151 GATA 8(5): 151-158, 1991

EMERGINGTECHNIQUES An Efficient Technique for Obtaining Sequences Flanking Inserted Retroviruses C L A R K S. H U C K A B Y , R I C H A R D E. K O U R I , M I C H A E L J. L A N E , S C O T T M. P E S H I C K , W I L L I A M T. C A R R O L L , S U S A N M. H E N D E R S O N , B R I A N D. F A L D A S Z , P. G R E G W A T E R B U R Y , and J O H N N. V O U R N A K I S

Genomic mapping studies frequently employ retrovirusmediated transfer of dominant selectable markers to specific target chromosomes. DNA probes containing sequences adjacent to inserted proviruses are valuable mapping tools in such studies. We have implemented a strategy for amplification of chromosomal sequences flanking the 5' LTR of MoMuL V-based vectors. Probes derived from these amplification products successfully differentiated murine versus human proviral localization in retrovirus-infected mouse-human chromosome

17q hybrid cells. Introduction Recent efforts to derive physical maps of mammalian genomes have introduced powerful cytogenetic techniques to manipulate large chromosomal domains, including the retrovirus-mediated transfer of dominant selectable markers to specific target chromosomes [1-6]. In such cases, it is often necessary to ascertain the chromosomal or subchromosomal location of the inserted provirus.

Insertion of rare cleavage sites [7, 8] into target chromosomes to facilitate mapping is an example of a strategy in which localization of vectors is important. Thus, genome mapping efforts have increasd the demand for rapid and accurate chromosomal localization of proviral inserts. One classic approach that can be employed for proviral localization is in situ hybridization. However, this strategy is not conducive to rapid screening when multiple cell lines are involved. Specialized selection and fusion regimes can, in some cases, determine linkage between introduced and endogenous markers, but such techniques yield low resolution and are labor intensive. Approaches that provide molecular probes for blot hybridization analysis are desirable; these approaches involve isolation of genomic sequences flanking the provirus. Flanking sequences have a variety of uses in addition to proviral localization, such as (a) delineation of novel sequence-tagged sites (STSs) [9], (b) starting points for chromosome walking and jumping, and (c) potential polymorphic markers for genetic studies. Until recently, most methods for isolating proviral flanking sequences involved plasmid rescue [1]. The advent of the polymerase chain reaction (PCR) [10] has led to more rapid alternatives. For example, the inverse PCR (IPCR) method [11-15] provides a strategy for isolating proviral flanking sequences. In this report, we describe an efficient IPCR-based strategy for generating genomic probes containing 5' flanking sequences of the inserted retroviral vector pZipNeo [16]. The strategy is applicable to any other vector based on MoMuLV [17]. We employed these probes to determine the murine versus human localization of proviruses in cloned microcell fusion hybrids prepared from pZipNeo-infected mouse fibroblast/ human chromosome 17q hybrid donors.

Materials and Methods Cell L i n e s

From Genmap Inc. (C.S.H., M.J.L., S.M.P., W.T.C., D.B.F., P.G.W., J.N.V.), New Haven, Connecticut; and the Division of Hematology/Oncoiogy (M.J.L.), Departments of Medicine and Microbiology, SUNY Health Sciences Center, Syracuse, New York; and BIOS C o r p o r a t i o n ( R . E . K . , S.M.H.), New Haven, Connecticut, USA. Address correspondence to John N. Vournakis, PhD, Genmap Inc., 291 Whitney Avenue, New Haven, CT 06511, USA. Received 7 March 1991; revised and accepted 28 March 1991.

HS68 human fibroblast and L M T K - mouse fibroblast cell lines were purchased from the ATCC. L1 cells [18], which are mouse fibroblasts containing human chromosome 17q, were a gift of E. V. Stubblefield. Microcell fusion hybrid clones (including 10B3, 6SP15, 9SP6, 9SP13, and 9SP19) containing single retroviral inserts were made as follows. L1 cells were infected with pZipNeo [16]

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152

GATA 8(5): 151-158, 1991

and selected for 2 weeks on media containing 800 p.g/ml G418. The resulting polyclonal mixture of infected cells was used as a source of microcells that were fused with LMTK cells using standard techniques [19]. Cloned fusion products containing both the proviral neo gene and the human TK gene were then obtained by coselection with 800 i~g/ml G418 and 1X HAT [18] for 3 weeks. Polyclonal pZipNeo-infected human lymphoblasts (IP404 cells) were prepared by pZipNeo infection of GM07048 cells (purchased from Coriell Institute) followed by selection on media containing 1 mg/ml G418.

C. S. H u c k a b y et al.

AGCTAGCTTGCCAAACCTAC-3') and C4 (5'GTAACGCCATTTTGCAAGGC-3' ; gel-purified synthetic oligonucleotides were purchased from Genosys Biotechnologies). The 100-~xl reactions contained 200 ng of template (approximate), 0.1 I~M each primer, 4 U Taq DNA polymerase (BIOS Corporation), 0.2 mM each dNTP, 1.5 mM MgCI 2, 50 mM KC1, 10 mM Tris pH 8.4, 0.01% gelatin, and 0.1% Triton X-100. Amplifications were carried out in a BIOSycler instrument (BIOS Corporation) programmed for 35 cycles of 92°C denaturation (20 s), 60°C annealing (20 s), and 72°C polymerization (35 s). In some cases, desired products were reamplified under similar conditions following isolation from agarose or polyacrylamide gels.

Genomic DNA Preparation Low-melt agarose-immobolized DNA inserts originally intended for pulse-field gel analysis were prepared as described [20]; each I00-~1 insert contained the DNA from ~ 1 0 6 cells. Single inserts were divided into small pieces, added to 4.5 ml STE (100 mM NaC1, 10 mM Tris pH 8, 1 mM EDTA), and melted several hours at 65°C. Purified DNA was then recovered by two phenol extractions, one chloroform extraction, and ethanol precipitation. Following resuspension in small volumes of TE (10 mM Tris pH 8, 1 mM EDTA), DNA was quantitated by fluorometry in a Hoefer TKO 100 instrument according to the manufacturer's instructions.

Template Preparation and PCR Five micrograms of each DNA sample were digested to completion with MboI (New England Biolabs), extracted with phenol and chloroform (once each) and ethanol precipitated [21]. Onetenth of each MboI-digested DNA preparation (-500 ng) was incubated with 400 units (U) T4 DNA ligase (New England Biolabs) in 100 I~1 500 mM Tris pH 7.6, I00 mM MgCI2, 100 mM DTT, 200 ~g/ml BSA, and 10 mM ATP. A parallel mock-ligation control reaction was run for each DNA sample under identical conditions except no DNA ligase was added. Following overnight incubation at 14°C, all reactions were heated to 65°C for 40 min; then 100 ILl 200 mM NaCI was added and the preparations were extracted with phenol and chloroform and precipitated with ethanol. E a c h ligated or m o c k - l i g a t e d D N A was s u b j e c t e d to P C R u s i n g p r i m e r s C3 (5'

Cloning and Sequencing Amplified DNA Ampified DNA was resolved by agarose gel electrophoresis and recovered using either the DEAE paper method [21] or the Gene Clean (BIO 101) method according to the manufacturer's instructions. Purified products were phosphorylated using T4 polynucleotide kinase (New England Biolabs) with routine reaction conditions [21]. The DNAs were then made blunt-ended using the single-strand-specific exonuclease activity of T4 DNA polymerase (New England Biolabs) according to reaction conditions previousty described [22]. DNA purification by phenol and chloroform extraction and ethanol precipitation was performed after each of these reactions. Bluntended, phosphorylated amplification products were then ligated into dephosphorylated, SmaIlinearized pBluescript SK ( + ) (Stratagene). Transformation of competent Escherichia coli DH5~ cells (Bethesda Research Laboratories) to ampicillin resistance was accomplished using the manufacturer's protocol. Positive clones were identified by restriction analysis of miniprep DNA [21] prepared from overnight cultures of random white colonies. Plasmid minipreps were treated with DNasefree ribonuclease (Boehringer-Mannheim) and extensively purified using organic extractions and multiple ethanol precipitations from 2 M ammonium acetate. Chain-termination sequencing [23] from pBluescript T7 and reverse primer sites was accomplished with Sequenase Version 2.0 (United States Biochemical) using the manufacturer's kit and protocol for sequencing double-stranded templates.

© 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

153 GATA 8(5): 151-158, 1991

Sequences Flanking Inserted Retroviruses

LTR )

Blot Hybridization Analysis

Q

Ten micrograms of appropriate genomic DNAs were digested completely with HindIII (Boehringer-Mannheim) and resolved on 0.8% agarose gels using s t a n d a r d t e c h n i q u e s [21]. Gelimmobolized DNA was depurinated for I0 min in 0.25 M HCI and treated in denaturing solution (0.5 M NaOH and 1.5 M NaC1) for 60 min. DNA was capillary transferred to Hybond-N (Amersham) filters using denaturing solution. Filters were rinsed in l0 x SSC(1 x SSC = 0,15 NaCI, 0.15 M sodium citrate, pH 7.0), air dried, baked at 65°C for 60 min, and irradiated at 0.3 J/cm 2 in a BIOSlink-312 instrument (BIOS Corporation). High-specific-activity 32P-labeled probes were prepared by random priming [24] of plasmid DNA preparations using a commercial kit (BoehringerMannheim). Prehybridization, hybridization, and normal stringency washing of filters were essentially as described [25].

Results and Discussion

Amplification Strategy The inverse PCR (IPCR) strategy used here for amplifying genomic sequences flanking integrated retroviruses (Figure 1) involves MboI cleavage, ligation under dilute conditions to form circular templates, and amplification using divergently oriented primers targeted to LTR U3 sequences. MboI was selected for genomic DNA digestion because (a) its 4-bp recognition sequence occurs at high frequency, yielding relatively short templates that are amenable to amplification; and (b) MboI ends are cohesive, resulting in efficient ligation. The priming sites and neighboring MboI sites occur in each provirai LTR; thus the strategy predicts that two PCR products will be generated from each ligated template preparation. The product of primary interest results from priming in the 5' LTR (Figure I); this product contains 90 bp of U3 together with genomic flanking sequences. The size of this product is variable among different clones of infected cells, due to variability in the location of the genomic MboI site nearest the 5' end of the provirus. The other PCR product is generated by priming in the 3' LTR and consists entirely of known proviral sequences. This second product is invariant in size (323 bp) and is

~

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Figure 1. Strategy for amplifying genomic sequences flanking proviruses. (a) Integrated pZipNeo provirus (box) with flanking genomic DNA (thick lines). (b) Expanded map showing relevant region of left LTR and flanking DNA. Mbol sites (M) occur in the flanking DNA and 93 bp from the 5' end of the LTR within the U3 region of the LTR. The thick line representing flanking DNA is broken to denote length variability between different clones of infected cells. DNA was digested with Mbol and ligated under conditions favoring circularization (dashed line between MboI sites). C3 and C4 are 20-mer PCR primers that match U3 sequences of the LTR at positions 16-35 and 39-58, respectively, from the 5' end of the integrated provirus; their 5' to 3' orientations (directions of arrows) are opposite and their 5' ends are separated by 3 bp. (c) Amplification product from template circularized at Mbol site. Guidelines drawn to b indicate the origin of the sequences in the product. The product contains a genomic flanking sequence whose length is determined by the position of the nearest genomic Mbol site, plus a total of 90 bp of LTR sequence distributed at the ends of the product. This procedure also results in a 323-bp product generated by priming in the 3' LTR and amplification of MoMuLV sequences 3' to an Mbol site 233 bp to the left of the 3' LTR (see the text); the black box beneath the right LTR in a indicates the origin of the amplified MoMuLV sequence.

useful as an internal positive control for successful template preparation and amplification. A more complex and related IPCR strategy recently developed by von Melchner et al. [15] used restriction digestion to cleave the proviral template containing 3' LTR priming sites. Such a strategy results in interruption of the desired template (containing the 5' LTR) if the unknown genomic sequences possess the appropriate restriction site. The strategy used in the present work avoids this possibility, but does have the potential for yielding comigrating products from 5' and 3' templates if the genomic MboI site is positioned appropriately. Potential additional problems common to all IPCR approaches are (a) the possibility of generating a template that is too long to amplify efficiently and (b) truncation of the genomic DNA too close to the provirus to yield a product of sufficient length.

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154 C. S. Huckaby et al.

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Generation and Sequencing of IPCR Products We s u b j e c t e d D N A from several p Z i p N e o containing cell lines to IPCR. All of the cell lines were cloned mouse/human microcell fusion hybrids except for IP404, which was a polyclonal mixture of pZipNeo-infected human lymphoblasts. The latter was included to provide a human control for subsequent hybridization experiments. DNA was digested with MboI and either ligated or mock-ligated under dilute conditions. As shown in Figure 2, PCR using the ligated template preparations yielded one or more amplification products detectable in an ethidium-bromidestained agarose gel. The generation of amplification products depended on prior ligation of the MboI-digested DNA. The predicted 323-bp allproviral product was obtained as one of the amplification products in all cell lines tested. Additionally, a second prominent ligation-specific product of variable size (containing flanking DNA) was observed for most of the cell lines. The polyclonal IP404 template yielded a faint array of such products, which was more easily observed by Figure 2. Products of IPCR reactions. Ten microliters of each are displayed in an ethdium-bromide-stained 4% Nusieve (FMC Corporation) agarose gel. MboI-digested DNA from the indicated cell lines were either mock-ligated ( - ) or ligated ( + ) prior to amplification. The provirus-derived 323-bp product common to all tigated templates is indicated. Other products contain genomic DNA and are variable in size. Markers (M) and 1 Ixg of a 123-bp ladder (BRL, Inc.). ,~. o

Ligation:

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polyacrylamide gel electrophoresis (data not shown); a randomly selected 250-bp product was isolated and reamplified for further analysis. The observed pattern of ligation-dependent constantand variable-size amplification products is consistent with predictions based on the IPCR strategy. To determine whether the amplification products were derived from the correct provial and genomic sites, the major products of interest were cloned into pBluescript and sequenced. A representative 323-bp product was compared with known MoMuLV sequences [17] and it proved to contain only proviral sequences in an arrangement predicted by the IPCR strategy (data not shown). Sequencing also confirmed that most of the variable-size products originated from the predicted sites (Figure 3). The proviral sequences shown in Figure 3a flank each of the different genomic sequences presented in Figure 3b. The proviral sequences, which terminate in the 20-bp PCR primer sites, match the appropriate published MoMuLV sequences [17] except for the adenine at position 48 of the 5' proviral sequence (originally reported as a guanosine). In contrast, a 285-bp amplification product representing 9SP19 contained a genomic DNA sequence flanked only by primer sequences (data not shown); this product is therefore a PCR artifact that originated by erroneous priming and was not included in further studies. Thus, four of the five. cloned amplification products derived from the hybrid cells were bone fide IPCR products. Sequencing revealed that an IPCR product representing IP404 (the polyclonal infected human lymphocytes) contained three MboI sites (Figure 3b). The 5' MboI site is consistent with the amplification strategy and was the only MboI site Ii

615

492 369 323 b p 246

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Figure 3. Sequences of IPCR products containing genomic prov'7"6"Vi~'~-flankingDNA. All sequences are presented in the orientation shown in Figure lc. Cumulative sequence lengths are shown at right in parentheses; those in boldface indicate the total length of the corresponding sequence. (a) Common proviral sequences that flank each genomic sequence. The 5' proviral sequence begins with the sequence of primer C4 (overlined) and the 3' proviral sequence terminates in the reverse complement of the sequence of primer C3 (underlined). All proviral sequences match previously reported MoMuLV sequences [17] except for the A marked by the arrow, which was previously reported as a G. (b) Genomic sequences that intervene between the proviral sequences shown in a. Sequences are identified by the designation of the plasmids containing the cloned IPCR products; the cell lines from which the IPCR products originated are given in parentheses. All MboI sites are boxed. Reliable sequence data were not obtained for approximately the middle third of the 9SP13 product, and are not shown.

© 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

155

Sequences Flanking Inserted Retroviruses

a

Common

5 '

(mrovirall

Common

3 '

TTTGCAAGGC

Common

sequence

TGAAAGACCC

b

CACCTGTAGG

Clone-snecific

1-4

seauences:

sequence

GTAACGCCAT

G A T A 8(5): 151-158, 1991

: ATGGAAAAAT

ACATAACTGA

GAATAGAAAA

: TTTGGCAAGC

TAGCT

(35)

sequences:

(IP404)

~GCTGT

TCTTTGGTAC

CAGTAATCCA

CATGTCAGCC

CCATTTTTCT

T G T A ~

CCAGCTGTTG

TTGTCAGCTT

CTACGATGT T

C TACAG~----~CACCAC

TACTCCATCC

AGCTTGGGTG

ACAGAAGAAG

ACCCTGTCCC

CCCTC

2-23

(55)

GTTCA

CCTAAGTCAC TG

(60 ) (120 ) ( i 65 )

(9SP13)

~--~GTTTG

TGCTCTCTGA

GCTCTTCTAC

TAATGCAATT

ATAAAGCACA

CATATTTGTT

(60 )

TGGCCTTGTA

TGTATAAATT

ACCATAATAA

TTCTGATACA

TATCACTGTG

GTATGTGTAT

(120 )

TAACACTTTG

ACATTTCTAT

TGTT

(approx.

ATGTTTTCAA

(276)

ATTTCTTAGG

TAAATATAAA

AGGGAGATGT

ACTACTAACA

GAAT TATTTT

(336 )

CAAATTTACT

AGGTCACTTC

ATGTTGCTCT

GAGAGGTATG

4-21

122 bp) ATATTTGCTG

(37 6 )

(9SP6)

~--~CGGCG

AGGTGACGTT

CCAGGTCTAC

TGAGACTCAT

TACCGATGGC

AGCACACAGT

(60)

CCGATAGCTT

GTCCCGGCCC

TCAGCATCCC

TCACTTCTGC

TCCTGCAGGC

GTCGTAGGTG

(120)

CACGGGCTGG

CTTACTTG

5-17

(138)

(6SP15)

~CAAAC

TGCTCTCCAC

ATTAGATAAT

GTTTATGTTG

GCTAAATATT

AGCACAAGTA

(60)

AGTCATTCAC

TTGGCACGTT

CACGGACAGG

GAAGCAGATG

GCTGGGCAGC

TATGCCATTG

(120)

AC

8-6

(122)

(10B3)

~--~GGAGT

CCAGCTTCTC

CAGGGCTACT

AGGAAGAAAC

TCAGTGTATA

GGGGCTTCTG

GATGTCATCT

GTTGTTCAGA

ATCACAGGCA

TGTTC

AGAGAGAGTT

(60) (105)

© 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

156 GATA 8(5): 151-158, 1991

C. S. Huckaby et al.

found in the other IPCR products. The two other MboI sites contained in the sequence of the IP404 IPCR product may have resulted from either (a) incomplete MboI digestion prior to template ligation or (b) coligation of unrelated small genomic MboI fragments during template preparation. Hybridization data (see next section) suggest that the latter is the case. In cases where the MboI site nearest the 5' proviral LTR is too close to allow efficient circularization [26], amplification of chimeric templates may be favored. Nevertheless, the genomic DNA content of the IP404 product must be entirely human; therefore, this product is an adequate human proviral insertion control for hybridization studies.

4a). The IPCR probes representing four microcell fusion hybrids identified positive HindlII fragments in only L! and L M T K - DNAs and not in HS68 DNA (Figure 4a, strips II-V). This indicates that these IPCR products contain only mouse genomic DNA and therefore pZipNeo is integrated into murine chromosomes in the 9SP13, 9SP6, 6SP15, and 10B3 cell lines. Each murine IPCR probe apparently contains single-copy sequences, except for the probe derived from 9SP6, which contains mouse-specific repetitive DNA. A computer search of current GenBank rodent sequences did not identify any significant matches with the sequence of the 9SP6 IPCR product, indicating that this product contains a novel murine repetitive element. As expected, the IP404 IPCR product hybridized only to human DNA (Figure 4a, strip I). However, three faint, yet distinct, bands were de-

H u m a n versus Murine Chromosomal Localization o f Proviral Inserts Southern blot hybridization analysis using cloned IPCR probes was performed to determine whether proviruses were inserted on human or murine chromosomes. HindlII-digested genomic DNA from HS68 (human fibroblast), L1 (hybrid mouse fibroblast containing human chromosome 17q), and L M T K - (mouse fibroblast) cells was electrophoresed and blotted. The blot was cut into strips that contained one lane of each of the three genomic DNAs, and each strip was hybridized to a different 32p-labeled cloned IPCR product (Figure

Figure 4. Southern blot analysis to determine human versus murine origin of IPCR products. (a) Hybridization of s2p_ labeled cloned IPCR products to HindIII-digested DNAs. HS68 is total human DNA, LMTK- is total murine DNA, and LI is total murine DNA plus human chromosome 17q. Markers (M) are HindIII-digested h DNA. The blot was divided into strips I-V, which were separately hybridized to the indicated probe plus s2P-labeled k DNA, and then reassembled for autoradiography. (b) Hybridization of s2P-labeled pBlueNeo to HindlIl-digested DNAs from infected cell lines. In addition to the prominent bands, faint bands (arrows) were detected in all cases except 10B3. See Figure 5 for a description of pBlueNeo and an explanation of these bands.

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DNA: ~ ~ ~ ~

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Origin of IPCR probe:

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© 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

Probe: pBlueNeo

157 GATA 8(5): 151-158, 1991

Sequences Flanking Inserted Retroviruses

tected. Since no HindlI1 sites were found in the sequence of the IP404 IPCR product, this hybridization pattern seems inconsistent with contiguous sequence representation in the probe. One possible explanation is that the probe sequence identifies a polymorphic locus in HS68 DNA. However, the two unexpected MboI sites in the probe sequence (Figure 3b) suggest that derivation of the probe from a chimeric template is a more likely explanation. This conclusion is strengthened by our observation that the different positive HindllI bands appear to localize to different human chromosomes, as judged by hybridization to somatic cell hybrid panels (BIOS Chromosome Blots, BIOS Corporation; data not shown). To confirm that the HindlII fragments identified by murine IPCR probes (Figure 4a) contain proviral insertion points, HindlII-digested DNA from the corresponding infected cell lines was hybridized to 32p-labeled pBlueNeo (Figure 4b). pBlueNeo contains proviral sequences spanning - 2 0 0 bp 5' and 2000 bp 3' to the single HindlII site in the provirus (Figure 5), Thus, two bands were detected in the DNA of most of the infected cells (Figure 4b). These bands have different intensities due to probe asymmetry; the faint band likely represents 5' sequences and the more prominent band likely represents 3' sequences (fragments B and C, respectively, Figure 5). If the IPCR probes hybridized to the correct HindlII fragments in DNA from uninfected cells (Figure 4a), then the size of those fragments should be 4.8 kb (the size of the provirus) less than the sum of the sizes of the two pBlueNeo-positive HindllI fragments in DNA from the corresponding infected cell line (Figure 4b). As described in the legend to Figure 5, the measured sizes of the relevant bands for 9SP13, 6SP15, and 10B3 meet these criteria. In the case of 9SP6, verification was not possible due to hybridization to repetitive sequences. Thus, we have confirmed the correct origin of the nonrepetitive IPCR products of the microcell hybrid cell lines. The initial objective in constructing our microcell hybrid cell lines was to obtain clones in which human chromosome 17q was marked with the pZipNeo provirus. We assumed that a proportion of the infected L1 microcell donors had proviral inserts on the human chromosome. Selection of microcell fusion products with both G418 (for the provirus) and HAT (for the human TK gene on chromosome 17q) was aimed at isolating such chromosomes. However, the cell lines studied

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Figure 5. Diagrammatic representation of Southern blot anal~ w n in Figure 4. Map at top shows an IPCR probe (black box labeled I) identifying a HindlII (H) fragment of length A in the DNA of an uninfected cell line; this corresponds to the experimental data shown in Figure 4a. The bottom map (which is aligned to the top map via broken lines) shows that the pBlueNeo probe (black box labeled N) hybridizes to two HindlII fragments of lengths B and C in the DNA of the corresponding infected cell lines; these data are shown in Figure 4b. The inserted provirus is 4.8 kb long and is represented by the box, over which the short arrows represent LTRs. The shaded portion of the provirus is the neomycin resistance (neo) gene, which contains the single HindlII (H) site in the provirus. The insert of pBlue Neo (N) contains all neo gene sequences plus ~800 bp of additional Zip-Neo sequences 3' to the neo gene (that is, the 2.2-kb XhoI fragment ofpZipNeo [16]). Fragment B is much fainter than C because of the asymmetry of this probe around the proviral HindllI site. To confirm that the IPCR products originated from proviral 5' flanking sequences, we noted that A + 4.8 = B + C. Measured fragment sizes in kilobases were as follows. 9SP13: A = 7.3, B = 4.9, and C = 7.4. 6SPI5:A = 2.75. B = 1.8, a n d C = 5.7. N o B b a n d w a s apparent for 10B3; however, we found that A = 12.5 and C = 8.7, therefore deduced that B = 8.6, and concluded that it was most likely unresolved from the C band. No single-copy A band was found for 9SP6 due to repetitive DNA hybridization, so 9SP6 was excluded from this analysis.

here did not contain any hybrids with provirusmarked human DNA. Recent work has shown that our hybrids generally contain small human subchromosomal regions in the immediate (within 10 Mb) vicinity of the TK gene [27]. It is thus likely that our microcell generation/fusion protocol favored the transfer of small subchromosomal pieces [e.g., 4] and that this minimized the probability of finding TK-proviral linkage in the fusion products. Under double selection, products were obtained that originated from introduction of multiple, unlinked chromosomal regions. For the purposes of this report, however, it is evident that IPCR probes derived by our strategy can successfully be employed as probes for pZipNeo chromosomal localization.

Conclusion An efficient IPCR-based strategy has been developed for isolating genomic DNA sequences adja-

© 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

158 C. S. Huckaby et al.

GATA 8(5): 151-158, 1991

cent to proviral inserts. Hybridization studies have shown that probes derived by this strategy can identify single-copy loci useful in determining the chromosomal location of proviral insertion. The proviral sequences contained in these probes do not cross-hybridize with any endogenous homologs under normal stringency conditions. The hybridization data are useful in understanding the performance of such cytogenetic methods as microcell-mediated chromosome transfer. The authors thank Gualberto Ruano (Yale University) and Dr. Marcia Lewis (BIOS Corporation) for helpful discussions, Dr. Suzanne Bradshaw (Yale University) for the GenBank search, Ann Smardon (SUNY Health Sciences Center at Syracuse) for provision of pBlueNeo, Thomas Brown for preparation of Figures 1 and 5, and Sandi Malek for manuscript preparation.

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