Advan. Enzyme Regul., Vol. 32, pp. 241-254, 1992 Printed in Great Britain. All rights reserved

0065-2571/92/$15.00 ~) 1992 Pergamon Press plc

REGULATION OFTHYMIDINE KINASE DURING GROWTH, CELL CYCLE AND DIFEERENTIATION E R H A R D WINTERSBERGER, HANS ROTHENEDER, MARTIN GRABNER, G E R H A R D BECK and CHRISTIAN SEISER Institute of Molecular Biology, University of Vienna, Wasagasse 9, A-1090 Vienna, Austria

INTRODUCTION

Thymidine kinase (TK; E.C. 2.7.1.21) catalyzes the phosphorylation of thymidine to thymidine monophosphate at the expense of ATP. This is a reaction of the salvage pathway of nucleotide biosynthesis. Two genes are present in most cells, one for the mitochondrial enzyme which is absolutely required for the provision of TTP for mitochondrial DNA replication, the other gene codes for the cytoplasmic enzyme, which seems to guarantee the fine regulation of the pool of thymidine precursors for nuclear DNA synthesis. It has been known for quite some time that the cytoplasmic TK is regulated with growth. Its activity is low in quiescent cells and increases at the beginning of the S phase (1-3); however, contrary to the expression of proliferation-dependent histone genes, induction of TK at the beginning and during S phase does not require ongoing DNA replication. TK activity is only high in replicating cells during the S phase. High TK activity in a population of cells, therefore, is indicative of rapid replication of a high percentage of these cells. Consequently TK activity is being used as one criterion for neoplastic transformation of cells. Particularly in some forms of leukemias and lymphomas but also in some other tumors activity of the enzyme was found to be unusually high (e.g., 4-6). There must exist regulatory processes assuring a turnoff of TK activity and expression past the S phase in the cell cycle and during growth arrest and differentiation. Despite the effort the cell takes for the control of TK, this enzyme is not required for cells growing in culture, and mutants lacking TK can be produced relatively easily from a variety of rodent and some human cell lines. Such TK- mutants usually grow normally; in many cases growth rates are indistinguishable from those of the parent cells. However, possible aberrations occurring in these cells which are not detrimental to growth of a cell population (for instance, increased mutation and/or recombination rates caused by an imbalance of the precursor pool) will hardly be recognized in cell lines grown in culture but may be crucial for cells in tissues and organs of the whole organism. 241

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The strong increase of TK at the G1/S border of the cell cycle and the ease with which the enzyme can be tested, together with the availability of TK- cell lines and a very potent selection system (HAT selection) for the TK + phenotype, made TK an ideal object to study regulatory events at the transition from the G1 phase to the S phase of the cell cycle. Since this is a decisive cell cycle- and growth-regulated step leading to the initiation of DNA replication, it is hoped that by studying TK regulation information can be gained on principles underlying this regulation which may in a similar fashion also apply to other enzymes and proteins involved in DNA synthesis and its control (for a recent review see ref. 7). Several years ago we have therefore initiated a study on cytoplasmic TK in mouse cells and very soon found that enzymes whose activity is easy to measure are not necessarily easy to purify. The main hindrance is the very small amount of enzyme protein present even in rapidly growing cells necessitating an estimated over 20,000-fold purification for obtaining reasonably pure protein. This could not be achieved with the cultured mouse cells. The availability of cDNA and genomic probes for the human TK (8), however, allowed us to proceed without the availability of pure enzyme. We and others (9-17) have thus cloned and analyzed mouse TK cDNA as well as genomic DNA including the upstream region with the promoter which permitted the initiation of studies on the regulation of TK gene expression. MATERIALS

AND METHODS

Cells. Mouse 3T3 or 3T6 fibroblasts, L cells or LMTK- cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing antibiotics and 10% fetal bovine serum. Cells were arrested by reduction of the serum concentration to 0.2% and keeping cells under these conditions for two or more days. Cloning techniques. The methods used for construction and screening of cDNA and genomic DNA clones carrying information for mouse TK were standard (18) and/or were described earlier (9, 10). DNA sequences were determined by the dideoxy method (19). Determination ofnuclease sensitivity. Nuclei were isolated from logarithmically growing or serum starved, arrested 3T3 mouse fibroblasts and incubated with various concentrations of DNAse I as described by LevyWilson et al. (20) followed by extraction of the DNA (18), restriction with EcoRI and separation of the fragments in 1% agarose gels. After transfer to nylon membranes, blots were hybridized with labeled probes from the 5' part of the TK gene and further upstream sequences. The hybridization solution contained 50% formamide, 5 x Denhardt's solution,

243

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1.5% sodium dodecylsulfate, 10 mM EDTA, 50 mM phosphate buffer (pH 8.0), 5 x SSC, 200/zg/ml of salmon sperm DNA and the labeled probe (around 50 x 106 cpm). Hybridizations were done at 43°C overnight. After autoradiography and prior to rehybridization with a different probe, filters were treated with 50% formamide in 10 mM phosphate buffer (pH 6.8) for 60 min at 70°C.

DNAse protection and gel mobility shift assays. Methods employed for these studies were essentially as described (21). Briefly, nuclei were prepared as follows: fibroblasts were scraped off the dishes, pelleted and suspended in buffer (40/zl/per cell equivalent from one 10 cm petri dish) consisting of 10% glycerol, 10 mM Hepes (pH 8.0), 0.5 mM spermidine, 0.15 mM spermine, 0.1 mM EDTA, 0.25 mM EGTA, 5 mM NaCI, 2 mM dithiothreitol and 2.5 units/ml of aprotinin. They were lysed by homogenization in a Dounce homogenizer, crude nuclei were pelleted, resuspended in the same buffer to which Triton X100 was added to 0.25% and purified by centrifugation through 30% sucrose in the same buffer. Nuclear extracts were prepared and used for DNAse protection and mobility shift experiments exactly as described (21). RESULTS AND DISCUSSION TRANSCRIPTIONAL REGULATION OFTHYMIDINE

KINASE

The genes coding for cytoplasmic TK in chicken, rodents and man are composed of 7 exons, which are separated by introns of variable sizes. The total length of the rodent or the human gene is about 10 kb, that of the chicken is somewhat shorter, due to a smaller size of the introns. The mRNA is about 1.2 kb long in all cases and codes for a protein of around 25 kDa molecular weight. Using TK cDNA as a probe it was observed that the steady state amount of TK mRNA (like the TK enzyme activity) rises during growth stimulation of cells and, furthermore, that transcriptional and post-transcriptional events must be involved in this regulation (9, 14-16, 22-24). Since post-transcriptional events do depend on the presence of a transcript to start with, and since transcriptional control was found to be important in case of the human TK (25), we began our analysis with a detailed study of the promoter activity of the mouse TK gene. Examination of the upstream region revealed binding sites for some known transcription factors (outlined in 10), notably two GC boxes as binding sites for transcription factor SP1. However, comparing the TK promoters of the mouse with that of the human and the hamster gene led to the surprising observation that these promoters have very little in common, except for the presence in all three of them of GC boxes and a very conserved sequence of 18 base pairs, which is at

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varying distance from the initiation codon (213 bp upstream of the ATG in the mouse, 244 bp in the hamster and 428 bp in the human TK gene) and is beyond the minimal promoter region required for growth regulated expression of a reporter gene; this region comprises about 180 bp of the human and the mouse TK upstream sequence (12, 14, 26, 27). The human TK promoter carries a TATA-like box and two reverse CCAAT boxes, at least one of which seems to be required for regulated gene expression (26); the hamster promoter has a TATA-like sequence and one reverse CCAAT box (28), the contribution of which to gene expression is so far unknown. The mouse promoter lacks both of these regulatory elements (10, 14, see Fig. 1); on the other hand, it contains sequence motives recently characterized as binding sites for a new transcription factor (HIP1) which may play a role in transcription from promoters lacking a TATA box (29). In agreement with other described cases of housekeeping genes whose control region has no TATA element, transcription of the mouse TK gene starts at many different sites, giving rise to a family of transcripts differing at the 5' end. Functional analysis of about 500 bp upstream of the initiation codon in the mouse TK gene revealed the presence of two, probably non-overlapping,

mouse

-200 -190 -180 -170 -160 TCTGGCCCGC AGAAAGGGAA GGAAACGCCA TGGCCAGATC CGGAGGGGAT

hamster

AGCCGGGTCC AGCTCAGAGG GCGAGCAGCT CGCTTTGCAG GGACAGGGGG

human

CCGGATTCCT CCCACGAGGG GGCGGGCTGC GGCCAAATCT CCCGCCAGGT

-140 -130 -120 -110 -100 -150 GGTCGAGCTC CAGGCTTTTC ACGTAGCTGA GAGGTGGGAC GAGTCTTGTC TTCGTCCCGC AGAGGGGCGG GGCCCACATG CGCCCTGGCC TTGGCACGCC TGCGTCTTCG GCTGCGATTG CAGCGGCCGG GCGCTGATTG GCCCCATGGC GGCGGGGCCG GCTCGTGATT GGCCAGCACG -90

-80

-70

-60

-50

-40

CCCTTTTGAG TTCGCGGGCA AATGCGAGCA GTAAGTCGAA ATTTTTCCAC CCACGGACTC GTCGGTGAGC TTTTAAAGGA AAGCGCGAAC CTGAGGACGC TTCCCACTCA CCGACCCCGG CCGTGGTTTA AAGCGGTCGG CGCGGGACCA GGGGCTTACT GCGGGACGGC CTTGGAGAGT -30 -20 -10 1 TCGGTGCTAA CTAAGGTTTG CACAGCAGCC ATG ACCTTGGCGC CTCAGGCTCG CACAGCCGCC ATG ACTCGGGTTC GTGAACTTCC CGGAGGCGCA ATG

FIG. 1. Comparison of the nucleotide sequence in the promoter region of the mouse, hamster and human TK gene. TATA boxes in the hamster and the human gene (TI"I'AAA) are indicated as well as the reverse CCAAT elements (underlined) and GC boxes (binding sites for transcription factor SP1, doubly underlined).

Exl

EC0 Rl

Ex 3 Nhel

Ex2

27a

910

150

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FIG. 2. Control regions at the 5’ end of the mouse TK gene. Top: Schematic drawing of the mouse TK genomic region up to intron 3. Numbers indicate nucleotides with reference to the A of the initiation codon which is nucleotide number 1. Nucleotides in the upstream region have a negative sign. A, B and C are diagnostic footprints showing protected sequences. (A) Protection of sequences within the promoter. Lane A/G shows the sequence bands, lane 1 shows bands of unprotected, DNAse-sensitive DNA, lanes 2 and 3 show regions within the DNA protected by incubation with 35 and 70 pg, respectively, of protein from a nuclear extract. The sequence from -80 to -100 carries the GC box, the second protected sequence (-30 to -50) comprises the AT rich region close to the transcription start sites. (B) The promoter proximal regulatory region within intron 2. Lanes A/G and A/C are sequencing lanes, lanes 1 and 2 show unprotected DNA, lanes 3 to 6 protection by, respectively, 6, 20, 35 and 70 pg of protein from a nuclear extract. The protected region between nucleotide 250 and about 285 is one of the novel regulatory sequences. (C) The promoter distal regulatory region within intron 2. Lane A/G shows the sequence bands, lane 1 shows unprotected DNA, lanes 2 to 4 results obtained by incubation of the DNA with, respectively, 20, 35 and 70 pg of protein from a nuclear extract. The sequence from nucleotide 908 to 945 is protected by high concentrations of nuclear protein. This region causes strong stimulation of transcription. There is a nuclease hypersensitive site at around nucleotide 925.

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promoters, active in opposite orientation (12). About 150 base pairs upstream of the ATG suffice for growth-regulated expression of the TK gene, whereas further upstream sequences have promoter activity in the opposite direction, controlling a hitherto unknown gene. So far we only know that this gene is not expressed in fibroblasts which do transcribe the TK gene but it is not known which cells express the gene and under which conditions. The diversely oriented part of the upstream region has a TATA box and several binding sites for known transcription factors. In assays employing chloramphenicol acetyltransferase (CAT) as a reporter gene, the presence of this sequence weakens the activity of the TK promoter (12). In vivo separation of the promoter activities must therefore be regulated possibly at the level of chromatin structure. This question was analyzed in fibroblasts initially by looking for DNAse I hypersensitive sites in the chromatin of the whole region. Such sites were indeed found and, in agreement with the gene activity, one of them was seen in that part of the regulatory sequence which controls the TK gene expression in close proximity to the initiation codon. Surprisingly, however, two additional DNAse I hypersensitive sites were found in the transcribed part of the TK gene, precisely at the beginning and at the end of intron 2 (Fig. 2). In order to characterize these sites more closely, in vitro footprints were done using cloned fragments encompassing either the promoter region or the transcribed part of the gene up to the beginning of exon 3. These fragments were incubated with nuclear extracts from growing or arrested 3T3 cells. Within the promoter sequence there was strong protection of and around the GC box (nucleotides -80 to -100) suggesting that binding of SP1 plus possibly other transcription factors interacting close by is required for mouse TK expression. In addition, several binding sites were discovered in the diverse promoter. No difference could be seen between extracts from growing or resting cells. In a recent study the group of Pardee likewise described protection around this same GC box, but in addition obtained evidence for growth-specific interaction at nucleotides upstream but adjacent to the GC box (30), an observation we were so far unable to confirm. Both groups also saw protection of sequences within or close to the AT-rich region around nucleotide -50 (see Fig. 2). Again, contrary to the claim of Dou et al. (30), we have no evidence for growth-dependent binding activity. In fact, this sequence includes a presumptive HIP1 binding site and may be covered by that protein. HIP1 does not appear to be growth controlled (29). There is no a priori reason to assume that protein factors binding to specific sites of a growth regulated promoter should only be present in growing cells or should at least exhibit growth regulated binding to the promoter, as many cases are already known where transcription is controlled by factors not directly binding to DNA. Provision of such factors in active form (which might depend on proper modification, in particular

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phosphorylation) could well be growth regulated and may not show up in footprinting experiments. Of special interest was our finding that specific sequences within intron 2 were also protected by protein binding. Two strongly protected regions coincide approximately with two nuclease sensitive regions in chromatin, namely those at the beginning and at the end of intron 2. In order to characterize these sites in more detail, the total sequence of intron 2 was determined, allowing precise definition of possible binding sites for regulators (H. Rotheneder, M. Grabner and E. Wintersberger, in preparation). The final proof for the existence of protecting proteins came through mobility shift experiments in which proteins were detected which bind to oligonucleotides corresponding to the two regulatory sites in intron 2. The question whether these binding sites contribute to the regulation of TK gene expression was studied using a reporter gene under the control 5' regions of the TK gene encompassing the promoter alone minus or plus additional coding regions. From these studies it became quite clear that the weak (but growth regulated!) minimal TK promoter can be modulated in a positive fashion by sequences within intron 2 whereby addition of the sequence close to the border to exon 3 rather dramatically increased the promoter strength. Fridovich-Keil et al. (31) also described that expressed parts of the murine TK gene are involved in promotion of transcription. Their constructs, however, ended with nucleotide 159, which is within exon 2 and, therefore, did not include the motives found by us. Also, contrary to our situation, the nucleotide sequences responsible for the effect have not been defined so far. It is worth mentioning that also the hamster TK gene expression is stimulated by intron sequences (28), whereas introns seem to be dispensable for the expression of the simpler chicken TK gene when transfected into mouse LMTK- cells (32). At any rate, it is interesting that in recent years several genes were found which coded for mRNAs whose expression is modulated by cis acting elements within the coding region of the gene (see, for instance, refs. 33-35). Many of these genes lack a TATA element in their promoter. Further studies must be aimed at unravelling the mechanism involved in the stimulation of TK gene expression by elements within the coding region. As a first step we have produced DNA clones carrying, on the one hand, the TK cDNA (without any intron) under the TK promoter and, on the other hand, "TK minigenes" (consisting of all exons plus introns 1 and 2) in order to test their expression after transfection into LMTK- cells. POST-TRANSCRIPTIONAL REGULATION OF THYMIDINE KINASE As briefly alluded to above, experiments with TK cDNAs of various different origins (chicken, hamster, human, mouse) strongly suggested

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247

that TK expression during growth stimulation of arrested cells is not only regulated at the transcriptional level but that also post-transcriptional events play an important role (9, 14, 23, 24). This became clear when it was observed that TK cDNA, under the control of a constitutive, heterologous promoter (e.g., the TK promoter from herpes simplex virus type I or the late promoter from Simian virus 40) stably transfected into TK- cells, gives rise to growth-dependent production of TK activity and, depending on the promoter used (36), also of TK mRNA. In such experiments, cells carrying a cDNA construct were made quiescent by serum withdrawal and then growth stimulated by serum addition. Under these conditions TK activity and mRNA appeared, as in the case of the expression of the endogenous gene, at the beginning of the S phase. How this regulation is achieved in the absence of the growth related TK promoter is not yet known. Other studies using different approaches yielded promising new insights. In these experiments cells were separated by centrifugal elutriation into populations at different stages within the cell cycle and regulation of TK activity, and TK protein and mRNA levels were measured in the various cell populations. Alternatively, growing cells capable of terminal differentiation were used and cells were growth arrested by applying conditions which induce differentiation. Using this latter approach, Merrill and coworkers (37) observed that although eventually TK gene transcription was shut off in differentiating cells, TK activity and TK protein disappeared more rapidly than transcription was shut off. Even in cells which still contained considerable amounts of TK mRNA TK activity and protein disappeared in parallel (38, 39). As was shown convincingly, this is due to control at the level of mRNA translation. The experimental system used by Merrill and coworkers consisted of TK- mouse myoblasts which were transformed to TK ÷ by transfection with several copies of the chicken TK gene. Chicken TK gene expression was therefore measured in a mouse cell background and considering the many steps involved in the regulation of TK gene expression, results obtained with this system could be criticized. However, the general conclusions from these experiments seem to hold because analogous experiments measuring regulation of the endogenous mouse gene in differentiating mouse F9 embryonal carcinoma cells (differentiation in this case was induced by addition of retinoic acid) gave very similar results (M. Kn6fler, C. Waltner, E. Wintersberger and E. Mfillner, in preparation). Again, TK activity disappeared much more rapidly than TK mRNA which was due to a rapid cessation of TK protein synthesis in cells committed to differentiation. It appears to be less important which type of terminal differentiation is induced and under which conditions cells are committed to differentiate, as there is little difference in the behavior of myoblasts versus F9 cells. Since quiescent cells have very low levels of TK mRNA, transcription is eventually shut off. In case of differentiation this appears

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to be a secondary effect, probably induced by growth arrest. A remaining question which so far was not studied in detail is whether growth arrest induced by removal of serum follows a similar mechanism or whether removal of growth factors turns transcription off more directly and the decrease of enzyme activity and protein is a consequence of the reduction of the level of mRNA. By analyzing cells at different stages of the cell cycle, Kelly and coworkers observed yet another system of TK regulation. In cycling cells, there appears to be little or no regulation at the mRNA level; all regulation is at the level of mRNA translation and/or TK protein stability. Studying the regulation of the human enzyme during the cell cycle in HeLa cells, Sherley and Kelly (40) found that TK mRNA is only very poorly translated during G1, while the synthesis of the enzyme increases about 10-fold when cells enter S phase and start DNA synthesis. Upon cell division, stability of TK decreases dramatically, so that the enzyme is rapidly cleared from newly divided cells. Extending this study, Kauffman and Kelly (41) showed that this regulation depends on a sequence within the carboxy-termina140 amino acids of the protein. Deletion of these amino acids has little or no effect on the specific activity of TK but abolishes cell cycle regulation; the truncated protein is stable throughout the cell cycle. The mouse enzyme may differ in this respect according to our experimental evidence. While trying to dissect the structure of the murine TK gene, we observed that the mouse genome contains two pseudogenes in addition to the gene for cytoplasmic TK. One of these pseudogenes was isolated, sequenced and studied in some detail (11, 42). It turned out to be a typical example of a processed pseudogene, lacking intron sequences but carrying an oligo(A) tract. Compared with the gene, the pseudogene has received a number of base changes, small deletions and insertions. The first deletion removes two bases after amino acid 177, altering the reading frame and creating a stop signal after addition of 12 wrong amino acids (11). Up to this point, the pseudogene differs only in three amino acids from the enzyme (all changes are conservative) while after that point more dramatic changes are apparent. Remarkably, the pseudogene is active upon transfection into LMTK- cells, in particular if brought under the control of a strong promoter. In some stably transfected cells the specific activity can reach values similar to those present in rapidly growing L cells (42). This indicates that the last 56 amino acids of mouse TK are not required for full enzyme activity and this agrees with the observations on human TK mentioned above. However, we observed that the mouse enzyme is still growth regulated (Fig. 3). Preliminary experiments in which mRNA levels were determined in Northern blots suggest that this regulation occurs at a level past mRNA synthesis. We constructed a mouse TK gene lacking the carboxy-terminal 50 amino acids and found that also the product of

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TK-PROTEIN

D

H

L

I

PSEUDO TK-PRODUCT

a) TK-cDNA

|

D

b) TK-cDNA H (truncal[md)

c) TK-cDNA O (truncated) "1600

1500

!

f 0~

~

arrested

growing

d) Pseudo TK

m'reotod

~

gnm~g

e) Pseudo TK D (truncated)

-

~

0

grovdng

f) L-Cells (control) -

)

)

i FIG. 3. Regulation of a truncated version of TK and of the product of the processed pseudogene. The top shows schematic drawings of the TK and the pseudogene product indicating points of truncation achieved by shortening of the cDNA or the pseudogene. The shaded part of the pseudogene product represents the 12 amino acids not related to the TK sequence. Below are examples of specific activities (cpm [3H]-TMP produced/p.g protein) measured in extracts from LMTK- cells transformed to TK ÷ by transfection of various cDNA or pseudogene DNA clones followed by selection in HAT medium. All the constructs were cloned in plasmid pSVL (Pharmacia) which contains the late promoter from SV40 followed by the intron from VP1 of SV40, then the coding sequence and at the 3' end the SV40 polyadenylation signal. Extracts were prepared from cells growing logarithmically (shaded columns) or arrested by reduction of the serum concentration to 0.2% for 3 days (filled columns). (a) Cells transformed by the cDNA coding for intact TK (233 amino acids, see Fig. 4). (b) Cells transformed by cDNA which was shortened at a HindlII site resulting in a truncated TK with 218 amino acids. (c) Cells transformed by cDNA which was shortened at a DralII site, resulting in a truncated TK with 182 amino acids. This as well as the HindlII digestion removes the non-translated 3' end of the cDNA, including the genuine polyadenylation signal of the TK gene. (d) Cells transformed by the pseudogene coding for 177 amino acids of TK which are followed by 12 unrelated amino acids. (e) Cells transformed by the pseudogene which was shortened at the DralII site. This removes 5 of the unrelated amino acids but leaves the TK coding part intact. Also removed by this digestion are non-translated regions at the 3' end of the pseudogene. (f) Shows as a control the specific activity of TK in extracts of L cells.

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this truncated gene like that of the pseudogene is highly active and its production is growth regulated (see Fig. 3). We must stress, however, that our results are not necessarily in conflict with those of Kauffman and Kelly (41). From the above discussion it is clear that the level of regulation of TK varies in different situations (quiescence vs growth, cell cycle, differentiation). Regulation of the truncated human enzyme was studied in elutriated cells at different phases of the cell cycle, while the mouse enzyme was studied in growing or quiescent cells. It is not known whether the truncated human enzyme is regulated with growth nor whether the mouse enzyme fails to be regulated during the cell cycle. One further point may be interesting, however; in several cases proteolytic degradation of rapidly turning over proteins was attributed to the presence of a sequence (PEST) in one or more copies (43). Such a sequence is present once in TK and is not removed in the truncated form (see Fig. 4 which compares the sequences of mouse, hamster and human TK). If stabilization of TK during the cell cycle by removal of around 40 to 50 amino acids from the

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V C M E C F R E A A Y T K R L G L E K E V E V I G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . .

y H S V C R L C Y F K K S . . . . . . V . . . . . . . . . . . . . . . . . . . A

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233 234 234

FIG. 4. Comparison of the amino acid sequences of mouse, hamster and human TK. Underlined are the amino acids making up the PEST structure characteristic of turning-over proteins. The asterisk (amino acid 177) shows the end of the TK sequence in the active product of the pseudogene.

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C terminus of the protein is a general phenomenon, and can be confirmed in different cell types, this indicates that the PEST sequence present in TK is not responsible for proteolytic degradation of this protein. Comparison of TK sequences furthermore reveals high conservation along a large part of the protein; interestingly, the dispensible carboxy-terminus is the most variable part of the protein. Earlier studies on the chicken TK provide additional evidence for the fact that the C-terminus is not necessary for enzyme activity. In this case, elimination of the last 12 (and the N-terminal 15) amino acids had no effect on enzyme activity (38). The effects of these changes on the regulation of the chicken enzyme are not known so far. CONCLUSIONS AND SUMMARY Thymidine kinase is an almost ubiquitous enzyme. It is present in most organisms from bacteria to man with the exception of some fungi (e.g., Saccharomyces cerevisiae) and it is coded for by a variety of DNA viruses from the T even phages to herpes and vaccinia viruses. While the viral enzymes could be imagined to function in the reutilization of nucleosides (produced by degradation of host DNA) for viral DNA replication, the precise role of the cellular enzymes (except for those of mitochondria and chloroplasts) is elusive. The most reasonable assumption is that the cytoplasmic enzyme is involved in the fine regulation of the precursor pool for DNA synthesis, and in line with this assumption is the observation that TK- cells exhibit higher mutation rates (44). Still, it is surprising that this enzyme is so precisely regulated at nearly every possible level. Resting cells have low enzyme activity and protein as well as low mRNA. Upon growth stimulation all three parameters increase at the G1/S boundary. This is due to transcriptional regulation most probably aided by some as yet unknown post-transcriptional event because the change in the rate of transcription cannot account for the increase in the steady state concentration of the mRNA. Cycling cells have high mRNA levels throughout the cell cycle but enzyme and protein levels are regulated such that both are low in freshly divided cells and in G1 but increase in S up to G2. This regulation is due to a change in the stability of the TK protein which becomes rapidly degraded shortly before and during mitosis. Low efficiency of translation of TK mRNA in G1 plays an additional role. The control of translation is of utmost importance in cells arresting by entry into a differentiation program, which is accompanied by growth arrest. Details of regulatory mechanisms are not known at any of the different levels. Because of the enormous current activity in the field of transcription regulation, this part of TK expression control can be expected to be disclosed rather soon. However, TK is but one of many genes whose transcription is being studied at present; this includes several growth controlled ones, and

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much similarity and overlap might be expected from these studies. As this regulation takes place by interaction with DNA (directly or indirectly) it may include elements also used in the initiation of DNA replication, in particular as there is increasing evidence for involvement of transcriptional regulators in the control of D N A replication (see refs. 7, 45, 46 for reviews of this topic). As regards detailed mechanisms at other levels of control, TK may indeed provide a very useful model system. This holds in particular for the mechanism of translational control and the modification of protein stability during the cell cycle. Many important and seminal results can be expected from analyses of these processes. ACKNOWLEDGEMENTS

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