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Journal of Bacteriology, June 2004, p. 3599-3608, Vol. 186, No. 11
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.11.3599-3608.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

DNA Binding Regions of Q Proteins of Phages and 80

Jingshu Guo^* and Jeffrey W. Roberts

Department of Molecular Biology and Genetics, Cornell University Ithaca, New York 14853

Received 3 December 2003/ Accepted 16 February 2004

	ABSTRACT

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Bacteriophage gene Q protein and the related proteins of other lambdoid phages are transcription antiterminators that interact both with DNA in the late gene promoter segment and with RNA polymerase subunits. Using hybrids between Q of and the related Q of phage 80, we characterized elements of both Q and DNA that contribute to the DNA binding function. In particular, we found a C-terminal segment of the protein that is responsible for binding specificity and an 15 residue segment on a predicted alpha helix within this segment at which alanine substitutions decrease DNA binding. We identified a six-nucleotide segment located between the –35 and –10 promoter elements that confers binding specificity and is the site of point mutants that impair binding, and we isolated suppressors in Q that restore binding function by increasing the overall binding affinity. We also identified putative zinc finger structures in both proteins.

	INTRODUCTION

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The Q proteins of phage and its relatives are transcription antiterminators that interact with DNA, with the RNA polymerase (RNAP) 70 initiation subunit, and with RNAP core subunits (11, 13, 14). Their role in phage growth is to modify transcription initiated at the promoter of a single long transcription unit for phage late genes (5) and, particularly, to extend transcription beyond a terminator immediately downstream of this promoter. Each Q protein acts at a promoter-associated engagement site (named qut), with a species specificity based at least in part on specific DNA binding, and becomes a subunit of the elongating complex (20).

The qut site consists of a DNA binding element, located between the –35 and –10 promoter elements (Fig. 1B), and a segment downstream of +1 containing a promoter –10 element-like sequence that serves to capture elongating RNAP 16 to 25 nucleotides from the RNA start site in a 70-induced paused state that is primed for Q recognition (12, 21). During this pause, Q binds DNA just upstream of the elongating enzyme (Fig. 1A); binding is sequence specific for various members of the Q family (19, 21). Phage Q (at least) simultaneously contacts region 4 of 70, displacing it forward to a promoter –35 element-like sequence located between the –2 and –7 positions of the promoter (11) (Fig. 1). Exchange of segments between qut sites of Q and phage 82Q shows that either pause-inducing sequence is functional in combination with either Q-binding element, which in turn confers Q specificity on the qut (3); we confirm this location of the specificity element for 80Q below. Although direct evidence is missing, essential contacts with the core enzyme likely also are made as Q binds, or at least during the early stages of RNAP emergence from the pause. Sometime during subsequent elongation 70 is likely released, leaving Q as a subunit of the elongating complex; in vitro analysis shows that Q of phage 82 becomes a firmly affixed subunit of the elongating core downstream of the promoter and that 70 is missing at this stage (20).

View larger version (110K): [in this window] [in a new window]   FIG. 1. Elements of phage and phage 80 Q function. (A) Diagram of the paused transcription complex before and after binding Q, showing the disposition of 70 subunits, and the displacement forward of 70 region 4 by Q (11, 12). (B) Sequence alignment of phage and phage 80 Q proteins (CLUSTAL, MegAlign, and DNASTAR). Predicted alpha-helical regions (PredictProtein; http://www.embl-heidelberg.de/predictprotein/predictprotein.html) are indicated by black underlines or overlines, and the determined region of specificity is shown as a red line between the sequences. Sites of cysteines of the putative zinc finger (black dots) and of strong (red triangles) and weak (open triangles) suppressors of the qut binding region mutations are shown. (C) The promoter and initial transcribed regions of qut and qut80, including the DNA binding element. The –35 and –10 promoter elements are shown as lines between the sequences. Overlines designate binding sites assigned for the phage 82 Q protein (9), which plausibly coincide with those of Q and 80Q (21). Dots are sites of ethylnitrosourea interference of Q binding (1), red bases are sites of a double-base-pair substitution that impairs Q function and DNA binding (21), and blue bases mark the main region of sequence difference between qut and qut80. The pause-inducing –10 promoter-like sequence is underlined (12). The initial transcribed base (designated +1) is known only for .

We have made mutants of both Q protein and its site of action in the DNA in order to understand the interacting elements of each. To guide the analysis, we used two Q proteins that are moderately but distinctly related, those of phages (22.5 kDa) and 80 (29.5 kDa); they are 47% identical over the span of Q, although 80Q has a nonhomologous insertion of ca. 80 residues in the middle (Fig. 1B). Each acts specifically on its own qut (Q utilization) site according to activity in a reporter system (see below). We identify specificity elements of the DNA binding element for each Q. We also show that the C-terminal region of each protein and, in particular, the segment underlined in red (Fig. 1B) is responsible for specificity. Structure prediction (PredictProtein; http://www.embl-heidelberg.de/predictprotein/predictprotein.html; Fig. 1B,black overline) of this region proposes nearly identical alpha-helical and loop regions, despite some substantial differences in amino acid sequence. We identify in the Q and 80Q proteins essential cysteines that may contribute to a zinc finger-like element.

	MATERIALS AND METHODS

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Strains, plasmids, and mutagenesis. The sequence of phage 80 gene Q and surrounding genes was obtained in this laboratory by L. Matthews and D. Kadosh (unpublished data). All plasmids were made with standard molecular biology techniques. For reporter construction, BamHI/SmaI fragments containing either qut or qut80 combined with the tandem terminators t₈₂t_o (6) were cloned into pRS528 (15). The qut point mutants and /80 qut fusions were made by PCR and cloned into p'RS528 so as to replace the promoter segment; p'RS528 is pRS528 [+49 (wt)t₈₂t_o] with the EcoRI site destroyed. The +49 qut fragment contains sequence from the HindIII site upstream of the late gene promoter pR' to nucleotide 49 of the late transcript, followed by an introduced EcoRI site (17). The qut-lacZ fusions in pRS528 were transferred to RS45 (15) and then in single copy to the chromosome of E. coli SG20250 as described previously (15); E. coli strain SG20250 [F^– ara139 (lacIPOZY)U169 strA thi] was from S. Gottesman (National Cancer Institute, National Institutes of Health, Bethesda, Md.). The parental reporter strains are designated JWR1002 (qut) and JWR1023 (qut80). In the absence of a Q source, these reporters gave background levels of ca. 20 to 30 ß-galactosidase units, and white color on MacConkey lactose plates after 16 h of incubation at 37°C. To generate templates for in vitro Q activity assay, qut fragments were subcloned into p'XY306, which carries +49 (wt)t₈₂t_o in the pXY306 backbone (17).

Q expression. pJG100 was constructed to express Q upon IPTG (isopropyl-ß-D-thiogalactopyranoside) induction for in vivo activity measurements. In brief, pJG100 is a low-copy plasmid conferring spectinomycin resistance and containing the phage T7 promoter A1 with two lac operators between –35 and +20 (from pUHE21-2; D. H. Bujard, unpublished data) and the lacI^q gene from pGEX-2T (Pharmacia Biotech) (4); a 760-bp fragment containing Q was cloned under control of the T7 A1 promoter. pJG102 is an equivalent construct expressing 80Q. Induction by IPTG of pJG100 transformed into JWR1002 gave 2,000 ß-galactosidase units and a deep red color on MacConkey lactose plates; pJG102 transformed into JWR1023 gave 500 ß-galactosidase units and a pink color.

To overexpress Q for purification, the gene was subcloned into p'QE-30, which was constructed from pQE-30 (Qiagen) to contain the lacI^q gene from pJG100 and to express native Q (lacking the histidine tag of the original pQE-30 vector).

ß-Galactosidase assay of Q function in vivo. ß-Galactosidase was assayed in permeabilized cells as described previously (10), after induction of Q by 1 mM IPTG for 1 h during exponential growth. Background activity without Q induction was negligible (2% of induced levels). Activity determinations generally represent an average of three to four independent experiments.

Construction of /80 fusion Q proteins and Q alanine scanning mutants. All and 80 fusion Q proteins were produced by a multistep PCR protocol. Briefly, a Q N-terminal or C-terminal PCR primer was used, along with an internal PCR primer, to make two overlapping Q fragments, which were then purified, combined in equimolar amounts, and used as a template in a further PCR in which appropriate C-terminal and N-terminal primers amplified the desired product; this was cloned into the pJG100 vector backbone, and its structure verified by sequencing.

Most of the 33 Q alanine scan mutants were made by the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) in the p'QE-30 vector; some were produced by the multistep PCR method described above. Point mutants were made by either procedure.

PCR mutagenesis of Q protein and screening. PCR mutagenesis was done separately on the N-terminal (bp 1 to 256) and C-terminal (bp 256 to 621) fragments by standard PCR. Twelve independent PCRs were pooled, ligated into the pJG100 backbone, and transformed into ElectroMAX DH10B cells (10¹⁰ transformants/µg of pUC19 DNA) to yield >10⁵ individual colonies. Plasmid DNA representing the Q mutant library was extracted from the pooled colonies. Then, 10 ng library DNA was electroporated into appropriate reporter cells to produce 10⁵ transformants for screening Q mutants. Defective Q mutants were identified as white or pink colonies with a large white halo on MacConkey Lac plates compared to the red-color phenotype of wild-type Q (expressed by pJG100) in wild-type qut reporter cells (JWR1002). Candidate clones were selected by colony color on the MacConkey lactose indicator plates and confirmed by streaking-out and reincubation. Q mutants were further characterized by ß-galactosidase assay on relevant reporter cells and by Western blot analysis to verify expression of the Q polypeptide. In the screen for defectives, 27 candidates from the 50,000 Q N-terminal mutagenized pool and 61 clones from the 50,000 Q C-terminal mutagenized pool were identified and sequenced. Q suppressors of qut mutants were identified to restore red color to the two qut mutant (–25G&;C and –22T&;G) reporter cells; seven potential Q suppressors were isolated from 500,000 Q C-terminal mutant colonies, and DNA sequencing identified five different suppressor mutations.

Q overexpression and purification. Q proteins were overexpressed to 10 to 20% of total cell protein upon IPTG induction of the p'QE-30 vector in E. coli BL21 cells and purified as described previously (18). Peak fractions from phosphocellulose chromatography were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pooled, dialyzed into Q storage buffer (10 mM K phosphate [pH 6.5], 1 mM dithiothreitol [DTT], 1 mM EDTA, 1 M KCl, 50% glycerol), and stored at –20°C.

Electrophoretic mobility shift assay of Q-DNA binding. A 115-bp 5'-end-labeled double-stranded DNA probe containing pR' –35 to +35 was made by PCR with a ³²-P-labeled primer and then gel purified. Q protein in 1 µl of Q buffer (10 mM Tris-HCl [pH 7.5], 1 mM DTT, 0.1 mM EDTA, 200 mM KCl, 50% glycerol, and 1 mg of bovine serum albumin/ml) was added to 0.01 nM purified probe (5,000 cpm) and a 100-fold excess of sonicated calf thymus DNA (competitor DNA) in a 25-µl binding reaction; the final buffer was 20 mM Tris-HCl (pH 8.0), 25 mM KCl, 12% glycerol, 1 mM DTT, 0.1 mM EDTA, and 40 µg of BSA/ml. Binding reactions were incubated 10 min at room temperature and stored on ice before loading onto a prechilled and prerun 5% polyacrylamide gel in 0.5x TAE (20 mM Tris-acetate [pH 8.5], 1 mM sodium EDTA) running buffer. Samples were electrophoresed at 4 to 8°C in a Bio-Rad Protean II xi apparatus (Bio-Rad, Hercules, Calif.) with a circulating ice-H₂O bath. Gels were dried and scanned with a PhosphorImager, and radioactive bands were quantified with ImageQuant software. The K_d is reported as the concentration of Q protein giving 50% shift.

In vitro transcription. RNAP and NusA protein were purified as described previously (21). Transcripts were made by preincubating 1 nM DNA template (280 bp, yielding a 113-nucleotide terminated transcript and a 204-nucleotide runoff transcript) and 20 nM RNAP (and 150 nM NusA for experiments with Q protein) for 5 min at 37°C in 20 mM Tris-HCl (pH 8.0); 0.1 mM EDTA; 1.0 mM DTT; 50 mM KCl; 10% glycerol; 200 µM ATP, CTP, and GTP; 50 µM UTP; and 0.5 µCi of [³²-P]UTP/µl in a 25-µl reaction volume. RNA synthesis was initiated by the addition of 2.5 µl of 40 mM MgCl₂ and 100 µg of rifampin/ml. In reactions where it was present, Q protein in 2.5 µl of Q buffer was added 30 s before the addition of MgCl₂ and rifampin. Reactions were stopped with 125 µl of stop buffer (12 mM EDTA, 600 mM Tris-HCl [pH 8.0], 120 µg of tRNA/ml) and extracted with 150 µl of phenol-chloroform-isoamyl alcohol (25:24:1). RNA was precipitated with 2.5 volumes of 100% ethanol and resuspended in 4 µl of 80% formamide, 1x Tris-buffered saline, and 0.05% bromophenol blue. RNA products were separated on a 12%, 8 M urea-polyacrylamide gel and scanned with a PhosphorImager. Percent terminator readthrough was calculated after quantification of the terminated and readthrough transcripts with ImageQuant software, considering the U content of the transcripts. The K_m is reported as the concentration of Q protein giving 50% of the maximum readthrough activity.

	RESULTS

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Mutational analysis of the Q binding regions. Previous work showed that Q function and DNA binding are impaired by the double point mutant –13A –15A of qut (1, 21) and by deletions of DNA farther upstream, although not by a substitution upstream of –26 that (necessarily) retains a functional –35 element (see Fig. 1C). The DNA binding function of qut in vitro is not impaired by substitution of sequences located between positions –12 and –1 (1). These results suggest that the essential Q DNA binding sequences are located approximately between positions –13 and –26, although sequences farther downstream (in addition to the pause-inducing sequences in the early transcribed region) still could be required for Q function. DNA footprinting experiments are consistent with this assignment; thus, Q proteins of phages , 82, and 21 protect the cognate qut DNA approximately between positions –30 and –10 against cleavage by MPE [methidium propyl EDTA · Fe(III)] (21), and the binding of Q is inhibited by the ethylnitrosourea reaction of phosphates in the vicinity of –12 (template), –15 (nontemplate), –22 (template), and –25 (nontemplate) (1).

The nontranscribed regions of qut80 and qut show the most extensive difference in the segment –22 to –28 (Fig. 1C), suggesting that this span may encompass contacts that are essential for binding specificity and that provide important binding energy. To investigate its function, we changed each base in the qut segment from –22 to –26 and assayed for Q function in vivo by using a reporter assay (7). Only three changes reduced Q-dependent reporter activity as much as twofold: –25C (25%), –22A (52%), and –22G (36%) (Table 1). As expected, if these mutations affect antitermination but not promoter function, background activity without induction of Q was not changed from that of the wild type (data not shown). We assayed mutant function by in vitro transcription and measured mutant effects on DNA binding by electrophoretic mobility shift assay, including also the previously characterized qut –15A –13A double mutant as a control. Representative experiments are illustrated in Fig. 2, and the results are summarized in Table 1. All of the mutants are about twofold defective in antitermination in vitro relative to the wild type and require an 10-fold-higher concentration of Q for maximum antitermination activity. Furthermore, all are defective in DNA binding; for the –22 mutants, essentially no binding occurred at 50 times the concentration required for maximal shift of wild-type DNA.

View this table: [in this window] [in a new window]   TABLE 1. Q Activity of mutant qut regionsa

View larger version (38K): [in this window] [in a new window]   FIG. 2. Defects of qut site mutations in Q function and DNA binding. (A) Gel analysis of Q antitermination activity on wild-type and two mutant qut DNAs: –13A/–15A (21) and –25C (the present study). Q was present at 2, 30, 50, 100, and 200 nM, and RNA synthesis was carried out for 2.5 min. The last lane shows the pattern of synthesis in the absence of Q, which is identical for all three DNAs. RT, readthrough RNA; t82, RNA stopped at the t82 intrinsic terminator in the template DNA. The pause, Q-dependent pause, and abortive products were as described previously (2). (B) Graph of the data in panel A. (C) Bandshift analysis of Q binding to wild-type and mutant DNAs. Q was present at 0, 1, 2, 5, 10, and 20 nM, except for the –13 –15 DNA, for which Q was present at 1, 2, 5, 10, 20, and 50 nM. (D) Graph of some of the data in panel C.

To determine whether the segment of greatest difference between qut and qut80 (positions –22 to –28) confers specificity for each Q, we made a set of exchanges in this segment between the two sites and assayed the hybrid sites for Q function in vivo and for DNA binding activity in vitro. Since we are unable to detect activity of purified 80Q in vitro, either in antitermination or DNA binding, we assayed only DNA binding by Q. Figure 3 summarizes the results of these exchanges. First note that each qut site shows the correct specificity for each Q (Fig. 3, lines 1 and 5). Second, replacement of the segment from –22 to –28 by that from 80 switches the specificity to 80 (line 2); thus, this segment confers specificity. However, the presence of this segment is not sufficient for activity because the converse replacement (line 6) shows no activity with Q, even though it binds Q approximately as well as do single point mutants of qut that impair Q function only two- to threefold. Note the lack of any simple relation of antitermination activity to Q binding activity in vitro; thus, qut80 is inactive with Q in vivo, but it binds Q as well as do point mutants of qut that still have substantial activity. Furthermore, replacement of qut80 bases with the critical –22T and –25G of qut increases the binding of Q to nearly equal that of qut, although this substitution has no activity with Q in vivo. This result confirms the role of these nucleotides, and this segment in Q binding and also the insufficiency of tight binding for Q activity.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Function of qut and qut80 hybrids recombinant in the segment from –28 to –22. sequences are shown in black or gray, and 80 sequences are indicated in red or pink. The DNAs were analyzed for activity in vivo as described in the text and for DNA binding in vitro by bandshift analysis.

Hybrids of Q and 80Q proteins identify a specificity region.

Exchange of flanking arms (Fig. 4, lines 3 and 4) showed clearly that the specificity resides in the C-terminal region. Fine structure mapping of this region by further reciprocal exchanges (examples shown in Fig. 4, lines 9 to 16) and then by exchange of a small segment defined by the activities of reciprocally exchanged hybrids (Fig. 4, lines 5 to 8) showed that the specificity resides in the segments from positions 155 to 181 of Q and positions 218 to 243 of 80Q; this segment is indicated by a red line in Fig. 1B. Structure prediction suggests that this region consists of two alpha helices separated by a loop of about five amino acids. Note that this is the segment of greatest sequence difference between the two proteins in the right half, a finding consistent with a role in binding specificity.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Function of hybrids between Q and 80Q. For each hybrid protein, the number above indicates the boundary residue from 80Q, and the number below indicates the boundary residue from Q. In vitro activity was determined at a saturating concentration of Q (500 nM).

Mutational analysis of the Q polypeptide. The region of the putative specificity element appeared prominently in a screen for defective Q function in a library of randomly mutagenized Q genes, which identified mutable sites throughout the gene (data not shown). Thus, we obtained defective mutants in 28 codons in the segment containing amino acids 87 to 206 and, of these, 14 codons were in the interval from positions 155 to 190 that covers the predicted helix-loop-helix segment. This mutagenesis was not saturated but was sufficiently dense so the distribution is likely to be meaningful: there were two occurrences in seven of the 28 codons, three occurrences in two of the codons, and five occurrences in one codon.

To determine in more detail regions active in DNA binding, we constructed point mutants by alanine scanning mutagenesis throughout the region of specificity. Figure 5 shows the result of in vivo and in vitro activity assays and DNA binding assays of purified proteins modified by alanine substitution. It is clear that the region of the second predicted helix (residues 173 to 190) and probably the adjacent upstream loop are required for both activity and DNA binding. Some of these substitutions probably disrupt protein structure, but others (e.g., S177, R178, and K181) are likely to remove solvent-exposed functional groups.

View larger version (61K): [in this window] [in a new window]   FIG. 5. Activity of alanine substitutions in the region of specificity of Q. The activity in vivo was determined as described. Activity in vitro was determined as the percent readthrough of the terminator in transcription by 500 nM purified Q, normalized to 100 for wild-type Q. DNA binding was measured by bandshift assay of purified protein and is given as 100 x (Kd with wild-type protein)/(Kd with mutant protein), except that the lowest values are plotted arbitrarily as 3 in order to indicate where the assays were done.

Mutations that increase Q activity and DNA binding.

View this table: [in this window] [in a new window]   TABLE 2. Activity of suppressor Q proteins on wild type and mutant quta

View larger version (23K): [in this window] [in a new window]   FIG. 6. Antitermination and DNA binding activity of suppressor Q. (A) Antitermination activity in vitro of wild-type Q on wild-type and mutant qut; (B) antitermination activity in vitro of E134K Q on wild-type and mutant qut; (C) DNA binding activity of E134K Q and H192Y Q on wild-type qut.

View this table: [in this window] [in a new window]   TABLE 4. Effects of substitutions in putative zinc finger residues and specificity element residues on DNA binding by Qa

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We can discern distinct DNA binding elements of the Q polypeptide and, possibly, of the qut site. The protein specificity regions, segments from positions 155 to 181 of Q and positions 218 to 243 of 80Q, likely contribute to the binding in the region of qut from positions –22 to –28: some mutations that change these segments of either Q or qut have no detectable DNA binding activity, and some combinations of a partially defective protein with mutant DNA give a greatly enhanced defect. The putative zinc finger mutants behave differently; they all have a modest effect on DNA binding, and they do not act synergistically with mutations in the Q binding element. We suggest that this element either affects overall structure of the DNA binding domain of Q or it contacts DNA at an unidentified locus to provide a small amount of binding energy. The location of two mutations increasing DNA binding (E134K and W136R) within the potential zinc binding motif, as well as their positive charge, is consistent with a distinct role in DNA binding.

The qut mutations at positions –13 and –15 may represent a binding site with a function distinct from that in the specificity segment from positions –22 to –28. First, the mutations at positions –13 and –15 change the footprint of Q on DNA in a qualitative manner: whereas Q protects wild-type DNA from cleavage by MPE approximately uniformly from positions –10 to –30, the –13/–15 double mutant DNA is protected only from positions –18 to –30 (21). Second, the –13 and –15 double mutation is suppressed poorly by substitutions screened to suppress mutations at –22 and –25, even though in vitro binding of Q is restored nearly to the wild-type binding constant. Thus, the conformation or persistence of the structure bound in the vicinity of positions –13 and –15 may be important independently of the residence of the whole Q polypeptide on DNA.

It remains to be shown whether a metal is in fact bound to the putative zinc finger domain, although treatment of Q with a chelating agent destroys DNA binding activity, and this can be restored by addition of zinc ions (data not shown); we could not, however, restore antitermination activity. Since the Q protein is prepared through steps of denaturation in guanidine and refolding in buffers without explicit addition of zinc, an essential metal would have to be a trace component of the buffers used.

The random library yielded some more information about the distribution of mutable sites in the Q polypeptide. In addition to the 28 mutated codons in the portion from residues 87 to 207, we obtained 12 mutated codons in the portion from positions 1 to 86; there were six single codon occurrences, four double occurrences, one triple occurrence, and one quadruple occurrence. Seven mutated codons were in the region from positions 48 to 58, a segment mostly of predicted alpha helix. Another notable feature of the mutant collection is the presence of lethal substitutions at either extreme of the polypeptide: both V6E and T206P gave ca. 5% wild-type activity. The functions of these terminal regions remain to be determined.

	ACKNOWLEDGMENTS

 
We thank members of the laboratory for advice and discussion. J.G. thanks Katherine F. Zhang for inspiration.

This was supported by grant GM 21941 from the National Institutes of Health.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853. Phone: (607) 255-2430. Fax: (607) 255-2428. E-mail: jwr7{at}cornell.edu.

Present address: Department of Genetics and Genomics, Roche Palo Alto, Palo Alto, CA 94304.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

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