Proc. Natl. Acad. Sci. USAVol. 85, pp. 7302-7306, October 1988Genetics

Coordinate regulation of two genes encoding gluconeogenicenzymes by the trans-dominant locus Tse-1

(tissue specificity/microceli hybrids/extinction)

JANIS LEM, ALLISON C. CHIN, MATHEW J. THAYER, ROBIN J. LEACH, AND R. E. K. FOURNIER*

Department of Microbiology and the Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, CA 90033

Communicated by Salome G. Waelsch, June 17, 1988

ABSTRACT Tissue-specific extinguisher-i (Tse-1) is amouse genetic locus that can repress liver-specific tyrosineaminotransferase gene expression in trans. To search for otherTse-l-responsive genes, hepatoma microcell hybrids retainingmouse chromosome 11 or human chromosome 17, containingmurine Tse-1 and human TSEI, respectively, were screened forexpression of liver-specific mRNAs. While most liver geneactivity was unaffected in such hybrids, phosphoenolpyruvatecarboxykinase and tyrosine aminotransferase gene expressionwas coordinately repressed in these clones. Extinction of bothgenes was apparently mediated by a single genetic locus thatresides on human chromosome 17.

Cellular differentiation is generally viewed as an orchestratedprocess in which specific gene sets are activated or repressedat defined developmental times. These events culminate inthe establishment of lineage-dependent patterns of transcrip-tion that are the basis of cell specialization. The molecularmechanisms that control these processes are poorly under-stood.

Tissue-specific genes are primarily regulated at the level oftranscription (1, 2), and discrete sequence elements arerequired in cis for proper developmental control (3). Theseobservations support the widely-held view that trans-actingfactors play key roles in regulating eukaryotic gene activity.Further analysis of this mechanism of gene control willrequire the characterization of specific regulatory factors inboth genetic and biochemical terms.The first clear evidence for trans-regulation of differenti-

ated functions in mammalian cells was reported by Davidsonet al. in 1966 (4). They observed that melanoma-fibroblasthybrid cells failed to produce the melanin pigment charac-teristic of their differentiated melanoma parent. This "ex-tinction" phenomenon proved to be both general and bidi-rectional: most stable hybrids formed by fusing distinctlydifferent cell types fail to express the tissue-specific productsof either parent (5, 6). However, extinguished traits can bereexpressed in hybrid segregants that have eliminated chro-mosomes of one of the parental cells (7-9). In reexpressingsegregants, heterologous gene activation may be observed(10, 11). Thus, expression of tissue-specific genes can bemanipulated experimentally in intertypic hybrids, and thisprovides a system with the potential to define genetic factorsthat regulate gene activity in trans.

Liver-specific gene expression in intertypic hepatomahybrids has been studied for many years, and, thanks largelyto the work of Weiss and coworkers (7-11), this remains themost comprehensively analyzed hybrid cell system to date.Two important facts about tissue-specific gene expression inthis system have recently been established. First, virtually allliver-specific gene activity is repressed in genotypically

complete rat hepatoma-mouse fibroblast hybrids, but reex-pression occurs upon loss of relatively few fibroblast chro-mosomes (12). Second, extinction of particular liver genes ismediated by discrete genetic loci that map to single murinefibroblast chromosomes (13, 14). These tissue-specific extin-guisher (Tse) loci affect expression of unlinked structuralgenes in trans.The studies described in this report were designed to

investigate the possibility that individual Tse loci might affectexpression of multiple liver-specific genes. We report that thegenes encoding two gluconeogenic enzymes, tyrosine ami-notransferase (TAT) and phosphoenolpyruvate carboxyki-nase (PEPCK), are coordinately regulated in trans by thepreviously defined locus, Tse-1 (13).

MATERIALS AND METHODSCell Lines and Culture Conditions. The rat hepatoma lines

FAO-1 (13) and FTO-2B (15) are derivatives ofH4IIEC3 (16).Mouse embryo fibroblast (MEF) cultures were prepared bystandard techniques (17). The isolation and characterizationof rat hepatoma-mouse fibroblast hybrid clones FF5-1 andFF3-3 have been described (13). Virtually all liver-specificgene activity is repressed in these karyotypically completehybrid clones (12).Rat hepatoma microcell hybrids were also used in these

studies (13). F(11)J, F(11)U, F(11)Y, and F(11)G are micro-cell hybrids that selectively retain mouse chromosome 11,while FB(11)J, FB(11)U, FB(11)Y, and FB(11)G are theirrespective back-selectants from which chromosome 11 hasbeen removed. Similarly, rat hepatoma microcell hybridsHF(17)E and HF(17)I selectively retain human chromosome17, while their back-selectants [HFB(17)E and HFB(17)I]have segregated that single human chromosome.The 7A-series clones are a set of deletion hybrids that

retain fragments of human chromosome 17. These lines wereconstructed by microcell fusion using donor cells [L(17n)C]in which the retroviral vector ZIPneoSV(X)1 had integratedinto human chromosome 17 (18). Fragments of that humanchromosome were transferred and fixed in hepatoma recip-ients by selecting for the G418-resistant phenotype encodedby the neo gene of the integrated viral vector. The isolationand characterization of this set of deletion hybrids will bedescribed in detail elsewhere (R.J.L., M.J.T., and R.E.K.F.,unpublished observations).

All cells were cultured in 1:1 (vol/vol) Ham's F12medium/Dulbecco's modified Eagle's medium with 10%(vol/vol) fetal bovine serum and without antibiotics asdescribed (13). FF-, F(11)-, and HF(17)-series hybrids werepropagated in medium supplemented with hypoxanthine/

Abbreviations: TAT, tyrosine aminotransferase; PEPCK, phospho-enolpyruvate carboxykinase.*Present address: Department of Molecular Medicine, Fred Hutch-inson Cancer Research Center, 1124 Columbia Street, Seattle, WA98104.

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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 85 (1988) 7303

aminopterin/thymidine (HAT), whereas the 7A-series cloneswas grown in medium containing 500 pmg of G418 per ml.RNA Blotting Analysis. Cytoplasmic RNA was isolated

from the various cell lines by sequential extraction withphenol and chloroform extraction (19). Blots were preparedby fractionation of the RNA samples on 1.2% agarose/formaldehyde gels followed by capillary transfer onto Zeta-bind (AMF-Cuno, Meriden, CT). Slot blots were also pre-pared on Zetabind membranes with serial dilutions of theRNA samples in 7.5 x standard sodium citrate solution (SSC;1 x SSC is 0.15 M sodium chloride/0.015 M sodiumcitrate)/4.3 M formaldehyde. All blots were UV-crosslinkedto immobilize the RNA and were prehybridized for severalhours at 420C in 1% bovine serum albumin/2 mM disodiumEDTA/500 mM sodium phosphate, pH 7.2/5% sodium do-decyl sulfate (SDS). Hybridizations were performed at 420Cin fresh buffer containing probe labeled with 32P by nick-translation (specific activity, 2-4 x 108 cpm/,4g) or byrandom hexamer-primed labeling (specific activity, 109cpm/,ug). Blots were washed sequentially in 2 x SSC/0.1%SDS (15 min at room temperature), 0.2 x SSC/0.1% SDS (15min at room temperature), and 0.2 x SSC/0.1% SDS (30-60min at 550C). Autoradiography was for 2 hr to several dayswith Kodak XAR or XRP film with a single intensifyingscreen at - 70'C. Densitometry was accomplished by usinga Hoefer scientific scanning densitometer (model GS300)with a Hewlett-Packard integrator (model 3392A).DNA Marker Analysis. High molecular weight cellular

DNA (20) was digested to completion with either EcoRI orHindIII (New England Biolabs), electrophoresed on 0.5%agarose gels, and transferred to Zetabind membranes by themethod of Southern (21). Plasmids pUC8TK (22), pTHH59(23), pMO4-6 (24), p10-3 (25), Hf677 (26), and phPKC-a7 (27),containing sequences from the human TK, THH59, HOX2,MYH, COLIA1, and PKCA loci (see Table 1), respectively,were oligolabeled by using random hexamer primers andwere hybridized to membrane-bound DNA as described (28).The filters were washed in 2 x SSC/0.1% SDS for 15 min atroom temperature and then in two changes of 0.1 x SSC/0.1% SDS for 30 min at 65°C. Autoradiography was for 24-72 hr at - 70°C with Kodak XRP film and a single screen.Under these conditions, the probes containing TK, THH59,and MYH sequences hybridized specifically to human DNA;no cross-hybridization to homologous rodent sequences wasobserved. For the HOX2 and COLIAI probes, hybridizationto both human and rat DNA occurred, but human-specificrestriction fragments could be obtained after digestion withHindIII or EcoRI, respectively. Human-specific PCKA re-striction fragments were obtained with either enzyme.

RESULTSHepatoma microcell hybrids that retain only mouse fibroblastchromosome 11 fail to express hepatic TAT activity or toaccumulate TAT mRNA, but removal of chromosome 11from the cells by back-selection results in reexpression of theTat-i gene product to full parental levels (13). In contrast,hepatoma hybrids containing a variety of other fibroblastchromosomes continue to express the Tat-i gene at levelscomparable to those of parental hepatoma cells (29). Thesedata indicate that extinction of Tat-i expression in hepatoma-fibroblast hybrids is a specific genetic effect mediated by adiscrete locus on mouse fibroblast chromosome 11. Thislocus, which can repress Tat-i activity in trans, has beendesignated tissue-specific extinguisher-i (Tse-i) (13).To identify other liver genes whose expression was regu-

lated by loci on mouse chromosome 11, hepatoma microcellhybrids retaining that single fibroblast chromosome werescreened for expression of liver-specific mRNAs. Cytoplas-mic RNAs from parental and hybrid cells were immobilized

on Zetabind, and the blots were probed with labeled cDNAclones. mRNAs encoding liver-specific TAT, PEPCK, trans-ferrin, serum albumin, alcohol dehydrogenase, and the prod-uct of pliv10 were all expressed in hepatoma cells (FTO-2B),but none of these sequences was detected in RNA preparedfrom mouse embryo fibroblasts (Fig. 1). Although all of thesetissue-specific mRNAs were extinguished in genotypicallycomplete hepatoma-fibroblast hybrids (12), most continuedto be expressed in monochromosomal clones that retainedonly fibroblast chromosome 11 (Fig. 1). For example, trans-ferrin, serum albumin, alcohol dehydrogenase, and pliv10mRNAs were expressed at similar levels in parental hepa-toma cells (FTO-2B), in monochromosomal hybrids [F(11)Jand F(11)U], and in hybrid back-selectants [FB(11)J andFB(11)U]. Other liver genes, including those encoding ala-nine aminotransferase, aldehyde dehydrogenase, sorbitaldehydrogenase, aldolase B, pyruvate kinase, a1-antitrypsin,and the products of pliv7, -9, and -10 showed a similar patternof continued expression in the F(11) clones (ref. 13; also datanot shown). Thus, extinction of these genes in intertypichepatoma hybrids (12) is mediated by genetic loci distinctfrom Tse-i.

In contrast to the continued expression of most liver genesin the F(11) hybrids, Tat-i and Pck-i (the gene encodingPEPCK) expression was specifically repressed in theseclones. For example, scanning densitometry indicated thatTAT mRNA levels in F(11)U and F(11)J were only 5% and11% of parental FTO-2B levels, but the respective back-selectants [FB(11)U and FB(11)J] reexpressed TAT mRNAto full hepatoma levels (Fig. 1). These results are in accordwith previously published observations (13, 29). Signifi-cantly, PEPCK mRNA levels were also depressed by factorsof 10-20 in the F(11) hybrids but were restored to parentalhepatoma levels upon segregation of fibroblast chromosome11 (Fig. 1). Thus, the genes encoding TAT and PEPCK werecoordinately extinguished and reexpressed in F(11) hybridsand their back-selectants.To verify that accumulation of TAT and PEPCK mRNAs

was specifically affected in the F(11) hybrids, blot-hybrid-ization experiments were performed (Fig. 2). RNA sampleswere fractionated on agarose/formaldehyde gels, transferred

TAT j Il IPEPCKg PI|

TRF

RSA

ADH

plivlO

IIlI l111111I11| ||II

egg 111,111 i1Ig I I I- ...____ II .......I._ _

ciTU I I

I LOpg RNA: u? c'

I I

<4Tt~~~~I--Ir1

FIG. 1. Expression of liver-specific mRNAs in parental cells,monochromosomal F(11) hybrids, and back-selectants. Concentra-tions of RNA in each slot are given in the key at the bottom of thefigure. TRF, transferrin; RSA, rat serum albumin; ADH, alcoholdehydrogenase; aTU, a-tubulin.

Genetics: Lem et al.

I

Proc. Natl. Acad. Sci. USA 85 (1988)

abc de fghi 1k Imno

A

Wp

B

VO W W 4WOP-WfO4

C

FIG. 2. Expression of TAT, PEPCK, and a-tubulin mRNAs inrat-mouse (lanes a-f) and rat-human (lanes g-j) microcell hybrids.Blots of cytoplasmic RNA were hybridized with labeled probes forTAT (A), PEPCK (B), or a-tubulin (C) as described. a, F(11)G; b,FB(11)G; c, F(11)J; d, FB(11)J; e, F(11)Y; f, FB(11)Y; g, HF(17)E;h, HFB(17)E; i, HF(17)I;j, HFB(17)I; k, FF5-1; 1, FF3-3; m, FAQ-1;n, FTO-2B; o, mouse embryo fibroblasts.

to Zetabind membranes, and probed with labeled TAT (Fig.2A), PEPCK (Fig. 2B), or a-tubulin (Fig. 2C) cDNA clones.

In agreement with the results shown in Fig. 1, TAT,PEPCK, and a-tubulin mRNAs were all expressed in parentalhepatoma cells (Fig. 2, lanes m and n), but only tubulintranscripts were detected in RNA from mouse embryofibroblasts (Fig. 2, lanes o). In karyotypically complete hep-atoma-fibroblast hybrids, neither TAT nor PEPCK mRNAwas expressed (Fig. 2 A and B, lanes k and 1); in fact,expression of at least 18 different liver-specific mRNAs was

extinguished in these clones (12, 13). Significantly, accumu-

lation of both the 2.4-kilobase (kb) TAT mRNA (Fig. 2A) andthe 2.8-kb PEPCK transcript (Fig. 2B) was depressed by a

factor of 10-20 in F(11) hybrids (Fig. 2, lanes a, c, and e)relative to their back-selectants (Fig. 2, lanes b, d, and f).

Coordinate extinction ofTAT and PEPCK mRNA expres-sion was also observed in hepatoma microcell hybrids thatselectively retained human chromosome 17. This chromo-some, which is genetically homologous to the distal portionof mouse chromosome 11 (30), carries human TSEI (13).Accordingly, TAT mRNA expression was extinguished inhepatoma microcell hybrids retaining human chromosome 17(Fig. 2A, lanes g and i) but was reexpressed in back-selectants(Fig. 2A, lanes h and j). Expression of PEPCK mRNA was

similarly affected in these clones (Fig. 2B, lanes g-j). Thus,TAT and PEPCK mRNA expression was coordinately re-

pressed by genetic loci on mouse chromosome 11 or humanchromosome 17.The results described above were consistent with the

possibility that TAT and PEPCK were extinguished by a

single genetic locus on mouse chromosome 11 or human 17.Alternately, TAT and PEPCK extinction could involve two

or more distinct loci that were coincidently located on thesame chromosome. To distinguish between these possibili-ties, deletion hybrids that retained only fragments of humanchromosome 17 were used.The 7A-series deletion hybrids were constructed by micro-

cell fusion using as donors hybrid clone L(17n)C: these aremouse L cells that contain a single human chromosome 17into which the retroviral vector ZIPneoSV(X)1 (31) is inte-grated. This cell line was prepared essentially as described(18). L(17n)C microcells were fused with PTK-A7A rathepatoma recipients (an FTO-2B derivative; see ref. 32), andselection was applied for the G418-resistant phenotype en-coded by the neo gene of the integrated viral vector. Clonesretaining the marked human chromosome 17 or particularfragments thereof were obtained.To identify the chromosome 17 fragments present in indi-

vidual clonal lines, the 7A-series hybrids were screened forretention of six DNA markers previously assigned to humanchromosome 17. Results of this analysis for 10 informativeclones are summarized in Table 1. As expected, each hybridretained the integrated neo gene whose expression was underselection. The simplest hybrid was 7AC-5: it contained the neovector and flanking human DNA sequences, but none of theother chromosome 17 markers were retained. The other ninehybrids had progressively more complex genotypes, retainingspecific subsets of the human markers assayed. In total, atleast 8 different fragments of human chromosome 17 werepresent in this collection of 10 hybrid lines.The 7A-series hybrids were screened for expression of

TAT and PEPCK mRNAs to determine whether theseextinction phenotypes could be dissociated. Hybrids thatexpressed PEPCK mRNA (Fig. 3A, lanes a-d) also expressedTATmRNA (Fig. 3B), whereas extinguished hybrids failed toaccumulate either mRNA (Fig. 3 A and B, lanes e-g). In nocase was expression of TAT and PEPCK discordant. As adiverse array of chromosome 17 fragments was present in7A-series clones, the coordinate extinction or expression ofTAT and PEPCK mRNAs in these hybrids suggests thathuman TSEJ is a discrete genetic locus that mediates bothphenotypic effects.

DISCUSSIONHybrid cells formed by fusing different parental cell typesgenerally fail to express the tissue-specific gene products ofeither parent, a well-known phenomenon termed extinction

Table 1. Retention of human chromosome 17 markers in7A-series deletion hybrids

Deletion Marker geneshybrid PKCA TKI THH59 neo COLIAJ HOX2 MYHJ TSEI

7AC-5 - - - + - -

7AE-3 - - + + - - - -

7AC-1 - + + + - - - -

7AD-7 - + + + - - - -

7AE-27 + + + + - - - +7AD-1 + + + + - - + +7AD-6 - - - + + + + +7AE-6 - - - + + + + +7AE-12 + + + + + + - +7AE-31 + + + + + + + +

Genomic Southern blots of hybrid cell DNAs were probed for theindicated markers as described in Materials and Methods. Thepresence of TSEI was inferred from the pattern ofTAT and PEPCKmRNA expression as shown in Fig. 3. PKCA encodes protein kinaseC a polypeptide; TKI, soluble thymidine kinase; THH59, an anon-ymous DNA segment; neo, viral G418-resistance gene; COLIAI,collagen type I, alpha 1; HOX2, homeobox region 2; MYHI, myosinheavy polypeptide of adult skeletal muscle.

7304 Genetics: Lem et al.

t v W. 0 ..-

Proc. Natl. Acad. Sci. USA 85 (1988) 7305

a bc de f gh i jA

B

w__

C

FIG. 3. RNA blot analysis ofPEPCK (A), TAT (B), and a-tubulin(C) mRNA expression in human 7A-series deletion hybrids. Lanes:a, 7AC-5; b, 7AC-1; c, 7AE-3; d, 7AD-7; e, 7AE-27; f, 7AD-6; g,7AE-12; h, L(17n)C; i, PTK-A7A; j, FTO-2B.

(4-14). Extinction is general in at least two respects: (i) itoccurs in virtually all crosses involving distinctly differentcell types of equal ploidy (5, 6), and (ii) essentially alltissue-specific gene activity is repressed (12). In addition,extinction is a quantitatively large effect, with tissue-specificmRNA expression being reduced by factors of500-1000 (12).The development of methods for constructing genotypically

simple hybrid cells that retain single, specific donor chromo-somes (18, 33) has allowed the genetic basis of this complexcellular phenotype to be explored. In two cases analyzed todate, extinction of particular liver genes in intertypic hepa-toma hybrids is the consequence of discrete genetic loci thatmap to single fibroblast chromosomes (13, 14). These datashow that extinction has a specific genetic basis and establisha genetic test for the definition of mammalian loci that regulatetissue-specific gene activity in trans.The studies described in this report show that a third liver

gene, that encoding the gluconeogenic enzyme PEPCK, isregulated by an extinguisher locus that maps to a singlemouse or human chromosome (autosome 11 or 17, respec-tively). Moreover, available evidence indicates that thisgenetic element is either linked to or identical with Tse-J, apreviously defined locus involved in TAT extinction (13).Hepatoma deletion hybrids retaining fragments of human

chromosome 17 were used in attempts to dissociate the TATand PEPCK extinction phenotypes. In 10 deletion hybridsdescribed in this report and in an extensive collection ofclones whose detailed characterization will be presentedelsewhere (R.J.L., M.J.T., and R.E.K.F., unpublished ob-servations), no dissociation of extinction phenotypes wasobserved. Although further studies will be required to deter-mine whether a single locus or multiple linked loci areinvolved, it does seem clear that TAT and PEPCK geneextinctions are genetically related events, and the loci me-diating those effects are not just coincidentally syntenic.The coordinate control of genes encoding tissue-specific

products of a particular cell lineage is a conceptually appeal-ing possibility, but data supporting this notion have beenobtained only recently (34, 35), and the underlying mecha-nisms have yet to be defined. In this context, coregulation ofTAT and PEPCK gene activities by a common (or related)

genetic factor is particularly intriguing. Of 14 liver-specificgenes assayed in our experiments, only Tat-i and Pck-Jappeared coordinately repressed by Tse-J. This is interestingbecause the genes encoding TAT and PEPCK share otherforms of gene control. For example, transcription of bothgenes is inducible by glucocorticosteroids and by cAMP (36,37), although insulin has opposite effects on gene activity.Furthermore, the developmental expression of Tat-i andPck-J is similar, with gene activity being induced shortly afterbirth. Finally, Tat-i and Pck-J are two of several liver geneswhose expression is deficient in hepatocytes of mutant micehomozygous for deletions around the albino (c) locus onchromosome 7 (38, 39). Further work will be required todetermine which features of TAT and PEPCK gene controlinvolve common regulatory factors.

Finally, the results presented here add to a growing bodyof evidence that indicates that negative as well as positivecontrols contribute to the maintenance of tissue-specificpatterns ofgene activity in mammalian cells (40-43). Multipletissue-specific extinguisher loci seem involved in the overallmechanism of gene repression, and individual extinguishersmay affect expression of several tissue-specific genes intrans.

These studies were supported by Grant GM26449 from theNational Institute ofGeneral Medical Sciences. R.J.L. is a LeukemiaSociety of America Fellow, and R.E.K.F. is the recipient of anAmerican Cancer Society Faculty Research Award.

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