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Free ribosomes during maturation of rat liver
Comp. Biochera. Physiol., 1963, Vol. 10, pp. 33 to 38. PergamonPressLtd. Printed in Great Britain
FREE RIBOSOMES DURING MATURATION OF RAT LIVER I. T . O L I V E R , W. F. C. B L U M E R * and I. J. W I T H A M Departments of Biochemistry and *Anatomy, University of Western Australia, Nedlands, Western Australia (Received 28 April 1963)
Abstract--1. A high concentration of free ribosomes has been found in homogenates of the liver of foetal and post-natal rats. The concentration is about 8 mg/g of liver cells until about 5 days after birth when it falls rapidly to the adult level of about 2 mg/g of liver cells. 2. In the 17-day foetal rat, 60 per cent of the liver is composed of haemopoietic and vascular tissue, but the percentage falls in an almost linear fashion to about 10 per cent 5 days after birth. After this time few haemopoietic cells can be seen in liver sections. 3. Evidence is presented for the view that the free ribosome fraction in these liver homogenates is almost exclusively derived from liver cells. INTRODUCTION IN A previous paper (Oliver et al., 1952) the concentrations of liver D N A t and RNA:~ during growth of the rat in the immediate postpartum period were shown to reach maximum levels at 5 days and 1S days after birth respectively. Further investigations of the cytoplasmic form of R N A in neo-natal rat liver have now shown that a large fraction of the total R N A is initially in the form of free ribosomes. T h e existence of such particles in liver and other tissues has been described before (see Barnum & Huseby, 1948; Palade, 1955; Peterman et al., 1953a, b, 1954, 1956) but in the present work quantitative data have been obtained on the concentration of free ribosomes (which are not attached to cytomembranes) during development of the rat liver through the late foetal and post-natal period. In addition, the amount of haemopoietic tissue present in the neonatal liver has been assessed by histological methods in order to ascertain the cytological origin of the free ribosomes. MATERIALS AND METHODS Measurement o f free ribosome concentration
T h e rats used in this study were of the Wistar albino strain, species Rattus All operations were performed in the cold-room at 0-4°C. Foetal animals were obtained from the uterus of the mother which was sacrificed by a blow on the head. T h e foetuses were washed in ice-cold distilled water, blotted on filter paper and weighed. T h e livers were removed, rapidly weighed and dropped norvegicus.
t Deoxyribonucleic acid. ~: Ribonucleic acid. 33
I . T . OLIVER,W. F. C. BLUMERAND I. J. WITHAM
34
into the ice-cold medium used for homogenization. Infant animals were sacrificed by decapitation and the livers similarly treated. T h e foetal age was determined from the time of appearance of a vaginal plug in mated females. T h e livers from seven to ten animals were pooled and homogenized in 3 vol of an ice-cold solution of sucrose (0-31 M) containing 0-02 M Tris*-HC1 buffer p H 7.4. T h e conditions of homogenization were standardized as far as possible by using Perspex coaxial homogenizers machined with standard clearance between pestle and wall and driven at the same speed, for the same number of up and down strokes. All homogenates were centrifuged at 6000 g for 20 rain at 0°C to remove nuclei and mitochondria, and the supernatants removed for ultracentrifugal analysis. Analyses were made in the Beckman Spinco Model E ultracentrifuge at 5-10°C in 12 m m aluminium centrepiece cells (4 ° sector), at 42,040 rev/min. T h e instrument was equipped with the R T I C system and phase-plate Schlieren optics. Ilford R40 panchromatic plates were used in photography. T h e microsome fraction sedimented during rotor acceleration and the ribosome boundaries appeared after the rotor reached 42,040 rev/min. T h e plates were analysed for sedimentation coefficients (S) by projection on a calibrated screen, and areas under the peaks were measured by summation of vertical measurements on tracings obtained from a photographic enlarger. In some experiments, a 1.0 ml sample of the 6000 g supernatant was incubated 24 hr at 0°C with 1 mg crystalline bovine pancreatic ribonuclease (Sigma Chemical Co., St. Louis, Missouri, U.S.A.) and then compared with duplicate samples incubated without ribonuclease. All the sedimenting boundaries in the range 30-100 S disappeared after ribonuclease treatment, but the control samples were unchanged. Area measurements were converted to concentration using a value of 1"87 x 10 3 for the specific refractive increment of rat liver ribonucleoprotein (Peterman & Pavlovec, 1963). T h e concentrations were converted to mg ribosomes/g of fresh liver. T h e addition of MgCI~ to the homogenization medium over the concentration range 10 .5 M - 1 0 -3 M did not significantly alter the yield or the relative proportions of the ribosome species. It was therefore omitted from routine runs.
Histology Liver obtained from two animals at each age as described above was sliced with a razor blade and fixed in either buffered 10% formalin or Zenkers fluid. T h e slices were dehydrated in 2-ethoxyethanol, cleared in toluene, embedded in paraffin and sectioned at 10/x. T h e sections were stained in Harris' haematoxylin and chromotrope 2R. T e n areas selected at random from several sections taken at each age were examined under oil immersion (magnification = 800 x ). In each of forty-eight squares in an eyepiece graticule, an estimate was made of the numbers of quarters of a square occupied by liver cell. T h e results were converted into the percentage * Tris(hydroxymethyl)amino methane.
FREE RIBOSOMES DURING MATURATION OF RAT LIVER
35
of liver not being composed of liver cells, that is, haemopoietic cells plus vascular tissue. In experiments designed to test the homogenization technique, sediments obtained by centrifugation at 6000g for 20 min were resuspended in 0.25 M sucrose, smeared on glass slides and dried in air at room temperature. The smears were fixed overnight in Heidenheim's Susa and stained as for liver sections. RESULTS The method used for the determination of ribosome concentration in liver was adopted in order to avoid losses. Preliminary experiments showed that the preparation of a ribosomal pellet by high-speed centrifugation, and its subsequent analysis in the analytical ultracentrifuge gave much lower values for concentration than did the analyses performed directly on 6000 g supernatants. This fact is probably due to agglutination of particles in the pellets or to difficulties of complete dispersion on resuspension of the pellets (Brown & Gaston, 1962: Peterman & Pavlovec, 1963). Table 1 shows the mean sedimentation coefficients ($20, w) obtained from fourteen experiments together with the standard errors of the mean. T A B L E 1 - - S E D I M E N T A T I O N COEFFICIENTS ($2o, W) OF MAJOR RIBONUCLEASE SENSITIVE BOUNDARIES IN LIVER HOMOGENATES
Mean $2o,w values Standard error of mean
87"5 + 0"9
58"2 + 0.6
47"3 _+0"6
34"0 + 0"8
All these boundaries disappeared after incubation with crystalline ribonuclease, but a 7 S boundary which is present in all liver extracts was unaffected. Hence, the former boundaries are due to ribonucleoproteins with sedimentation coefficients close to those usually ascribed to liver ribosomes (Peterman et al., 1953a, b, 1956). Figure 1 shows the concentration of ribosomes in mg/g fresh weight of liver, at different ages in the rat. The results have been calculated per gramme of whole liver and have also been corrected to a value per gramme of liver cell using the quantitative histological data obtained from liver sections. On the same figure is plotted the percentage of the organ occupied by haemopoietic cells and vascular space. After 5 days post-partum, few haemopoietic cells were ever seen in liver sections and the small value shown on the graph after this age represents vascular tissue. It is noteworthy that the concentration of ribosomes does not follow the same time course as the fraction of liver occupied by haemopoietic cells. In smears prepared from the nuclear plus mitochondrial fraction of foetal and early post-natal livers (6000g pellet), the presence of haemopoietic cells was readily detectable. They were smaller than the isolated liver cell nuclei and showed an intense basophilic nucleus, with, in most cases, a thin rim of acidophilic cytoplasm. They appeared to be undamaged by the "standard" homogenization procedure, and preparations showing obviously damaged haemopoietic cells (free
I . T . OLIVER,W. F. C. BLUMER AND I. J. WITHAM
36
nuclei, cytoplasmic ruptures) could only be obtained by repeating the homogenization procedure once or twice more. Even in preparations from foetal liver, in which haemopoietic cells form a large proportion of the total tissue, the majority of haemopoietic ceils survived undamaged when the "standard" homogenization was carried out as many as three times. I0
8
~
livercells
~4 2
8O
0
g \
6
60
Ribosomes/g liver
\ \
'S' volue
4
40 u 20
2
0
22
16 Foetal
6 Post- natal
12 18 Age, days
H 24
88 " A
E a:
FIG. 1. Concentration of free ribosomes and haemopoietic tissue in neo-natat rat liver. Concentrations of ribosomes were determined in liver homogenates by ultracentrifugal analysis (see text for details). T h e proportions of various components distinguished by their S values are indicated on the lower figure. • - - • = per cent of liver sections composed of haemopoietic plus vascular tissue. DISCUSSION
The presence of free ribosomes in foetal liver has been previously detected by electron m i c r o s c o p y (Palade, 1955) and in regenerating rat liver b y the ultracentrif u g e studies of Peterman and her colleagues (Peterman et al., 1953b, 1956),
but as far as we are aware no quantitative estimates of their concentration have been made during liver development. Since a large fraction of the immature rat liver consists of haemopoietic cells, the possibility must be considered that some or all of the free ribosomes in liver homogenates were derived from these cells. The histological examination of
FREE RIBOSOMES DURING MATURATION OF RAT LIVER
37
"nuclear" fractions of liver homogenates indicates that haemopoietic cells are undamaged by the "standard" homogenization used in this work. Although no quantitative statement is possible on this point, the results shown in Fig. 1 make it unlikely that a significant proportion of the ribosomes were derived from nonhepatic cells, since the concentration changes in the maturing liver are not directly related to the changes measured in cytological terms. Furthermore, from the relative cytoplasmic volume of haemopoietic cells and liver cells assessed from sections, it can be calculated that even when 50 per cent of the liver is composed of haemopoietic cells, they would contribute less than 1 per cent of the final cytoplasmic extract if they were all broken by homogenization. It has been calculated from previous data on the concentration of liver RNA during growth (Oliver et al., 1962) that the ribosomes found in immature rat liver account for up to 60 per cent of the total RNA. The presence of high concentrations of such particles is of considerable interest, since a number of enzyme systems appear in the organ for the first time at about the time of birth, and increase rapidly in activity thereafter (Brown & Zuelzer, 1958; Dawkins, 1961; Ballard & Oliver, 1962, 1963). The free ribosome fraction might be expected to play an important role in the synthesis of such intracellular protein. The well-recognized role of the endoplasmic reticulum and its attached ribosomes in the synthesis of plasma proteins raises yet another question. In the young rat, the plasma protein concentration remains at a low level until about 10 days after birth, whereafter it rises rapidly (Oliver et al., 1962). However, the free ribosome concentration falls to low levels at this period, and the possibility that the free ribosomes become attached to cytomembranes to form the functional endoplasmic reticulum cannot be dismissed. (See also Burraston & Pollack, 1961.) REFERENCES BALLARDF. J. & OLIVERI. T. (1962) The appearance of fructose-l,6-diphosphatase in post-natal rat liver. Nature, Land. 195, 498--499. BALLARDF. J. & OLIVERI. T. (1963) Glycogen metabolism in embryonic chick and neonatal rat liver. Biochira. Biophys. Acta 71, 578-588. BARNUMC. P. & HUSEBYR. A. (1948) Some quantitative analyses of the particulate fractions from mouse liver cell cytoplasm. Arch. Biochem. 19, 17-23. BROWNA. K. & ZtmLZ~ W. W. (1958) Studies on the neo-natal development of the glucuronide conjugating system. J. Clin. Invest. 37, 332-340. BROWND. D. & GASTONJ. D. (1962) Biochemistry of amphibian development. I. Ribosome and protein synthesis in early development of Rana pipiens. Devel. Biol. 5, 412-434. BURRASTON J. & POLLACKJ. K. (1961) Amino acid incorporation into embryonic rat liver. Exp. Cell. Res. 25, 687-690. DAWKINSM. J. R. (1961) Changes in glucose-6-phosphatase activity in liver and kidney at birth. Nature, Land. 191, 71-73. OLIVER I. T., BALLARDF. J., SHIELDJ . & BENTLEYP. J. (1962) Liver growth in the early post-partum rat. Devel. Biol. 4, 108-116. PALADEG. (1955) A small particulate component of the cytoplasm. 3t. Biophys. Biochem. Cytol. 1, 59-68. Pn.amRMANM. L. & HAMILTONM. G. (1953a) An ultracentrifugal analysis of the macromolecular particles of normal and leukaemic mouse spleen. Cancer Res. 12, 373-378.
38
I. T. OLIVER,W. F. C. BLUMERAND I. J. WITHAM
PETERMAN M. L., MIZEN N. A. & HAMILTON M. G. (1953b) The macromolecular particles of normal and regenerating rat liver. Cancer Res. 13, 372-375. PETERMAN M. L., HAMILTON M. G. & MIZEN N. A. (1954) Electrophoretic analysis of the macromolecular nucleoprotein particles of mammalian cytoplasm. Cancer Res. 14, 360366. PETERMAN M. L., MIZEN N. A. & HAMILTON M. G. (1956) The cytoplasmic nucleoproteins of azo dye-induced rat liver tumours. Cancer Res. 16, 620-627. PETERMAN M. L. & PAVLOVECA. (1963) Ribonucleoprotein from a rat tumour, the Jensen Sarcoma. III. Ribosomes purified without deoxycholate but with bentonite as RNA-ase inhibitor. J. Biol. Chem. 238, 318-323.
FREE RIBOSOMES DURING MATURATION OF RAT LIVER I. T . O L I V E R , W. F. C. B L U M E R * and I. J. W I T H A M Departments of Biochemistry and *Anatomy, University of Western Australia, Nedlands, Western Australia (Received 28 April 1963)
Abstract--1. A high concentration of free ribosomes has been found in homogenates of the liver of foetal and post-natal rats. The concentration is about 8 mg/g of liver cells until about 5 days after birth when it falls rapidly to the adult level of about 2 mg/g of liver cells. 2. In the 17-day foetal rat, 60 per cent of the liver is composed of haemopoietic and vascular tissue, but the percentage falls in an almost linear fashion to about 10 per cent 5 days after birth. After this time few haemopoietic cells can be seen in liver sections. 3. Evidence is presented for the view that the free ribosome fraction in these liver homogenates is almost exclusively derived from liver cells. INTRODUCTION IN A previous paper (Oliver et al., 1952) the concentrations of liver D N A t and RNA:~ during growth of the rat in the immediate postpartum period were shown to reach maximum levels at 5 days and 1S days after birth respectively. Further investigations of the cytoplasmic form of R N A in neo-natal rat liver have now shown that a large fraction of the total R N A is initially in the form of free ribosomes. T h e existence of such particles in liver and other tissues has been described before (see Barnum & Huseby, 1948; Palade, 1955; Peterman et al., 1953a, b, 1954, 1956) but in the present work quantitative data have been obtained on the concentration of free ribosomes (which are not attached to cytomembranes) during development of the rat liver through the late foetal and post-natal period. In addition, the amount of haemopoietic tissue present in the neonatal liver has been assessed by histological methods in order to ascertain the cytological origin of the free ribosomes. MATERIALS AND METHODS Measurement o f free ribosome concentration
T h e rats used in this study were of the Wistar albino strain, species Rattus All operations were performed in the cold-room at 0-4°C. Foetal animals were obtained from the uterus of the mother which was sacrificed by a blow on the head. T h e foetuses were washed in ice-cold distilled water, blotted on filter paper and weighed. T h e livers were removed, rapidly weighed and dropped norvegicus.
t Deoxyribonucleic acid. ~: Ribonucleic acid. 33
I . T . OLIVER,W. F. C. BLUMERAND I. J. WITHAM
34
into the ice-cold medium used for homogenization. Infant animals were sacrificed by decapitation and the livers similarly treated. T h e foetal age was determined from the time of appearance of a vaginal plug in mated females. T h e livers from seven to ten animals were pooled and homogenized in 3 vol of an ice-cold solution of sucrose (0-31 M) containing 0-02 M Tris*-HC1 buffer p H 7.4. T h e conditions of homogenization were standardized as far as possible by using Perspex coaxial homogenizers machined with standard clearance between pestle and wall and driven at the same speed, for the same number of up and down strokes. All homogenates were centrifuged at 6000 g for 20 rain at 0°C to remove nuclei and mitochondria, and the supernatants removed for ultracentrifugal analysis. Analyses were made in the Beckman Spinco Model E ultracentrifuge at 5-10°C in 12 m m aluminium centrepiece cells (4 ° sector), at 42,040 rev/min. T h e instrument was equipped with the R T I C system and phase-plate Schlieren optics. Ilford R40 panchromatic plates were used in photography. T h e microsome fraction sedimented during rotor acceleration and the ribosome boundaries appeared after the rotor reached 42,040 rev/min. T h e plates were analysed for sedimentation coefficients (S) by projection on a calibrated screen, and areas under the peaks were measured by summation of vertical measurements on tracings obtained from a photographic enlarger. In some experiments, a 1.0 ml sample of the 6000 g supernatant was incubated 24 hr at 0°C with 1 mg crystalline bovine pancreatic ribonuclease (Sigma Chemical Co., St. Louis, Missouri, U.S.A.) and then compared with duplicate samples incubated without ribonuclease. All the sedimenting boundaries in the range 30-100 S disappeared after ribonuclease treatment, but the control samples were unchanged. Area measurements were converted to concentration using a value of 1"87 x 10 3 for the specific refractive increment of rat liver ribonucleoprotein (Peterman & Pavlovec, 1963). T h e concentrations were converted to mg ribosomes/g of fresh liver. T h e addition of MgCI~ to the homogenization medium over the concentration range 10 .5 M - 1 0 -3 M did not significantly alter the yield or the relative proportions of the ribosome species. It was therefore omitted from routine runs.
Histology Liver obtained from two animals at each age as described above was sliced with a razor blade and fixed in either buffered 10% formalin or Zenkers fluid. T h e slices were dehydrated in 2-ethoxyethanol, cleared in toluene, embedded in paraffin and sectioned at 10/x. T h e sections were stained in Harris' haematoxylin and chromotrope 2R. T e n areas selected at random from several sections taken at each age were examined under oil immersion (magnification = 800 x ). In each of forty-eight squares in an eyepiece graticule, an estimate was made of the numbers of quarters of a square occupied by liver cell. T h e results were converted into the percentage * Tris(hydroxymethyl)amino methane.
FREE RIBOSOMES DURING MATURATION OF RAT LIVER
35
of liver not being composed of liver cells, that is, haemopoietic cells plus vascular tissue. In experiments designed to test the homogenization technique, sediments obtained by centrifugation at 6000g for 20 min were resuspended in 0.25 M sucrose, smeared on glass slides and dried in air at room temperature. The smears were fixed overnight in Heidenheim's Susa and stained as for liver sections. RESULTS The method used for the determination of ribosome concentration in liver was adopted in order to avoid losses. Preliminary experiments showed that the preparation of a ribosomal pellet by high-speed centrifugation, and its subsequent analysis in the analytical ultracentrifuge gave much lower values for concentration than did the analyses performed directly on 6000 g supernatants. This fact is probably due to agglutination of particles in the pellets or to difficulties of complete dispersion on resuspension of the pellets (Brown & Gaston, 1962: Peterman & Pavlovec, 1963). Table 1 shows the mean sedimentation coefficients ($20, w) obtained from fourteen experiments together with the standard errors of the mean. T A B L E 1 - - S E D I M E N T A T I O N COEFFICIENTS ($2o, W) OF MAJOR RIBONUCLEASE SENSITIVE BOUNDARIES IN LIVER HOMOGENATES
Mean $2o,w values Standard error of mean
87"5 + 0"9
58"2 + 0.6
47"3 _+0"6
34"0 + 0"8
All these boundaries disappeared after incubation with crystalline ribonuclease, but a 7 S boundary which is present in all liver extracts was unaffected. Hence, the former boundaries are due to ribonucleoproteins with sedimentation coefficients close to those usually ascribed to liver ribosomes (Peterman et al., 1953a, b, 1956). Figure 1 shows the concentration of ribosomes in mg/g fresh weight of liver, at different ages in the rat. The results have been calculated per gramme of whole liver and have also been corrected to a value per gramme of liver cell using the quantitative histological data obtained from liver sections. On the same figure is plotted the percentage of the organ occupied by haemopoietic cells and vascular space. After 5 days post-partum, few haemopoietic cells were ever seen in liver sections and the small value shown on the graph after this age represents vascular tissue. It is noteworthy that the concentration of ribosomes does not follow the same time course as the fraction of liver occupied by haemopoietic cells. In smears prepared from the nuclear plus mitochondrial fraction of foetal and early post-natal livers (6000g pellet), the presence of haemopoietic cells was readily detectable. They were smaller than the isolated liver cell nuclei and showed an intense basophilic nucleus, with, in most cases, a thin rim of acidophilic cytoplasm. They appeared to be undamaged by the "standard" homogenization procedure, and preparations showing obviously damaged haemopoietic cells (free
I . T . OLIVER,W. F. C. BLUMER AND I. J. WITHAM
36
nuclei, cytoplasmic ruptures) could only be obtained by repeating the homogenization procedure once or twice more. Even in preparations from foetal liver, in which haemopoietic cells form a large proportion of the total tissue, the majority of haemopoietic ceils survived undamaged when the "standard" homogenization was carried out as many as three times. I0
8
~
livercells
~4 2
8O
0
g \
6
60
Ribosomes/g liver
\ \
'S' volue
4
40 u 20
2
0
22
16 Foetal
6 Post- natal
12 18 Age, days
H 24
88 " A
E a:
FIG. 1. Concentration of free ribosomes and haemopoietic tissue in neo-natat rat liver. Concentrations of ribosomes were determined in liver homogenates by ultracentrifugal analysis (see text for details). T h e proportions of various components distinguished by their S values are indicated on the lower figure. • - - • = per cent of liver sections composed of haemopoietic plus vascular tissue. DISCUSSION
The presence of free ribosomes in foetal liver has been previously detected by electron m i c r o s c o p y (Palade, 1955) and in regenerating rat liver b y the ultracentrif u g e studies of Peterman and her colleagues (Peterman et al., 1953b, 1956),
but as far as we are aware no quantitative estimates of their concentration have been made during liver development. Since a large fraction of the immature rat liver consists of haemopoietic cells, the possibility must be considered that some or all of the free ribosomes in liver homogenates were derived from these cells. The histological examination of
FREE RIBOSOMES DURING MATURATION OF RAT LIVER
37
"nuclear" fractions of liver homogenates indicates that haemopoietic cells are undamaged by the "standard" homogenization used in this work. Although no quantitative statement is possible on this point, the results shown in Fig. 1 make it unlikely that a significant proportion of the ribosomes were derived from nonhepatic cells, since the concentration changes in the maturing liver are not directly related to the changes measured in cytological terms. Furthermore, from the relative cytoplasmic volume of haemopoietic cells and liver cells assessed from sections, it can be calculated that even when 50 per cent of the liver is composed of haemopoietic cells, they would contribute less than 1 per cent of the final cytoplasmic extract if they were all broken by homogenization. It has been calculated from previous data on the concentration of liver RNA during growth (Oliver et al., 1962) that the ribosomes found in immature rat liver account for up to 60 per cent of the total RNA. The presence of high concentrations of such particles is of considerable interest, since a number of enzyme systems appear in the organ for the first time at about the time of birth, and increase rapidly in activity thereafter (Brown & Zuelzer, 1958; Dawkins, 1961; Ballard & Oliver, 1962, 1963). The free ribosome fraction might be expected to play an important role in the synthesis of such intracellular protein. The well-recognized role of the endoplasmic reticulum and its attached ribosomes in the synthesis of plasma proteins raises yet another question. In the young rat, the plasma protein concentration remains at a low level until about 10 days after birth, whereafter it rises rapidly (Oliver et al., 1962). However, the free ribosome concentration falls to low levels at this period, and the possibility that the free ribosomes become attached to cytomembranes to form the functional endoplasmic reticulum cannot be dismissed. (See also Burraston & Pollack, 1961.) REFERENCES BALLARDF. J. & OLIVERI. T. (1962) The appearance of fructose-l,6-diphosphatase in post-natal rat liver. Nature, Land. 195, 498--499. BALLARDF. J. & OLIVERI. T. (1963) Glycogen metabolism in embryonic chick and neonatal rat liver. Biochira. Biophys. Acta 71, 578-588. BARNUMC. P. & HUSEBYR. A. (1948) Some quantitative analyses of the particulate fractions from mouse liver cell cytoplasm. Arch. Biochem. 19, 17-23. BROWNA. K. & ZtmLZ~ W. W. (1958) Studies on the neo-natal development of the glucuronide conjugating system. J. Clin. Invest. 37, 332-340. BROWND. D. & GASTONJ. D. (1962) Biochemistry of amphibian development. I. Ribosome and protein synthesis in early development of Rana pipiens. Devel. Biol. 5, 412-434. BURRASTON J. & POLLACKJ. K. (1961) Amino acid incorporation into embryonic rat liver. Exp. Cell. Res. 25, 687-690. DAWKINSM. J. R. (1961) Changes in glucose-6-phosphatase activity in liver and kidney at birth. Nature, Land. 191, 71-73. OLIVER I. T., BALLARDF. J., SHIELDJ . & BENTLEYP. J. (1962) Liver growth in the early post-partum rat. Devel. Biol. 4, 108-116. PALADEG. (1955) A small particulate component of the cytoplasm. 3t. Biophys. Biochem. Cytol. 1, 59-68. Pn.amRMANM. L. & HAMILTONM. G. (1953a) An ultracentrifugal analysis of the macromolecular particles of normal and leukaemic mouse spleen. Cancer Res. 12, 373-378.
38
I. T. OLIVER,W. F. C. BLUMERAND I. J. WITHAM
PETERMAN M. L., MIZEN N. A. & HAMILTON M. G. (1953b) The macromolecular particles of normal and regenerating rat liver. Cancer Res. 13, 372-375. PETERMAN M. L., HAMILTON M. G. & MIZEN N. A. (1954) Electrophoretic analysis of the macromolecular nucleoprotein particles of mammalian cytoplasm. Cancer Res. 14, 360366. PETERMAN M. L., MIZEN N. A. & HAMILTON M. G. (1956) The cytoplasmic nucleoproteins of azo dye-induced rat liver tumours. Cancer Res. 16, 620-627. PETERMAN M. L. & PAVLOVECA. (1963) Ribonucleoprotein from a rat tumour, the Jensen Sarcoma. III. Ribosomes purified without deoxycholate but with bentonite as RNA-ase inhibitor. J. Biol. Chem. 238, 318-323.