Vol. 7, 251-261, February 1996 Cell Growth & Differentiation 251

Expression of Thyroid Transcription Factor I GeneCan Be Regulated at the Transcriptional andPosttranscriptional Levels’

Renata Lonigro,2 Mario De Felice, EIio Biffali,Paolo Emilio Macchia, Giuseppe Damante,Carmela Asteria, and Roberto Di LauroDepartment of Science and Biomedical Technology, University ofUdine, Via Gervasutta 48, 33100 Udine [A. L, G. D.]; StazioneZoologica A. Dohm, Villa Comunale 1 Napoli [M. D. F., E. B., P. M.,C. A., R. D. L]; and Multidisciplinary Institute of General Pathology,Immunology Section, Faculty of Medicine, University of Messina, viaCannizzaro, Messina [M. D. F.], Italy

Abstract

The complete structure of the gene for thyroidtranscription factor I (TTF-I), both in rats and humans,has been determined. The rat 7TF-1 gene shows threetranscriptional start sites and contains two introns, oneof which is alternatively spliced. Nuclear run-on andtransient transfection experiments indicate that 7TF-1

gene expression can be controlled at different levels.Using thyroid and nonthyroid cell lines, it can be shownthat transcriptional mechanisms are involved incontrolling thyroid-specific expression of the TTF-1

gene. In contrast, in thyroid cells expressing anactivated Ki-ras oncogene, the steady-state level ofTTF-I mRNA is greatly reduced, while transcription ofthe TTF-1 gene is only moderately affected, suggestingthat the accumulation of TrF-I mRNA can be regulatedby a posttranscriptional, Ras-sensitive mechanism.

Introduction

Homeodomain-containing proteins are transcriptional regu-

lators that play a central role in embryonic development and

cell differentiation (i-3). A major feature of this class ofproteins is the temporal and spatial restriction of their ex-

pression pattern (4-9), which is achieved mostly by tran-scriptional mechanisms (8, 9). Hence, it is necessary to study

the promoters of the corresponding genes to understand this

important aspect of differentiation and development. TT3

is a homeodomain-containing protein expressed in thyroid

Received 5/18/95; revised 10/26/95; accepted 11/30/95.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this tact.1 This work was supported in part by a grant from Commission of Euro-pean Community (to R. L) and by the Associazione Italiana per Ia Ricercasul Cancro.2 To whom requests for reprints should be addressed. Phone: +39 432547301; Fax: +39 432 547210.3 The abbreviations used are: TTF-1 , thyroid transcnptiontactor-i ; GPDH,glyceraldehyde-3-phosphate dehydrogenase; BS, Bluescript; RSV, Roussarcoma virus; CAT, chloramphenicol acetyltransterase.

follicular cells, in the lung epithelium, and in restricted areasof the developing diencephalon (1 0, 1 i). In thyroid follicular

cells, TTF-i has been implicated in the transcriptional acti-

vation of genes encoding thyroglobulin, thyroperoxidase,

and TSH receptor (12-i 4), whereas in the lung, TTF-i has

been suggested to positively regulate transcription fromgenes encoding several surfactant proteins (1 5, 1 6). Since

1TF- 1 gene expression can be detected at the onset of

organogenesis in both thyroid and lung development, theidentification of mechanisms involved in TTF-i transcrip-

tional regulation could provide insights into the molecularevents responsible for the determination of thyroid and lungprecursor cells. The rat 1TF- 1 gene has already been pro-

posed as a candidate target for regulation by Hox proteins(1 7). Moreover, 1TF- 1 gene expression shows an interestingsensitivity to tumoral transformation, because TTF-i mRNA

is present in thyroid papillary carcinoma, but it is absent inanaplastic carcinomas of the thyroid gland (1 8). Thus, 1TF-1

gene expression appears to be sensitive to events that are

specific of undifferentiated thyroid cancer.

In this study, we report the complete structure of rat andhuman 1TF-1 genes. Furthermore, studies on 1TF-1 gene

transcription have been carried out. The results obtained,

comparing the measurement of TTF-i steady-state mRNA

levels in several different cell lines, suggest that 1TF-1 geneexpression can be regulated both at the transcriptional andat the posttranscriptional level. Interestingly, the negativeeffect of the activated ras oncogene on 1TF- 1 gene expres-

sion (i9-2i) appears to be contributed mostly by interler-

once with posttranscriptional mechanisms.

ResultsStructure of Rat TTF-1 Gene. Screening of rat and human

genomic libraries with a probe derived from the TTF-i cDNA(see “Materials and Methods”) resulted in the isolation of twophages (ARGTTF-i .1 and AHGTTF-i .1) containing the entire

TTF-i coding sequence within a genomic insert of approxi-

mately i 2 kb. The partial restriction map of the rat gene isshown in Fig. 1 . Hybridization with probes derived from the

TTF-i cDNA (1 0), subcloning and sequencing of the relevantfragments (see below), and comparison with the cDNA se-

quence (10) allowed us to deduce that the 1TF-1 gene is

made of three exons separated by two introns (Fig. 1). To

map the transcription start site(s), the oligonucleotide El (Fig.1) was synthesized and used in a primer extension assay withpoIy(A)� RNA from several tissues as a template. The results

of such assays are shown in Fig. 2B and schematized in Fig.2A. Two main transcription start sites located at 198/200 and1 77/1 81 nucleotides upstream of the putative translationinitiation codon (ATG in Fig. 1 ; double underlined in Fig. 4),

1 2 3

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RNA PROTECTION PROBES

252 Ras Effect on TTF-i Gene Expression

1000 bp

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Av

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Fig. 1. 1TF-1 gene structure. A partial restriction map of the 12-kb rat genomic fragment encompassing TTF-1 gene is represented (top panel). Boxes 1,2, and 3 are exons; the black box at the 5’ portion of exon 3 indicates the homeobox. Exon 1 is 90 or 71 bp long, depending on the transcription start sites1 and 2, respectively (Figs. 2B and 38). Exon 2 is 372 bp long and containstheATG almost atthe 5’ end. Exon 3 is 1988 bp long and containsthe homeoboxsequence (blackbox) in the 5’ coding portion, plus 1210 bp of the 3’ untranslated sequence otthe gene. The position otthe translational stop codon ffGA)is indicated. A Hindlll-Apal fragment is enlarged, and the El oligonucleotide (arrow) used for the primer extension assay, as well as the three probes usedfor the RNase protection assay, is indicated (see Figs. 2 and 3 legends). mt. i and lnt.2 are introns. Intron 1 Is 98 bp long In the 5’ untranslated region. Intron2 is 897 bp long. A, ApaI; Av, Aval; Aw, AIwNI; B, BamHl; E, EcoRI; H, Hindlll; n, Nail; S. Sail. #{176},the Sail sites belong to the EMBL-3 vector polylinker.

respectively, were identified (starts 1 and 2 in Figs. 2A and3A, respectively). Identical signals are present when lung orthyroid poly(A)� RNA are used, whereas no extensions ofsimilar sizes are observed when poIy(A)� RNAS from tissuesknown not to contain any TTF-i mRNA are used. A longer(about 255 nucleotides long), thyroid-specific extension (Fig.2B, well evident in Thy lane) is also occasionally observed;since a transcription start site at this position has not beenconfirmed by RNase protection assays (see below), we in-terpret it as an experimental artifact, presumably due to areverse transcriptase strong stop in a much longer TTF-l

transcript. To confirm and extend the observations made byprimer extension, an approach based on the RNase protec-

tion assay was used. Three genomic fragments, designated

probes 1 (378-bp long), 2 (223-bp long), and 3 (1 24-bp long)in Figs. 1 and 3A, were subcloned into vectors suitable forthe preparation of RNA probes. Each probe included vectorsequences, which served as a control for RNase digestion in

the case of full protection (see below). The probes werehybridized with either thyroid poly(A)� RNA(Fig. 3B, Lanes 3,

4, and 9) or control RNA(Fig. 3B, Lanes 2, 5, and 8), digested

with RNases and fractionated on denaturing PAGE togetherwith an undigested probe (Fig. 3B, Lanes 1, 6, and 7). In Fig.3A, a schematic interpretation of the results obtained with

each probe is shown. The RNA fragments of 234, 21 2, and

171 bp with probe 1 and of 212 and 1 71 bp with probe 2 are

in agreement with the presence of TTF-1 mRNAs initiated attranscription start sites, as determined by primer extension.

Furthermore, among the fragments obtained with probe 1,after hybridization to thyroid RNA and RNase digestion, is anRNA fragment of 378 bp that is the result of full probe iprotection (the reduction in size compared to the entireprobe is due to the digestion of vector sequences includedinto the probe); to explain the origin of this fragment, wepostulate the presence of another start site upstream of thethree determined by primer extension assays. Such a start

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Cell Growth & Differentiation 253

I

� L�AFig. 2. Determination of rat 1TF-1 gene transcription start sites: primerextension assay. A, schernatization of the results. White boxes, the twomajor extended signals; black box, intron 1 . Arrows, the transcription startsites 1 and 2. B, primer extension assay performed with the El oligonu-cleotide and poly(A)� RNA from different tissues. Thy, thyroid; Lun, lung;Thm, thyrnus; Int, intestine; Tes, testis. The sequence on the left part hasbeen performed on a Bluescript-TTF-1 genomic subclone using the sameEl oligonucleotide as primer. Arrows, the major signals obtained with thethyroid and lung RNAs. The 255-nucleotide-long band has not beenconfirmed by the RNase protection assay (see text).

site is confirmed by the full protection observed with probe 3

(again, the decrease in size is due to digestion of transcribed

vector sequences). Finally, other RNA fragments are ob-

served when probes 1 and 2 are used. As schematically

indicated in Fig. 3A, these fragments are consistent with the

presence of hF-i mRNAs from which a 98-nucleotide longintron, spanning from 1 1 1 to 1 3 nucleotides upstream of the

putative translation initiation site, has been removed (Fig. 3A,

230-, 86-, 65-, and 50-bp fragments with probe 1 ; 75-, 65-,

and 50-bp fragments with probe 2). The existence of two

populations of hF-i mRNAs, with or without such intron

sequence, has been confirmed by cloning (Ref. 22 and this

study; data not shown). Furthermore, the 1 71 -bp protected

fragment observed either with probes 1 or 2 indicates an-

other transcription start site located at -130 bp from the

ATG (start 3 in Fig. 3A). This transcription start site is not

observed in the primer extension assay, probably because

the signal due to the extended fragment is hidden by the

smear of the El oligonucleotide (Fig. 2B). The 1TF-1 gene

shows multiple transcriptional start sites which, together with

the alternative splicing of the 5’ small intron, result in a family

of TTF-l mRNAs molecules. Although most TTF-l mRNAs

encode the same protein, transcripts from the upstream start

site could encode for a larger protein, since the removal of

the alternatively spliced intron results in the fusion of a small

open reading frame to that of the mature, predominant TTF-l

protein. However, antibodies to this putative peptide failed to

reveal any protein product, leaving unanswered the question

of as to whether such an extension could have any physio-

logical relevance (data not shown).

Both rat (4308 bp) and human (3923 bp) 1TF- 1 genes and

flanking regions have been sequenced. In Fig. 4, the two

sequences have been aligned. Rat and human 1TF-1 genes

show a high sequence similarity in the area 5’ to the mapped

transcription start sites (89.4%) and in intron 1 (98%). In the

5’ region, the only remarkable difference between the two

sequences is a TC dinucleotide repeat located at -382 bp

from the ATG, which is 70 bp long in the rat gene, whereas

the human is much shorter (14 bp). In intron 2, the similarity

between the two genes is reduced to about 60%, while

stretches of very high similarity are present in the 3’ untrans-

lated region. Interestingly, in both the rat and human genes,

two TTF-l consensus sequences are present in the 5’ region,

at - 1 77 and -350 bp from the ATG, respectively. Gel-

retardation assay experiments indicate that in vitro TTF-l is

able to recognize these sequences, albeit with an affinity

lower than that observed for the C site in the thyroglobulin

promoter (Ref. 12; data not shown). The role of TTF-l protein

on the transcriptional activity of its gene remains to be

verified.

Transcriptional Mechanisms Are Responsible for Cell-specific 1TF-1 Gene Expression. Rat 7TF-1 gene expres-

sion has been studied by nuclear run-on and mRNA levels

experiments in: (a) the rat follicular differentiated thyroid cells

FRTL-5 (23); (b) the Kirsten ras-transformed thyroid cells,

FRTL-5 KiMol, where the thyroid-differentiated phenotype

has been irreversibly lost (24); (c) the rat fibroblastic cell line

Rat-i ; and (d) the cell line FRT, a rat thyroid-derived cell line

that shows no expression of the thyroid differentiated phe-

notype. To measure transcription of the 1TF-1 gene in these

cell lines, nascent RNA chains, labeled in vitro using purified

nuclei, were hybridized to blots containing TTF-l genomic

fragments, in addition to positive (GPDH) and negative (BS)

controls (Fig. 5). As shown in this figure, normal FRTL-5 cells

show easily detectable 1TF-1 gene transcription, while none

or very little transcription could be detected in Rat-i and FRT

cells. These results are in agreement with the absence of

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254 Ras Effect on TTF-l Gene Expression

Fig. 3. Determination of rat TTF- 1 gene transcription start sites. RNase protection assay. A, schematization of the results. Darkilne, the length of the probesused with the relative restriction sites. In probes 1 and 2, the black box indicates the position of the 5’ intron (int.i). Arrows, the transcription start sites.The white boxes below each probe indicate the protected fragments and correspond to the length of the bands visible in B. See text for detailed explanationot signals. B, RNase protection assay performed with thyroid RNA and probe 1 (Lane 1, Hindlll-AlwNl fragment), probe 2 (Lane 6, Aval-AIwNI fragment),and probe 3 (Lane 7, HindIll-NarI fragment). These three probes were hybridized with tRNA as negative control (Lanes 2, 5, and 8) and with thyroid RNA(Lanes 3, 4, and 9). M, molecular weight marker. Arrows, all the signals in the Lanes 3, 4, and 9.

TTF-1 mRNA in both Rat-i and FRT cells (data not shown)

and suggest that 1TF- 1 gene expression can be down-reg-

ulated at transcriptional level. Interestingly, nascent RNA

chains from FRTL-5 cells hybridize, in addition to TTF-l

genomic fragment 2, which contains the three transcription

start sites precisely mapped by primer extension (see previ-

ous paragraph), also to genomic fragment 1 . Thus, this result

confirms the existence of an additional, as yet unidentified,

7TF-1 gene promoter. In the nuclear run-on assay, Ki-ras-transformed thyroid cells also show detectable levels of

1TF- 1 gene transcription, albeit with a modest reduction

from the FRTL-5 levels; we have reported previously that

these cells contain greatly reduced levels ofTTF-i mRNA, as

also shown by an RNase protection experiment carried out

with a probe corresponding to the SfiI-ApaI (i82-bp) coding

fragment indicated in Fig. 5. Such evidence suggests that

Ki-ras reduces steady-state hF-i mRNA levels, acting

mostly at the posttranscriptional level.

The ras Oncogene Down-Regulates TTF-1 Gene Ex-pression at Posttranscnptional Levels. The nuclearrun-on experiments performed on FRTL-5 KiMol cells (Figs.

5 and 6A) show that 1TF-1 gene transcription is reduced

2-3-fold in this Kirsten ras-transformed cell line compared tothe transcription observed in the differentiated FRTL-5 cells

(Table 1). On the contrary, TTF-i mRNA levels in KiMol cellsare almost 30 times lower than those observed in nontrans-

formed cells (Figs. 5 and 6; Table 1). These results strongly

suggest that activated p21 ras affects 1TF-1 gene expres-

sion, mainly at the posttranscriptional level. To obtain further

support for this interpretation, cell lines transformed with a

Kirsten murine sarcoma virus carrying a temperature-sensi-tive Ki-ras allele have been used (25). These cell lines lose the

Cell Growth & Differentiation 255

transformed phenotype but do not recover differentiation

when grown at the nonpermissive temperature (39#{176}C).It hasbeen reported that treatment of these cells with the DNAdemethylating agent 5-azacytidine (20) increases the fre-quency of finding cellular clones showing a partial recoveryof the differentiated phenotype. One such clone (Ats-aza)was used to verify the transcriptional and posttranscriptionaleffect of ras oncogene on 1TF-1 gene transcription andTTF-i mRNA levels. Ats-aza cells were grown for 4 weeks at

39#{176}Cor for 4 weeks at 33#{176}Cand were used to measure TTF- 1

gene transcription or TTF-i mRNA levels in either condition

(Fig. 6). In this experiment, 1TF-1 gene transcription wasmeasured by hybridization to an Xbal-Styl fragment, whoseposition within the 1TF-1 gene is illustrated at the bottom ofFig. 6A. A DNA fragment derived from a cDNA clone for rat

thyroglobulin (26), the most abundantly expressed thyroid-specific mRNA known, was run in the same lane as the TTF-i

genomic fragment to provide an internal control of the ox-pression of the differentiated phenotype in the cells analyzed(indicated as TG in Fig. 6A). As shown in Fig. 6A, thyroglob-

ulin transcription is only detected in FRTL-5 or in the Ats-aza

cells grown at 39#{176}C,while it is absent in FRTL-5 KiMol orAts-aza grown at 33#{176}C,thus demonstrating the dependenceof Tg gene transcription on the temperature treatment in theAts-aza cells. Conversely, 1TF-1 gene transcription seems tobe very similar in all cell lines analyzed, except for the FRTL-5cells that show a little higher transcription. The strong Blue-script signal (BS arrow in Fig. 6A) present in Ats-aza cellstranscriptional assay is most probably due to a pTg-NEO

plasmid that was stably inserted into the genome of this cell

line (20). Accurate quantitation of relative TTF-i mRNA levels

was carried out by RNase protection assay as shown in Fig.6B, using a standard curve constructed with FRTL-5 cyto-

plasmic RNA (see Fig. 6B legend for a detailed description ofthis experiment). The results shown in Fig. 6 are expressed ina more quantitative manner in Table 1 . These data confirmthat cells harboring an active Ki-ras oncogene (FRTL-5 KiMoland Ats-aza 33#{176}C)have levels ofTTF-i mRNA much reducedwhen compared to those measured in cells that either do not

express (FRTL-5) or express an inactive version of the sameoncogene (Ats-aza, 39#{176}C).In contrast, 7TF- 1 gene transcrip-

tion is either unaffected (compare Ats-aza at 33#{176}Cand 39#{176}C)

or only moderately influenced. Hence, it can be concludedthat TTF-i mRNA accumulation can be regulated at a Ras-sensitive, posttranscriptional step.

TrF-1 Promoter Activity in a ras-transformed ThyroidCell Line. To further investigate the transcriptional regula-tion of the 1TF-1 gene, we compared levels of transcriptionfrom a genomic fragment containing hF-i transcription start

sites. Two overlapping, 2i 03- and 243-bp 5’-flanking DNA

fragments encompassing the major transcription start sites

of the rat 1TF-1 gene were cloned in front of the luciferasereporter gene in the pBS-Luc2 expression vector to obtainpTTF-i -Luc �-2OOO and pTTF-i -Luc i�-l 42, respectively.These two constructs were transiently transfected in FRTL-5,Rat-i , and FRTL-5 KiMol cell lines in transient transfectionassays, together with a RSV-CAT reporter construct (RSVpromoter + CAT cDNA) that was used to normalize for thetransfoction efficiency. In Fig. 7 are shown the results ob-

tamed. The TTF-i genomic fragments used appear to retain,

at least in part, the information necessary for tissue-specificexpression, since they both show a transcriptional activitymuch higher in FRTL-5 than in the Rat-i cells. Interestingly,promoter activity by the same fragments in FRTL-5 KiMol iscloser to that observed in FRTL-5 cells. These results are inagreement with those obtained with the nuclear run-on as-

says and again support the idea that the effect of ras onco-

gene on 7TF-1 gene expression is mostly due to interferencewith posttranscriptional mechanisms.

DiscussionThe data presented in this paper demonstrate that the ox-prossion of the gene encoding TTF-i can be regulated bothat a transcriptional and at a posttranscriptional level. Run-onassays show the absence of TTF-1 gene transcription in the

fibroblastic cell line Rat-i and in the dedifferentiated thyroid

cell line FRT. Thus, specific transcriptional regulatory mech-

anisms must exist that allow hF-i expression in thyroidcells. Since 1TF-1 gene expression is first detected at day

9.5 post coitum of mouse development, in the precursorsof thyroid follicular cells, as well as in two other cell types

(1 1), these regulatory mechanisms must be operating be-

fore that date in development. Interestingly, the mRNA forPax-8, another developmentally regulated transcriptionfactor that is expressed at the same time in developmentand in the same thyroid cells precursors as TTF-i , is

present in FRT cells (27), suggesting that different mech-

anisms control the thyroid-restricted expression of TTF-iand Pax-8.

Sequence comparison of the rat and human TTF- 1 genepoints out a very high similarity in the genomic region up-stream of the ATG. This homology is suggestive for thepresence of conserved regulatory elements. In spite of thefact that some TTF-i consensus sequences exist in the 5’

untranslated region of the TTF- 1 gene, we have been unableto demonstrate a functional relevance of such sites, either by

mutagenesis or by cotransfection in nonthyroid cells, of aTTF-i expression vector together with constructs containingthe TTF-i promoter fused to reporter genes (data notshown). Nevertheless, the near wild-type level of 1TF-1 genetranscription, observed in the Ki-ras-transformed cell lines inthe presence of greatly reduced TTF-i mRNA level, suggeststhat TTF-i protein does not exert an important role on tran-scription of its own gene.

Thyroid cell transformation by the v-Ki-ras oncogene re-suIts in a dramatic reduction of Tg and TPO gene transcrip-

tion (28) that correlates with a substantial decrease in hF-i

mRNA levels. In contrast, as we have shown in this study, inKi-ras-transformed thyroid cell lines, a significant 7TF-1 genetranscription persists, indicating that the reduction in hF-imRNA concentration must be explained by posttranscrip-tional mechanisms.

Data obtained using a cell line carrying a temperature-sensitive, activated Ki-ras allele (Ats-aza) show that hF-imRNA levels are much lower when Ats-aza cells are grownat 33#{176}C,a temperature permissive for Ki-ras activity. Con-

versely, no major difference can be detected between the

amount of TTF-1 gene transcription at permissive versus

256 Ras Effect on TTF-i Gene Expression

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H �I I I I II I I I I II I I I II I I I I II I I I II I I I I II I I I I II I I Ill I I I I III 11111 III II I I I II I I I I II I I I I II I I I II I I I I I I II I I II

R � 134

HII I I II I I I I II I I II I I I I I I II I I I I Ill I I I II I I I II I 11111 II I II I I I I II I I I II I I I I I I I I I I II I I I II I I I I II I I I II I I III

R � 234

HI I I I II I I I II I I I II I I I I II I I I II I I I I II Ill 11111 I 1111111111 I II I I I I II I I I I I I I II I I I I II I I I II I I Ill I I I II I I I

R � 334

HlIIlllllllllIlllllllllllllllllIlllllIIlIIlIllllllIIlIllllll I II II II II 1111111111 1111

R GCCC�GMrACGGcX3CCAACCCAGATCCGCGcTI�CCCGCCAgtaagtgaggccaccccgccgcggg . . gccacgggccgagcaccggaggcgcggt 432

H gagagccgtccagaaggcgcggcgccggcaggctgcgcgctgggcatcagggagggcggcccggcagcggcgccagggacttgggtgcgg . .gactgggg

1111111 lIllIllIlIllI II llllllllllllllIIllll Ill I IllIllIllIll I IllR cagcgcggcccgctaggtgcggcgcccgccagtt . . gcactaggcatcggggtgggcagccgaacagtctcctataggagtggggtgcgggcgcttggac 530

H atgcttccccctgctcggctgggggtccaagaacaggcacttggtagcgctggggtcctgcggtcagatgcggtactcgcgtctcctagcgcggtggact

11111 11111111 11111111 lIlllIlllIIIllllllIllllllllllllll I Ill I IIR tttcttcgtcccgctgggcaagggtttcaa . atgaggcacttagaagagctggggtcctgcggacaggtgctggcacccggccac tgcaggcgc 623

H ggcagctctgctcggcgcagaagacctccggggagccaagggaagcgaccccga gctcaaggagcaggggcgagcagaggctagaccgggcca

liii IllIlIlIllIll 1111 1111111 1111 I 11111 II 111111 1111R caacaacaggcttgatgctgaagacttcagggagctgagggaagc�cacccggagttaggggctcgggccacaggagttagaggagaccaggagggacaa 723

Fig. 4. Human and rat TTF-l gene sequence from - 1064 to +3249 of the rat gene. The coding portion is in capital characters, while the intron sequencesas well as the 5’ noncoding and the 3’ untranslated sequences are in small characters. The smailarrows (numbered 1 , 2, and 3) indicate the three rat genetranscription start sites experimentally defined. The long 5’ pointed arrow underlines the sequence of the El oligonucleotide used for the primer extensionassay. The ATG is double underlined and the translational stop codon (TGA) is in bold.

�cta�

�ccta

�cctcac1 �ggcc

�tagt

�aqaaqcc

agag� saac �ga#{231}

gtctgggggctccagcttttggaa . . . gtc

I II III I:ggggacgtttgaaagtgaa

B H ggagggag .gctgccctgttgggaggCactcgagCgcccggcccggccctIIIIIIIIIIIIIIIIIIIII I III Ill I 1111 I Ill II II IR gtagagagagccgctctgctgggacgcatcccggcccggctgcctggatttctaaaaatctctgggc�gttgggtgggctctaaatg.ggtcgggcggat 823

H gaggggacl �gggtccgagcaggtg( :g#{231} �tgacaggaL :aagaggc� aagc

I I I III I I I 11111 III I II II I 111111 11111111 IIR �ggtgccctc� �agctcca� ttcgccct� :gagctcgcaag ia g� �gcc g� gcgcctggaagtt

H aacaccccctctcctaacctctccaaactggggtctaccgtaggaccccagctccggcctgagccagttcgccgcctgtggccagctaatcctaatgctc

III Ill I I I I II I 1111111111 I 1111111111 I I I I II I IR aactcccttttgctaagctcctgaagttgggggtctaccttcccaccccagctctctgcacccctgggtttcccacctgg 1003

H tgaccggctggcacgaaaggagcagaagcggcc . tttcccccactgcgtcttttggttcgaaaga�gaactgagactgagggagggcagccagggttggIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 11111111111 1111111111 1111111 III

R cttgggcacgaaaggagccgaagcgcccttttccctcactgcacattctgggtctagaga . . .cactgacaccgatgaggagtagctagacttga 1095

H ggctgtgagcgctccagtacagccccctcgacggtacggcctggggcaggcgctggcagttcccc

I I I I IIIIIIIIIIIIIIIIIIIIIIII 11111 1111 II Ill III IllR acccct taagcagttcgacggcacggcccggagaaggcactagccactccccagctagctgagcctctttcggggaaagagcactgg 1182

H gctgcctgggtcaggagggcgccgtcggttggggcgggccgggcgggccaatggcgcggaaaacaggggtggcctggctcggcctggccccggccgacgc

I IlIllIlIllIll 111111 III IIIIIIIIIIIIIIIIIIIII 11111111 IR gtggcctaggtgaggaggatgtcgtcg . . aaaattggggttggcgggccaagggcacagaaaccgggcttgccctggc . . . . ctcggccccagccgacgc 1276

H �11111111111 IIIIIlIIlllIIIIIIIlllllIIIIlIllIllllIIIIlI llIIIIIlIlIlllIllIIIIllIl

R � 1376

H �

IlIllIllIllIllIll 11111 II Ill 1111111 IIIIIIIIIIIIII lIIllIIlIllIlllIllIl IllIlIllIllIllIll 1111111R � 1476

H �

IIIIIIIIIIIIIIIIIIIIIIIII IllIllIlIllIll 11111111 IlllIIlIIIIIIIIIllllIIlIIIlIllIlIIIlIllIl 11111111R � 1576

H CCAGGCCAAGGACAAOGCGGcOCAGCAOCAACI�3CAGCAGG1iCAGcGGcGGCGGC. . . GGGOC�GOOGOG0C�OOGTG�OCAG�AGCMCAGGCF

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IlllIIllIIIIlIIlIlllllIIllIIIIll IllR CCAG0CGAAGGP�CAA0GCCGc0C�GC�OCAAC1OC�GCAGGACA.OCGGCGOCGGOGGA.OGCOGcOOcO0OGOc0C0OG’1”rGcccOCAOCAOCAGCAAGCT 1676

HII 11111 IlIllIlIIIIlIllIIIIlllIIIllll 11111 11111 11111 II II II 11111111111 1111

R � 1776

H

R � 1876

H �11111111111 11111111111 11111 IllllIllIIlIlIIlIlIl 11111111111 IllIllIllIllIllIl II 11111 II II Ill

R � 1976

H � gaggacgccgggccggccctagcccagcgctctgIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII II Ill I I I I

R � gatg tgggacgcgcctgagccctgtgcga 2071

H cctcaccgcttccctcctgcccgccacacagaccacca . tccaccgctgctccacgcgcttcgacttttcttaacaacctggccgcgtttagaccaagga11111111111 Il II III II II 111111111 II IIIIIIIIIIIIII 1111 111111 I I

R cctcaccgctttccctctaccctccgcaaagaccaccattcgcccgctgctccacgcccttctactttt ttctcatgtttagaccaagg . 2160

H acaaaaaaaccacaaaggccaaactgctggacgtctttctttttttc cccccctaaaattt

1111 llIlIlIlllIlIlIlIIIllllII 111111 II 111111111

R . . . aaaagtacacaaagaccaaactgctggacttcttcttcttcttcttcttttttccttcatccttctccctttcctttctctttcctacctaaaactt 2557

H gtggtttttt aaaaagaaatgaaaagaaccaagcgcatccaatc . . . tcaaggaatctttaagcagagaagggcataaaacagctttggg

1111 1111 1111 1111 1111 11111 1111111 IIIIIIIIIIIIIIIIIIIIIIIIIII 111111R gtggacttttttttaacataaaaagaaaatagaaacagtcaagcaaatccaacccctttacgaattctttaaccagagaaggacaaagaacaaatttggg 2657

H gtgtctttttttggtgattcaaatgggttttccacgctagggcggggcacagattggagagggctctgtgctgacatggctctggactctaaagac . .

I 11111111 IIIIIIIIIIIIIII 1111 II I 11111 111111111 IIIIIIIIIIIIIIIII IR g . . .gtctttctggtagttcaaatgggttctcaagcttaggcatggcacaattttggggcctgctctatgcttccacggccctgaactctg�agc�ggaa 2754

H aacttcactctgggcacactctgccagcaaagaggactcgcttgtaaataccaggatt tttttttttttttgaagggaggacgggagctggggag

11111111111 lllllllIIIIlIlIlIl IIIIIIIIIIIIIIIIIIIII 111111111 11111111111 111111111R aacttccctgtggatgcactctgccagcaaagagagcttgcttgtaaataccaggatttttcgtttgtttgtttcagaagggaggacagaaactggagag 2854

H aggaaagagtcttcaacataacccacttgtcactgacacaaaggaagtgccccctccccggcaccctctggccgcctggctcagccggcgaccgccctccIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 1111111111 III 111111

R aggaaagattcttcagcataacccacttgtccctgacacaaaggaagtgtcccctcccaggtgccctctggccctataggttcagtccaggctggccttt 2954

H gcgaaaatagttt . . .gtttaatgtgaacttgtagctgtaaaatgctgtcaaaagttggactaaatgcctagtttttagtaatctgtacattttgttgta111111 III 1111 IIIIIIIIIllllllllllIIlIlIlllIIIllIIIlIlIllIIllllIIIllIlIIIllI 111111111 1111111

R cagaaaattgttttaggtttgatgtgaacttgtagctgtaaaatgctgtcaaaagttggactaaatgcctagtttttagtaacctgtacattatgttgta 3054

H aaaagaaaaaccactcccagtccccagcccttcacattttttatggg. . .cattgacaaatctgtgtatattatttggcagtttggtatttgcggcgtca1111111 lIllIllIllIll IllIllIllIll 1111 11111111 IllIllIllIllI lIllIllIllIllIll II

R aaaag. aactccagtcccagtccctggcccctcactttttaaaagggcatcatcgacaagcctgtgtatattactccacggtttggtatttgcagcacca 3153

H gt ctttttctgttgtaacttatgtagatatttggcttaaatatagttcctaagaagcttctaataaattatacaaattaaaaagattct

II IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 1111 IIR gttgccccctcccttttttctgttgtaacttatgtagatatttggcttaaatatagttcctaagaagcttctaataaattatacgaattggaattc . . . . 3249

Cell Growth & Differentiation 257

agttcca

923

Fragments used to detecttranscrIption

,, 0’1 2

TTF-iGENOMIC CLONE

nonpermissive temperature. Hence, we suggest that the

activated Ki-ras oncogene is responsible for the observed

reduction in hF-i mRNA steady-state levels. In this re-

spect, it is of groat interest that in the attempt to identify

proteins involved in the control of p2i ras activity via

GAPs, and/or vice versa, a number of phosphotyrosine

containing proteins have been identified (29-3i), including

the p190 and p62 proteins (32). In light of our results, p62

is of particular interest since, given its homology to a

putative hnRNP protein and its ability to bind single-

stranded DNA and RNA, it has been suggested that this

protein could have a role in RNA processing and transla-

tion (33). It is conceivable that hF-i mRNA stability could

be regulated by c-Ki-ras via p62 or a p62-like protein. The

presence of high levels of a constitutively activated Ki-ras

protein could then result in abnormal metabolism of hF-i

transcripts, with a consequent reduction in the steady-

state concentration of its mRNA.

258 Ras Effect on TTF-l Gene Expression

TRANSCRIPTION FRTL-5 FRTL-5 RAT-i FRTKIM0I

mRNALEVEL

GPDH-�O ..:. #{149}B5 -0 �1_0.I �

(TTF-1) 2 -0#{176} � - �,,. .

TTF-1 -0.

GPDH � p

Transformation of FRTL-5 cells by an activated Ha-ras

oncogene results in cell lines containing nearly wild-type

levels of hF-i mRNA and protein (28). The phenotype ob-

tamed upon transformation with Ki-ras, as detailed in this

study, consists of reduction of hF-i mRNA levels and con-

sequent, substantial decrease in intracellular concentration

of the hF-i protein. We do not believe that this difference is

due to clonal effects, since the Ki- and Ha-ras phenotypehave been observed consistently, with different cell lines

transformed by either oncogenes. It is conceivable that the

differential effects elicited by two very similar oncogenes on

a defined biochemical marker, such as hF-i , could help in

elucidating the biochemical mechanisms underlying their

activities.

Materials and Methods

T

Probe used forRNase protection

Fig. 5. Rat TTF-l gene transcription and TTF-l mRNA levels in differentcell lines. To measure transcription, 32P-labeled nRNA, derived from thecell lines indicated at the top of the figure, was hybridized to Southernblots containing appropriately cleaved plasmids carrying either a TTF-lgenomic insert or GPDH cDNA that was used as a positive control. Arrows1 and 2, TTF-l genomic fragments HindIll-HindlIl (3000 bp) and Hindlll-Xhol (1 790 bp), respectively (see diagram at the bottom of the figure). Theposition of the GPDH cDNA, which is positive with all RNAs, and of the BSplasmid (BS), which does not hybridize with any of the RNAs, is alsoindicated. 1TF-l mRNA levels were measured by RNase protection, using32P-labeled RNA corresponding to the Sfil-Apal (1 80-bp) fragment and 60�.Lg of cytoplasmic RNA obtained from the different cell lines. In thediagram at the bottom of the figure, the black box represents the ho-meobox, and the thin line in the HindIll-XhoI fragment indicates intron 2 ofthe TTF- 1 gene. TTF-l transcription start sites 1 , 2, and 3 are indicated bya single arrow. 1 and 2, genomic fragments 1 and 2 described before.TTF-l -specific signals are indicated. The data shown in this figure arerepresentative of three run-on assays and three RNase protection assays.

Plasmids and Probes. All of the plasmid vectors carrying 1TF- 1 genefragments have been constructed using the Bluescript KS� (BS) from

Stratagene. To perform the RNase protection assays, pRGTTF-1 a,pRGTTF-l b, and pRGTTF-l c plasmids have been used. pRGTTF-l a con-tains the 378-bp HindlII-AlwNI fragment of the 1TF-1 gene (probe 1).

pRGTTF-l b contains the 223-bp Aval-A!wNI fragment (probe 2) and wasderived from pRGTTF-i a by AvaI cleavage and filling with Klenow poly-merase. pRGTTF-l c contains the 124-bp HindIII-Narl fragment (probe 3).The pRGTTF-l HA plasmid, containing the 673-bp Hindlll-ApaI fragment

(-344 bp/+329 bp from the ATG) of the TTF-1 gene has also been used.To perform the nuclear run-on assays, the following plasmids have been

used: pRGTTF-i .1 containing 8 kb (SaIl-EcoRl) of the rat TTF-1 gene;pBs-THA containing the 2.5 kb of rat TTF-l cDNA; pRT36-1 containing

the rat thyroglobulin cDNA sequence (26); and pGPDH1 (gift from P.

Chamay) containing the 1 .3-kb PstI fragment of GPDH cDNA. To performthe TTF-l promoter transfection assay, the following plasmids have been

constructed. pTTF-l -Luc �-2000 was obtained by inserting a 2i03-bpSacl-SspI fragment of the rat hF-i genomic upstream sequence, ex-

tending to position + 1 03 in its 3’ end, into the Sad and Hindlll sites of thepBS-Luc2 luciferase reporter plasmid (1 3). PTTF-1 -Luc �-i 42 was ob-tamed by internal deletion of the TTF-i sequences upstream of the HindIIIsite at position - 142 of the rat TTF- 1 gene.

Rat and Human 7TF-1 Gene Cloning. A rat liver genomic library in AEMBL3 vector and a human liver genomic library in A FixIl vector were

screened using the El oligonucleotide (5’-GGTGTTACCTTAACGC-

CGATCTTG-3’) derived from the 5’ nontranslated sequence of the hF-icDNA (-1 26/- 102 bp from the ATG) and using the EcoRI 625-bp trag-ment derived from the 3’ nontranslated region of the same hF-i clone

(10). The El oligonucleotide was 32P end-labeled, while the TTF-l 3’double-strand fragment was [a-32P]CTP-ATP labeled using the randomoligo priming technique. Library screening and DNA extraction and se-

quencing have been performed following standard protocols (34). Rat andhuman positive clones, of approximately 12 kb, were obtained (phages

ARGTTF-l .1 and AHGTTF-i .1 , respectively). A Sall-EcoRI 8-kb rat frag-ment and a EcoRI 10-kb human fragment containing the TTF- 1 gene weresubcloned to generate the pRGTTF-l .1 and the pHGTTF-l .1 plasmids,respectively. Both rat and human clones were sequenced by the dideoxymethod, and the rat TTF- 1 gene structure has been determined (seebelow).

Determination of the Rat TTF-1 Gene Transcription Start Sites.The rat HF-i gene transcription start sites were determined using corn-bined results of the primer extension and the RNase mapping techniques.

RNA isolation was carried out from different tissues (thyroid, lung, thymus,intestine, and testis) and from the FRTL-5 cell line by the acid guani-

dinium-thiocyanate-phenol procedure (35). Poly(A)� RNA was prepared

by oligo(dT) cellulose chromatography. The primer extension assay wasperformed according to Bensi et a!. (36) using 5 �tg of poly(A)� RNA fromthe different tissues and the El oligonucleotide as primer in the Moloneyreverse transcriptase (Pharmacia) elongation reaction. The RNase protec-tion assay was performed according to Keohavong et a!. (37), using 2 �gof thyroid poly(A)4 RNA and three different TTF-l antisense riboprobes

generated from pRGTTF-la, pRGTTF-l b, and pRGTTF-lc plasmids, re-

mRNALEVEL

, I � �

GPDH � j� %. � �, B � FRTL�S � FRTL� 3�’1:”�’�.c.

as-.- � ., . V I I ‘H *�.

TTF�1-�. l#{243} � � w � .� I I I 0 ei I

�I�TF-1 -� #{149} I #{149} I 1.5 6 15 30 60 60 60 60 pg cyt.RNA

� #{149}rrF�i

GPDH -� I �t I I 25

2O

P N

. -l9Obp

C -160 bp

‘5

10

FRTL-S

1.5 6 15

a Ats..za33�C.

Mgcyt.RNA

Cell Growth & Differentiation 259

A TRANSCRIPTION FRTL-5 FRTL-5KIMoI

Ats-aza

�a#{176}c

Fragment used to detecttranscription

17F-i T1�-� _ TcDNA CLONE

sf/\71

Probe used forRNase protection

0

kiM�I

60

Fig. 6. Rat 1TF-1 gene transcription and hF-i mRNA levels in ras-transtormed cell lines. A, cell lines are indicated at the top of the figure. At the bottom,the cDNA XbaI-StyI (2300-bp) fragment used to detect 1TF-1 gene transcription and the Sfil-ApaI (l80-bp)tragment used to detect TTF-l mRNA levels areindicated. Black box, the homeobox; arrow, the start of cDNA clone (thick line). TG, thyroglobulin gene. TTF-l -specific signals are indicated. UndigestedDNA is responsible for a signal on the top of lanes. B, quantitation otTTF-i mRNA levels in the cell lines indicated at the top of the panel. A RNase protectionassay (upperpart) was performed using a l80-bp SfiI-Apal hF-i cDNA fragment as probe. The TTF-l signal was detected in various amounts of FRTL-5cytoplasmic RNA and in 60 �g of KiMol and Ats-aza cell cytoplasmic RNA. t, tRNA; P, probe; M, molecular weight marker. The TTF-l -specific signal isindicated, whereas the 1 90-nucleotide-long band is nonspecific, as demonstrated by the fact that its intensity does not depend on the quantity of RNA usedin the assay. The hF-i signal present in the ras-transformed cell lines was referred to the titration curve shown below the RNase protection assay. Eachautoradiographic signal was measured at the phosphorimager computer, and the TTF-l values were normalized for the GPDH values obtained with 5 �gof the corresponding RNA (GPDH signals are not shown). The data shown in this figure are representative of two run-on assays and two RNase protectionassays.

Table 1 Quantitation of 1TF-1 gene transcription and TTF-lcytoplasmic mRNA levels in FRTL-5, FRTL-5 KiMol, and Ats-aza celllines

Ras+ and Ras- in ras-transformed cells indicate when the oncogene isactive and when it is not, respectively. hF-i expression has been nor-malized for GPDH expression and in all the ras-transformed cells has beenreported as a percentage of the levels detected in the normal and differ-entiated cell line (FRTL-5).

Cell lines Transcription TTF-l mRNA

FRTL-5 100.0 100.0

FRTL-5 KiMol

(Ras+)

38.0 2.9

Ats-aza 39#{176}C(Ras-)

38.8 78.2

Ats-aza33#{176}C

(Ras+)

30.3 13.5

spectively. These three plasmids were linearized in Kpnl site and tran-scribed by the U RNA polymerase (Boehringer) in the presence of[a-32P]UTP (Amersham).

Cell Cultures and Nuclei and Cytoplasmic RNA Preparations.FRTL-5 and FRTL-5 KiMol cell lines were grown routinely at 37#{176}Cin

chemically defined Coon’s modified Fi2 medium (Sigma Chemical Co.)supplemented with 5% calf serum and six hormones, according to Amb-

esi-Impiombato et a!. (38). The Ats-aza cells were grown in the same

medium at 33C or 39#{176}C.Nuclei were prepared as follows. For each cell

line, 5 X 150-mm diameter dishes (4 x 107.108 cells) were used, andice-cold solutions and glassware were used. The cells were washed twice

with 6 ml of Rinsing RSB buffer [1 0 m� Tris-HCI (pH 7.4), 1 0 mt�i NaCI, and

3 mM MgCl�] and then scraped with 2 mVplate of lysing RSB buffer(Rinsing RSB plus 0.5% NP4O, 2.75 m� DTT, and 1000 units/mI RNAsin).

Cells were homogenized into a cold Dounce homogenizer with the tighterpestle (3-4 strokes) and then centrifuged at 4#{176}Cfor 5 mm at 1000 x g in

a Sorvall swinging bucket HB-4 rotor. The supematant was carefully

removed, the nuclear pellet was resuspended in 10 ml of washing RSB

buffer (Lysing RSB without NP4O), and centrifuged again as above. Thesupematants from both centritugations were collected and used to pre-

pare the cytoplasmic RNA (39). The nuclear pellet was resuspended in 0.6

ml of Keller storage buffer [5 m� MgCI2, 10 m�i Tris-HCI (pH 7.5), 0.5 M

D-501bitOl, 2.5% FicoIl, 0.008% spermidine, 1 m� DTT, and 50% glycerol

50] and stored at -80#{176}Cuntil use.

Nuclear Run-On and RNase Protection Assays. Each transcriptionreaction was performed at 27#{176}Cfor 35 mm in a total volume of 400 �l with

200 pJ of nuclear suspension, 0.6 m� ATP, 0.3 m� GTP, 0.3 m� CTP (pH7.0), 20 �.tI [a-32P]UTP (400-600 Ci/mmol), 100 pA of 4x salt solution [160

mM Tris-HCI (pH 8.3), 600 m� NH4CI, and 30 m� MgCI2], and 200 units/mI

of RNAsin. The DNA template was removed by adding 40 units of RNase-

free DNasel (RQ1 -Promega) at 27#{176}Cfor 1 0 mm. The hot, elongated nRNA

was purified by adding 133 pJ otextraction buffer[lO m� Tris-HCI(pH 7.4),

15 mM EDTA, 3% SDS, 2 mg/mI Proteinase-K, and 3 mg/mI heparin] at42#{176}Cfor 3 h, and phenol-chloroform was extracted three times before to

be ethanol precipitated twice with 2.5 M ammonium acetate. The specific

nuclear in vitro transcribed RNAS were detected using specific probes.

These probes were cut with appropriate restriction enzymes, separated by

PROMOTERACTIVITY(LUC/CAT)

400

300

200

100

TE1

� FRTL-5 KiMol

I RAT-i

pTTF-i-Luc pTTF.1-Luc�-2000 �-i42

Ras Effect on hF-i Gene Expression

pTTF.1.Luc �#{149}2OOO

pTTF-1.Luc �-142

Sad BamHI HindIll

�L��LrE�EJHindlll

L�:j

Fig. 7. Transient expression assay of TTF-lgene promoter in normal and ms-transformedcell lines. Top, schematic representation of thetwo hF-i promoter constructs in a luciferasegene expression vector. In the pTTF-l -Luci�-2000 construct, the luciterase gene transcnp-tion is driven by 2000 bp of the rat 1TF-1 gene,5, to the transcription start site no. 1 . In thepTTF-l-Luc �-l42 construct, the most 3’ 142bp of the �-2000 construct drives the luciterasegene expression. Bottom, hF-i promoter ac-tivity in FRTL-5, FRTL-5 KiMol, and Rat-l cells.Bars, the mean values of six independent trans-tection assays. The promoter activity is ex-pressed in light units. The absolute light unitsderived from the luciterase activity (Luc) of eachtranstected construct have been normalized forthe percentage of the CAT conversion derivedfrom a cotranstected RSv-CAT plasmid.

agarose gel, and blotted on nylon membranes (Hybond). Precisely, 3 �gof pBs-THA plasmid (XbaI-Styl-EcoRI cut) or pRGTTF-l .1 plasmid (Hin-dIII-Xhol-EcoRl cut) were used to detect 1TF-1 gene transcription; 1 �g ofpRT31-6 plasmid (EcoRI linearized) was used to detect thyroglobulmn

gene transcription, and 1 �g of PGPDH plasmid (Hindlll linearized) wasused to detect GPDH gene transcription. The membrane-immobilizedprobes were incubated with the hot RNAS (1 .5 x 106 cpm/m� producedin the run-on reaction for 3 days at 42#{176}Cin the hybridization solution (50%

tormamide, 5x SSC (8.76 g NaCI, 4.4 g sodium citrate, and 1 liter H20; pH7.0), 5x Denhardt’s, 1 % SDS, and 1 mg/mI of tRNA). Filters were washed

three times for 10 mm each time at room temperature in 2x SSC and 2times for 30 mm each time at 60#{176}Cin 0.1 x SSC and 0.5% SDS. Specific

signals were detected by autoradiography. TTF-l mRNA levels weremeasured by RNase protection assays (37) using 60 �.tg each of thecytoplasmic RNA obtained from the different cell lines and 0.5 x l0� cpm

of the [a-32P]UTP-labeled riboprobe obtained by the T3 RNA polymerase

transcription of pRGTTF-i HA plasmid. This plasmid was linearized at the

Sf1 site internai at the hF-i sequence to obtain a riboprobe protecting180 bases of the translated hF-i mRNA. Signal quantitation in thenuclear run-on assay as well as in the RNase protection assay wasobtained at the phosphorimager computer (Molecular Dynamics).

Transfection Assay. FRTL-5 and FRTL-5 KiMol cells were cultured in

the same medium and conditions (38). Rat-i cells were cultured as de-scribed previously (40). For hF-i promoter activity assay, 9-cm disheswere transfected by calcium phosphate-mediated transfection (41) with10 p� of pTTF-i-Luc �-2000 or pTTF-i-Luc �-i42 plasmid and 2 �g ofRSV-CAT plasmid (pRSV-CAT; a gift from G. Morrone, University of Reg-

gio Calabria, Italy) to monitor for transfection efficiency. Cell extracts wereprepared 48 h after transfection, and the CAT (42) and Iuciterase (43)activities were determined. The light units from the luciterase assay were

normalized to the percentage of the CAT conversion.

AcknowledgmentsWe thank Professor Franco Quadritoglio and Dr. Carlo Pucillo for criticalreading of the manuscript.

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