387
Biochimica et Biophysica A cta, 565 (1979) 387--390 © Elsevier/North-Holland Biomedical Press
BBA Report BBA 91491
SIMILAR DISTRIBUTIONS OF REPAIRED SITES IN CHROMATIN OF NORMAL AND XERODERMA PIGMENTOSUM VARIANT CELLS DAMAGED BY ULTRAVIOLET LIGHT
JAMES E. CLEAVER
Laboratory of Radiobiology, University of California, San Francisco, CA 94143 (U.S.A.) (Received May 28th, 1979) Key words: Chromatin damage; DNA excision repair; Ultraviolet irradiation; (Xeroderma pigmentosum)
Summary Excision repair of damage from ultraviolet light in both normal and xeroderma pigmentosum variant fibroblasts at early times after irradiation occurred preferentially in regions of DNA accessible to micrococcal nuclease digestion. These regions are predominantly the linker regions between nucleosomes in chromatin. The alterations reported at polymerization and ligation steps of excision repair in the variant are therefore not associated with changes in the relative distributions of repair sites in linker and core particle regions of DNA.
Damaged sites in DNA are not uniformly accessible to repair enzymes in vivo [1,2]. In general, those regions of DNA within chromatin that are more accessible to exogenous micrococcal nuclease are also the regions that are more rapidly repaired [3--6]. Both nonuniform distributions of damaged sites and nonuniform rates of repair contribute to these observations. After damage from ultraviolet light, pyrimidine dimers are uniformly distributed in chromatin [6], and the observed nonuniform distribution of repaired sites is probably due largely to the relative access of,repair enzymes to damaged sites [1,2]. The human repair-deficient disease xeroderma pigmentosum has several genetically distinct complementation groups that represent different defects in excision repair [7]. One group, the xeroderma pigrnentosum variant, has an apparently normal amount of excision repair, as judged by unscheduled DNA synthesis and repair replication [8], but has subtle alterations in the rate at which excision gaps are sealed [9,10] and in the sensitivity of repair polymerization to cytosine ambinoside [11,12]. From data on the induction of muta-
388 tions and cell killing by ultraviolet light it can also be argued that the xeroderma pigmentosum variant has an alteration that increases the error rate of excision repair but does not impair cell viability [13]. The xeroderma pigmentosum variant and all other groups investigated also have altered semiconservative DNA replication after irradiation [12,14--17]. However, because of the intimate relationship between damage, excision repair, and DNA replication [16--18] it is not known which alterations represent the primary defect in these diseases. In view of the data suggesting a subtle defect in the polymerization steps of excision repair in xeroderma pigmentosum variants [9--12], I investigated the distribution of excision repair sites within chromatin of normal and xeroderma pigmentosum variant cells at early times after irradiation, when the higher rates of repair in nuclease-accessible sites allow a simple discrimination between repaired sites in different regions. Normal human fibroblasts (strain E l l ) and xeroderma pigmentosum variant fibroblasts (XP4BE) were grown in Eagle's minimal essential medium with 10% fetal calf serum. Cells were labelled by growing in 0.01 pCi/ml of [14C]thymidine (spec. act., 56.4 Ci/mol) in 90-mm Petri dishes until confluence had been reached and mitotic figures were no longer detectable. Cultures were then rinsed and grown for 24 h in unlabelled medium before being irradiated with 13 J/m 2 ultraviolet light (254 nm, at a dose rate of 1.3 J/m 2 per s). They were then labelled with [3H] thymidine (10 ~Ci/ml, 64 Ci/mmol), h y d r o x y u r e a (1" 10 - 3 M), and fluorodeoxyuridine ( 2 . 1 0 -6 M) for 30 min, harvested immediately by trypsinization for 5 min, and then chilled on ice. Under these conditions there is negligible semiconservative replication during the time of irradiation and labelling [4]. Therefore, the 14C activity is uniformly distributed throughout the DNA but the 3H activity is confined to the short patches involved in repair of ultraviolet damage [19]. Nuclei were isolated by hypotonic t r e a t m e n t and Dounce homogenization in 1% Triton X-100 as previously described [4]. Equal numbers of washed nuclei were then distributed into incubation tubes containing 2 ~l of micrococcal nuclease (Worthington Biochemical Corp., U.S.A., approx. 40 units to 1.104 nuclei) and incubated for various times at 37°C. After enz y m e digestion nuclei were harvested, precipitated onto glass fiber filters by 4% perchloric acid (4°C), and counted in toluene-based scintillation fluid. Nuclei labelled with [ ~4C]thymidine alone were used as a ~4C scintillation standard to estimate channel ratios. The fractions of the initial a m o u n t of acidinsoluble radioactivity remaining after various times of digestion were calculated. Repaired (3H-labelled) DNA was digested from nuclei by micrococcal nuclease at a faster rate than the uniformly distributed ~4C-labelled DNA in both cell types (Figs. 1 and 2). Previous results have shown that after very brief labelling periods of 10 min the majority of the 3H-labelled DNA is digested completely within 30--60 rain whereas about 70% of bulk ~4Clabelled DNA remains [4]. At labelling times of 60--90 min the 3H-labelled DNA remaining is close to bulk ~4C-labelled DNA either because of eventual repair of bulk regions of chromatin [4,6] or perhaps because of redistribution of labelled sites [5]. The choice of a 30 min labelling period in the current
389 |.0 O.VI
)
a>_- 0.8 ~o.~ ~Q o.6
_z 0.3 I
I
I
60
I
120 MIN
NUCLEA,SE INCUBATION TIME
60 .46/.66 - 0.731 120 .315/.44= 0.72 J
0.01S SE
Fig. I . P e r c e n t a g e o f 14C- a n d 3 H - l a b e l l e d D N A r e m a i n i n g a c i d i n s o l u b l e a f t e r d i g e s t i o n o f n o r m a l cell n u c l e i w i t h m i c r o c o c c a l n u c l e a s e f o r v a r i o u s t i m e s a t 3 7 ° C. C u l t u r e s w e r e u n i f o r m l y l a b e l l e d w i t h [14C]t h y m i d i n e , i r r a d i a t e d w i t h 1 3 J / m 2 u l t r a v i o l e t light, a n d l a b e l l e d f o r 3 0 m i n w i t h [ 3 H ] t h y m i d / ~ n e , 1 • 1 0 - ~ M h y d r o x y u r e a , a n d 2 • 1 0 - 6 M f l u o r o d e o x y u r i d i n e . • , [14 C] t h y m i d i n e r a d i o a c t i v i t y ; o , [ SH] thymidine radioactivity.
experiments (Figs. 1 and 2) strikes a balance between labelling of nucleaseaccessible and -inaccessible regions by repair and would permit detection of differences between normal and xeroderma pigmentosum variant cell types. Both cell types, however, showed similar increased digestibility of SH-labelled DNA. The mean ratios of SH/14C acid-insoluble radioactivity after 10--120 min of enzyme digestion were 0.71 + 0.01 (S.E.) for normal cells and 0.65 + 0.06 for xeroderma pigrnentosum variant cells, indicating that there is no significant difference in the nuclease sensitivity o f SH-labelled DNA in the two cell types. hO
•
0.9
~.
0.8
0.6 0.1
0.~ i
I
I
I
NUCLEASE INCUBATION TIME TIMES 15 30 60 120
.62/.911•0.'- --I .5$/.69u0.7976.~. ~ 0,65'1"0,12SO .32/.b2 -0,516 0.06 ~E ,29/,44= 0.
Fig. 2. P e r c e n t a g e o f 14C- a n d SH-labelled D N A r e m a i n i n g a c i d i n s o l u b l e a f t e r d i g e s t i o n o f x e r o d e r m a p l g m e n t o s u m v a r i a n t cell n u c l e i w i t h m i c r o c o c c a l n u c l e a s e f o r v a r i o u s t i m e s a t 3 7 ° C . C u l t u r e s w e r e u n i formly labelled with [14C]thymidine, irradiated with 13 J/m 2 ultraviolet light, and labelled for 30 min with [SH]thymidine, I'I0 -s M hydroxyurea, and 2 "10 -~ M fluorodeoxyuridine, o, [14C]thymldine r a d i o a c t i v i t y ; o, [ s H ] t h y m i d i n e r a d i o a c t i v i t y .
390
Previous studies of nuclease digestion [3--6] have shown that the conditions used here result in rapid digestion o f linker regions of D N A between nucleosomes and a slow digestion of nucleosomes to core particles. Our o w n gel analyses of the products of enzyme digestion [20] are consistent with this interpretation. Recent observations that regions of D N A around replication origins are also exposed [21,22] imply that a small proportion of rapidly digested D N A may be from regions other than linker DNA. The present observations indicate that there is no difference in the distribution of repaired D N A in linker and core particle D N A of normal and xeroderma pigmentosum variant fibroblasts. Similar observations obtained under different labelling conditions have been reported for both xeroderma pigmentosum group E and variant [6]. Therefore, the subtle alterations in excision repair observed in the xeroderma pigmentosum variant [9--12,23] do not appear to alter the relative distribution of repaired sites in linker and core particle DNA. Whether other groups of xeroderma pigmentosum (e.g., groups C and D, which perform 10--20% of normal levels of unscheduled D N A synthesis [7] ) have altered distributions of repaired sites will be more difficult to resolve by these techniques; however, this is under investigation because it is conceivable that the residual levels of repair in those groups could occur in the more accessible linker regions of D N A [2]. I am grateful for the assistance and comments of G.H. Thomas, W.J. Bodell and J.I. Williams during these investigations. This work is supported by the U.S. Department of Energy. References 1 Wilkins, R.J. and Hart, R.W. (1974) Nature 247, 35--36 2 Mortelmans, K., Friedbezg, E.C., Slot, H., Thomas, G. and Cleaver, J.E. (1976) Proe. Natl. Acad. Sei. U.S.A. 73, 2757--2761 3 Bodell, W.J. (1977) NucL Acids Res. 4, 2 6 1 9 - - 2 6 2 8 4 Cleaver, J.E. (1977) Nature 270, 451--453 5 Smerdon, M.J. and Lieberman, M.W. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4238--4241 6 Williams, J.L and Friedberg, E.C. (1979) Biochemistry 18, 3 9 6 5 - - 3 9 7 2 7 Cleaver, J.E. and Bootsma, D. (1975) Annu. Rev. Oen. 9, 19--38 8 Cleaver, J.E. ( 1 9 7 2 ) J . Invest. Derm. 58, 124--128 9 Dingman, C.W. and Kakunaga, T. (1976) Int. J. Radiat. Biol. 30, 55--66 10 Fornace, A.J., Jr., Kohn, K.W. and Kann, H.E., Jr. (1976) Proc. Natl. Acad. ScL U.S.A. 73, 39--43 11 Dunn, W.C. and Regan, J.D. (1979) Mol. Pharm. 15, 367--374 12 Cleaver, J.E., Thomas, G.H. and Park, S.D. (1979) Biochim. Biophys. A c t a 564, 122--131 13 Maher, V.M., Ouellettc, L.M., Curren, R.D. and McCormick, J.J. (1976) Nature 2 6 1 , 5 9 3 - - 5 9 5 14 Lehmarm, A.R., Kirk-Bell, S., Arlett, C.F., Paterson, M.C., Lohma n, P.H.M., de WeerdKastelein, E.A. and Bootsma, D. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 219--223 15 Lehmann, A.R., Kirk-BelL S., Arlett, C.F., Harcourt, S.A., de Weerd-Kastelein, E.A., Keijzer, W. and Hail-Smith, P. (1977) Cancer Res. 37, 9 0 4 - - 9 1 0 16 Park, S.D. and Cleaver, J.E. (1979) NucL Acids Res. 6, 1151--1159 17 Park, S.D. and Cleaver, J.E. (1979) Proc. NatL Acad. Sei. U.S.A~ 76, 3927--3931 18 Cleaver, J.E. (1979) Biochim. Biophys. Acta 5 1 6 , 4 8 9 - - 5 1 6 19 Cleaver, J.E. (1975) in Methods in Cancer Research (Busch, H., ed.), Vol. 9, PP. 123--165, Academic Press, New Y o r k 20 Cleaver, J.E. and Bodell, W.J. (1979) Biochemistry, in the press 21 Varshavsky, A.J., Sundin, O. and Bohn, M.J. (1978) Nucl. Acids Res. 5, 3 4 6 9 - - 3 4 7 7 22 Varshavsky, A.J., Sundin, O. and Bohn, M. (1979) Cell 16, 453--466 23 Netrawali, M.S. and Cerruttl, P.A. (1979) Biochem. Biophys. Res. Commun. 87, 8 0 2 - - 8 1 0
Biochimica et Biophysica A cta, 565 (1979) 387--390 © Elsevier/North-Holland Biomedical Press
BBA Report BBA 91491
SIMILAR DISTRIBUTIONS OF REPAIRED SITES IN CHROMATIN OF NORMAL AND XERODERMA PIGMENTOSUM VARIANT CELLS DAMAGED BY ULTRAVIOLET LIGHT
JAMES E. CLEAVER
Laboratory of Radiobiology, University of California, San Francisco, CA 94143 (U.S.A.) (Received May 28th, 1979) Key words: Chromatin damage; DNA excision repair; Ultraviolet irradiation; (Xeroderma pigmentosum)
Summary Excision repair of damage from ultraviolet light in both normal and xeroderma pigmentosum variant fibroblasts at early times after irradiation occurred preferentially in regions of DNA accessible to micrococcal nuclease digestion. These regions are predominantly the linker regions between nucleosomes in chromatin. The alterations reported at polymerization and ligation steps of excision repair in the variant are therefore not associated with changes in the relative distributions of repair sites in linker and core particle regions of DNA.
Damaged sites in DNA are not uniformly accessible to repair enzymes in vivo [1,2]. In general, those regions of DNA within chromatin that are more accessible to exogenous micrococcal nuclease are also the regions that are more rapidly repaired [3--6]. Both nonuniform distributions of damaged sites and nonuniform rates of repair contribute to these observations. After damage from ultraviolet light, pyrimidine dimers are uniformly distributed in chromatin [6], and the observed nonuniform distribution of repaired sites is probably due largely to the relative access of,repair enzymes to damaged sites [1,2]. The human repair-deficient disease xeroderma pigmentosum has several genetically distinct complementation groups that represent different defects in excision repair [7]. One group, the xeroderma pigrnentosum variant, has an apparently normal amount of excision repair, as judged by unscheduled DNA synthesis and repair replication [8], but has subtle alterations in the rate at which excision gaps are sealed [9,10] and in the sensitivity of repair polymerization to cytosine ambinoside [11,12]. From data on the induction of muta-
388 tions and cell killing by ultraviolet light it can also be argued that the xeroderma pigmentosum variant has an alteration that increases the error rate of excision repair but does not impair cell viability [13]. The xeroderma pigmentosum variant and all other groups investigated also have altered semiconservative DNA replication after irradiation [12,14--17]. However, because of the intimate relationship between damage, excision repair, and DNA replication [16--18] it is not known which alterations represent the primary defect in these diseases. In view of the data suggesting a subtle defect in the polymerization steps of excision repair in xeroderma pigmentosum variants [9--12], I investigated the distribution of excision repair sites within chromatin of normal and xeroderma pigmentosum variant cells at early times after irradiation, when the higher rates of repair in nuclease-accessible sites allow a simple discrimination between repaired sites in different regions. Normal human fibroblasts (strain E l l ) and xeroderma pigmentosum variant fibroblasts (XP4BE) were grown in Eagle's minimal essential medium with 10% fetal calf serum. Cells were labelled by growing in 0.01 pCi/ml of [14C]thymidine (spec. act., 56.4 Ci/mol) in 90-mm Petri dishes until confluence had been reached and mitotic figures were no longer detectable. Cultures were then rinsed and grown for 24 h in unlabelled medium before being irradiated with 13 J/m 2 ultraviolet light (254 nm, at a dose rate of 1.3 J/m 2 per s). They were then labelled with [3H] thymidine (10 ~Ci/ml, 64 Ci/mmol), h y d r o x y u r e a (1" 10 - 3 M), and fluorodeoxyuridine ( 2 . 1 0 -6 M) for 30 min, harvested immediately by trypsinization for 5 min, and then chilled on ice. Under these conditions there is negligible semiconservative replication during the time of irradiation and labelling [4]. Therefore, the 14C activity is uniformly distributed throughout the DNA but the 3H activity is confined to the short patches involved in repair of ultraviolet damage [19]. Nuclei were isolated by hypotonic t r e a t m e n t and Dounce homogenization in 1% Triton X-100 as previously described [4]. Equal numbers of washed nuclei were then distributed into incubation tubes containing 2 ~l of micrococcal nuclease (Worthington Biochemical Corp., U.S.A., approx. 40 units to 1.104 nuclei) and incubated for various times at 37°C. After enz y m e digestion nuclei were harvested, precipitated onto glass fiber filters by 4% perchloric acid (4°C), and counted in toluene-based scintillation fluid. Nuclei labelled with [ ~4C]thymidine alone were used as a ~4C scintillation standard to estimate channel ratios. The fractions of the initial a m o u n t of acidinsoluble radioactivity remaining after various times of digestion were calculated. Repaired (3H-labelled) DNA was digested from nuclei by micrococcal nuclease at a faster rate than the uniformly distributed ~4C-labelled DNA in both cell types (Figs. 1 and 2). Previous results have shown that after very brief labelling periods of 10 min the majority of the 3H-labelled DNA is digested completely within 30--60 rain whereas about 70% of bulk ~4Clabelled DNA remains [4]. At labelling times of 60--90 min the 3H-labelled DNA remaining is close to bulk ~4C-labelled DNA either because of eventual repair of bulk regions of chromatin [4,6] or perhaps because of redistribution of labelled sites [5]. The choice of a 30 min labelling period in the current
389 |.0 O.VI
)
a>_- 0.8 ~o.~ ~Q o.6
_z 0.3 I
I
I
60
I
120 MIN
NUCLEA,SE INCUBATION TIME
60 .46/.66 - 0.731 120 .315/.44= 0.72 J
0.01S SE
Fig. I . P e r c e n t a g e o f 14C- a n d 3 H - l a b e l l e d D N A r e m a i n i n g a c i d i n s o l u b l e a f t e r d i g e s t i o n o f n o r m a l cell n u c l e i w i t h m i c r o c o c c a l n u c l e a s e f o r v a r i o u s t i m e s a t 3 7 ° C. C u l t u r e s w e r e u n i f o r m l y l a b e l l e d w i t h [14C]t h y m i d i n e , i r r a d i a t e d w i t h 1 3 J / m 2 u l t r a v i o l e t light, a n d l a b e l l e d f o r 3 0 m i n w i t h [ 3 H ] t h y m i d / ~ n e , 1 • 1 0 - ~ M h y d r o x y u r e a , a n d 2 • 1 0 - 6 M f l u o r o d e o x y u r i d i n e . • , [14 C] t h y m i d i n e r a d i o a c t i v i t y ; o , [ SH] thymidine radioactivity.
experiments (Figs. 1 and 2) strikes a balance between labelling of nucleaseaccessible and -inaccessible regions by repair and would permit detection of differences between normal and xeroderma pigmentosum variant cell types. Both cell types, however, showed similar increased digestibility of SH-labelled DNA. The mean ratios of SH/14C acid-insoluble radioactivity after 10--120 min of enzyme digestion were 0.71 + 0.01 (S.E.) for normal cells and 0.65 + 0.06 for xeroderma pigrnentosum variant cells, indicating that there is no significant difference in the nuclease sensitivity o f SH-labelled DNA in the two cell types. hO
•
0.9
~.
0.8
0.6 0.1
0.~ i
I
I
I
NUCLEASE INCUBATION TIME TIMES 15 30 60 120
.62/.911•0.'- --I .5$/.69u0.7976.~. ~ 0,65'1"0,12SO .32/.b2 -0,516 0.06 ~E ,29/,44= 0.
Fig. 2. P e r c e n t a g e o f 14C- a n d SH-labelled D N A r e m a i n i n g a c i d i n s o l u b l e a f t e r d i g e s t i o n o f x e r o d e r m a p l g m e n t o s u m v a r i a n t cell n u c l e i w i t h m i c r o c o c c a l n u c l e a s e f o r v a r i o u s t i m e s a t 3 7 ° C . C u l t u r e s w e r e u n i formly labelled with [14C]thymidine, irradiated with 13 J/m 2 ultraviolet light, and labelled for 30 min with [SH]thymidine, I'I0 -s M hydroxyurea, and 2 "10 -~ M fluorodeoxyuridine, o, [14C]thymldine r a d i o a c t i v i t y ; o, [ s H ] t h y m i d i n e r a d i o a c t i v i t y .
390
Previous studies of nuclease digestion [3--6] have shown that the conditions used here result in rapid digestion o f linker regions of D N A between nucleosomes and a slow digestion of nucleosomes to core particles. Our o w n gel analyses of the products of enzyme digestion [20] are consistent with this interpretation. Recent observations that regions of D N A around replication origins are also exposed [21,22] imply that a small proportion of rapidly digested D N A may be from regions other than linker DNA. The present observations indicate that there is no difference in the distribution of repaired D N A in linker and core particle D N A of normal and xeroderma pigmentosum variant fibroblasts. Similar observations obtained under different labelling conditions have been reported for both xeroderma pigmentosum group E and variant [6]. Therefore, the subtle alterations in excision repair observed in the xeroderma pigmentosum variant [9--12,23] do not appear to alter the relative distribution of repaired sites in linker and core particle DNA. Whether other groups of xeroderma pigmentosum (e.g., groups C and D, which perform 10--20% of normal levels of unscheduled D N A synthesis [7] ) have altered distributions of repaired sites will be more difficult to resolve by these techniques; however, this is under investigation because it is conceivable that the residual levels of repair in those groups could occur in the more accessible linker regions of D N A [2]. I am grateful for the assistance and comments of G.H. Thomas, W.J. Bodell and J.I. Williams during these investigations. This work is supported by the U.S. Department of Energy. References 1 Wilkins, R.J. and Hart, R.W. (1974) Nature 247, 35--36 2 Mortelmans, K., Friedbezg, E.C., Slot, H., Thomas, G. and Cleaver, J.E. (1976) Proe. Natl. Acad. Sei. U.S.A. 73, 2757--2761 3 Bodell, W.J. (1977) NucL Acids Res. 4, 2 6 1 9 - - 2 6 2 8 4 Cleaver, J.E. (1977) Nature 270, 451--453 5 Smerdon, M.J. and Lieberman, M.W. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4238--4241 6 Williams, J.L and Friedberg, E.C. (1979) Biochemistry 18, 3 9 6 5 - - 3 9 7 2 7 Cleaver, J.E. and Bootsma, D. (1975) Annu. Rev. Oen. 9, 19--38 8 Cleaver, J.E. ( 1 9 7 2 ) J . Invest. Derm. 58, 124--128 9 Dingman, C.W. and Kakunaga, T. (1976) Int. J. Radiat. Biol. 30, 55--66 10 Fornace, A.J., Jr., Kohn, K.W. and Kann, H.E., Jr. (1976) Proc. Natl. Acad. ScL U.S.A. 73, 39--43 11 Dunn, W.C. and Regan, J.D. (1979) Mol. Pharm. 15, 367--374 12 Cleaver, J.E., Thomas, G.H. and Park, S.D. (1979) Biochim. Biophys. A c t a 564, 122--131 13 Maher, V.M., Ouellettc, L.M., Curren, R.D. and McCormick, J.J. (1976) Nature 2 6 1 , 5 9 3 - - 5 9 5 14 Lehmarm, A.R., Kirk-Bell, S., Arlett, C.F., Paterson, M.C., Lohma n, P.H.M., de WeerdKastelein, E.A. and Bootsma, D. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 219--223 15 Lehmann, A.R., Kirk-BelL S., Arlett, C.F., Harcourt, S.A., de Weerd-Kastelein, E.A., Keijzer, W. and Hail-Smith, P. (1977) Cancer Res. 37, 9 0 4 - - 9 1 0 16 Park, S.D. and Cleaver, J.E. (1979) NucL Acids Res. 6, 1151--1159 17 Park, S.D. and Cleaver, J.E. (1979) Proc. NatL Acad. Sei. U.S.A~ 76, 3927--3931 18 Cleaver, J.E. (1979) Biochim. Biophys. Acta 5 1 6 , 4 8 9 - - 5 1 6 19 Cleaver, J.E. (1975) in Methods in Cancer Research (Busch, H., ed.), Vol. 9, PP. 123--165, Academic Press, New Y o r k 20 Cleaver, J.E. and Bodell, W.J. (1979) Biochemistry, in the press 21 Varshavsky, A.J., Sundin, O. and Bohn, M.J. (1978) Nucl. Acids Res. 5, 3 4 6 9 - - 3 4 7 7 22 Varshavsky, A.J., Sundin, O. and Bohn, M. (1979) Cell 16, 453--466 23 Netrawali, M.S. and Cerruttl, P.A. (1979) Biochem. Biophys. Res. Commun. 87, 8 0 2 - - 8 1 0
