- Email: [email protected]
Globin synthesis by landschutz ascites ribosomes
448
BIOCHIMICAET BIOPHYSICAACTA
BBA 97436
G L O B I N S Y N T H E S I S BY LANDSCHUTZ ASCITES RIBOSOMES
BRIAN B. COHEN Medical Research Council, Clinical and Population Cytogenetics Unit, Western General Hospital, Crew Road, Edinburgh, EH4 2 X U (Great Britain)
(Received May 26th, 1972)
SUMMARY The requirements for the translation of globin m R N A by ascites ribosomes were studied. It was found that globin was synthesised by a system derived entirely from ascites cells, but the level of synthesis could be increased by the addition of the KC1 wash of reticulocyte ribosomes.
INTRODUCTION The m R N A coding for globin has been isolated and translated in the following cell-free systems: reticulocytes 1, ascites cells *-4, muscle cells (S. M. Heywood, unpublished result) and liver cells 5. It has also been translated after injection into amphibian eggs 6. In the non-reticulocyte systems (apart from liver) translation was accomplished without the addition of any factors prepared from reticulocytes. In the liver system Prichard et al. 5 have reported that translation requires the addition of a specific factor (M3) contained in the wash removed from rabbit reticulocyte ribosomes b y KClflreatment. This KC1 wash of reticulocyte ribosomes has been shown to contain factors required for polypeptide chain initiation 7. However, in the systems where translation of the globin m R N A was achieved without the addition of reticulocyte factors no report was made of whether the KC1 wash of reticulocyte ribosomes enhanced globin synthesis. This paper describes experiments which determine in more detail the requirements for translation of globin m R N A by ascites ribosomes.
MATERIALS AND METHODS Landschutz ascites cells were grown in Porton strain mice and removed 11-13 days after inoculation. The following materials were prepared as described previously 8 ascites ribosomes, ascites enzyme as the concentrated post-ribosomal supernatant, KC1 wash of ascites ribosomes ("KC1 extract" or "KC1 factors"), reticulocyte enzyme as the 40-70 % (NH4)~SO4 precipitate of the post-ribosomal supernatant, and KC1 wash of reticulocyte ribosomes. Globin m R N A was prepared by phenol extraction of the sub-particle removed from rabbit reticulocyte ribosomes after treatment with E D T A 8. The activity of the m R N A was the same whether unwashed or KCl-washed ribosomes were used and for economy KCl-washed ribosomes were preferred. Bioehim. Biophys. Acta, 28i (1972) 448-452
449
GLOBIN SYNTHESIS BY ASCITES RIBOSOMES
Amino acid incorporation The reaction mixture used was that described previously a. [14C]Valine of a spec. act. of 12 Ci/mole was used except in Expt 3 (see Results) where the spec. act. was 270 Ci/mole. Saturating amounts of KC1 extracts, enzyme fractions and mRNA were added where indicated. The products of translation were analysed by column chromatography on CM-cellulose (see Fig. I).
fl
03r
(a) 1
(b) 16
~
0.1 0 8
0.4
(c)
(d)
0.3
6
0.2
4~
A (M
x
E
2~ 8
0.1 E
o o' c~ '=c
12 o
~= 0
(f)
(e)
10
0.3 0.2
4
0.1
60
~
~o
~
60 Fraction No.
65
70
75
Fig. i. A n a l y s i s of t h e p r o d u c t s of t r a n s l a t i o n of globin m R N A - d i r e c t e d s y n t h e s i s . 0.2 m l of t h e i n c u b a t i o n m i x t u r e s of E x p t 3 (Table I) were m i x e d w i t h I ml r a b b i t h a e m o g l o b i n a t A4t5 nm r e a d i n g of 57- T h e f r a c t i o n s were e x t r a c t e d w i t h a c i d - a c e t o n e to p r e p a r e globin a n d applied to a 2 c m x 42 c m W h a t m a n CM52 CM-cellulose c o l u m n a n d e l u t e d w i t h a 33o-ml linear g r a d i e n t of o.2 M to 2.0 M formic acid in 0.02 M p y r i d i n e a c c o r d i n g to t h e m e t h o d of D i n t z i s 9. F r a c t i o n s of 3.6 m l were collected e v e r y i o min. 0.25 m l w a s applied to W h a t m a n G F / B discs, dried in h o t air a n d t h e r a d i o a c t i v i t y m e a s u r e d . (a) r e t i c u l o c y t e KC1 e x t r a c t ; (b) ascites e n z y m e ; (c) reticuloc y t e e n z y m e ; (d) r e t i c u l o c y t e KC1 e x t r a c t + a s c i t e s e n z y m e ; (e) r e t i c u l o c y t e KC1 e x t r a c t + r e t i c u l o c y t e e n z y m e ; (f) r e t i c u l o c y t e K C l - w a s h e d r i b o s o m e s + r e t i c u l o c y t e e n z y m e + r e t i c u l o c y t e KCI e x t r a c t . 0 - 0 , A~80 nm; O - O r a d i o a c t i v i t y .
Biochim. Biophys. Acta, 281 (1972) 448-452
450
B . B . COHEN
RESULTS
Factors reouired ]or the translation o] globin mRNA by ascites ribosomes The amino acid incorporation b y ascites ribosomes was measured in the presence and absence of globin mRNA. Fractions prepared from reticulocytes and ascites cells were tested for their ability to stimulate amino acid incorporation (Table I).
TABLE
I
AMINO ACID INCORPORATION BY ASCITES RIBOSOMES o.36-ml &liquots of t h e s t a n d a r d i n c o r p o r a t i o n m i x t u r e w e re i n c u b a t e d a t 37 °C. A f t e r I h, o.I m l w a s r e m o v e d a n d t h e a m i n o acid i n c o r p o r a t i o n m e a s u r e d . W h e r e s h o w n t h e m i x t u r e c o n t a i n e d o.2 m g of riboso mes, 0.04 m l KC1 e x t r a c t , 5 m g r e t i c u l o c y t e e n z y m e fra c t i on, 5.4 m g of a s c i t e s enzyme fraction and IO/tg globin mRNA.
Conditions of incubation
Incorporation (nmoles valine/mg of ribosomes) Expt z
R i b o s o m e s alone A s c i t e s KCI e x t r a c t R e t i c u l o c y t e s KC] e x t r a c t Ascites enzyme Reticulocyte enzyme A s c i t e s KC1 e x t r a c t + ascites enzyme A s c i t e s KC1 e x t r a c t + reticulocyte enzyme R e t i c u l o c y t e KC1 e x t r a c t + ascites enzyme R e t i c u l o c y t e KC1 e x t r a c t + reticulocyte enzyme
Expt 2
Expt 3
--mRNA
+mRNA
--mRNA
+mRNA
+mRNA
o.22 o.16 o.33 0.29 0.34
o.32 o.18 2.77 1.75 1.52
o.13 o.15 o.21 0.35 o.31
o.12 o.31 1.65 1.43 0.72
o.81 0.77 I.OI
o.16
0.27
o.21
0.55
o. 17
o. 29
o.21
o.56
o.33
3.32
o.28
2.19
1.o6
0.65
3.98
0.57
2.29
1.96
In almost every case m R N A stimulated synthesis. The highest incorporation was obtained when reticulocyte KC1 extract was present with either reticulocyte or ascites enzyme fractions. Incorporation was reduced b y 50 % when the extract was absent, and was very low in the absence of both enzyme and extract. In contrast the ascites KC1 extract had a marked inhibitory effect when present. The high incorporation obtained using reticulocyte KC1 extract alone is probably due to its contamination with enzyme fraction since it has been previously shown to contain elongation factors as well as initiation factors 1°. The reaction mixtures in E x p t 3 were analysed to determine the products of translation. In every case all of the incorporation was into 0¢- and fl-globin. An incubation using reticulocyte KCl-washed ribosomes with attached m R N A (from which the m R N A was prepared) was also analysed for comparison, and showed the products were the same regardless of which type of ribosomes were translating the message. The incomplete separation of the x- and r-chains probably results from using the eluting gradient as it first appeared in the original paper by DintzisL (This was apparently later corrected from o.2 M formic acid and o.02 M pyridine to be 2 M formic acid, 0.2 M pyridine.) Biochim. Biophys. Acta, 281 (1972) 448-452
451
GLOBIN SYNTHESIS BY ASCITES RIBOSOMES DISCUSSION
The results show that ascites ribosomes can synthesise globin without the addition of reticulocyte KC1 extract (initiation factors). This differs from the results obtained by Prichard et al. 5 who found that liver ribosomes required a reticulocyte initiation factor, but does agree with the results of Matthews et al. ~ and Housman et al. ~ using ascites ribosomes, and Gurdon et al. 6 using amphibian oocytes. However, none of these latter gioups report the effect of adding reticulocyte KC1 extract, and the results in this paper show that the addition of extract produces a 2-3-fold increase in synthesis in the presence of reticulocyte enzyme fraction and a 1.4-I.q-fold increase using the ascites enzyme. Essentially, the two results to be explained are (a) the reason why systems derived entirely from ascites cells synthesis globin in the absence of reticulocyte factors and (b) why do reticulocyte factors stimulate these systems? There are three more obvious explanations. (i) These systems are artificial in that the mRNA exists in a purified unbound state which may not be the state in which it exists in the cell and under these conditions initiation can occur in the absence of initiation factors. (ii) Initiation factors exist in the ascites enzyme fraction. These are of low activity either because they are present in small amounts or else because the globin mRNA requires specific reticulocyte factors. (iii) The ascites ribosomes form a heterogeneous population of ribosomes in different stages of the ribosomal cycle, so that ribosomes in one stage can initiate and synthesise globin without the addition of factors and those in other stages require factors. This explanation would also include the possible requirement of factors for re-initiation after one round of synthesis without factors. With all three explanations the addition of reticulocyte factors would increase globin synthesis in these systems.
ADDENDUM
After this paper was submitted similar results were reported using Krebs ascites cell-free systems n. These showed that systems containing ascites cell lysate (post-mitochondrial supernatant) and globin mRNA could synthesis globin and that the addition of reticulocyte KC1 extract stimulated synthesis. The stimilation reported was much higher than that found in this study, possibly because unconcentrated cell lysate was used and not concentrated enzyme fraction and isolated ribosomes.
ACKNOWLEDGEMENT
I wish to thank Katrine Lindsay for technical assistance.
REFERENCES I R. L o c k a r d a n d J. B. Lingrel, Biochem. Biophys. Res. Commun., 37 (1969) 2o4. 2 M. B. M a t t h e w s , M. O s b o r n a n d J. B. Lingrel, Nature, 233 (1971) 2o6. 3 B. B. Cohen, Biochim. Biophys. Acta, 247 (1971) 133.
Biochim. Biophys. Acta, 281 (1972) 4 4 8 - 4 5 2
452
B . B . COHEN
4 D. t t o u s m a n , R. P e m b e r t o n and R. Taber, Proc. Natl. Acad. Sci. U.S., 68 (1971) 2716. 5 P. M. Prichard, D. J. picciano, D. G. Laycock and W. F. Anderson, Proc. Natl. Acad. Sci. U.S., 68 (1971 ) 2752. 6 J. B. Gurdon, C. D. Lane, H. R. Woodland and G. Marbaix, Nature, 233 (1971) 177. 7 P. M. Prichard, J. M. Gilbert, D. A. Shafritz and W. F. Anderson, Nature, 226 (197 o) 511. 8 R. Williamson, G. L a n y o n and J. paul, Nature, 223 (1969) 628. 9 H. M. Dintzis, Proc. Natl. Acad. Sei. U.S., 47 (I96I) 247. IO D. A. Shafritz, P. M. Prichard, J. M. Gilbert and W. F. Anderson, Biochem. Biophys. Res. Commun., 38 (197 o) 721. II S. Metafora, M. Terada, L. W. Dow, P. A. Marks a n d A. Bank, Proc. Natl. Aead. Sci. U.S., 69 (1971) 1299.
Biochim. Biophys. Acta, 281 (1972) 448-452
BIOCHIMICAET BIOPHYSICAACTA
BBA 97436
G L O B I N S Y N T H E S I S BY LANDSCHUTZ ASCITES RIBOSOMES
BRIAN B. COHEN Medical Research Council, Clinical and Population Cytogenetics Unit, Western General Hospital, Crew Road, Edinburgh, EH4 2 X U (Great Britain)
(Received May 26th, 1972)
SUMMARY The requirements for the translation of globin m R N A by ascites ribosomes were studied. It was found that globin was synthesised by a system derived entirely from ascites cells, but the level of synthesis could be increased by the addition of the KC1 wash of reticulocyte ribosomes.
INTRODUCTION The m R N A coding for globin has been isolated and translated in the following cell-free systems: reticulocytes 1, ascites cells *-4, muscle cells (S. M. Heywood, unpublished result) and liver cells 5. It has also been translated after injection into amphibian eggs 6. In the non-reticulocyte systems (apart from liver) translation was accomplished without the addition of any factors prepared from reticulocytes. In the liver system Prichard et al. 5 have reported that translation requires the addition of a specific factor (M3) contained in the wash removed from rabbit reticulocyte ribosomes b y KClflreatment. This KC1 wash of reticulocyte ribosomes has been shown to contain factors required for polypeptide chain initiation 7. However, in the systems where translation of the globin m R N A was achieved without the addition of reticulocyte factors no report was made of whether the KC1 wash of reticulocyte ribosomes enhanced globin synthesis. This paper describes experiments which determine in more detail the requirements for translation of globin m R N A by ascites ribosomes.
MATERIALS AND METHODS Landschutz ascites cells were grown in Porton strain mice and removed 11-13 days after inoculation. The following materials were prepared as described previously 8 ascites ribosomes, ascites enzyme as the concentrated post-ribosomal supernatant, KC1 wash of ascites ribosomes ("KC1 extract" or "KC1 factors"), reticulocyte enzyme as the 40-70 % (NH4)~SO4 precipitate of the post-ribosomal supernatant, and KC1 wash of reticulocyte ribosomes. Globin m R N A was prepared by phenol extraction of the sub-particle removed from rabbit reticulocyte ribosomes after treatment with E D T A 8. The activity of the m R N A was the same whether unwashed or KCl-washed ribosomes were used and for economy KCl-washed ribosomes were preferred. Bioehim. Biophys. Acta, 28i (1972) 448-452
449
GLOBIN SYNTHESIS BY ASCITES RIBOSOMES
Amino acid incorporation The reaction mixture used was that described previously a. [14C]Valine of a spec. act. of 12 Ci/mole was used except in Expt 3 (see Results) where the spec. act. was 270 Ci/mole. Saturating amounts of KC1 extracts, enzyme fractions and mRNA were added where indicated. The products of translation were analysed by column chromatography on CM-cellulose (see Fig. I).
fl
03r
(a) 1
(b) 16
~
0.1 0 8
0.4
(c)
(d)
0.3
6
0.2
4~
A (M
x
E
2~ 8
0.1 E
o o' c~ '=c
12 o
~= 0
(f)
(e)
10
0.3 0.2
4
0.1
60
~
~o
~
60 Fraction No.
65
70
75
Fig. i. A n a l y s i s of t h e p r o d u c t s of t r a n s l a t i o n of globin m R N A - d i r e c t e d s y n t h e s i s . 0.2 m l of t h e i n c u b a t i o n m i x t u r e s of E x p t 3 (Table I) were m i x e d w i t h I ml r a b b i t h a e m o g l o b i n a t A4t5 nm r e a d i n g of 57- T h e f r a c t i o n s were e x t r a c t e d w i t h a c i d - a c e t o n e to p r e p a r e globin a n d applied to a 2 c m x 42 c m W h a t m a n CM52 CM-cellulose c o l u m n a n d e l u t e d w i t h a 33o-ml linear g r a d i e n t of o.2 M to 2.0 M formic acid in 0.02 M p y r i d i n e a c c o r d i n g to t h e m e t h o d of D i n t z i s 9. F r a c t i o n s of 3.6 m l were collected e v e r y i o min. 0.25 m l w a s applied to W h a t m a n G F / B discs, dried in h o t air a n d t h e r a d i o a c t i v i t y m e a s u r e d . (a) r e t i c u l o c y t e KC1 e x t r a c t ; (b) ascites e n z y m e ; (c) reticuloc y t e e n z y m e ; (d) r e t i c u l o c y t e KC1 e x t r a c t + a s c i t e s e n z y m e ; (e) r e t i c u l o c y t e KC1 e x t r a c t + r e t i c u l o c y t e e n z y m e ; (f) r e t i c u l o c y t e K C l - w a s h e d r i b o s o m e s + r e t i c u l o c y t e e n z y m e + r e t i c u l o c y t e KCI e x t r a c t . 0 - 0 , A~80 nm; O - O r a d i o a c t i v i t y .
Biochim. Biophys. Acta, 281 (1972) 448-452
450
B . B . COHEN
RESULTS
Factors reouired ]or the translation o] globin mRNA by ascites ribosomes The amino acid incorporation b y ascites ribosomes was measured in the presence and absence of globin mRNA. Fractions prepared from reticulocytes and ascites cells were tested for their ability to stimulate amino acid incorporation (Table I).
TABLE
I
AMINO ACID INCORPORATION BY ASCITES RIBOSOMES o.36-ml &liquots of t h e s t a n d a r d i n c o r p o r a t i o n m i x t u r e w e re i n c u b a t e d a t 37 °C. A f t e r I h, o.I m l w a s r e m o v e d a n d t h e a m i n o acid i n c o r p o r a t i o n m e a s u r e d . W h e r e s h o w n t h e m i x t u r e c o n t a i n e d o.2 m g of riboso mes, 0.04 m l KC1 e x t r a c t , 5 m g r e t i c u l o c y t e e n z y m e fra c t i on, 5.4 m g of a s c i t e s enzyme fraction and IO/tg globin mRNA.
Conditions of incubation
Incorporation (nmoles valine/mg of ribosomes) Expt z
R i b o s o m e s alone A s c i t e s KCI e x t r a c t R e t i c u l o c y t e s KC] e x t r a c t Ascites enzyme Reticulocyte enzyme A s c i t e s KC1 e x t r a c t + ascites enzyme A s c i t e s KC1 e x t r a c t + reticulocyte enzyme R e t i c u l o c y t e KC1 e x t r a c t + ascites enzyme R e t i c u l o c y t e KC1 e x t r a c t + reticulocyte enzyme
Expt 2
Expt 3
--mRNA
+mRNA
--mRNA
+mRNA
+mRNA
o.22 o.16 o.33 0.29 0.34
o.32 o.18 2.77 1.75 1.52
o.13 o.15 o.21 0.35 o.31
o.12 o.31 1.65 1.43 0.72
o.81 0.77 I.OI
o.16
0.27
o.21
0.55
o. 17
o. 29
o.21
o.56
o.33
3.32
o.28
2.19
1.o6
0.65
3.98
0.57
2.29
1.96
In almost every case m R N A stimulated synthesis. The highest incorporation was obtained when reticulocyte KC1 extract was present with either reticulocyte or ascites enzyme fractions. Incorporation was reduced b y 50 % when the extract was absent, and was very low in the absence of both enzyme and extract. In contrast the ascites KC1 extract had a marked inhibitory effect when present. The high incorporation obtained using reticulocyte KC1 extract alone is probably due to its contamination with enzyme fraction since it has been previously shown to contain elongation factors as well as initiation factors 1°. The reaction mixtures in E x p t 3 were analysed to determine the products of translation. In every case all of the incorporation was into 0¢- and fl-globin. An incubation using reticulocyte KCl-washed ribosomes with attached m R N A (from which the m R N A was prepared) was also analysed for comparison, and showed the products were the same regardless of which type of ribosomes were translating the message. The incomplete separation of the x- and r-chains probably results from using the eluting gradient as it first appeared in the original paper by DintzisL (This was apparently later corrected from o.2 M formic acid and o.02 M pyridine to be 2 M formic acid, 0.2 M pyridine.) Biochim. Biophys. Acta, 281 (1972) 448-452
451
GLOBIN SYNTHESIS BY ASCITES RIBOSOMES DISCUSSION
The results show that ascites ribosomes can synthesise globin without the addition of reticulocyte KC1 extract (initiation factors). This differs from the results obtained by Prichard et al. 5 who found that liver ribosomes required a reticulocyte initiation factor, but does agree with the results of Matthews et al. ~ and Housman et al. ~ using ascites ribosomes, and Gurdon et al. 6 using amphibian oocytes. However, none of these latter gioups report the effect of adding reticulocyte KC1 extract, and the results in this paper show that the addition of extract produces a 2-3-fold increase in synthesis in the presence of reticulocyte enzyme fraction and a 1.4-I.q-fold increase using the ascites enzyme. Essentially, the two results to be explained are (a) the reason why systems derived entirely from ascites cells synthesis globin in the absence of reticulocyte factors and (b) why do reticulocyte factors stimulate these systems? There are three more obvious explanations. (i) These systems are artificial in that the mRNA exists in a purified unbound state which may not be the state in which it exists in the cell and under these conditions initiation can occur in the absence of initiation factors. (ii) Initiation factors exist in the ascites enzyme fraction. These are of low activity either because they are present in small amounts or else because the globin mRNA requires specific reticulocyte factors. (iii) The ascites ribosomes form a heterogeneous population of ribosomes in different stages of the ribosomal cycle, so that ribosomes in one stage can initiate and synthesise globin without the addition of factors and those in other stages require factors. This explanation would also include the possible requirement of factors for re-initiation after one round of synthesis without factors. With all three explanations the addition of reticulocyte factors would increase globin synthesis in these systems.
ADDENDUM
After this paper was submitted similar results were reported using Krebs ascites cell-free systems n. These showed that systems containing ascites cell lysate (post-mitochondrial supernatant) and globin mRNA could synthesis globin and that the addition of reticulocyte KC1 extract stimulated synthesis. The stimilation reported was much higher than that found in this study, possibly because unconcentrated cell lysate was used and not concentrated enzyme fraction and isolated ribosomes.
ACKNOWLEDGEMENT
I wish to thank Katrine Lindsay for technical assistance.
REFERENCES I R. L o c k a r d a n d J. B. Lingrel, Biochem. Biophys. Res. Commun., 37 (1969) 2o4. 2 M. B. M a t t h e w s , M. O s b o r n a n d J. B. Lingrel, Nature, 233 (1971) 2o6. 3 B. B. Cohen, Biochim. Biophys. Acta, 247 (1971) 133.
Biochim. Biophys. Acta, 281 (1972) 4 4 8 - 4 5 2
452
B . B . COHEN
4 D. t t o u s m a n , R. P e m b e r t o n and R. Taber, Proc. Natl. Acad. Sci. U.S., 68 (1971) 2716. 5 P. M. Prichard, D. J. picciano, D. G. Laycock and W. F. Anderson, Proc. Natl. Acad. Sci. U.S., 68 (1971 ) 2752. 6 J. B. Gurdon, C. D. Lane, H. R. Woodland and G. Marbaix, Nature, 233 (1971) 177. 7 P. M. Prichard, J. M. Gilbert, D. A. Shafritz and W. F. Anderson, Nature, 226 (197 o) 511. 8 R. Williamson, G. L a n y o n and J. paul, Nature, 223 (1969) 628. 9 H. M. Dintzis, Proc. Natl. Acad. Sei. U.S., 47 (I96I) 247. IO D. A. Shafritz, P. M. Prichard, J. M. Gilbert and W. F. Anderson, Biochem. Biophys. Res. Commun., 38 (197 o) 721. II S. Metafora, M. Terada, L. W. Dow, P. A. Marks a n d A. Bank, Proc. Natl. Aead. Sci. U.S., 69 (1971) 1299.
Biochim. Biophys. Acta, 281 (1972) 448-452