INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

As a step toward a high-resolution functional map of the complete human immunodeficiency virus type 1 (HIV-1) genome, we carried out a detailed study of a 1-kb segment at the 5' end of the viral RNA genome. This portion of the viral genome was chosen for two reasons. First, it contains many previously identified functional elements (see Fig. 2), including several cis-acting elements and sequences encoding the matrix and capsid proteins. Comparison of our results to previous studies of this region served as a means to validate our approach. Second, several of the functions attributed to this region have not been mapped to particular sequences, suggesting that much remains to be discovered in this segment of the genome.

We used a genetic footprinting (26) method to construct and analyze a large number of mutants distributed in a relatively random fashion over the selected segment of the genome. A library of 15-bp insertion mutants was constructed in vitro using MuA transposase and then selected en masse for the ability to carry out various phases of the viral life cycle. Each mutant contained a single insertion, which included a restriction endonuclease recognition sequence.

Nucleic acid samples of the library taken before and after each functional test were analyzed to assess the fitness and recovery of each mutant. Mutants defective for a given phase of the viral life cycle were relatively depleted at that step. This mutagenesis approach permitted the efficient functional classification of defects in viral replication caused by individual mutations. Although our findings are for the most part in accordance with results of previous studies, we were able to identify several novel functional features of the HIV genome.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. The replication-defective HIV-1 proviral clone mutagenized in this report (HIVpuro) was derived from pHIV-APenvVifVpr (27) and subcloned into either Bluescript KS+ (Stratagene) or pBS-Kan (a Bluescript KS+-derived vector in which the ampicillin resistance gene was replaced by the kanamycin resistance gene). pHIV-APenvVifVpr was constructed from HIV-AP, an HIV-1 proviral clone containing the human placental alkaline phosphatase gene in place of nef (13), by making multiple deletions to eliminate most of env, vif, and vpr. To construct HIVpuro, the human placental alkaline phosphatase gene was replaced by the puromycin acetyltransferase gene driven by the simian virus 40 promoter (19). In addition, host DNA sequences flanking the proviral sequences were eliminated. PCR mutagenesis was used to eliminate the five BsgI sites originally present in the plasmid (GC at position 1186, CG at position 2538, AC at position 4820, AT at position 5719, and AC at position 5848). These changes did not detectably affect viral replication as measured by endpoint titration (data not shown). A fragment of HIVpuro was subcloned into Bluescript KS+, mutagenized (see below), and subsequently cloned back into HIVpuro to generate a library of mutant proviruses.

Mutagenesis. The mutagenesis procedure was a modification of the method described by Singh et al. (26). MuA transposase was purified as described elsewhere (1). The double-stranded oligonucleotide (Not15) used for mutagenesis was made by annealing Not15A (5'-TGCGGCCGCGCACGAAAAACGCGAAAGCGTTTCACGATAAATGCGAAAAC-3') and Not15B (5'-GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCGGCCGCA-3') in 50 mM NaCl. This oligonucleotide contains recognition sequences for both MuA transposase and the NotI restriction endonuclease. The integration reaction was performed by incubating the Not15 duplex oligonucleotide, target plasmid, and MuA transposase at 30°C for 1 h (25). The five-nucleotide gaps resulting from the integration events were repaired by Taq DNA polymerase-mediated nick translation. The products of these nick translation reactions were then digested with NotI and recircularized by ligation. A detailed description of reaction conditions for the mutagenesis can be found at our website (http://cmgm.stanford.edu/pbrown/footprint.html).

Cell culture. 293, 293T, and HOS cells were grown in Dulbecco's modified Eagle's medium containing 4.5 g of glucose/liter and 10% defined fetal calf serum (HyClone). 293T cells were used for all transient transfection experiments. 293 cells were used for all stable transfection experiments and transductions by virions produced by transient transfection. HOS cells were used for transductions by virions produced from transduced or stably transfected cells. Cells were grown at 37°C in 5% CO₂. Puromycin selection was performed with puromycin (Sigma) at 2.5 µg/ml for 293 cells and 5 µg/ml for HOS cells.

Transfections and transductions. Transient and stable transfections using a Lipofectamine Plus kit (Gibco/BRL) were performed according to the recommended protocol; 30 µg of total plasmid DNA, 60 µl of Plus reagent, and 40 µl of Lipofectamine were used for each 15-cm-diameter tissue culture dish. For stable transfections, puromycin selection was initiated 48 h posttransfection. For transient transfections, the medium was changed 48 h posttransfection and virus was harvested 72 h posttransfection. Viral stocks were diluted to the desired titer in medium containing Polybrene (4 µg/ml; Sigma) and used to transduce cells for 2 h at 37°C. Puromycin selection was initiated 48 h after transduction.

Nucleic acid preparation and manipulation. Plasmid DNA was purified using a Qiagen plasmid DNA kit and subsequently banded in a cesium chloride gradient (24). A Qiagen blood and cell culture genomic DNA kit was used to prepare genomic DNA from tissue culture samples. Total cellular RNA was prepared using a Qiagen RNeasy total RNA kit. Viral RNA was prepared by pelleting virions by ultracentrifugation (28,000 rpm for 2 h at 4°C in a Beckman SW28 rotor), decanting the supernatant, resuspending the viral pellet in the residual medium, and using a Qiagen Oligotex direct mRNA kit.

Pretreatment of streptavidin-agarose beads. Streptavidin-agarose beads (Sigma) were incubated for 1 h in the presence of poly(dl-dC) (200 µg per ml of streptavidin agarose slurry) in 1× binding buffer (12% [vol/vol] glycerol, 12 mM HEPES [pH 7.9], 4 mM Tris-HCl [pH 8.0], 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol), washed extensively, and resuspended in 1× binding buffer as a 50% slurry.

Footprinting. Initial amplification of nucleic acid samples was performed using the cold PCR protocol with HIV-specific primers HIV37 (5'-TGGAAGGGCTAATTCACTCCCAAAG-3'), HIV493 (5'-TCTCTCTGGTTAGACCAGATCTG-3'), HIV521 (5'-GGGAGCTCTCTGGCTAACTAGGG-3'), and HIV1573r (5'-CATCCTATTTGTTCCTGAAGGG-3'); 10 ng of plasmid samples, 1/10 of the products of RT reactions (equivalent to 10 ng of input RNA), or 0.5 µg of genomic DNA samples was used as the template; 10 ng of cold PCR products was used for hot PCRs. For hot PCRs, one HIV-specific primer was 5' end labeled with [-³²P]ATP and T4 DNA polynucleotide kinase (New England Biolabs), while the other primer was 5' biotinylated (Operon). High-specific-activity [-³²P]ATP (160 µCi/mmol, 23 pmol/µl; ICN) was used for radiolabeling at a stoichiometry of 1 pmol of ATP/1 pmol of oligonucleotide. Hot PCR products were treated with single-stranded affinity matrix (Clontech), purified, and adsorbed to 50 µl of pretreated streptavidin-agarose beads in 1× binding buffer for 1 h at 25°C. The beads were then washed twice with 0.5 ml of 1× binding buffer for 15 min at 25°C, washed once with 0.5 ml of 1× restriction enzyme buffer 3 (New England Biolabs), and incubated in 50 µl of 1× restriction enzyme buffer 3 with 20 U of restriction enzyme NotI for 1 h at 37°C. The supernatant from this digestion step was separated from the beads by centrifugation through a Micro Bio-spin column (Bio-Rad) and ethanol precipitated. Samples were analyzed by denaturing polyacrylamide-urea electrophoresis.

Sources of variability in the data. One kilobase of the HIV genome was analyzed in 10 overlapping intervals of 200 to 300 bp by using 10 primer pairs. Each mutant was thus examined using at least two primer pairs for amplification. Moreover, each series of transfection-transduction experiments was carried out in its entirety in triplicate. It was therefore necessary to develop a normalization procedure so that data from separate gels and different replicates could be combined to determine the quantitative effect of each mutation.

Quantitation. Autoradiographs were scanned using a flatbed scanner (Hewlett-Packard ScanJet IIc) at a resolution of 300 dots/inch resolution, with brightness and contrast set at 125 (50%), using the DeskScan II utility (Hewlett-Packard). Scanned images were read into a Matlab-based application (15; http: //cmgm.stanford.edu/pbrown/footprint.html) by which individual bands were selected and quantitated for peak intensity values.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Constructing a library of insertion mutants. The objective was to make a large number of mutations of the same type at diverse positions in a 1-kb segment of the HIV genome and to assess the performance of each mutant at several points in the viral replication cycle. An in vitro transposition reaction was used to introduce a 15-bp insertion mutation into a replication-defective HIV-1 proviral genome in which the env gene had been replaced by the puromycin acetyltransferase gene. The mutations were made in the segment of the HIV proviral genome extending from nucleotide positions 1 to 1514. This segment included the 5' long terminal repeat (LTR), the 5' untranslated region, the complete matrix gene, and the 5' half of the capsid gene. Mutants and mutations are numbered according to the nucleotide position immediately 5' to the insertion.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Mutagenesis scheme using MuA tranposase. Oligonucleotides used for mutagenesis are represented by bold lines (), the target DNA plasmid is represented by thin lines, and the 5-bp sequences in the target DNA that were duplicated during mutagenesis are represented by open boxes ().

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Map of the mutations evaluated in this study. Previously described features, indicated on the map, are TAR, the polyadenylation signal (poly-A), the att site (att), the primer-binding site (PBS), the kissing loop domain (KLD), the major splice donor (sd), two Gag-binding stem-loop structures (SL), alpha helices 1 to 5 (H1 to H5) of matrix, the basic region of matrix (BR), the beta hairpin of capsid (), helices 1 to 4 (H1 to H4) of capsid, and the cyclophilin A-binding region of capsid (CyPA). Numbers indicate nucleotide positions at the borders of major regions in the HIV genome. Arrows indicate positions of insertional mutations evaluated in this study.

Sampling populations of mutants at different steps in the viral replication cycle. To identify insertions that disrupted cis-acting elements (e.g., transcriptional modulators, the packaging sequence, and the viral att site), the library was either transiently or stably transfected into producer cells. A plasmid encoding vesicular stomatitis virus G protein (VSV-G) was transiently transfected into the producer cells to pseudotype the env-defective mutant virions. Virions were harvested from these cells and used to transduce fresh HOS cells. Nucleic acid samples were collected at various steps during this experiment (Fig. 3). Depletion of mutants at each of these steps in the viral replication cycle was followed by analysis of these nucleic acid samples by genetic footprinting.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Relationship of nucleic acid samples analyzed by genetic footprinting to specific steps in the life cycle. A decrease in the abundance of a given mutant between two of the indicated nucleic acid samples implies that the mutant is defective in one of the intervening steps of the viral life cycle.

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Design of transfection and transduction experiments. The library of mutagenized proviruses was either transiently or stably transfected into cells to produce populations of mutant virions. In our experiments, the viral genomes of mutants defective in trans-acting factors were efficiently rescued during the transient transfection by phenotypic mixing but were not detectably rescued during the stable transfection. The virus produced in the transient transfection experiment was used to transduce fresh, untransduced cells at a low multiplicity, resulting in a population of producer cells that contained a single provirus per cell. Symbols represent wild-type viral genome (), replication-defective viral genome with mutation in trans-acting factor (), wild-type viral protein (), and mutant viral protein ().

Footprinting analysis procedure. Samples of genomic DNA from producer cells or transduced cells, mRNA from producer cells, or genomic RNA from virions were subjected to an initial round of amplification by either PCR (for DNA samples) or RT-PCR (for RNA samples). PCR was then performed on these preamplified samples, using one ³²P-labeled DNA primer and one biotinylated DNA primer (Fig. 5). The primers were complementary to HIV sequences and flanked the region to be analyzed. The products of this second PCR were treated with a single-stranded nucleic acid-binding resin to remove incomplete extension products, bound to streptavidin-agarose beads, and digested with NotI. The released radioactive products were concentrated and subjected to electrophoresis on denaturing polyacrylamide-urea gels. This assay generates a radioactive product of unique length for each mutation; the length depends only on the position of the insertion in the HIV sequence. Figure 6 shows a typical genetic footprinting gel. Adjacent to each band, the position of the insertion sequence, quantitative measurements of recovery in several nucleic acid samples, and local nucleic acid sequences for the corresponding mutant are indicated.

View larger version (31K): [in this window] [in a new window]   FIG. 5.   Genetic footprinting scheme using flanking PCR and restriction digestion. A collection of insertion mutants is subjected to PCR using one radioactively labeled primer () and one biotinylated primer (). The PCR products are captured by streptavidin-agarose resin () and digested with a restriction enzyme that recognizes a site in the insertion sequence. The radioactively labeled ends of the PCR products are released.

View larger version (54K): [in this window] [in a new window]   FIG. 6.   Genetic footprinting of library of mutagenized proviruses and nucleic acid samples from the transient transfection experiment, second round (the uncomplemented round) of viral production and transduction (cellular RNA, viral RNA, and transduced cell genomic DNA). Numbers directly to the left of the gel indicate exact positions of insertions. In this numbering convention, the first nucleotide of the HIV provirus is at position 1. Quantitative values for recovery of the mutants represented by each band, in each sample, averaged from normalized measurements are also given to the left of the gel. The nucleic acid sequences of the mutants represented by each band are written to the right of the gel. The sequences derived from the insertion oligonucleotide are boxed, and the target sequence duplications are underlined. Eighty-nine percent of 19 individually sequenced mutants contained the expected precise 5-bp duplication flanking the 9-bp insertion.

cis-acting versus trans-acting elements. Mutations that disrupt cis-acting sequences can be distinguished from those that disrupt trans-acting sequences by their inability to be complemented by wild-type viral genomes.

View larger version (39K): [in this window] [in a new window]   FIG. 7.   Percent recovery of mutants through single-cycle transductions. Data are not shown for mutants for which the coefficient of variation between triplicate experiments was greater than 0.5, except in cases where the observed phenotypes were confirmed by reanalysis. Ordinate values are plotted on a logarithmic scale. Tick marks indicate mutants that display severe depletions (<45% recovery). Mutations that compromise replication in both the presence and the absence of complementation are considered to be located in cis-acting sequences, while those that affect only uncomplemented replication cycles are considered to be located in trans-acting sequences. (A) Percent recovery of mutants after a single round of transduction in the presence of complementation. Data are from the transient transfection experiment, first round of transduction. (B) Percent recovery of mutants after a single round of transduction in the absence of complementation. Data are from the transient transfection experiment, second round of transduction. Data are not given for mutants whose abundance after the first round of infection was too low to allow accurate quantitation of further depletion.

Mutations that impair viral RNA transcription or stability. The four mutations in the TAR region (positions 456 to 506) severely compromised replication (Fig. 8). These mutants are probably defective for Tat-cyclin T1 binding, which would result in diminished transcription.

View larger version (20K): [in this window] [in a new window]   FIG. 8.   Genetic footprinting of library of mutagenized proviruses and infected cell genomic DNA sample from the transient transfection experiment, first round (the complemented round) of viral production and transduction. Numbers directly to the left of the gel indicate exact positions of insertions. In this numbering convention, the first nucleotide of the HIV provirus is at position 1. The extent of the TAR region is indicated to the right of the gel.

View larger version (19K): [in this window] [in a new window]   FIG. 9.   Effects of selected mutations in cis-acting sequences in the transient transfection experiment. These mutants were replication competent in the first round of transduction but defective for transcript formation in the second round, possibly indicating that it is the copy of these cis-acting sequences in the 3' LTR which is active.

View larger version (24K): [in this window] [in a new window]   FIG. 10.   Recovery of genomes carrying mutations in cis-acting sequences, at several steps of a single-cycle uncomplemented transduction. Samples were collected from the stable transfection experiment. Percent recovery was calculated by dividing the abundance of a mutant in a given nucleic acid sample by the abundance of that mutant in the genomic DNA sample from the stable transfection. Data are not shown for points where the abundance of a particular mutant was very low in the preceding nucleic acid sample or where the coefficient of variation between triplicate experiments was greater than 0.5. Graphs are plotted on a log scale. Tick marks indicate mutants that display severe depletions (<50% of preceding nucleic acid sample). Mutants which were depleted in the cellular RNA sample are considered to be defective in transcript formation, mutants which were depleted in the virion RNA sample are considered to be defective in packaging, and mutants which were depleted in the transduced cell genomic DNA sample are considered to be defective in an early step in viral transduction.

Mutations in cis-acting sequences that affect viral assembly. A cis-acting RNA packaging signal has previously been mapped to the few hundred base pairs around the 5' splice donor site and the 5' end of gag. This region includes the "kissing loop" dimerization and packaging signal, the 5' splice donor site, and two stem-loop structures which have been found to bind in vitro to Gag and nucleocapsid proteins (2, 3, 6, 23). We found that in all experiments, most genomes with mutations in the interval between positions 666 and 810 were underrepresented in virions compared to the abundance of the corresponding RNA in the producer cells (Fig. 10).

cis-acting mutations that impair steps between virus production and integration. Mutations at positions 571 to 618 and 691 to 712 resulted in a reduction in fitness for the portion of the replication cycle that includes viral entry, uncoating, RT, nuclear entry, and integration (Fig. 10). The segment between positions 571 and 618 is in U5, just 5' to the att site. Although no specific function for this region of U5 has previously been defined, its proximity to the att site raises the possibility that sequences in this region might affect recognition of the viral genome by integrase. Alternatively, the proximity of this interval to the primer-binding site suggests a possible role in initiation of RT (16). The second group of mutations, between positions 691 and 712, is in the kissing loop motif. A role for sequences in the 5' stem-loop of the kissing loop motif (nucleotides 692 to 738) in the synthesis of proviral DNA (in addition to the expected role in genomic RNA packaging) has been described elsewhere (20).

Mutations in matrix. Sequences at the 5' end of the matrix coding sequence (positions 791 to 802) appeared to contribute to viral RNA packaging in cis (see above). A few mutations near the 5' end of the matrix gene (positions 840 to 893) appeared to impair the production of stable transcripts (Fig. 11). These trans-acting mutations, which could be rescued by complementation, were located in the sequences that encode the C-terminal end of helix 1, a loop between helix 1 and helix 2, and the N-terminal half of helix 2. This region contains many basic residues and is at the edge of the globular domain of matrix that faces away from the trimer interfaces. The phenotype of these mutants suggests that the matrix domain of the Gag polyprotein might have a role in enhancing transcription or stabilizing the viral RNA genome in the producer cell prior to budding. Supporting this possibility, it was first proposed (5) and subsequently demonstrated in vitro (17) that the matrix protein has RNA-binding activity.

View larger version (43K): [in this window] [in a new window]   FIG. 11.   Recovery of mutations in matrix and capsid at several steps of a single-cycle uncomplemented transduction. Samples were collected from the transient transfection experiment, second round of viral production and transduction. Percent recovery was calculated by dividing the abundance of a mutant in a given nucleic acid sample by the abundance of that mutant in the transduced cell genomic DNA sample from the first round of transduction. Data are not shown for points where the abundance of a particular mutant was very low in the preceding nucleic acid sample or where the coefficient of variation between triplicate experiments was greater than 0.5. Graphs are plotted on a log scale. Tick marks indicate mutants that display severe depletions (<45% of preceding nucleic acid sample). Mutants which were depleted in the cellular RNA sample are considered to be defective in transcript formation, mutants which were depleted in the virion RNA sample are considered to be defective in assembly, and mutants which were depleted in the transduced cell genomic DNA sample are considered to be defective in an early step in viral replication. All mutations in capsid that affected viral replication showed their effects in trans.

Mutations in capsid. In agreement with other reports (8, 18), we found that mutations in the sequence encoding the N-terminal half of the capsid protein severely impaired viral replication (Fig. 7B). Capsid mutants with insertions between positions 1240 and 1428 were defective both at a step in viral production (assembly or budding) and at an early step in transduction of new host cells (Fig. 11 and 12). This result is particularly notable for the contrast to most of the matrix mutants, which appeared to be specifically defective at the assembly/budding step (Fig. 12).

View larger version (29K): [in this window] [in a new window]   FIG. 12.   Percent recovery of matrix and capsid mutants through the viral assembly process and the early steps of the viral life cycle. Data are derived from single-round uncomplemented viral production and transduction cycles. A schematic of the phases of the life cycle tested is drawn above the graphs. Numbers to the right of each point indicate the number of mutants from which data were averaged. Below each data point is a schematic of the region of matrix or capsid evaluated. Error bars indicate 95% confidence intervals. (A) Data for the matrix protein. Data are shown for insertions in the complete matrix protein ( [amino acids 1 to 132; nucleotides 792 to 1187]), the N-terminal region ( [amino acids 1 to 35; nucleotides 792 to 896]), the central region ( [amino acids 36 to 102; nucleotides 897 to 1097]), and the C-terminal region ( [amino acids 104 to 130; nucleotides 1101 to 1181]). (B) Data for the N-terminal half of the capsid protein. Data are shown for insertions in the N-terminal half ( [amino acids 1 to 101; nucleotides 1188 to 1490]), the N-terminal beta hairpin ( [amino acids 1 to 15; nucleotides 1188 to 1232]), the central helical region ( [amino acids 17 to 81, nucleotides 1236 to 1430]), and the cyclophilin A-binding region ( [amino acids 85 to 96; nucleotides 1440 to 1475]).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In the experiments described here, a large number of mutants with insertions at diverse positions in the 5' 1-kb segment of the HIV-1 genome were followed en masse through two rounds of replication. This strategy permitted a comprehensive, comparative examination of the effects of each mutation on viral replication. Selection and analysis of the mutants in parallel provided built-in internal controls for variables such as sample recovery and efficiency of analysis procedures. Retroviruses, particularly HIV, have been extensively studied using a variety of techniques. As a result, the basic mechanisms of major steps in the retroviral life cycle have been elucidated and many functional features of the retroviral genome have been mapped. Yet numerous important questions about retroviral replication remain to be answered. There is, therefore, still much to be learned from a systematic dissection of the HIV genome.

Mutations of any type can produce unpredictable consequences. For example, results from a previous genetic footprinting experiment (26) suggest that 36-bp insertion mutations may in some cases be less disruptive to the structure of a small RNA molecule than 12-bp substitution mutations. The mutation introduced in this study, a 15-bp insertion mutation, was designed to be disruptive enough to affect reproductive fitness of mutant viruses yet subtle enough to produce partially active mutants. The observation that many mutants with insertions at diverse locations had partially defective phenotypes demonstrates the usefulness of this type of mutagenesis strategy. Interestingly, these partially active mutants contained insertions in known essential sequences, as well as in sequences not previously noted to be important for viral replication.

The three-dimensional structures of matrix, capsid, and many HIV RNA elements have revealed much about the properties of these proteins and RNAs, and much directed mutagenesis has been done based on these structures. However, in our studies, the severity of the effects of mutations does not predictably correlate with the locations of insertions with respect to known secondary or tertiary structures. This observation suggests that important functional data can still be derived by thorough, nondirected mutagenesis approaches, at least until we know better how to predict function based on structural information.

Expected results and novel observations. The experiments defined several functional features in the HIV-1 genome, some of which have not previously been described. We mapped three kinds of cis-acting sequences: sequences that affect transcription or transcript stability, sequences involved in viral RNA packaging, and sequences important for an early step in viral transduction. Some of these sequences were found in intervals previously implicated in these functions (e.g., TAR and the kissing loop motif), and others were found in previously unrecognized locations.

Discrepancies with previously published results. The inconsistencies between our results and previous reports can be grouped into two classes. First, mutations at 25 positions in the matrix gene resulted in severe defects in replication in our study, while mutations disrupting some of the same sites were reportedly tolerated in a previous study (10). Second, we found that many mutations in the N-terminal half of capsid impaired viral assembly. Previous studies have mapped the residues important for Gag multimerization and viral assembly to the C-terminal domain of capsid (8, 14, 21, 29), while viruses with mutations in the N-terminal domain of capsid were found to be competent for viral assembly, although sometimes abnormal in core morphology (8, 9, 21, 22, 31).

Possible extensions of this work. In the original report describing the genetic footprinting technique, this method was used to generate a high-resolution functional map of a small (200-bp) gene encoding an RNA molecule (26). Modifications to the genetic footprinting procedure permitted us to analyze a much larger (1,000-bp) stretch of nucleic acid, including both cis-acting and protein-coding sequences. The experiments presented here involved the isolation and analysis of complex nucleic acid samples, including cellular RNA, virion RNA, and genomic DNA samples from a selection scheme in eukaryotic cells. Thus, genetic footprinting could be used to map the functional features in any DNA sequence if an appropriate selection scheme were available.