, Sakaguchi AY, Shows TB (1984): Gene mapping by in situ hybridization. Cytogenet Cell Genet 37:615-616. 38. Eccles MR, Millow LJ, Wilkins RJ, Reeve AE (1984): Harvey-ras allele deletion detected by in situ hybridization to Wilms' tumor chromosomes. Hum Genet 67:190-192. 39. Neel BG, Jhanwar SC, Chaganti RSK, Hayward WS (,1982): Two h u m a n c-onc genes are located on the long arm of chromosome 8. Proc Natl Acad Sci USA 79:7842-7846. 40. Diaz MO, Le Beau MM, Rowley JD, Drabkin H, Patterson D (1985): The role of the c-mos gene in the 8;21 translocation in h u m a n acute myeloblastic leukemia. Science 229:767769. 41. Schwab M, Varmus HE, Bishop JM, Grzeschik KH, Naylor SL, Sakaguchi AY, Brodeur G, Trent J (1984): Chromosome localization in normal h u m a n cells and neuroblastomas of a gene related to c-myc. Nature 308:288-291. 42. Davis M, Malcolm S, Hall A, Marshall CJ (1983): Localization of the h u m a n N-ras oncogene to chromosome lcen-p21 by in situ hybridization. EMBO J 2:2281-2283. 43. Rabin M, Watson M, Barker PE, Ryan J, Breg WR, Ruddle FH (1984): NRAS transforming gene maps to region p l l - p 1 3 on chromosome I by in situ hybridization. Cytogenet Cell Genet 38:70-72. 44. Munke M, Lindgren V, de Martinville B, Francke U (1984): Comparative analysis of mouse--human hybrids with rearranged chromosomes 1 by in situ hybridization and Southern blotting: High-resolution mapping of NRAS, NGFB, and AMY on h u m a n chromosome I. Somat Cell Mol Genet 10:589-599. 45. Bonner T, O'Brien SJ, Nash WG, Rapp UR, Morton CC, Leder P (1984): The h u m a n homologs of the raf (rail) oncogene are located on h u m a n chromosomes 3 and 4. Science 223:71-74. 46. Bartram CR, de Klein A, Hagemeijer A, Grosveld G, Heisterkamp N, Groffen J (1984): Localization of the h u m a n c-sis oncogene in Phl-positive and phi-negative chronic myelocytic leukemia by in situ hybridization. Blood 63:223-225. 47. Le Beau MM, Westbrook CA, Diaz MO, Rowley JD (1984): Evidence for two distinct c-src loci on h u m a n chromosomes 1 and 20. Nature 312:70-71. 48. Emanuel BS, Cannizzaro LC, Magrath I, Tsujimoto Y, Nowell PC, Croce CM (1985): Chromosomal orientation of the lambda light chain locus: Vk is proximal to Ck in 22q11. Nucl Acids Res 13:381-387. 49. Le Beau MM, Diaz MO, Karin M, Rowley JD (1985):Metallothionein gene cluster is split by chromosome 16 rearrangements in myelomonocytic leukaemia. Nature 313:709-711.
ABSTRACT: In situ hybridization offers a direct approach for localization and quantitation of nucleic acid sequences in cellular preparations. Recent improvements in technology and methodology make possible the detection of DNA and RNA of relatively low copy number. For example, development of in situ hybridization methods for detection of single copy DNA sequences on mitotic chromosomes has led to general use of this technique for gene mapping of the human genome. More recently, improvements in methodology for detection of low abundancy RNA make possible a facilitated analysis of gene expression, both from cellular genes and exogenous sequences, such as viral genomes. In situ hybridization is now a powerful method for studying nucleic acid organization and function in normal cells, as well as in malignant cells, which should contribute to better understanding of the cell transformation process.
INTRODUCTION
In situ hybridization allows the visualization of nucleic acid sequences directly on cytologic preparations. Initially described in 1969 for chromosomal localization of DNA sequences of relatively high copy n u m b e r [1, 2], in situ hybridization n o w can be used to detect chromosomal sequences present in only one or two copies per cell [3, 4] or RNA present at less than 20 transcripts per cell [5] after short autoradiographic exposure. High sensitivity is attained by hybridization of cloned nucleic acid probes radiolabeled with 3H or 35S using high specific activity nucleotide triphosphates. Slide preparations are coated with nuclear track emulsion, exposed for several days to several weeks, developed, and visualized by light microscopy. In situ hybridization offers several advantages as a technique for detecting and/ or localizing nucleic acid sequences in eukaryotic cells. In situ hybridization is a direct method, is relatively rapid, and requires small tissue samples. Low abundancy sequences can be detected readily and, u n d e r similar conditions of probe hybridization and exposure, the n u m b e r of sequences can be reasonably quantitated. In situ hybridization as a gene m a p p i n g technique results in an immediate regional or band sublocalization, directly visualizes sequence relocation as a result of chromosomal rearrangement, and can detect breakpoints w i t h i n defined sequences.
From the Laboratoryof Tumor Cell Biology,NationalCancer Institute, Bethesda, MD. Address requests for reprints to Dr. Mary E. Harper, Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, MD 20205. Received August 5, 1985; accepted August 19, 1985.
73 ©1986ElsevierScience PublishingCo., Inc.
52VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet19:73-80 (1986) 0165-4608/86/$03.50
M. E. Harper and L. M. Marselle
74 CHROMOSOMAL
MAPPING
OF SINGLE
COPY SEQUENCES
Several years ago, improvements were introduced into the in situ hybridization methodology for mapping genes on chromosomes in order to allow higher sensitivity and detection of single copy sequences on mitotic chromosomes [3, 41. According to this method, cloned DNA probes are 3H-labeled by nick translation and denatured, generating randomly cleaved, single-stranded fragments. Hybridization to high quality chromosome preparations, previously ribonuclease-treated and denatured, is carried out in the presence of 10% dextran sulfate, which promotes the formation of DNA networks among the partially complementary fragments [6]. Careful control of temperature and pH results in efficient hybridization and preservation of chromosome morphology. Following autoradiography, chromosome preparations are G-banded directly by several series of staining-destaining-restaining with Wright’s stain [7]. Labeled G-banded chromosomes are visualized directly in the microscope and grain locations are recorded and analyzed. Probes are generally hybridized at a concentration resulting in visualization of 1-5 grains per cell (typically lo-200 ngiml). Hybridization at increasing DNA concentrations results in concordant nonspecific binding, manifested as increasingly frequent chromosomal grains and/or grain clusters. Hybridized preparations are analyzed by compilation of grains, or labeled sites consisting of multiple grains, on all chromosomes within multiple mitotic spreads. Assigned localizations should be based on a significant site or sites of labeling throughout the entire karyotype. MAPPING
ONCOGENES
BY IN SITU
HYBRIDIZATION
Oncogenes represent particular DNA sequences believed to affect normal cellular events and exert a direct role in cell transformation. Such sequences include cellular equivalents of viral one genes (called c-one genes), which may correspond to genes coding for certain growth factors and cell surface receptors, as well as genes defined by their ability to induce transformation of nonneoplastic cells upon DNA transfection [for review, see ref. 81. Recently, several oncogenes have been found to be associated with the consistent, nonrandom chromosomal aberrations observed in specific human neoplasms and believed to play an important role in the genesis or progression of malignancy. For example, the consistent translocations t(8;14), t(2;8), or t(8;22) observed in Burkitt’s lymphoma place the c-myc gene into close proximity with one of the immunoglobulin gene loci (heavy, kappa or lambda chain genes) [9, lo]. Also, in all Ph-positive chronic myelogenous leukemia (CML) cases studied to date, the c-abl gene is translocated from chromosome #9 to chromosome #22 [ll], which results in altered transcription of the gene [12, 131. In order to further investigate relationships between oncogenes and nonrandom chromosomal rearrangement, a direct approach is to determine oncogene positions within the normal genome, as compared with transformed genomes. In the case of the c-myb gene, in situ hybridization was carried out to sublocalize the gene, previously assigned to chromosome #6 [l4]. Hybridization of the ‘H probe resulted in significant label on 6q, at a position slightly distal to the midportion of the arm (Fig. 1). Of 163 cells analyzed, 50 (30.7%) exhibited grains on region q2 of one or both chromosomes #S. These sites represented 7.4% (511685)of all labeled sites throughout the 163 cells. Compilation of data from a large number of labeled chromosomes #6 (88) confirmed the hybridization to 6q2 and sublocalized c-myb to 6q22-24 (Fig. 2) [15]. These results suggested that c-myb may be associated with a nonrandom chromosomal abnormality consistently observed in certain human malignancies. Deletion or translocation of the distal half of 6q has been observed in a significant num-
75
w
|
|
Figure 1 Representative human mitotic cell hybridized with the 3H-labeled c-myb-specific probe, demonstrating typical labeling of the long arm of chromosome #6 (arrow).
re 2 Distribution of labeled sites on 88 lachromosomes #6 following hybridization the c-myb probe, indicating sublocalization c-myb gene to 6q22-24.
6
76
M.E. Harper and L. M. Marselle ber of cases of acute lymphocytic leukemia (ALL) [16, 17], ovarian carcinoma [18], and melanoma [19-21]. In addition, elevated levels of c-myb RNA have been detected in primary ALL cells, as well as in two cell lines (Molt-4 and CEM) established from ALL patients [22]. Interestingly, Molt-4 cells exhibit a deletion/translocation of the q21-qter segment of one chromosome #6 [23]. Localization studies on c-myb in transformed cells containing the 6 q - chromosome, in conjunction with analyses of quantitative and qualitative changes in c-myb expression, are needed to determine the relationship of this oncogene to the chromosomal rearrangements. In situ hybridization has been utilized to sublocalize a variety of oncogenes within the human genome (Table 1). As these studies indicate, it has become increasingly apparent that other cellular oncogenes may be associated with specific chromosomal rearrangements observed in particular human neoplasms. The in situ hybridization technique allows direct examination of the position of any given cellular sequence in normal and transformed cells and also may reveal translocations not detected by standard kayotypic analysis.
DETECTION OF CELLULAR RNA BY IN SITU HYBRIDIZATION
Investigation of the expression of particular genes is important in determining the function of such genes in normal development, as well as their role in disease states such as malignancy. In situ hybridization offers several advantages as a method for studying the level and distribution of RNA transcription (see INTRODUCTION).In addition, in situ hybridization can be used to assay a wide variety of tissues for specific transcripts, and gives information regarding the type(s), as well as distribution, of cells expressing the gene within a particular tissue. In our laboratory, use of previously described methods for RNA in situ hybridization [24, 25] did not detect low abundancy RNA after short autoradiographic exposure (several days). Therefore, modifications were introduced in order to detect less than 100 copies of RNA per cell after 2-days exposure. This method makes use of 3~S-RNA probes with high specific activity (10 9 cpm/~g) synthesized from specific DNA sequences inserted into transcription vectors such as pSP64. Several pretreatment steps, stringent washing, and ribonuclease treatment following hybridization result in minimal background labeling [5]. DETECTION OF HTLV R N A BY IN SITU HYBRIDIZATION
Human T-lymphotropic virus (HTLV) represents a family of human retroviruses that share an increasing number of striking biological and biochemical properties [26]. Recent serologic, viral isolation, and nucleic acid studies have shown that HTLV-III is the etiologic agent of the acquired immunodeficiency syndrome (AIDS] [reviewed in ref. 26]. Using the improved in situ hybridization method, primary mononnclear cell preparations from peripheral blood obtained from AIDS and AIDS-related complex (ARC) patients were examined for the presence of HTLV-III RNA. Hybridization was carried out with 106 dpm 35S-RNA probe specific for HTLV-III; slides were autoradiographed and exposed for 2 days. Analysis of hybridized slides showed labeling of a very low percentage of cells (typically less than 0.01%) in the samples studied. The HTLV-III-infected cells exhibited morphologic characteristics consistent with that of lymphocytes, and contained approximately 20-100 grains per cell (Fig. 3). Mononuclear cell preparations from the same patients hybridized with a lambda-specific control probe were consistently negative. Similarly, mononuclear cells from normal individuals were negative when hybridized with the HTLV-III probe [5]. Specificity of hybridization was further shown by use of the T-cell line H9, a
77 Table 1
Oncogenes sublocalized in the h u m a n genome by in situ hybridization
Oncogene
Location
Ref.
ABL BLYM ERBA1
9q34 lp32 17q21-q22 17q11-q12 11q23-q25 11q23 15q25-q26 15q26.1 14q21-q31 11p14.1 11p15 11p13 12p12.1,12q24.2 8q22 6q22-q24 8q24 2p23-p24 1cen-p21 lp11-p13 lp22 3p25 22q13.1 22q12.3-q13.1 lp36.3,20q13.3
[11, 29] [30] [31] [32] [33] [34] [15] [29] [35] [36] [37] [38] [36] [39, 40] [15] [10, 39, 40] [41] [42] [43] [44] [45] [29] [46] [47]
ETS FES FOS HRAS1
KRAS2 MOS MYB MYC NMYC NRAS
RAF1 SIS SRC
Figure 3 Mononuclear cell preparation from uncultured peripheral blood of ARC patient hybridized with HTLV-III probe, demonstrating presence of rare infected lymphocyte expressing viral RNA.
78
M . E . Harper and L. M. Marselle neoplastic a n e u p l o i d line significantly resistant to the cytopathic effects of HTLVIII [27]. W h e n h y b r i d i z e d with the HTLV-III probe, uninfected H9 cells exhibited no grains, whereas, a clone of H9 cells infected with HTLV-III was highly labeled (average of 150 grains per cell). Comparison of n u m b e r of grains observed over the infected H9 cells with Northern blot analysis of cells from the same culture indicated a p p r o x i m a t e equivalency b e t w e e n grain n u m b e r and RNA c o p y n u m b e r when slides were exposed for 2 days [5]. The in situ h y b r i d i z a t i o n m e t h o d is n o w being used to detect the presence of HTLV-III viral sequences in a w i d e variety of tissues from infected individuals to determine the extent of cells and tissues infected. For example, hybridization to frozen sections detected HTLV-III RNA in brain tissue from AIDS patients with encephalopathy. However, the infected cells d i d not exhibit the cellular morphology of l y m p h o c y t e s [28]. Further use of in situ hybridization, in conjunction with i m m u n o c y t o c h e m i s t r y for detection of specific cellular antigens, should help to further define and characterize infected cells in HTLV-III disease.
APPLICATIONS OF IN SITU HYBRIDIZATION In situ h y b r i d i z a t i o n is a powerful technique for studying nucleic acid sequence organization and function in a w i d e variety of cell types and tissues. In addition to chromosomal m a p p i n g of DNA sequences, this m e t h o d has the capability to directly detect repositioning of sequences w i t h i n the karyotype as a result of chromosomal rearrangements. Also, small rearrangements not detectable by standard karyotypic analysis m a y be uncovered by direct h y b r i d i z a t i o n of relevant probes to chromosome spreads. A n o t h e r a p p l i c a t i o n is detection and characterization of breakpoints using probes for defined sequences; for example, the breakpoint on chromosome #22 in the variant t(8;22) in Burkitt's l y m p h o m a interrupts the l a m b d a chain variable region genes [48], and the m e t a l l o t h i o n e i n gene cluster is split by chromosome #16 rearrangements in m y e l o m o n o c y t i c l e u k e m i a [49]. Use of in situ hybridization for detection of cellular RNA should yield useful information regarding cellular specificity, quantity, and quality of expression of a w i d e variety of gene sequences. Furthermore, c o m b i n e d use of the two types of methods should contribute greatly to u n d e r s t a n d i n g the m o d e of action of oncogenes as well as other genes important in h u m a n malignancies.
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