extrachromosomal elements (amplisomes) containing the amplified

Transcription

1 Proc. Nti. Acd. Sci. USA Vol. 87, pp , April 199 Genetics Erly ppernce nd long-term persistence of the sumicroscopic extrchromosoml elements (mplisomes) contining the mplified DHFR genes in humn cell lines (cytogenetic normlities/drug resistnce/pulsed-field gel electrophoresis) GIOVANNI PAULETTI, ERIC LAI*, AND GIUSEPPE ATTARDIt Division of Biology, Cliforni Institute of Technology, Psden, CA Contriuted y Giuseppe Attrdi, Jnury 16, 199 ABSTRACT Sumicroscopic extrchromosoml elements (mplisomes) contining mplified dihydrofolte reductse (DHFR) genes hve een investigted in methotrexteresistnt derivtive of the humn cell line HeL BU25, 1OB3, y field-inversion gel electrophoresis. The mount nd kinetics of formtion of these elements hve een correlted with the level nd time course of overll DHFR gene mplifiction. The mplisomes ccount for the gret mjority nd possily the totlity of the mplified DHFR genes in 1OB3 cells. They pper very erly during the development of methotrexte resistnce nd increse in prllel with the mplified genes. These oservtions suggest tht these elements re involved in n erly event, possily the first event, of gene mplifiction in this system. Amplisomes tend to e lost from 1OB3 cells in the sence of selective pressure, lthough much more slowly thn expected from simple dilution of nonreplicting elements. Surprisingly, under selective pressure, these elements hve shown no tendency to ecome integrted into chromosomes or to generte minute chromosomes over period of lmost 1 yer, in contrst to wht hs een descried in other systems. Besides the previously descried cytogenetic chnges ccompnying gene mplifiction (1, 2) [i.e., "extended chromosome regions" nd "doule minute chromosomes" (DMs)], sumicroscopic extrchromosoml elements contining mplified genes hve een identified. These structures were discovered in Leishmni species s 3-kilose (k) supercoiled-circulr DNA molecules contining mplified copies of the gene for the ifunctionl protein dihydrofolte reductse-thymidylte synthetse (3, 4). Similr ut lrger sumicroscopic elements (12-75 k) hve een identified in mmmlin cells nd shown to crry mplified copies of the DHFR gene in methotrexte (MTX)-resistnt derivtives of HeL BU25 nd VA2B cells (5), the multiple drug resistnce gene (mdrl) in vinlstine-resistnt KB-V1 cells (6), the MYC gene in HL6 nd COL32 DM tumorderived cells (7), or the trnsfected CAD gene in CHO cells (8). The sumicroscopic elements re clerly distinct from the minute chromosomes; they re not only much smller ut lso strikingly homogeneous in size. We propose the term "mplisomes" (i.e., odies crrying mplified genes) to designte these sumicroscopic extrchromosoml elements. This term includes the reference to gene mplifiction ut voids the impliction tht the element cn lternte etween integrted nd free sttes (9), n impliction tht ws inherent in the previous term "episome" (8) nd property tht hs not een shown s yet to pply to the sumicroscopic elements. In the present work, we hve investigted the kinetics of ppernce of mplisomes in HeL BU25-1B3 (5), correlted the kinetics of formtion of the mplisomes The puliction costs of this rticle were defryed in prt y pge chrge pyment. This rticle must therefore e herey mrked "dvertisement" in ccordnce with 18 U.S.C solely to indicte this fct. with the process of overll DHFR gene mplifiction, nd nlyzed their long-term ehvior in the presence or sence of selective pressure. MATERIALS AND METHODS Cell Lines. The humn cell line HeL BU25 nd its MTXresistnt vrints lob1, 1OB2, nd 1OB3 hve een descried (1). Briefly, clonl isolte of HeL BU25 cells dpted to grow in 9 nm DL-MTX ws split into three cultures, which were then sujected to three regimens of drug dministrtion up to.18 mm DL-MTX, thus producing the three lob vrints ove. In ddition, the 1OB3 cell line ws dpted to grow in suspension (referred to s 1OB3S). Preprtion of DNA. Totl DNA for dot-lot nlysis ws prepred from cell cultures y the method ofdvis et l. (11). An utomted extrctor (Applied Biosystems model 34A) ws used to prepre high moleculr weight DNA for Southern lot nlysis. The preprtion of DNA in grose locks ws performed s descried (5), except for the omission of the RNse tretment nd the reduction of proteinse K to 1 mg/ml. Sl I digestion ofdna in grose slices ws prepred ccording to stndrd procedures (3) fter proteinse K inctivtion y phenylmethylsulfonyl fluoride. After incution for 1 hr with the enzyme, the slices were further incuted for 2 hr t 5 C with proteinse K t 1 mg/ml nd then extensively wshed in field-inversion gel electrophoresis (FIGE) uffer (see elow). FIGE. A horizontl chmer (3 x 3 cm) connected to n electromechnicl rely controlled y computer ws employed for FIGE nlysis (12). The 1% grose gels (SeKem, ME; FMC) were cst nd electrophoresed t 14 C in 45 mm Tris/45 mm orte/2 mm EDTA, ph 8. A rmped-pulse regime from 3-sec to 5-sec switching cycles, with constnt 3:1 rtio etween the forwrd nd the reverse intervl, ws pplied for 2 hr with voltge grdient of 1 V/cm. Southern nd Dot Blot Anlysis. Ethidium romide (EtdBr)- stined gels were UV-irrdited t 254 nm for 6 sec t flux intensity of 2 mw/cm2. After trnsferring the DNA frgments to nylon memrne (Zet-Proe, Bio-Rd) y cpillrity under lkline conditions (13), for 2 dys for FIGE gels nd 16 hr for conventionl grose gels, the memrnes were neutrlized, wshed with 2x SSC (lx SSC =.15 M NCl/.15 M sodium citrte, ph 7.), UV-irrdited t 312 nm for 5 min, nd ked t 8 C for 1 hr. For dot-lot nlysis, smples of totl DNA in solution or derived from grose slices, melted nd diluted in 5 vol of H2, were Arevitions: DHFR, dihydrofolte reductse; DMs, doule minute chromosomes; MTX, methotrexte; FIGE, field-inversion gel electrophoresis; EtdBr, ethidium romide. *Present ddress: Deprtment of Phrmcology, University of North Crolin, Chpel Hill, NC tto whom reprint requests should e ddressed. 2955

2 2956 Genetics: Puletti et l. shered y severl pssges through 25-guge needle nd sujected to RNse A digestion (5 /Lg/ml). After incution in.4 M NOH for 2 hr t 68C, the smples were cooled nd pplied onto Zet-Proe memrne using commercil slot-lot pprtus (Schleicher & Schuell). As proes, the insert of the recominnt plsmid phd84, contining humn DHFR cdna (14), nd chromosome lp-specific clone, pynz2 (15), rdioleled y extension of rndom hexnucleotide primers (16), were used for high-stringency hyridiztion. y-irrdition. The 'Co single-point source t the Jet Propulsion Lortory (Cliforni Institute of Technology, Psden, CA) ws employed to irrdite the smples. Agrose slices were plced in wells of 24-well microwell plte nd irrdited t room temperture under conditions ensuring uniform rdition flux to ll the smples, Before nd fter irrdition, the smples were stored in 1 mm TrisHCl, ph 8./1 mm EDTA t 1'C. FIGE frctiontion ws performed within 2 hr of irrdition. RESULTS DHFR Gene Copy Numer of HeL BU25 MTX-Retnt Vrints. The MTX-resistnt derivtive HeL BU25-1B3, which ws shown to hror DHFR gene-contining mplisomes (5), nd two relted MTX-resistnt vrints, HeL BU25-1B1 nd -1B2, lcked ny ovious "extended chromosoml regions" nd hd smll numer of DMs per cell (1). Fig. l shows Southern lot of HeL Bu25-1OB1, -1B2, nd -1OB3 cell DNA nd, for comprison, HeL S3 nd HeL BU25 DNA, digested with EcoRV nd proed with phd84. The EcoRV frgments contining DHFR gene segments re much more undnt in the MTX-resistnt vrints thn in HeL BU25, =25 times in lob1 nd 1OB3 nd =16 times in 1OB2, s estimted y densitometry. These dt for the reltive DHFR gene copy numer in the three MTXresistnt HeL BU25 derivtives gree remrkly well with the previous estimtes of enzyme ctivity in the three cell lines (1). The EcoRV frgments tht re not mplified presumly contin DHFR pseudogenes or frgments thereof (17). /ig $ LO) N O O mou~j IOB3 ro - O o O Ln ~U!) (D II-) 4), N -" o o U O O O WAMM ui m I-. :. Xl- IX-$ III VI -. FIG. 1. Southern lot nlysis of DNA from the HeL S3, HeL BU25, HeL BU25-1OB1, -1B2, nd -1B3 cell lines, using the phd84 proe contining humn DHFR cdna. In, the indicted mounts of EcoRV-digested totl DNA were frctionted y conventionl electrophoresis in.7% grose gel; in, groseemedded totl DNA from 2 x 16 cells ws nlyzed y FIGE. The size mrkers of were derived from HindIII digest of A DNA. In, the romn numerls on the left indicte yest chromosomes (Scchromyces cerevisie strin YNN295). I nd II on the right of indicte mplisome nds I nd II. I -I -]I As shown in Fig. l, frctiontion of lob1, 1OB2, nd 1OB3 DNA y FIGE, followed y DNA-trnsfer hyridiztion with the phd84 proe, reveled two nds of similr intensity (nds I nd II). The fster moving nd II corresponds to the 65-k elements identified in 1OB3 DNA (5). The moility of these DNA molecules in pulsed-field gel electrophoresis or in FIGE is ffected y the pulse time s is the moility of liner yest chromosomes, suggesting tht the 65-k nd elements re liner (5). The slower moving nd I, in turn, corresponds to nother type of extrchromosoml element contining DHFR-specific sequences tht were chrcterized in 1OB3 DNA y moility tht ws reltively insensitive to the pulse time (5). This ehvior in pulsed-field gel electrophoresis commonly indictes supercoiled-circulr DNA structure (4, 18). When rerun, most elements of nd I migrted in FIGE s the 65-k liner mplisomes (nd II) (dt not shown). This oservtion supports the proposl tht the two types of elements re interrelted, representing two topologicl forms of the sme elements or the sme form in two sttes due to rtifcts resulting from FIGE. In the present nlysis, the sumicroscopic elements contined in the two nds will e considered together. Kinetics of Appernce ofdhfr Ampsomes. As shown in Fig. 2, mplisomes cn e seen in 1OB3 cultures resistnt to 1.8 AM DL-MTX (see lso elow). Despite some fluctution in the signl intensity, due to vrition in cell concentrtion in the grose slices of different smples (see Fig. 2), there is trend towrd n increse in the mount of mplisomes t successive stges of MTX selection with no chnge in size. Fig. 3 shows densitometric dt for mplisome nds from cultures t vrious times during the selection. Dt were corrected for vrition in input DNA y dot-lot hyridiztion with the single-copy proe pynz2. The DNA ws otined from slices of the sme grose gel plugs prepred I- Proc. NtL Acd. Sci. USA 87 (199) mn i2 II m " FIG. 2. FIGE nlysis of the time course of ppernce ofdhfr mplisomes in HeL BU25-1B3 cells t vrious stges during step-wise selection in the presence of MTX. () Totl DNA from grose-emedded cells growing t vrious DL-MTX concentrtions. Lnes: 1, 18jtM;2,9nM;3, 1.8 tm;4, j.m;6nd 3.6 um; 5, 7.2 7, 14 AM; 8, 29 IAM; 9',58 A.M; 1,.12 mm; 11,.18 mm. The numer of cells emedded in ech slice were -2 x 16 except for lne 4 (-4 x 16 cells), lne 6 (-5 x 16 cells), lne 7 (-2.5 x 16 cells), nd lne 1 (-8 x 16 cells). Rom'n numerls on the left indicte the upper (I) nd lower (II) mplisome nds. () DNA/grose inserts from the sme preprtions used for the first five lnes (BU25 smple inclusive) of the gel in were y-irrdited t 6 Gy. Lne 4* represents n unirrdited control of the smple in lne 4. The segment of the gel contining the DNA/grose slices ws excised nd lotted seprtely to prevent diffusion of smll DNA frgments (presumly single strnded, relesed y the lkli tretment of the UV-irrdited gel) into the upper portion of the gel during lotting.

3 Genetics: Puletti et l. Proc. Ntl. Acd. Sci. USA 87 (199) 2957 IS ls {S I! I- lo ls 7 TQ2 x x x x x x x x x +MTX O 5 I1 -MTX X-roys 14' D Z1 x Z) z CL PI Totl DHFR genes_../ 5-r / Extrchrom- DHFR PI genes.1. A O,&_I--C--I---H DAYS FIG. 3. Time course of increse in the mount of DHFR genes ssocited with mplisomes nd with totl DNA in HeL BU25-1B3 cells during step-wise selection in the presence of MTX. The densitometric dt of Southern lot nd dot-lot nlysis hve een normlized for vrition in input DNA content of the slices on the sis of hyridiztion with the pynz2 proe. The numers t the top indicte the molr concentrtion of DL-MTX to which the cells were exposed, nd the rrowheds indicte the time limits of exposure of the cells to ech concentrtion. for FIGE. The dt re expressed s percent of the vlue found for cells resistnt to.18 mm DL-MTX. Also shown in the digrm is the totl DHFR gene content determined y reproing the stripped dot lots with the insert of phd84 nd normlizing the dt for input DNA vrition. A similr curve for the totl DHFR gene content ws determined with dot-lotted smples of totl DNA extrcted directly from the cell cultures. The increse in the mount of mplisomes nd the increse in totl DHFR gene numer re prllel throughout the gene mplifiction process. Instility of DHFR Amplisomes in the Asence of Selective Pressure nd their Long-Term Persistence Under Selection. A culture of 1B3 cells resistnt to.18 mm DL-MTX ws grown for out 9 months in the sence of the drug, wheres prllel culture ws mintined in the presence of. 18 mm DL-MTX. As shown in Fig. 4, during growth in the sence of the drug, 1B3 cells exhiit reltively slow loss of mplisomes (7%o loss in :3 genertions) for 11 weeks, fter which mplisomes ecome rely perceptile. These dt confirm previous results from this lortory (1), showing the instility of DHFR ctivity in 1B3 cells in the sence of selective pressure. By contrst, over period of 4 weeks, there is no detectle loss of mplisomes in the culture under constnt selective pressure. Quntittion of the mplisomes in the two cultures normlized for vrition in the level of DNA in the grose slices (crried out s descried ove) is shown in Fig. 5. The curve includes dt up to 4 weeks of drug withdrwl. The reltive mounts of totl DHFR genes in the DNA-contining grose (DNA/ grose) plugs of the sme cell smples re lso shown for comprison. Here gin, prllel exists etween loss of mplisomes nd loss of totl DHFR genes in the sence of selection. Similrly, the numer of mplisomes nd the totl numer ofdhfr genes re unchnged over 4-week period in the presence of MTX. Extensive Trpping of Amplisomes t the FIGE Origin. Sucrose grdient sedimenttion nlysis of metphse chromosome preprtion from 1B3 cells (5) nd the sence of n pprecile numer of microscopiclly recognizle DMs in these cells (5, 1) suggested tht most of the mplified DHFR genes in this cell line re ssocited with sumicroscopic elements. Therefore, the fct tht only minor frc- FIG. 4. Quntittive ehvior of DHFR mplisomes in HeL BU25-1B3 cells fter removl of MTX. () DNA/grose inserts from -2 x 16 cells were prepred t regulr intervls fter withdrwing the drug nd sujected to FIGE. Numerls (lne lels) indicte weeks of culture without MTX (-MTX) or with MTX (+MTX). () FIGE of y-irrdited DNA/grose inserts prepred from cells grown for 14, 17, nd 4 weeks in sence of MTX. A nonirrdited 14-week (-MTX) smple ws lso sujected to FIGE frctiontion. tion ofdhfr-specific DNA sequences moved into the gel y FIGE or pulsed-field gel electrophoresis (5) indicted tht the migrtion of the mjority of these elements ws prevented either y their structurl fetures or y some form of trpping y the chromosoml DNA. In conventionl grose gel electrophoresis, relxed-circulr molecules lrger thn criticl size hve negligile moility nd stop t the origin, the criticl size depending minly on the field strength nd the grose concentrtion (19). The sme phenomenon hs lso een oserved in FIGE (2). On the other hnd, trpping of the sumicroscopic elements y the chromosoml DNA in the grose lock ppered to e distinct possiility. If the opertion of inverting DNA/grose slice nd sujecting it gin to FIGE is repeted severl times, ech time n mount of DHFR mplisomes equl to or greter thn in the first ck z C-, 5 z Lu llj CL D -MTX, totl DHFR genes 1if +MTX, extrochrom. DHFR genes +MTX, totl DHFR genes -MTX, extrchrom. DHFR genes un I - -?I %-l' U ' q- - I(,= I 2'' 3 DAYS FIG. 5. Quntittive ehvior of the DHFR genes ssocited with mplisomes nd of totl DHFR genes in HeL BU25-1B3 cells grown in the sence of MTX (lower curves) or in the presence of.18 mm DL-MTX (upper curves). The dt hve een derived nd normlized s explined in Fig. 3. The dotted line represents n extension of the -MTX curve for the extrchromosoml elements joining the 1-week nd 4-week points (no precise quntittion ws done for this portion of the curve).

4 2958 Genetics: Puletti et l. FIGE migrted into the gel (dt not shown). This oservtion is consistent with trpping of mplisomes y the chromosoml DNA. The Mjority of the Amplified DHFR Genes Cn Be Accounted for y Amplisomes. To provide quntittive estimte of this phenomenon, y-irrdition, which is known to produce single-strnd nd doule-strnd DNA reks (21), ws employed. As shown y the EtdBr-stined pttern in Fig. 6, when grose slices contining 1B3S cell DNA (grown for 1 months in the presence of MTX) were exposed to incresing doses of y-irrdition, susequent FIGE showed progressive rekge of the DNA with the resulting heterogeneously sized frgments. As determined y densitometric mesurements, -95% of the chromosoml DNA migrted into the gel fter irrdition with 1 Gy. By contrst, proing of the DNA with the phd84 cdna insert reveled striking progressive increse in the intensity of DHFR mplisome nds I nd II. In prticulr, there ws no increse in phd84-rective mteril in the rod nd of heterogeneous frgments contining DHFR gene sequences t the ottom of the gel. Only t higher doses (5 nd 1 Gy) of y-irrdition ws there some evidence of smer of mteril recting with the phd84 proe, which moved fster thn the 65-k mplisomes, suggesting the eginning of mplisome degrdtion. At 18 Gy nd ove, degrdtion ecme significnt (dt not shown), producing continuous smer throughout the lne. By densitometric nlysis of pproprite exposures of the utordiogrm, we estimted tht the DHFR-specific DNA sequences ssocited with mplisomes t 1 Gy represented up to 4% the DHFR-specific sequences remining in the DNA/grose slices t the origin. Due to the difficulty of estimting the efficiency of trnsfer nd hyridiztion of the chromosoml DNA remining in the slices t the origin, the ove quntittion cn only e useful for compring the effects of different doses of y-irrdition. EtdBr Autorod. EtdBr lutord. vico C C. cj:: ct n.~ CT N co 3 CN 't GO FIG. 6. y-irrdition or Sl I digestion of 1B3 DNA/grose slices reduces trpping of mplisomes t the FIGE origin. () FIGE of 1B3 DNA/grose inserts from -2 x 16 1B3S cells (grown in suspension for 1 months in.18 mm DL-MTX) sujected to incresing doses of -irrdition, s indicted in Grys (Gy): EtdBr-stining pttern nd DNA lot proed with phd84 (Autord.). () FIGE of 1OB3S DNA/grose inserts (sme preprtion s ove) sujected to Sl I digestion t three enzyme concentrtions, s indicted in units (U)/ml. EtdBr-stining pttern nd DNA lot proed with phd84 (Autord.) re shown. Lnes:, smple pretreted s the Sl I-digested smples;, Mg2 smple pretreted nd incuted t 37C s the SlI-digested smples, except without the restriction enzyme. The smer in the upper portion of the first three lnes in the utordiogrm of is due to diffusion of smll DNA frgments from the gel slices t the origin during lotting (see Fig. 2). Proc. Ntl. Acd. Sci. USA 87 (199) However, the results clerly show the progressive relese of mplisomes from the slices y y-irrdition. An lterntive pproch for disrupting the chromosoml DNA network in the grose slice ws suggested y the finding tht the restriction enzyme Sl I does not cut 1OB3 mplisomes; thus presumly intct elements cn e relesed from digested totl DNA smples. As shown y the EtdBrstined pttern in Fig. 6, digestion of the DNA/grose slices with high concentrtions of Sl I prior to FIGE resulted in mssive relese of heterogeneously sized chromosoml DNA frgments. It ws determined tht >95% of the chromosoml DNA digested y Sl I t 8 units/ml migrted into the gel y FIGE. In contrst to the totl chromosoml DNA, the DHFR-specific DNA sequences relesed y Sl I digestion gin migrted only with nds I nd II of mplisomes, which were incresed significntly in mount reltive to the untreted smple. The level of intensity of these nds ws pproximtely equivlent to tht of the DHFR sequences left in the DNA/grose slices fter digestion with Sl I t 2 units/ml nd remined reltively constnt t higher concentrtions of the enzyme. Therefore, the relesed mplisomes did not rech the level otined with the highest doses of y-irrdition. A drmtic dditionl relese of mplisomes (-2% of those still remining in the DNA/grose slice) ws otined y -irrdition of Sl I-digested DNA/grose slices sujected once to FIGE (dt not shown), confirming the much greter effectiveness of y-irrdition in relesing the trpped mplisomes, due to the direct rekge of these structures. The closer migrtion of the upper nd lower nds contining such structures s well s the somewht lower moility of the heterogeneously sized frgments contining DHFR-specific sequences ner the ottom of the gel (phenomen lredy noticed fter y-irrdition) re presumly the consequence of mssive mounts of chromosoml DNA in the gel. The oservtion tht, in the control smple incuted without enzyme in Mg2+-contining uffer, there ws sustntil increse in the intensity of nd II suggests tht endogenous nucleses my hve een ctivted, with resulting prtil digestion of the chromosoml DNA meshwork nd relese of linerized mplisomes. As illustrted in Fig. 2, y-irrdition of the DNA/grose slices from the erliest time points in the step-wise selection of 1OB3 cells clerly showed relese of mplisomes tht migrted with the fster moving nd when the DNA smple ws from cells resistnt to 1.8,uM DL-MTX. There is hint of these elements lso in the smple from cells resistnt to 9 nm DL-MTX nd possily lso in smples from cells resistnt to 18 nm DL-MTX nd from the prentl HeL BU25 cells. A similr nlysis (Fig. 3) reveled the presence of mplisomes, migrting minly with the lower nds, in the DNA smples from cells grown for 14 or 17 weeks in the sence of MTX. DISCUSSION Severl min conclusions hve een derived from this work. First, mplisomes could e recognized, during the step-wise selection in MTX of 1OB3 cells, s erly s the first detectle mplified genes. This oservtion is consistent with the possiility tht the relese of the first of these elements from the chromosoml DNA [presumly from the origin locus in chromosome 5 (17)] ws, in this cell line, the initil or very erly event in the gene mplifiction process. Furthermore, the finding tht, in cells exmined t vrious stges of MTX resistnce, the DHFR gene numer nd the mplisome numer incresed in prllel supports the hypothesis tht the repliction of mplisomes ws the only or min mechnism underlying gene mplifiction in this system. The results of y-irrdition or Sl I digestion of the DNA/grose slices prior to FIGE lso rgue strongly tht very few, if ny, of the

5 Genetics: Puletti et l. DHFR genes in 1B3 cells re not ssocited with mplisomes. The oservtion tht y-irrdition is more effective thn Sl I digestion in relesing mplisomes from the grose slice is consistent with these elements hving circulr structure. The elements tht remined trpped in the gel slices fter the most extensive y-irrdition or Sl I digestion my represent circulr molecules tht ecme nicked s result of y-irrdition or of Mg2" ctivtion of endogenous nucleses during Sl I digestion or represent oligomeric or conctented circles. The surprising oservtion tht, even fter extensive y-irrdition, the rod nd of heterogeneously sized frgments contining DHFR-specific sequences migrting ner the ottom of the gel in FIGE did not increse in intensity suggests tht these frgments my not rise from rtifctul degrdtion of mplisomes during preprtion. An interesting possiility is tht these frgments re growing DNA chins relesed from replictive intermedites. The DHFR gene mplifiction system of HeL BU25-1B3 cells, descried ove, exhiits some similrities to tht of the extrchromosoml elements of Leishmni (3, 22, 23). Whether the first mplisome in 1B3 cells ws derived from the chromosoml copy y repliction, s hs een proposed for the R circles (4) nd H circles (22) of Leishmni, or y excision, s hs een suggested for the CAD mplisomes in CHO cells (24), hs to e determined. The present oservtions suggest tht, during development of MTX resistnce, there ws no pprecile integrtion of mplisomes into the chromosomes or conversion of mplisomes to DMs. In fct, the ltter hve een found not to enter the grose gel during FIGE under the conditions used in the present work (5). The pprent filure of the DHFR mplisomes in 1B3 cells to integrte into chromosomes either during the course of gene mplifiction or during long-term growth of 1B3 cells in the presence of high concentrtion of MTX is intriguing nd in contrst to the generl tendency tht hs een oserved in vitro for DMs (23). This property mkes such elements potentilly useful for the construction of mmmlin expression vectors. The filure of the mplisomes to e converted to DMs ws confirmed y the finding tht no ovious chnge in their size ws oserved during 9 months of exposure of 1B3 cells to incresing concentrtions of MTX or fter 1-month exposure of 1OB3S cells to the mintennce concentrtion of MTX. The ehvior of the DHFR mplisomes in 1B3 cells differs in this respect from tht which hs een reported for the CAD mplisomes (24). On the other hnd, kryotypic evidence hs een otined (5, 1) tht 1B3 cells contin very smll verge numer of DMs. It is, therefore, possile tht these rose y different mechnism not involving mplisomes. The instility of the DHFR mplisomes oserved in the sence of selective pressure indictes tht these elements re centromeric, s ws shown for the DMs (24) nd for the CAD mplisomes (8). On the other hnd, the finding tht the rte of loss of the DHFR mplisomes in the sence of selective pressure ws much slower thn expected from dilution of nonreplicting DNA molecules suggests tht they replicte utonomously, s ws reported in other systems (6-8). Proc. NtL. Acd. Sci. USA 87 (199) 2959 This work ws supported y grnt from the Mrgret E. Erly Medicl Reserch Trust, y Ntionl Institutes of Helth grnt (GM11726) to G.A., nd y Fellowship (AIFCR 88-14) of the Americn-Itlin Foundtion for Cncer Reserch to G.P. 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