5-Responsive Expression of the Chinese Hamster Thymidine Kinase

Vol. 6, No. 6

MOLECULAR AND CELLULAR BIOLOGY, June 1986, p. 2262-2266 0270-7306/86/062262-05$02.00/0 Copyright © 1986, American Society for Microbiology

Genetic Determinants of Growth Phase-Dependent and Adenovirus 5-Responsive Expression of the Chinese Hamster Thymidine Kinase Gene Are Contained within Thymidine Kinase mRNA Sequences JOHN A. LEWISt* AND DIANA A. MATKOVICH Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 Received 15 October 1985/Accepted 21 February 1986

We have constructed a chimeric thymidine kinase (TK) minigene, pHeA6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transformants is growth phase dependent, responsive to adenovirus 5 infection, and indistinguishable in character under a variety of cell culture conditions from the pattern of TK gene expression in rat 4 cells transformed to Tk+ with the genomic Chinese hamster TK gene clone XHaTK.5. We are led to the conclusion that the genetic elements which mediate growth phase-dependent TK gene expression are contained entirely within the sequences of the mature cytoplasmic hamster TK mRNA.

growth phase-dependent accumulation of cytoplasmic TK mRNA through a systematic in vitro construction of mutant and chimeric TK genes whose pattern of expression could be studied upon their stable introduction into any of a variety of Tk- cell lines. As an indispensable first step in this experimental approach, we cloned and characterized by nucleotide sequencing a nearly full-length Chinese hamster TK cDNA, XcTK.90, described in the accompanying manuscript (21), and we used these sequence data to resolve the structure of the Chinese hamster TK gene. Since the genetic determinants which interest us might be contained within 5' promoter or promoter-proximal sequences, within exon or intron sequences, or within 3' flanking DNA, a genetic distance we have shown to exceed 11.2 kilobases, we began our search for the determinants of growth phase-dependent TK gene expression in a simple way by analyzing first only TK gene exon sequences for their presence. We constructed the chimeric TK gene we designate pHeA6Ha (Fig. 1) by combining the complete coding and 3' noncoding sequences of the Chinese hamster TK cDNA (XcTK.90) with the promoter and 5' noncoding region of the TK gene of herpes simplex virus (HSV) type 1. Since TK gene expression in Tk- cells transformed to Tk+ with the HSV TK gene is growth phase independent (22, 25, 38), we reasoned that a growth phase-dependent pattern of TK gene expression in cells transformed to Tk+ with pHeA6Ha would reflect the presence of the genetic determinants of growth phasedependent TK expression within the exon sequences of the Chinese hamster TK gene. As a Tk- recipient cell line for our analysis, we used the rat 4 cell line, a Tk- derivative of the parental line rat 52 described by Topp (43). The rat 4 cell line grows with a doubling time of approximately 18 to 20 h and is effectively growth arrested at a saturation density of 3 x 106 cells per 60-mm dish, reaching this density 90 to 120 h after a seeding of 2 x 105 cells in Dulbecco modified Eagle medium plus 10% fetal calf serum in a 5% C02, humidified atmosphere. Rat 4 cells are viable as growth-arrested monolayers without medium refreshment for as long as 144 h, after which the cells of these cultures can be induced by serum refreshment to reenter the cell division cycle with insignificant losses in

The expression of the thymidine kinase (TK) gene in higher eucaryotic cells in culture has been understood since the work of Kit et al. (14, 15) and Littlefield (26, 27) in 1965 to be growth phase dependent, i.e., to be maximal as asynchronously growing cell cultures are plated at low density and divide logarithmically and then minimal as cultures reach stationary confluence and cells withdraw from the cell division cycle (1, 5, 12). With the development of techniques to prepare synchronously dividing in vitro cell cultures, TK gene expression was shown to be cell-cycle S phase specific, to be scarcely measurable in cultures of G1-phase cells, to rise sharply as DNA synthesis is initiated in S-phase cultures, and then to decline as cell cultures progress through the G2 and M phases of the cell division cycle (4, 16, 17, 30, 33, 40, 41). In this light, the growth phase dependence of TK gene expression is understood to reflect a withdrawal of cells from the division cycle as asynchronous cultures grow to stationary confluence. The genetic determinants which govern the growth phase dependence of TK gene expression have yet to be characterized in any final detail, though they are thought to be linked closely to TK structural gene sequences since TK gene expression in mouse Ltk- cells transformed to Tk+ with a variety of mammalian genomic DNAs or with a cloned human TK gene (3, 38) is cell cycle and growth phase dependent. The recent clonings of the chicken (35), hamster (22), and human (2, 19, 24) TK genes have advanced the understanding of growth phase-dependent TK gene expression by providing molecular probes for Northern blot analyses which have established that growth phase-dependent TK gene expression, assayed as TK enzyme activity, is mediated by growth phase-dependent changes in the level of cytoplasmic poly(A)+ TK mRNA (22, 39). We have previously described our experiments leading to the molecular cloning of the Chinese hamster TK gene (22), which were undertaken with the notion in mind that we might define the genetic determinants which govern the Corresponding author. t Present address: Departmeuit of Virus and Cell Biology, Merck, Sharp & Dohme Research Laboratories, West Point, PA 19486. *

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NOTES

VOL. 6, 1986 A.

tranislation.

HeA6Ha

B.

GCGCCTTGTAGAAGCG 500

CCTCGAGG

CCGCCATG

510

He

Xho I

Ho

FIG. 1. Structure of the chimeric TK gene pHeA6Ha. (A) pHeA&6Ha was constructed by combining a 900-bp BamHIIXhoI fragment, containing the promoter and 113 bp of 5' noncoding. sequence of the HSV type 1 TK gene from the plasmid pThx81 (18), with the BamHIIXhoI vector fragment of the plasmid pHaTK.1A6. pHaTK.1A6 was constructed by combining a 330-bp XhoI/EcoRI fragment of the exonuclease III-S1 deletion mutant pHaTK.2A6 with the XhoI-partial EcoRI vector fragment of pHaTK.1 (see reference 21 for details of plasmids pHaTK.1 and pHaTK.2A6). HSV TK sequences are shown (relatively to scale) as a stippled box; Chinese hamster TK cDNA sequences are indicated by the open box. (B) Nucleotide sequence at the junction of the HSV and Chinese hamster cDNA XcTK.90 sequences in the 5' noncoding region of pHeA6Ha. The numbering of the HSV TK sequences is according to Wagner et al. (44). The 5' noncoding region of the pHeA6Ha minigene is composed of 113 bp of HISV TK sequence, an 8-bp XhoI linker (18), and 5 bp of noncoding sequence from the Chinese hamster cDNA XcTK.90.

plating efficiency. The rat 4 cell line can be efficiently transformed to Tk+ with a variety of TK genes by using the standard CaPO4 precipitation method. For these reasons, the line has proven to be ideally suited to studies of growth phase-dependent TK gene expression. We introduced the chimeric HSV/hamster TK gene pHeA6Ha into rat 4 cells by using the CaPO4 precipitation method described by Wigler et al. (46) and established rat 4 Tk+ cell lines of polyclonal origin by pooling the hypoxanthine-aminopterin-thymidine-resistant colonies which appeared after 17 days of selection (15 to 30 colonies per 20 ng of plasmid DNA). We derived, using similar methods, polyclonal rat 4 Tk+ cell lines transformed with either the Chinese hamster TK gene contained in XHaTK.5 (21) or the HSV TK gene in the plasmid pTK.2 (51) (15 to 30 colonies per 2 ng of XHaTK.5 or pTK.2 DNA). We analyzed the growth phase dependence of TK gene expression in these various rat 4 Tk+ cell lines by establishing replica cultures of 2 x i05 cells in 60-mm dishes, harvesting these cultures at 24-h intervals as these cultures grew through mid-log phase

2263

(day 1/day 2) to stationary confluence (day 5), and assaying TK enzyme activity as an indirect reflection of cytoplasmic TK mRNA levels (Table 1). As we expected, the specific TK activity in the line rat 4.34, transformed to Tk+ with the XHaTK.5 clone, was strictly growth phase dependent, being maximal in cell cultures harvested in mid-log phase and minimal (20% mid-log value) in cell cultures harvested at stationary confluence. By contrast, as we expected as well, the specific TK activity in the rat 4.121 line transformed to Tk+ with the HSV TK gene was growth phase independent, being largely invariant in replica cell cultures harvested either in mid-log phase or at stationary confluence. Importantly, we found that TK gene expression in the rat 4.113 cell line transformed to Tk+ with pHeA6Ha was growth phase dependent, qualitatively indistinguishable in character from the pattern of TK gene expression described above for the rat 4.34 (AHaTK.5) cell line. We similarly analyzed the pattern of TK gene expression in the clonally derived cell lines rat 4.26A and rat 4.26B transformed to Tk+ with pHeA6Ha, and we found it to be growth phase dependent as well, being maximal in mid-log-phase cultures and minimal (20% of mid-log value) in stationary-phase cultures. These experiments with the rat 4.26A and rat 4.26B cell lines convinced us of the validity of our conclusions about growth phase-dependent TK gene expression drawn from experiments with polyclonally derived Tk+ cell lines. We analyzed the growth phase dependence of TK gene expression in our various rat 4 Tk+ cell lines further by assaying TK enzyme activity at 12-h intervals as 5-day-old monolayer cultures were induced, by either serum refreshment or infection with adenovirus 5, to reenter the cell division cycle (9, 12, 13, 14, 20, 36, 37, 42, 49, 50). Our experiments with adenovirus 5 were prompted by the recent observation of Liu et al. (28) that the infection of serumarrested cell cultures with adenovirus 2 activates only a subset of those genes activated by serum refreshment. We were interested, therefore, in asking whether TK gene expression in stationary-phase cultures of cells transformed with pHeA6Ha could be influenced differentially by serum refreshment or adenovirus infection. A differential response to these factors would distinguish the genetic determinants which govern growth phase-dependent TK expression from those which govern the TK gene response to adenovirus infection. The results of these experiments (Tables 2 and 3) were consistent with the conclusion that the pHeA6Ha TK gene carries genetic determinants which impose a growth phase-dependent and adenovirus-responsive character on its expression. Although the absolute level of TK activity in the rat 4.113, rat 4.26A, and rat 4.26B cell lines was only 20% of the level of TK activity in the rat 4.34 cell line (AHaTK.5), the relative increases in TK activity at 24 h in the pHeA6Haderived lines in response to serum refreshment (2.5- to 6-fold), in response to replating and refeeding (6- to 8-fold), and at 36 h in response to adenovirus 5 infection (6- to 15-fold) were strictly proportional to the relative TK activity increases in the rat 4.34 cell line in response to the same culture variables (2.5-fold, 7-fold, and 7-fold, respectively). The specific TK activity in the rat 4.121 line, derived with the HSV TK gene, varied little under both conditions of cell culture. These experiments led us to the conclusion that the genetic determinants of the growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster TK gene are contained entirely within the 1,199 base pairs (bp) of the mature TK mRNA. It remains to be established whether the determinants which govern growth phase-

2264

MOL. CELL. BIOL.

NOTES TABLE 1. TK activity in rat 4 cell lines transformed to Tk+ with various TK genes'

Cell line

Transfected gene TK

3 days in culture: 2 TK sp actb (x103) after

1

5

4

25.1 22.9 25.5 50.7 59.8 61.7 8.5 10.9 8.1 4.1 9.7 12.1 6.5 6.1 10.5 a Replica cultures of 2 x 105 cells were established in 60-mm dishes (Falcon) in Dulbecco modified Eagle medium (GIBCO Laboratories, Grand Island, N.Y.) + 10o fetal calf serum (GIBCO) with 100 U of penicillin and 100 ,ug of streptomycin (GIBCO) per ml and harvested at successive 24-h intervals by trypsinization and centrifugation. Cell pellets were washed once with phosphate-buffered saline and held at -70°C until assayed. All samples from a given experiment were assayed for TK activity simultaneously, using the method described by Wilkie et al. (47), and assayed for protein concentration using a Bio-Rad dye-binding assay as described by Zipser et al. (51). The data presented in Tables 1, 2, and 3 are derived from one experiment. The experimental protocols of Tables 1, 2, and 3 have been repeated at least three times with similar results. b Counts per minute of [3H] thymidine phosphorylated per 50 ,ug of protein per 30 min.

HSV XHaTK.5 pheA6Ha pheA6Ha pheA6Ha

Rat 4.121 Rat 4.34 Rat 4.113 Rat 4.26A Rat 4.26B

24.1 283.0 40.0 30.4 22.5

26.8 156.0 37.4 51.3 18.1

dependent expression are the same determinants which govern the response to adenovirus infection. The conclusions we draw here are similar to those described previously by Merrill et al. (30, 31), who used the approach of HSV/chicken chimeric TK gene construction to demonstrate that the genetic determinants which mediate the extinction of TK activity in differentiating cultures of mouse myoblasts transformed to Tk+ with the chicken TK gene are contained not within promoter sequences, but within either exon or intron sequences of the chicken TK gene. At present we do not know whether the determinants which govern this developmental aspect of TK gene regulation are those which govern the growth phase dependence of TK gene expression.

Although we have considerably narrowed the scope of our search for these determinants, we do not yet understand their function. Although the location of these determinants within TK mRNA sequences might suggest that they govern TK gene expression through posttranscriptional processes, it remains a formal possibility that these determinants function as growth phase-specific enhancers of TK gene transcription. Groudine and Casimir (8) have recently described TABLE 2. TK activity in stationary-phase Tk+ rat cell cultures after serum refreshment or replatinga Cell line

Transfected TK gene


HSV XHaTK.5 pHeA6Ha

Rat 4.121 Refeeding and replating Rat 4.34

HSV XHaTK.5 pHeA6Ha

Treatment

Refeeding

Rat 4.113 Rat 4.26A Rat 4.26B

pHeA6Ha pHeA6Ha

pHeA6Ha pHeA6Ha

TK sp actb (x103) at time (h) after treatment: 24 12 0 36

29.7 52.5 10.8 15.8 10.2

25.1 50.7 8.5 16.5 6.5

26.1 91.8 23.5 35.0 17.2

25.1 50.7 8.5 16.5 6.5

24.0 35.2 28.4 82.0 389.6 332.7 19.6 65.5 35.8 63.4 134.8 111.6 11.7 41.2 31.1

39.0 120.8 30.5 34.9 38.3

a Five-day-old cultures of stationary-phase Tk+ rat 4 cell lines established 165 cells per 60-mm dish, were either refed with 4 ml of Dulbecco modified Eagle medium plus 109% fetal calf serum (Refeeding) or trypsinized and passaged at a 1:5 split ratio and refed with 4 ml of Dulbecco modified Eagle medium plus 10% fetal calf serum (Refeeding and replating). Replica cultures were harvested at 12-h intervals after treatment as described in Table 1 footnote . b Counts per minute of [3H] thy;nidine phosphorylated per 50 p.g of protein per 30 min.

at 2 x

experiments using sersitive hybridization methods which failed to measure differences in the rate of TK gene transcription in stationary-phase or actively dividing cell cultures of chicken erythrocytes, and they argued therefore that growth phase-dependent chicken TK gene expression is accomplished largely through a posttranscriptional nmechanism(s). Leys and Kellems (23) have similarly argued the importance of posttranscriptional mechanisms governing the growth phase dependence of dihydrofolate reductase gene expression since they were unable, using brief pulse-labeling protocols, to measure differences in the rate of gene transcription of the amplified dihydrofolate reductase gene loci in cultures of logarithmically growing or stationary-phase, antifolate-resistant murine fibroblasts. More recently, however, Farnham and Schimke (6) have reported a striking increase in dihydrofolate reductase gene transcription as synchronized cell cultures progress from the G1 to S phase, a result similar to data reported previously by Johnson et al. (10, 11). However these inconsistencies are finally reconciled, it will be of obvious interest to continue to characterize the genetic determinants which underlie the growth phase dependence of TK gene expression and to compare these elements with those determinants defined for other growth phase-regulated genes which encode enzymes important to nucleotide biosynthesis, e.g., dihydrofolate reductase (10, 11, 29, 45, 48) and thymidylate synthetase (7, 34), whose

TABLE 3. TK activity in stationary-phase Tk+ rat transformed cell lines after infection with adenovirus 5" TK sp actb (x 103) at time (h) postinfection: Transfected TK Cell line gene


0

12

24

36

26.6 31.9 376.7 179.7 50.5 153.4 82.2 139.3 pHeA6Ha 37.0 29.1 pHeA6Ha a Five-day-old cultures of stationary-phase rat 4 Tk+ cell lines, established at 2 x 10W cells per 60-mm dish, were infected with 100 PFU of CsCl-banded adenovirus 5 per cell in 1 ml of spent medium at 37°C for 1 h with gentle

HSV XHaTK.5 pHeA6Ha

25.1 50.7 8.5 16.5 6.5

27.9 45.6 19.4 33.7 15.1

intermittent rocking. After the viral adsorption period, cell cultures were washed once with phosphate-buffered saline and refed with 4 ml of spent culture mediaum removed originally from these cultures. Cell cultures were harvested at 12-h intervals as previously described. b Counts per minute of [3H] thymidine phosphorylated per/50 p.g of protein per 30 min.

VOL. 6, 1986

NOTES

isolation and detailed characterization permit such an experimental approach. This research was supported by a National Science Foundation grant to J.A.L. (PCM 8309360). We thank Mike Wigler and David Kurtz for their support and encouragement, Bill Topp for the rat 4 cell line, Bruce Stillman for stocks of adenovirus 5, Jesse Kwoh and David Zipser for the HSV TK gene mutant pThx8l, and Lynne Bonde for her invaluable help in manuscript preparation. LITERATURE CITED 1. Bello, L. J. 1974. Regulation of thymidine kinase synthesis in human cells. Exp. Cell Res. 89:263-274. 2. Bradshaw, H. D., Jr. 1983. Molecular cloning and cell cyclespecific regulation of a functional human thymidine kinase gene. Proc. Natl. Acad. Sci. USA 80:5588-5591. 3. Bradshaw, H. D., Jr., and P. L. Deininger. 1984. Human thymidine kinase gene: molecular cloning and nucleotide sequence of a cDNA expressible in mammalian cells. Mol. Cell. Biol. 4:2316-2320. 4. Brent, T. P., J. A. V. Butler, and A. R. Crathorn. 1965. Variations in phosphokinase activities during the cell cycle in synchronous populations of HeLa cells. Nature (London)

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42.

43. 44. 45.

46.

NOTES

activity in colcemid synchronized fibroblasts. Exp. Cell Res. 48:652-656. Takahashi, M., S. Ueda, and T. Ogina. 1966. Enhancement of the thymidine kinase activity of human embryonic kidney cells and newborn hamster kidney cells by infection with human adenoviruses types 5 and 12. Virology 30:742-743. Topp, W. C. 1981. Normal rat cell lines deficient in nuclear thymidine kinase. Virology 113:408-411. Wagner, M. J., J. A. Sharp, and W. C. Summers. 1981. Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1. Proc. Natl. Acad. Sci. USA 78:1441-1445. Wiedemann, L. A., and L. F. Johnson. 1979. Regulation of dihydrofolate reductase synthesis in an overproducing 3T6 cell line during transition from resting to growing state. Proc. Natl. Acad. Sci. USA 76:2818-2827. Wigler, M., A. Pellicer, S. Silverstein, and R. Axel. 1978. Biochemical transfer of single copy eucaryotic genes using total cellular DNA as donor. Cell 14:725-731.

MOL. CELL. BIOL. 47. Wilkie, N. M., J. B. Clements, W. Boll, N. Mantei, D. Lonsdale, and C. Weissman. 1979. Hybrid plasmids containing an active thymidine kinase gene of herpes simplex virus 1. Nucleic Acids Res. 7:859-877. 48. Wu, J. S. R., and L. F. Johnson. 1982. Regulation of dihydrofolate reductase gene transcription in methotrexate resistant mouse fibroblasts. J. Cell Physiol. 110:183-189. 49. Yoder, S. S., and S. M. Berget. 1985. Posttranscriptional control of DHFR gene expression during adenovirus 2 infection. J. Virol. 54:72-77. 50. Yoder, S. S., B. L. Robberson, E. J. Leys, A. G. Hook, M. Al-Ubaidi, C.-Y. Yeung, R. E. Kellems, and S. M. Berget. 1983. Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2. Mol. Cell. Biol. 3:819-828. 51. Zipser, D., L. Lipsich, and J. Kwoh. 1981. Mapping functional domains in the promoter region of the HSV thymidine kinase gene. Proc. Natl. Acad. Sci. USA 78:6276-6280.