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Vol. 2. 67-76. february 1991 Cell Growth & Differentiation 67 Growth-responsive Expression from the Murine Thymidine Kinase Promoter: Genetic Analysis of DNA Sequences’ Judith 1. Fridovich-Keil,2 Jean M. Gudas, Qing-Ping Dou, Isabeile Bouvard, and Arthur B. Pardee Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School [J. L. F-K., J. M. G., Q-P. D., A. B. P.], and Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, [J. 1. F-K., J. M. G., Q-P. D., I. B., A. B. P.], Boston, Massachusetts 02115 Abstract As a first step toward elucidating the biochemical basis of gene regulation at the G1-S boundary of the cell cycle, we have identified regions of the murine thymidine kinase (TK) promoter sufficient to confer appropriately growth-responsive expression to a heterologous gene. Using a series of 1K promoter- chioramphenicol acetyltransferase (CAT) gene fusion constructs, we have identified sequences located between -1 74 base pairs upstream and +1 59 base pairs downstream of the 1K translation initiation site that are sufficient to drive efficient S phase-specific expression of the CA T reporter gene in transfeded murine fibroblasts. Both deletion analysis and site- specific mutagenesis experiments indicated that an Spi consensus binding site is critical to the adivity of this promoter. Synchronized populations of BALB/c 313 cells stably transfeded with either 1K promoter-CAT fusion construds or 1K promoter-9-globin fusion constructs expressed their respedive reporter genes in an S phase-specific manner following serum stimulation. In each case, reporter gene expression was reduced during quiescence and G1 and rose upon entry of cells into S phase. The TK sequences included in these construds therefore contained information sufficient to confer S phase-specific regulation to these two reporter genes. These results set the stage for a more detailed analysis of the sequences and trans- acting factors responsible for regulating murine TK gene expression and may lead to insights into the control of proliferation in normal and transformed cells. Introduction Proliferation is a very tightly regulated process in normal mammalian cells (1, 2) and is often poorly regulated in cancer cells. The biochemical basis for this difference may involve changes in any of the key factors or prod- esses associated with normal control, including produc- tion and response to growth factors, signal transduction, and cell cycle-regulated gene expression. At least two Received 9/27/90. This work was supported by U5PHS Grant CA22427 (to A. B. P.) and National Research Fellowship Award CA08317-03 (to I. M. C.). 2 To whom requests for reprints should be addressed, at Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, 44 Binney 5t., Boston, MA 02115. systems of signal transduction have been postulated to direct the progression of normal cells through the cell cycle. The first system regulates the transition of cells from quiescence (G0) into G1 and involves the induction of many new mRNAs such as the protooncogenes c-los and c-myc (3). The induction of these new messages is necessary to bring cells to a state of “competence” but is not sufficient to initiate the DNA synthesis required for cell proliferation. A second system of signal transduction is required to take stimulated, competent cells through the final stages of C1 and across the G1-S boundary (2). Little is currently known about regulation of this portion of the cell cycle, although some recent reports indicate that the retinobiastoma gene product and members of the cdc2 and cyclin families may play key roles in this process (4-6). Transit across the G,-S boundary is less tightly regu- lated in cancer cells than in normal cells (7, 8). Although most normal cells require growth factors and efficient protein synthesis to progress through late C1 phase, transformed cells do not (9, 10). These findings under- score the point that a greater understanding of this por- tion of the cell cycle is fundamental to our understanding of cancer. In the experiments described here, we have used the murine thymidine kinase gene as a model for addressing the issue of gene regulation at the G1-S boundary of the cell cycle. Concomitant with the onset of S phase, the expression of mRNAs and protein products for histones and many of the enzymes associated with DNA synthesis increases (1 , 2). Among these enzymes are thymidine kinase (which catalyzes the phosphorylation of thymidine to thymidyl- ate), thymidyiate synthase, dihydrofolate reductase, and DNA polymerase a. The tight coupling of induction of TK3 with the onset of S phase suggests that some prior common mechanism may trigger both events, either directly or indirectly. Previous work from our laboratory demonstrated that both DNA synthesis and TK activity are sensitive to pulses of cycloheximide administered late in C1 (7), suggesting that the synthesis of some labile protein is required at this point to trigger both events. Subsequent research (1 1 ) demonstrated that the induc- tion of TK activity (as well as DNA synthesis) is dependent on insulin-like growth factor 1, further implicating a re- quirement for expression of new genes late in G,. The regulation of TK gene expression operates on multiple levels, including transcriptional, posttranscrip- tional, and translational controls (12-26). The tnanscnip- tional component of this regulation was first demon- strated by Stewart et a!. (24) and by Coppock and Pardee (12), using nuclear run-on experiments to indicate that, in both CV1 cells and BALB/c 313 cells, respectively, the 3 The abbreviations used are: 1K, thymidine kinase; bp, base pair(s); CAT, chloramphenicol acetyltransferase; cDNA, complementary DNA; PBS, phosphate-buffered saline.

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Page 1: Growth-responsive Expression fromtheMurine Thymidine ...cgd.aacrjournals.org/cgi/reprint/2/2/67.pdf · Growth-responsive Expression fromtheMurine Thymidine KinasePromoter: Genetic

Vol. 2. 67-76. february 1991 Cell Growth & Differentiation 67

Growth-responsive Expression from the Murine ThymidineKinase Promoter: Genetic Analysis of DNA Sequences’

Judith 1. Fridovich-Keil,2 Jean M. Gudas,Qing-Ping Dou, Isabeile Bouvard, and Arthur B. Pardee

Department of Biological Chemistry and Molecular Pharmacology,Harvard Medical School [J. L. F-K., J. M. G., Q-P. D., A. B. P.], andDivision of Cell Growth and Regulation, Dana-Farber Cancer Institute,

[J. 1. F-K., J. M. G., Q-P. D., I. B., A. B. P.], Boston, Massachusetts 02115

AbstractAs a first step toward elucidating the biochemical basisof gene regulation at the G1-S boundary of the cellcycle, we have identified regions of the murinethymidine kinase (TK) promoter sufficient to conferappropriately growth-responsive expression to aheterologous gene. Using a series of 1K promoter-chioramphenicol acetyltransferase (CAT) gene fusionconstructs, we have identified sequences locatedbetween -1 74 base pairs upstream and +1 59 basepairs downstream of the 1K translation initiation sitethat are sufficient to drive efficient S phase-specificexpression of the CA T reporter gene in transfededmurine fibroblasts. Both deletion analysis and site-specific mutagenesis experiments indicated that an Spiconsensus binding site is critical to the adivity of thispromoter. Synchronized populations of BALB/c 313cells stably transfeded with either 1K promoter-CATfusion construds or 1K promoter-�9-globin fusionconstructs expressed their respedive reporter genes inan S phase-specific manner following serumstimulation. In each case, reporter gene expression wasreduced during quiescence and G1 and rose upon entryof cells into S phase. The TK sequences included inthese construds therefore contained informationsufficient to confer S phase-specific regulation to thesetwo reporter genes. These results set the stage for amore detailed analysis of the sequences and trans-acting factors responsible for regulating murine TKgene expression and may lead to insights into thecontrol of proliferation in normal and transformedcells.

IntroductionProliferation is a very tightly regulated process in normalmammalian cells (1, 2) and is often poorly regulated incancer cells. The biochemical basis for this differencemay involve changes in any of the key factors or prod-esses associated with normal control, including produc-tion and response to growth factors, signal transduction,and cell cycle-regulated gene expression. At least two

Received 9/27/90.This work was supported by U5PHS Grant CA22427 (to A. B. P.) and

National Research Fellowship Award CA08317-03 (to I. M. C.).2 To whom requests for reprints should be addressed, at Division of Cell

Growth and Regulation, Dana-Farber Cancer Institute, 44 Binney 5t.,

Boston, MA 02115.

systems of signal transduction have been postulated todirect the progression of normal cells through the cellcycle. The first system regulates the transition of cellsfrom quiescence (G0) into G1 and involves the inductionof many new mRNAs such as the protooncogenes c-losand c-myc (3). The induction of these new messages isnecessary to bring cells to a state of “competence” but isnot sufficient to initiate the DNA synthesis required forcell proliferation. A second system of signal transductionis required to take stimulated, competent cells throughthe final stages of C1 and across the G1-S boundary (2).Little is currently known about regulation of this portionof the cell cycle, although some recent reports indicatethat the retinobiastoma gene product and members ofthe cdc2 and cyclin families may play key roles in thisprocess (4-6).

Transit across the G,-S boundary is less tightly regu-lated in cancer cells than in normal cells (7, 8). Althoughmost normal cells require growth factors and efficientprotein synthesis to progress through late C1 phase,transformed cells do not (9, 10). These findings under-score the point that a greater understanding of this por-tion of the cell cycle is fundamental to our understandingof cancer. In the experiments described here, we haveused the murine thymidine kinase gene as a model foraddressing the issue of gene regulation at the G1-Sboundary of the cell cycle.

Concomitant with the onset of S phase, the expressionof mRNAs and protein products for histones and manyof the enzymes associated with DNA synthesis increases(1 , 2). Among these enzymes are thymidine kinase (whichcatalyzes the phosphorylation of thymidine to thymidyl-ate), thymidyiate synthase, dihydrofolate reductase, andDNA polymerase a. The tight coupling of induction ofTK3 with the onset of S phase suggests that some priorcommon mechanism may trigger both events, eitherdirectly or indirectly. Previous work from our laboratorydemonstrated that both DNA synthesis and TK activityare sensitive to pulses of cycloheximide administeredlate in C1 (7), suggesting that the synthesis of some labileprotein is required at this point to trigger both events.

Subsequent research (1 1 ) demonstrated that the induc-tion of TK activity (as well as DNA synthesis) is dependenton insulin-like growth factor 1, further implicating a re-quirement for expression of new genes late in G,.

The regulation of TK gene expression operates onmultiple levels, including transcriptional, posttranscrip-tional, and translational controls (12-26). The tnanscnip-tional component of this regulation was first demon-strated by Stewart et a!. (24) and by Coppock and Pardee(12), using nuclear run-on experiments to indicate that,in both CV1 cells and BALB/c 313 cells, respectively, the

3 The abbreviations used are: 1K, thymidine kinase; bp, base pair(s); CAT,chloramphenicol acetyltransferase; cDNA, complementary DNA; PBS,phosphate-buffered saline.

Page 2: Growth-responsive Expression fromtheMurine Thymidine ...cgd.aacrjournals.org/cgi/reprint/2/2/67.pdf · Growth-responsive Expression fromtheMurine Thymidine KinasePromoter: Genetic

68 DNA Sequences Regulating Murine TK Gene Expression

rate of TK transcription increased by 2- to 7-fold as cellsleft G1 and entered S phase. We were particularly inter-ested in identifying those regions of the munine TK pro-moter essential for gene expression and regulation, be-cause the murine sequence lacks both TATA and CCAATbox elements (19, 27)that have been identified as criticalfor expression from the human TK promoter (20, 22, 28).Lieberman et a!. (19) identified a 291-bp fragment fromthe 5’ end of the murine TK gene which had promoterfunction in the context of a TK minigene, but they didnot uncouple these sequences from the rest of the geneor examine them for the presence of growth-responsiveregulatory elements. Our results presented here bothconfirm and extend their findings.

Cell cycle regulation of the human TK promoter hasbeen studied in a variety of ways by different groups. Forexample, Knight et a!. (1 7) reported that the pattern ofprotein binding to a 67-bp fragment of the human TKpromoter changed as cells crossed the G1-S phaseboundary. More recently, Travali et a!. (26) reported thata 471-bp fragment of the human TK promoter was suffi-cient to direct growth-responsive expression of the bac-teniai CAT gene in stably-transfected mouse BALB/c 3T3cells. Similarly, both Kim et a!. (29) and Roehi and Conrad(22) have reported that fragments of human TK upstreamsequence are sufficient to confer cell cycle regulation tothe otherwise constitutive expression of a neo reportergene.

We have used both deletion analysis and site-specificmutagenesis to delineate small regions of mouse TK 5’sequence that are essential for the efficient and regulatedexpression of a heterologous gene. We have found that,in addition to upstream regions, sequences locateddownstream of the TK translation initiation site are im-portant for efficient S phase-regulated expression. TK

promoter sequences derived from both upstream anddownstream regions together were sufficient to conferappropriate serum-responsive regulation to the expres-sion of two different reporter genes (CAT and �3-globin).When the upstream sequences alone were linked to areporter gene, expression was only partially regulated.TK downstream sequences therefore contributed to theobserved regulation. The results of our studies presentedhere set the stage for a more detailed investigation (usingboth genetic and biochemical techniques) into the natureand mediators of the S phase-specific regulation ofTK gene expression in normal and transformed cells (3#{216})�4

ResultsDeletion Analysis of the Murine TK Promoter Sequence.To delineate regions of munine TK promoter sequencerequired for efficient expression of a heterologous gene,we generated and analyzed a family of nested 5’ dele-tions carrying different amounts of TK sequence linkedto a CAT reporter gene (Fig. 1 ). We used transient expres-sion transfection analysis of mouse L929 cells to deter-mine the relative activity of each fusion gene construct.To control for varying transfection efficiencies betweenplates, all values were normalized according to theexpression of a cotransfected �3-galactosidase-encoding

plasmid (see “Materials and Methods”). As the data pre-sented in Fig. 1 illustrate, TK sequences located between...,-1000 bp and -557 bp of the TK translation initiation

site were deleted with virtually no effect on CAT activity.Truncation of the 5’ sequence to -1 74 bp resulted in aslight decrease in CAT activity, whereas truncation to-143 bp caused CAT activity to drop almost 3-fold.When the TK promoter sequence was truncated to -24bp, however, CAT activity dropped more than 30-fold.These results indicate that, although some promoter ele-ments may be located as far upstream as -557 bp, themost critical elements are located downstream of -174bp and include essential sequences located between-143 bp and -24 bp.

An examination of the nucleotide sequence in thisregion (1 9, 27) indicated a perfect consensus binding sitefor the transcription factor Spi (31) located between -90bp and -100 bp. DNase I footprint analysis, performedusing both crude nuclear extracts (prepared from mouseBALB/c 3T3 clone A31 cells) and purified human Spiprotein (kindly provided by Drs. S. Jackson and R. Tjian),also indicated binding at this site.4 To test the contnibu-tion of this Spi site to TK promoter activity in our CATexpression system, we introduced a single point mutationinto pACATm(-1 74)A at position -93 bp, changing a Cto C, creating pACATm.MT1m1. This mutation, which waspreviously shown to lower the binding of Spi protein inan in vitro assay by at least 30-fold relative to binding ata wild-type consensus sequence (32), prevented thebinding of either purified human Spi protein or mousenuclear extract in DNase I protection and band-shiftassays of the murine TK promoter region.4 This singlepoint mutation also virtually eliminated CAT expressionin our system (Fig. 1), suggesting that Spi plays a criticalrole in determining expression from the murine TK pro-moter.

All of the TK-CAT fusion genes described here in-cluded sequences derived from downstream as well asupstream of the TK translation initiation site, linked in-frame to the CAT coding sequence. These constructswere prepared and analyzed because analogous plasmidslacking the TK downstream sequences promoted little,if any, CAT activity relative to the promoterless con-trol. Each of the constructs described here [exceptpACATm(+28)A] may encode a TK-CAT fusion proteininitiated at the translation initiation site for TK. To testwhether the properties of this fusion protein would differsignificantly from those of wild-type CAT protein in ourassays, we generated a control TK-CAT plasmid in whichthe TK translation initiation codon was mutated, so thatthe first in-frame ATG in the CAT transcription unit wasthe ATG for CAT. Cells transfected with this plasmidexhibited levels of CAT activity virtually indistinguishablefrom those in cells transfected with the analogous wild-type plasmid (data not shown). We therefore concludethat if a TK-CAT fusion protein is encoded by the con-structs described here, it has properties similar to thoseof wild-type CAT in our transfection assay system.

The results of both nuclease protection assays andprimer extension reactions5 using endogenous mouse 1KRNA have indicated 5’ ends that map to greater than 20

4 Q-P. Dou, J. 1. Fridovich-Keil, and A. B. Pardee. Inducible proteinsbinding to the murine thymidine kinase promoter in late Cl/S phase.

Proc. NatI. Acad. Sd. USA, in press, 1991.

S j, M. Gudas, M. W. Datta, J. L. Fridovich-Keil, and A. B. Pardee. Exon-intron structure and analysis of multiple transcription start sites in themunine thymidine kinase gene. In preparation.

Page 3: Growth-responsive Expression fromtheMurine Thymidine ...cgd.aacrjournals.org/cgi/reprint/2/2/67.pdf · Growth-responsive Expression fromtheMurine Thymidine KinasePromoter: Genetic

.557 �p X �Z�Z�SS�

�: upstream sequencs

= = exon sequence

-= Intron sequence

-= CAT sequence

.174bp X

0% 100% (n)

pACATm(.1000)A I 100 (6)

pACATm(.557)A I 103 (4)

pACATm(.174)A . I is (6)

�I 36 (2)

� 3 (4)

I � (4)

I 6 (4)

� I (4)

-93bp

pACATm.MTI ml

Cell Growth & Differentiation 69

.�1OOObp X

X =polyA site trlmer

.ll4bp X

.lbp +159bp

-143 bp � �-_____ pACATm(.143)A

.24 bp x �- pACATm(.24)A

+28 bp X �-.----- pACATm(+28)A

X I- pA.promoterl.ss

acetylated T �P.chloramphenicol I

L #{149}�.

unacetylatedchloramphenicol

NormalizedCAT

Activity

0 R � cv, �. m ,-0 It) N. �‘ C”l c�i �0 It) ,-. ,- ..!� + l�,;. � � E ‘�‘ .

�< C) C.) U < �O 0.0 < < < 0.� << 0. 0. 0.0.

fig. 1. 5’ deletion analysis and site-specific mutagenesis of murine TK 5’ sequences. A, nested 5’ end deletions were generated using Exolll/Sl digestion

of a mouse 1K/CAT fusion gene, as described in “Materials and Methods.’ Deletion end points are indicated to the left of each diagram. Each constructcontains a trimer cassette of the 5V40 major late polyadenylation site inserted just upstream of the TK promoter region to block background read-through

CAT expression. +1, the murine TK translation initiation site. pACATm.MT1” is identical to pACATm(-174(A except for a single point mutation (at -93bp) in an Spl consensus binding site. The relative activity of each construct was assayed using transient transfection of mouse 1.929 cells; respectivevalues normalized for transfection efficiency (according to the expression of a cotransfected fl-galactosidase-encoding plasmid( are presented to the right

of each diagram. The actual numbers presented here were normalized to the expression of pACATm(-l000(A (100%). Bars, SD. (n(, the number of timeseach transfection was repeated. B, representative CAT assays of extracts prepared from cells transiently expressing the mouse TK/CAT fusion geneconstructs illustrated in A.

different sites, spanning a region of more than 100 basesat the 5’ end of the mouse TK gene. Although unusual,this result is similar to that recently reported by Blasbandet a!. (33) from experiments with transcripts of the rattransforming growth factor a gene. Both of these genes(mouse TK and rat transforming growth factor a) have

promoters that lack canonical CCAAT or TATA se-quences. To avoid confusion due to the multiplicity ofmouse 1K RNA 5’ ends, we have listed all positionsrelative to the TK translation initiation site, rather than atranscription initiation site. Preliminary primer extensiondata from experiments using TK reporter gene RNAs

Page 4: Growth-responsive Expression fromtheMurine Thymidine ...cgd.aacrjournals.org/cgi/reprint/2/2/67.pdf · Growth-responsive Expression fromtheMurine Thymidine KinasePromoter: Genetic

C.)

>

104 20 30

cells begIn to enter S phase

Hours FollowIng Serum Stimulation

pCATm(-174)A

acetylated r #{149}#{149}..S�chloramphenlcol L

unacetylated rchloramphenicol L #{149} � #{149}#{149}

0 2 6 12 16 25 30

Hours Following Serum Stimulation

fig. 2 Seruni.responsive expression of CAT in cells stably expressing amurine TK/CAT fusion gene. A. pooled populations of BALB/c 3T3 ellsstably expressing different promoter/CAT fusion constructs were syn-(hronize(l in quies(ence by serum starvation, restimulated with lO%

serum, and harvested at the indicated tinies. Percentage of labeled nuclei(dashed line( demonstrated that cells began entering S phase after 12 h;

by 16 h, more than 35% of the ells had entered 5, and by 25 h, virtuallyall (ells had entered S phase. Equivalent amounts (�g protein( of extractsfrom ea h time point were analyzed for CAT activity; the results ofrepresentative experiments are presented. PFC700 is a fos-CAT construct,pBfCAT2 carries the HSVIk pronloter, pCATni(-174(A carries niurine TK

sequences 1 74 1)1) to + 1 59 1)1), and I)BLCAT3 is a promoterless control.It is important to note that these extracts have not been normalized toa ( ount for transfe lion elfx iency, so that absolute value comparisonsare valid (inly within ((‘II lines and not between cell lines. These data arepresented Ii) indicate patterns of expression in response to serum slim-

ulation rather than absolute levels of reporter gene expression. B, repre-sentative CAT assay data from cells expressing the murine TK/CAT

construct pCATm(- 1 74(A. These extracts, prepared from cells harvestedat the times indicated, were normalized acording to protein concentra-

tion.

70 DNA Soqut�nes Regulating Murine TK Gene Expression

indicate that the 5’ ends of these fusion RNAs do mapwithin the appropriate region.

In order to block any background expression of CATdue to transcripts initiated within the plasmid backboneof our constructs (outside of the TK promoter region),we inserted a previously characterized trimer “cassette”(34) of the SV4O major late polyadenylation site justupstream of the TK promoter sequence in each plasmid(Fig. 1). This cassette effectively eliminates background

CAT expression in this system (data not shown). Indeed,as mentioned above, preliminary primer extension data

indicate that reporter genes expressed from this modified

plasmid backbone do initiate transcription within theappropriate TK promoter region.5

Domains of TK Sequence Important for S Phase-spe-.� cific Regulation of Reporter Gene Expression. We per-� formed cell synchrony experiments with populations of

2 mouse BALB/c 3T3 clone A31 fibroblasts stably express-.� ing different TK-CAT or TK-/3-globin constructs to deter-

:� mine which TK promoter sequences were sufficient to.! confer serum-regulated expression to a reporter gene.� Briefly, cells were synchronized in quiescence by serum

starvation, restimulated with high-serum-containing me-dium, and harvested at the times indicated in Figs. 2, 4,and 5. Parallel cultures of cells incubated in the presenceof [3H]thymidine were analyzed by autoradiography as ameasure of cell synchrony and time of entry into S phase(Fig. 2, dashed line). At each time point, cell extracts wereanalyzed for CAT enzyme activity, CAT mRNA level, or�3-gIobin mRNA level. As a positive control, endogenousTK mRNA levels were monitored in parallel with reportergene message levels (Figs. 4 and 5). It is important tonote that the absolute abundance of CAT activity or f3-globin message detected in each of these cell lines wasnot normalized to account for transfection efficiency, sothat absolute value comparisons are valid only within

each cell line, and not between lines. These data arepresented to indicate patterns of reporter gene expres-sion in response to serum stimulation, rather than abso-lute levels of expression. It is also important to note that,due to the stability of both CAT protein and fl-globinmRNA, these cell synchrony experiments were wellsuited to detect cumulative increases, not decreases inreporter gene expression levels.

Cells transfected with the construct pCATm(-174)Aexpressed low levels of CAT activity during quiescenceand G1 and higher levels following entry into S phase(Fig. 2). TK promoter sequences located between -174bp and +159 bp were therefore sufficient to drive CATexpression in these cells in an S phase-specific, serum-responsive manner. As controls, populations of cells ex-pressing CAT transcribed from either the constitutiveHSVtk promoter (pBLCAT2, designated HSVtk-CAT) (35)or the early serum-responsive c-los promoter (pFC700,designated los-CAT) (36) were also examined. HSVtk-CAT-transfected cells expressed CAT activity in a virtuallyconstitutive manner (Fig. 2), whereas cells stably trans-fected with los-CAT expressed CAT at levels that wererelatively low in quiescence and rose sharply within 2 hfollowing serum stimulation. Cells stably transfected withthe promoterless control plasmid pBLCAT3 (designatedpromoterless-CAT) expressed little CAT activity regard-less of growth status. The differences between thesepatterns of CAT expression served to confirm the impor-tance of promoter sequences in determining the regula-tion of CAT reporter gene expression in each of thesedifferent cell lines.

To determine whether the observed rise in CATenzyme activity reflected a corresponding rise inCAT mRNA levels, we isolated total RNA from synchro-nized populations of cells expressing the constructpACATm(-174)A. Northern blot analysis of these sam-pies indicated that the level of CAT mRNA detected inthese cells was lowest in quiescent and G cells and roseas the cells entered S phase (data not shown). As hasbeen reported for the expression of endogenous TK

Page 5: Growth-responsive Expression fromtheMurine Thymidine ...cgd.aacrjournals.org/cgi/reprint/2/2/67.pdf · Growth-responsive Expression fromtheMurine Thymidine KinasePromoter: Genetic

- . � b.�b... . #{216}.gI�b� 0’ ...d 3’�’.�.d�g ,.ge...

� : Tk � � #{149}. � �.di.g

� : TX �dW�g �q..... - - - . ..q...�.

- = my.. ..q..... #{149}. H$VIk �

‘Vt’,,

‘.4’

P.’�,*

�‘: �. .�,‘

‘H�.

4��

� #{149}

Cell Growth & Differentiation 71

murki#{149}TX ..qu.nc. �.gIo�n s.qunc.

,�----�----� �

-

.-�\ \1,.,.,J�’-J-J’J-.,.,J�A � �

-lMbp .ibp .iMb�

, , ,

-il4bp -4bp

, ,

c- pft.gIoU(.557)A

\\�__ p�5.gloM(.557)B

\ \�- pft.gIoNl.144)A

0\\L��___ p�5.g�M(.l44I8

pHSVIk.Itg)o

Fig. 3. /.�-Globin expression vectors. Diagrams of representative $-globin expression vectors introduced into mouse BALB/c 3T3 cells. p�l-gloM(-557(Aand pfl-gloM(-144(A contain mouse 1K sequences derived from both upstream and downstream of the TK translation initiation site (+1) as indicated;

pf3-gloM(-557(B and pf3-gloM(-1 44(B each contain only 1K sequences derived from upstream of +1 ; pHSVtk-�iglo carries the constitutive HSVtk promoter.

Diagrams are not drawn to scale.

�I�1 p�-gloM(-557)A EI�1p�-gloM(-557)B �1

�-gIobin �

endogenous TK

microglobulin

0 812162024 0 812162024 0 6 9121518

Hours Following Serum Stimulation

Fig. 4. Northern blot analysis of total RNA from cells stably expressing murine TK//.�-globin fusion gene constructs. Cells were synchronized in quiescenceby serum starvation, restimulated with 10% calf serum-containing medium, and harvested at the indicated times. Twenty �g of total RNA were loaded in

each well of a formaldehyde-agarose gel, electrophoresed, and transferred to a nylon membrane for filter hybridization with the probes indicated at left.Samples from these different cell lines were not normalized relative to one another to account for transfection efficiency, so that absolute valuecomparisons are valid only within each cell line and not between lines. These data are presented to indicate patterns of expression in response to serum

stimulation rather than absolute levels of reporter gene expression. The bands representing full-length �i-globin message are indicated with arrowheads.A, cells stably expressing pf3-gloM(-557(A, which includes TK sequences -557 bp to +159 bp. B, cells stably expressing p�l-gloM(-557(B, which includes

1K sequences -557 bp to -4 bp. C, cells stably expressing pHSVtk-�lglo, which includes the HSVtk promoter.

Page 6: Growth-responsive Expression fromtheMurine Thymidine ...cgd.aacrjournals.org/cgi/reprint/2/2/67.pdf · Growth-responsive Expression fromtheMurine Thymidine KinasePromoter: Genetic

‘It’,,

#{149}01*t4

‘N,

‘Is”

. 0#{149}1

N.,.

72 DNA Sequences Regulating Murine TK Gene Expression

1I�1p�-gIoM(-144)A II�1p�-gIoM(-144)B

�-globin �

endogenous TK

32-

microglobulin

08121621 exp 0 812162024

Fig. 5. Northern blot analysis

of total RNA from cells stablyexpressing the constructs

p$-gloM(-144)A and pfl-gloM(-144)B. Samples were prepared

and analyzed as described inthe legend to Fig. 4. p$-gloM(-144)A includes mouse 1K

sequences -144 bp to +159 bp

(A); pfl-gloM(-144)B includesmouse 1K sequences -144 bp

to -4 bp (B). exp, RNA harvestedfrom cells during exponentialgrowth.

Hours Following Serum Stimulation

mRNA and enzyme levels in these cells (37), the rise inCAT mRNA levels preceded the rise in CAT enzymeactivity by several hours.

As an independent test of the regulatory capacity ofthe murine TK promoter, we linked these sequences tothe human fl-globin gene and generated a collection ofcell lines that stably expressed these genes (Fig. 3). Theseconstructs and the cell lines expressing them were de-signed to investigate the relative contributions of bothTK distal upstream sequences and TK downstream se-quences on the serum-responsive regulation of fl-giobinreporter gene expression. Two sets of constructs en-coded fl-globin driven off TK promoter sequences ex-tending to -557 bp, whereas another two carried TKpromoter sequences extending only to -144 bp. Withineach of these sets, one construct included both TK up-stream and downstream sequences (extending to +159bp), whereas the other carried TK sequences truncatedat -4 bp. As controls, both a promoterless-�-globin plas-mid and pHSVtk-$gio, a construct encoding f3-globindriven off the constitutive HSVtk promoter, were gener-ated and analyzed. Cells transfected with the promoter-less control plasmid expressed little, if any, detectable [3-

globin message (data not shown). Cell lines stably ex-

pressing each of the other constructs were synchronizedin quiescence by serum starvation and then restimulatedwith serum and harvested at the indicated times (Figs. 4and 5). Total RNA prepared from these cells was sizeseparated by formaldehyde-agarose gel electrophoresisand transferred to nylon filters for Northern blot analysis.

By hybridizing these filters first with a human /3-globincDNA probe and then stripping and rehybridizing eachwith a mouse TK cDNA probe, we were able to visualize

and compare expression of the [3-globin reporter genewith that of the endogenous TK gene. Subsequent hy-bridization of each filter with a mouse fl2-microglobulinprobe was used to indicate comparable loading of lanes.The bands representing full-length [3-globin message areindicated with arrowheads (Figs. 4 and 5). The upperband apparent in each of these blots was consistentlyobserved in all of our [3-globin reporter gene experi-ments, even those involving the promoterless controlplasmid. Due to its large size, constitutive appearance,and insensitivity to changes in the promoter region ofthe reporter gene constructs, we presume that it mayrepresent transcription of the antisense strand, or someother form of read-through expression.

We found that cell lines expressing /3-globin from a TK

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Cell Growth & Differentiation 73

promoter including both upstream and downstream se-quences expressed the reporter gene in an S phase-specific manner, with kinetics similar to those seen forthe endogenous TK gene (Figs. 4 and 5). The absolutelevel of [3-globin mRNA induction was less than that seenfor the endogenous TK mRNA, presumably because onlya subset of the transcriptional and/or posttranscriptionalcontrols responsible for regulating endogenous TK

expression were conferred to the reporter gene by thepresence of the TK 5’ sequences. The presence or ab-sence of the TK distal upstream sequences (-557 bp to-145 bp) made little, if any, difference in the observedregulation of reporter gene expression. In contrast, bothcell lines expressing [3-globin from a TK promoter includ-ing only the upstream sequences and lacking the down-stream sequences (-4 bp to +159 bp) showed onlypartial induction of reporter gene expression with theonset of S phase. In these cells, although some inductionof [3-globin mRNA was observed, the pattern of inductiondid not closely parallel that of the endogenous TK. Asexpected, the endogenous TK gene exhibited a strikingS phase-specific induction of expression in each of thesecell lines.

Together, these results demonstrate that TK promotersequences located between -144 bp and +159 bp weresufficient to confer S phase-specific expression to a[3-globin reporter gene. In contrast, cells expressing/3-globin driven off the constitutive HSVtk promotershowed virtually constitutive expression of [3-globinmRNA, although the endogenous TK mRNA levels inthese cells increased as expected as the cells entered Sphase. These findings both confirm and extend thoseobtained using the CAT reporter gene system (Fig. 2).Furthermore, these results indicate that sequences lo-cated downstream ofthe TKtranslation initiation site playan important role in the regulation of TK mRNA levelsfollowing serum stimulation. Additional experiments arerequired to determine the mechanism of that regulation.

Discussion

The results presented here demonstrate two main find-ings: (a) that sequences located between -1 74 bp and-24 bp are essential for the efficient expression of a CAT

reporter gene from the murine TK promoter, and thatbinding of the transcription factor Spi likely plays acritical role in the function of the promoter, and (b) thatTK promoter upstream and downstream sequences to-gether are sufficient to confer S phase-specific expressionto both CAT and [3-globin reporter genes. These conclu-sions were derived from experiments in which a combi-nation of techniques, including analysis of CAT enzymeactivity levels, CAT mRNA levels, and f3-globin mRNAlevels, were used to assess reporter gene expression intransfected cells.

The observation that sequences located between-1 74 bp and -24 bp of the TK translation initiation siteare essential for efficient expression of the CAT reportergene implies that specific cis-acting elements essentialfor expression of the gene are found in this region.Indeed, in vitro analyses of DNA-protein interactionshave shown that nuclear proteins do bind to this regionand, moreover, that at least one of these proteins (notSpi) binds in an S phase-specific manner.4 Perhaps mostinteresting, comparisons between transformed and non-

transformed cells indicate striking differences in the sta-bility and cell cycle regulation of this DNA binding activ-ity (30). A computer-based search for transcription factorconsensus binding sites within the murine TK promoter[using the system of Ghosh (38)] revealed a multitude offull or partial matches both upstream and downstream ofthe TK translation initiation site (data not shown). Thesignificance of many of these sequence homologies re-mains to be determined.

At least one match, a perfect Spi consensus bindingsite located between -90 bp and -100 bp, was criticalfor activity of the mouse TK promoter. A single pointmutation introduced at this site both interrupted proteinbinding4 and lowered CAT reporter gene expression bygreater than 1 order of magnitude. This result is reminis-cent of those of both McKnight and Kingsbury (39) andJones et a!. (40), who demonstrated the importance ofSpi binding sites in the viral HSVtk promoter using linkerscanning mutagenesis and single point mutations.

Our second principal finding is that TK promoter se-quences located between -1 74 bp and +1 59 bp aresufficient to confer appropriately S phase-specific regu-lation to the expression of two different reporter genes(CAT and [3-globin) and that the downstream portion ofthis sequence (-4 bp to +159 bp) appears to play someimportant role in this process. Our results imply thatmouse TK upstream sequences alone do not fully conferS phase-specific regulation to reporter gene expression,although they certainly may contribute to that regulation,as suggested by mutagenesis experiments and the in vitroDNA-protein binding studies4 mentioned earlier (30).These data also further emphasize the difference be-tween murine and human TK promoter sequences, sincehuman TK upstream sequences alone were reported tobe sufficient to confer S phase-specific regulation to theexpression of an otherwise constitutive reporter gene(22, 26, 29).

Our finding that downstream as well as upstream TKpromoter sequences contribute to the regulation of re-porter gene expression is striking although not withoutprecedent. Recent reports indicate that a small but grow-ing class of genes requires sequences downstream aswell as upstream of the transcription initiation site forproper expression and/or regulation. Our observationsare also consistent with the findings of Lewis and Mat-kovich (18), Lieberman et a!. (19), and Stewart et a!. (24),who reported that hamster, mouse, and human TK cDNAsequences (respectively) themselves were sufficient toconfer growth-responsive regulation to their own expres-sion from an otherwise constitutive promoter.

Examples from the literature indicate that internal reg-ulatory elements may influence gene expression in anyof a number of ways, including general or tissue-specificenhancer effects (41 -43), effects on transcription initia-tion (44-46), transcription elongation (47-51), mRNAstability (52), or translational efficiency (53). Further ex-periments are required to determine the actual mecha-nism by which murine 1K downstream sequences influ-ence gene expression in this system.

The regulation of gene expression at the G1-S phaseboundary of the cell cycle has been studied for a numberof genes other than TK, including histones (54, 55), di-hydrofolate reductase (41, 56), proliferating cell nuclearantigen (57, 58), and thymidylate synthase (59). Thepatterns of regulation for each gene studied appear to

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74 DNA Sequences Regulating Murine TK Gene Expression

be complex, and the possibility remains that commonsequences and/or factors may coordinately regulatesome or all of these genes. Such sequences and factorsmay represent components of the biochemical machin-ery responsible for controlling not only cell cycle-regu-lated gene expression, but also cell proliferation. Corn-

parative studies of these factors in transformed and non-transformed cells, therefore, may lead to a greaterunderstanding not only of normal cell growth, but alsoof the aberrant growth that is characteristic of cancer.

Materials and Methods

Plasmid Construction. All murine TK promoter/CAT fu-sion gene constructs were generated by inserting variousdomains of TK sequence into the polylinker region of thepromoterless CAT vector pBLCAT3 (35). Where mdi-cated, a Hindlll fragment containing a trimer cassette (34)of the SV4O major late polyadenylation site was alsoinserted into the plasrnid backbone, just upstream of thepromoter sequence. This cassette has been demon-strated to block effectively background expression dueto transcripts initiated within the plasmid backbone, out-side of the designated promoter region (34). All manip-ulations were performed according to standard recom-binant techniques (60). All constructs were verified byrestriction endonuclease mapping, dideoxy sequencing,or both. Unless otherwise noted, all positions are givenrelative to the TK translation initiation site (+1) to avoidconfusion caused by the multiplicity of transcription startsites in the murine TK gene.5

pACATrn(-1000)A includes the polyadenylation tn-mer cassette inserted just upstream of TK sequenceswhich extend from -�--1000 bp (the Psti site) to +159 bp;the 3’ end of the TK sequence in this construct was gen-erated by cutting at the Apal site, rendering the endblunt with DNA polymerase I large fragment (Klenow),and then ligating Xhol 8-mer linkers to it. This Xhol cutend was then ligated to the XhoI site in pBLCAT3.pACATm(-557)A is identical to pACATm(-1000)Aexcept that it carries TK sequences truncated at -557bp (an EcoRl site). Similarly, pACATm(-174)A carriesTK sequences truncated at -174 bp (a Ncol site).pACATm(- 1 43)A, pACATm(-24)A, and pACATm(+28)Awere all generated from pACATm(-557)A using an Exolii/Si deletion protocol (Promega’s Erase-a-Base system) andcarry TK sequences truncated at positions -143 bp, -24bp, and +28 bp, respectively. pACATm.MT1m1 is identi-cal to pACATm(-1 74)A except for a single point mutation(C to C) at position -93 bp. To introduce this mutation,appropriate sequences were transferred to a phagemidvector backbone (pGEM5) and subjected to oligonucle-otide-directed mutagenesis (using the synthetic oligonu-cleotide 5’-CTCAAAAGGGGGGGGGACGA-3’) as de-scribed by McClary et a!. (61). This mutation was con-firmed by dideoxy sequencing. The mutated sequencewas then returned to its original position within the CATexpression vector by standard techniques. pA-promot-erless was generated from pBLCAT3 by inserting thepolyadenylation site trimer cassette Hindlli fragment intothe HindIll site of the polylinker.

The [3-globin expression vectors used in these studieswere generated as follows: p/3-gloM(-557)A includes mu-rine TK promoter sequences -557 bp to +159 bp, whichwere excised from pCATm(-557)A by digestion with

Xhoi, blunted with Kienow, and Hindlil linkered beforeligation into the Hindili site of a Gem 7 backbone thatalready carried the (promoterless) human /3-globin gene(62). This ligation placed the murine TK promoter in thecorrect orientation directly upstream of the [3-globin se-quence. p/3-gloM(-557)B is similar to pf.3-gloM(-557)Aexcept that it includes only TK sequences -557 bp to -4bp. The TK sequences in this plasmid were truncated at-4 bp by cutting the DNA with Fnu4Hl and bluntingwith Klenow, followed by ligation to a Hindlll site inpGEM5 which had also been blunted with Klenow. ABamHl site adjacent to the blunted Hindill site in thepolylmnker was used to remove the TK fragment fromGem5. pf3-gloM(-144)A and pf3-gloM(-144)B are similarto p[3-gloM(-557)A and B, respectively, except that eachcarries TK promoter sequences truncated at the SstI sitelocated at -144 bp. pHSVtk-figlo carries the same HSVtkpromoter sequences as pBLCAT2 inserted just upstreamof the /3-globin reporter gene. All constructs were con-firmed by restriction enzyme digestion and/or dideoxysequencing.

Cell Culture and DNA Transfection. Mouse BALB/c313 clone A31 cells were used for all stable transfectionassays and were cultured in 10% CO2 in air at 37#{176}CinDulbecco’s modified Eagle’s medium supplemented with4 mM glutamine, 10 units/mI penicillin, 10 zg/ml strep-tomycin, and 10% bovine calf serum (Hyclone). We usedthe mouse fibroblast line L929 (generously donated byDrs. Ann Georgi and Bernard Fields) for all transientexpression assays because they gave a much strongerreporter gene signal than did the 313 cells, perhapsreflecting a higher efficiency of transfection. These cellswere cultured in 6% CO2 in air at 37#{176}Cin Dulbecco’smodified Eagle’s medium supplemented with glutamine

and antibiotics, as above, and 10% fetal bovine serum(Hyclone).

One day prior to transfection, 1 x 10� cells were platedonto 100-mm dishes for transient expression assays, or 5x iO� cells/plate for stable expression transfections. Cal-cium phosphate transfection was performed essentiallyas described by Ausubel et a!. (63). Fifteen �zg of super-coiled DNA were used per plate for each transfection.Transient transfections were performed using 10 zg ofCAT-construct DNA, 2 �zg of p[3AclacZ DNA (64), and 3� of pGEM5 DNA (Promega) as carrier. Stable transfec-tions were performed using 10 �zg of CAT-construct or [3-

globin-construct DNA, 1 �g of pSV2-Neo DNA (65), 2 �zgof p/3AclacZ DNA, and 2 �g of pG EMS.

Two days following transfection, cells were either har-vested for analysis (transient expression) or trypsinizedand replated for selection at dilutions of between 1 :5 and1:10 in culture medium containing 1 mg/mI (w/v) G418(GIBCO). Cells under selection were refed fresh mediumcontaining G418 every 3 days until colonies were clearlyvisible (9 to 12 days). In an effort to minimize copynumber and position effects, stable transfectants weretrypsinized as populations of colonies and maintained aspopulations under selection by G418. Each pooled pop-ulation was composed of cells derived from at least 100individual colonies, originating from the combination of

two independent transfection experiments. Becauseeach population was analyzed shortly following its initialselection, the possibility of overgrowth by nonrepresen-tative clones is unlikely.

Cell Synchrony Experiments. Stable transfectants were

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Cell Growth & Differentiation 75

plated at a density of S x iO� cells/iSO-mm plate (Lux)in culture medium containing 10% calf serum and al-owed to grow for 3 days. The subconfluent, monolayer

cultures were then washed twice with culture medium

containing low serum (0.4% calf serum) and incubatedin this medium for 60 h. Medium was removed from thequiescent cultures and replaced with culture mediumcontaining 10% calf serum. Plates of cells were harvestedat the indicated times.

We used the incorporation of [3H]thymidine as anindicator of entry into S phase. Cells were plated at adensity of 5 X iO� cells/well in 24-well plates (Falcon)and treated as described above. Quiescent cells wererefed either fresh low-serum medium containing 1 zCi/ml [3H]thymidine (NEN/DuPont) or 10% calf serum me-dium containing 1 �zCi/ml [3Hjthymidine. Cells refed low-serum medium were incubated for 12 h before fixing. Allother cells were fixed at the appropriate times and pre-pared for autoradiography essentially as described byMoscovitis and Pardee (66).

Analysis of CAT Adivity. Cells were harvested andanalyzed for CAT activity essentially as described byGorman et a!. (67). Briefly, cells were washed twice inice-cold PBS, harvested by scraping with a rubber police-man in 1 ml of cold PBS, transferred to pre-chilled Ep-pendorftubes, and spun briefly at 4#{176}Cto pellet the cells.The PBS supernatant was removed, and cells were resus-

pended in 150 zl of ice-cold 0.25 M Tris, pH 7.8-15%glycerol. Cell suspensions were cycled through fourrounds of freeze/thaw and then centrifuged at 10,000rpm in an Eppendorf Microfuge (4#{176}C)for 5 mm to pelletnuclei and insolubles. Supernatants were removed andtransferred to pre-chilled tubes. Two jzl of each samplewere removed for protein determination (Bio-Rad).

Extracts from stable expression transfectants were nor-malized for analysis of CAT activity according to proteinconcentration. Extracts from transient expression trans-fectants were normalized according to /3-galactosidaseactivity, reflecting expression of the cotransfected/3AclacZ plasmid, thereby minimizing the effects of vary-ing transfection efficiencies between plates (usually lessthan 2- or 3-fold). /3-Galactosidase activity was measuredusing a colorimetric assay similar to that described by Anet a!. (68). Briefly, 100 �g of cell extract were added tolac Z buffer (60 mM Na2HPO4-40 m�i NaH2PO4, pH 7.0-10 mM KCI-1 mM MgSO4-50 m�i /3-mercaptoethanol) toa final volume of 450 zl. Fifty .zl of 50 mr�i chlorophenolred-[3-D-galactopyranoside (Boehringer Mannheim) were

then added, and the mixture was incubated at 37#{176}Cforup to 1 h. Absorbance at 574 nm was measured for eachsample against a parallel blank reaction containing extractfrom mock-transfected cells, thereby subtracting out anycontribution from endogenous [3-galactosidase activity.

The relative CAT activity in each sample was quanti-tated by liquid scintillation counting of excised thin-layerchromatography spots in Econofluor (NEN/DuPont). Rel-ative activities were calculated as percentage of totalcpm shifted from the unacetylated spot to the acetylatedspots.

Northern Blot Analysis. Total cellular RNA was isolatedfrom populations of cells and characterized by Northernblot analysis as described previously (14). In brief, 20 �zgof total RNA were loaded into each well of a formalde-hyde-agarose gel, size fractionated by electrophoresis,and transferred to a Nytran filter for subsequent hybrid-

ization with a series of three different probes (human [3-

globin cDNA, mouse TK cDNA, and a fragment of mouse132-microglobulin sequence).

Acknowledgments

We thank Drs. Pin-Fang l.in and Frank Ruddle for providing the mouse1K cosmid DNA, Dr. Deborah zajchowski for many helpful discussions,Drs. George Vasios, Lorraine Cudas, and Victor Rivera for plasmids, andDr. Erik Flemington and Forrest Nelson for technical advice. We also

thank Drs. Dennis Lynch, John Boylan, Jeffrey Goliger, Bruce Dezube,

Neil Freedman, and Linnea Hansen for critical reading of the manuscript.

References

1 . Baserga, R. The Biology of Cell Reproduction. Cambridge: HarvardUniversity Press, 1985.

2. Pardee, A. B. Cl events and regulation of cell proliferation. Science

(Wash. DC), 246: 603-608, 1989.

3. Rollins, B. J., and Stiles, C. D. Serum-inducible genes. Adv. CancerRes., 53: 1-31, 1989.

4. DeCaprio, J. A., Ludlow, J. W., Lynch, D., Furukawa, Y., Griffin, I..Piwnica-Worms, H., Huang, C-M., and Livingston, D. M. The product ofthe retinoblastoma susceptibility gene has properties of a cell cycleregulatory element. Cell, 58: 1085-1095, 1989.

5. Murray, A. W., and Kirschner, M. W. Dominoes and clocks: the unionoftwo views of the cell cycle. Science (Wash. DC), 246: 614-621, 1989.

6. Richardson, H. E., Wittenberg, C., Cross, F., and Reed, S. I. An essential

Cl function for cyclin-like proteins in yeast. Cell, 59: 1 127-1 133, 1989.

7. coppock, D. L., and Pardee, A. B. Regulation of thymidine kinase

activity in the cell cycle by a labile protein. I. Cell. Physiol., 124: 269-

274, 1985.

8. Pardee, A. B. Molecules involved in proliferation of normal and cancercells: presidential address. Cancer Res., 47: 1488-1491, 1987.

9. Medrano, E. E., and Pardee, A. B. Prevalent deficiency in tumor cellsof cycloheximide-induced cycle arrest. Proc. NatI. Acad. Sci. USA, 77:4123-4126, 1980.

10. Rossow, P. W., Riddle, V. C. H., and Pardee, A. B. Synthesis of labile,serum-dependent protein in early Cl controls animal cell growth. Proc.

NatI. Acad. Sci. USA, 76: 4446-4450, 1979.

1 1 . Yang, H. C., and Pardee, A. B. Insulin-like growth factor I regulationof transcription and replicating enzyme induction necessary for DNA

synthesis. J. Cell. Physiol., 127: 410-416, 1986.

1 2. Coppock, D. L., and Pardee, A. B. Control ofthymidine kinase mRNA

during the cell cycle. Mol. Cell. Biol., 7: 2925-2932, 1987.

13. Cross, M. K., and Merrill, C. F. Thymidine kinase synthesis is re-pressed in nonreplicating muscle cells by a translational mechanism that

does not affect the polysomal distribution of thymidine kinase mRNA.Proc. NatI. Acad. Sci. USA, 86: 4987-4991, 1989.

14. Cudas, J. M., Knight, C. B., and Pardee, A. B. Nuclear posttranscrip-tional processing of thymidine kinase mRNA at the onset of DNA synthe-sis. Proc. NatI. Acad. Sci. USA, 85: 4705-4709, 1988.

1 5. Hofbauer, R., Mullner, E., Seiser, C., and Wintersberger, E. Cell cycleregulated synthesis of stable mouse thymidine kinase mRNA is mediatedby a sequence within the cDNA. Nucleic Acids Res., 15: 741-752, 1987.

16. Ito, M., and Conrad, S. E. Independent regulation ofthymidine kinase

mRNA and enzyme levels in serum-stimulated cells. J. Biol. Chem., 265:

6954-6960, 1990.

17. Knight, C. B., Cudas, J. M., and Pardee, A. B. Cell-cycle-specificinteraction of nuclear DNA-binding proteins with a CCAAT sequencefrom the human thymidine kinase gene. Proc. NatI. Acad. Sci. USA, 84:8350-8354, 1987.

18. Lewis, J. A., and Matkovich, D. A. Genetic determinants of growthphase-dependent and adenovirus 5-responsive expression of the Chinese

hamster thymidine kinase gene are contained within thymidine kinase

mRNA sequences. Mol. Cell. Biol., 6: 2262-2266, 1986.

19. Lieberman, H. B., Lin, P-F., Yeh, D-B., and Ruddle, F. H. Transcrip-

tional and posttranscriptional mechanisms regulate murine thymidinekinase gene expression in serum-stimulated cells. Mol. Cell. Biol., 8:5280-5291, 1988.

20. Lipson, K. E., Chen, S-I., Koniecki, J., Ku, D-H., and Baserga, R. S-phase-specific regulation by deletion mutants of the human thymidinekinase promoter. Proc. NatI. Acad. Sci. USA, 86: 6848-6852, 1989.

21. Merrill, C. F., Hauschka, S. D., and McKnight, S. L. 1k enzymeexpression in differentiating muscle cells is regulated through an internal

Page 10: Growth-responsive Expression fromtheMurine Thymidine ...cgd.aacrjournals.org/cgi/reprint/2/2/67.pdf · Growth-responsive Expression fromtheMurine Thymidine KinasePromoter: Genetic

76 DNA Sequences Regulating Murine TK Gene Expression

segment of the cellular tk gene. Mol. Cell. Biol., 4: 1777-1784, 1984.

22. Roehl, H. H., and conrad, S. E. Identification of a Cl-S-phase-regulated region in the human thymidine kinase gene promoter. Mol.

Cell. Biol., 10: 3834-3837, 1990.

23. Sherley, J. L., and Kelley, I. J. Regulation of human thymidine kinaseduring the cell cycle. I. Biol. Chem., 263: 8350-8358, 1988.

24. Stewart, C. I. Ito, M., and Conrad, S. E. Evidence for transcriptionaland post-transcriptional control of the cellular thymidine kinase gene.Mol. cell. Biol., 7: 1 1 56- 1 1 63, 1987.

25. Stuart, P., Ito, M., Stewart, C., and Conrad, S. E. Induction of cellularthymidine kinase occurs at the mRNA level. Mol. Cell. Biol., 5: 1490-1497, 1985.

26. Travali, S., Lipson. K. E., Jaskulski, D., Laurel, E., and Baserga, R. Roleof the promoter in the regulation ofthe thymidine kinase gene. Mol. Cell.Biol., 8: 1551-1557, 1988.

27. Seiser, C., Knofler, M., Rudelstorfer, I., Haas, R., and Wintersberger,E. Mouse thymidine kinase: the promoter sequence and the gene andpseudogene structures in normal cells and in thymidine kinase deficientmutants. Nucleic Acids Res., 17: 185-195, 1989.

28. Arcot, S. S., Flemington, E. K., and Deininger, P. L. The humanthymidine kinase gene promoter. I. Biol. Chem., 264: 2343-2349, 1989.

29. Kim, Y. K., Wells, 5, Lau, Y-F. C., and Lee, A. S. Sequences containedwithin the promoter of the human thymidine kinase gene can direct cell-

cycle regulation of heterologous fusion genes. Proc. NatI. Acad. Sd. USA,85: 5894-5898, 1988.

30. Bradley, D. W., Dou, Q-P., Fridovich-Keil, I. L., and Pardee, A. B.Transformed and non-transformed cells differ in stability and cell cycleregulation of a binding activity to the thymidine kinase promoter. Proc.NatI. Acad. Sd. USA, 87: 9310-9314, 1990.

31. Jones, K. A., and Tjian, R. Spl binds to promoter sequences andactivates herpes simplex virus ‘immediate-early” gene transcription invitro. Nature (Lond.), 317: 179-182, 1985.

32. Letovsky, J., and Dynan, W. S. Measurement of the binding oftranscription factor Spl to a single CC box recognition sequence. NucleicAcids Res., 17: 2639-2653, 1989.

33. Blasband, A. J., Rogers, K. I., Chen, X., Azizkhan, J. C., and Lee, D.C. Characterization of the rat transforming growth factor alpha gene andidentification of promoter sequences. Mol. Cell. Biol., 10: 2111-2121,

1990.

34. Maxwell, I. H., Harrison, C. S., Wood, W. M., and Maxwell, F. ADNA cassette containing a trimerized SV4O polyadenylation signal whichefficiently blocks spurious plasmid-initiated transcription. Biotechniques,

7: 276-280, 1989.

35. Luckow, B., and Schutz, C. CAT constructions with multiple uniquerestriction sites for the functional analysis of eukaryotic promoters and

regulatory elements. Nucleic Acids Res., 15: 5490, 1987.

36. Fisch, I. M., Prywes, R., and Roeder, R. C. c-fos sequences necessaryfor basal expression and induction by epidermal growth factor, 12-0-tetradecanoylphorbol-l 3-acetate, and the calcium lonophore. Mol. Cell.Biol., 7: 3490-3502, 1987.

37. Cudas, J. M., Knight, C. B., and Pardee, A. B. The cell cycle andrestriction point control. In: E. I. Frel led.), The Regulation of Proliferationand Differentiation in Normal and Neoplastic Cells, pp. 3-20. San Diego:Academic Press, 1989.

38. Chosh, D. A relational database of transcription factors. NucleicAcids Res., 18: 1749-1756, 1990.

39. McKnight, S. L., and Kingsbury, R. Transcriptional control signals ofa eukaryotic protein-coding gene. Science (Wash. DC), 217: 316-324,1982.

40. Jones, K. A., Yamamoto, K. R., and Tjian, R. Two distinct transcriptionfactors bind to the HSV thymidine kinase promoter in vitro. Cell, 42: 559-572, 1985.

41. Farnham, P. J., and Means, A. L. Sequences downstream of thetranscription initiation site modulate the activity of the murine dihydro-folate reductase promoter. Mol. Cell. Biol., 10: 1390-1398, 1990.

42. Cillies, S. D., Morrison, S. L., Oi, V. I., and Tonegawa, S. A tissue-specific transcription enhancer element is located in the major intron ofa rearranged immunoglobulin heavy chain gene. Cell, 33: 71 7-728, 1983.

43. Queen, C., and Baltimore, D. Immunoglobulin gene transcription isactivated by downstream sequence elements. Cell, 33: 741-748, 1983.

44. Ayer, D. E., and Dynan, W. S. Simian virus 40 major late promoter:a novel tripartite structure that includes intragenic sequences. Mol. Cell.

Biol., 8: 2021-2033, 1988.

45. Ayer, D. E., and Dynan, W. S. A downstream-element-binding factorfacilitates assembly of a functional preinitiation complex at the simianvirus 40 major late promoter. Mol. Cell. Biol., 10: 3635-3645, 1990.

46. Krauskopf, A., Resnekov, 0., and Aloni, Y. A cis downstream elementparticipates in regulation of in vitro transcription initiation from the p38promoter of minute virus of mice. J. Virol., 64: 354-360, 1990.

47. Bentley, D. L., and Croudine, M. A block to elongation is largely

responsible for decreased transcription of c-myc in differentiated HL6Ocells. Nature (Lond.), 321: 702-706, 1986.

48. Kao, S-Y., Calman, A. F., Luciw, P. A., and Peterlin, B. M. Anti-termination of transcription within the long terminal repeat of HIV-l bytat gene product. Nature (Lond.), 330: 489-493, 1987.

49. Miller, H., Asselin, C., Dufort, D., Yang, J-Q., Gupta, K., Marcu, K.B., and Nepveu, A. A cis-acting element in the promoter region of themurine c-myc gene is necessary for transcriptional block. Mol. Cell. Biol.,

9: 5340-5349, 1989.

50. Nepveu, A., and Marcu, K. B. Intragenic pausing and anti-sensetranscription within the murine c-myc locus. EMBO J., 5: 2859-2865,1986.

51 . Yang, J-Q., Remmers, E. F., and Marcu, K. B. The first exon of the c-myc proto-oncogene contains a novel positive control element. EMBOJ.,5: 3553-3562, 1986.

52. Ceballe, A. P., Spaete, R. R., and Mocarski, E. S. A cis-acting elementwithin the 5’ leader of a cytomegalovirus � transcript determines kinetic

class. Cell, 46: 865-872, 1986.

53. Rao, C. D., Pech, M., Robbins, K. C., and Aaronson, S. A. The 5’-untranslated sequence of the c-sis/platelet derived growth factor 2 tran-script is a potent translational inhibitor. Mol. Cell. Biol., 8: 284-292, 1988.

54. Heintz, N. Temporal regulation of gene expression during the mam-malian cell cycle. Curr. Opinion Cell Biol., 1: 275-278, 1989.

55. Schumperli, D. Multilevel regulation of replication-dependent his-tone genes. Trends Genet., 4: 187-191, 1988.

56. Farnham, P. J., and Schimke, R. I. Transcriptional regulation of mousedihydrofolate reductase in the cell cycle. J. Biol. Chem., 260: 7675-7680,1985.

57. Ottavio, 1., Chang, C-D., Rizzo, M-G., Travali, S., Casadevall, C., andBaserga, R. Importance of introns in the growth regulation of mRNA levelsof the proliferating cell nuclear antigen gene. Mol. Cell. Biol., 10: 303-309, 1990.

58. Chang, C-D., Ottavio, L., Travali, S., Lipson, K. E., and Baserga, R.Transcriptional and posttranscriptional regulation of the proliferating cellnuclear antigen gene. Mol. Cell. Biol., 10: 3289-3296, 1990.

59. Jenh, C-H., Geyer, P. K., and Johnson, 1. F. Control of thymidylatesynthase mRNA content and gene transcription in an overproducingmouse cell line. Mol. Cell. Biol., 5: 2527-2532, 1985.

60. Sambrook, J., Fritsch, E. F., and Maniatis, I. Molecular Cloning: ALaboratory Manual, Ed. 2. Cold Spring Harbor, NY: Cold Spring HarborLaboratory, 1989.

61 . McClary, J. A., Witney, F., and Geisselsoder, J. Efficient site-directedin vitro mutagenesis using phagemid vectors. Biotechniques, 7: 282-289,1989.

62. Lawn, R. M., Efstratiadis, A., O’Connell, C., and Maniatis, T. Thenucleotide sequence of the human �-globin gene. Cell, 21: 647-651,1980.

63. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J.C., Smith, J. A., and Struhl, K. Current Protocols in Molecular Biology.New York: John Wiley & Sons, 1989.

64. Vasios, G. W., Gold, I. D., Petkovich, M., Chambon, P., and Cudas,1. J.A retinoic acid-responsive element is present in the 5’ flanking regionofthe laminin Bi gene. Proc. NatI. Acad. Sci. USA, 86: 9099-9103, 1989.

65. Southern, P. 1.. and Berg, P. Transformation of mammalian cells toantibiotic resistance with a bacterial gene under control of the SV4O early

region promoter. J. Mol. AppI. Genet., 1: 327-341, 1982.

66. Moscovitis, C., and Pardee, A. B. Citric acid arrest and stabilizationof nucleoside incorporation into cultured cells. Anal. Biochem., 101: 221-224, 1980.

67. Gorman, C. M., Moffat, L. F., and Howard, B. H. Recombinantgenomes which express chloramphenicol acetyltransferase in mammaliancells. Mol. Cell. Biol., 2: 1044-1051, 1982.

68. An, C., Hidaka, K., and Siminovitch, L. Expression of beta-galactosid-ase in animal cells. Mol. Cell. Biol., 2: 1628-1632, 1982.