The turnover of deoxyuridine triphosphate during the HeLa cell cycle

Printed in Sweden Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/80/010087-08$02.0010

Experimental

THE

TURNOVER

OF DEOXYURIDINE THE

SUNEETA Departments

Cell Research 125 (1980) 87-94

HeLa

MAHAGAOKAR,’

of LDevelopmental Therapeutics Center, M. D. Anderson Hospital

TRIPHOSPHATE

CELL

ANTONIO

DURING

CYCLE ORENGO* and POTU N. RAOL *

and 2Biochemistry, The University of Texas System and Tumor Institute, Houston, TX 77030, USA

Cancer

SUMMARY The synthesis and breakdown of deoxyuridine triphosphate (dUTP) was studied to determine whether a dUTP pool is present at any stage of the HeLa cell cycle. Although cell extracts were found to be capable of phosphorylating dUMP to dUTP, only minimal quantities of intracellular dUMP, dUDP or dUTP could be detected. When thymidylate synthetase was blocked with FUdR the dUMP pool increased but no substantial increase in dUDP or dUTP was seen. A powerful and specific dUTP nucleotidohydrolase (dUTPase, EC3.6.1.23) which hydrolyses dUTP to dUMP and PPi was detected. The activity of this enzyme as well as that of the dUTP synthesizing enzymes was low in Gl, rose through S and G2 and reached a maximum just prior to cell division. Pulsing experiments with [VH]UdR and [IIC]TdR suggest that the size of the dUTP pool is 1% of the dTTP pool.

The fact that uracil has not been found in the DNA of most organisms has been the object of many investigations. Studies performed in vitro revealed that DNA polymerase can incorporate deoxyuridine triphosphate (dUTP) into DNA [2]. Uracil residues are, however, immediately cleaved by a uracil-DNA-glycosidase, thereby initiating DNA-strand breaks [lo, 111. The degree of uracil incorporation into bacterial DNA appears to be dependent upon the size of the dUTP pool which is effectively controlled by the enzyme dUTPase (EC3.6.1. 23). The importance of this pyrophosphatase seems to be demonstrated by the increased incorporation of uracil residues in dUTPase-deficient mutants of Escherichia coli (dut-). Such mutants transiently accumulated short (4-5s) DNA fragments due to the excision-repair of misincorporated uracil residues [23, 241.

The ability of mammalian cells to similarly incorporate dUTP and subsequently to excise uracil residues from their DNA has been demonstrated in isolated nuclei undergoing DNA replication in the presence of exogenous dUTP [S, 8, 251. Furthermore, the enzyme uracil-DNA-glycosidase has been isolated and characterized from human placenta and fibroblasts in culture [19]. Thus, it appears that the size of the dUTP pool plays a critical role in the rate of incorporation of uracil into DNA of mammalian cells. We therefore decided to investigate the synthesis and degradation of dUTP in synchronous cultures of HeLa cells in the hope that we might gain some insight into the mechanism of uracil exclusion from mammalian DNA. * To whom reprint requests may be sent. Exp CdRes

125(1980)

88

Mahagaokar, MATERIALS

Orengo and Rao AND

METHODS

Nucleotides All unlabeled nucleotides were from P. L. Biochemicals. [5-3H]dUMP (12.7 Ci/mmoIe), [5-3H]dUTP (3.3 Cilmmole), [methylJH]dTMP (18. Ci/mmoIe), [Y-S‘H]ATP (59 Cilmmole) were purchased from Amersham. (Methyl-3H]TdR (6.7 Cilmmole), [methylSH]dTTP (18.58 Cilmmole), [5,6-$H]UTP (37 Ci/ mmole), [8,5’JH]dGTP (29.8 Ci/mmole), [5-3H]dCTP (25.46 Cilmmole), [3H]dATP (11.96 Cilmmole). and [2-‘YZ]TdR (62 mCi/mmole) were bought from New England Nuclear. [2-W]UdR (23 mCi/mmole) was purchased from SchwarzlMann. [VH]UdR was prepared by dephosphorylation of [VH]dUMP as follows: 250 ~1 of [5-3H]dUMP (12.7 Cilmmole) was lyophilized to dryness. This was then suspended in 100 ~1 of 0.1 M Tris-Cl (pH 8.0) and incubated with alkaline phosphatase (1 l.U.) for 15 min at 37°C. The phosphatase was inactivated by boiling for 10 min and the solution was brought to 250 ~1 with distilled water. Paper electrophoresis of the mixture revealed that 99 % of the label co-migrated with UdR and 0.4 % with dUMP. 5-Fluoro-2’deoxyuridine (5FUdR) was from Roche Laboratories, and 5-fluoro-2’ deoxyuridine-5’-phosphate (FdUMP) was from Calbiochem.

Cells and cell synchrony HeLa cells were grown as spinner cultures at 37°C in Eagle’s minimal essential medium (MEM) supplemented with 1% each of non-essential amino acids, sodium pyruvate, glutamine, and penicillin-streptomycin mixture, and 10% (v/v) heat-inactivated fetal calf serum (FCS). These cells had a generation time of about 22 h consisting of 10.5 h of Gl, 7.0 h of S, 3.5 h of G2, and 1.0 h of mitosis. For synchronization. a sninner culture in exnonential growth was first partially’ synchronized in S ‘phase by mcubation with TdR (2.5 mM) for 24 h. Soon after removal of TdR the cells were plated in several 150 mm plastic culture dishes and incubated at 37°C in an atmosphere of 95 % air, 5% CO,. Three hours later the unattached cells were removed bv changing the medium, and the cultures were then-subjected to a N,O block (80 p.s.i. at 37°C for 10 h), as described earlier [18]. After the release of the N,O block, the rounded and loosely attached mitotic cells were collected by selective detachment. Some were used immediately as the mitotic sample, and the rest were incubated in a spinner flask at 37°C as described above. At appropriate time intervals, cells were collected in the Gi, S, and G2 nhases. Colcemid (0.05 iug/ml) was added to the spinner 10 h after reversal ofthe N20 block to prevent cells from dividing and also to monitor the degree of mitotic synchrony toward the end of the first cycle. For biochemical analysis, aliquots of 3-4X 10’ cells were taken at regular intervals after the reversal of the N,O block, centrifugated at 1000 g for 10 min at CC, and the cell pellets were stored at -20°C until needed. Simultaneously, samples of 5~ 105 cells were incubated with [3H]TdR (1 &i/ml; spec. act. 6.7 Ci/ mmole) for 20 mitt, deposited on slides by the use of cytocentrifuge, fixed, processed for autoradiography, and scored for labeling and mitotic indices in order to monitor progression of cells through the cell cycle. Erp

Cell

Rcs 125 (1980)

Preparation of cell extracts for enzyme assays Cell extracts were prepared by thawing the cell pellets and lysing them by cavitation (800 p.s.i. of N, for 10 mitt, followed by sudden release) in a buffer of 50 mM KCI, 50 mM Tris-Cl (pH 7.5), 10 mM dithiothreitol (DTT). If some cells were still found intact, homogenization was completed by forcing the extract through a 25-nature svrinne needle. The volume was then ahjusted 70 Il.0 ml wzh the above buffer, and the extract was centrifugated at 135 000 t! for 45 min at 4°C in a Beckman Modei L5-65 ultracemifuge. The supernatant was used for enzyme determinations.

Enzyme assays All reactions were carried out at 37°C for varying lengths of time so that no more than 15% of the substrate was converted to the product. Reactions were terminated by quick freezing in a dry ice-acetone bath, following the addition of 20 ~1 of a solution containing the appropriate nucleoside and its mono-, di-, and triphosphate, each at a concentration of 10 mM. Since the electrophoretic system does not distinguish between the deoxy- and ribonucleotides, a mixture of the corresponding ribonucleotides, which are less expensive, was used as a carrier.

Assay for dUTPase

activity

A radiochemical assay that measures the production of [$H]dUMP and r3H]dUDP from [3H]dUTP was used. The standard reaction mixture for the assay contained: 167 mM Tris-Cl (pH 7.5), 50 pM[3H]dUTP (15.9 &i/pmole) enzyme and water to a final volume of 60 ~1. In some of the experiments, [3H]dTTP, r3H]UTP, [3H]dATP, [3H]dCTP or r3H]dGTP was used as a substrate in the elate of 13HldUTP to determine whether they were s:milarly hydrolysed to their mono- and diphosphates.

Demonstration [5-W]dUMP

of dUTP synthesis from

To prove that HeLa cell extracts could phosphorylate [5-3H]dUMP to [5-3H]dUTP, excess unlabeled dUTP was added to the reaction mixture to reduce hydrolysis of the [5-3H]dUTP which might have been synthesized. The reaction mixture contained 69 mM KPG, buffer (pH 7.5), 10.3 mM MgCl,, 17.2 mM DTT, 8.3 mM ATP, 2.75 pM[5-3H]dUMP (12.7 Cilmmole), 3.4 mM FdUMP, 0.65-3.2 mM dUTP, mitotic extract, and water to a final volume of 145 ~1.

Measurement dUMP

of dUTP formation

from

An indirect method which measures the amount of r3H]ATP hydrolysed by cell extracts as a result of the addition of dUMP to the reaction mixture was used. To monitor the hvdrolvsis of ATP due to other ATP-consuming enzymic activities identical reactions were carried out in the absence of dUMP. These

Turnover values were subtracted from the ATP hydrolysed in the presence of dUMP. The reaction mixture contained 100 mM Tris-CI (pH 7.5), 6.0 mM rYH]ATP (2.45pCi/pmole), 15.0 mM MgCI,, 25 mM DTT, 50 mM dUMP, cell extract, and water to a final volume of 100 /.Ll. In order to be certain that our assay conditions suvvorted dUMP-devendent ATP hvdrolvsis at the maximum velocity, initial assays were performed with increasing concentrations of dUMP. Thereafter, all extracts were assayed at concentrations of 25 and 50 mM dUMP, which were found to be sufficiently high to assure zero-order kinetics.

Separation of nucleotides by paper electrophoresis For the separation of mono-, di-, and triphosphates of all the nucleotide studies, 30 ~1 of the reaction mixture was spotted on a sheet of Whatman No. 3M paper (57x27 cm), which had been dampened with 50 mM citric acid-citrate buffer (pH 5.2). Electrophoresis was run at 2500 V, 50 mA at 10°C. The duration of the run was 2 h for the separation of pyrimidine nucleotides and 2.75 h for purine nucleotides. The nucleotide spots localized under UV light were cut and then transferred to vials containing 20 ml of Packard Permafluor 1, which had been diluted 25-fold with toluene. Samvles were counted in a Packard Tri-carb liquid scintillation spectrometer (Model 3255).

Measurement of the rate of phosphorylation of TdR and UdR during the HeLa cell cycle Synchronized or random populations of HeLa cells (6~ 106) were pulsed separately with [5-3H]UdR (88.18 @i/pmole), [2-‘*C]TdR (40.75 @i/pmole), or [2-14C]UdR (43 $.Zi/~mole), for either 5 or 10 min at 37°C. At the end of the incubation period, cells and medium were separated by centrifugation, and both were precipitated with TCA. The samples were treated as described by Al-Bader et al. [l], and the amount of radioactivity recovered as the nucleoside, and its mono-, di-, and triphosphates in the TCA-soluble fraction was estimated after their separation by paper electrophoresis.

Assay for the activity of TdRIUdR kinase in HeLa cell extracts A radiochemical assay that measured the conversion of [3H]TdR to [3H]dTMP and [5-3H]UdR to [5-3H]dUMP was used. The reaction mixture contained 62.5 mM KPO, buffer (pH 7.5) 9.4 mM MgCl,, 15.6 mM DTT, 5.6 mM ATP, either 0.14 mM [3H]TdR (43.6 &i/pmole) or 0.13 mM [5-3H]UdR (88.18 &i/pmole), mitotic extract, and water to a final volume of 160 ~1.

Assay for dTMP kinase

In order to determine whether dTMP and dUMP are phosphorylated by a common kinase, the phosphorylation of [3H]dTMP was studied in the presence of vary-

of dUTP during cell cycle

89

ing concentrations of unlabeled dUMP. Reaction mixtures for assaying dTMP kinase activity contained 100 mM KPO, buffer (pH 7.5), 15 mM MgCl,, 3.0 mM ATP, 25 mM DTT, 9.27 mM [3H]dTMP (2.13 $Zi/ pmole), HeLa cell extract and water to a final volume of 100 PI.

RESULTS Specificity of dUTPase

dUTP was hydrolysed by HeLa cell extracts, yielding dUMP (81%) and dUDP Partially purified (19%) as products. dUTPase, however, produced equimolar amounts of dUMP and PPi demonstrating that the enzyme is a pyrophosphatase (dUTPase). None of the naturally occurring deoxynucleoside triphosphates nor UTP was hydrolysed to the monophosphatate by cell extracts (1.22 pg protein), indicating that this pyrophosphatase is highly specific for dUTP. With an increased amount of protein (10 pg), the assay detected a phosphatase which converts all the other nucleoside triphosphates to diphosphates. Only with dATP as a substrate 12 % of the counts were recovered as dAMP. The addition of excess amounts of unlabeled dUMP, UTP, or dTTP (10, 20, 50 nmoles) to the reaction mixture had minimal effect on the hydrolysis of dUTP to dUMP, suggesting that dUTPase binds poorly to these nucleotides. A 17-fold excess of dUMP, UTP or dTTP over [3H]dUTP yielded a reduction in dUMP formation of 23, 29 and 31%, respectively. dUTPase activity during the HeLa

ceLl cycle

The results shown in fig. 1 indicate that the breakdown of dUTP was lowest during Gl and gradually increased as the cells progressed through S and G2, reaching a maximum during mitosis. There appears to be no correlation between dUTPase activity and DNA synthesis. A low concentra-

90 350

Mahagaokar,

Orengo and Rao

r

-100

200

80

150

60 100 40 50

, i 0

..l

,/ 2

4

__,__,__ a-.." 6

8

10

12

14

16

18

20 o

20

time after mitosis (hours); ordinate: product formed/h/l@ cells; (right) labeling or mitotic index. Pattern of hydrolysis (0- - -0) and synthesis (0-O) of dUTP in a synchronized population of HeLa cells. Labeling (W---m) and mitotic (O---U) indices were measured at each time point. Fig. 1. Abscissa: (left) nmoles

tion of [3H]dUTP was used in dUTPase assays to select for dUTPpyrophosphatase, which bears a high affinity for dUTP, and to minimize the hydrolysis of dUTP by nonspecific phosphatases.

Can HeLa cells synthesize dUTP?

The failure to detect [5-3H]dUTP after incubation of HeLa cell extracts with [5-3H]dUMP could either be due to the inability of HeLa cells to phosphorylate dUMP to

dUTP or to the rapid dephosphorylation of dUTP. Excess unlabeled dUTP was added to the reaction mixture in order to saturate the dUTPase and reduce the hydrolysis of C3H]dUTP that may be formed after incubation of the cell extracts with [5-3H]dUMP. Table 1 indicates that under these conditions [5-3H]dUTP could be detected. Therefore, the failure to observe dUTP formation is due to the presence of a powerful dUTP dephosphorylating activity. Since HeLa cells are capable of synthesizing dUTP, we measured its synthesis as a function of cell cycle by estimating the amount of [3H]ATP hydrolysed after the addition of dUMP to cell extracts. Two molecules of ATP are hydrolyzed for every molecule of dUMP phosphorylated to dUTP. Although these measurements are subject to the disadvantages of using cell extracts as an enzymic source, they were found to be reproducible and thus give a good approximation of the capability of HeLa cells to synthesize dUTP. The rate of synthesis of dUTP during the cell cycle exhibited a pattern similar to that of dUTPase activity, i.e. low during G 1, with a progressive increase through S and G2, and reaching a peak during mitosis, as seen in fig. 1. The capacity of cells to synthesize dUTP appeared to be of the same order of magnitude as that of dUTPase.

Table 1. Proof of dUTP synthesis by HeLa cell extract Products, in pmoles

Blank (no extract) Extract+05 nmoles Extract+470 nmoles Extract+05 nmoles Extract+05 nmoles Extract+O.S nmoles

FdUMP (no dUTP FdUMP+94 FdUMP+282 FdUMP+470

UdR

dUMP

dUDP

unlabled dUTP)

2.4 3.2 4.1

396.2 395.6 365.2

0.8 1.2 6.2

nmoles of dUTP nmoles dUTP nmoles dUTP

3.2 3.2 3.7

322.8 343.6 354

6.4 7.6

All reaction mixtures contained 400 pmoles of [5-3H]dUMP. Exp Cell Res 125 (1980)

8.1

dUTP 0.3 240.: 6716 45.6 34.0

Turnover

Table 2. Comparison

of the uptake and metabolism by randomly growing HeLa cells

[2-%]TdR Isotope used for pulselabeling [VH]UdR [2-“‘C]UdR [2-‘4C]TdR

ofdUTP

during cell cycle

of [.5-3H]]UdR,

[2-Y]UdR,

91 and

pmoles recovered % in cell

Nucleoside

Monophosphate

Diphosphate

Triphosphate

8.2 9.8

2 673

19.4 8.9

2.5 3.8 24.4

3.5 56.6 366.0

5.5

1814 1 113

14.8

Rate of synthesis and breakdown of dUTP during the HeLa cell cycle

under identical conditions produced labeled dTMP dTDP, and dTTP as expected, indicating that the kinases involved were functional under the experimental conditions described. In order to estimate the amount of dUTP present in HeLa cells in relation to the pool size of dTTP we carried out pulse experiments with high specific activity [5-3H]UdR and [2-14C]TdR. After 5 min of exposure to the isotope at 37°C the cells were separated from the medium, lysed and the amount of radioactivity recovered as dUTP and dTTP was measured. It was found that the ratio of labeled dUTP (3.5 pmoles to labeled dTTP (366 pmoles) recovered from 6~ lo6 cells was approx. 1 : 100 (table 2). Pulsing with [2-14C]UdR revealed that TdR is a better precursor of dTTP than

To study the phosphorylation of UdR to its nucleotides in vivo, cells taken at different points in the cycle were incubated with [S3H]UdR for 10 min at 37”C, and TCA-soluble fractions were analysed for the presence of labeled dUMP, dUDP, and dUTP. [5-“H]Ud,R was used because the methylation of dUMP to dTMP causes a complete loss of the tritium on C5 of the pyrimidine ring and therefore, any radioactivity recovered can be ascribed only to deoxyuridine nucleotides. The results of these experiments indicated that only minimal radioactivity was recovered as dUMP, dUDP, or dUTP. However, during mitosis 3% of the radioactive UdR which entered the cell was recovered as dUMP. Incubation of cells with [methyl-3H]TdR Table 3. Phosphorylation of [Methyl-3H]TdR

and [5-3H]UdR by HeLa cell extract Products formed, in nmoles

Substrate

22.7 22.7 22.7 20 20 20 20 20

nmoles nmoles nmoles nmoles nmoles nmoles nmoles nmoles

TdR TdR TdR UdR UdR UdR UdR UdR

Addition of

Monophosphate

Diphosphate

Triphosphate

0 189 nmoles UdR 945 nmoles UdR 0 20 nmoles TdR 60 nmoles TdR 125 nmoles TdR 249 nmoles TdR

7.3 6.17 4.3 5.5 0.21 0.04 0.08 0

0.068 0.045 0.045 0.016 0.005 0.004 0.03 0.03

0.704 0.14 0.39 0 0 0 0 0

Exp Cell

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Mahagaokar,

Orengo and Rao

UdR (table 2). Studies with HeLa cell extracts indicate that UdR can be phosphorylated to dUMP as effectively as TdR is converted into dTMP, as shown in table 3. Inhibition of the phosphorylation of [5-3H]UdR by equimolar amounts of unlabeled TdR indicated that the same kinase is involved in the phosphorylation of UdR and TdR. Further, competition studies indicated that excessively large doses of unlabeled UdR were necessary to cause a small decrease in the phosphorylation of [3H]TdR. These results suggest that the kinase has a greater affinity for TdR than for UdR. Even during the subsequent steps of phosphorylation, i.e. from mono- to triphosphate, the kinases have a greater preference for dTMP than dUMP. A 5-fold excess of dUMP over r3H]dTMP reduced the recovery of labeled dTDP and dTTP only 40% in respect to the control. Therefore, the presence of even small amounts of TdR or dTMP could effectively inhibit the phosphorylation of UdR or dUMP. To determine whether the inhibition of thymidylate synthetase by SFUdR facilitates the synthesis of dUTP, random population of HeLa cells were pretreated with SFUdR (1 PM) for 16 h. Separate experiments indicated that such an exposure to 5FUdR almost completely blocked thymidylate synthetase, confirming the data of Elford et al. [6]. At the end of the treatment, 5FUdR was removed by washing the cells with fresh medium and the cells were pulsed for 10 min with 10, 20 and 100 nmoles of [5-3H]UdR (88.18 &i/pmole). FUdR was removed to avoid any competition with [5-3H]UdR for the nucleoside kinase. No increase in dUTP could be detected. However, a dramatic increase in the counts recovered as dUMP was noted. The amount of dUMP found in the cytosol of 6~10~ cells pulsed for 10 min with 10 and

20 nmoles of UdR was 2300 pmoles and 3 180 pmoles respectively. A pulse with 100 nmoles of UdR did not reveal any additional increase in the count recovered as dUMP. DISCUSSION The results of this study indicate the presence of a powerful and specific dUTPase in the cytosol of HeLa cells. This enzymatic activity appears to be low in Gl and increases progressively as the cells traverse through S, and G2 to mitosis. This pattern appears to be similar to that of other enzymes involved in deoxyribonucleotide synthesis such as TdR kinase, dTMP kinase [4], dCMP deaminase [7], ribonucleotide reductase [15] and dCMP kinase [9]. dUTPase appears to be extremely specific for dUTP. According to our estimate, the approximate size of the dUTP pool in HeLa cells is 1% of that of dTTP. Therefore, the possibility exists that one molecule of dUTP could be inserted per 350 nucleotides that are incorporated into DNA. We have based our calculation on the (A+T) content of HeLa DNA being 58 %. Incorporation of dUMP into DNA followed by its excision may therefore contribute to the formation of Okazaki fragments during the replication of mammalian DNA. This possibility has been suggested by several authors [5, 17, 20, 22, 231. Further, Brynolf et al. [5] have shown that DNA fragments produced by uracil incorporation into polyoma DNA replicating in animal cell nuclei can be chased into longer molecules. HeLa cells are capable of synthesizing dUTP as can be seen in fig. 1 and in table 1. Therefore, the question arises as to what purpose this cycle of dUTP synthesis and breakdown serves in mammalian cells. As Shlomai & Komberg [20] have pointed out

Turnover

of dUTP during cell cycle

93

to determine the pathway of choice in various malignant cells and in the host tissues. An intracellular inhibition of dUTPase could be selectively used to fragment the DNA of tumor cells utilizing the dUTPase pathway if the tissue of the host derives its dTTP through the deamination of dCMP. Fig. 2. Pathways for pyrimidine metabolism and their regulation. Solid lines, known pathways; dotted lines with spiral arrows at the end, stimulation if the arrow is pointing in the same direction as the reaction, inhibition, if the arrow is pointing in the opposite direction.

a controlled incorporation of uracil may facilitate recombination and other genetic mechanisms which may be advantageous to the cell. In addition, this cycle may provide a basis for the production of dUMP required for dTTP synthesis, by a metabolic pathway that is more direct than the one involving the deamination of dCMP by dCMP deaminase, as outlined in fig. 2. Since ribonucleotide reductase catalyses the reduction of all four ribonucleoside diphosphates [13] and since the uridine nucleotides are abundant within the cell [12], dUDP can be produced. The lack of specificity [14, 161 and high activity of nucleoside diphosphate kinase favors the formation of any nucleoside triphosphate and therefore of dUTP. dUTP can then be efficiently hydrolysed to dUMP, and hence the dUTP pyrophosphorylase may play an important role in furnishing dUMP for the de novo synthesis of dTTP. Although dUMP is commonly thought to be produced by the deamination of dCMP, there are reports that some rapidly proliferating tumors exhibit very low levels of dCMP deaminase [21, 31. These tissues must obtain dUMP by some other route during their DNA synthesis. It may be of great interest

This work was submitted to the University of Texas Graduate School of Biomedical Sciences at Houston, by S. M. in partial fulfillment of the degree of Doctor of Philosophy. This investigation was supported by USPHS Grants CA-11520 and CA-14528, CA-23879 from the NC1 and GM-23252 from the Institute of General Medical Sciences to P.N.R., and USPHS Grant RR5511 to A.O.

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24. Tye, B K, Nyman, P 0, Lehman, I R, Hochhauser, S & Weiss, B, Proc natl acad sci US 74 (1977) 154. 25. Wist, E, Unhjem, 0 & Krokan, H, Biochim biophys acta 520 (1978) 253. Received April 30, 1979 Revised version received July 23, 1979 Accepted August 1, 1979