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{71t lSe-tt130187). ONCOGENES ACTWE IN COLON CANCER 5O188959

.'Ibis proposal presents e.xp~rlm~nts c~s%~n~ ~o allc~w ~be :-.en~1flcar, on. Conln~ anc~ cl~racterlzation of ct~co~enes which may t=e revolved in the ~;enes~s of human colon cancer and to study their role in the regulation of cell prolifera~on. Towards this end we have developed a sensitu,-e new assay capable of detemng oncogenes by virtue of their abilit,,¢ to induce cell proliferation in a defined scram-free medium in the absence of normally essential gro~,~h factors. The original l:m~osai emmc~a:ed three spe~fic a~ms: fl) to screen DNA ~solated malignant, premaltgnant arid normal tissue of colon cancer patients for DNA sequences that allow N I H3T3 cells to prolffe~te in the absence of [Blatelet-denved growth factor (P]aGF} and fibroblast growth factor (FGF); {2) to determine if the detected human D:~A sequences are related to any known oncogene; and {3~ to isolate any novel genes detected by ,.molecular cloning, The first two aims have been accomplished and our efforts are now fc~ased u~on the ciontng and charactenzabon of two [0otentially he:,..x.'enes Our orlsj~al proposal e,~.~s~one~ a two-staged apporach. First, to use the serum-free. growth-factor dependent ass,~:; systems to examine the patterns of oncogene activation in human colcm carcinoma. In this regard, we ~]anned to studv 32 DNA prel~araticms isolated from .carcino,-~as. polyps, and apparenily normal muc.~sa by ~wo types of a7 r4~ transformation assay systems, the traditional dense-focus assay, and the newly devek:rped det~ned serum-free media assay. Such a screen of primary human tisssue would also allow us ~o achieve our second goal, an evaluation of the defined serum- free media assay~ Ovr previous studies have indicated that the serum-flee selection ~s capable of detecting classes of oncogenes that ,~ail to respond in the serum-based focus assay. "['hus this system should be useful for uncovenng new classes of oncogenes, or other genes involved in intracellular slgnaling~ There are three steps to this approach. First, DNA isolated from normal, premalignant or mahgnant hssue is examined for the abihtv to reduce either dense loci on cells gro,~,~ng =n serum-supplemented medium, or ~o]onies of pru]iferat~ng cells in a serum-~ree medium from which a s=n~le essential gro~,~h-factor has been omitted lsee below) Secondary transfection and selection experiments are peffo.."meal to confirm that the appn.~.Donate phenotype can be passa~3ed ~ z,~ cellular DNA.."~..e res[:x'mse of each preparation ~s recorded m the Umvers~tv oi ~]tt~bur_~'s Human Colon C.arc~noma I~, ~..le~-'~ i~,-ICCP~ data base for ~ater correia:~ve studies ~h patholosy and prod.moses Stage two involves analv~s ~f the h~man sequences in the selected lines b~." restncuon enzyme anal~'s=s and Southern tr.~nsfer and hybridization With the appropnate repetitive sec~uence probe. ]n ~me cases hybridizations will be pefforrn,,ed to determine ~ the selected hvman sequences share homology' to t:~'eviously ider.t:,ffed oncogenes. [f stage ~. and stage 2 anal~ sesm..,.a,~ "~ ..... that a new human gene ~s camecl by one of the selected !rues, or a novel ~..~e o1 activation of a know ~,n¢o~ene. ~he gene will be ~solated by gene ciomng. Thus three ~il revolve t~e :s.~;a::on and charactenza=~on o~ selected genes. 50188960

',Ve ~.a,;e m~t!ated tl~s .Dro.~ram v.~th the h~Ip cf seed pa~d;n~ tr~m the t'nl'.~r~.'v of .Fittsburgh, the Pit~.shurgh Cancer Inst:m.~e. and a grant CTR~g6, (this proposal} IY~rn. the Council i:orTnh%cco [<¢.¢earc.h %~'hat I~o]|ows 1-~ a summary of our progr~s throu~h .November. !9S7 Screening Primary, Human Tissue for Onco~enes (a) !solfftk~n t~f.DNA. For our initial studies we planned to analyze approximately 32 different DNA preparations, including I~. ~omas. 6 pol~s. 6 nodal colonic mu~a, 6 ~ l~phc~es, an~ 2 metastases to lhe lun~..~I tissues have been obl~ed ~m the HCCF Tumor Bank. H~ mol~lar wei~t DNA is prepared ~om this p~a~ ~ssue by standard methods. Frozen tissue is homogenized in a dounce h~ogeniz~ in the p~ence of 1% SDS, and treated ~th Drote~nase K. ~e l)~te is e~ed successively ~ith ph~ol~hlomfo~, and chlorofo~ alone, then diai~ e~ensively a~mst Tds- EDTA ~ffe~ {TE). Fo!lo~ing ~ea~ent ~th dbonudease and a s~nd round of ~nic e~ons, the DXA ~s a~ain dialled against ~ and if necessa~, ~c~t~t~. (b) DNA Transfectk~n High molecular ~'ei~,t DNA is introduced into NIH3T3 cells by the calcium phosphate precipitation method of Craham and van der E'b as modified by Wi~er et ,~/. A CaPO4 prempitate containing 2.5 or 75 I~g of human DNA and 5-I0 l~g of the plasm~d p~. HL72.. which confers res~stani:e to the dru~ G418 in mammalian cells, is added to 5 × 105 cells growing in a I0 crn culture dish. Following a shock treatment ~,,~th ~Jycerol, the cells are refed with serum-supplemented medium and maintained at 37oC. ior 48 hr. The cells are passed I to 3 to fresh 10 crn dishes and those'cells which have taken up exogenously added D,N'A are selected for by the ~ddRion of CA18. Follo~,ng t~o weeks of selection in the presence of the d~, G41S resistant colonies are counted and pooled. (c) Focus-formal'ion Assay. Cells co~ransfected ~.~th high molecular weight human DNA and pSH L72 and preselected ~or resistance to G4~, 8 are pooled so as to rep~ese~;t a population of greater than 500 G418-resistant colonies. The cells (approximately t001 ~rom this pool are distributed to 10 cm dishes and maintained in medium supplemented ,.vith 5% calf serum. The medium is changed every 3 to 4 days. Dishes are sc~red for the presence of foci of dense-multilayer~d cells approximately two week~ afte~the cells teach confluence. Since cells carrying human DNA are preselected and allo~,ed to proceed through several rounds of division dunng maintenance in the pn~sence of G41,8, the transfer of a single dominant gene inducing focus formation should result in multiple foc~ on each dish. Samples are considered positive for .~ocus formation if at least 3 loci/d~sh are observed. ld} Serun~-free Assay. in '.his assay cells are selected on the basis of their ability proliferate in the absence of a specific ~7owth factor We have sho~,,'n that .Nlh3"l.'3 cells ~'ow. in a serum-.fi-ee basal nutnent medmm supplemented ,,~,ith fibn.-mecttn ( 50188981

transfernn (TF), el=Ic~errnal ~'~':,'t.h fa~c]r (EGI=). Insulin {In~. ants platelet derived gro~,,'th factor (PDGF). The~e ceils either fail to ~ or i~ro~," e.'ca'emely ~lowly in serum-free medium missing e~ther In, EGF, or PDGF. but containing the other four factors. Furthermore, we have shov~ that different oncogenes affect the gro~th factor requirements of these cells in specific ways. Similar resuRs have recently been reported by other ~ For example in our system, cells expressing the SV40 large T antigen no longer require In, EGF, or PDGF t~ pn~liferation. Cells expressing an activated H-raspmduct no longer require EGF or I"DGF, but still'do require normal amounts of In, while cells overexpressing the mouse c-m.kcgene have reduced insulin - and EGF requirements, but s'cill require PDGF. Thus we conclude that maintenance of cells in these different media provide different selec..Eve pressures w~th regards to cell proliferation. We predict that these wdl allow the detection of oncogenes not detected by the focus-formation assay. Cells representing at least 500 C__.-418 resistant colonies are collected by t~,psin~zation and pooled. Our ex'penence has shown that sing!e-copy genes can be detected v,~th th~s number of colonies. TDpsin is inactivated by addition of purified soybean trypsin inhibitor. The cells are.pelleted and washed with a 1:1 mixture of FI2 Hams medium ~and Dulbecco's .Modified EagJe medium (basal medium) to remove residual serum and the inactwated t .rypsin. The cells are counted, and diluted to a density of 5 x 103 • ce~s/ml in basal medium, and plated onto 35 mm dishes, 2 ml per dish (/e. ate density of I x 103 cells/cm2). The appropriate growth factors are added to the dishes at final concentrations o~ l~bronectin 10 pg/ml, transferrin 25 pg/ml, insulin 10 ~g/ml, EG'F 10 ng/ml, and PDGF ! half-maximal unit per mL Ten dishes of each selection condition are plated in each experiment and the media is chan~l every 3 - 4 days. After two weeks of selection, the dishes are scored. If discrete colonies of actively droving cells represent the majority of living cells on the dishes, the cells are passed to mecltum containing serum, amplified and stored in liquid nitrogen. If the dishes ' contain many living cells not assignable to discrete colonies, the cells are trypsinized, .~oled and ~lated once again at ! x 103cells/cm2 and subjected to another 2 weeks ei selection for i~rov,%h factor independence. (e} AnaK..~is of Data. Each DNA preparation is scored forits ability to induce a res[~onse in the focus-formation or the serum-free assay. We originally anticipated We types of results: (ll ~egative on 10c~th focus assay and In all serum-free selections. In this case the negative results are recorded for later pc~sstble correlation w~th tissue- .type. and ~o further experiments are done with this sample. {2) Positive in the focus formation assay and forms colonies of transformed cel[,~ under one or more of the serum-free assay conditions. In this case we pursue study of the preparation by focus.mrmation in serum-supplemented medium. This assumes tha~ a single oncogene in the D.~A preparation is responsible for the response in bo~h assa3: systems. 50188962

(3) Pos~t~'~ ~n tl~e ~cc~s (orrnatI~n assay and ne~a~ve in a!l serum-nee se:e~lons. The transforrnin~ se~ences ~}} be ~ursued b.V the locus-assa.~: (4) .Ne~-abve In the ~¢c~s-furmat~on ,~ssay, but posltlve in one of the serum-free conditions. "['nis ts potent~aIly the most important type ef result It may indlcate the pre~ence of a~ cmcogene that does not respond in the tradltiona[ focus assay. Such cases have been idenhfied (see below) and are being pursued using the serum-free system. (5} Negative in the focus-formation assay, but positive under two or more serum-free selections. A~ we have indicated above, it is possible for a sin~le oncogene to alter the cellular requlrements for.multiple growth factors. If such cases arise we v,~ll tentatively conclude that the responses are due to the presence of a single 0ncogene in the preparation, and this gene will be pursued in one of the serum-flee conditions, chosen on the basis of economics and/or technical grounds. if) Seconda .ry and Tertian- Transfections. High molecular weight DNA ts prepared from cell lines expanded from ioci or colonies isolated in the assays described above. These D~N'As are examined by Southern transfer and hybridization for the presence of distinct sequences, homologous to the human repetitive a/usequences present in the BLUR8 probe (75}. Based on our previous experience, we expect all of the selected lines to contain som~ human DNA, but they van] wide.h,, in amount. ~ollowing confirmation" of the presence of human sequences, those D.NAs will be introduced to normal cells by transfechon and placed under selective conditions identical to the one used originally. Secondary transforrnants will be isolat~l, cloned, and expanded as described for pnman/, transformants. Cellular D.~Ais prepared and examined for the presence ot sequences homologous to the BLUR8 probe. In some cases the DNA will be passed through a third round of transfection and select'ion. If a DNA preparation scores positive through t~'o or more rounds of transfection/sele~.~on, and if the resulting cell line contains a simphfied pattern of human repetitive D.~A. we conclude that St contains a human gene of interest. Such samples are pursued to identify the gene and examine its arrangement in normal and malignant tissue. {g) Preliminary. Res~ We have initiated stage I, the screen~n8 of primary human tissue for the presence o(oncogenes, and are proceed:ng to analyze the results o~ .~hese initial exi~enments. NIH3T3 cells were cotransfected v.~th DNA isolated from tissue and the bactmial plasmid pSHL72. C,418-resistant colonies were !~oled and maintained either in medium supplemented ~,,~th 5% calt serum and scored ior the presence of loci, or in a serum-free medium, and scored for colonies or populations of proliferating cells. Four types of selective serum-free conditions were employed, the complete medium (supplemented with all five gro~h ~actors). -PDGF (comp]ete medium lacking PDG~, -EGF, and medium missing both insulin and EGF. 50188983

"I'nus tear D.~A prepared from ten adenocarc~ncmas, one villous adenoma, one lung metastasis, ~ur ap1~arently normal mucosa, one mucosa from a case of Cardner s~'ndrome as well as l:rom Iyrnphoc~es of this same patient, and one mucosa fi-om a case of dlverttcu!itis, are at venous stages of ana[ysis` The response ot~ these pmparaticms in each of the m ~,#rob~olo~ical assay systems tested is summarized in Table I. Of ten carcinomas examined, three gave a strong positive response ~n the serum-based focus formation assay, the remaining scoring either marShal.or negative, DNA isolated from the apparently normal mucosa of a patient ~th Gardner syndrome also scored positive in this assay. DNA isolated from a single polyp, a single lung metastasis, and four out the five apparently normal mucosa tested also scored negative. Limitations of time and ~unding have nol~ allowed us to pursue samples that induce loci on N[H3T3 cells. Rather we have given priority to samples that respond in the serum-free system. Initially we concentrated on seven cell lines which were derived from these primary. transfection experiments. WTSci-P1) was selected as a population of cells proliferating in the absence of PDGF, follo~,~ng transfection w~th DNA isolated from an adenocarcinoma. Thig sample does not respond in the focus formation assay and the cell line selected in the absence of growt.h-factor retains a flat morphology, line ISPA" was isolated m a s;m~lar manner. Two lines, 11CA and IICB, were o .brained .by selection as rapidly proliferating colonies in the complete serum-free medium although this same sample also induced the proliferation of cells in the absence of PDGF. A single line, 13EA, was isolated as a population of cells proliferating in the absence of EGF. Two otherlines are of special interesL I}oth were derived from the same Gardner syndrome patient. DNA isolated from the apparently normal mucosa of this patient responded strongly in the focus formation assay. Tl~Is same sample also induced the proliferation of cells in the absence of EGF/in. The cell line PDm(-IEI} was isolated from this serum-free selection, but has a highly re~:ractile, transformed morpholo~;y. The line Pdl(-FI) was isolated as a population of cells proliferating in the absence of PDGF. This line was derived by transfection of cells with DNA isolated from the lymphocytes of the Gardner syndrome patient~ This line is of special mteres( because: the DNA sample does not give a response in the focus assay, the selected cells retain a.flat morphology, and the sample was derived from the somatic tissue of ,~ patient with a known genetic predisposition to colon cancer. DNA was prepared from each of these primary transformants and used in seconda;', transfection experiments. The results of these studies are summarized in Table 2 Based on the strength of the phenotype of the selected lines and on the presence or ,~ simplil~cl human-specific alu repetitive pattern as judged by Southern transfer hybridization to the BLUR8 probe, three of these secondary transfectant l;nes, i'4& ,~.~ and P40 have been chosen for further study. The human sequences present in P48 originally derived ~:rom the colonic mucosa ot., patient ~,~,th Gardner s,,,ndrome. The primary line, PDm{-IEI) was selected m the absence of both insulin and EGF. However, this sample also responded strongl.~ ~n the focus ~orrnation assay. B.oth of these pl~enot~,~es were passed dunngsecondar.." 50188964

transfect~ns, suEE,~stm~ that the)' a:-e lhe resu:,, o! lhe ~,c~c~n o£ a smgI~ selected gene or multiple genes c!osely linked. The secondary hnes P~6 and ~4D have similar properhes but were derived from different tlss~e-types in separate transfection experiments. Both lines retain a fiat morphology ~ ant capable of proliferation in the absence of the essentlal factor, PDGF. "I'he htrman sequences present in Pt~6 were ori~inally den~,ed fi'om the somatic tissue (lymphocytes) of a patient ,,~ath Gardner syndrome, v,:hlle those camed by ceIlline ~,0 were i.,;olated from a case of apparently spo~dic carcinoma. ~ example of the g~,~wth propemes o~ the ~6 and P,10 cell lines is sho~vn In these e~e~men~. I x ~ D~ cells we~ se~ed into 33ram dish~ and mainlined ~or ~ wee~ ~h~ in the complete se~m-Hee medium, or in medium missing PDGE ~e ~ intimate ~h.~t bo~h the P4O and the ~6 lines: (I) p~te ~ual~/well in ~e p~ence or absence ~f PDGE and (2) proliferate better than the parental ~IH3~ l~e or line 17 which ~as t~ansfected ~th salmon spe~ DN~ in both the presence ~ abs~ce of PDGF. t~3. sed on these expenments ~.ve conclude that we have selected at least three cell lines c~ntabling h~.man DNA ~th a reduced requirement for ~Io~h factors. Stage I[ analysis involves the prehminary characterization of the human sequences present i~ these lines. Characterization of Sequences Present in Selected Lines Line P48 ,c~nt.ains the human H- FaS ~en~. The p~'operties of hne P48, described above, are indicative of the beha'.nor of NIH3T3 ceils transformed b.v an activated H-ras gene. This possibility was examined by ~uthern transfer hybridization o, xpeffments using the I~rified insert ,h~,~m the Dlasmsd pEJ6.6 (76) "[l~e results of this e~eriment, sho~,m in Fibre 2, confirm ~he p~e~ence of multiple cc~p~es of the human H-rasl locus. ~,;'e ~o ~ot yet k"no,N ~f the ras gene in ~his line has been mutat~onally activated, nor ha~e we examined the statu~ of the ras gene in DNA derived from primary Ij.ne t~6 c~,~ntains h~_m_ap ,-; _e~.uences. Southern transfer and hybndization v,~th BLURS demonstrated :he presence of human repetm~e DXA in th~s hne (Fi&,u,-e ?,', Based on the ,,'~zes ol ;he observed fraI~ments, ;~e esbmate that thls line contatr~s at. least 35 Kb of human sequences. [n separate hyb.ndlzauon experiments we have shown that these sequences are not related to the human H-ras or .~-ras loci. Furthermore, examination c'..~ :he EcoRI restnc'bon enz~,Tne d~sest patterns. demonstrates that these sequences are not related to the human K-ras gene. Ci, onm~ ex1~ments with this line are currently under,.vay (see below). Line P40 c~ntams h_u,q'.:O..,_q~.~'!.c~,S.2[IRLaE~u_13r.elated to those pre~ent in FF;6..trace the prot~erties of line 240 ;~,ere s~milar to those of ["80, it is possible we have detected the same genem two ,~.de~e~.dent transfection experiments. However, the data 50188985

presentecl In Fl~n.tre 4 incltcates that thls ts n~t the cas~_ Tlaese I:nes have clearly distinct restricbon frn~nent patterns when hybridized v.~th a human rel~titwe sequenea-sFeeifi~" probe. Examination of the digest patterns using several different restrt~on en ,zymes confirms this observation tdata not shov, r~}. Further experiments. with this line are discussed below. Clomng Genes from Selected Lines Cur clccning experiments have been directed towards isolating the human sequences presentin P86 and P40. A library was constructed from EcoR1 digested P86 DNA m the larnbda vector EMBL4 (78). Approximately 450,0130 plaques were screened for homolo~ to human repeti~,ive sequences using a human a/t,,-spedfic probe. £!even positive plaques were ~lcked Anal)~is of these clones by restriction enzyme digestion. showed that 9 of the I 1 contained the same 12 Kb insert. Subsequently we have cloned this insert into the p.SF,322 denvitive, pKF772. This plasmid is called p6D-23. We • have prepared an independent hbrary from ]:'86 in EMBI_3 using a partial MboI digest of cellular DNA. Of 600.000 plaques screened, 29 were identified as carrying inserts homologous to the human alurepeat. So far, eis~at more inserts have been identified and partially mapped. Frehm~nary maps of the human inserts present ~ p6D-2.3 and the lambda clones listed above, ~,lth the approximate locations of the sequences homologous with the BLURB probe are shown in Figure 5. From line P40 four d~fferent alu containing sequences ha~e been cloned into EMBL3 {lambda 4A, 5B, 7B, and IIA). r':Yeliminary analysis indicates that not all the human sequences in P40 have been cioned. Two of these clones also contain mouse repetitive sequences (IIA and 713) while the other two do not. Expression of Human Sequences in P40 Some of the clon~s described above have been used as probes to detect human-specific ILNA isolated from F40 and P86. Northern blot analysis of RNA samples from cell lines P40 and NIH3T3 were isolated under the following conditions: (1) serum star',cal. (2) serum-stimulated; and (3) defined serum-free medium eontainirig all essentaai growth factors. Two bands of approximately 2 Kb and 4Kb were e:<press.ed in P40 cells growing in serurn-,i'ee, but not serum-containing or serum-starved cells. These RNAs were not detected in NIH3I'3 cells under any of the conditions tested. We are currently pursuing these observations. 50188966

T~le 1 P=ima=y Transfection Assays~ pipas, J~s H. Selected Sample. Tissue2 Colonies cell-line s5 TGc-35 C - 394 ÷ TGc(-:--I) WTSc-62 C 710 - WTSc (-PI) JBc-29 C ND - T3(~) C Iii0 +/- IICA, IICB ". SN(~) "C 1260 - 13EA FH(c) C 420 - 1SPA SS(c) C 270 + WS(c) C 520 +/- WT(c) C 996 +/- WTSw-61 M ND - TNm-76 M 1137 - SN(DM) M 1040 - WS(DM) .M 834 + WT(DM) M 1137 - :TB(p) P 1180 - ECc-13 LM ND - .PDI-14 L 117 - • PDI (-PI), PDm-5 M ND ÷ PDm (-IEI) PDm-78 M 1045 + Salmon sperm 430 - NIH3T3 786 - Se!eculon~ ND - I~D ND +/- ND +l- ~D. - +I- + -- +/- I%I'D ND _6 ND + ND +I- +I- +I- ~ +/- ND ND ÷ ND ~ "+6 23 50188987

Origin of tissue from which ~ wa, isolated. Rder~c~rcino,~o, C; M, (~entlsl normal colonic mJcoso; P, polyp; LM, r~tosto$i$ to lung; ! u~h~cy re. Total number of G418r~ colonies plated in ~elective ~edia. I~, r~t detor~ined. Scruples were scored l~i tiv@ fop fccu,J-for~<~tio~ i f )6 f~ilcel I ~puloti~ ~i~ int~iote ~s ~e listed ~ +/-. S~i~ were ol~ s~ f~ tM ~e of collies of ~olif~oti~ cells in t~ ~lete ~i~, ~ in ~di~ fr~ ~i~ ~, E~ ~ ~n ~itt~. ~le ~e 5 Cell line~ derived fros pri~:r~ tronsf$ctions that ~eee used to isolate Ot4R for r~c~'K~-y trc~fection oxp~rim~nt~. • ~is s~lectio~ w~s also ~ in tM ¢~b~ of in~mJl~n. 50188988

?i~as, Ja=es .~i. Table 2 Secondary Transfecfion Assays Selection1 G418r Colonies Tested focus. Comvlete -PDGF Selected -EGF cell lines PDI (-P1) 1055 ND ND + W'I'S c (-P1) 500 ND ND +/- PDm (-IE1) ND + ND ND llCA 490 +/- +/- 11CB 650 - - + 13EA 1000 - ND ND 1SPA 490 + +/- + ND 40 ND 41 +/- 42 N'D 43 26 50188989

~-alls / dish 50188970

Legend to Figure 1 Cells were seeded at 1 x 10~ cells/3s mm dish and maintained in the complete serum-free medium (black bars), or in medium lacking PDGF (stipled bars). Cell numbers were determined 9 days (A) or 14 days (B) after.plating. 27 50188971

Pipas, J~es ~I. 29. 50188972

Pip~s, Legend to Figure 2 Cell l~ne P48 is a secondary transfectant derived from a focus induced by DNA isolated from the a~pparently normal mucosa of a patient with Gardner syndrome. DNA from the P48 line was d~gested with EcoRl, BamHl, ¢~r Hind/I/ and electrcphoresed through a 1% agarose gel, transferred to nitrocellulose and hybridized against a human H~'as specific probe. The two lanes on the right show hybridization to a BamH1 digest of normal human DNA and to NIH3T3 cell DNA. 3O 50188973

Pipas, Ja~es ~. ~ 1.3 kb 2 31 50188974

LEGEND TO FIGURE 3 Lymphocyte DNA isolated from a patient with Gardner syndrome was used to transfect NIH3T3 cetls. DhlA Lsolated from colonies grown under selective conditions was used to obtain secondary transformants. 20 p.g of DNA from two secondary transformants was digested with EcoR/, resolved on ~ 0.8% agarose gel, and transferred to nitrocellulose. The filter was hybridized to & human a/u-specific probe labeled and by nick translation to a specific activity of 2.2 x 1G3 dprrdlJ.g. The filter was washed in conditions corresponding to Tm-27°C., and exposed for 20 hours on XAR-5 film with a Lighting Plus intensifying screen. 3-" 50188975

1 2 :33 50188976

Pipaa, Ja~.s ~. Legend to Figure 4 Southern hybrldizat~on of high molecular weight DNA from serum-free selected ceil lines to the human Alul repetitive sequence. DNA was digested with EcoRI, • eiectrophoresed on a 1% agarose gel, transferred to nitrocellulose and hybridaized to the human.specific sequences present in the BLUR8 probe. Lane 1 and 2 contain DNA from cell line 40 and P86 respectively. 50188977

RH II H S S H R I I ! I I p6D-23 R R R R ! I I / A26 R R R R ! I,, ,' '~ )~ IOA R R R R I I I I I R R I I .I 2 4 & e .10 I2 I~t 16 size ~n kb Fig. 5 : Maps of genomic clones of P86. Shown are restrictionmaos of the inserts of four of the five clones derivecl from P86. The fifth clone, 15B is not included in the diagram. The EcoRl fragments of clone 13A have not been ordered, and therefore are diagramed discon- tinuously. Heavy lines designate fragments that contain human ah/ sequences. R, EcoR1 site; H, Hind Ill site; S;Sacl site. The spatial relationship between the four fragment~ has not yet been determined. 35 50188978