Kinetics of amino acid uptake and protein synthesis in neurospora

BIOCHIMICA ET BIOPHYSICA ACTA

423

K I N E T I C S O F AMINO ACID U P T A K E AND P R O T E I N S Y N T H E S I S IN N E U R O S P O R A M. ZALOKAR*

Microbiology Department, Yale University, New Haven, Conn. (U.S.A.) (Received July i8th, 196o)

SUMMARY

The incorporation of L-[14Clproline into Neurospora mycelium was studied at short time intervals. Proline was taken into the cell immediately at a linear rate and reached a concentration exceeding the outside concentration. The incorporation rate of proline into proteins (trichloroaeetic acid insoluble material) increased gradually during the first minute and became linear thereafter. The initial lag could have been due either to the presence of an intermediary pool of proline in the cell or to the time needed to synthesize a molecule of protein. The free proline pool of mycelia was found to be greater than the pool necessary to explain the lag in protein synthesis. The pool size which would explain the lag was calculated to be between 0.05 and 0.3 mg/g dry weight. When the rate of uptake of proline into the cell was varied by changing the external concentration or temperature, the incorporation into proteins followed a curve which was consistent with the presence of a pool of the calculated size. These experiments indicated that the absorbed proline enters the intermediary or internal pool and is taken from it for protein synthesis. Pretreatment of the mycelia with non-radioactive proline showed that the absorbed proline, after mixing with the internal pool, is diverted into a reservoir, the expandable pool. The presence of an intermediate pool accounts entirely for the lag in the incorporation of proline into proteins, so that it could be inferred that the time necessary to make a molecule of protein (polypeptide chain) is negligible, of the order of magnitude of a few seconds or less.

INTRODUCTION

For a precise understanding of protein synthesis in living cells, it is important to know the rates of uptake of amino acid precursors into the cell, the size and disposition of internal pools and the time needed to produce new protein molecules. Recently, several studies concerned with these problems have been reported. In bacteria, it has been shown that added amino acids are immediately taken into the cell and eventually reach a concentration several hundred times higher than the external concentration 1-s. Inside the cell the amino acids form a pool, out of which they are removed for protein synthesis 2. Exogenous amino acids are used for protein synthesis in preference to the endogenously formed amino acids and often suppress the production of the latter 3,4. * Present address: D e p a r t m e n t of Biology, University of California, La Jolla, Calif. (U.S.A.).

Biochim. Biophys. Acta, 46 (I96I) 423-432

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In Saccharomvces, the exogenous amino acids are used for protein synthesis without passing through an established pool 5. In Candida, COWIE AND McCLURE~ demonstrated the presence of two pools, an expandable pool where exogenous amino acids enter first and are accumulated, and an "internal" pool from which the precursors are taken directly into protein-synthesizing sites. There is a free exchange between the external precursors and the expandable pool, while the internal pool takes up amino acids only at the rate of their use in protein synthesis. While these studies did not include calculations of the time necessary for the biosynthesis of the protein molecule, it can be inferred from the data presented that this time was negligible. In a later work, McQuILLEN AND ROBERTS7 reported that this time was of the order of 5 sec. The study of the time factor in protein biosynthesis was approached more directly in animal tissuesS, 9. In liver, it was reported that several minutes were needed to synthesize a molecule of the protein ferritin 9. This material had the advantage that measurements could be made for a specific protein, ferritin, isolated in crystalline form, but exact measurements of precursor uptake rates and of amino acid pools were not possible. In this paper, investigations on kinetics of amino acid incorporation in Neurospora are reported. Techniques have been developed for measuring precursor uptake into the cell and into proteins at very short time intervals. From experimental data and the information obtained on internal pools, the time necessary to assemble a molecule of protein could be calculated. MATERIALS AND METHODS

Wild type Neurospora crassa, strain 5297a was used in all experiments. Mycelium was grown on 25 ml of Fries minimal medium in i25-ml Erlenmeyer flasks at 3 °0 for two days. A drop of Tween 80 was added to the medium to prevent conidiation 1°. The resulting mycelial pad was filtered over a Buchner funnel with gentle suction and washed with fresh medium. The pad was then placed on a filter paper, and discs cut from it with a cork-borer of 13 mm diameter. These mycelial discs were put on a moistened filter paper (Whatman No. I, 7 cm diameter, plus 0.6 ml medium) in a Petri dish and incubated for i h, to allow the mycelium to recover from the shock of filtering and cutting. These mycelia were in the "cube-root" growth phase 11 which corresponds to the "logarithmic" phase of unicellular organisms. Mycelial discs can be handled like pieces of filter paper, and this makes it possible to use extremely short feeding times. Drops of medium containing radioactive precursor (o.o4 ml each) were arranged on a Petri dish coated with silicon grease. When mycelial discs were put into these drops, they became soaked with the medium immediately. At appropriate times, the discs were transferred to a Buchner filter to remove excess medium. When total uptake of amino acids into the cell was studied, the discs were washed with a continuous stream of ice-cold nutrient medium, containing non-radioactive precursor, for 2o to 3o sec. This, it was found, removed all extraneous tracer, but did not remove any tracer which was already taken into the cell. The discs were then mounted directly onto planchettes; they were covered with filter paper and pressed onto the planchettes with a rubber stopper. After being dried on a hot plate at IOO°, the discs adhered to the planchettes and were ready for the counting of radioactivity. Biochim. Biophys. Acta, 46 (1961) 423 432

AMINO ACID UPTAKE IN

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425

When the uptake into proteins was measured, the discs were filtered as before, then plunged into hot 5 % trichloroacetic acid containing non-radioactive precursor. The trichloroacetic acid was changed three times, at room temperature, then the discs were washed twice in 95 % alcohol and once in acetone. After this, they were dried and mounted on planchettes. In order to insure good adherence, a drop of I °/o gelatine fixed in formol was added and the excess liquid removed by compressing the disc onto the planchette, using filter paper and rubber stopper, as above. Uniformily labeled L-I14C]proline (Schwartz Co.) was used as a tracer amino acid, since it was shown b y VOGEL12 (personal communication) that the external proline is used preferentially to the endogenous and since proline enters specifically into proteins without being considerably otherwise metabolized. Proline was purified b y paper chromatography before use and dissolved in Fries minimal medium at a concentration of 400 t~g/ml, unless otherwise specified. This rather high concentration was chosen to minimize tile effect of internal pools and supplies. Relatively low specific activity (1. 3/~C/mg) was used in most experiments except for very short time experiments where ten times higher specific activity (50 t~C/mg diluted four times with equal concentrations of "cold" proline) was used. Radioactivity was measured in mycelial discs with a windowless flow counter. The unextracted discs weighed around 2 mg, and measured I cm 2, giving about 25 % self-absorption. The weight and the self-absorption varied only slightly in all preparations, not affecting the relative values of results significantly. After extraction, the discs lost about one-half of their weight, thus decreasing the self-absorption to 12 ~o. The protein content of discs of 2 mg dry wt. was 0.7 mg. The precursor incorporation is expressed in tables and graphs as mg/g dry wt. of mycelium. To convert counts to mg of precursor, a known amount of the precursor was added to non-radioactive mounted mycelial discs and the measured radioactivity used for necessary calculation. Theoretical considerations The kinetics of the uptake of radioactive material into the cell has been treated mathematically by several authors TM. Calculations have also been developed to estimate the time necessary to synthesize a molecule of protein 8, 9. Similar theoretical considerations and calculations are being used in the present study. If a precursor (amino acid) of specific activity a is fed to a cell in a steady state, it will be absorbed and incorporated into proteins at a constant rate. If the precursor is used directly for protein formation, and if no time is needed to assemble a protein molecule, the total radioactivity (y) of newly formed proteins, which can be measured, would increase linearly from the beginning, according to the formula y ~ abt, where b is the rate of incorporation oi the precursor into protein and t is time. If time is needed to assemble a protein molecule, the radioactivity of newly formed proteins will increase only gradually to a constant rate, according to the following consideration: The cell contains polypeptide chains in different states of completion; new residues are incorporated at regular time intervals (At), and upon the addition of labeled precursor, they are all derived from it. At every time interval, another polypeptide chain is completed, containing an increasing proportion of radioactive residues. Finally, all the new polypeptide chains will be built entirely from the radioactive precursor. This will indicate the time necessary to make a molecule of protein (x). The specific activity of the protein will remain constant from this point Biochim. Biophys. Acta, 46 (1961) 423-432

426

M. ZALOKAR

on. It is immaterial whether amino acids are assembled linearly or some other way. In a population of polypeptides (incomplete proteins) of all sizes, residues are incorporated at all times, so that the interval At can be taken as very small and the increase of total activity of new proteins can be expressed with the formulas: y--

t2 ab-2X

fort = o t o x , a n d y

abx = abt ....

2

fort ~> x

A similar increase of specific activity of new proteins would occur if there is an internal pool with which the extraneous precursor mixes before being used for protein synthesis. In this case, assuming that no time is necessary to assemble a protein molecule, the specific activity of new proteins will be proportional to the specific activity of the pool. The latter will increase with the uptake of extraneous tracer. To calculate the activity of the pool, two extreme conditions can be considered. In the first case, the absorbed tracer is simply added to the pool, thus increasing its size. The change in pool specific activity can be expressed b y the formula: 3'

kt a - p +kt

where y is specific activity of the pool; a, specific activity of the precursor; k, rate of absorption of the precursor; p, initial pool size ; t, time. In the second case the pool size remains constant, i . e . , as much material is taken out (after mixing), as there was added. The specific activity of the pool will increase in this case faster, according to the formula:

In reality, an intermediate situation probably exists, with slight increase of pool size due to the feeding of a precursor, which would result in a state somewhere between the two conditions formulated. I f proteins are made by using precursors from these pools, their tota 1radioactivity will increase according to the integrals of the preceding formulas, multiplied by the rate of protein synthesis. Curves calculated with the preceding formulas are shown in Fig. i. If we know the rate of uptake of a precursor and the rates of protein formation, we can calculate theoretical curves for different sizes of the pool. If the pool size is known, the theoretical curves should coincide with the measured curve, unless there is an additional lag due to the time necessary to form a molecule of protein. The third factor determining the specific activity of newly formed proteins is the internal supply of the precursor. If this supply remains unchanged and constant after feeding the precursor, then the effect on pools and newly formed proteins will be a constant dilution of the absorbed material, resulting in a constant dilution of specific radioactivity of the precursor. ROBERTS et a l . 4 showed that the external supply of amino acid is used preferentially to the internal supply and that it can suppress the internal production of the precursor. No information is available on how fast after feeding of a precursor this suppression occurs. If there is a short period during which the internal supply diminishes, the dilution of specific radioactivity of the precursor would diminish, and as a result, the specific activity of proteins formed would increase. The total activity of new proteins will then increase along a curve Biochim. Biophys.~4cla, 4 6 ( i 0 6 t ) 423 43-"

AMINO ACID UPTAKE IZ~

Neurospora

427

similar to the ones calculated in previous cases, but steeper at the beginning. Since the internal supply in a steady state can not be greater than the amount used in protein synthesis and since the outside precursor is absorbed at a much higher rate, this possibility can be neglected in the evaluation of results. t y,(~bt //

1.0

//

k

o7

0.8

D

cz

02

Q8

kt p kt

.>

t~ (16

ti

"~ 0,5

.2

~ 0.4

b

•o 0.4

o.3

O.2 1

2

3

4

'0.2

Time Fig. I. Rise of specific a c t i v i t y of a p r e c u r s o r pool a f t e r feeding a r a d i o a c t i v e p r e c u r s o r of a specific a c t i v i t y I. T h e u p t a k e a t t h e t i m e i is e q u a l to t h e size of t h e pool. A, calculated for c o n s t a n t size of t h e pool; B, c a l c u l a t e d for e x p a n d i n g pool; C, t o t a l labeling of p r o t e i n s (arbitrary rate) in t h e a b s e n c e of a pool; D, t o t a l labeling of p r o t e i n s if a c o n s t a n t size pool is present.

01

2

6 8 10 12 Minutes Fig. 2. Total uptake of L-~14Clproline into Neurospora, a n d i n c o r p o r a t i o n into cell p r o t e i n s at

3 o°.

4

P r e c u r s o r c o n c e n t r a t i o n 4oo/~g/ml, specific a c t i v i t y I. 3 /~C/mg.

RESULTS

When Neurosporamycelium was put into the medium containing radioactive proline (1.3 FC/mg; 40o/~g/ml), the uptake of proline into the cell started immediately and continued at a constant rate for the first 16 min (Fig. 2). After this, the uptake rate slowed down and after 32 min became parallel to the rate of incorporation into proteins. After I h, the uptake rate declined further because of progressive exhaustion of the medium, which consisted of only 0.05 ml in these experiments. The total amount of L-proline absorbed in 32 min amounted to 4 mg/g dry wt., of which 2.4 mg/g were not bound to proteins. Since I g dry wt. of the mycelium corresponds approx, to 6 g fresh weight, the absorbed proline equaled or exceeded the external concentration. The incorporation of radioactivity into trichloroacetic acid insoluble material increased gradually to a constant rate (Fig. 2) which remained so for the next 32 min. The use of L-proline of higher specific activity (12. 5 FC/mg) allowed a closer study of the initial lag, with points obtained at short intervals. The uptake rate approached a constant value in I min (Fig. 3). The straight part of the uptake curve was considered to express the rate of precursor incorporation into proteins and was used for the calculations of the increase of radioactivity in proteins in the presence of precursor pools of different sizes. In order to affirm the protein nature of the material whose radioactivity was Biochim. Biophys. Acta, 46 (1961) 423-432

428

M. ZALOKAR

measured, myce]ia were extracted with I N KOH, and the proteins reprecipitated with trichloroacetic acid. The radioactivity of these proteins was at all times a constant proportion of values obtained before extraction, indicating a proportional loss of 20 o/ /0" If the precursors were incorporated into proteins directly, without passing through a pool, these data would indicate that the m a x i m u m time necessary to assemble a molecule of protein is of the order of i min. A pool of proline could, however, be demonstrated in Neurospora by direct assay of cold perchloric acid soluble extracts of the mycelium. Proline was measured b y bio-assay with proline-less mutant of E. coli*. These assays were made on hyphae, in the logarithmic growth phase (io h old), on mycelia, grown in the same way as the ones used for feeding experiments (48 h old), and on parts of four day old mycelia which could be split into "young" and "old" halves 14 (Table I). A substantial pool was found in all these mycelia, indicating that even if there were age inequalities in the hyphae of 48 h old mycelium, these hyphae would contain a pool which could be expected to remain within the limits measured for young and old hyphae. If the value for the proline pool of 48-h mycelium (0.86 mg/g dry wt.) and the information about proline uptake into the cell are used for calculation of the increase of radioactivity of proteins, the theoretical curve would show that proteins shou]d get labeled much more slowly than observed. The intermediate pool must then have been smaller than the one measured b y extracting the mycelium. The best approach to the experimental curve was obtained by a theoretical curve calculated for a pool of 0.05 mg/g (Fig. 3). Pool size varied from experiment to experiment; the highest

1

2 Minutes

3

4

Fig. 3. I n c o r p o r a t i o n of L-[14C]proline (400 /~g/ml, 12, 5 #C/mg) into Neurospora proteins at 3 ° ° . Theoretical curve for the incorporation in the presence of an intermediate pool consisting of total free proline p r e s e n t in the cell was calculated from the total u p t a k e rate and protein f o r m a t i o n rate. The latter was inferred f r o m the s t r a i g h t u p p e r p a r t of the experimental curve. * The a u t h o r is indebted to Dr. T. YURA for these analyses.

Biochim. Biophys..4cta, 46 (1961) 423-432

AMINO ACID UPTAKE IN

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429

TABLE I L-PROLINE POOL OF Neurospora MYCELIUM E x t r a c t i o n w i t h cold o.i N perchloric acid a n d m e a s u r e d b y bio-assay, u s i n g proline-less E. coli (T. Y U R A ) .

I o - h old h y p h a e 48-h old m y c e l i u m 4 - d a y s old m y c e l i u m upper layer lower l a y e r

0.45 m g / g d r y wt. 0.86 m g / g d r y wt. 0.38 m g / g d r y wt. i .45 m g / g d r y wt.

value obtained was o.3 mg/g (see Fig. 5). This introduced an uncertainty about the pool; if the intermediate pool was smaller than the measured pool of free proline in the cell, it could as wed be non-existent. Other experiments were needed to determine the presence and the size of an intermediate pool. This could be done by varying the uptake rate of the precursor, without changing its specific activity. If there was no intermediate pool, variation of the uptake rate should have no effect on incorporation into proteins, as long as the external supply remained in excess. If there was a pool, a decrease of uptake rate would result in a slower increase in the specific activity of the pool and, therefore, in a slower increase of total activity of new proteins. The uptake rate could be varied by changing the concentration of the precursor in the medium. Uptake into the cell and into proteins was measured for four different concentrations of exogenous precursor (Fig. 4). Theoretical curves for the increase in protein labeling at the different uptake rates were calculated by assuming the pool size of 0.25 mg/g. It can be seen in Fig. 5 that the measured curves agree reasonably

00/ug/rnl 1.o

.~0.8

a20~

/

[

/

o.18t

200

06 0.2

0.16 0.14~

/

~ o.1o

E 008 Minutes Fig. 4. Total uptake at different external 006 concentrations of L- F]4C]proline (I.3 #g/rag). 004 Fig. 5. - - , I n c o r p o r a t i o n of L- [x4C] proline (I .3/~C/mg) into p r o t e i n s a t different e x t e r n a l concentrations; , theoretical c u r v e s c a l c u l a t e d f r o m t o t a l u p t a k e rate, a s s u m i n g a pool of a size of 0.25 m g / g d r y wt.

/

z/

0.02 2

4 Minutes

6

8

Biochim. Biophys. Acta, 46 (1961) 423-432

43 °

M. ZALOKAR

well with the theoretical ones, thus indicating the presence of an intermediate pool of the assumed order of magnitude. The uptake rate of precursor could be varied also with temperature. Since the rate of protein synthesis changes with temperature also, the calculations had to take this into account. At 20 °, the rate of uptake into the cell was about 3 times slower than at 3 °0 . The rate of formation of proteins decreased by about one-half, and calculations show that the initial slow labeling of proteins could be explained entirely by the slower increase of the labeling in the pool (Fig. 6). Since the measured amounts of free proline in the mycelium exceeded that calculated for the intermediate pool, there must be two pools of proline in the cell. Is the precursor taken into both? If non-radioactive proline is fed to the mycelium, it will be taken into the pool and the amount absorbed can be determined from the TABLE I[ L-[14CIPROLINE UPTAKE BY UPPER AND LOWER LAYERS OF A THREE DAY OLD MYCELIUM Mycelium was fed L-~14C]proline (I .3/zC/mg) for 2 min, washed with distilled w a t e r and m o u n t e d . Counts/rain

Split mycelium

I

Split mycelium 2

" / /

Mounted Mounted / Upper Lower Upper Lower

upright upside-down half half half half

Dry wt.

398 246 41I 20 449 29

]"

5.0 5.0 3-3 3.o 2. 7 3.4

/

(,

(x4

c~

mg mg mg mg mg mg

/

? d

/

// / /j / Q1

/

0.1

2 Minutes

Fig. 6. Total u p t a k e of L- [1~C]proline (400/~g/ml, 1.3 #C/mg) and incorporation into proteins at 3 °o a n d at 2o °. The curve for 2o °, agreeing closely w i t h experimental points, is calculated b y a s s u m i n g a pool of a size of o. 25 m g / g d r y wt., as inferred f r o m the experimental curve at 3 o°.

4

6

8 Minutes

10

12

14

16

Fig. 7. Incorporation of L-~14C]proline 0-3 /~C/mg, 400 #g/ml) into proteins after p r e v i o u s feeding of non-radioactive L-proline for o,4, and 8 min. Theoretical curves for 4 and 8 min were calculated for the case where all a b s o r b e d L-proline remained in t h e pool.

Bivchim. Biophys. Acta, 46 0961) 423 432

AMINO ACID UPTAKE IN Neurospora

431

knowledge obtained by feeding equal concentrations of radioactive precursor. If a radioactive precursor is added subsequently, it will be diluted by the established pool and the proteins should become labeled more slowly than in the case where radioactive precursor was added directly. Mycelium was fed 400 t~g/ml of "cold" proline for 4 and 8 min, washed with minimal medium for 30 sec, and then fed radioactive proline, and fixed at short intervals. The resulting increase of radioactivity in proteins was considerably faster than could be calculated under the assumption that the "cold" proline taken up was present in the intermediate pool (Fig. 7)- Proline must have been shifted into another pool, the "expandable" pool. The apparent existence of twc pools m a y be due to the fact that the experimental mycelium is not composed of a uniform cell population. The measured proline uptake could have happened in old cells with low protein formation activity, while the actively protein producing hyphae do not accumulate precursor. In a 3 day old mycelium, the young cells are at the surface, the old ones at the bottom of the culture and can be separated by splitting the mycelial pad 13. When such a mycelium was fed radioactive proline, the radioactivity was higher when the upper surface was turned towards the counter than when the lower one was (Table II). As the self-absorption of these preparations, weighing around 5 mg/cm2, was over 50 %, it was obvious that the upper face must have taken much more precursor than the lower one. In split mycelia, the upper half absorbed about 20 times as much radioactivity as the lower half. Most of the precursor was therefore taken in by young cells, actively synthetizing protein, and the two pools must have been established in the same cell. CONCLUSION When L-[14Clproline was added to the medium, Neurospora mycelium took it up immediately. The uptake rate was proportional to the outside concentration and was temperature dependent. The uptake therefore could not be a passive diffusion, particularly since the inside concentration eventually exceeded the outside one. The precursor once taken in, was not freely exchangeable with the subsequently added exogenous precursor. The knowledge of the total uptake rate into the cell and into proteins made it possible to demonstrate the presence of at least two pools of proline in the cell. The intermediate pool corresponds to the "internal" pool of COWlE AND McCLURE 6, the other pool to their "expandable" pool. Newly added precursor passes through the "internal" pool into the "expandable" pool, where it is accumulated. Since feeding non-radioactive precursors before adding tracer delayed labeling of proteins to some extent, the internal pool must have been also slightly increased, although most of the precursor passed quickly into the "expandable" pool. Amino acids of the internal pool are used for protein formation. I t is interesting to speculate about the intracellular sites of these two pools. The speculation can be aided by the knowledge of the fine structure of Neurospora cells obtained from electron microscopy 15 (unpublished observation of the author). The amino acid enters the cell through the cell wall and cytoplasmic membrane, probably directly into the water c o m p a r t m e n t of the liquid cytoplasm. There it is directly available to ribosomes, for protein synthesis. This must constitute the internal pool. The expandable pool consists either of the adsorption of amino acid into some

Biochim. Biophys. Acta, 46 (i961) 423-432

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M. ZALOKAR

cell components, or of their storage in separate liquid compartments. Two structures of the cytoplasm could serve as a possible reservoir. First are the submicroscopic vesicles, "the endoplasmic reticulum", which constitute about one-fourth of the volume of the protoplast and which move into the supernatant "enchy]ema" layer when living cells are centrifuged 16. Second are vacuoles, which, especially in older cells, occupy the major part of the cell volume. Since it was shown that old vacuolated cells are less active in amino acid uptake than young ones, the second possibility seems to be less probable. The knowledge of uptake rates and of pool sizes made it possible to estimate the time necessary to assemble a molecule of protein in NeurosporaIv. All the delay in the increase of radioactivity in newly synthesized proteins could be explained by the presence of pools, and it could be concluded that the formation time of a protein molecule is negligible, of the order of few seconds or less. This time did not increase measurably b y lowering the temperature at 20 ~. The estimation refers to an average of all cell proteins, although some m a y take slightly longer, some shorter times. Under protein we understand hot trichloroacetic acid insoluble, proline containing, material which m a y include incomplete proteins in the form of polypeptide chains long enough to be insoluble. It would be naturally desirable to supplement this work with the extraction of a crystalline protein, which, in Neurospora would be a difficult task. We can, therefore, not reject on the basis of our experiments the estimates published for the time necessary to produce a molecule of ferritin 9, or for hemoglobin is, both counted in minutes. It m a y be possible that a complete complex protein requires more time for its formation, but the difference cannot be due to the time necessary to assemble amino acids into a polypeptide chain, since the time required by bacteria 7, yeastS, 6 and Neurospora was shown to be very short. ACKNOWLEDGEMENT

This research was supported by a grant from the National Institutes of Health.

REFERENCES E, F. GALE, J. Gen. Microbiol., I (1947) 53. 2 R. J. BRITTEN, R. B. ROBERTS AND E. F. FRENCH, Proc. Natl. Acad. Sei. U.S., 41 (1955) 863. 3 G. N. COHEN AND H. V. RICKENBERG, Ann. Inst. Pasteur, 91 (1956) 693. 4 1{. B. ROBERTS, P. n . ABELSON, D. B. COWIE, E. T. BOLTON AND R. J. BRITTEN, Studies o[ Biosynthesis in E s c h e r i c h i a coli, Carnegie Inst. of W a s h i n g t o n , 1957. 5 H. O. HALVORSON AND G. N. COHEN, Ann. Inst. Pasteur, 95 (1958) 72. 6 D. ]3. CowIE AND F. T. MCCLuRE, Biochim. Biophys. Acta, 31 (1959) 236. 7 McQuILLEN, R. I3. ROBERTS AND R. J. BRITTEN, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1437. s C. E. DALGLIESH, Science, 125 (1957) 271. 9 R. ]3. LOFTFIELD AND E. A. EIGNER, J. Biol. Chem., 231 (1958) 925. 10 M. ZALOKAR, Arch. Biochem. Biophys., 50 (1954) 71. 11 S. EMERSON, J. Bacteriol., 60 (195 o) 221. 12 R. H. VOGEL AND M. J. I~OPAC, Biochim. Biophys. Acta, 36 (1959) 505 • is j . S. ROBERTSON, Physiol. Revs., 37 (1957) 133. 14 M. ZALOKAR, Am. J. Botany, 46 (1959) 555. 15 A. J. SHATKIN AND E. L. TATUM, J. Biophys. Biochem. Cytol., 6 (1959) 423 . 16 M. ZALOKAR, Exptl. Cell Research, 19 (196o) 114. 17 M. ZALOKAR, Federation Proc., 18 (1959) 358. 18 H. i . DINTZIS, H. BORSOOK AND T. VINOGRAD, in R. B. ROBERTS, Microsomal Particles and Protein Synthesis, P e r g a m o n Press, New Y o r k , 1958. I

Biochim. Biophys. Acta, 46 (1961) 423-432