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[28] Isolating Cytochrome P450 c D N A and Genomic Clones: Library Screening with Synthetic D N A Oligomers By CHRISTOPHER HASSETT, RICHARD RAMSDEN, and CURTIS J. OMIECINSKI
Introduction Cytochrome P450 enzymes are encoded by a large and complex superfamily of genes. Current P450 gene pools are believed to be the products of extensive duplication events, ultimately descended from one or more ancestral genes. Notable regions of conservation as well as divergence are apparent within P450 structures. For example, each microsomal P450 protein which has been characterized contains a hydrophobic leader sequence of approximately 20 amino acids which is believed to anchor the enzyme to the endoplasmic reticulum. Amino acid sequence conservation is found near a carboxy-terminal cysteine residue; this region is thought to function as the ligand to heme at the enzyme active site. Additionally, it is likely that conserved amino acids will be identified elsewhere in these proteins, for instance, at the region(s) that facilitates electron transfer from NADPH-cytochrome-P450 oxidoreductase. Global alignment of P450 protein sequences also reveals divergent regions which are likely important to specify substrate interaction. The existence of both conserved and divergent sequence elements within the P450 superfamily can be exploited to effect isolation of members from cDNA or genomic libraries using synthetic DNA oligomer probes. Traditional methods of library screening fall within two categories: (1) antigenic detection using antibodies and (2) homology to radiolabeled nucleic acid probes. Identification of target cDNAs by the interaction of antigens with antibody probe requires that the cloned DNA be inserted into an expression vector; the insert DNA must be in the correct orientation and reading frame. If the antibody is specific for an N-terminal epitope, the eDNA must encode this region (i.e., the clone must be nearly full length). In addition, this screening approach requires antibody of sufficient purity and specificity to maintain an acceptable signal-to-noise ratio (usually problematic only when using polyclonal antibodies). Other concerns relate to the preservation within bacterial hosts of the protein epitope(s) necessary for antibody interaction, and the variable stability of foreign proteins in Escherichia coli-based expression systems. Consequently, the efficiency of detecting cloned inserts with an expression system screening METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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approach can be decreased markedly relative to nucleic acid probe methods. Numerous reports testify that library screening with antibodies is effective, but very specific reagents and considerable experience in their preparation may be required (e.g., monoclonal antibodies). Library screening using cloned nucleic acids or synthetically prepared oligomers obviates certain difficulties inherent to antibody screening. In addition, oligomers offer a number of potential advantages compared to cloned DNA probes. Chemical synthesis of oligomers is rapid, inexpensive, and does not require the previous isolation of a cDNA clone. Knowledge of short amino acid regions of the target molecule is often sufficient for oligomer design. Because oligomers have minimal sequence complexity relative to longer cDNA probes, they hybridize with target DNA at a much faster rate. Also, under optimal conditions, oligomer hybridization to complementary molecules can be controlled with a very high degree of specificity. This specificity of oligomers can be exploited to great advantage when isolating unique or related members of a multigene family, such as cytochrome P450. In our laboratory, we have employed a variety of oligomer probes to screen both cDNA and genomic libraries. By targeting conserved sequences among rat, rabbit, and human P450 genes in the 2B and 2C families, we have used an oligomer screening approach to isolate a battery of related cDNAs. One clone from the rabbit (Cyp2C16) ~,2 and another from the human 3 P450 2C subfamily are novel isolates. We also have designed oligomers specific for hypervariable regions in the rat P450 2B genes which distinguish highly related subfamily members; this latter approach has aided the isolation of the corresponding genomic clones. 4,5 (Other papers which describe the utility of oligomer probes for library screening purposes are available. 6,7) Design of Probes Although hybridization and washing conditions will ultimately direct the interaction of any probe with its respective target molecule, the construction of the oligonucleotide with respect to length and sequence coral C. Hassett and C. J. Omiecinsld, Biochem. Biophys. Res. Commun. 149, 326 (1987). 2 C. Hassett and C~ J. Omiecinski, Nucleic Acids Res. 18, 1429 (1990). 3 K. Sommer, C. Hassett, and C. J. Omiecinski, unpubfished observations (1990). 4 C. M. Giachelli, J. Lin-Jones, and C. J. Omiecinski, J. Biol. Chem. 264, 7046 (1989). 5 R. Ramsden and C. J. Omiecinski, unpubfished observations (1988). 6 R. B. Wallace and C. G. Miyada, this series, Vol. 152, p. 432. 7 S. V. Suggs, T. Hirose, T. Miyake, E. H. Kawashima, M. J. Johnson, K. Itakura, and R. B, Wallace, in "Developmental Biology Using Purified Genes" (D. D. Brown, ed.), p. 683. Academic Press, New York, 1981.
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position clearly will be an important determinant of probe specificity. The logical path to choosing the sequence of an oligonucleotide probe begins with an analysis of the relevant sequence information already available. This path quickly becomes branched depending on the following considerations: (1) whether full or partial DNA sequence data are available for the exact molecule of interest; (2) whether DNA sequence data for related molecules are available; and (3) whether only amino acid sequence data are accessible. In practice, a few basic principles of probe design will typically enable the construction of functional oligonucleotide probes. At least two computer programs are also available for those who wish more objective guidance with respect to probe selection criteria. 8'9 Some general guidelines for the design of oligomers appear in the paragraph below, followed by more specific examples of probe selection. Especially with the advent of polymerase chain reaction (PCR) procedures, oligomer probes and primers tend to accumulate rapidly in the laboratory. Therefore it is preferable to choose a common theme of oligomer design. By standardizing length and GC composition among probes, highly similar sets of hybridization/annealing and washing conditions can be derived and utilized for many different oligomers. In general, the design rules we follow are relatively simple. We have found that oligomer probes 18-22 bases in length and with a 60% GC composition will result in successful library screening. Oligomers should be avoided that contain low GC composition, long stretches of a particular nucleotide, or sequence elements that have the potential to form stable hairpin structures. The presence of these sequence residues tends to promote either nonspecific interactions or a reduction in the efficacy of probe-target interaction. Once selected, the candidate oligomer sequence should be computer analyzed for specificity against a DNA sequence bank (e.g., GenBank or EMBL). For a 20-mer, one should scan the database at a level permitting hits at least 14 of the 20 residues. It may not be necessary to screen oligomer sequences against the entire DNA sequence database, but at the very least it is important to check homology against as many related sequences as possible, for example, against a P450 sequence database. Maximal hybridization specificity is attained when nucleotide mismatches between the oligomer probe and competing target are located internally, rather than in proximity to the termini of the oligomer molecule. Discrimination between two closely related sequences clearly requires at least one mismatch between target and nontarget molecules in order to 8 W. Rychlik and R. E. Rhoads, Nucleic Acids Res. 17, 8543, (1989). 9 T. Lowe, J. Sharefkin, S. Q. Yang, and C. W. Dieffenbach, Nucleic Acids Res. 18, 1757 (1990).
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achieve specificity of probe interaction. Under these circumstances, however, it is much more desirable to program at least 3 internal base substitutions in a 20-mer probe sequence. In hybridization reactions requiring the discrimination of two or more highly related sequences, internal mismatches should be positioned within an oligomer so as to minimize the length of contiguousregions available for hydrogen bonding with nontarget molecules. For primer specificity in PCR experiments, the above considerations also apply, but, in addition, it is especially important to program mismatches at the 3' terminus of the oligomer. In PCR reactions, oligomers function as primers for DNA polymerase as well as "hybridization probes." Because polymerase extends from the Y-most residue of the oligomer, "wobbles" or mismatches in this 3' region effectively inhibit the enzyme from efficient interaction with the primer-template hybrid. In instances when full or partial DNA sequence data are available for a molecule, it is a relatively straightforward project to utilize the data to construct a specific oligomer. In addition to utility in hybridization experiments (e.g., with Northern blots), appropriately designed oligomer probes provide a means to access full-length cDNA or corresponding genomic derivatives of the respective molecule. By targeting oligomer sequences to distinct 5' or 3' regions, for example, it is feasible to utilize oligomer probes for "walking" through a library and isolating sequences not previously available. A more challenging use of oligomer probes involves searching for novel clones whose molecular identities have not been characterized. In some instances, the sequences of related P450 gene family members may be known. If there is reason to predict that additional related genes or gene products also exist, oligomer probes can be designed to permit their isolation. To this end, conserved regions of DNA sequence are evaluated and exploited to produce a "consensus" probe. A computer program, CLUSTAL, 1° is a very useful aid for comparing related sequences. It facilitates such analyses by generating an overlay of multiple sequence alignments, identifying regions that are conserved or divergent. Library screening with probes directed against conserved regions of sequence has proved useful in isolating previously uncharacterized P450 cDNAs.I-3 In other situations, only protein sequence data may be available without the corresponding DNA sequence. An oligomer specifying a coding domain of the relevant peptide can be generated and used as a probe for cDNA or genomic clone isolation. It is important to target amino acids 1o D. G. Higgins and P. M. Sharp, Gene 73, 237 (1988).
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that offer the least redundant codon choices. If related protein sequences are known, for example, from orthologous proteins in different species, alignments can also be performed to highlight amino acids that offer the greatest degree of conservation as well as the least codon redundancy. The latter criteria should be employed to aid in identifying the most appropriate sites for targeting oligomer probes. Two general strategies for designing oligomers targeted to the coding regions of proteins are typically employed. One scheme is to design an oligomer based on preferred codon usage frequencies, thereby minimizing DNA sequence degeneracy in the probe, u-~3 Another approach is to program the oligomer sequence with some or all of the unknown bases (corresponding to the third base "wobble" position of each codon), thereby synthesizing a probe mixture. In the former scheme, probe mixtures or redundancies are minimized, but it is likely that some of the programmed "guesses" will be incorrect. Nevertheless, depending on the inherent sequences, it is possible that appropriate hybridization conditions can be ascertained which are permissive for a certain degree of hybrid mismatch while retaining relative specificity of interaction. In the latter scheme, redundancies along the length of an oligomer mount geometrically. Thus, even though one of the probes in the mixture may be the correct sequence, its proportion of the total probe could be minor, and its resulting contribution to hybridization signal strength quite small. Furthermore, some of the alternative probes in the mixture may give rise to an intolerable background of nonspecific hybridization. With these caveats in mind, the choice in probe design is not necessarily clear. Successful isolations have been achieved by both approaches, and the choice between strategies does not have to be mutually exclusive. For example, we designed an oligomer probe based on amino acid sequence to isolate cDNAs encoding rabbit microsomal epoxide hydrolase. 14Twelve amino acids were targeted for probe design; although redundancy in codon choice was minimal for most of these residues, some assumptions based on preferred codon usage were considered necessary to minimize probe redundancy. For additional details regarding oligomer probe design, the reader is referred to other chapters in this series. 6,15 II R. Lathe, J. Mol. Biol. 183, 1 (1985). 12 K. Wa~a, S. Aota, R. Tsuchiya, F. Ishibashi, T. Gojobori, and T. Ikemura, Nucleic Acids Res. 18, 2367 (1990). 13 R. Grantham, C. Gautier, and M. Gouy, Nucleic Acids Res. 8, 1893 (1980). t4 C. Hassett, S. M. Turnblom, A. DeAngeles, and C. J. Omiecinski, Arch. Biochem. Biophys. 271, 380 (1989). ts W. I. Wood, this series, Vol. 152, p. 443.
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Library Plating/Screening Bacteriophage, plasmid, or cosmid libraries are cultured according to a variety of established protocols. Once plated, however, subsequent methods to screen for a eDNA or gene of interest are very similar. A set of six plates is conveniently processed, each 150-mm petri dish containing 20,000 to 30,000 colonies or plaques 0.5-1.0 mm in diameter. This number, near the maximum density before confluency is reached, should be derived beforehand by plating serial dilutions of the library. Distinct, round (nonconfluent) colonies or plaques will yield the clearest signals following autoradiography. After being chilled thoroughly at 4 °, the plates are ready to be placed in contact with the transfer membrane. When performing lifts on plates which have a top agar coating (e.g., a phage library screen), we find it helpful to prewet the transfer membrane on a sterile L plate in order to avoid adhering top agar to the membrane. While in contact with the chilled plate, the disk is punctured asymmetrically with a needle to facilitate future reorientation. The membrane is peeled away and placed colony/ plaque side up in a 1.5 ml pool or 0.5 N NaOH on plastic wrap to denature the duplex DNA. After 3 min, the disk is blotted on filter paper, then similarly dipped and blotted again. The DNA fixed to the membrane is then neutralized for 3 minutes in 1.5 ml of 0.5 M Tris-HC1, pH 7.5, blotted, and this step repeated a second time. Nylon membranes are dried at room temperature or under a heat lamp; nitrocellulose filters must be baked at 80 ° in a vacuum oven. Nylon is the preferred transfer membrane because its binding capacity is greater than nitrocellulose. Increased retention of bound DNA translates into more target for probe hybridization, and consequently a stronger autoradiographic signal. Nylon can be UV irradiated to cross-link fixed DNA to the membrane, which has been reported to increase subsequent signal detection levels.16 Nylon is also more durable, which is an important consideration if the membranes are to be probed multiple times.
Hybridization Conditions Our standard hybridization buffer is composed of 5 x or 6 × SSC (see below), 1% (w/v) sodium dodecyl sulfate (SDS), 25 mM sodium phosphate, pH 6.5, 1 x Denhardt's solution, and 10/~g/ml polyadenylic acid (I × SSC is 0.15 M NaCI, 15 mM sodium citrate; 1 x Denhardt's solution contains 0.02% each of Ficoll, poiyvinylpyrrolidone 360, and bovine serum albumin). Dried membranes are placed in heat-sealed bags, two disks per bag 16 E. W. Khandijian, Bio Technology 5, 165 (1987).
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with colony/plaque sides facing outward. Then 15 to 20 ml of hybridization buffer is added to each bag, and prehybridization proceeds for at least 1 hr at the incubation temperature. Oligomers are radiolabeled at the 5' terminus using T4 polynucleotide kinase using standard conditions,17 with a 1.5 × molar excess of [~/-32p]ATP in the labeling reaction (relative to the oligomer concentration). The radiolabeled probe is separated from radioactive precursor in a 20% polyacrylamide gel and purified by elution. Oligomer probe is added at a final concentration of 1-2 × 106 disintegrations per minute (dpm) per milliliter buffer. Hybridization is performed overnight in a moderately shaking water bath. The appropriate salt concentration and hybridization temperature are crucial for successful library screening with oligomers. These parameters will vary with the length and GC content of the oligomer, as well as the stringency desired. For relatively short oligomers, a formula has been described 6,7 for estimating the Td, or temperature at which one-half of the duplexes will dissociate from target DNA in 6 x SSC: Td(°C) = 4(G + C) + 2(A + T). However, optimal hybridization conditions often must be derived empirically, for example, using results from Northern blots as a guide. For library screening experiments in our laboratory, we have found that hybridization in 5 × SSC at a temperature of 54° has worked well for 20-base oligomers with 60% GC content, or 52° for 18-base oligomers with 66% GC. When using degenerate oligomer probes, conditions will often be more variable. We have screened cDNA libraries with a degenerate 18met (termed the PBmer), which consists of 8 distinct oligomer sequences ranging in GC content from 44 to 61%. 1-3 When using this probe, we relax the hybridization stringency to 42° in 5 x SSC buffer in order to accommodate each of the 8 oligomers in the mix. We also have isolated cDNA clones using shorter and more complex probes. For instance, two oligomers were employed to identify cDNAs encoding rabbit serum paraoxonase (arylesterase).18 This enzyme, in concert with P450-mediated catalysis, metabolizes organophosphate pesticides and other substrates. Based on rabbit peptide sequence information, a 17-mer (64-fold degenerate; GC content ranging from 29 to 47%) and a 15-mer (256-fold degeneracy; GC content 27-60%) were synthesized and used sequentially in the screening process) s Because of the wide GC content variation in these oligomers, it was necessary to decrease the hybridization temperature to 37° and increase the salt buffer to 6 × SSC. 17j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. ts C. Hassett, R. J. Richter, R. Humbert, C. Chapline, J. W. Crabb, C. J. Omiecinski, and C. E. Furlong, Biochemistry, in press (1991).
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Washing Conditions After overnight hybridization, filters are nonstringently washed twice at room temperature (5-10 min each time) in 5 × SSC/0.1% SDS buffer. This is followed by two changes in this solution at the hybridization temperature, each wash being for 20 min. At least 100 ml of wash solution is recommended per 137-ram membrane. High stringency is attained using a solution containing 3 M tetramethylammonium chloride, once at 37° (to remove Na + ions) and twice at a temperature 3° below the dissociation temperature.19 When Na ÷ in the wash solution is replaced with tetramethylammonium chloride salts, the Td is dependent on length of the oligomer probe and independent of base composition. This feature becomes especially valuable when screening libraries with degenerate probes of variable GC content, but uniform length. Washed membranes are heat-sealed in plastic bags and taped to an intensifying screen (or exposed film), colony/plaque side up. After placing in a cassette, X-ray film is carefully aligned to the mounting surface and taped to the intensifying screen to ensure subsequent alignment. Filters from a bacteriophage or plasmid library screen should be exposed overnight at - 8 0 ° with at least one intensifying screen. A positive signal on the autoradiogram appears as a distinct black dot. Longer exposures may be necessary under some circumstances, such as when screening cosmid libraries which contain fewer oligomer target sites per colony or when screening with degenerate probes in which only a subpopulation of oligomer probe binds to the target sequence. Verification of Positive Signals We apply various approaches to confirm that an autoradiographic signal from the initial round of library screening represents the desired clone. When possible, we exploit the same oligomer(s) used in library screening as an adjuvant in the verification process. Verifying signals will be more convincing and easier if information is available to permit the design of more than one oligomer specific for the clone of interest. The standard procedure we employ when screening libraries with oligomer probes requires harvesting the colonies or plaques associated with an autoradiographic signal, and replating in such a manner as to amplify the positive signal. After successive rounds of replating at lower density, the specific colony/plaque hybridizing to the oligomer probe can be clearly distinguished and isolated. Alternatively, one can pick all colonies/plaques 19 W. I. Wood, J. Gitschicr, L. A. Lasky, and R. M. Lawn, Proc. Natl./*cad. Sci. U.S.A. 82, 1585 0985).
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surrounding the autoradiographic signals and individually transfer them in grid fashion to a fresh agar plate; obviously many will not be the clone of interest. Bacterial colonies are transferred by sterile toothpick or inoculating loop. Phage suspensions (-5/zl), which should be amplified for several hours in liquid culture, are dotted onto a lawn of susceptible cells. The grid is transferred to a nylon membrane as described above, and probed with the oligomer probe. This process usually reduces the number of successive rounds of rescreening and also verifies, in a relatively rapid manner, whether the clones being pursued are likely to be true positives. More recently, we have employed a PCR-based approach to verify positive signals when screening phage libraries. This method also employs the oligomer hybridization probe, but as a primer component in PCR amplification. A plug encompassing the autoradiographic signal is cored from the agar plate using the wide end of a Pasteur pipette. The total number of plaques within the plug apparently is unimportant. The plug is suspended in 1 ml of elution buffer and placed at room temperature for several hours. Five microliters of this eluate is added to a (total volume) 100/zl PCR reaction 2°which contains two cDNA-specific oligomer primers (each could also be useful in the initial library screening). The two oligomers must flank a portion of the insert to be amplified (Fig. 1A), each one being complementary to opposing strands (i.e., one oligomer is sense, the other antisense). If a specific oligomer is available for only one region of the cDNA, the PCR primer pair can be completed using the universal forward or reverse primer specific for the cloning vector (see Fig. 1B). In this instance, two separate PCR reactions must be performed which combine the cDNA-specific primer with each universal primer, since the orientation of the insert in the vector is unknown. The presence of a discrete PCR product in an agarose gel is suggestive that the clone of interest has been identified, since a successful PCR reaction requires that the cDNAspecific oligomer hybridize to the insert. Following isolation in low temperature gelling agarose, the PCR product can be sequenced without further manipulation. 21 Subsequent rounds of rescreening are usually required to isolate the cDNA, but one can be confident that this effort will lead to the characterization of a desired clone. However, isolation may also be possible using a second cDNA-based primer (complementary to the one initially employed) to amplify the entire cloned insert. If the oligomer sequence contains a suitable restriction site, or if one prefers to use the complementary regions directly as sites for 20 R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. A. Arnheim, Science 230, 1350 (1985). 21 K. A. Kretz, G. S. Carson, and J. S. O'Brien, Nucleic Acids Res. 17, 586 (1989).
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A I
L\\N,\\\\\\\\\\
\1
I%\\\\\\\\\\\\N]
B [~\\\\\\\\L\~'~
~P
N\\\\\\\\\\\\'Q
I FB-,t~ -.q--RP
+
I> ,\\\\\\\\\\\\'t
/~\\?~\\\\\\"
I
I
F]o. 1. (A) PCR amplification strategy with two eDNA-specific oligomers. Each oligomer (indicated by the symbols *, *') must be complementary to opposite strands of the cDNA. (B) PCR amplification strategy with only one cDNA-specific oligomer. The primer pair necessary for amplification is completed using either the forward or reverse universal primer, which is vector specific. Because the orientation of the insert in the vector is unknown, two reactions must be performed, although only one combinationof primers will yield a product.
PCR extension, 22it should also be feasible to fuse the respective 5' and 3' PCR products, thereby creating a full-length construct. After a single colony/plaque has been isolated which hybridizes to the oligomer probe(s), the vector/insert DNA can be prepared by standard methods. Purified clones can be categorized by restriction mapping and characterized by DNA sequence analysis and in vitro expression. Conclusions
Identification and characterization of cytochrome P450 cDNAs and genes can be complicated owing to the size of this superfamily, as well as to the considerable similarity among many family members. We have 22R. M. Horton, H. D. Hunt, S. N. Ho, J. K. Pullen, and L. R. Pease, Gene 77, 61 (1989).
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found library screening with oligomers to be a very effective method for identifying novel and previously characterized cloned DNAs. With proper control of oligomer design and hybridization/washing conditions, autoradiographic signals obtained from library screening will be very convincing with little or no interference from background noise. Furthermore, oligomer/PCR-based strategies provide unique opportunities for the rapid characterization of clones, circumventing the need to isolate and categorize cloned DNAs by traditional approaches. Acknowledgments Research in our laboratory has been supported by Grants GM-32281, ES-046%, and ES04978 from the National Institutesof Health.
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[28] Isolating Cytochrome P450 c D N A and Genomic Clones: Library Screening with Synthetic D N A Oligomers By CHRISTOPHER HASSETT, RICHARD RAMSDEN, and CURTIS J. OMIECINSKI
Introduction Cytochrome P450 enzymes are encoded by a large and complex superfamily of genes. Current P450 gene pools are believed to be the products of extensive duplication events, ultimately descended from one or more ancestral genes. Notable regions of conservation as well as divergence are apparent within P450 structures. For example, each microsomal P450 protein which has been characterized contains a hydrophobic leader sequence of approximately 20 amino acids which is believed to anchor the enzyme to the endoplasmic reticulum. Amino acid sequence conservation is found near a carboxy-terminal cysteine residue; this region is thought to function as the ligand to heme at the enzyme active site. Additionally, it is likely that conserved amino acids will be identified elsewhere in these proteins, for instance, at the region(s) that facilitates electron transfer from NADPH-cytochrome-P450 oxidoreductase. Global alignment of P450 protein sequences also reveals divergent regions which are likely important to specify substrate interaction. The existence of both conserved and divergent sequence elements within the P450 superfamily can be exploited to effect isolation of members from cDNA or genomic libraries using synthetic DNA oligomer probes. Traditional methods of library screening fall within two categories: (1) antigenic detection using antibodies and (2) homology to radiolabeled nucleic acid probes. Identification of target cDNAs by the interaction of antigens with antibody probe requires that the cloned DNA be inserted into an expression vector; the insert DNA must be in the correct orientation and reading frame. If the antibody is specific for an N-terminal epitope, the eDNA must encode this region (i.e., the clone must be nearly full length). In addition, this screening approach requires antibody of sufficient purity and specificity to maintain an acceptable signal-to-noise ratio (usually problematic only when using polyclonal antibodies). Other concerns relate to the preservation within bacterial hosts of the protein epitope(s) necessary for antibody interaction, and the variable stability of foreign proteins in Escherichia coli-based expression systems. Consequently, the efficiency of detecting cloned inserts with an expression system screening METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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[28]
approach can be decreased markedly relative to nucleic acid probe methods. Numerous reports testify that library screening with antibodies is effective, but very specific reagents and considerable experience in their preparation may be required (e.g., monoclonal antibodies). Library screening using cloned nucleic acids or synthetically prepared oligomers obviates certain difficulties inherent to antibody screening. In addition, oligomers offer a number of potential advantages compared to cloned DNA probes. Chemical synthesis of oligomers is rapid, inexpensive, and does not require the previous isolation of a cDNA clone. Knowledge of short amino acid regions of the target molecule is often sufficient for oligomer design. Because oligomers have minimal sequence complexity relative to longer cDNA probes, they hybridize with target DNA at a much faster rate. Also, under optimal conditions, oligomer hybridization to complementary molecules can be controlled with a very high degree of specificity. This specificity of oligomers can be exploited to great advantage when isolating unique or related members of a multigene family, such as cytochrome P450. In our laboratory, we have employed a variety of oligomer probes to screen both cDNA and genomic libraries. By targeting conserved sequences among rat, rabbit, and human P450 genes in the 2B and 2C families, we have used an oligomer screening approach to isolate a battery of related cDNAs. One clone from the rabbit (Cyp2C16) ~,2 and another from the human 3 P450 2C subfamily are novel isolates. We also have designed oligomers specific for hypervariable regions in the rat P450 2B genes which distinguish highly related subfamily members; this latter approach has aided the isolation of the corresponding genomic clones. 4,5 (Other papers which describe the utility of oligomer probes for library screening purposes are available. 6,7) Design of Probes Although hybridization and washing conditions will ultimately direct the interaction of any probe with its respective target molecule, the construction of the oligonucleotide with respect to length and sequence coral C. Hassett and C. J. Omiecinsld, Biochem. Biophys. Res. Commun. 149, 326 (1987). 2 C. Hassett and C~ J. Omiecinski, Nucleic Acids Res. 18, 1429 (1990). 3 K. Sommer, C. Hassett, and C. J. Omiecinski, unpubfished observations (1990). 4 C. M. Giachelli, J. Lin-Jones, and C. J. Omiecinski, J. Biol. Chem. 264, 7046 (1989). 5 R. Ramsden and C. J. Omiecinski, unpubfished observations (1988). 6 R. B. Wallace and C. G. Miyada, this series, Vol. 152, p. 432. 7 S. V. Suggs, T. Hirose, T. Miyake, E. H. Kawashima, M. J. Johnson, K. Itakura, and R. B, Wallace, in "Developmental Biology Using Purified Genes" (D. D. Brown, ed.), p. 683. Academic Press, New York, 1981.
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position clearly will be an important determinant of probe specificity. The logical path to choosing the sequence of an oligonucleotide probe begins with an analysis of the relevant sequence information already available. This path quickly becomes branched depending on the following considerations: (1) whether full or partial DNA sequence data are available for the exact molecule of interest; (2) whether DNA sequence data for related molecules are available; and (3) whether only amino acid sequence data are accessible. In practice, a few basic principles of probe design will typically enable the construction of functional oligonucleotide probes. At least two computer programs are also available for those who wish more objective guidance with respect to probe selection criteria. 8'9 Some general guidelines for the design of oligomers appear in the paragraph below, followed by more specific examples of probe selection. Especially with the advent of polymerase chain reaction (PCR) procedures, oligomer probes and primers tend to accumulate rapidly in the laboratory. Therefore it is preferable to choose a common theme of oligomer design. By standardizing length and GC composition among probes, highly similar sets of hybridization/annealing and washing conditions can be derived and utilized for many different oligomers. In general, the design rules we follow are relatively simple. We have found that oligomer probes 18-22 bases in length and with a 60% GC composition will result in successful library screening. Oligomers should be avoided that contain low GC composition, long stretches of a particular nucleotide, or sequence elements that have the potential to form stable hairpin structures. The presence of these sequence residues tends to promote either nonspecific interactions or a reduction in the efficacy of probe-target interaction. Once selected, the candidate oligomer sequence should be computer analyzed for specificity against a DNA sequence bank (e.g., GenBank or EMBL). For a 20-mer, one should scan the database at a level permitting hits at least 14 of the 20 residues. It may not be necessary to screen oligomer sequences against the entire DNA sequence database, but at the very least it is important to check homology against as many related sequences as possible, for example, against a P450 sequence database. Maximal hybridization specificity is attained when nucleotide mismatches between the oligomer probe and competing target are located internally, rather than in proximity to the termini of the oligomer molecule. Discrimination between two closely related sequences clearly requires at least one mismatch between target and nontarget molecules in order to 8 W. Rychlik and R. E. Rhoads, Nucleic Acids Res. 17, 8543, (1989). 9 T. Lowe, J. Sharefkin, S. Q. Yang, and C. W. Dieffenbach, Nucleic Acids Res. 18, 1757 (1990).
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achieve specificity of probe interaction. Under these circumstances, however, it is much more desirable to program at least 3 internal base substitutions in a 20-mer probe sequence. In hybridization reactions requiring the discrimination of two or more highly related sequences, internal mismatches should be positioned within an oligomer so as to minimize the length of contiguousregions available for hydrogen bonding with nontarget molecules. For primer specificity in PCR experiments, the above considerations also apply, but, in addition, it is especially important to program mismatches at the 3' terminus of the oligomer. In PCR reactions, oligomers function as primers for DNA polymerase as well as "hybridization probes." Because polymerase extends from the Y-most residue of the oligomer, "wobbles" or mismatches in this 3' region effectively inhibit the enzyme from efficient interaction with the primer-template hybrid. In instances when full or partial DNA sequence data are available for a molecule, it is a relatively straightforward project to utilize the data to construct a specific oligomer. In addition to utility in hybridization experiments (e.g., with Northern blots), appropriately designed oligomer probes provide a means to access full-length cDNA or corresponding genomic derivatives of the respective molecule. By targeting oligomer sequences to distinct 5' or 3' regions, for example, it is feasible to utilize oligomer probes for "walking" through a library and isolating sequences not previously available. A more challenging use of oligomer probes involves searching for novel clones whose molecular identities have not been characterized. In some instances, the sequences of related P450 gene family members may be known. If there is reason to predict that additional related genes or gene products also exist, oligomer probes can be designed to permit their isolation. To this end, conserved regions of DNA sequence are evaluated and exploited to produce a "consensus" probe. A computer program, CLUSTAL, 1° is a very useful aid for comparing related sequences. It facilitates such analyses by generating an overlay of multiple sequence alignments, identifying regions that are conserved or divergent. Library screening with probes directed against conserved regions of sequence has proved useful in isolating previously uncharacterized P450 cDNAs.I-3 In other situations, only protein sequence data may be available without the corresponding DNA sequence. An oligomer specifying a coding domain of the relevant peptide can be generated and used as a probe for cDNA or genomic clone isolation. It is important to target amino acids 1o D. G. Higgins and P. M. Sharp, Gene 73, 237 (1988).
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that offer the least redundant codon choices. If related protein sequences are known, for example, from orthologous proteins in different species, alignments can also be performed to highlight amino acids that offer the greatest degree of conservation as well as the least codon redundancy. The latter criteria should be employed to aid in identifying the most appropriate sites for targeting oligomer probes. Two general strategies for designing oligomers targeted to the coding regions of proteins are typically employed. One scheme is to design an oligomer based on preferred codon usage frequencies, thereby minimizing DNA sequence degeneracy in the probe, u-~3 Another approach is to program the oligomer sequence with some or all of the unknown bases (corresponding to the third base "wobble" position of each codon), thereby synthesizing a probe mixture. In the former scheme, probe mixtures or redundancies are minimized, but it is likely that some of the programmed "guesses" will be incorrect. Nevertheless, depending on the inherent sequences, it is possible that appropriate hybridization conditions can be ascertained which are permissive for a certain degree of hybrid mismatch while retaining relative specificity of interaction. In the latter scheme, redundancies along the length of an oligomer mount geometrically. Thus, even though one of the probes in the mixture may be the correct sequence, its proportion of the total probe could be minor, and its resulting contribution to hybridization signal strength quite small. Furthermore, some of the alternative probes in the mixture may give rise to an intolerable background of nonspecific hybridization. With these caveats in mind, the choice in probe design is not necessarily clear. Successful isolations have been achieved by both approaches, and the choice between strategies does not have to be mutually exclusive. For example, we designed an oligomer probe based on amino acid sequence to isolate cDNAs encoding rabbit microsomal epoxide hydrolase. 14Twelve amino acids were targeted for probe design; although redundancy in codon choice was minimal for most of these residues, some assumptions based on preferred codon usage were considered necessary to minimize probe redundancy. For additional details regarding oligomer probe design, the reader is referred to other chapters in this series. 6,15 II R. Lathe, J. Mol. Biol. 183, 1 (1985). 12 K. Wa~a, S. Aota, R. Tsuchiya, F. Ishibashi, T. Gojobori, and T. Ikemura, Nucleic Acids Res. 18, 2367 (1990). 13 R. Grantham, C. Gautier, and M. Gouy, Nucleic Acids Res. 8, 1893 (1980). t4 C. Hassett, S. M. Turnblom, A. DeAngeles, and C. J. Omiecinski, Arch. Biochem. Biophys. 271, 380 (1989). ts W. I. Wood, this series, Vol. 152, p. 443.
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Library Plating/Screening Bacteriophage, plasmid, or cosmid libraries are cultured according to a variety of established protocols. Once plated, however, subsequent methods to screen for a eDNA or gene of interest are very similar. A set of six plates is conveniently processed, each 150-mm petri dish containing 20,000 to 30,000 colonies or plaques 0.5-1.0 mm in diameter. This number, near the maximum density before confluency is reached, should be derived beforehand by plating serial dilutions of the library. Distinct, round (nonconfluent) colonies or plaques will yield the clearest signals following autoradiography. After being chilled thoroughly at 4 °, the plates are ready to be placed in contact with the transfer membrane. When performing lifts on plates which have a top agar coating (e.g., a phage library screen), we find it helpful to prewet the transfer membrane on a sterile L plate in order to avoid adhering top agar to the membrane. While in contact with the chilled plate, the disk is punctured asymmetrically with a needle to facilitate future reorientation. The membrane is peeled away and placed colony/ plaque side up in a 1.5 ml pool or 0.5 N NaOH on plastic wrap to denature the duplex DNA. After 3 min, the disk is blotted on filter paper, then similarly dipped and blotted again. The DNA fixed to the membrane is then neutralized for 3 minutes in 1.5 ml of 0.5 M Tris-HC1, pH 7.5, blotted, and this step repeated a second time. Nylon membranes are dried at room temperature or under a heat lamp; nitrocellulose filters must be baked at 80 ° in a vacuum oven. Nylon is the preferred transfer membrane because its binding capacity is greater than nitrocellulose. Increased retention of bound DNA translates into more target for probe hybridization, and consequently a stronger autoradiographic signal. Nylon can be UV irradiated to cross-link fixed DNA to the membrane, which has been reported to increase subsequent signal detection levels.16 Nylon is also more durable, which is an important consideration if the membranes are to be probed multiple times.
Hybridization Conditions Our standard hybridization buffer is composed of 5 x or 6 × SSC (see below), 1% (w/v) sodium dodecyl sulfate (SDS), 25 mM sodium phosphate, pH 6.5, 1 x Denhardt's solution, and 10/~g/ml polyadenylic acid (I × SSC is 0.15 M NaCI, 15 mM sodium citrate; 1 x Denhardt's solution contains 0.02% each of Ficoll, poiyvinylpyrrolidone 360, and bovine serum albumin). Dried membranes are placed in heat-sealed bags, two disks per bag 16 E. W. Khandijian, Bio Technology 5, 165 (1987).
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with colony/plaque sides facing outward. Then 15 to 20 ml of hybridization buffer is added to each bag, and prehybridization proceeds for at least 1 hr at the incubation temperature. Oligomers are radiolabeled at the 5' terminus using T4 polynucleotide kinase using standard conditions,17 with a 1.5 × molar excess of [~/-32p]ATP in the labeling reaction (relative to the oligomer concentration). The radiolabeled probe is separated from radioactive precursor in a 20% polyacrylamide gel and purified by elution. Oligomer probe is added at a final concentration of 1-2 × 106 disintegrations per minute (dpm) per milliliter buffer. Hybridization is performed overnight in a moderately shaking water bath. The appropriate salt concentration and hybridization temperature are crucial for successful library screening with oligomers. These parameters will vary with the length and GC content of the oligomer, as well as the stringency desired. For relatively short oligomers, a formula has been described 6,7 for estimating the Td, or temperature at which one-half of the duplexes will dissociate from target DNA in 6 x SSC: Td(°C) = 4(G + C) + 2(A + T). However, optimal hybridization conditions often must be derived empirically, for example, using results from Northern blots as a guide. For library screening experiments in our laboratory, we have found that hybridization in 5 × SSC at a temperature of 54° has worked well for 20-base oligomers with 60% GC content, or 52° for 18-base oligomers with 66% GC. When using degenerate oligomer probes, conditions will often be more variable. We have screened cDNA libraries with a degenerate 18met (termed the PBmer), which consists of 8 distinct oligomer sequences ranging in GC content from 44 to 61%. 1-3 When using this probe, we relax the hybridization stringency to 42° in 5 x SSC buffer in order to accommodate each of the 8 oligomers in the mix. We also have isolated cDNA clones using shorter and more complex probes. For instance, two oligomers were employed to identify cDNAs encoding rabbit serum paraoxonase (arylesterase).18 This enzyme, in concert with P450-mediated catalysis, metabolizes organophosphate pesticides and other substrates. Based on rabbit peptide sequence information, a 17-mer (64-fold degenerate; GC content ranging from 29 to 47%) and a 15-mer (256-fold degeneracy; GC content 27-60%) were synthesized and used sequentially in the screening process) s Because of the wide GC content variation in these oligomers, it was necessary to decrease the hybridization temperature to 37° and increase the salt buffer to 6 × SSC. 17j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. ts C. Hassett, R. J. Richter, R. Humbert, C. Chapline, J. W. Crabb, C. J. Omiecinski, and C. E. Furlong, Biochemistry, in press (1991).
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Washing Conditions After overnight hybridization, filters are nonstringently washed twice at room temperature (5-10 min each time) in 5 × SSC/0.1% SDS buffer. This is followed by two changes in this solution at the hybridization temperature, each wash being for 20 min. At least 100 ml of wash solution is recommended per 137-ram membrane. High stringency is attained using a solution containing 3 M tetramethylammonium chloride, once at 37° (to remove Na + ions) and twice at a temperature 3° below the dissociation temperature.19 When Na ÷ in the wash solution is replaced with tetramethylammonium chloride salts, the Td is dependent on length of the oligomer probe and independent of base composition. This feature becomes especially valuable when screening libraries with degenerate probes of variable GC content, but uniform length. Washed membranes are heat-sealed in plastic bags and taped to an intensifying screen (or exposed film), colony/plaque side up. After placing in a cassette, X-ray film is carefully aligned to the mounting surface and taped to the intensifying screen to ensure subsequent alignment. Filters from a bacteriophage or plasmid library screen should be exposed overnight at - 8 0 ° with at least one intensifying screen. A positive signal on the autoradiogram appears as a distinct black dot. Longer exposures may be necessary under some circumstances, such as when screening cosmid libraries which contain fewer oligomer target sites per colony or when screening with degenerate probes in which only a subpopulation of oligomer probe binds to the target sequence. Verification of Positive Signals We apply various approaches to confirm that an autoradiographic signal from the initial round of library screening represents the desired clone. When possible, we exploit the same oligomer(s) used in library screening as an adjuvant in the verification process. Verifying signals will be more convincing and easier if information is available to permit the design of more than one oligomer specific for the clone of interest. The standard procedure we employ when screening libraries with oligomer probes requires harvesting the colonies or plaques associated with an autoradiographic signal, and replating in such a manner as to amplify the positive signal. After successive rounds of replating at lower density, the specific colony/plaque hybridizing to the oligomer probe can be clearly distinguished and isolated. Alternatively, one can pick all colonies/plaques 19 W. I. Wood, J. Gitschicr, L. A. Lasky, and R. M. Lawn, Proc. Natl./*cad. Sci. U.S.A. 82, 1585 0985).
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surrounding the autoradiographic signals and individually transfer them in grid fashion to a fresh agar plate; obviously many will not be the clone of interest. Bacterial colonies are transferred by sterile toothpick or inoculating loop. Phage suspensions (-5/zl), which should be amplified for several hours in liquid culture, are dotted onto a lawn of susceptible cells. The grid is transferred to a nylon membrane as described above, and probed with the oligomer probe. This process usually reduces the number of successive rounds of rescreening and also verifies, in a relatively rapid manner, whether the clones being pursued are likely to be true positives. More recently, we have employed a PCR-based approach to verify positive signals when screening phage libraries. This method also employs the oligomer hybridization probe, but as a primer component in PCR amplification. A plug encompassing the autoradiographic signal is cored from the agar plate using the wide end of a Pasteur pipette. The total number of plaques within the plug apparently is unimportant. The plug is suspended in 1 ml of elution buffer and placed at room temperature for several hours. Five microliters of this eluate is added to a (total volume) 100/zl PCR reaction 2°which contains two cDNA-specific oligomer primers (each could also be useful in the initial library screening). The two oligomers must flank a portion of the insert to be amplified (Fig. 1A), each one being complementary to opposing strands (i.e., one oligomer is sense, the other antisense). If a specific oligomer is available for only one region of the cDNA, the PCR primer pair can be completed using the universal forward or reverse primer specific for the cloning vector (see Fig. 1B). In this instance, two separate PCR reactions must be performed which combine the cDNA-specific primer with each universal primer, since the orientation of the insert in the vector is unknown. The presence of a discrete PCR product in an agarose gel is suggestive that the clone of interest has been identified, since a successful PCR reaction requires that the cDNAspecific oligomer hybridize to the insert. Following isolation in low temperature gelling agarose, the PCR product can be sequenced without further manipulation. 21 Subsequent rounds of rescreening are usually required to isolate the cDNA, but one can be confident that this effort will lead to the characterization of a desired clone. However, isolation may also be possible using a second cDNA-based primer (complementary to the one initially employed) to amplify the entire cloned insert. If the oligomer sequence contains a suitable restriction site, or if one prefers to use the complementary regions directly as sites for 20 R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. A. Arnheim, Science 230, 1350 (1985). 21 K. A. Kretz, G. S. Carson, and J. S. O'Brien, Nucleic Acids Res. 17, 586 (1989).
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A I
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F]o. 1. (A) PCR amplification strategy with two eDNA-specific oligomers. Each oligomer (indicated by the symbols *, *') must be complementary to opposite strands of the cDNA. (B) PCR amplification strategy with only one cDNA-specific oligomer. The primer pair necessary for amplification is completed using either the forward or reverse universal primer, which is vector specific. Because the orientation of the insert in the vector is unknown, two reactions must be performed, although only one combinationof primers will yield a product.
PCR extension, 22it should also be feasible to fuse the respective 5' and 3' PCR products, thereby creating a full-length construct. After a single colony/plaque has been isolated which hybridizes to the oligomer probe(s), the vector/insert DNA can be prepared by standard methods. Purified clones can be categorized by restriction mapping and characterized by DNA sequence analysis and in vitro expression. Conclusions
Identification and characterization of cytochrome P450 cDNAs and genes can be complicated owing to the size of this superfamily, as well as to the considerable similarity among many family members. We have 22R. M. Horton, H. D. Hunt, S. N. Ho, J. K. Pullen, and L. R. Pease, Gene 77, 61 (1989).
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found library screening with oligomers to be a very effective method for identifying novel and previously characterized cloned DNAs. With proper control of oligomer design and hybridization/washing conditions, autoradiographic signals obtained from library screening will be very convincing with little or no interference from background noise. Furthermore, oligomer/PCR-based strategies provide unique opportunities for the rapid characterization of clones, circumventing the need to isolate and categorize cloned DNAs by traditional approaches. Acknowledgments Research in our laboratory has been supported by Grants GM-32281, ES-046%, and ES04978 from the National Institutesof Health.
