MOLECULAR AND CELLULAR BIOLOGY, May 1988, p. 1863-1867 Vol. 8, No. 50270-7306/88/051863-05$02.00/0Copyright © 1988, American Society for Microbiology

A Fragile Site in the Human U2 Small Nuclear RNA Gene ClusterIs Revealed by Adenovirus Type 12 Infection

DIANE M. DURNAM,* JOAN C. MENNINGER, SUSAN H. CHANDLER, PATRICIA P. SMITH,AND JAMES K. McDOUGALL

Program in Experimental Pathology, The Fred Hutchinson Cancer Research Center, Seattle, Washington 98104

Received 9 December 1987/Accepted 22 January 1988

Using in situ hybridization, we found that the U2 small nuclear RNA gene cluster mapped very close to andwas frequently disrupted by the gaps and breaks induced specifically in the human 17q21-q22 region by highlyoncogenic adenovirus type 12 (Adl2). Restriction mapping revealed no structural alterations in the U2 genelocus as a result of Adl2 infection. Likewise, no Adl2-induced alterations in U2 RNA levels were detected. Weestimate that the maximum size of the region specifically disrupted by this virus was less than 350 to 700kilobases. A comparison of these data with similar data regarding biochemically induced fragile sites was made.

It has been known for over 20 years that viruses can causechromosomal damage both in vivo and in cultured cells.Most viruses cause random chromosomal damage, whichcan vary in severity from single gaps or breaks to completegenomic pulverization (13). Highly oncogenic human adeno-virus type 12 (Ad12), however, induces chromosomal dam-age which in many respects resembles damage inducedbiochemically at fragile sites. Like damage at fragile sites,the damage induced by Ad12 occurs in very specific chro-mosomal regions (10, 26). The primary site affected by Ad12is located in the 17q21-q22 region; secondary sites arelocated in the p36, q21, and q42-q43 regions of chromosome1. When examined by light microscopy, both fragile-site andAd12-induced damages appear as nonstaining gaps orbreaks. Examination by electron microscopy, however, re-veals that both are actually regions of uncoiled or decon-densed DNA (2, 5). Both types of damaged sites are highlysusceptible to breakage and rearrangement in cultured cells(12, 22). Finally, both Ad12-induced damage and fragile sitesmap in chromosomal regions which are frequently rear-ranged in human cancers (16, 25). This latter observation hasled to the hypothesis that regions of chromosomal fragilitypredispose the chromosome to cancer-related rearrange-ments (25).The similarity between Adl2-induced damage and damage

induced biochemically at fragile sites suggests that the twophenomena may also be mechanistically similar. Analysis ofthe mechanisms governing biochemically induced fragilesites has been inhibited by the fact that no site has beencloned, and genetic linkage mapping has progressed slowlyin most cases. Likewise, little is known about the mecha-nism or chromosomal sequences which facilitate Adl2-induced chromosomal damage. McDougall et al. (12) dem-onstrated that Adl2-induced breakage in the 17q21-q22region could occur at sites both proximal and distal to thethymidine kinase gene, suggesting that the Ad12-sensitiveregion might span a relatively broad domain. Lindgren et al.(8) postulated that small RNA genes might be the cellulartarget for Adl2-induced damage because small RNA genesmap at several of the Adl2-specific sites of damage. In thestudy presented here, we tested the latter hypothesis byconducting in situ hybridization on Adl2-infected cells witha probe specific for the U2 small nuclear RNA gene (U2

* Corresponding author.

gene) region. The U2 gene locus consists of 10 to 20 copiesof a 6-kilobase (kb) repeat arranged tandemly in a single genecluster within the 17q21-q22 region (8). Our data indicatethat the U2 genes map very close to the site of Adl2-induceddamage in that region and are frequently disrupted by thedamage. Adl2-induced effects on U2 gene structure andtranscriptional activity were also assessed.

MATERIALS AND METHODSCell culture and chromosome preparation. All cells were

grown in Dulbecco modified Eagle medium supplementedwith 10o fetal bovine serum. Colcemid (0.01 p.g/ml) wasadded to the culture medium 5 h before chromosome har-vest. Chromosomes were prepared by incubation of trypsi-nized cells in a hypotonic solution consisting of 40 mM KCI,0.5 mM EGTA [ethylene glycol-bis(,-aminoethyl ether)-N,N,N',N'-tetraacetic acid], and 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7) for20 min at 37°C, followed by fixation in methanol-acetic acid(3:1) for at least 24 h. Chromosome preparations were storedin fixative until they were analyzed.

Virus infection. Adl2 was a gift from M. Green. Virusstocks were amplified by infecting human embryo kidneycells at a multiplicity of infection of 1 to 10. After 2 days, thecells were freeze-thawed three times, the cell debris wasremoved by centrifugation, and the virus-containing super-natant was quick frozen and stored at -70°C. For chromo-some analysis, human embryo lung (HEL) cells were plated24 h before infection and washed three times with phos-phate-buffered saline before the addition of virus at a multi-plicity of infection of 1 to 10. Virus was adsorbed for 90 minbefore the readdition of culture medium. Chromosomeswere prepared 16 to 24 h postinfection.

In situ hybridization. The plasmid pU2.6/H1 was a giftfrom Gunnar Westin and contains the entire U2 structuralgene in addition to 285 and 94 base pairs of 5'- and 3'-flankingsequences, respectively (23). For in situ hybridization, theU2 probe was labeled by nick translation in the presence ofthree 3H-labeled deoxynucleotides (dATP, dCTP, and TTP)to a specific activity of 2 x 107 to 6 x 107 cpm/,lg, asdescribed by Deeb et al. (1).Chromosome preparations were dropped on acid-cleaned

slides, aged, and treated with RNase as described elsewhere(4). Chromosomes were denatured in 2x SSC (lx SSC is0.15 M NaCl plus 0.015 M sodium citrate) (pH 12.5) for 2 min

1863

1864 DURNAM ET AL.

TABLE 1. Location of grains relative to Adl2-induced 17q21-q22 damage on slides probed with a U2 gene probe

No. of cells No. of cells with 17q21-q22 damage No. of grains which were:Expt examined Nonhybridized Hybridized Proximal Distal Splita Centralb

1 230 26 10 8 1 0 12 538 38 20 14 6 0 03 391 26 32 15 13 2 24 585 6 38 25 2 6 5

a Grains present on both sides of the gap or break.b Grain(s) present within the gap or break.

at room temperature and then were immediately plungedinto ice-cold 70% ethanol for 2 min, dehydrated, and airdried. The U2 probe was diluted to 30 to 100 ng/ml in 50%formamide-10% dextran sulfate-2x SSC-10x Denhardt so-lution-sonicated herring sperm DNA (0.1 mg/ml). Afterdilution, the U2 probe was denatured at 70°C for 10 min andplaced on ice for 5 min before being placed on slides (50pLA/cm2). Hybridizations were carried out for 16 h at 42°C.Slides were washed in four changes of 2 x SSC at roomtemperature for a total of 16 min and then were washed oncein lx SSC-50% formamide at 45°C for 12 min, twice in 2xSSC at 45°C, twice at room temperature in 2x SSC, andfinally once in 2x SSC at 4°C with stirring for 4 h. Slideswere subsequently dehydrated, air dried, coated with NTB-2emulsion (Eastman Kodak Co., Rochester, N.Y.), and de-veloped after 3 to 5 weeks of exposure at 4°C. Chromosomeswere stained in 4% Giemsa (Biomedical Specialties, SantaMonica, Calif.) in Gurr buffer (Biomedical Specialties) for2.5 min at room temperature.Northern (RNA) blot analysis. Total RNA from Adl2-,

Ad2-, and mock-infected HEL cells was prepared 16 to 24 hpostinfection by the guanidinium-cesium chloride method asdescribed elsewhere (9). RNAs were electrophoresedthrough 2.2 M formaldehyde gels of 1.2% agarose (9), blottedonto nitrocellulose in 20x SSC, hybridized, and washed asdescribed elsewhere (15). The probes, i.e., pU2.6/H1 and a32-microglobulin cDNA clone (20), were nick translated in

the presence of [32P]dCTP and [32P]TTP to specific activitiesof 0.5 x 109 to 1.0 x 109 cpm/,ug (15).

Southern blot analysis. DNA from Adl2- and mock-in-fected HEL cells was prepared 16 to 24 h postinfection asdescribed elsewhere (2). DNAs were digested with enzymes(see Fig. 2) according to the specifications of the manufac-turer (Bethesda Research Laboratories, Inc., Gaithersburg,Md.) and electrophoresed through 0.7% agarose gels. Blot-ting, hybridization, and washing were conducted as de-scribed elsewhere (15). The plasmid, pU2.6/H1, was labeledby nick translation (-5 x 108 cpm/,lg) as described else-where (15).

RESULTS

In situ hybridization. To determine the relative map posi-tions of the U2 gene cluster and the site of Adl2-inducedchromosome damage in the 17q21-q22 region, we conductedin situ hybridization on Adl2-infected cells with a 3H-labeledprobe, pU2.6/H1, which is specific for the U2 gene region(23). HEL cells were infected with Adl2 at a multiplicity ofinfection of -10 for 16 to 24 h before chromosome harvest.Only preparations with >30% of the metaphases showing17q21-q22 damage and little damage elsewhere in the gen-ome were selected for further study. The results of fourhybridization experiments are summarized in Table 1. Of

1,744 metaphases examined, 196 (11%) showed Adl2-in-duced damage in the 17q21-q22 region. This level of damagewas lower than we had observed in the preparations beforehybridization; we believe that this difference may haveoccurred as a result of the masking of narrow gaps by theemulsion or silver grains. Of the 196 spreads with observed17q21-q22 damage, 100 (51%) showed U2-specific hybridiza-tion in the 17q21-q22 region. Of the 381 silver grains, 237(62%) were located in the 17q21-q22 region of these cells.Surprisingly, the U2 gene cluster did not routinely map in thesame location relative to the gaps and breaks induced byAdl2. Of the cells, 62% showed grain(s) proximal to the siteof Adl2 damage; 22% showed grain(s) distal to the site ofdamage (Ho proximal = distal; x2 = 19.0 on 1 df; P < 0.001)(Table 1). The remaining 16% were divided evenly betweencells with grains mapping within the gapped or broken regionand cells with grains on both sides of the damaged region.Examples of U2-specific hybridization are shown in Fig. 1.Some variation in the number of proximal and distal grainswas noted among the four experiments (Table 1). Thesedifferences may reflect genetic polymorphisms, becauseprimary cells from different individuals were used in eachexperiment. Alternatively, slight variations in probe concen-tration, hybridization conditions, or exposure times mayhave contributed to this variability.Concerned about the resolution and scatter of silver grains

in our in situ assay, we paid particular attention to how closethe grains mapped to the damage on 17q. In the majority ofcells, the proximal and distal grains were located precisely atthe edge of the gap or break (Fig. 1). Little scatter onto otherregions of the chromosome was observed, and scatter to theside of the chromosome seldom occurred. Both proximaland distal hybridization was observed in cells in which thewidth of the gaps and breaks exceeded the maximum dis-tance we observed for scatter (Fig. 1). We conclude fromthese data that the U2 gene locus maps very close to the siteof Adl2-induced chromosomal damage and is disrupted bythis damage in at least the 8% of the cells showing splithybridization (Table 1).

Analysis of U2 RNA levels. Because of the proximity of theU2 gene cluster to t-he site of Adl2-induced damage, wedetermined whether U2 gene transcription was specificallyaffected by Adl2. These studies were conducted by compar-ing the amounts of U2 RNA in mock-, Ad2-, and Adl2-infected HEL cells. Unlike Adl2, Ad2 causes random chro-mosomal damage and serves as a control for any nonspecificeffects resulting from viral infection. RNA was isolated 16 to24 h postinfection. Analysis of chromosomes isolated fromparallel preparations showed that at least 30% of the Adl2-infected cells had 17q21-q22 damage; mock- and Ad2-in-fected cells showed no preferential damage in the region.Northern blot analysis of these RNAs showed that there islittle or no difference in the amount of U2 RNA in the

MOL. CELL. BIOL.

U2 GENES MAP AT Adl2-INDUCED 17q21-q22 DAMAGE SITE

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FIG. 1. In situ hybridization of chromosomes from Adl2-infected cells with a U2 gene probe. Examples of proximal (a and d), distal (band e), and split (c and f) hybridizations are shown. Hybridized chromosomes with Adl2-induced damage are indicated by arrowheads inpanels a, b, and c.

samples isolated from Adl2- and Ad2-infected cells (Fig.2A). Reprobing of the same blot with a probe for theP2-microglobulin gene, a gene not present in an Adl2-sensitive region, shows that equal amounts of RNA wereloaded in all three lanes (Fig. 2B). We routinely noted aslight decrease in U2 RNA levels in the virally infectedsamples relative to those in the mock-infected sample. Thisdecrease may reflect the fact that the relative amount ofcellular RNA in these lanes is decreased by the presence ofviral RNA. Alternatively, it is well known that adenovirusescan affect the transcription rates of cellular genes (6), andthus it is possible that Ad2 and Adl2 both decrease thetranscription of U2 genes. The fact that our data showed noAdl2-specific effects on the levels of U2 RNA suggests,however, that the disruption of the region by Adl2 has littleor no effect on the transcriptional activity of U2 genes.

Analysis of U2 gene structure. Structural alterations withinthe U2 gene cluster resulting from Adl2 infection were alsoassessed. Restriction mapping of DNA isolated from Adl2-and mock-infected cells showed no alteration in either frag-ment size or the intensity of U2-specific bands in Adl2-infected cells relative to those of the mock-infected controlcells (Fig. 3). Long exposures of blots (up to 2 weeks) wereconducted to allow detection of a change in a single U2 gene.These data provide no evidence of a major structural rear-rangement occurring within the U2 gene cluster as a result ofAdl2 infection. In addition, it is unlikely that insertion of

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__V2FIG. 2. Northern blot analysis of U2 RNA from Adl2-, Ad2-,

and mock-infected cells. (A) Hybridization with the U2 gene-specific probe, pU2.6/H1. Lambda DNA digested with HindIll isshown in the left lane. (B) The same blot reprobed with a 02-microglobulin (B2M) cDNA clone (20) to ensure that an equalamount of RNA was loaded in all three lanes. j2-Microglobulin waschosen as a control because it does not map in an Adl2-sensitiveregion.

b

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1865VOL. 8, 1988

I

1866 DURNAM ET AL.

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FIG. 3. Southern blot analysand mock-infected (C) HEL cellthe enzymes indicated. The molindicated.

viral DNA occurs at any poirsitu hybridization with Adl2as cloning of U2 genes fronunpublished observation) haintegration at the site of 17q

Finally, we assessed whe'increased sensitivity to nuclcdensation of the region bynuclei were isolated from Acells and subjected to digestwas subsequently purified,zyme, and analyzed by Scprovide no evidence of increity as a result of Adl2 intherefore conclude that the sAdl2 in the 17q21-q22 regionchanges leading to the incrserved by others (19).

DISC

We have shown that Adl.in the 17q21-q22 region canand frequently within the Ualone, however, do not alloAdl2-sensitive region lies diror whether the region disrupof the 17q21-q22 region whicSupport for the latter hypot]tions. First, detailed restriccluster shows that the 6-kb r(

24). We predict, therefore, tifrequency within any of theU2 gene repeat, equal numbeand distally to the site of daiin situ data, however, does n0.001). Second, the observati

Adl2-induced breakage and rearrangement of 17q sequencesin mouse-human hybrid cells can occur on either side of the

.s. '% * \thymidine kinase gene suggests that, like the U2 genes, the° X@ .St ct thymidine kinase gene also lies within the 17q region dis-

w~> rupted by Adl2. These data are not consistent with thehypothesis that the Adl2-sensitive region is limited to the U2

C AC A C A C A gene region and support the idea that the Adl2-sensitive23 kb region spans a broader domain within the 17q21-q22 region.

- 23 k b Assuming that the 10 to 20 tandem copies of the U2 geneare located in a single cluster (21, 24), the entire U2 gene

- 9.4 locus would include 60 to 120 kb of DNA. If we assume thatgaps and breaks can occur with equal frequency throughout

__w- 6.6 the Adl2-sensitive region, then our data showing split hy-bridization (i.e., physical disruption of the U2 gene cluster)

-4A in 8% of the cells allow us to estimate that the maximal sizeof the region sensitive to Adl2 is 750 to 1,500 kb. In fact, webelieve that this is an overestimate, because only 46% of theanalyzed spreads showed multiple grains and were thus

-2.3 capable of showing disruption of the U2 gene region. It is-2.0 likely, therefore, that the maximum region sensitive to

disruption by Adl2 is less than 350 to 700 kb.On the basis of our data, we suggest that the data

regarding the mapping of the human metallothionein, hapto-globin, and adenine phosphoribosyltransferase genes rela-

is of U2 genes in Adl2-infected (A) tive to the fragile sites at 16q (3, 17, 18) should be reevalu-s. DNA samples were digested with ated. As in our experiments, these in situ hybridizationlecular weights of size markers are analyses showed grains mapping both proximally and dist-

ally to the region disrupted at the fragile sites. However,those authors discounted these observations because statis-

nt within the U2 gene cluster. In tical analysis did not indicate an equal distribution of prox-'-specific probes (11, 26) as well imal and distal grains. We believe, however, that their datani Adl2-infected cells (D.M.D., may indicate that the region disrupted at biochemicallyne also failed to indicate viral induced fragile sites also spans a broad domain.21-q22 damage. The possibility of a relationship of fragile sites and cancer-ther the U2 gene region shows related chromosomal abnormalities is supported by both thezase attack as a result of decon- correlation of their map positions (25) and the fact thatAdl2. For these experiments, normal, unaffected cells of some cancer patients have fragiled12-, Ad2-, and mock-infected sites which map at the sites of karyotypic abnormalitiesion by DNase I (19). The DNA present in their malignant cells (7). Environmental or geneticdigested with a restriction en- factors which might account for the fact that only a fractionuthern blotting. These results of fragile-site carriers develop such malignancies remain toased U2 gene nuclease sensitiv- be determined. If, as our data suggest, chromosomal break-fection (data not shown). We age and rearrangement can occur over a broad domain at,tructural alterations induced by fragile-site loci, then it is possible that only a fraction of thei occur at a level independent of rearrangements lead to cancer-related abnormalities.reased nuclease sensitivity ob- Our Northern blot analyses showed that Adl2 does not

specifically alter U2 RNA levels. Preliminary experimentsdesigned to give more direct measurements of U2 gene

'USSION transcription have also failed to show Adl2-specific effectson U2 gene expression (D.M.D., unpublished data). These

2-induced chromosome damage data suggest that decondensation alone may be insufficient tooccur proximally and distally to cause aberrant gene expression and may explain why, with2 gene cluster. Our in situ data the exception of the fragile X chromosome, which is asso-lw us to determine whether the ciated with X-linked mental retardation, most fragile sitesrectly within the U2 gene cluster have no phenotypic penetration (14).)ted by Adl2 spans a broad part It is also interesting to recall the observation of Lindgrenh merely includes the U2 genes. et al. (8) that the Adl2 modification sites on chromosome 1hesis comes from two observa- map in the region of Ul RNA genes and class 1 Ul RNAwtion mapping of the U2 gene pseudo genes. The fact that the Ul pseudo genes areepeats are highly conserved (21, transcriptionally inactive may suggest that the structure ofhat if damage occurs with equal small RNA genes rather than their transcriptional activity10 to 20 identical copies of the predispose these loci to viral damage. Experiments are in~rs of grains will map proximally progress to more precisely define the chromosomal se-mage. Statistical analysis of our quences required for Ad12-induced damage. It is our hopelot support this hypothesis (P < that by analysis and comparison of the chromosomal se-ion by McDougall et al. (12) that quences required for both Adl2- and biochemically induced

MOL CELL B IOL .

14

U2 GENES MAP AT Adl2-INDUCED 17q21-q22 DAMAGE SITE

damage, insight will be gained about both the mechanism bywhich chromosomes break and rearrange and the relation-ship between such rearrangements and cancer.

ACKNOWLEDGMENTS

We thank Christine Disteche for her help and advice during thepreliminary stages of this work and Steven Self for his help withstatistical analysis of the data. The U2 gene probe, pU2.6/H1, was agenerous gift from Gunnar Westin; the 32-microglobulin cDNAclone was a gift from K. Itakura.

This work was supported by grant CD-301 from the AmericanCancer Society.

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