- Email: [email protected]
R-Loop analysis of procollagen messenger RNA for the assessment of human collagen mutations
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Future Directions
Once a DNA fragment can be localized that potentially harbors a point mutation resulting in an abnormality in splicing, then direct DNA sequencing of the genomic fragment could reveal the precise mutation. Because there are two alleles represented within any restriction fragment, it will be necessary to identify the normal and abnormal allele before the DNA sequence data can be confidently interpreted. This choice would be strengthened if the intron were demonstrated to be resistant to mammalian splicing signals. We are currently evaluting the feasibility of using a murine retroviral shuttle vector as a test vector for an intron containing a mutation which alters normal splicing. 7 Genomic DNA is cloned into this vector and then transfected into a helper cell line. These cells give rise to an infectious RNA virus that has undergone splicing using mammalian splicing signals. When the RNA is eventually rescued as a bacterial plasmid, it will contain a fully spliced copy of the genomic DNA, unless there is a mutation which interferes with splicing. This intron-containing plasmid would be readily detected from the control DNA. Finally, DNA sequencing of the cDNA containing the intron will indicate the precise mutation, when it is compared to the corresponding intron from control genomic DNA. 7 C. L. Cep ko, B. E. Roberts, and R. C. Mulligan,
Cell 37,
1053 (1984).
[11] R - L o o p A n a l y s i s o f P r o c o l l a g e n M e s s e n g e r R N A for t h e Assessment of Human Collagen Mutations By
WOUTER J. DE WET
As described earlier in this section, molecular defects involving type I procollagen have been uncovered by direct analysis on the protein level of procollagen synthesized by fibroblast cultures from patients with the heritable diseases of connective tissues, osteogenesis imperfecta (OI), Marfan syndrome (MS), and the Ehlers-Danlos syndrome (EDS). The different types of mutations include substitutions and insertions or deletions of amino acid sequences. Largely because of the relative ease with which changes in the electrophoretic mobility of procollagen a chains or peptide fragments of collagen chains can be detected in SDS-polyacrylamide gels, a disproportionate number of the known structural mutations involve in-frame deletion of a large number of amino acid residues. Such inMETHODS IN ENZYMOLOGY, VOL. 145
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
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flame deletions may be the result of either complex gene rearrangements or splicing abnormalities of the very large and highly interrupted type I procollagen genes. Recent advances in the isolation and characterization of the human type I procollagen genes 1-3 have opened the way for the elucidation of the exact nature of mutations which result in in-flame collagen deletions. Because of the large size of the collagen genes and their unusual exon-intron structure, a refined determination of the position of a molecular defect is a prerequisite for cloning and/or analysis of the affected genomic region. This may be achieved by either extensive peptide analysis, blot analysis of restricted genomic fragments, or mRNA mapping. Advances in methods for the formation, stabilization, and detection of D N A - R N A hybrids have made possible the high-resolution mapping of low-abundance eukaryotic mRNAs. 4 This chapter describes and explains a specialized application of R-loop analysis for the assessment of structural abnormalities in large human mRNA transcripts. Principle of the Method D N A - R N A hybridization techniques are based on the fact that a given RNA will form a specific hybrid only with that DNA segment on one of the DNA strands which served as a template for its synthesis. Thus, when a RNA molecule and a denatured single-stranded DNA molecule with partial complementarity are hybridized, homologous regions will reanneal to form double-stranded or heteroduplex regions, whereas regions which are not complementary in base sequence do not pair and remain single-stranded. Traditional hybridization techniques, such as those that involve fixation of DNA or RNA to a solid support, are based on the formation of such D N A - R N A heteroduplexes. These hybrids are sometimes referred to as single-stranded R-loops. 5 However, when single-stranded RNA is incubated with double-stranded DNA containing sequences complementary to the specific RNA, a characteristic triple hybrid is formed, called a R-loop. 6,7 As illustrated in Fig. 1, the RNA hybridizing to its complementary sequence displaces the noncomplemeni M.-L. Chu, W. de Wet, M. Bernard, J.-F. Ding, M. Morabito, J. Myers, C. Williams, and F. Ramirez, Nature (London) 310, 337 (1984). z j. C. Myers, L. A. Dickson, W. J. de Wet, M. P. Bernard, M.-L. Chu, M. Di Liberto, G. Pepe, F. O. Sangiorgi, and F. Ramirez, J. Biol. Chem. 258, 10128 (1983). 3 W. de Wet, L. Dickson, M.-L. Chu, M. Bernard, D. Weil, and F. Ramirez, in preparation. 4 D. B. Kaback, L. M. Angerer, and N. Davidson, Nucleic Acids Res. 6, 2499 (1979). 5 C. Brack, CRC Crit. Rev. Biochem. 10, 113 (1981). 6 R. L. White and D. S. Hogness, Cell 10, 177 (1977). 7 M. Thomas, R. L. White, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 73, 2294 (1976).
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FIG. 1. R-looping analysis of the 3'-untranslated region of the human pro-al(I) collagen gene. The EcoRI 6.0-kb fragment of the genomic clone RMS-7 was subcloned in pBR322, linearized with the enzyme PvuI, and cross-linked with trimethylpsoralen prior to R-loop hybridization (also see Fig. 4). The DNA was hybridized to human fibroblast pro-ctl(I) mRNA (A), and the resultant hybrids were stabilized and spread for visualization in the electron microscope as described in Methods. The R-loop of 342 -+ 52 bp (B) represents a D N A - R N A hybrid of 314 bp. [M.-L. Chu, W. de Wet, M. Bernard, and F. Ramirez, J. Biol. Chem. 260, 2315 (1985).] Note the 3' poly(A) tail extending from the R-loop structure (C). Both single-stranded virion DNA (D) and the nicked double-stranded replicative form DNA (E) of phage ~bX174 were used as length standards. Bar, 1.0 kb.
tary DNA strand to form a bubblelike structure comprised of a D N A RNA heteroduplex region and a displaced single-stranded DNA of equal size, flanked on both sides by double-stranded DNA. In contrast to singlestranded R-loops, structures like these are more readily visualized in the electron microscope. Also, the edges of regions of homology between DNA and RNA molecules are more clearly demarcated. The kinetics of formation of heterotriplex R-loop hybrids are more complex than heteroduplex hybridization and have been extensively studied and reviewed by a number of authors. 4-9 A crucial observation was that D N A - R N A hybrids have a higher thermal stability than the corresponding DNA/RNA duplexes in high formamide concentrations of 708 j. Casey and N. Davidson, Nucleic Acids Res. 4, 1539 (1977). 9 Y.-H. Chien and N. Davidson, Nucleic Acids Res. 5, 1627 (1978).
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Consequently, the maximal rate of R-loop formation occurs at temperatures higher than the thermal denaturation temperature (Tm) of D N A - D N A hybrids, but lower than the Tm of DNA-RNA heteroduplexes. Subsequent incubation at temperatures lower than the Tm of the D N A - D N A duplex regions allows rapid renaturation of any unhybridized DNA sequences to form bona fide R-loops. Also, advanced mounting procedures, allowing discrimination between double- and single-stranded sequences, 1° made it possible to detect deletion loops in DNA-RNA hybrids when spliced RNAs were hybridized to DNA. 6 These deletion loops correspond to sequences present in DNA, but absent from RNA. The discovery of intervening sequences and the interrupted nature of eukaryotic genes greatly broadened the application of R-looping. 5 This visual technique permits mapping of exons and introns distributed over many kilobases of chromosomal DNA which otherwise would have been characterized by more tedious and time-consuming mapping techniques. An example illustrating the power of the electron microscope in analyzing complex mammalian genes is shown in Fig. 2. Hybridization of human fibroblast pro-a2(I) mRNA to the genomic clone NJ-1, 2 resulted in the formation of an elaborate DNA-RNA hybrid comprised of 26 regions of homology (exons) interdispersed by 25 displacement loops of DNA (introns). Therefore, the clone NJ-1 represents 16 kb of genomic sequences containing exons 25-50 of the human a2(I) collagen gene. These 26 exons with a combined length of 2308 bp account for 54% of the 4300-nucleotidelong mRNA. 2,3 The short single-stranded RNA segment extending from the 5' end of the R-loop contains sequences of upstream exons, which are not represented in the genomic clone. As was the case for the R-loop shown in Fig. 1, the poly(A) tail of the mRNA, together with the RNA sequences complementary to exons 51 and 52, can be seen protruding from the other end of the D N A - R N A hybrid. Protruding strands of RNA, like these, greatly aid clear discrimination between the ends of heteroduplex regions and the flanking double- or single-stranded vector sequences. Electron microscopy of such DNA-RNA hybrids not only maps the body of genomic clones, but also positions all the exon sequences present in the mRNA which are complementary to the genomic probe. As illustrated in Fig. 2, mRNA sequences corresponding to successive exons are clearly delineated by displacement loops. Also, the generation of a characteristic pattern of introns in such elaborate R-loop structures allows the positive identification of specific exonic sequences in the mRNA. As a result, heteroduplexing of large spliced mRNAs to genomic clones yields 80%. 6-8
~oR. W. Davis, M. Simon, and N. Davidson, this series, Vol. 21, p. 413.
[11]
R-LOOP ANALYSIS OF m R N A
29
239
40
0•4:14a 50
45 32
33
47
49
/
k_-
34
FIG. 2. Electron micrograph of D N A - R N A hybrid formed between the human pro-a2(1) genomic clone N J-1 and pro-a2(I) mRNA from control fibroblasts. Below the micrograph is the interpretive tracing. The numbers correspond to exons, beginning at the 5' end of the gene. D N A - R N A duplex regions are indicated by heavy lines, and single-stranded DNA by light lines. Broken lines represent unhybridized 5' and 3' ends of pro-a2(I) mRNA. Conditions for hybridization, stabilization, and spreading are described in Methods. Bar, 400 bp.
highly informative structures from which detailed maps of the mRNAs may be derived. In sharp contrast, individual molecules of unhybridized large mammalian mRNAs appear as noninformative tangled masses of collapsed bushlike structures in the electron microscope (Fig. 1). The generation of elaborate hybrid structures upon hybridization of spliced mRNA to genomic DNA clones has been utilized for the assessment of major structural abnormalities in human type I collagen mRNAs. H,12 As diagrammatically illustrated in Fig. 3A, deletion, substiH W. de Wet, M. Sippola, G. Tromp, D. Prockop, M.-L. Chu, and F. Ramirez, J. Biol. Chem. 261, 3857 (1986). ~2 W. J. de Wet, unpublished observations, 1985.
240
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A
Genomic clone b
(3
c
d
e
mRNA
¥ t3
]E
II
t3
B
cDNA clone e
b
c
d
e
f
mRNA
LLt b
c
d
e
f
o
LL
b~e
V a
f
b
c
d
e
f
Lv
,~/d - m
o b c|e f o b c FIG. 3. Schematic representation of five different types of D N A - R N A hybrids that may be formed between control or mutated m R N A and the corresponding genomic DNA (A) or c D N A (B). Structures I and i depict hybridization to control mRNA. The following anomalous hybridization patterns represent m R N A mutations involving one or more exons: Structures II and ii, deletion of exon d; structures Ill and iii, deletion of exons c and d; structures IV and iv, inversion or substitution of exon d; structures V and v, insertion or duplication of exonic sequences between exons c and d. mRNAs are indicated by heavy lines, and D N A is indicated by light lines (introns in A, displaced exons in B) or boxes (exons a - f ) .
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tution, inversion, insertion, or duplication in mutant mRNA of sequences corresponding to one or more exons will result in the formation of three different types of aberrant D N A - R N A heteroduplexes. 1. Compared to the normal hybrid (structure I), deletion in the mRNA of the sequences corresponding to exon d will lead to a hybrid (structure II) containing four, instead of the normal five displacement loops. Also, exon d will not be visualized, but will be absorbed into a larger displacement loop between exons c and e. Consequently, the measured length of the DNA strand displaced between exons c and e should correspond to the combined lengths of intron c, exon d, and intron d. Structure III reflects the deletion of more than one consecutive exons. 2. Inversion or substitution of the sequences corresponding to exon d in mutant mRNA will produce a slightly different abnormal hybrid (structure IV) containing an asymmetrical bubblelike nonhomologous region between the homologous regions c and e. Again, exon d will not be visualized, but will be contained in the larger displacement loop between exons c and e. 3. Insertion or duplication of exonic sequences between exons c and d, on the other hand, will result in the appearance of the normal six regions of homology (structure V). However, the homologies corresponding to exons c and d will be interrupted by an additional displacement loop in the RNA strand. The deletion loop of RNA will coincide with the intronic loop between exons c and d, to form an asymmetrical bubble structure in that region. On the basis of the different scenarios depicted in Fig. 3A, this type of Rloop analysis can be used as a sensitive tool for detecting not only the position, but also the extent and nature of mutations involving the sequences of one or more exons. A more traditional approach to heteroduplex mapping of the above-mentioned types of mutations would involve hybridization of the mutant mRNA to normal cDNA clones. 10,~3For comparison, a similar set of possible hybrid structures is shown in Fig. 3B. Although widely used for nuclease $1 mapping, 14'~5 hybridization of mutant mRNA to cDNA will generate a visually less recognizable pattern of small displacements. Therefore, the more traditional heteroduplex mapping procedures clearly do not allow the assessment of the different types of exonic mutations with a comparable sensitivity or accuracy in the electron microscope. 13 B. C. Westmoreland, W. Szybalski, and H. Ris, Science 163, 1343 (1969). 14 p. A. Sharp, A. J. Berk, and S. M. Berget, this series, Vol. 65, p. 750. 15 T. Pihlajaniemi and J. C. Myers, this volume [9].
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Materials and Reagents
Nucleic Acids Preparation of RNA. Control fibroblasts (GM3348 and GM970) and fibroblasts from a lethal variant of OI (GM2962) were purchased from the Human Genetic Mutant Cell Repository (Camden, New Jersey). Fibroblast cultures are grown in 175-cm2 flasks under standard conditions in Dulbecco's modified Eagle's minimum essential medium containing 10% fetal bovine serum. At confluency, total poly(A)-enriched RNA is isolated according to the method of Burnett and Rosenbloom. 16 Isolation ofDNA. Six human genomic clones have thus far been used for the detection of mutations in type I collagen mRNAs. 1H2 Two of these, RMS-1 and RMS-8, code for the pro-al(I) collagen chain. 1The proa2(I) chain, except for the carboxy-terminal 126 amino acid residues, is encoded by the clones NJ-l, NJ-3, NJ-9, and NJ-27. 2,3 These genomic clones were kindly made available by Francesco Ramirez (UMDNJRutgers Medical School, Piscataway, New Jersey). Amplification of the genomic clones in phage Charon 4A and purification of phage DNA are performed according to standard procedures.~7 Standards for Length Calibration. Nicked double-stranded form DNA (5386 bp) as well as single-stranded virion DNA (5386 nucleotides) of phage ~bX174 were from New England Biolabs. Both forms of tbX174 are routinely included in all spreads as internal length standards for the determination of absolute molecular sizes.18 Materials Formamide (99%, Fluka or Matheson, Coleman, and Bell) is recrystallized as described by Casey and Davidson, 8and stored at -80°C. Glyoxal (40%, Matheson, Coleman, and Bell) Piperazine-N,N'-bis-2-ethanesulfonic acid (PIPES) and cytochrome c (type VI) were purchased from Sigma. Copper 200-mesh grids (GC 200), tungsten wire (diameter 0.025 in.), platinum-paladium (80:20) wire (diameter 0.008 in.), and uranyl acetate were from Ted Pella, Inc. Nitrocellulose (1% in amyl acetate, Ernest F. Fullam, Inc.) lntramedic polyethylene tubing (internal diameter 1.14 mm, Clay Adams) 16 W. Burnett and J. Rosenbioom, Biochem. Biophys. Res. Commun. 86, 478 (1979). 17 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982. is D. Stfiber and H. Bujard, Mol. Gen. Genet. 154, 299 (1977).
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Pyrex disposable microsampling pipettes (Coming Glass Works) and 6 x 50-mm borosilicate glass tubes (Kimble) are rendered nucleasefree by baking at 200° for 2 hr. Only fresh double-glass distilled H20 is used.
Specialized Preparatory Procedures Ultracleaning of Dialysis Tubing. Dialysis tubing (1 in., Sigma) is cut into 12- to 18-in.-long pieces and boiled in 1.5% (w/v) NaHCO3 and 0.5% (w/v) EDTA for 30 min. The tubing is then rinsed with copious amounts of water, and the boiling and rinsing are at least repeated twice. After another boiling step in water only, the tubing is autoclaved in water for 20 min, and finally stored in 1 mM EDTA (pH 7.0) at 4 °. Ultracleaning of Glass Slides. After treatment with chromic acid overnight, borosilicate glass slides are extensively washed with copious amounts of water. Care should be taken to handle the slides only with a forceps. The washed slides are placed in a near vertical position in a covered glass beaker, containing a circular pad of Whatman paper, to drain and dry completely. Only completely clean and dry glass slides should be used. Coating of Grids with Nitrocellulose. The following standard procedure rendered very strong supporting films on copper electron microscope grids. A carefully cleaned rectangular glass trough is filled with water within 1-2 cm of the top. In the following step, about four drops of a 1% nitrocellulose solution in amyl acetate are spread on a glass slide. Excess solution is vertically drained on blotting paper, and the uniform film of nitrocellulose is left to air-dry for about 3-5 min. A razor is used to cut the film along the sides and edges of the slide. While this is in progress, the surface of the water in the glass trough is cleaned by dropping a few drops of nitrocellulose solution onto the water. After a few minutes, the polymerized film is skimmed with an open forceps. At this stage, the nitrocellulose-covered glass slide is slowly submersed at an angle of about 45 ° into the water, allowing the film to gradually disengage. As soon as the film almost completely floats on the surface, the slide is gently dropped to fully separate the floating film from the slide. Copper grids are rinsed by two quick sequential immersions in acetone and air-dried on Whatman paper. The cleaned grids are, dull side down, carefully dropped in a regular pattern, close together, on the most uniform spots of the floating film. Areas with a yellowish or bluish appearance should be ignored. Using a forceps, a slightly larger piece of Whatman No. 1 paper is dropped on top of the grid-carrying film. The forceps is quickly repositioned to the other end of the filter paper and the whole
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array is lifted in one sweeping motion, blotted dry from the back, and finally baked at 65° for 10 min in a glass Petri dish. At least five batches of covered grids should be available for the mounting of nucleic acids for electron microscopy. Custom-Made Teflonware. At least two rectangular Teflon bars (15 × 1 × 0.7 cm) are used for spreading. Custom-made ultramicro Teflon dialysis holders greatly facilitate dialysis of small volumes. A circular recess with a diameter of 9 mm and a depth of 4 mm is machined into one side of a Teflon disk (16 x 8 mm). A groove, to accommodate an O ring, is cut into the circumference, and the outer edge of the disk is rounded to facilitate mounting of the O ring. For dialysis, a rectangular piece of dialysis tubing is cut along one edge, opened on a clean surface (unused aluminum foil), blotted, and draped over the well in the disk. The dialysis membrane is finally mounted by pushing an O ring over the rounded edge into the groove. During dialysis, the sample should be in contact with the membrane. For removal of the sample following dialysis, the holder is (membrane up) centrifuged at low speeds for a few seconds. A small hole is cut in the dialysis membrane and the sample is removed.
Solutions R-loop stock buffer: PIPES, 1 M; EDTA, 0.I M adjusted to pH 7.2 at 20° with concentrated NaOH. Dialysis buffer: NaCI, 0.5 M; EDTA, I mM; Tris 10 mM adjusted to pH 7.2 at 20 ° with HCI Spreading stock buffer: Tris, 0.5 M; EDTA, 0.05 M adjusted to pH 8.5 at 20° with HCI Cytochrome c solution: cytochrome c is dissolved at a concentration of 1 mg/ml in buffer containing Tris-HCl, 0. I M; EDTA, I0 mM adjusted to pH 8.5. The solution should be stored at 4° in a dark container. Uranyl acetate stock solution: uranyl acetate, 0.05 M; HCI, 0.05 M in 90% ethanol. The solution should be stored at 4° in a dark container. Methods In order to cover fully the procedure for effective identification of mRNA mutations by R-loop mapping, the methodology is divided into three sections dealing with (1) R-loop formation and stabilization, (2) processing of R-loops for electron microscopy, and (3) electron microscopy and interpretation of results.
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R-Loop Formation and Stabilization A typical 50-/.d hybridization mixture contains 6/.~g/ml of phage DNA (of maximum sequence complexity of 50 kb) and 20-40/~g/ml of fibroblast total poly(A)-enriched RNA in 70% (v/v) recrystallized formamide, 0.1 M PIPES (pH 7.2), 0.01 M EDTA, and 0.3 M NaCI (a final [Na +] of about 0.46 M including the contribution of the PIPES~9). R-loop mixtures are constituted by first mixing 3/~1 of 5 M NaCI, 5/~1 of R-loop stock buffer, and 35/~1 of recrystallized formamide. This is best done in 6 x 50 mm glass tubes using Pyrex microsampling pipettes. Phage DNA, followed by the RNA, is then added to a final volume of 50/~1. To minimize evaporation during prolonged incubation at high temperatures, the R-loop mixtures are covered by 50-100/A of Paraffin Oil and the tubes are well sealed with Parafilm. Incubation at the optimal temperature is a critical aspect of R-loop formation. In principle, the optimal incubation temperature is limited by two parameters. These are the irreversible melting temperature (Tss) of the DNA sequence participating in R-loop formation and the higher melting temperature of the specific D N A - R N A heteroduplex. 7,8 In high formamide concentrations, the difference between these two parameters varies. 8 Furthermore, depending on the GC content, the Tss values of most DNA sequences vary between 45 and 58° (in 70% formamide and about 0.5 M Na+). 7 This may imply that the efficient formation of R-loops requires determination of the T~s of every DNA sequence that participates in R-loop formation, especially in genes containing GC-rich clusters such as the collagen genes. In order to obviate difficult and tedious procedures 2° to accommodate the widely different denaturation temperatures of different DNA sequences, the following protocol is employed to enhance R-loop formation. R-loop mixtures containing DNA that code for procollagen triple-helical sequences are first incubated at 57° for 12 hr. The temperature is then decreased to 53° over a 2-hr period, and the incubation is continued for another 3 hr. On the other hand, hybridization to DNA clones encoding nontriple-helical propeptide regions is performed first at 52 ° for 12 hr, followed by an incubation at 48° for 4 hr. Incubation at the initial higher temperatures results in complete dissociation of DNA duplexes, allowing hybridization of mRNA with single-stranded DNA. Furthermore, incubation over the indicated range of temperatures is very 19 D. S. Holmes, R. H. Cohn, H. H. Kedes, and N. Davidson, Biochemistry 16, 1504 (1977). 20 G. Vogeli, H. Ohkubo, V. E. Avvedimento, M. Sullivan, Y. Yamada, M. Mudryj, I. Pastan, and B. de Crombrugghe, Cold Spring Harbor Syrup. Quant. Biol. 45, 777 (1980).
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FIG. 4. Visualization of psoralen cross-links in DNA. The genomic subclone containing the 3'-untranslated region of the human pro-al(I) collagen gene (Fig. 1) was linearized with the enzyme PvuI and cross-linked with trimethylpsoralen. An aliquot of the cross-linked material was subjected to glyoxal denaturation and spread for electron microscopy. Crosslinking and the determination of the cross-linking frequency were performed according to the procedure of D. B. Kaback, L. M. Angerer, and N. Davidson, Nucleic Acids Res. 6, 2499 (1979). Ideally two random cross-links (denoted by arrows) should be present. Bar, 2.0 kb.
likely to result in association of DNA and RNA, regardless of the base composition of the interacting nucleic acids. Under certain circumstances, formation of bona fide R-loop structures are beneficial for the analysis of D N A - R N A hybrids (Fig. 1). Introduction of a few random trimethylpsoralen-induced cross-links 2~ in vector sequences flanking the region of interest prior to R-looping prevents complete strand separation. The two complementary DNA strands are held in register (Fig. 4). This greatly facilitates snapback of any unhybridized DNA sequences on both sides of the DNA-RNA hybrid region. 4 However, trimethylpsoralen cross-linking does not necessarily enhance analysis of elaborate D N A - R N A heteroduplexes of the type shown in Fig. 2. Moreover, introduction of random cross-links in large cloned regions of the collagen genes may seriously distort interaction between DNA and RNA in D N A - R N A heteroduplexes. The technique, therefore, has a limited application in R-loop analysis and should only be attempted when the formation of bona fide R-loops is essential. 21 R. S. Cole, Biochim. Biophys. Acta 217, 30 (1970).
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In fact, visual identification of an elaborate pattern of deletion loops in D N A - R N A hybrids is considerably simplified by electron microscopy of single-stranded R-loop structures such as the example shown in Fig. 2. Presence of displaced DNA strands in large bona fide R-loops may result in branch migration, random displacement of RNA sequences, entangling with and masking of deletion loops, and even formation of triple hybrids. The latter is the result of snapback of complementary intronic DNA sequences 5 that may greatly distort D N A - R N A heteroduplexes. Rapid lowering of the temperature at the end of the incubation usually does not allow extensive renaturation of any unhybridized DNA sequences and results in the appearance of single-stranded R-loops. Although R-loops, once formed, are quite stable, branch migration at the ends of D N A - R N A segments frequently induces partial displacement of RNA sequences and selective loss of smaller R-loops. 4'7'22 This especially occurs when the formamide concentration and temperature are decreased during the process of mounting nucleic acids for electron microscopy. Spreading from high-formamide solutions may reduce branch migration, but frequently may result in poor contrast. In an important modification of the R-loop method, Kaback et al. 4 stabilized R-loops by treatment with glyoxal at 12°. Under these conditions, the denaturing agent specifically modifies only unhybridized guanine residues, thus minimizing branch migration even in the absence of formamide. At the end of the incubation, the R-loop mixtures are quickly chilled, separated from the overlaying Parafin Oil, and transferred to separate 6 × 50 mm glass tubes. Complete separation of the two phases is easily done by rapid uptake of the lower hydrophilic phase in a 20-cm-long piece of polyethylene tubing, attached to a micropipette suction apparatus of the Clay Adams type. The distal end of the tubing containing the oil phase is simply snipped off with a razor. To stabilize the R-loops, 40% glyoxal is added to a final concentration of 1.0 M (1/7 vol), 4 and the solution is incubated for 2 hr at 11°. Extreme care should be taken to prevent warming of glyoxal-containing material above 12°. To remove free glyoxal, the R-loops are dialyzed against the dialysis buffer at 4°.
Processing of R-Loops for Electron Microscopy A simplified version of the original Kleinschmidt monolayer technique 23for the mounting of nucleic acids will be described. To constitute a 50-t~l spreading solution containing about 50 ng of DNA, 25 ~1 of recrystallized formamide are mixed with 10 ~1 of spreading stock buffer in a 6 × 22 C. S. Lee, R. W. Davis, and N. Davidson, J. Mol. Biol. 48, 1 (1970). 23 A. K. Kleinschmidt, this series, Vol. 12, p. 361.
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50 mm glass tube. For length calibration, 2/.d of 1/zg/ml double-stranded qbX174 DNA and 2/zl of 0.5/zg/ml single-stranded ~bX174 virion DNA are added. At this stage a 90-ram square plastic Petri dish is filled with hypophase until the hypophase is well above the edges, being held there by surface tension. Although freshly made 15% (v/v) commercial formamide in 10 mM Tris-HC1 (pH 8.5)-1 mM EDTA is frequently used as a hypophase, I prefer water. A rectangular Teflon bar is placed in front of the hypophase surface so that the bar is supported by the edges of the tray. The bar is then gently pushed to the rear over about two-thirds of the length of the dish. Another Teflon bar is placed just in front of the first bar and drawn forward over about one-third of the length of the dish. This leaves the central region of the surface of the hypophase wiped twice. A dry glass slide, serving as a ramp, is then inserted at an angle of about 30° into the hypophase between the two Teflon bars, so that the upper onethird of the slide protrudes above the surface and rests against the rear Teflon bar. No talc should be dusted on the surface of the hypophase. In the next step, 10/xl of the dialyzed R-loop solution is mixed with the spreading solution, followed by 2/zl of the cytochrome c solution. The spreading solution is immediately and continuously spread from a 50-/zl glass microsampling pipette onto the glass ramp, about 1 cm above the surface of the hypophase. After about 40 sec, the monomolecular film is picked up onto three covered grids, preferably from different batches, at a distance of about 1 cm from the slide-solution boundary. The grids are stained for 30 sec in diluted uranyl acetate, freshly prepared (within 2 hr) by diluting 10/~1 of the stock solution with 5 ml of 90% ethanol. After staining, the grids are submersed for 10 sec in 90% ethanol. To enhance the poor contrast obtained with staining only, the specimens are rotary-shadowed essentially as described. 23 Shadowing with PtPd (80 : 20) at a distance of about 15 cm and an angle of 9° is performed in a high vacuum. About 16 turns of the Pt-Pd wire around a suitably bent tungsten wire electrode yield a satisfactory result. Slow melting and evaporation of the Pt-Pd through at least two spark events is crucial.
Electron Microscopy and Interpretation of Results R-loop structures are visualized and photographed on electron image film (3¼ × 4 in.) at a screen magnification of 18,000 and 36,000 times and 60-kV accelerating voltage, z4 Only structures should be photographed in which the intronic deletion loops are sufficiently unambiguous to permit 24 R. Schleif and J. Hirsh, this series, Vol. 65, p. 885.
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249
immediate identification by visual inspection in the electron microscope. Whenever possible, double- and single-stranded forms of ~bX174 DNA should appear in the same electron micrograph as the R-loop of interest (Fig. 1). The negatives are enlarged 5 times, and the images are printed on high-contrast photographic paper. Following identification of the various exonic and intronic regions of D N A - R N A heteroduplexes, molecular lengths of these regions are obtained by comparative contour length measurements, relative to the internal double- or single-stranded DNA standards, ~°,18 using an electronic digitizer. At least 20 molecules should be digitized. An example of the use of R-loop mapping for the assessment of human collagen mutations is shown in Fig. 5. Cultured skin fibroblasts from a perinatal lethal variant (GM2962) of OI were previously found to synthesize pro-a2(I) chains shortened by about 20 amino acids in o~2(I)-CB3,5 A, a fragment containing amino acids 358-775 of the a2(I) chain. 25 In comparison with D N A - R N A heteroduplexes formed between the human proot2(I) genomic clone NJ-1 and pro-ot2(I) mRNA from control fibroblasts (Fig. 2), abberant R-loop structures were obtained upon hybridization of the variant's pro-t~2(I) mRNA to the same genomic clone (Fig. 5). Instead of the normal 26 exons and 25 introns, only 25 exons and 24 introns were visualized. The abnormal distribution of exons and introns shown in Fig. 5, indicates that the sequences corresponding to exon 28, a 54-bp exon encoding amino acids 448-465, did not hybridize to the variant's pro-a2(I) mRNA. In fact, a larger intron of 408 --- 28 bp can be seen between exons 27 and 29. In control R-loops, exons 27 (59 --+ 15 bp), 28 (53 -+ 7 bp), and 29 (69 - 13 bp) are interrupted by two relatively small introns, respectively, 114 --- 18 bp and 223 +-- 34 bp in size (Fig. 2). The length of the DNA strand displaced between exons 27 and 29 in the aberrant D N A - R N A heteroduplexes, therefore, closely correspond to the combined lengths of intron 27, exon 28, and intron 28. Also, no additional displacement loop in the RNA strand between exons 27 and 29 can be seen (Fig. 5). On this basis, heteroduplexes formed between the genomic clone NJ-1 and the variant's pro-t~2(I) mRNA resemble the deletion hybrid (structure II) shown in Fig. 3A. Absence of hybridization to exon 28 was only observed with mutant pro-et2(I) mRNA, and exon 28 was the only exon for which loss of hybridization was visualized. Moreover, that the sequences corresponding to exon 28 were absent in the mutated pro-a2(I) mRNA was independently substantiated by conventional nuclease S~ analysis, n 25 W. J. de Wet, T. Pihlajaniemi, J. Myers, T. E. Kelly, and D. J. Prockop, J. Biol. Chem. 258, 7721 (1983).
250
GENETICANOMALIES .
.
.
.
.
.
.
.
.
[1 1] .
.
i
27.-~ (-~ ~ 26
^N. I IJ / .32
I /4d~ ; u--.. U~! (}oIj,o45 (,'-, 50
FIG. 5. R-loop mapping of pro-a2(I) mRNA from a lethal variant of OI. Poly(A)-enriched RNA from OI fibroblasts was hybridized to the human pro-a2(I) genomic clone NJ-1, and the resultant hybrids were stabilized and spread as in Methods. Below the micrograph is the interpretative tracing. The numbers correspond to exons, beginning at the 5' end of the gene. D N A - R N A duplex regions are indicated by heavy lines, and single-stranded DNA is indicated by light lines. Unhybridized 5' and 3' ends of the pro-a2(I) mRNA are also shown. The asterisk (*) indicates the position of the altered region in R-loops formed with the mutated mRNA. Bar, 400 bp.
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Comments
1. Because the R-loop method is based on the generation of an abnormal pattern of introns and exons upon hybridization of mutated mRNA to a normal genomic clone, the validity of the approach is determined by the specificity and stability of exon hybridization. Absence of hybridization to an exon should not be found for any other exon except in the mutated sequences. R-loop mapping of 138 exons in genomic clones covering the pro-otl(I), 1pro-ct2(I), 2,3 and pro-ctl(III) 26 genes showed specific and stable hybridization to exons, ranging in size from 35 bp to more than 1000 bp under the conditions for R-loop formation, hybridization, and spreading described here. This was especially the case for the hybridization of procollagen mRNAs to the numerous triple-helical domain exons. However, these conditions did not allow visualization of two extremely small exons of 11 and 15 bp, respectively, in the N-propeptide region of the human pro-a2(I) gene. 3 2. Although it is not known what degree of sequence mismatch will result in the absence of hybridization to a specific exon, the R-loop method will only be applicable for the mapping of mutations that cover a significant part of an exon. The technique is not suitable for the detection of small regions of mismatch such as the 4-bp frameshift deletion recently found in the pro-a2(I) gene. 27 3. In contrast to the widely used nuclease $1 mapping technique, 14,15 the R-loop method is based on hybridization to genomic clones. Except for the example shown in Fig. 5, deletion at the mRNA level of exon sequences have been visually detected for exon 6 and exon 11 of the proa2(I) gene, as well as for exon 6 of the pro-ctl(I) gene. 11,12Because these mutations fall beyond the reach of any of the existing human procollagen cDNA clones, it was not possible to employ nuclease S~ analysis. Also, other nucleic acid mapping techniques such as Southern blot analysis were not sensitive enough to detect the mutations. 1~R-loop hybridization did allow the identification of anomalies in the hybridization pattern of these human collagen gene mutations with a high degree of accuracy. 4. Although R-looping can be used as a powerful and sensitive tool for detecting collagen mutations leading to abnormal mRNA transcripts, the technique does not provide a definite answer about the exact cause of rearrangements at the gene level. Direct sequencing of the affected alleles is required to distinguish between genomic deletion or splicing defects. 26 M.-L. Chu, D. Weil, W. de Wet, M. Bernard, M. Sippola, and F. Ramirez, J. Biol. Chem. 260, 4357 (1985). 27 T. Pihlajaniemi, L. A. Dickson, F. M. Pope, V. R. Korhonen, A. Nicholls, D. J. Prockop, and J. C. Myers, J. Biol. Chem. 259, 12941 (1984).
252
GENETIC ANOMALIES
[1 1]
Acknowledgments I wish to thank Dr. F. Ramirez for his kind gift of the human collagen genomic clones, Dr. D. J. Prockop for his enthusiastic support, Dr. J. Coetzee and Mr. C. van der Merwe (Electron Microscope Unit, University of Pretoria) for kindly providing facilities, and most of all Dr. David Kaback for his invaluable advice and kindness in introducing me to R-looping. I also gratefully acknowledge the invaluable help of Ms. Isobel de Beer and Ms. Rina Kroeze in preparing the manuscript. This work was supported by a grant from the South African Medical Research Council.
R-LooP ANALYSISOF mRNA
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Future Directions
Once a DNA fragment can be localized that potentially harbors a point mutation resulting in an abnormality in splicing, then direct DNA sequencing of the genomic fragment could reveal the precise mutation. Because there are two alleles represented within any restriction fragment, it will be necessary to identify the normal and abnormal allele before the DNA sequence data can be confidently interpreted. This choice would be strengthened if the intron were demonstrated to be resistant to mammalian splicing signals. We are currently evaluting the feasibility of using a murine retroviral shuttle vector as a test vector for an intron containing a mutation which alters normal splicing. 7 Genomic DNA is cloned into this vector and then transfected into a helper cell line. These cells give rise to an infectious RNA virus that has undergone splicing using mammalian splicing signals. When the RNA is eventually rescued as a bacterial plasmid, it will contain a fully spliced copy of the genomic DNA, unless there is a mutation which interferes with splicing. This intron-containing plasmid would be readily detected from the control DNA. Finally, DNA sequencing of the cDNA containing the intron will indicate the precise mutation, when it is compared to the corresponding intron from control genomic DNA. 7 C. L. Cep ko, B. E. Roberts, and R. C. Mulligan,
Cell 37,
1053 (1984).
[11] R - L o o p A n a l y s i s o f P r o c o l l a g e n M e s s e n g e r R N A for t h e Assessment of Human Collagen Mutations By
WOUTER J. DE WET
As described earlier in this section, molecular defects involving type I procollagen have been uncovered by direct analysis on the protein level of procollagen synthesized by fibroblast cultures from patients with the heritable diseases of connective tissues, osteogenesis imperfecta (OI), Marfan syndrome (MS), and the Ehlers-Danlos syndrome (EDS). The different types of mutations include substitutions and insertions or deletions of amino acid sequences. Largely because of the relative ease with which changes in the electrophoretic mobility of procollagen a chains or peptide fragments of collagen chains can be detected in SDS-polyacrylamide gels, a disproportionate number of the known structural mutations involve in-frame deletion of a large number of amino acid residues. Such inMETHODS IN ENZYMOLOGY, VOL. 145
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
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flame deletions may be the result of either complex gene rearrangements or splicing abnormalities of the very large and highly interrupted type I procollagen genes. Recent advances in the isolation and characterization of the human type I procollagen genes 1-3 have opened the way for the elucidation of the exact nature of mutations which result in in-flame collagen deletions. Because of the large size of the collagen genes and their unusual exon-intron structure, a refined determination of the position of a molecular defect is a prerequisite for cloning and/or analysis of the affected genomic region. This may be achieved by either extensive peptide analysis, blot analysis of restricted genomic fragments, or mRNA mapping. Advances in methods for the formation, stabilization, and detection of D N A - R N A hybrids have made possible the high-resolution mapping of low-abundance eukaryotic mRNAs. 4 This chapter describes and explains a specialized application of R-loop analysis for the assessment of structural abnormalities in large human mRNA transcripts. Principle of the Method D N A - R N A hybridization techniques are based on the fact that a given RNA will form a specific hybrid only with that DNA segment on one of the DNA strands which served as a template for its synthesis. Thus, when a RNA molecule and a denatured single-stranded DNA molecule with partial complementarity are hybridized, homologous regions will reanneal to form double-stranded or heteroduplex regions, whereas regions which are not complementary in base sequence do not pair and remain single-stranded. Traditional hybridization techniques, such as those that involve fixation of DNA or RNA to a solid support, are based on the formation of such D N A - R N A heteroduplexes. These hybrids are sometimes referred to as single-stranded R-loops. 5 However, when single-stranded RNA is incubated with double-stranded DNA containing sequences complementary to the specific RNA, a characteristic triple hybrid is formed, called a R-loop. 6,7 As illustrated in Fig. 1, the RNA hybridizing to its complementary sequence displaces the noncomplemeni M.-L. Chu, W. de Wet, M. Bernard, J.-F. Ding, M. Morabito, J. Myers, C. Williams, and F. Ramirez, Nature (London) 310, 337 (1984). z j. C. Myers, L. A. Dickson, W. J. de Wet, M. P. Bernard, M.-L. Chu, M. Di Liberto, G. Pepe, F. O. Sangiorgi, and F. Ramirez, J. Biol. Chem. 258, 10128 (1983). 3 W. de Wet, L. Dickson, M.-L. Chu, M. Bernard, D. Weil, and F. Ramirez, in preparation. 4 D. B. Kaback, L. M. Angerer, and N. Davidson, Nucleic Acids Res. 6, 2499 (1979). 5 C. Brack, CRC Crit. Rev. Biochem. 10, 113 (1981). 6 R. L. White and D. S. Hogness, Cell 10, 177 (1977). 7 M. Thomas, R. L. White, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 73, 2294 (1976).
[11]
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237
FIG. 1. R-looping analysis of the 3'-untranslated region of the human pro-al(I) collagen gene. The EcoRI 6.0-kb fragment of the genomic clone RMS-7 was subcloned in pBR322, linearized with the enzyme PvuI, and cross-linked with trimethylpsoralen prior to R-loop hybridization (also see Fig. 4). The DNA was hybridized to human fibroblast pro-ctl(I) mRNA (A), and the resultant hybrids were stabilized and spread for visualization in the electron microscope as described in Methods. The R-loop of 342 -+ 52 bp (B) represents a D N A - R N A hybrid of 314 bp. [M.-L. Chu, W. de Wet, M. Bernard, and F. Ramirez, J. Biol. Chem. 260, 2315 (1985).] Note the 3' poly(A) tail extending from the R-loop structure (C). Both single-stranded virion DNA (D) and the nicked double-stranded replicative form DNA (E) of phage ~bX174 were used as length standards. Bar, 1.0 kb.
tary DNA strand to form a bubblelike structure comprised of a D N A RNA heteroduplex region and a displaced single-stranded DNA of equal size, flanked on both sides by double-stranded DNA. In contrast to singlestranded R-loops, structures like these are more readily visualized in the electron microscope. Also, the edges of regions of homology between DNA and RNA molecules are more clearly demarcated. The kinetics of formation of heterotriplex R-loop hybrids are more complex than heteroduplex hybridization and have been extensively studied and reviewed by a number of authors. 4-9 A crucial observation was that D N A - R N A hybrids have a higher thermal stability than the corresponding DNA/RNA duplexes in high formamide concentrations of 708 j. Casey and N. Davidson, Nucleic Acids Res. 4, 1539 (1977). 9 Y.-H. Chien and N. Davidson, Nucleic Acids Res. 5, 1627 (1978).
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Consequently, the maximal rate of R-loop formation occurs at temperatures higher than the thermal denaturation temperature (Tm) of D N A - D N A hybrids, but lower than the Tm of DNA-RNA heteroduplexes. Subsequent incubation at temperatures lower than the Tm of the D N A - D N A duplex regions allows rapid renaturation of any unhybridized DNA sequences to form bona fide R-loops. Also, advanced mounting procedures, allowing discrimination between double- and single-stranded sequences, 1° made it possible to detect deletion loops in DNA-RNA hybrids when spliced RNAs were hybridized to DNA. 6 These deletion loops correspond to sequences present in DNA, but absent from RNA. The discovery of intervening sequences and the interrupted nature of eukaryotic genes greatly broadened the application of R-looping. 5 This visual technique permits mapping of exons and introns distributed over many kilobases of chromosomal DNA which otherwise would have been characterized by more tedious and time-consuming mapping techniques. An example illustrating the power of the electron microscope in analyzing complex mammalian genes is shown in Fig. 2. Hybridization of human fibroblast pro-a2(I) mRNA to the genomic clone NJ-1, 2 resulted in the formation of an elaborate DNA-RNA hybrid comprised of 26 regions of homology (exons) interdispersed by 25 displacement loops of DNA (introns). Therefore, the clone NJ-1 represents 16 kb of genomic sequences containing exons 25-50 of the human a2(I) collagen gene. These 26 exons with a combined length of 2308 bp account for 54% of the 4300-nucleotidelong mRNA. 2,3 The short single-stranded RNA segment extending from the 5' end of the R-loop contains sequences of upstream exons, which are not represented in the genomic clone. As was the case for the R-loop shown in Fig. 1, the poly(A) tail of the mRNA, together with the RNA sequences complementary to exons 51 and 52, can be seen protruding from the other end of the D N A - R N A hybrid. Protruding strands of RNA, like these, greatly aid clear discrimination between the ends of heteroduplex regions and the flanking double- or single-stranded vector sequences. Electron microscopy of such DNA-RNA hybrids not only maps the body of genomic clones, but also positions all the exon sequences present in the mRNA which are complementary to the genomic probe. As illustrated in Fig. 2, mRNA sequences corresponding to successive exons are clearly delineated by displacement loops. Also, the generation of a characteristic pattern of introns in such elaborate R-loop structures allows the positive identification of specific exonic sequences in the mRNA. As a result, heteroduplexing of large spliced mRNAs to genomic clones yields 80%. 6-8
~oR. W. Davis, M. Simon, and N. Davidson, this series, Vol. 21, p. 413.
[11]
R-LOOP ANALYSIS OF m R N A
29
239
40
0•4:14a 50
45 32
33
47
49
/
k_-
34
FIG. 2. Electron micrograph of D N A - R N A hybrid formed between the human pro-a2(1) genomic clone N J-1 and pro-a2(I) mRNA from control fibroblasts. Below the micrograph is the interpretive tracing. The numbers correspond to exons, beginning at the 5' end of the gene. D N A - R N A duplex regions are indicated by heavy lines, and single-stranded DNA by light lines. Broken lines represent unhybridized 5' and 3' ends of pro-a2(I) mRNA. Conditions for hybridization, stabilization, and spreading are described in Methods. Bar, 400 bp.
highly informative structures from which detailed maps of the mRNAs may be derived. In sharp contrast, individual molecules of unhybridized large mammalian mRNAs appear as noninformative tangled masses of collapsed bushlike structures in the electron microscope (Fig. 1). The generation of elaborate hybrid structures upon hybridization of spliced mRNA to genomic DNA clones has been utilized for the assessment of major structural abnormalities in human type I collagen mRNAs. H,12 As diagrammatically illustrated in Fig. 3A, deletion, substiH W. de Wet, M. Sippola, G. Tromp, D. Prockop, M.-L. Chu, and F. Ramirez, J. Biol. Chem. 261, 3857 (1986). ~2 W. J. de Wet, unpublished observations, 1985.
240
[ 11]
GENETIC ANOMALIES
A
Genomic clone b
(3
c
d
e
mRNA
¥ t3
]E
II
t3
B
cDNA clone e
b
c
d
e
f
mRNA
LLt b
c
d
e
f
o
LL
b~e
V a
f
b
c
d
e
f
Lv
,~/d - m
o b c|e f o b c FIG. 3. Schematic representation of five different types of D N A - R N A hybrids that may be formed between control or mutated m R N A and the corresponding genomic DNA (A) or c D N A (B). Structures I and i depict hybridization to control mRNA. The following anomalous hybridization patterns represent m R N A mutations involving one or more exons: Structures II and ii, deletion of exon d; structures Ill and iii, deletion of exons c and d; structures IV and iv, inversion or substitution of exon d; structures V and v, insertion or duplication of exonic sequences between exons c and d. mRNAs are indicated by heavy lines, and D N A is indicated by light lines (introns in A, displaced exons in B) or boxes (exons a - f ) .
[11]
R-LooP ANALYSISOF mRNA
241
tution, inversion, insertion, or duplication in mutant mRNA of sequences corresponding to one or more exons will result in the formation of three different types of aberrant D N A - R N A heteroduplexes. 1. Compared to the normal hybrid (structure I), deletion in the mRNA of the sequences corresponding to exon d will lead to a hybrid (structure II) containing four, instead of the normal five displacement loops. Also, exon d will not be visualized, but will be absorbed into a larger displacement loop between exons c and e. Consequently, the measured length of the DNA strand displaced between exons c and e should correspond to the combined lengths of intron c, exon d, and intron d. Structure III reflects the deletion of more than one consecutive exons. 2. Inversion or substitution of the sequences corresponding to exon d in mutant mRNA will produce a slightly different abnormal hybrid (structure IV) containing an asymmetrical bubblelike nonhomologous region between the homologous regions c and e. Again, exon d will not be visualized, but will be contained in the larger displacement loop between exons c and e. 3. Insertion or duplication of exonic sequences between exons c and d, on the other hand, will result in the appearance of the normal six regions of homology (structure V). However, the homologies corresponding to exons c and d will be interrupted by an additional displacement loop in the RNA strand. The deletion loop of RNA will coincide with the intronic loop between exons c and d, to form an asymmetrical bubble structure in that region. On the basis of the different scenarios depicted in Fig. 3A, this type of Rloop analysis can be used as a sensitive tool for detecting not only the position, but also the extent and nature of mutations involving the sequences of one or more exons. A more traditional approach to heteroduplex mapping of the above-mentioned types of mutations would involve hybridization of the mutant mRNA to normal cDNA clones. 10,~3For comparison, a similar set of possible hybrid structures is shown in Fig. 3B. Although widely used for nuclease $1 mapping, 14'~5 hybridization of mutant mRNA to cDNA will generate a visually less recognizable pattern of small displacements. Therefore, the more traditional heteroduplex mapping procedures clearly do not allow the assessment of the different types of exonic mutations with a comparable sensitivity or accuracy in the electron microscope. 13 B. C. Westmoreland, W. Szybalski, and H. Ris, Science 163, 1343 (1969). 14 p. A. Sharp, A. J. Berk, and S. M. Berget, this series, Vol. 65, p. 750. 15 T. Pihlajaniemi and J. C. Myers, this volume [9].
242
GENETICANOMALIES
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Materials and Reagents
Nucleic Acids Preparation of RNA. Control fibroblasts (GM3348 and GM970) and fibroblasts from a lethal variant of OI (GM2962) were purchased from the Human Genetic Mutant Cell Repository (Camden, New Jersey). Fibroblast cultures are grown in 175-cm2 flasks under standard conditions in Dulbecco's modified Eagle's minimum essential medium containing 10% fetal bovine serum. At confluency, total poly(A)-enriched RNA is isolated according to the method of Burnett and Rosenbloom. 16 Isolation ofDNA. Six human genomic clones have thus far been used for the detection of mutations in type I collagen mRNAs. 1H2 Two of these, RMS-1 and RMS-8, code for the pro-al(I) collagen chain. 1The proa2(I) chain, except for the carboxy-terminal 126 amino acid residues, is encoded by the clones NJ-l, NJ-3, NJ-9, and NJ-27. 2,3 These genomic clones were kindly made available by Francesco Ramirez (UMDNJRutgers Medical School, Piscataway, New Jersey). Amplification of the genomic clones in phage Charon 4A and purification of phage DNA are performed according to standard procedures.~7 Standards for Length Calibration. Nicked double-stranded form DNA (5386 bp) as well as single-stranded virion DNA (5386 nucleotides) of phage ~bX174 were from New England Biolabs. Both forms of tbX174 are routinely included in all spreads as internal length standards for the determination of absolute molecular sizes.18 Materials Formamide (99%, Fluka or Matheson, Coleman, and Bell) is recrystallized as described by Casey and Davidson, 8and stored at -80°C. Glyoxal (40%, Matheson, Coleman, and Bell) Piperazine-N,N'-bis-2-ethanesulfonic acid (PIPES) and cytochrome c (type VI) were purchased from Sigma. Copper 200-mesh grids (GC 200), tungsten wire (diameter 0.025 in.), platinum-paladium (80:20) wire (diameter 0.008 in.), and uranyl acetate were from Ted Pella, Inc. Nitrocellulose (1% in amyl acetate, Ernest F. Fullam, Inc.) lntramedic polyethylene tubing (internal diameter 1.14 mm, Clay Adams) 16 W. Burnett and J. Rosenbioom, Biochem. Biophys. Res. Commun. 86, 478 (1979). 17 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982. is D. Stfiber and H. Bujard, Mol. Gen. Genet. 154, 299 (1977).
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Pyrex disposable microsampling pipettes (Coming Glass Works) and 6 x 50-mm borosilicate glass tubes (Kimble) are rendered nucleasefree by baking at 200° for 2 hr. Only fresh double-glass distilled H20 is used.
Specialized Preparatory Procedures Ultracleaning of Dialysis Tubing. Dialysis tubing (1 in., Sigma) is cut into 12- to 18-in.-long pieces and boiled in 1.5% (w/v) NaHCO3 and 0.5% (w/v) EDTA for 30 min. The tubing is then rinsed with copious amounts of water, and the boiling and rinsing are at least repeated twice. After another boiling step in water only, the tubing is autoclaved in water for 20 min, and finally stored in 1 mM EDTA (pH 7.0) at 4 °. Ultracleaning of Glass Slides. After treatment with chromic acid overnight, borosilicate glass slides are extensively washed with copious amounts of water. Care should be taken to handle the slides only with a forceps. The washed slides are placed in a near vertical position in a covered glass beaker, containing a circular pad of Whatman paper, to drain and dry completely. Only completely clean and dry glass slides should be used. Coating of Grids with Nitrocellulose. The following standard procedure rendered very strong supporting films on copper electron microscope grids. A carefully cleaned rectangular glass trough is filled with water within 1-2 cm of the top. In the following step, about four drops of a 1% nitrocellulose solution in amyl acetate are spread on a glass slide. Excess solution is vertically drained on blotting paper, and the uniform film of nitrocellulose is left to air-dry for about 3-5 min. A razor is used to cut the film along the sides and edges of the slide. While this is in progress, the surface of the water in the glass trough is cleaned by dropping a few drops of nitrocellulose solution onto the water. After a few minutes, the polymerized film is skimmed with an open forceps. At this stage, the nitrocellulose-covered glass slide is slowly submersed at an angle of about 45 ° into the water, allowing the film to gradually disengage. As soon as the film almost completely floats on the surface, the slide is gently dropped to fully separate the floating film from the slide. Copper grids are rinsed by two quick sequential immersions in acetone and air-dried on Whatman paper. The cleaned grids are, dull side down, carefully dropped in a regular pattern, close together, on the most uniform spots of the floating film. Areas with a yellowish or bluish appearance should be ignored. Using a forceps, a slightly larger piece of Whatman No. 1 paper is dropped on top of the grid-carrying film. The forceps is quickly repositioned to the other end of the filter paper and the whole
244
GENETIC ANOMALIES
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array is lifted in one sweeping motion, blotted dry from the back, and finally baked at 65° for 10 min in a glass Petri dish. At least five batches of covered grids should be available for the mounting of nucleic acids for electron microscopy. Custom-Made Teflonware. At least two rectangular Teflon bars (15 × 1 × 0.7 cm) are used for spreading. Custom-made ultramicro Teflon dialysis holders greatly facilitate dialysis of small volumes. A circular recess with a diameter of 9 mm and a depth of 4 mm is machined into one side of a Teflon disk (16 x 8 mm). A groove, to accommodate an O ring, is cut into the circumference, and the outer edge of the disk is rounded to facilitate mounting of the O ring. For dialysis, a rectangular piece of dialysis tubing is cut along one edge, opened on a clean surface (unused aluminum foil), blotted, and draped over the well in the disk. The dialysis membrane is finally mounted by pushing an O ring over the rounded edge into the groove. During dialysis, the sample should be in contact with the membrane. For removal of the sample following dialysis, the holder is (membrane up) centrifuged at low speeds for a few seconds. A small hole is cut in the dialysis membrane and the sample is removed.
Solutions R-loop stock buffer: PIPES, 1 M; EDTA, 0.I M adjusted to pH 7.2 at 20° with concentrated NaOH. Dialysis buffer: NaCI, 0.5 M; EDTA, I mM; Tris 10 mM adjusted to pH 7.2 at 20 ° with HCI Spreading stock buffer: Tris, 0.5 M; EDTA, 0.05 M adjusted to pH 8.5 at 20° with HCI Cytochrome c solution: cytochrome c is dissolved at a concentration of 1 mg/ml in buffer containing Tris-HCl, 0. I M; EDTA, I0 mM adjusted to pH 8.5. The solution should be stored at 4° in a dark container. Uranyl acetate stock solution: uranyl acetate, 0.05 M; HCI, 0.05 M in 90% ethanol. The solution should be stored at 4° in a dark container. Methods In order to cover fully the procedure for effective identification of mRNA mutations by R-loop mapping, the methodology is divided into three sections dealing with (1) R-loop formation and stabilization, (2) processing of R-loops for electron microscopy, and (3) electron microscopy and interpretation of results.
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245
R-Loop Formation and Stabilization A typical 50-/.d hybridization mixture contains 6/.~g/ml of phage DNA (of maximum sequence complexity of 50 kb) and 20-40/~g/ml of fibroblast total poly(A)-enriched RNA in 70% (v/v) recrystallized formamide, 0.1 M PIPES (pH 7.2), 0.01 M EDTA, and 0.3 M NaCI (a final [Na +] of about 0.46 M including the contribution of the PIPES~9). R-loop mixtures are constituted by first mixing 3/~1 of 5 M NaCI, 5/~1 of R-loop stock buffer, and 35/~1 of recrystallized formamide. This is best done in 6 x 50 mm glass tubes using Pyrex microsampling pipettes. Phage DNA, followed by the RNA, is then added to a final volume of 50/~1. To minimize evaporation during prolonged incubation at high temperatures, the R-loop mixtures are covered by 50-100/A of Paraffin Oil and the tubes are well sealed with Parafilm. Incubation at the optimal temperature is a critical aspect of R-loop formation. In principle, the optimal incubation temperature is limited by two parameters. These are the irreversible melting temperature (Tss) of the DNA sequence participating in R-loop formation and the higher melting temperature of the specific D N A - R N A heteroduplex. 7,8 In high formamide concentrations, the difference between these two parameters varies. 8 Furthermore, depending on the GC content, the Tss values of most DNA sequences vary between 45 and 58° (in 70% formamide and about 0.5 M Na+). 7 This may imply that the efficient formation of R-loops requires determination of the T~s of every DNA sequence that participates in R-loop formation, especially in genes containing GC-rich clusters such as the collagen genes. In order to obviate difficult and tedious procedures 2° to accommodate the widely different denaturation temperatures of different DNA sequences, the following protocol is employed to enhance R-loop formation. R-loop mixtures containing DNA that code for procollagen triple-helical sequences are first incubated at 57° for 12 hr. The temperature is then decreased to 53° over a 2-hr period, and the incubation is continued for another 3 hr. On the other hand, hybridization to DNA clones encoding nontriple-helical propeptide regions is performed first at 52 ° for 12 hr, followed by an incubation at 48° for 4 hr. Incubation at the initial higher temperatures results in complete dissociation of DNA duplexes, allowing hybridization of mRNA with single-stranded DNA. Furthermore, incubation over the indicated range of temperatures is very 19 D. S. Holmes, R. H. Cohn, H. H. Kedes, and N. Davidson, Biochemistry 16, 1504 (1977). 20 G. Vogeli, H. Ohkubo, V. E. Avvedimento, M. Sullivan, Y. Yamada, M. Mudryj, I. Pastan, and B. de Crombrugghe, Cold Spring Harbor Syrup. Quant. Biol. 45, 777 (1980).
246
GENETIC ANOMALIES
[1 1]
FIG. 4. Visualization of psoralen cross-links in DNA. The genomic subclone containing the 3'-untranslated region of the human pro-al(I) collagen gene (Fig. 1) was linearized with the enzyme PvuI and cross-linked with trimethylpsoralen. An aliquot of the cross-linked material was subjected to glyoxal denaturation and spread for electron microscopy. Crosslinking and the determination of the cross-linking frequency were performed according to the procedure of D. B. Kaback, L. M. Angerer, and N. Davidson, Nucleic Acids Res. 6, 2499 (1979). Ideally two random cross-links (denoted by arrows) should be present. Bar, 2.0 kb.
likely to result in association of DNA and RNA, regardless of the base composition of the interacting nucleic acids. Under certain circumstances, formation of bona fide R-loop structures are beneficial for the analysis of D N A - R N A hybrids (Fig. 1). Introduction of a few random trimethylpsoralen-induced cross-links 2~ in vector sequences flanking the region of interest prior to R-looping prevents complete strand separation. The two complementary DNA strands are held in register (Fig. 4). This greatly facilitates snapback of any unhybridized DNA sequences on both sides of the DNA-RNA hybrid region. 4 However, trimethylpsoralen cross-linking does not necessarily enhance analysis of elaborate D N A - R N A heteroduplexes of the type shown in Fig. 2. Moreover, introduction of random cross-links in large cloned regions of the collagen genes may seriously distort interaction between DNA and RNA in D N A - R N A heteroduplexes. The technique, therefore, has a limited application in R-loop analysis and should only be attempted when the formation of bona fide R-loops is essential. 21 R. S. Cole, Biochim. Biophys. Acta 217, 30 (1970).
[ l 1]
R-LOOP ANALYSIS OF m R N A
247
In fact, visual identification of an elaborate pattern of deletion loops in D N A - R N A hybrids is considerably simplified by electron microscopy of single-stranded R-loop structures such as the example shown in Fig. 2. Presence of displaced DNA strands in large bona fide R-loops may result in branch migration, random displacement of RNA sequences, entangling with and masking of deletion loops, and even formation of triple hybrids. The latter is the result of snapback of complementary intronic DNA sequences 5 that may greatly distort D N A - R N A heteroduplexes. Rapid lowering of the temperature at the end of the incubation usually does not allow extensive renaturation of any unhybridized DNA sequences and results in the appearance of single-stranded R-loops. Although R-loops, once formed, are quite stable, branch migration at the ends of D N A - R N A segments frequently induces partial displacement of RNA sequences and selective loss of smaller R-loops. 4'7'22 This especially occurs when the formamide concentration and temperature are decreased during the process of mounting nucleic acids for electron microscopy. Spreading from high-formamide solutions may reduce branch migration, but frequently may result in poor contrast. In an important modification of the R-loop method, Kaback et al. 4 stabilized R-loops by treatment with glyoxal at 12°. Under these conditions, the denaturing agent specifically modifies only unhybridized guanine residues, thus minimizing branch migration even in the absence of formamide. At the end of the incubation, the R-loop mixtures are quickly chilled, separated from the overlaying Parafin Oil, and transferred to separate 6 × 50 mm glass tubes. Complete separation of the two phases is easily done by rapid uptake of the lower hydrophilic phase in a 20-cm-long piece of polyethylene tubing, attached to a micropipette suction apparatus of the Clay Adams type. The distal end of the tubing containing the oil phase is simply snipped off with a razor. To stabilize the R-loops, 40% glyoxal is added to a final concentration of 1.0 M (1/7 vol), 4 and the solution is incubated for 2 hr at 11°. Extreme care should be taken to prevent warming of glyoxal-containing material above 12°. To remove free glyoxal, the R-loops are dialyzed against the dialysis buffer at 4°.
Processing of R-Loops for Electron Microscopy A simplified version of the original Kleinschmidt monolayer technique 23for the mounting of nucleic acids will be described. To constitute a 50-t~l spreading solution containing about 50 ng of DNA, 25 ~1 of recrystallized formamide are mixed with 10 ~1 of spreading stock buffer in a 6 × 22 C. S. Lee, R. W. Davis, and N. Davidson, J. Mol. Biol. 48, 1 (1970). 23 A. K. Kleinschmidt, this series, Vol. 12, p. 361.
248
GENETIC ANOMALIES
[11]
50 mm glass tube. For length calibration, 2/.d of 1/zg/ml double-stranded qbX174 DNA and 2/zl of 0.5/zg/ml single-stranded ~bX174 virion DNA are added. At this stage a 90-ram square plastic Petri dish is filled with hypophase until the hypophase is well above the edges, being held there by surface tension. Although freshly made 15% (v/v) commercial formamide in 10 mM Tris-HC1 (pH 8.5)-1 mM EDTA is frequently used as a hypophase, I prefer water. A rectangular Teflon bar is placed in front of the hypophase surface so that the bar is supported by the edges of the tray. The bar is then gently pushed to the rear over about two-thirds of the length of the dish. Another Teflon bar is placed just in front of the first bar and drawn forward over about one-third of the length of the dish. This leaves the central region of the surface of the hypophase wiped twice. A dry glass slide, serving as a ramp, is then inserted at an angle of about 30° into the hypophase between the two Teflon bars, so that the upper onethird of the slide protrudes above the surface and rests against the rear Teflon bar. No talc should be dusted on the surface of the hypophase. In the next step, 10/xl of the dialyzed R-loop solution is mixed with the spreading solution, followed by 2/zl of the cytochrome c solution. The spreading solution is immediately and continuously spread from a 50-/zl glass microsampling pipette onto the glass ramp, about 1 cm above the surface of the hypophase. After about 40 sec, the monomolecular film is picked up onto three covered grids, preferably from different batches, at a distance of about 1 cm from the slide-solution boundary. The grids are stained for 30 sec in diluted uranyl acetate, freshly prepared (within 2 hr) by diluting 10/~1 of the stock solution with 5 ml of 90% ethanol. After staining, the grids are submersed for 10 sec in 90% ethanol. To enhance the poor contrast obtained with staining only, the specimens are rotary-shadowed essentially as described. 23 Shadowing with PtPd (80 : 20) at a distance of about 15 cm and an angle of 9° is performed in a high vacuum. About 16 turns of the Pt-Pd wire around a suitably bent tungsten wire electrode yield a satisfactory result. Slow melting and evaporation of the Pt-Pd through at least two spark events is crucial.
Electron Microscopy and Interpretation of Results R-loop structures are visualized and photographed on electron image film (3¼ × 4 in.) at a screen magnification of 18,000 and 36,000 times and 60-kV accelerating voltage, z4 Only structures should be photographed in which the intronic deletion loops are sufficiently unambiguous to permit 24 R. Schleif and J. Hirsh, this series, Vol. 65, p. 885.
[11]
R-LOOP ANALYSISOF mRNA
249
immediate identification by visual inspection in the electron microscope. Whenever possible, double- and single-stranded forms of ~bX174 DNA should appear in the same electron micrograph as the R-loop of interest (Fig. 1). The negatives are enlarged 5 times, and the images are printed on high-contrast photographic paper. Following identification of the various exonic and intronic regions of D N A - R N A heteroduplexes, molecular lengths of these regions are obtained by comparative contour length measurements, relative to the internal double- or single-stranded DNA standards, ~°,18 using an electronic digitizer. At least 20 molecules should be digitized. An example of the use of R-loop mapping for the assessment of human collagen mutations is shown in Fig. 5. Cultured skin fibroblasts from a perinatal lethal variant (GM2962) of OI were previously found to synthesize pro-a2(I) chains shortened by about 20 amino acids in o~2(I)-CB3,5 A, a fragment containing amino acids 358-775 of the a2(I) chain. 25 In comparison with D N A - R N A heteroduplexes formed between the human proot2(I) genomic clone NJ-1 and pro-ot2(I) mRNA from control fibroblasts (Fig. 2), abberant R-loop structures were obtained upon hybridization of the variant's pro-t~2(I) mRNA to the same genomic clone (Fig. 5). Instead of the normal 26 exons and 25 introns, only 25 exons and 24 introns were visualized. The abnormal distribution of exons and introns shown in Fig. 5, indicates that the sequences corresponding to exon 28, a 54-bp exon encoding amino acids 448-465, did not hybridize to the variant's pro-a2(I) mRNA. In fact, a larger intron of 408 --- 28 bp can be seen between exons 27 and 29. In control R-loops, exons 27 (59 --+ 15 bp), 28 (53 -+ 7 bp), and 29 (69 - 13 bp) are interrupted by two relatively small introns, respectively, 114 --- 18 bp and 223 +-- 34 bp in size (Fig. 2). The length of the DNA strand displaced between exons 27 and 29 in the aberrant D N A - R N A heteroduplexes, therefore, closely correspond to the combined lengths of intron 27, exon 28, and intron 28. Also, no additional displacement loop in the RNA strand between exons 27 and 29 can be seen (Fig. 5). On this basis, heteroduplexes formed between the genomic clone NJ-1 and the variant's pro-t~2(I) mRNA resemble the deletion hybrid (structure II) shown in Fig. 3A. Absence of hybridization to exon 28 was only observed with mutant pro-et2(I) mRNA, and exon 28 was the only exon for which loss of hybridization was visualized. Moreover, that the sequences corresponding to exon 28 were absent in the mutated pro-a2(I) mRNA was independently substantiated by conventional nuclease S~ analysis, n 25 W. J. de Wet, T. Pihlajaniemi, J. Myers, T. E. Kelly, and D. J. Prockop, J. Biol. Chem. 258, 7721 (1983).
250
GENETICANOMALIES .
.
.
.
.
.
.
.
.
[1 1] .
.
i
27.-~ (-~ ~ 26
^N. I IJ / .32
I /4d~ ; u--.. U~! (}oIj,o45 (,'-, 50
FIG. 5. R-loop mapping of pro-a2(I) mRNA from a lethal variant of OI. Poly(A)-enriched RNA from OI fibroblasts was hybridized to the human pro-a2(I) genomic clone NJ-1, and the resultant hybrids were stabilized and spread as in Methods. Below the micrograph is the interpretative tracing. The numbers correspond to exons, beginning at the 5' end of the gene. D N A - R N A duplex regions are indicated by heavy lines, and single-stranded DNA is indicated by light lines. Unhybridized 5' and 3' ends of the pro-a2(I) mRNA are also shown. The asterisk (*) indicates the position of the altered region in R-loops formed with the mutated mRNA. Bar, 400 bp.
[11]
R-LOOP ANALYSISOF mRNA
251
Comments
1. Because the R-loop method is based on the generation of an abnormal pattern of introns and exons upon hybridization of mutated mRNA to a normal genomic clone, the validity of the approach is determined by the specificity and stability of exon hybridization. Absence of hybridization to an exon should not be found for any other exon except in the mutated sequences. R-loop mapping of 138 exons in genomic clones covering the pro-otl(I), 1pro-ct2(I), 2,3 and pro-ctl(III) 26 genes showed specific and stable hybridization to exons, ranging in size from 35 bp to more than 1000 bp under the conditions for R-loop formation, hybridization, and spreading described here. This was especially the case for the hybridization of procollagen mRNAs to the numerous triple-helical domain exons. However, these conditions did not allow visualization of two extremely small exons of 11 and 15 bp, respectively, in the N-propeptide region of the human pro-a2(I) gene. 3 2. Although it is not known what degree of sequence mismatch will result in the absence of hybridization to a specific exon, the R-loop method will only be applicable for the mapping of mutations that cover a significant part of an exon. The technique is not suitable for the detection of small regions of mismatch such as the 4-bp frameshift deletion recently found in the pro-a2(I) gene. 27 3. In contrast to the widely used nuclease $1 mapping technique, 14,15 the R-loop method is based on hybridization to genomic clones. Except for the example shown in Fig. 5, deletion at the mRNA level of exon sequences have been visually detected for exon 6 and exon 11 of the proa2(I) gene, as well as for exon 6 of the pro-ctl(I) gene. 11,12Because these mutations fall beyond the reach of any of the existing human procollagen cDNA clones, it was not possible to employ nuclease S~ analysis. Also, other nucleic acid mapping techniques such as Southern blot analysis were not sensitive enough to detect the mutations. 1~R-loop hybridization did allow the identification of anomalies in the hybridization pattern of these human collagen gene mutations with a high degree of accuracy. 4. Although R-looping can be used as a powerful and sensitive tool for detecting collagen mutations leading to abnormal mRNA transcripts, the technique does not provide a definite answer about the exact cause of rearrangements at the gene level. Direct sequencing of the affected alleles is required to distinguish between genomic deletion or splicing defects. 26 M.-L. Chu, D. Weil, W. de Wet, M. Bernard, M. Sippola, and F. Ramirez, J. Biol. Chem. 260, 4357 (1985). 27 T. Pihlajaniemi, L. A. Dickson, F. M. Pope, V. R. Korhonen, A. Nicholls, D. J. Prockop, and J. C. Myers, J. Biol. Chem. 259, 12941 (1984).
252
GENETIC ANOMALIES
[1 1]
Acknowledgments I wish to thank Dr. F. Ramirez for his kind gift of the human collagen genomic clones, Dr. D. J. Prockop for his enthusiastic support, Dr. J. Coetzee and Mr. C. van der Merwe (Electron Microscope Unit, University of Pretoria) for kindly providing facilities, and most of all Dr. David Kaback for his invaluable advice and kindness in introducing me to R-looping. I also gratefully acknowledge the invaluable help of Ms. Isobel de Beer and Ms. Rina Kroeze in preparing the manuscript. This work was supported by a grant from the South African Medical Research Council.