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The human mdr1 (multidrug-resistance) gene harbours a long homopyrimidine·homopurine sequence next to a cluster of Alu repeated sequences in intron 14
Gene, 153 (1995) 299-300 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
299
GENE 08505
The human mdrl (multidrug-resistance) gene harbours a long homopyrimidine- homopurine sequence next to a cluster of Alu repeated sequences in intron 14" (P-glycoprotein; repetitive., sequences; nucleotide sequence; gene cloning; recombinant DNA; gene mapping)
Marc Pauly a, Isabelle Kayser a, Martine Schmitz", Fernand Ries b, Frangois Hentges b and Mario Dicato b aLaboratoire de Recherche sur le Cancer et les Maladies du Sang, B~timent des Sciences, Centre Universitaire de Luxembourg, L-1511 Luxembourg, Grand Duchy of Luxembourg; and b Service de M~decine Interne, Centre Hospitalier de Luxembourg, L-1210 Luxembourg, Grand Duchy of Luxembourg. Tel. (352) 466644-432.
Received by G. Bernardi: 12 August 1994; Accepted: 14 September 1994; Received at publishers: 13 October 1994
SUMMARY
In order to identify specific DNA sequences useful as 'genetic landmarks' in the construction of a complete map of the human mdrl (multidrug-resistance) gene, we investigated the introns in the central region. In intron 14, we identified a long stretch of a homopyrimidine.homopurine sequence most probably adopting an unconventional DNA conformation, followed by a cluster of three Alu repeated sequences in an inverted orientation. Here, we describe the structure, formation and nucleotide sequence of these DNA elements.
The human multidrug-resistance-encoding gene mdrl plays a major role in the aquisition of cellular resistance against several drugs simultaneously, which frequently leads to failure of anticancer chemotherapy (Shen et al., 1986; Ueda et al., 1987). The underlying mechanism has been attributed to an increased synthesis of its product, the P-glycoprotein, a 170-kDa TM protein acting as a drug-transporting efflux pump (Chen et al., 1986). Human mdrl extends for more than 100 kb on the long arm of chromosome 7 (band position 7q21.1) and its structural organization encompasses 28 introns (Chen et al., 1990), of which only very few have been explored Correspondence to: Dr. M. Pauly, Laboratoire RCMS, Bht. Sciences, C.
Uni., Av. Fa'iencerie 162A, L-1511 Luxembourg, Grand Duchy of Luxembourg. Tel. (352) 466644-432; Fax (352) 466644-436. *On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations:bp, base pair(:~);kb, kilobase(s) or 1000bp; MCS, multiple cloning site (polylinker); mdr, multidrug-resistance-encoding gene; mdrl, first member of the mdr family; nt, nucleotide(s); poly(A), polyadenosine; poly(T), polythymidine; TM, transmembrane;Y.R, homopyrimidine.homopurine. SSDI 0378-1119(95)00716-0
so far. We investigated the introns in the central region of the gene in order to identify specific sequences which could be useful as 'genetic landmarks' in the construction of a complete map of human mdrl. In the first half of intron 14, we identified a homopyrimidine'homopurine (Y'R) sequence with a length of 410-bp encompassing various minisatellite-like repeated motifs (Fig. 1, nt position 935-1344). As we found during DNA sequencing at this site (Sanger et al., 1977), interpretable nt sequence data could only be obtained after separation of both DNA strands by subcloning this region into vector types generating singlestranded DNA, like the pGEM phagemid series (see legend to Fig. 1). Using double-stranded DNA as sequencing templates always lead to premature arrests of elongation in all four sequencing reactions, even when using thermostable DNA polymerases at high temperatures in the presence of denaturing agents such as formamide or dimethylsulfoxide. These observations strongly suggest that, at least in vitro, the Y.R sequence adopts an unconventional DNA conformation preventing the progression of DNA polymerases, as has been shown for the same type of DNA sequence elsewhere (Liu and Chan, 1990).
300 1 ~ e~a~e,~ie~~ 911
~
c,/x~Araa~/aaAe,~w.~i. . . . . . . . . .
TC~"~'~C.:I~TT T G C T T ~ ~ GTtT,A.TT T C A T C T C T GT GTT T T T C~ TT T 2 C ' Z T T ~ T T ~ T >>>>>>>>>>>>>>>t *et ee*~*****~*c,****et t e****t *t ~ t t t
971 2 C C T T C C T TCCTT C C T T C C C T C C C T CCCT C.AATCCCT C C C T C T CT ~ GCT CT~CCT CT TCC 1031 T T T C C T T T C T T T C C T T ~ C C T T T C C T C ~ A C C ~ T C C C T T C C T T T C A T T T C C T T T C C C T T C C C T 1091 T C C C T T T C T T T C C C T T C C C T T C C C T T C C T 2 T C C C T T C C C ~ T C C C T T C C T T T C C C T ~ C C C T 1151 T CCCT T C C T T T C C C T C ~
2CCCTTCCCTCCCCT TCCCTTCCCTCCCCTT CCCTCCCCT
1211 T C C C T C C C C T T C C C T C C C C T C C C A T C C C C ~ C C C C T C C C ~ T T T T C T T T T T C T T T T T T C T C T 1271 T C T C T T C T C T T C C T C T C C T C T C C T G T C T T T T T C T T T T T C T T T T C T T A T C T T A T C T T T T C T 1331 TT T C T T G T T T CT T T T C T C A E T CT GTCACC.~.GGCTGAAGT GCAGT TGTGCCATC2KTC-C~CA e*~e****e*ee*e .............................................. 1391 C T A C A A C C T C T G C T G C C C A ~ T C T C A A G T G A ~ C C T C C C A C C T ~ C A C A A G T ~ GG ............................................................ 1451 G A C ~ GTGT G C C A C C A T G C ~ T TTT T T T T T T T2 T T T 2TTTT T T T G A G A T G G 1511 A G T C T C G C T C T G T C G C C C A G G C T C-GAGT G C A G T ~ A D . A ~ T C T
TGGCT C A C T G C A A C C T C
1571 TG~CTCCTGCCTCADd~CTCCT G A G T A G C 2 A G G A T ~ G C A E C G C C A C A C C T
GGC
1631 TAA.TTTTTGTATTTTTAG2AAAC~F.AC~GGTTTCGTCA~GTGGCCCACGCTGGTCTTGAAC 1691 T CCT GACCT ~
GAT CCACC~G(2CT C ~ C C C A A A G T
GCT G G G A T T A C A C G C GT GA
1751 G C T A C C G T G C C C A G C ~ T T T G A 2 A ~ A 2 T G T A G A G A . T G A G G T
CTCACTA:TCTT
1811 C,C C C C ~ C T GGTCT ~ T CCT G G G C T ~ ~ C C G C C T C A G C C T CCC.AAAGT GC ............................................................ 1871 T GGAAT TACACda.GT G A G C C A C T G C T T A C T G T T T GCT TAT CT GT GTTT CC .............................. >>>>>>>>>>>>>> 2731 TT GGT T T G T ~ G A 2 T A 2 T A T T A T T T T T ~ T A ~ T T T T T A T 2 T T T T TTTT TT G A G A >>>>>>>>>>>>>>>>" 2791 T GGAGT CT CACT CT G2 C A C C C A G G C T G G A G T G C A G T G G T GCGAT CTT GC-CT CACT C-CAAG 2851 CT C T A C C T C C C A A G T A G C G G G G A C T A C A G G C A C C C G C C A C C A 2 2911 TAT T T T T A G T A G A G A T GGGGT T T C A C T GT G 2971 T GT GAT C ~ C T
T
T
~
G C C C A G C T A A T TT T TT G
GGT CT T GAT CT CCTGACCT
T G G C C T C C C A A A G T GCT G G G A T T A C A G G C G T G A C G ~ C C A C G C C C
3031 A G C ~ C C C T CIATTAT T A C T G C A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~>>>>>>>>>>>>>>>> 3301 A T T T C T C T C T C T T T A G
Fig. 1. Structure and nt sequence of the Y.R and Alu repeats in intron 14 of human mdrl. The nt position of intron 14 (GenBank/EMBL accession No. X78081, total length 3316 nt), starting with + 1 (5'-end of the intron, coding strand), is indicated at the left-hand margin. The Y-R sequence is underlined with asterisks (***), Alul with broken lines, Alu2 with a double broken line, Alu3 with a full line and the flanking direct repeats with arrowheads (> > >). Methods: Gene cloning and DNA isolation were performed according to Sambrook et al. (1989). A positive kCharon40 clone was isolated by screening a human leucocyte genomic library with fragment 5A of the human mdrl cDNA as a probe (Ueda et al., 1987). The Sau3AI-BamHI insert of the clone (about 17 kb) was digested with XbaI and the resulting fragments subcloned in the MCS of plasmid vector pUC19 (Pharmacia). For further isolating the Y.R sequence region, the respective XbaI fragment was digested by EcoRI (nt 734) and SphI (nt 1610) and subcloned in vector pGEM7Z f (+) (Promega). The results of DNA sequencing (Sanger et al., 1977) with T7 DNA polymerase (Pharmacia) were analysed using the Hibio DNAsis software (Hitachi).
In the second half of i n t r o n 14, we discovered a cluster of three Alu r e p e a t e d sequences, which we n a m e d Alul (nt 1345-1479 a n d 1767 1900, Fig. l), Alu2 (nt 1480-1766) a n d Alu3 (nt 2757-3034). Typical Alu repeats are flanked on b o t h sides b y direct repeats a n d encompass a 280-bp h e a d - t o - t a i l d i m e r of two h o m o l o g o u s parts, followed at the 3'-end by a p o l y ( A ) stretch of variable length. As r e t r o p o s o n s , they very likely derive from R N A i n t e r m e d i a t e s by reverse t r a n s c r i p t i o n a n d integr a t i o n of the respective c D N A into the p r i m a t e g e n o m e (Rogers, 1985). T h e Alu repeats in i n t r o n 14 of h u m a n mdrl closely follow the c o m m o n structural o r g a n i z a t i o n , sharing a high degree of h o m o l o g y (Alul, 71%; Alu2, 80%; Alu3, 81%) with a consensus sequence (Britten et al., 1988), but they are all in the inverted orientation, with a p o l y ( T ) stretch at the 5'-end. T h e Alul dimer, however, is d i s r u p t e d in its central region by the insertion
of Alu2, a n d is l o c a t e d next to the Y-R sequence, which a p p e a r s as a substitute for the p o l y ( T ) stretch (Fig. 1). F u r t h e r m o r e , the lack of direct repeats flanking Alu2 a n d the presence of such repeats i m m e d i a t e l y u p s t r e a m from the Y.R sequence a n d d o w n s t r e a m from Alul suggest that the Y.R sequence a n d the Alul-Alu2 cluster might have been t r a n s p o s e d t o g e t h e r as a single i n t e g r a t i o n block, i n d e p e n d e n t l y of Alu3. Based on previous o b s e r v a t i o n s a n d suggestions (Liu a n d Chan, 1990), we p r o p o s e that the Y.R sequence generates a histone-free site easily accessible to the integr a t i o n of t r a n s p o s a b l e D N A elements in the h u m a n g e n o m e a n d that it might, therefore, have f a v o u r e d the g e n e r a t i o n of a cluster of multiple Alu repeats in its close vicinity. We t h a n k Dr. I. P a s t a n ( N I H , Bethesda, M D , U S A ) and Prof. P. Reizenstein ( K a r o l i n s k a Institute, S t o c k h o l m , Sweden) for the gift of p r o b e 5A; Dr. A. Steinmetz, Prof. E. Westhof, J.-M. G a r n i e r a n d Prof. P. C h a m b o n ( C N R S , Strasbourg, France) for the gift of the h u m a n g e n o m i c l i b r a r y a n d helpful suggestions. This w o r k was s u p p o r t e d by the Centre de Recherche P u b l i c de la Sant6 a n d the F o n d a t i o n de Recherche C a n c e r et Sang of L u x e m b o u r g .
REFERENCES Britten, R.J., Baron, W.F., Stout, D.B. and Davidson, E.H.: Sources and evolution of human Alu repeated sequences. Proc. Natl. Acad. Sci. USA 85 (1988) 4770 4774. Chen, C.J., Chin, J.E., Ueda, K., Clark, D.P., Pastan, I., Gottesman, M.M. and Roninson, I.B.: Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein) gene from multidrug-resistant human cells. Cell 47 (1986) 381-389. Chen, C.J., Clark, D., Ueda, K., Pastan, I., Gottesman, M.M. and Roninson, I.B.: Genomic organization of the human multidrug resistance (mdrl) gene and origin of P-glycoproteins. J. Biol. Chem. 265 (1990) 506-514. Liu, Q.R. and Chan, P.K.: Identification of a long stretch of homopurine-homopyrimidine sequence in a cluster of retroposons in the human genome. J. Mol. Biol. 212 (1990) 453-459. Rogers, J.H.: The origin and evolution of retroposons. Int. Rev. Cytol. 93 (1985) 187-279. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463 5467. Shen, D.W., Fojo, A., Roninson, I.B., Chin, J.E., Soffir, R., Pastan, I. and Gottesman, M.M.: Multidrug resistance of DNA-mediated transformants is linked to transfer of the human rndrl gene. Mol. Cell. Biol. 6 (1986) 4039-4044. Ueda, K., Cardarelli, C., Gottesman, M.M. and Pastan, I.: Expression of a full-length cDNA for the human "mdrl" gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc. Natl. Acad. Sci. USA 84 (1987) 3004-3008.
299
GENE 08505
The human mdrl (multidrug-resistance) gene harbours a long homopyrimidine- homopurine sequence next to a cluster of Alu repeated sequences in intron 14" (P-glycoprotein; repetitive., sequences; nucleotide sequence; gene cloning; recombinant DNA; gene mapping)
Marc Pauly a, Isabelle Kayser a, Martine Schmitz", Fernand Ries b, Frangois Hentges b and Mario Dicato b aLaboratoire de Recherche sur le Cancer et les Maladies du Sang, B~timent des Sciences, Centre Universitaire de Luxembourg, L-1511 Luxembourg, Grand Duchy of Luxembourg; and b Service de M~decine Interne, Centre Hospitalier de Luxembourg, L-1210 Luxembourg, Grand Duchy of Luxembourg. Tel. (352) 466644-432.
Received by G. Bernardi: 12 August 1994; Accepted: 14 September 1994; Received at publishers: 13 October 1994
SUMMARY
In order to identify specific DNA sequences useful as 'genetic landmarks' in the construction of a complete map of the human mdrl (multidrug-resistance) gene, we investigated the introns in the central region. In intron 14, we identified a long stretch of a homopyrimidine.homopurine sequence most probably adopting an unconventional DNA conformation, followed by a cluster of three Alu repeated sequences in an inverted orientation. Here, we describe the structure, formation and nucleotide sequence of these DNA elements.
The human multidrug-resistance-encoding gene mdrl plays a major role in the aquisition of cellular resistance against several drugs simultaneously, which frequently leads to failure of anticancer chemotherapy (Shen et al., 1986; Ueda et al., 1987). The underlying mechanism has been attributed to an increased synthesis of its product, the P-glycoprotein, a 170-kDa TM protein acting as a drug-transporting efflux pump (Chen et al., 1986). Human mdrl extends for more than 100 kb on the long arm of chromosome 7 (band position 7q21.1) and its structural organization encompasses 28 introns (Chen et al., 1990), of which only very few have been explored Correspondence to: Dr. M. Pauly, Laboratoire RCMS, Bht. Sciences, C.
Uni., Av. Fa'iencerie 162A, L-1511 Luxembourg, Grand Duchy of Luxembourg. Tel. (352) 466644-432; Fax (352) 466644-436. *On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations:bp, base pair(:~);kb, kilobase(s) or 1000bp; MCS, multiple cloning site (polylinker); mdr, multidrug-resistance-encoding gene; mdrl, first member of the mdr family; nt, nucleotide(s); poly(A), polyadenosine; poly(T), polythymidine; TM, transmembrane;Y.R, homopyrimidine.homopurine. SSDI 0378-1119(95)00716-0
so far. We investigated the introns in the central region of the gene in order to identify specific sequences which could be useful as 'genetic landmarks' in the construction of a complete map of human mdrl. In the first half of intron 14, we identified a homopyrimidine'homopurine (Y'R) sequence with a length of 410-bp encompassing various minisatellite-like repeated motifs (Fig. 1, nt position 935-1344). As we found during DNA sequencing at this site (Sanger et al., 1977), interpretable nt sequence data could only be obtained after separation of both DNA strands by subcloning this region into vector types generating singlestranded DNA, like the pGEM phagemid series (see legend to Fig. 1). Using double-stranded DNA as sequencing templates always lead to premature arrests of elongation in all four sequencing reactions, even when using thermostable DNA polymerases at high temperatures in the presence of denaturing agents such as formamide or dimethylsulfoxide. These observations strongly suggest that, at least in vitro, the Y.R sequence adopts an unconventional DNA conformation preventing the progression of DNA polymerases, as has been shown for the same type of DNA sequence elsewhere (Liu and Chan, 1990).
300 1 ~ e~a~e,~ie~~ 911
~
c,/x~Araa~/aaAe,~w.~i. . . . . . . . . .
TC~"~'~C.:I~TT T G C T T ~ ~ GTtT,A.TT T C A T C T C T GT GTT T T T C~ TT T 2 C ' Z T T ~ T T ~ T >>>>>>>>>>>>>>>t *et ee*~*****~*c,****et t e****t *t ~ t t t
971 2 C C T T C C T TCCTT C C T T C C C T C C C T CCCT C.AATCCCT C C C T C T CT ~ GCT CT~CCT CT TCC 1031 T T T C C T T T C T T T C C T T ~ C C T T T C C T C ~ A C C ~ T C C C T T C C T T T C A T T T C C T T T C C C T T C C C T 1091 T C C C T T T C T T T C C C T T C C C T T C C C T T C C T 2 T C C C T T C C C ~ T C C C T T C C T T T C C C T ~ C C C T 1151 T CCCT T C C T T T C C C T C ~
2CCCTTCCCTCCCCT TCCCTTCCCTCCCCTT CCCTCCCCT
1211 T C C C T C C C C T T C C C T C C C C T C C C A T C C C C ~ C C C C T C C C ~ T T T T C T T T T T C T T T T T T C T C T 1271 T C T C T T C T C T T C C T C T C C T C T C C T G T C T T T T T C T T T T T C T T T T C T T A T C T T A T C T T T T C T 1331 TT T C T T G T T T CT T T T C T C A E T CT GTCACC.~.GGCTGAAGT GCAGT TGTGCCATC2KTC-C~CA e*~e****e*ee*e .............................................. 1391 C T A C A A C C T C T G C T G C C C A ~ T C T C A A G T G A ~ C C T C C C A C C T ~ C A C A A G T ~ GG ............................................................ 1451 G A C ~ GTGT G C C A C C A T G C ~ T TTT T T T T T T T2 T T T 2TTTT T T T G A G A T G G 1511 A G T C T C G C T C T G T C G C C C A G G C T C-GAGT G C A G T ~ A D . A ~ T C T
TGGCT C A C T G C A A C C T C
1571 TG~CTCCTGCCTCADd~CTCCT G A G T A G C 2 A G G A T ~ G C A E C G C C A C A C C T
GGC
1631 TAA.TTTTTGTATTTTTAG2AAAC~F.AC~GGTTTCGTCA~GTGGCCCACGCTGGTCTTGAAC 1691 T CCT GACCT ~
GAT CCACC~G(2CT C ~ C C C A A A G T
GCT G G G A T T A C A C G C GT GA
1751 G C T A C C G T G C C C A G C ~ T T T G A 2 A ~ A 2 T G T A G A G A . T G A G G T
CTCACTA:TCTT
1811 C,C C C C ~ C T GGTCT ~ T CCT G G G C T ~ ~ C C G C C T C A G C C T CCC.AAAGT GC ............................................................ 1871 T GGAAT TACACda.GT G A G C C A C T G C T T A C T G T T T GCT TAT CT GT GTTT CC .............................. >>>>>>>>>>>>>> 2731 TT GGT T T G T ~ G A 2 T A 2 T A T T A T T T T T ~ T A ~ T T T T T A T 2 T T T T TTTT TT G A G A >>>>>>>>>>>>>>>>" 2791 T GGAGT CT CACT CT G2 C A C C C A G G C T G G A G T G C A G T G G T GCGAT CTT GC-CT CACT C-CAAG 2851 CT C T A C C T C C C A A G T A G C G G G G A C T A C A G G C A C C C G C C A C C A 2 2911 TAT T T T T A G T A G A G A T GGGGT T T C A C T GT G 2971 T GT GAT C ~ C T
T
T
~
G C C C A G C T A A T TT T TT G
GGT CT T GAT CT CCTGACCT
T G G C C T C C C A A A G T GCT G G G A T T A C A G G C G T G A C G ~ C C A C G C C C
3031 A G C ~ C C C T CIATTAT T A C T G C A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~>>>>>>>>>>>>>>>> 3301 A T T T C T C T C T C T T T A G
Fig. 1. Structure and nt sequence of the Y.R and Alu repeats in intron 14 of human mdrl. The nt position of intron 14 (GenBank/EMBL accession No. X78081, total length 3316 nt), starting with + 1 (5'-end of the intron, coding strand), is indicated at the left-hand margin. The Y-R sequence is underlined with asterisks (***), Alul with broken lines, Alu2 with a double broken line, Alu3 with a full line and the flanking direct repeats with arrowheads (> > >). Methods: Gene cloning and DNA isolation were performed according to Sambrook et al. (1989). A positive kCharon40 clone was isolated by screening a human leucocyte genomic library with fragment 5A of the human mdrl cDNA as a probe (Ueda et al., 1987). The Sau3AI-BamHI insert of the clone (about 17 kb) was digested with XbaI and the resulting fragments subcloned in the MCS of plasmid vector pUC19 (Pharmacia). For further isolating the Y.R sequence region, the respective XbaI fragment was digested by EcoRI (nt 734) and SphI (nt 1610) and subcloned in vector pGEM7Z f (+) (Promega). The results of DNA sequencing (Sanger et al., 1977) with T7 DNA polymerase (Pharmacia) were analysed using the Hibio DNAsis software (Hitachi).
In the second half of i n t r o n 14, we discovered a cluster of three Alu r e p e a t e d sequences, which we n a m e d Alul (nt 1345-1479 a n d 1767 1900, Fig. l), Alu2 (nt 1480-1766) a n d Alu3 (nt 2757-3034). Typical Alu repeats are flanked on b o t h sides b y direct repeats a n d encompass a 280-bp h e a d - t o - t a i l d i m e r of two h o m o l o g o u s parts, followed at the 3'-end by a p o l y ( A ) stretch of variable length. As r e t r o p o s o n s , they very likely derive from R N A i n t e r m e d i a t e s by reverse t r a n s c r i p t i o n a n d integr a t i o n of the respective c D N A into the p r i m a t e g e n o m e (Rogers, 1985). T h e Alu repeats in i n t r o n 14 of h u m a n mdrl closely follow the c o m m o n structural o r g a n i z a t i o n , sharing a high degree of h o m o l o g y (Alul, 71%; Alu2, 80%; Alu3, 81%) with a consensus sequence (Britten et al., 1988), but they are all in the inverted orientation, with a p o l y ( T ) stretch at the 5'-end. T h e Alul dimer, however, is d i s r u p t e d in its central region by the insertion
of Alu2, a n d is l o c a t e d next to the Y-R sequence, which a p p e a r s as a substitute for the p o l y ( T ) stretch (Fig. 1). F u r t h e r m o r e , the lack of direct repeats flanking Alu2 a n d the presence of such repeats i m m e d i a t e l y u p s t r e a m from the Y.R sequence a n d d o w n s t r e a m from Alul suggest that the Y.R sequence a n d the Alul-Alu2 cluster might have been t r a n s p o s e d t o g e t h e r as a single i n t e g r a t i o n block, i n d e p e n d e n t l y of Alu3. Based on previous o b s e r v a t i o n s a n d suggestions (Liu a n d Chan, 1990), we p r o p o s e that the Y.R sequence generates a histone-free site easily accessible to the integr a t i o n of t r a n s p o s a b l e D N A elements in the h u m a n g e n o m e a n d that it might, therefore, have f a v o u r e d the g e n e r a t i o n of a cluster of multiple Alu repeats in its close vicinity. We t h a n k Dr. I. P a s t a n ( N I H , Bethesda, M D , U S A ) and Prof. P. Reizenstein ( K a r o l i n s k a Institute, S t o c k h o l m , Sweden) for the gift of p r o b e 5A; Dr. A. Steinmetz, Prof. E. Westhof, J.-M. G a r n i e r a n d Prof. P. C h a m b o n ( C N R S , Strasbourg, France) for the gift of the h u m a n g e n o m i c l i b r a r y a n d helpful suggestions. This w o r k was s u p p o r t e d by the Centre de Recherche P u b l i c de la Sant6 a n d the F o n d a t i o n de Recherche C a n c e r et Sang of L u x e m b o u r g .
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