Assay of Protamine Messenger RNA from Rainbow Trout Testis*

A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [*‘C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by lo-’ M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation. Starch gel electrophoresis showed that labeled arginine was incorporated in vitro into products identical with both authentic protamine and histones as found previously (Gilmour, R. S., and Dixon, G. H. (1972) J. Bid. Chem. 247, 4621-4627). The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of [“Clarginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine. A 4 to S was also the postribosomal supernatant of trout testis cells which, when in the ascites cell-free [“Clarginine into acid-insoluble material. The

A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [*'C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by lo-' M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation.
The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of ["Clarginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine.
A 4 to 6 S RNA fraction was also prepared from the postribosomal supernatant of trout testis cells which, when assayed in the ascites cell-free system, stimulated ["Clarginine incorporation into acidinsoluble material. The polypeptide product synthesized in response to this RNA fraction was analyzed by starch gel electrophoresis and carboxymethylcellulose (CM52) chromatography. Chromatography on CM52, which allows the separation of the three components of trout testis protamine, C,, C,,, and C,, , (Ling, V., Jergil, B., and Dixon, G. H. (1971) J. Biol. Chem. 246, 1168-1176 as individual peaks, showed that ("Clarginine was incorporated into all three components. A comparison of the incorporation of ["Clarginine and ["Clleucine in the presence of either trout testis sRNA or rabbit globin mRNA showed that while the incorporation of both labeled amino acids was stimulated by rabbit globin mRNA, there was no ["Clleucine incorporation in the presence of trout testis sRNA, although ["Clarginine was extensively incorporated. Since leucine is absent from trout testis protamine (Ando, T., and Watanabe, S. (1969) Znt. J. Protein Res. 1, 221-224), this finding suggests that the major mRNA activity in trout testis sRNA is that for protamine. The involvement of methionyl-tRNA in the initiation of protamine synthesis (Wigle, D. T., and Dixon, G. H. (1970) Nature 227, 676-680) was confirmed by the observation that the testis sRNA fraction containing protamine mRNA was able to form an 80 S initiation complex in a rabbit reticulocyte lysate system (Darnbrough, C., Legon, S., Hunt, T., and Jackson, R. J. (1973) J.

Mol.
Biol. 76,. In addition, polysome profiles of the fractionated Krebs II ascites S-30 in the presence of sRNA showed that 70% of the nascent protamine was associated with the monoribosome peak and 30% with the disome peak.
Protamines are a group of highly basic nuclear proteins bound to the DNA of sperm cells of most higher organisms. Protamine from rainbow trout (Salmo gairdnerii) has a molecular weight of close to 5000 and it has been shown that total protamine can be resolved by chromatography on CM-cellulose columns (1) into three components with different amino acid compositions. From the amino acid sequences of these compo-*This work was generously supported by the Medical Research Council of the United Kingdom.
$ British Council Predoctoral Fellow. 5 Address to which correspondence concerning this paper and requests for reprints should be sent. nents (2) it was evident that of the total of 31 to 33 amino acids, 21 to 23 are arginine together with a limited range of neutral amino acids; proline, serine, valine, alanine, and isoleucine but no leucine. There is strong evidence that this unique spermspecific nuclear protein is synthesized by the conventional mechanisms of protein synthesis in the cytoplasm (3) of early and middle spermatids (4). The synthesis seems to occur mainly on the disomes (1) and is inhibited (5) by puromycin and cycloheximide, but not by actinomycin D, over 6 to 10 hours suggesting its synthesis is in response to a stable mRNA. The involvement of methionine as the initiating amino acid both in uiuo (6) and in vitro (7)  The addition of both low molecular weight polysomal RNA and supernatant RNA from trout testis to a preincubated Krebs II ascites S-30 caused a stimulation of the incorporation of ["Clarginine into hot trichloroacetic acid-tungstate-insoluble material (Fig. 1, a and b). The stimulation of ["Clarginine incorporation by both RNA preparations was routinely found to be between 2-and 3-fold. In a second experiment (Table I) it was observed that the addition of trout testis supernatant RNA to a preincubated Krebs II ascites S-30 stimulated ["Clarginine incorporation into hot trichloroacetic acid-tungstateinsoluble material but had no effect on the incorporation of ["Clleucine.
On the other hand, rabbit globin mRNA stimulated both ["Clarginine and ["Clleucine incorporation. This result suggests that part of the stimulation observed in the presence of trout testis supernatant RNA is due to the synthesis of protamine since the amino acid sequence of trout testis protamine (2) shows that arginine comprises 2/3 of the total residues and leucine is absent. As expected, rabbit globin mRNA caused a stimulation in the incorporation of both ["C lleucine and ["C larginine since rabbit globin (Y and fi chains contain both these residues (the ratio of leucine to arginine in combined LY and /3 chains is 6.3).

Characterization of Product Synthesized in Krebs ZZ Ascites S-30 inPresence of Either Supernutant
RNA or Low Molecular Weight Polysomal RNA from Trout Testis In order to demonstrate the presence of protamine mRNA, the products synthesized in the ascites S-30 in the presence of the RNA fractions and ["Clarginine were studied. Two methods of analysis are reported here. Firstly, the acid-soluble products were analyzed by electrophoresis on starch gels. After termination of the in vitro reaction, a mixture of unlabeled trout testis protamine and histones were added and the incubation mixtures extracted with 0.2 M sulfuric acid and the extracts run on starch gels as described under "Materials and Methods.". In the absence of added RNA, one peak of radioactivity was observed which migrated on the starch gel in the same position as free arginine (Fig. 2). In the presence of supernatant RNA from trout testis, there were two peaks of radioactivity.
The faster moving peak co-migrated with free arginine and the slower with the carrier protamine which was detected by staining one portion of the gel with Amido black prior to slicing the remainder for radioactive counting (Fig. 2). In the presence of low molecular weight polysomal RNA from trout testis, three peaks of radioactivity were observed (Fig.  3o). The slowest moving peak corresponded to the area of the gel in which marker histones migrated. The major peak of radioactivity migrated in the protamine region and there was a third peak which migrated in the free arginine region. When only the 4 to 6 S region of the low molecular weight RNA, which had been separated on a sucrose density gradient, was added to a Krebs II ascites S-30, the synthesis of histones was abolished presumably due to the removal of the larger species of RNA containing histone mRNA (Fig. 36).
Secondly, the cell-free products synthesized in the presence of supematant RNA were analyzed by CM-cellulose chromatography using a lithium chloride/acetate gradient (1) nents of protamine from trout testis, Ci, C,,, and C,,,, could be separated as individual peaks. It can be seen (Fig. 4)  which co-chromatographed with C,, C,,, and C,,,. It can also be seen that these three components are not synthesized in equal amounts, there being more incorporation into C,,, than C,, with very little into C,.

Partial Characterization of Protamine mRNA on Sucrose Gradients
Samples of both low molecular weight polysomal RNA and supernatant RNA from trout testis were analyzed by sucrose density centrifugation.
After centrifugation under the conditions given in the legend of Fig. 5, RNA was extracted from various regions of the sucrose gradient of the low molecular weight polysomal RNA (Fig. 5a) and the 4 S region from the supernatant RNA (Fig. 5b). These RNA fractions were assayed conditions of chromatography were such that three compo-for mRNA activity in the Krebs II ascites cell-free system. bulk of the protamine mRNA activity was found at the top of the gradient in the 4 to 6 S region. A small amount of activity was seen in the 6 to 18 S region as well, however, there was no protamine mRNA activity in the 18 S region of the gradient (data not shown).

Characterization of Protamine mRNA Translation in Krebs ZZ Ascites Cell-free System
Time Course and mRNA Concentration- Fig.  6 shows the effect of adding increasing amounts of sRNA to an ascites S-30 system. The stimulation of ["Clarginine incorporation reaches a plateau at 4 pg/30 ~1 reaction mixture (120 &ml) of sRNA. Similar amounts of sRNA could also saturate the fractionated Krebs II ascites S-30 (Fig. 7a) Fig. 7b shows the rate of incorporation of [Wlarginine in a fractionated Krebs II ascites S-30 as directed by sRNA. The rate of incorporation was linear for the first 10 min and then declined. In the presence of trout testis low molecular weight polysomal RNA, incorporation of ["Clarginine by the ascites S-30 remains linear for the first 30 min (Fig. la).
Effect of Adding Transfer RNA-It has been reported recently that the translation of a number of messenger RNAs including EMC RNA (23) and rabbit globin mRNA (24) in the ascites S-30 is dependent on, or can be stimulated by, the addition of tRNA. It was therefore worthwhile to test the effect of the addition of tRNA on protamine mRNA translation.
The addition of either ascites tRNA, reticulocyte tRNA, or trout testis tRNA (purified on BD-cellulose) to a preincubated ascites S-30 had no effect on the incorporation of [W Jarginine.  In the presence of sRNA or low molecular weight polysomal RNA, the addition of tRNA caused a 25% increase in the incorporation of [Wlarginine when compared to the value obtained in the presence of the RNA alone (Table II). It appears therefore that the translation of protamine mRNA in the ascites S-30 used here is only slightly stimulated by the addition of tRNA so that none of the tRNAs required for the insertion of the limited range of amino acids present in protamine are limiting in the Krebs II ascites S-30 system. Effect of Aurintricarboxylic Acid-Aurintricarboxylic acid is an inhibitor of protein synthesis which acts at the level of initiation in eukaryotic systems. Addition of this compound in optimal amounts to a cell-free system should therefore inhibit the translation of exogenous mRNA. In general, a concentration of aurintricarboxylic acid of IO-' M has been shown to be optimal in inhibiting the initiation of protein synthesis in mammalian systems (25, 26). Fig. 8 shows that the stimulation of incorporation of ['Flarginine incorporation by low molecular weight polysomal RNA is completely abolished by the addition of lo-' M aurintricarboxylic acid, an observation which further supports the idea that the observed stimulation is due to the synthesis of protamine directed by protamine mRNA. Similar observations have been made with testis sRNA containing protamine mRNA activity. * "Shift" Experiment-It is now generally accepted that the initiation of polypeptide synthesis in both prokaryotic and eukaryotic cells involves the incorporation of a methionyl residue by a special initiating tRNA which binds to the initiation AUG codon in the messenger RNA. The synthesis of protamine both in uiuo (6) and in oitro (7) has been shown to involve the incorporation of an unblocked methionyl residue at the NH,-terminal position of the nascent protamine chain. Recently Darnbrough et al. (20) have developed a system in which they can study the mechanism of initiation of protein synthesis in a rabbit reticulocyte lysate. This system can be used to detect the presence of a messenger RNA by following the "shift" in methionine label ( somycin to prevent movement of the 80 S ribosome along the messenger, thus, there is no translation and initiation stops at the formation of this 80 S complex. In Fig. 9, it can be seen that in the absence of any exogenous mRNA, a peak of radioactivity is associated with the small 40 S ribosomal subunit of the sucrose gradient (Fig. 9a). The addition of either rabbit globin mRNA (Fig. 96) or trout testis sRNA (Fig. 9c) causes a change in the radioactivity distribution leading to an increase of label in the 80 S region at the expense of label associated with the 40 S ribosomal subunit. There is a larger shift of labeled [YS]Met-tRNArMet in the presence of rabbit globin mRNA than in the case of trout testis sRNA so that is likely that the concentration of trout testis sRNA used did not saturate the system. Since this assay depends on the presence of an AUG codon at the initiation site of mRNA, this observation of the "shift" of [%]Met-tRNArMet from the 40 S to the 80 S complex in the presence of trout testis sRNA indicates the presence of an mRNA containing an AUG-initiating codon. The coding portion of the protamine message would be expected to contain 96 to 99 nucleotides, a region approximately one-fifth the size of the coding portion of the globin mRNA. The average number of ribosomes associated with globin message in reticulocyte polysomes is five (16) and since protamine mRNA is one-fifth the size, it would be predicted that with the same packing, only a single ribosome at a time would be associated with a monocistronic protamine mRNA. Ling et al. (3) discussed the apparent discrepancy between this extrapolation from globin-synthesizing polysomes and the observed appearance of nascent protamine chains on diribosomes in intact trout testis spermatid cells in terms of either a tighter packing or the presence of a dicistronic messenger for protamine but could not distinguish between these possibilities.
This question was, therefore, re-examined in the heterologous translation system described above of the Krebs II ascites S-30 programmed with trout testis sRNA containing messenger activity for protamine. Polysome Profiles of Fractionated Ascites S-30 System after Addition of sRNA Containing Protamine Message-Trout testis sRNA was added in increasing concentrations to a fractionated ascites S-30 system with very low endogenous and after 5-and lo-min incubation, the polysomes were fractionated on sucrose gradients and the distribution of [Wlarginine in the nascent protamine associated with the polysomes was determined. Fig. 10, A and D, show that there was no ["Clarginine label associated with ribosomes after either 5 and 10 min of incubation in the absence of added mRNA. In the presence of 7.5 rg of sRNA, a small amount of label was observed in the monosome region (Fig. 1OB) after 5 min of incubation and there was a significant increase at 10 min (Fig. 1OC).
At 15 pg of sRNA, there was much more label associated with the monosome peak at 5 min and this increased at 10 min (Fig. 10,E and 8'). In contrast to the results of Ling et al. (3) the major peak of the newly synthesized protamine is found associated with the monosomes and a minor peak in the disome region (70% of the radioactivity on the monosome and 30% on the disome).

DlSCUSSlON
An important consideration in studies of the translation of an isolated messenger RNA is the selection of an appropriate translation system. Gilmour and Dixon (7) used a preincubated trout liver ribosome system to detect the presence of the messenger RNA for protamine in a low molecular weight RNA fraction prepared from trout testis polysomes. This system had the advantage of being derived from an easily available tissue in the same animal but one which does not synthesize protamine endogenously and hence has a very low background incorporation.
However, the activity of the preincubated trout liver ribosomes was low and somewhat variable. Mathews and Korner (15)  virus, bacteriophage Q& and reovirus have been translated in the Krebs II system. This cell-free system, which has a low background incorporation of ["Clarginine has proved both sensitive and convenient for detecting and assaying protamine mRNA. Addition of low molecular weight RNA prepared both from trout testis polysomes (Fig. la) and postribosomal supernatant (Fig. lb) to the Krebs II ascites S-30 leads to marked stimulation of incorporation of [Wlarginine into hot trichloroacetic acid-tungstate-precipitable material. At low concentrations of added low molecular weight RNA, the increase in [Wlarginine incorporation was linearly related to RNA concentration but the system became saturated as the RNA concentration was increased (Figs. 6 and 7a). Gilmour and Dixon (7) showed with the trout liver ribosome cell-free system that methionine was incorporated into the NH,-terminal position of nascent protamine chains, an observation consistent with the NH,-terminal incorporation of methionine into protamine by intact trout testis cells (6). This NH,-terminal methionine incorporation in vitro indicated that de nouo initiation rather than chain elongation was taking place in the trout liver cell-free system. A sensitive assay for initiation is the effect of aurintrisarboxylic acid upon in vitro incorporation of ["Clarginine into protamine. As seen in Fig. 8, aurintricarboxylic acid at 1 x lo-' M completely inhibits protamine synthesis by the Krebs II ascites S-30 and since it is known to affect the initiation step specifically, this total inhibition indicates that substantial de nouo initiation must be taking place in the ascites S-30. The ["Clarginine-labeled product of synthesis in the presence of added low molecular weight RNA was further characterized by starch gel electrophoresis as described by Gilmour and Dixon (7) and Fig. 2a shows both the resolution of ["Clarginine-labeled protamine from free ["Clarginine and the strong dependence of the incorporation upon added protamine mRNA. As shown by Gilmour and Dixon (7) 5  ID  I5  20  25  5  lo  I5  20  25  5  IO  I5  20  2 both protamine and histones and we have confirmed this finding with the Krebs II ascites system (Fig. 3~). However, when the low molecular weight RNA is further purified by sucrose density gradient centrifugation (Fig. 5~) and the 4 to 6 S RNA peak assayed, the protamine mRNA activity is retained ( Fig. 3b and Table II) but the histone mRNAs are removed (Fig. 3b). This finding is consistent with the larger size of the histone messenger RNAs (7 to 9 S) which would not be included in the 4 to 6 S peak of the gradient.
A rather surprising finding in the present study is the presence of a considerable amount of protamine messenger RNA in the postribosomal supernatant solution from trout testis ( Fig. lb and Table II). The RNA from this postribosomal supernatant shows a peak sedimenting at 4 to 6 S in the sucrose density gradient (Fig. 5b) and produced a marked stimulation of incorporation of ["Clarginine into hot trichloroacetic acidtungstate-precipitable material. Control incubations containing ascites tRNA (Table II) or trout testis tRNA purified on BD-cellulose showed no such stimulatory effect.
In Fig. 2, the product of testis postribosomal sRNA translation in the ascites S-30 is shown to undergo co-electrophoresis with carrier protamine on starch gels while in Fig. 4 the product is further characterized by ion exchange chromatography on a carboxymethylcellulose column eluted with a gradient of lithium chloride in the presence of lithium acetate/acetate acid buffer, pH 5.0 (1). Three subcomponents of trout testis protamine, C,, C,,, and C,,, are resolved by this procedure and it is clear that ["Clarginine is incorporated into each. However, Cl,, shows the highest incorporation and FRACTION NUMBER d---SEDIMENTATION possesses the greatest specific activity, thus the three components are not labeled equally or in proportion to the amounts of each present in the cold carrier protamines extracted from trout testis nuclei. There are at least two possible explanations for this result; either the sRNA fraction was isolated at a stage of testis development when the tissue is richest in the C,,, message or the Krebs II ascites S-30 is able to translate the protamine mRNA fractions for the three components with differing efficiencies. In the case of (Y-and @-globin mRNA, it is well known that there can be considerable variation in the rates of (Y to fi chain synthesis in different cell-free translation systems which may be related to the presence or absence of initiation factors specific for one message or the other (28-30). We have observed wide variations in the distribution of incorporation into the three protamine components and those studies will be reported in a subsequent communication.
Protamine has a very limited range of amino acids in its structure and it is possible to compare the incorporation of an amino acid such as leucine which is not present in the known sequence, with one that is very frequent such as arginine. Such an experiment is described in Table I where the incorporation of ["Clleucine and ["Clarginine is compared when the ascites S-30 is programmed with either rabbit reticulocyte globin mRNA, trout testis sRNA, or a mixture of both. Leucine is a major constituent of both (Y and B chains and is extensively incorporated in response to globin mRNA but not at all in response to testis sRNA. This lack of stimulation of ["Clleutine incorporation by testis sRNA indicates that the amounts of mRNAs coding for leucine-containing proteins is undetectable in the total testis sRNA. In contrast, there is strong mRNA stimulation of [l'C]arginine incorporation by the testis sRNA but rather weak stimulation by globin mRNA; this is consistent with the presence of only 3 arginine residues out of 141 to 143 in both rabbit a-and @globins as compared with 21 arginines out of 31 residues in the protamines. When both globin mRNA and testis sRNA are present simultaneously there is a slight decrease in the leucine and arginine incorporation, respectively, indicating a slight degree of competition between the two messages at the concentrations employed. Another useful assay for the presence of messenger RNA is the "shift" assay described by Darnbrough et al. (20) which depends upon the formation of a complex between [ssS]Met-tRNArM" and the small 40 S ribosome subunit in the presence of initiation factors. When mRNA containing the initiation codon, AUG, is present together with the large 60 S ribosomal subunit, then there is a shift of radioactivity from the 40 S region to the 80 S region. Since the "shift" assay is done in the presence of sparsomycin, a potent inhibitor of elongation, no polysomes are formed and the 3JS label remains associated with the 80 S monoribosome peak. The occurrence of the expected shift in the presence of both globin m'RNA and sRNA containing protamine mRNA shows that both possess the initiating codon characteristic of natural messenger RNA. In addition, the shift of [86S]methionyl-tRNA in the presence of protamine mRNA confirms the involvement of methionyl-tRNA in the initiation of protamine synthesis as previously reported in oioo by Wigle and Dixon (6) and in oitro by Gilmour and Dixon (7). When the exogenous testis sRNA is added to Krebs II ascites S-30 in the presence of ["Clarginine and the polysome profile examined at 5 min (Fig. 10, C and D) and 10 min (Fig. 10, E and F), it is seen that there is a stimulation of incorporation of ["Clarginine into nascent protamine associated largely (70%) with the monoribosome peak but with some label (30%) in the diribosome peak. This result is somewhat different to that in viuo in trout testis cells (3) where the predominant site of nascent protamine synthesis appeared to be the diribosome peak. The basis of this difference is not clear at the moment, but the efficiency of the heterologous in vitro protein synthesis system is so much reduced compared to the in oiuo situation that the smaller polysome size in vitro may reflect the greatly reduced efficiency of mRNA translation under these artificial conditions. In an accompanying paper, Gedamu et al. (31) have shown that two editions of protamine mRNA exist in testis cells, one possessing a poly(A) region which binds tightly to oligodeoxythymidylate cellulose columns and one which does not. The postribosomal supernatant appears to be somewhat richer in the species of mRNA lacking poly (A) and since in the experiment depicted in Fig. 10, the source of the mRNA was postribosomal supernatant (sRNA), it is possible that the predominant location of nascent protamine on the monoribosome may be related to the presence of an appreciable amount of protamine mRNA lacking in poly(A)region.
Further experiments are under way to examine this possibility.
From the amino acid sequence of the rainbow trout protamines (2), it can be calculated that 3 x 31 to 33 = 93 to 96 nucleotides plus the initiating and terminating codons for a total of 99 to 102 nucleotides, would be required to code for protamine.
This would represent the minimum size for the protamine mRNA. However, by analogy with other messenger RNAs (32) which are usually significantly larger than their predicted coding sizes, it would be expected that protamine would correspond to an S value of 5 to 6. In the accompanying paper (31), it is shown that poly(A) containing protamine mRNA which can be purified by oligo(dT)-cellulose chromatography followed by sucrose density gradient centrifugation has an S value of 5.7 corresponding to about 165 nucleotides. A significant proportion of the extra 63 to 66 nucleotides over the minimum coding size of 99 to 102 must be poly(A) but the exact size of this region is not yet known.
The sucrose density gradient centrifugation of sRNA (Fig.  5b) shows a single peak in the 4 to 6 S region; the great majority of the material must be tRNA but the total absence of 18 and 28 S ribosomal RNA indicates that the messenger activity present in the postribosomal supernatant cannot be due to contaminating polysomes containing bound mRNA but must represent a distinct postribosomal form of protamine mRNA.