Proc. Nail. Acad. Sci. USAVol. 89, pp. 4840-4844, June 1992Medical Sciences

Expression pattern of the RARcr-PML fusion gene in acutepromyelocytic leukemia

(reciprocal translocation/leukemogenesis/transcription factors)

MYRIAM ALCALAY*, DANIELA ZANGRILLI*t, MARTA FAGIOLI*, PIER PAOLO PANDOLFI*,AMEDEA MENCARELLI*, FRANCESCO Lo Cocof, ANDREA BIONDI§, FAUSTO GRIGNANI*,AND PIER GIUSEPPE PELICCI*¶*Istituto Clinica Medica I, University of Perugia, Policlinico Monteluce, 06100 Perugia, Italy; tDipartimento di Medicina Interna, Cattedra di Ematologia, IIUniversity of Rome, 00100 Rome, Italy; tDipartimento di Biopatologia, Divisione di Ematologia, I University of Rome, 00161 Rome, Italy; and §ClinicaPediatrica, University of Milan, Ospedale S. Gerardo, 20052 Monza, Italy

Communicated by Renato Dulbecco, January 21, 1992

ABSTRACT Two chimeric genes, PML-RARa andRARa-PML, are formed as a consequence of the acute pro-myelocytic leukemia (APL)-specific reciprocal tranlocation ofchromosomes 15 and 17 [t(15;17)]. PML-RARa is expressed asa fusion protein. We investigated the organization and expres-sion pattern of the RARa-PML gene in a series ofAPL patientsrepresentative of the molecular heterogeneity of the t(15;17)and found (i) two types of RARa-PML mRNA junctions(RARa exon 2/PML exon 4 orRARa exon 2/PML exon 7) thatmaintain the RARa and PML longest open reading framesaligned and are the result of chromosome 15 breaking at twodifferent sites; and (us) 10 different RARe-PML fusion tran-scripts that differ for the assembly of theirPML coding exons.ARARa-PML transcript was present in most, but not all, APLpatients.

A reciprocal translocation involving chromosomes 15 and 17[t(15;17)] is a characteristic feature of acute promyelocyticleukemia (APL) in humans (1). It is usually the only karyo-typic aberration present and is detectable cytogenetically in70%o of APLs and molecularly in 100% (1-3). This constantand exclusive association suggests that the t(15;17) is deter-minant in the pathogenesis of APLs.The two chromosome breakpoints have recently been

mapped to the PML ("promyelocytes") gene on chromo-some 15 and the retinoic acid receptor a (RARa) gene(referred to here as RARa; RARA in human gene mappingnomenclature) on chromosome 17 (4-7). RARa is one of theintracellular receptors that functions to regulate gene expres-sion in response to the binding ofretinoic acid (8, 9), and thereis growing evidence that it is involved in the control ofterminal myeloid differentiation (10). PML is also a putativetranscription factor: it contains a cysteine-rich motif thatresembles the zinc finger DNA-binding domain common toseveral classes of transcriptional factors (11, 12). The phys-iological role of PML is unknown.Two fusion genes, PML-RARa and RARa-PML, are

formed as a result of the translocation. PML-RARa genesgenerate fusion mRNAs that encode chimeric PMLRARaproteins (11-13). We demonstrated that chromosome 15breakpoints cluster within three regions of the PML gene:intron 6 (breakpoint cluster region 1, or bcrl), exon 6 (bcr2),and intron 3 (bcr3) (14, 15). The heterogeneity of the chro-mosome 15 breakpoint accounts for the diverse architectureof the PMLRARa mRNAs isolated from different APLpatients, and the alternative splicings ofPML exons give riseto multiple isoforms of the PML-RARa mRNAs even withina single patient (14, 15).

Less information is available on the RARo-PML gene.Kakizuka et al. (11) reported the cloning of a RARa-PMLtranscript in which RARa exon 2 was correctly spliced toPML exon 4 and the longest PML and RARa open readingframes (ORFs) aligned. It is not clear whether a RARa-PMLmRNA is present in all APLs and whether it is differentlyorganized in cases with chromosome 15 breakpoints in bcrl,bcr2, and bcr3, as would be expected if the reciprocity of thetranslocation was maintained at the molecular level. Theanalysis of the organization of RARo-PML genes in APLpatients with chromosome 15 breakpoints in bcrl, bcr2, andbcr3, and ofRARa-PML transcripts in a large series ofAPLpatients is reported here.

MATERIALS AND METHODSIsolation and Analysis of RARct-PML A Genomic Clones.

Genomic libraries were constructed from APL patients 22,28, and 8 by inserting partially Mbo I-digested DNA into theXho I site of the A FIX II vector (Stratagene) by the Xho Ihalf-site method (filling in the first two nucleotides of bothMbo I and Xho I sites, leaving compatible overhangs).RARo-PML genomic clones were isolated with the aid of thePP, RH15, and L7ASH probes (Fig. 1) and were analyzed byrestriction enzyme mapping, and fragments containingRARa-PML junctions were subcloned in plasmid vectorpGEM-3 (Promega) and sequenced by using the Sangerdideoxynucleotide chain-termination method.

Northern Blot Analysis. Samples (10 rug) of total RNA wereanalyzed by electrophoresis on a 1% agarose gel containing2.2 M formaldehyde and transferred to a nylon membranefilter. Filters were hybridized with the PML probe C2AB.PCR Amplification of RAR&-PML mRNA Junctions. Sin-

gle-stranded cDNAs were generated from 1 ,gg of APL totalRNA by using a commercial kit (Promega RiboClone cDNASynthesis System) and amplified in a 40-cycle PCR. Theprimers used were as follows: R18, 5'-TGGACAGCAGCTC-CAGGACA-3'; R2, 5'-GCTCTGACCACTCTCCAGCA-3';SM1, 5'-TCTTCCGAGCTGCTGATCA-3'; SM2, 5'-GAGT-CCTGGCCTGAACTTCTG-3'; M48, 5'-GGACTCAGA-GACTAAATTAG-3'; and M15, 5'-ACCGCAGCCAGCTG-GCTA-3'. PCR-amplified fragments were cloned in thepCR1000 vector by using the TA cloning system (Invitrogen,San Diego).

RESULTSIsolation ofRARe-PML Genes. RARa-PML genes of APL

patients with chromosome 15 breakpoints that had been

Abbreviations: RARa, retinoic acid receptor a; APL, acute promy-elocytic leukemia; bcr, breakpoint cluster region; ORF, open readingframe.$To whom reprint requests should be addressed.

4840

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 89 (1992) 4841

RAR-a1E X

1 kb

As-.2

FPP

E

APL #22 (brlo)

APL #28 (bcr2)

APL #8 (bcr3) iF F

APL breakpoints*C D E3 4 56

XX EXiEii I I' II2 7

ATG TGA

2 6 7

a b

14+'4"'EHXE

5IA

PHL

bcr3 bcr2bcrl

P Cl-C2 C3 L- Z C-H S.~~~~~~~~~

iTr 2 3 4 5 6 TGAala va t I

W7ASH RB115a b

FIG. 1. Organization ofRARa-PML genes. A limited restriction enzyme map of the 5' portion of the RARa locus is shown in the upper partof the figure (7). Black boxes indicate RARa exons. The exons encoding the RARa A to E functional domains are indicated. A limited restrictionenzyme map of the normal PML locus (15) is shown at the bottom of the figure. Hatched boxes indicate PML exons. The exons coding for theP, C1, C2, C3, aH, L-Z, and S domains are indicated above the map. The regions involved in chromosome 15 breakpoints (bcrl, bcr2, bcr3)are indicated with arrows above the map. PML and RARa probes used in this study are indicated as solid bars below the maps. The RARo-PMLfusion genes cloned from APL patients 22, 28, and 8 are shown in the middle, aligned with the PML map. Vertical arrows indicate RARa-PMLgenomic junctions. B, BamHI; E, EcoRI; H, HindIII; X, Xba I; P, Pst I; K, Kpn I; Hc, HincII; Sc, Sac I; kb, kilobases.

mapped by Southern blot experiments to bcrl (APL 22), bcr2(APL 28), and bcr3 (APL 8) (data not shown) were clonedfrom genomic A phage libraries with PML or RARa probesthat had disclosed rearrangements (Fig. 1).The 5' regions of the three RARa-PML genes included

RARa exon 2 and/or variable portions of intron 2, in agree-ment with our findings that all APL chromosome 17 break-points lie within RARa intron 2 (14). The PML portions of theRARa-PML genes differed, as predicted from Southern blotanalysis. The chromosome 15 breakpoint lay within intron 6,102 base pairs (bp) 5' to PML exon 7 in APL 22 (bcrl); withinexon 6, 65 bp 5' to the PML intron 6 donor splicing consensus[nucleotide position 1665 of the reported PML cDNA se-quence (13)] in APL 28 (bcr2); and within intron 3, 468 bp 5'to PML exon 4 in APL 8 (bcr3).

Northern Blot Analysis of PML mRNA Expression. ThePML mRNA expression pattern was preliminarily evaluatedby Northern blotting of RNAs from 15 APL patients repre-sentative of the various chromosome 15 breakpoints with aprobe derived from the most 3' PML exon, exon 9, which wasretained in all three RARa-PML genes isolated (probe C2A,Fig. 1). A 4.6-kb band, which corresponded to the PML4transcript (15), was detected in all mRNAs examined (Fig. 2).

bcrl

4.6 ----I3 . 8 kb-E

0 (N Xl Ed LA Om m0 \ (N cn s

_ -4 N IA IAZ In rf) IAv .-m _4 CO 1U * * * s * * * % * * * * ** _.A An additional hybridizing transcript of approximately 3.8 kb

was detected in 7/10 APL patients with chromosome 15breakpoints in bcrl, in 2/2 patients with breakpoints in bcr2,but in 0/3 patients with breakpoints in bcr3. We conclude thatan abnormal mRNA containing the PML 3' portion is ex-pressed in the majority of APL patients.PCR Analysis of RARa-PML Junctions. To determine

whether the RARa-PML gene is transcribed into a fusionmRNA that corresponds to the aberrant PML transcript, aseries ofAPLs were examined by reverse PCR withRARa andPML primers. mRNAs from four patients with chromosome15 breakpoints in bcrl (nos. 22, 10, 14, and 55), three withbreakpoints in bcr2 (nos. 28, 13, and 32), and two withbreakpoints in bcr3 (nos. 8 and 34) were amplified by usingoligonucleotide R18, derived from RARa exon 2, and oligo-nucleotide SM1, from PML exon 7a (which is common to allPML isoforms) (Fig. 3). APL patients 22 and 14 (bcrl) andpatients 28, 13, and 32 (bcr2) gave a 359-bp amplificationproduct (not shown). Patients 10 and 55 (bcrl) yielded noamplification products (not shown). The last two patientsdisplayed no PML aberrant transcripts in Northern blot anal-ysis (Fig. 2). Cloning and nucleotide sequencing of one bcrl(APL 22) and all bcr2 (APLs 28, 13, and 32) amplification

--bcr2----- bcr3

FIG. 2. Northern blot analysis of PML ex-pression in APLs. RNA samples from 15 APLpatients and one AML-M2 cell line (C) wereanalyzed by Northern blotting using the C2Aprobe. APL cases are indicated by numbersabove the blot. Lengths of hybridizing frag-ments are indicated on the left. Arrows on theblot indicate PML aberrant transcripts.

Medical Sciences: Alcalay et al.

8 TGA 9H TGA

a C2A

4842 Medical Sciences: Alcalay et al.

products demonstrated that, in all cases, RARa exon 2 wasjoined to PML exon 7 by a correct splicing event that did notalter the PML ORF (Fig. 3). The RARa-PML junction gen-erated an AAG codon that encoded a lysine residue which wasnot part of the corresponding RARa or PML sequences. Theportion of PML exon 6 retained in the RARa-PML gene ofAPL patients with chromosome 15 breakpoints in bcr2 was notpresent in the transcript, probably due to the fact that the 5'portion ofPML exon 6 remained on the 15q+ derivative andthe translocated 3' end lacked a splicing acceptor site (Fig. 3).PCR amplification of the RARa-PML junction in two APL

patients with chromosome 15 breakpoints in bcr3 (nos. 8 and 34)revealed three amplification products of 833, 689, and 430 bp(data not shown). Cloning and nucleotide sequencing of theamplification products from patient 8 disclosed that thejunctionwas the same in all three transcripts (RARa exon 2/PML exon4). The junction maintained the alignment of the RARa andPML longest ORFs (Fig. 3) and generated a novel asparagineresidue. The multiplicity of the products was due to alternativesplicings ofPML exons 5 and 6. The 833-bp product retainedPML exons 4, 5, 6, and 7; the 689-bp product, exons 4, 6, and7; and the 430-bp product, exons 4 and 7 (Fig. 3).The correlation between PCR amplification of the RARa-

PML mRNAjunctions and Northern blotting identification ofthe PML aberrant transcripts in APLs with chromosome 15breakpoints in bcrs 1 and 2 suggests that the PML aberranttranscripts correspond to the RARa-PML transcripts. More-over, the size of the aberrant PML transcript (approximately3.8 kb) coincides with the size of the expected RARa-PMLtranscripts as calculated from the PCR-isolated RARa-PMLjunctions [3806 bp, not considering the poly(A) tail]. Theapparent discrepancy between the PCR amplification ofRARa-PML transcripts and the lack of expression of aber-rant PML transcripts in the two APLs with chromosome 15breakpoints in bcr3 may have been due to comigration of thenormal and aberrant PML mRNAs.

It, therefore, appears that (i) a RARa-PML transcript ispresent in the majority, but not all, of APL cases; (ii) twotypes of RARa-PML mRNA junctions are generated, de-pending on the location of the chromosome 15 breakpoint;(iii) the RARa and PML longest ORFs are aligned in bothjunctions; and (iv) alternative splicings ofPML exons 5 and6 are responsible for the additional heterogeneity of theRARa-PML fusion transcripts in APLs with chromosome 15breakpoints in bcr3.

Analysis of the 3' Portion of the Chimeric RARa-PMLTranscripts. We have identified fourPML isoforms that differin the use of alternative 3' coding exons 7 (PML1 and PML2),8 (PML3), or 9 (PML4). To ascertain whether the RARa-PML transcripts displayed the same variations in 3' exonconformations as the normal PML transcripts, PCR analysiswas carried out on mRNAs from one APL patient with thechromosome 15 breakpoint in bcrl (no. 22), three in bcr2(nos. 13, 28, and 32), and two in bcr3 (nos. 8 and 34). PrimerR2 was always used as the 5' primer, while the 3' primerswere SM2, from PML exon 7, M48 from PML exon 8, andM15 from PML exon 9 (Fig. 4). APLs with chromosome 15breakpoints in bcrl and bcr2 had identical RARa-PMLmRNA junctions and will be discussed together.PCR amplification of APLs 22, 13, 28, and 32 (bcrl and

bcr2) yielded a single amplification product that varied in sizeaccording to the 3' PML primer used: 145 bp, which corre-sponds to the expected junction between RARa exon 2 andPML exon 7, with the SM2 primer; 317 bp, which corre-sponds to the junction between RARa exon 2 andPML exons7a and 8, with the M48 primer; and 293 bp, which correspondsto the junction between RARa exon 2 and PML exons 7a, 8a,and 9, with the M15 primer (Fig. 5). The three RARa-PMLtranscripts revealed by the amplification experiments (RM7,RM8, and RM9) are shown in Fig. 4B.

bcrl

APL #22(DNA)

APLB #22(RNA)

bcr21665

RARaA46 7 PHIAPL #28(DNA) t1kb

APLS #28,#13, #32 AM 359 bp(RNA) 4 i 39b

bcr3PML

D a Do%%M 2 4 5 6 7

APL #8

(DNA) aAScAL a 1 kb

j1. I|> ! 833 bpCCAGCCGM lC '

APLs #8 ProAlaAAlaAla(RNA) | 689 bp

TGA

| 430 bp

FIG. 3. RARa-PML mRNA junctions. (Top) Schematic repre-sentation of the RARa-PML bcrl genomic junction ofAPL 22. Thenucleotide sequences of the RARa donor and the PML acceptorinvolved in the mRNA junction are indicated below the map. Theexon and intron sequences are shown in uppercase and lowercase,respectively, and the predicted amino acid residues are given un-derneath. The gt and ag splicing consensus dinucleotides are boxed.The position and orientation ofthe R2 and SM1 primers are indicatedby arrowheads. A diagram of the exon assembly of the 359-bp PCRamplification product is shown below the map (splicing is shown bythe zig-zag broken line). The sequence of the RARa-PML mRNAjunction and predicted protein is shown below the broken line. Thearrow indicates the junction; the nonhomologous lysine residue isunderlined. (Middle) Schematic representation ofRARa-PML bcr2junction of APL 28. The nucleotide sequence of the 5' end of theresidual portion ofPML exon 6 is given below the map. A diagramof the exon assembly of the 359-bp PCR amplification product andthe sequence ofthe RARa-PML mRNAjunction ofAPLs 28,13, and32 are shown below the map (identical to the junction describedabove forAPL 22). (Bottom) Schematic representation ofthe RARa-PML bcr3junction ofAPL 8. The nucleotide sequences of the RARadonor and the PML acceptor involved in the mRNA junction areindicated below the map. A diagram ofthe exon assembly ofthe 833-,689-, and 430-bp PCR amplification products and the sequence of theRARa-PML mRNA junction are shown below the map. The non-homologous asparagine is underlined.

Proc. Natl. Acad. Sci. USA 89 (1992)

ATG

k II -

Proc. Natl. Acad. Sci. USA 89 (1992) 4843

Patients 8 and 34 (bcr3) gave three amplification products:619, 475, and 216 bp (corresponding to transcripts RM4-5-6-7,RM4-6-7, and RM4-7 in Fig. 4A) with the SM2 primer; 791,647, and 388 bp (corresponding to transcripts RM4-5-6-7-8,RM4-6-7-8, and RM4-7-8 in Fig. 4A) with the M48 primer; and767, 623, and 364 bp (corresponding to transcripts RM4-5-6-7-8-9, RM4-6-7-8-9, and RM4-7-8-9 in Fig. 4A) with the M15primer. The PCR blots are shown in Fig. 5. The sizes of thebands of each experiment agreed with the previously de-scribed alternative splicings of PML exons 5 and 6.

In conclusion, the three alternative 3' PML exon confor-mations investigated were present in RARa-PML transcripts.Modular Organization of RARa-PML Isoforms. Fig. 6

shows diagrams ofRARa-PML proteins deduced from all theRARa-PML junctions sequenced and shows the putativePML and RARa domains retained in each.The RARa protein can be divided into six regions, A-F, on

the basis of sequence homology with other members of thenuclear receptor superfamily (16, 17). The C region is aDNA-binding domain, the E region is a ligand-binding do-main, and the A/B region has transcriptional activationfunctions with cell-type and promoter specificity. The PMLproteins contain the cysteine-rich motif (subdivided intothree clusters, C1, C2, and C3), an N-terminal proline-richmotif (P), an a-helix domain (aH) that includes a segmentwith homology to the leucine-zipper region of the fos family(LZ), a C-terminal serine/proline-rich domain (S) (11), andfour alternative C termini.Ten different RARa-PML putative proteins are encoded by

the mRNAs described in this report. Fusion transcripts iso-lated from APL patients with chromosome 15 breakpoints in

A RAR-a1 2

ATG

.I .. .I21 kb s

bcr314

bcrl and bcr2 potentially encode three isoforms (RM7, RM8,RM9) made up ofthe RARa A domain, the PML S region, andthe alternative C termini. Seven isoforms are encoded byfusion transcripts isolated from patients with breakpoints inbcr3. All contain the RARa A domain, variable portions of thePML aH region, and, except for the RM4-7 protein, the PMLS region. The common features of all the RARa-PML putativeproteins are, therefore, the RARa A domain, the PML Sregion, and the variable PML C termini.

DISCUSSIONRARa-PML genes differ in the site of their chromosome 15breakpoint. RARa exons 1 and 2 and PML exons 7, 8, and 9appear to be constant features of the RARa-PML genestructure. RARa-PML transcripts were detected in most, butnot all, APL patients. Depending on the site of the chromo-some 15 breakpoint, there were two types of RARa-PMLmRNA junctions: RARa exon 2/PML exon 7 (in bcrl andbcr2) andRARa exon 2/PML exon 4 (in bcr3). Bothjunctionsmaintained the alignment of the longest RARa and PMLORFs. The 3' conformation of the RARa-PML transcriptswas identical to that of normal PML. Multiple RARa-PMLtranscripts, which differed in their 3' exons (7, 8, or 9), werefound in all APLs. Additional internal splicings (PML exons5 and 6) generated further heterogeneity in patients withchromosome 15 breakpoints in bcr3. One of the PML splic-ings yielded an exon 4/exon 7 junction that did not maintainthe alignment of the PML longest ORF. A TGA codon wassituated 15 bp 3' to this junction, within the portion of exon7 common to all PML transcripts. All the alternative splicingsidentified are also present in the normal PML transcripts (15).

PML5 6

ATGRM4-5-6-7 I I

ATGRM4-6-7 I I

ATGRM4-7 I I

ATGRM4-5-6-7-8 I a

ATG

RM4-6-7-8 I IATG

RM4-7-8 I IATG

RM4-5-6-7-8-5 IATG

RM4-6-7-8-9 I IAG

RM4-7-8-9 I a

B RAR-a1 2

ATG

I I-1 kb >

R2

RM7 I

RM8 I

RM9 I

ATG

I

ATG

ATG

I1

bcr2 bcrl6 47

tTGA

SM2

TGA

a b

a

a..-a

7TGAa. I.-

%S12TGA

TGA

TGA*

a

TGA*

.-TGA***

a

8 9TGA TGA

8Mil5

TGA

IaTGA

TGA

a

PML FIG. 4. Alternatively spliced RARa-PML8 9 transcripts. (A) A representative RARa-PMLTGA TGA bcr3 genomic junction is shown at the top. Thei .7 position and orientation of the R2. SM2, M48, and5 < M15 primers are indicated by arrowheads. Dia-M48 M15 grams of the exon assembly of the RARa-PML

mRNAs predicted from the PCR experiments aregiven below. (B) A representative RARa-PMLbcrl-bcr2 genomic junction is shown at the top.The position and orientation ofthe R2. SM2., M48.and M15 primers are indicated by arrowheadsbelow the map. Diagrams of the exon assembly of

a the RARa-PML mRNAs RM7, RM8. and RM9.TGA predicted from the PCR experiments. are given/:~4~ below. The RARa-PML mRNA junction is the

a same in all transcripts (see Fig. 3).

Medical Sciences: Alcalay et al.

I

V

i.

4844 Medical Sciences: Alcalay et al.

R2/SM2 (RARa exon 2/PML exon 7)N (n CD N4 IN _N - 6m

1W.:~ -- ;m I-619 bp

145 bp ->s a _mi

R2/M48 (RARa exon 2/PML exon 8)N m co N NN -4 N C) a (n

* 7> 6 4 87 b

31b>-- 000-A < 388 bp

R2/M15 (RARa exon 2/PML exon 9)a N

*- *' *0 *

< 767 bp- 623 bp

< 364 bp

FIG. 5. PCR analysis of the 3' portion of RARa-PML mRNAs.PCR blots of six representative APL patients (APL 22 has thechromosome 15 breakpoint in bcrl; 13, 28, and 32, in bcr2; and 8 and34, in bcr3) were hybridized to the SM1 oligonucleotide from PMLexon 7a. The primers used for each experiment, and their respectiveexon derivation, are indicated above each blot. Molecular sizes ofhybridizing bands are given.

It is difficult to predict the role of the putative RARa-PMLproteins in the pathogenesis of APL. However, some of ourfindings suggest that they may not be directly involved: (i)RARa-PML transcripts never appeared alone but were al-ways associated with PML-RARa transcripts (data notshown); (ii) APL patients who did not display the RARa-

" RM7

';I: RMB

ARM9

RM4-5-6-7

RM4-6-7

m RM4-5-6-7-8

Q RM4-6-7-8

RM4-5-6-7-8-9

RM4-6-7-8-9

Sjl MW 12. 4

A SQ MW 15

S 4 MW 42.5

xaH MW 28.9

A aiH S54l mw 23.9

a cH S MW 31. 4

axH SQ MW 26.5

IAaH S 4 MW 59

coBUS 4 MW 54

RM -7 11aHI MW 9

FIG. 6. Modular organization of RARa-PML proteins. Symbolsfor RARa (A) and PML (aH, S) are as in Fig. 1. Boxes 1, 3, and 4indicate the PML1, PML3, and PML4 alternative C termini. Molec-ular weights x 10-3 are given on the right.

PML transcripts by both Northern blot and PCR analysis allexpressed the PML-RARa transcript (data not shown); (iii)the RARa DNA-binding and retinol-binding domains and thePML DNA-binding domain became part of the PML-RARafusion proteins. The RARa-PML putative proteins seem tobe made up of at least the RARa A domain, which hastranscriptional activation functions in the RARa protein, thePML S region, thought to be involved in the regulation of thephosphorylation of the PML protein (11), and the alternativePML C termini, whose function is unknown.On the other hand, the fact that, regardless of the variabil-

ity in the chromosome 15 breakpoint, the RARa-PMLmRNA always encodes a putative fusion protein suggeststhat there may be selective pressure for chromosome trans-locations that lead to the generation ofboth RARa-PML andPML-RARa functional transcripts. Two events, consideredessential to the multistep process of carcinogenesis in mostother tumors, do not occur in APL: (i) additional chromo-some abnormalities and (ii) mutations in ras and p53 genes(unpublished data). The single chromosome abnormality[t(15;17)] that appears to lead to the generation of RARa-PML and PML-RARa proteins may be all that is required forthe APL target cell to become fully transformed.

This research was supported by grants from the AssociazioneItaliana per la Ricerca sul Cancro and the Consiglio Nazionale delleRicerche Oncologia to P.G.P. and F.G.

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14. Pandolfi, P. P., Alcalay, M., Fagioli, M., Zangrilli, D., Men-carelli, A., Diverio, D., Biondi, A., Lo Coco, F., Rambaldi, A.,Grignani, F., Rochette-Egly, C., Gaub, M.-P., Chamboh, P. &Pelicci, P. G. (1992) EMBO J. 11, 1397-1407.

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16. Evans, R. M. (1988) Science 240, 889-895.17. Green, S. & Chambon, P. (1988) Trends Genet. 4, 309-314.

Proc. NatL Acad. Sci. USA 89 (1992)

- < 216 bp

2 9 3 bp > doft moo. am-