ANALYTICAL
BIOCHEMISTRY
69, 84-91 (1975)
Temperature of Acrylamide Electrophoretic Mobilities
Polymerization and of Nucleic Acids
J. GRESSEL, A. ROSNER, AND NOA COHEN Department of Plant Genetics, Weizmann Institute of Science, Rehovot, Israel Received December 12, 1974; accepted June 4, 1975 The electrophoretic mobilities of DNA, ribosomal RNAs, and pulse-labeled RNAs were compared on polyacrylamide gels polymerized at temperatures from 4 to 35°C and subjected to electrophoresis at a fixed temperature. DNA migrated the same distance irrespective of polymerization temperature, the ribosomal RNAs, and the major pulse-labeled species (a putative rRNA precursor) migrated more rapidly in gels polymerized at higher temperatures. The linearity of the migration versus the log of the molecular weight remained for the five rRNA species used, but the extrapolated molecular weight of the putative precursor ranged from 1.8 X 106 to 2.5 X 106 depending on polymerization temperatures. By varying polymerization temperatures, the optimal resolution of various groups of RNA species can be obtained. The results are explained in terms of polymerization temperature effects on gel structure as well as nucleic acid conformation.
Although the chemistry of acrylamide-methylene bis acrylamide cross linked polymerization is known (1,2) the tertiary structure of the gel is a matter of conjecture and model building (3,4). Studies on the theoretical aspects of electrophoretic mobility of proteins (2-6) and nucleic acids (7) in polyacrylamide gel electrophoresis have carefully limited themselves to polymerization at a single temperature (4,5). Temperature effects have been measured on protein mobility, but the effects of temperature on polymerization and on electrophoresis were not tested separately, i.e., the polymerization and the electrophoresis were at identical temperatures (45). Different electrophoresis temperatures were found to differently affect the mobility of nucleic acids of different conformations (8) which could be minimized by denaturing (opening) the secondary structure of nucleic acids (e.g., 9). Various laboratories in our Institution were arriving at different apparent molecular weights for a pulse-labeled RNA species, thought to be a precursor of rRNA. Differences in mobilities and resolution of ribosomal RNAs also were noted, even though the linear correlation between mobility and the logarithm of the molecular weight remained. These discrepancies could only be pinpointed to the differences in laboratory temperatures during polymerization; when the laboratories ex84 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
GEL
POLYMERIZATION
AND
NA
MOBILITY
85
changed gels, electrophoresis temperature had no effect. As many studies of rRNA processing depend on polyacrylamide gel electrophoresis to estimate the sizes of precursors and intermediates (lo-13), we deemed it necessary to more fully estimate the effects of polymerization temperature on the mobility of the RNAs. Data presented below will be of assistance in choosing the optimal conditions for best resolution of RNAs of various molecular weights. MATERIALS
AND
METHODS
Gel pofymeriz&on. Acrylamide (BDH) was recrystallized from chloroform and N,N-methylene-bis-acrylamide (Eastman) from acetone as outlined by Loening (12). The mixture consisting of 2.4% acrylamide, 0.125% N,N-methylene-bis-acrylamide, 36 mM tris(hydroxymethy1) aminomethane, 30 mM NaH,PO,, 1 mM disodium ethylenediaminetetraacetate was degassed and apportioned to tubes at different temperatures. N,N,N’,N’-tetramethylethylenediamine (TEMED) (0.5 ,&/ml) and ammonium persulfate (final concentration 0.08%) were added and the mixture was divided into acrylic plastic electrophoresis tubes which were preequilibrated to the temperature of polymerization. The solution was overlayed with water. The reaction was allowed to go to completion by polymerizing overnight, as temperature affects the rate of polymerization. The persulfate concentration was in excess for the same reason. Better than 96% polymerization would be expected under all conditions (6). RNA preparation. RNA from the duckweed Spirodela oligorrhiza was used, both for marker rRNA peaks as well as for the pulse-labeled putative precursor. [ S3H] Uridine (35 &i/ml, 19 Ci/mmole) (Nuclear Research Centre, Negev, Israel) was added to cultures for 2 hr prior to harvesting to specifically label the RNA (and not DNA). The cultures were maintained, harvested, and the nucleic acids extracted, and fractionated as outlined previously (13). RESULTS
Sample results of an experiment in which gels were polymerized between 4 and 35°C and run together at 22°C are summarized in Fig. 1. The solid line represents the ultraviolet absorbancy having peaks at apparent molecular weights of 1.3 X lo6 (cytoplasmic heavy subunit rRNA), 1.05 X lo6 (chloroplast heavy subunit rRNA), 0.7 X lo6 (cytoplasmic light subunit rRNA), 0.56 X 106 (chloroplast light subunit rRNA), and 0.42 X lo6 (a nicked product of the 1.05 x lo6 chloroplast rRNA). The processing of these materials is discussed in (14). The broken line with points represents the radioactivity of a 2-hr pulse with [3H]uridine. The points are connected in the area of the high molecular
FIG.
axenically performed molecular
I
20
1.3 I
II I
07 056
0.42 03 I
0
4.01
2
30I
201
4
I.3
6
I.1 I
Weight
Mlgroted(cm)
c
22oc
Molecular
Distance
Apparent
x I#
0.7056 I,
8
042 (
C
I
lcn80 I ,
2
60 I 35'C
4
40 30 1 ,
20 ,
6
1311 1 ,
8
,
a7cm5042 , , ,
,
1. Mobility of RNAs in gels polymerized at different temperatures. About 50 pg of RNA (from Duckweed plants labeled for 2 hr with [3H]-uridine) were applied to gels polymerized at 4, 22, 35°C as described in Materials and Methods. Electrophoresis was at 22°C for 3.5 hr at 50 V. Absorbancy scans and radioactivity determinations were done as described in (13). The apparent weight of the precursor was determined by extrapolation from the rRNAs standards.
04-
30
GEL
POLYMERIZATION
AND
NA
MOBILlTY
87
weight RNA. This material is considered to be a precursor for rRNA ( 10) and our kinetic data support this assumption (13). There is a large amount of polydisperse pulse-labeled RNA on the gels, as well as rapidly labeling material with apparent molecular weights of 1.4, 1.2, 0.7, 0.5 x 1Ofi which are discussed elsewhere ( 13 ). The nucleic acids were treated with DNase in this experiment so that the DNA would not interfere with the interpretation of the pulse-labeled material (see below). All of the gels shown in Fig. 1 were run together for the same length of time at the same temperature. Note that the rRNAs move more rapidly and the peaks remain closer together in the gels polymerized at higher temperatures. According to extrapolations of the data, RNAs of higher apparent molecular weights should enter the gels polymerized at higher temperatures. The apparent extrapolated molecular weight of the precursor in this experiment varies from 2.0 X 1O6in gels polymerized at 4°C to 2.5 X 10” in gels polymerized at 35°C. These values varied slightly from experiment to experiment. In another experiment, the DNA was not digested and the mobilities were similarly ascertained following polymerization at additional temperatures. The data are plotted as mobility (distance of migration) versus the logarithm of the molecular weight. The validity of such plots for RNA is discussed in references (7,15), among others. Empirically, it is quite clear that the molecular weights of the rRNAs remain linear following the various polymerization conditions (Fig. 2). The DNA moved essentially the same distance into the gels following all polymerization temperatures in spite of the changing mobilities of the rRNAs and the putative precursor. The extrapolated apparent molecular weight of the precursor in this experiment varied from 1.85 to 2.4 X lo6 as the temperature of polymerization increased (Figs. 2A and B). The apparent molecular weights of the pulse-labeled RNAs - 1.4, 1.2, 0.5 X 10” -were not significantly affected (not shown). In the experiments discussed above, the gels polymerized at different temperatures were run together at the same temperature (22°C) for the same length of time. It could be suggested that longer durations of electrophoresis might cause the RNAs to penetrate further into the low temperature polymerized gels which would then approximate the conditions of the high temperature polymerized gels. We performed experiments to clarify this possibility and part of the results are summarized in Fig. 3. It is clear that the duration of electrophoresis does not modify the apparent molecular weights. To further ascertain the effects of the polymerizing temperature on the mobility of RNAs, linear regression plots (16) were made from the same data from which Fig. 2 was prepared, and the lines of best fit were drawn (Fig. 4). It is clear that the temperature of gel polymerization dif-
60
A ’
I
I
I
’
B
2.4
n’.-:’ IO Polymerization
\
L
20 Tempemtd
: 30
I
I 3
W
\
0.2)
I I 123456785
I
I
I
I
I
Distance Migrated (cm ) 2. The relationship between nucleic acid mobility, apparent molecular weight, and the temperature of acrylamide polymerization. (A) The migration of the rRNAs standards was plotted against the logarithm of their known molecular weights. The migrations of the putative rRNA precursor (A) and the DNA (0 are shown). (B) The apparent molecular weight of the putative precursor was determined by extrapolation from the data in 2A and plotted against the respective polymerization temperature of the gel (2B). Experimental procedures as in Fig. 1. FIG.
I
2
I
I
I
I
4 6 Distance Migratedkm)
,
I
8
FIG. 3. The effect of duration of electrophoresis on the relative migration of the RNA fractions. Experimental details as in Fig. 1 except that the duration of electrophoresis was 3.5 and 5.5 hr for RNA in gels polymerized at 4°C (0) and 35°C (A). The plot is similar to that of Fig. 2.
GEL
POLYMERIZATION
AND
NA
89
MOBILITY
m= 0.060 -------
i
m=O.O63
I
IO
I
15 Polymerization
I
20
I
25
1
I
30
TemperatureF’C)
FIG. 4. Linear regression analysis of the effect of polymerization temperature on the mobility of each nucleic acid species. The data of migration distances of RNAs in gels polymerized at various temperatures were taken from Fig. 2 and replicate data, and analyzed according to Snedcor (16) to obtain the linear regressions.
ferently affects each of the five rRNA species; the heavier the rRNA, the greater the slope; from 0.60 mm/C to 0.85 mm/C (Fig. 4). Peculiarly, the change in mobility of the putative precursor is more similar to light rRNA than to the heavier rRNAs. The mobility of the DNA was hardly affected by the temperature of polymerization of the gel. DISCUSSION
In order to understand the differences in mobility in gels prepared at different temperatures we have to try and visualize the effect of polymerization temperature on the structure of the gels. The gels were prepared under identical concentrations of acrylamide, bis-acrylamide, activator, and catalyst. Thus, the volume occupied by the polyacrylamide should be the same under all conditions. The only variable was the temperature. The gel point in any polymer system is inversely proportional to the molecular weight (length) of the polymer chain. As the polymerization temperature decreases, the rate of initiation decreases and the molecular weight increases, i.e., the gel point at low temperatures is achieved at a much lower degree of conversion of acrylamide. As the polymerization continues to completion, further polymer chains will create more interpenetrating polymer networks decreasing the effec-
90
GRESSEL,
ROSNER
AND
COHEN
tive pore size of the gel. One could thus predict the lower RNA mobility in the gels prepared at lower temperatures (Fig. 4).’ By modifying the temperature of polymerization of the gels, gel systems can be designed to best suit one’s purposes; polymerization at low temperatures for maximal resolution of the rRNA species and polymerization at higher temperatures for best separation of high apparent molecular weight RNAs. The data strongly indicate the idiosyncracies and drawbacks of using rRNA molecular weights to estimate the molecular weights of RNA species with apparently different tertiary conformations from rRNA (see also 8,17). The data also indicate the inherent dangers of constructing processing pathways based on extrapolated molecular weights ( 10). The tertiary structure of rRNA, based on its partly base paired, partly open secondary structure is probably analogous to the classically random coiled proteins, which are used for theoretical studies on gel filtration and electrophoresis. The DNA molecules are probably most similar to rigid rods and putative precursor to open structured single-stranded RNA. It is quite clear from the literature that the relative mobilities of the rRNAs are least affected by changing the parameters of electrophoresis (e.g., 8,15,17). Actually, by changing conditions such as electrophoresis temperature and voltage gradients, Fisher and Dingman (8) were able to demonstrate differences in the absolute mobilities of single and double stranded viral RNAs compared to rRNA standards. Their data brought them to suggest that “the mobilities reflect molecular radii rather than molecular weights,” but this does not completely explain the differential effects. Acrylamide concentration Gas also found to differentially affect the mobility of “single” versus double-stranded RNA (17) but had little effect on the extrapolated molecular weight of putative rRNA precursors (15,17). This supports that what we have is not a change in effective gel concentration, but an effect on the configuration of the fiber maze forming the pores. It is possible that the greater protein mobility found with high running temperatures (6) reflects the higher gel polymerization temperatures and not the running temperatures. The molecular weight of the precursor varied from 2.0 to 2.5 X lo6 with gels prepared from 4 to 35°C (Fig. 1). This is probably due to the fact that one is measuring an extrapolated value and not an intrapolated value, involving a consideration uncertainty. From Fig. 3 one can see that the extrapolation to zero distance of migration at 4°C is 3. I X 1O6 ’ One reviewer offers the following interesting explanation. Shorter polymer chains generated at higher temperatures offer less resistance to the “ballistic” migration of random coiled RNA than longer chains generated at lower temperatures. The effect should be accentuated in direct relation to molecular weight.
GEL
POLYMERIZATION
AND
NA
MOBILITY
91
MW which is not very far from the value of 2.5 X 10” estimated for the precursor in gels polymerized at 35°C. Yet, the extrapolated molecular weight of the origin remains constant despite increases in the duration of electrophoresis. Obviously if we could try a sample of molecular weight 3.1 X lo6 it would move to a certain degree and not stay at zero distance, as we are dealing with an average pore size and not with absolute values. As the relative mobility of the putative precursor varied relative to the five rRNA markers, one can assume that its secondary and tertiary structures differ from that of rRNA. The fact that the mobility of the DNA molecules is unaffected by the pore size [or acrylamide concentration (15)], is probably connected to its rigid structure which enables it to move at an equal rate through decreasingly smaller pores than the open more single stranded RNA. It has been suggested that the DNA aligns parallel to the direction of migration (8). ACKNOWLEDGMENTS We gratefully acknowledge the discussions and cooperation of Yaffa Nevo, who first called our attention to the discrepancies between laboratories. The technical assistance of Ofra Kirsten in part of these experiments helped us immensely. We thank Prof. Moshe Levy of the Plastic Research Department of this institution for his valuable contribution to the part of the discussion of our results concerning the theoretical basis of gel polymerization. An interesting useful correspondence with Dr. A. Chrambach is gratefully acknowledged.
REFERENCES 1. Raymond, S., and Wang, Y. (1960) A&. Biochem. 1, 391. 2. Allen, R. C.. and Maurer, H. R. (eds.) (1974) Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel, 3 16 pp.. deGruyter. Berlin and New York. 3. Fawcett. J. S., and Morris, C. J. 0. R. (1966) Sepur. Sci. 1, 9. 4. Chrambach, A., and Rodbard, D. ( 1971) Science 172,440. 5. Rodbard, D., and Chrambach, A. (1971) Anal. Biochem. 40, 95. 6. Chrambach, A., and Rodbard, D. (1972) Sepur. Sci. 7, 663. 7. Richards, E. G., and Lecanidou, R. (1971) Anal. Biochem. 40, 43. 8. Fisher, M. P. and Dingman, C. W. (1971) Biochemistry 10, 1895. 9. Reijnders, L., Sloof, P.. Sival. J., and Borst, P. (1973) Biochim. Biophys. Actu 324, 320. 10. Grierson, D., and Loening, U. E. ( 1974) Eur. J. B&hem. 44, 501. 11. Weinberg, R. A., Loening, U. E.. Willems. M.. and Penman, S. (1967) Proc. Nat. Acud.
12. 13. 14. 15. 16. 17.
Sci. USA
58. 1088.
Loening, U. E. (1967) Biochem. J. 102, 25 1. Rosner, A., Posner, H. B., and Gressel, J. (1973) Plant Cell Physiol. 14, 555. Rosner. A., Porath, D., and Gressel, J. (1974) Plunt Cell Physiol. 15, 891. Loening, U. E. (1969) Biochem. J. 113, 131. Snedecor, G. W. (1946) Statistical Methods, p. 103, Iowa State College Press. Harley, E. H.. White. J. S., and Rees. K. R. (1973) B&him. Bioplrvs. Actn 299, 253.
BIOCHEMISTRY
69, 84-91 (1975)
Temperature of Acrylamide Electrophoretic Mobilities
Polymerization and of Nucleic Acids
J. GRESSEL, A. ROSNER, AND NOA COHEN Department of Plant Genetics, Weizmann Institute of Science, Rehovot, Israel Received December 12, 1974; accepted June 4, 1975 The electrophoretic mobilities of DNA, ribosomal RNAs, and pulse-labeled RNAs were compared on polyacrylamide gels polymerized at temperatures from 4 to 35°C and subjected to electrophoresis at a fixed temperature. DNA migrated the same distance irrespective of polymerization temperature, the ribosomal RNAs, and the major pulse-labeled species (a putative rRNA precursor) migrated more rapidly in gels polymerized at higher temperatures. The linearity of the migration versus the log of the molecular weight remained for the five rRNA species used, but the extrapolated molecular weight of the putative precursor ranged from 1.8 X 106 to 2.5 X 106 depending on polymerization temperatures. By varying polymerization temperatures, the optimal resolution of various groups of RNA species can be obtained. The results are explained in terms of polymerization temperature effects on gel structure as well as nucleic acid conformation.
Although the chemistry of acrylamide-methylene bis acrylamide cross linked polymerization is known (1,2) the tertiary structure of the gel is a matter of conjecture and model building (3,4). Studies on the theoretical aspects of electrophoretic mobility of proteins (2-6) and nucleic acids (7) in polyacrylamide gel electrophoresis have carefully limited themselves to polymerization at a single temperature (4,5). Temperature effects have been measured on protein mobility, but the effects of temperature on polymerization and on electrophoresis were not tested separately, i.e., the polymerization and the electrophoresis were at identical temperatures (45). Different electrophoresis temperatures were found to differently affect the mobility of nucleic acids of different conformations (8) which could be minimized by denaturing (opening) the secondary structure of nucleic acids (e.g., 9). Various laboratories in our Institution were arriving at different apparent molecular weights for a pulse-labeled RNA species, thought to be a precursor of rRNA. Differences in mobilities and resolution of ribosomal RNAs also were noted, even though the linear correlation between mobility and the logarithm of the molecular weight remained. These discrepancies could only be pinpointed to the differences in laboratory temperatures during polymerization; when the laboratories ex84 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
GEL
POLYMERIZATION
AND
NA
MOBILITY
85
changed gels, electrophoresis temperature had no effect. As many studies of rRNA processing depend on polyacrylamide gel electrophoresis to estimate the sizes of precursors and intermediates (lo-13), we deemed it necessary to more fully estimate the effects of polymerization temperature on the mobility of the RNAs. Data presented below will be of assistance in choosing the optimal conditions for best resolution of RNAs of various molecular weights. MATERIALS
AND
METHODS
Gel pofymeriz&on. Acrylamide (BDH) was recrystallized from chloroform and N,N-methylene-bis-acrylamide (Eastman) from acetone as outlined by Loening (12). The mixture consisting of 2.4% acrylamide, 0.125% N,N-methylene-bis-acrylamide, 36 mM tris(hydroxymethy1) aminomethane, 30 mM NaH,PO,, 1 mM disodium ethylenediaminetetraacetate was degassed and apportioned to tubes at different temperatures. N,N,N’,N’-tetramethylethylenediamine (TEMED) (0.5 ,&/ml) and ammonium persulfate (final concentration 0.08%) were added and the mixture was divided into acrylic plastic electrophoresis tubes which were preequilibrated to the temperature of polymerization. The solution was overlayed with water. The reaction was allowed to go to completion by polymerizing overnight, as temperature affects the rate of polymerization. The persulfate concentration was in excess for the same reason. Better than 96% polymerization would be expected under all conditions (6). RNA preparation. RNA from the duckweed Spirodela oligorrhiza was used, both for marker rRNA peaks as well as for the pulse-labeled putative precursor. [ S3H] Uridine (35 &i/ml, 19 Ci/mmole) (Nuclear Research Centre, Negev, Israel) was added to cultures for 2 hr prior to harvesting to specifically label the RNA (and not DNA). The cultures were maintained, harvested, and the nucleic acids extracted, and fractionated as outlined previously (13). RESULTS
Sample results of an experiment in which gels were polymerized between 4 and 35°C and run together at 22°C are summarized in Fig. 1. The solid line represents the ultraviolet absorbancy having peaks at apparent molecular weights of 1.3 X lo6 (cytoplasmic heavy subunit rRNA), 1.05 X lo6 (chloroplast heavy subunit rRNA), 0.7 X lo6 (cytoplasmic light subunit rRNA), 0.56 X 106 (chloroplast light subunit rRNA), and 0.42 X lo6 (a nicked product of the 1.05 x lo6 chloroplast rRNA). The processing of these materials is discussed in (14). The broken line with points represents the radioactivity of a 2-hr pulse with [3H]uridine. The points are connected in the area of the high molecular
FIG.
axenically performed molecular
I
20
1.3 I
II I
07 056
0.42 03 I
0
4.01
2
30I
201
4
I.3
6
I.1 I
Weight
Mlgroted(cm)
c
22oc
Molecular
Distance
Apparent
x I#
0.7056 I,
8
042 (
C
I
lcn80 I ,
2
60 I 35'C
4
40 30 1 ,
20 ,
6
1311 1 ,
8
,
a7cm5042 , , ,
,
1. Mobility of RNAs in gels polymerized at different temperatures. About 50 pg of RNA (from Duckweed plants labeled for 2 hr with [3H]-uridine) were applied to gels polymerized at 4, 22, 35°C as described in Materials and Methods. Electrophoresis was at 22°C for 3.5 hr at 50 V. Absorbancy scans and radioactivity determinations were done as described in (13). The apparent weight of the precursor was determined by extrapolation from the rRNAs standards.
04-
30
GEL
POLYMERIZATION
AND
NA
MOBILlTY
87
weight RNA. This material is considered to be a precursor for rRNA ( 10) and our kinetic data support this assumption (13). There is a large amount of polydisperse pulse-labeled RNA on the gels, as well as rapidly labeling material with apparent molecular weights of 1.4, 1.2, 0.7, 0.5 x 1Ofi which are discussed elsewhere ( 13 ). The nucleic acids were treated with DNase in this experiment so that the DNA would not interfere with the interpretation of the pulse-labeled material (see below). All of the gels shown in Fig. 1 were run together for the same length of time at the same temperature. Note that the rRNAs move more rapidly and the peaks remain closer together in the gels polymerized at higher temperatures. According to extrapolations of the data, RNAs of higher apparent molecular weights should enter the gels polymerized at higher temperatures. The apparent extrapolated molecular weight of the precursor in this experiment varies from 2.0 X 1O6in gels polymerized at 4°C to 2.5 X 10” in gels polymerized at 35°C. These values varied slightly from experiment to experiment. In another experiment, the DNA was not digested and the mobilities were similarly ascertained following polymerization at additional temperatures. The data are plotted as mobility (distance of migration) versus the logarithm of the molecular weight. The validity of such plots for RNA is discussed in references (7,15), among others. Empirically, it is quite clear that the molecular weights of the rRNAs remain linear following the various polymerization conditions (Fig. 2). The DNA moved essentially the same distance into the gels following all polymerization temperatures in spite of the changing mobilities of the rRNAs and the putative precursor. The extrapolated apparent molecular weight of the precursor in this experiment varied from 1.85 to 2.4 X lo6 as the temperature of polymerization increased (Figs. 2A and B). The apparent molecular weights of the pulse-labeled RNAs - 1.4, 1.2, 0.5 X 10” -were not significantly affected (not shown). In the experiments discussed above, the gels polymerized at different temperatures were run together at the same temperature (22°C) for the same length of time. It could be suggested that longer durations of electrophoresis might cause the RNAs to penetrate further into the low temperature polymerized gels which would then approximate the conditions of the high temperature polymerized gels. We performed experiments to clarify this possibility and part of the results are summarized in Fig. 3. It is clear that the duration of electrophoresis does not modify the apparent molecular weights. To further ascertain the effects of the polymerizing temperature on the mobility of RNAs, linear regression plots (16) were made from the same data from which Fig. 2 was prepared, and the lines of best fit were drawn (Fig. 4). It is clear that the temperature of gel polymerization dif-
60
A ’
I
I
I
’
B
2.4
n’.-:’ IO Polymerization
\
L
20 Tempemtd
: 30
I
I 3
W
\
0.2)
I I 123456785
I
I
I
I
I
Distance Migrated (cm ) 2. The relationship between nucleic acid mobility, apparent molecular weight, and the temperature of acrylamide polymerization. (A) The migration of the rRNAs standards was plotted against the logarithm of their known molecular weights. The migrations of the putative rRNA precursor (A) and the DNA (0 are shown). (B) The apparent molecular weight of the putative precursor was determined by extrapolation from the data in 2A and plotted against the respective polymerization temperature of the gel (2B). Experimental procedures as in Fig. 1. FIG.
I
2
I
I
I
I
4 6 Distance Migratedkm)
,
I
8
FIG. 3. The effect of duration of electrophoresis on the relative migration of the RNA fractions. Experimental details as in Fig. 1 except that the duration of electrophoresis was 3.5 and 5.5 hr for RNA in gels polymerized at 4°C (0) and 35°C (A). The plot is similar to that of Fig. 2.
GEL
POLYMERIZATION
AND
NA
89
MOBILITY
m= 0.060 -------
i
m=O.O63
I
IO
I
15 Polymerization
I
20
I
25
1
I
30
TemperatureF’C)
FIG. 4. Linear regression analysis of the effect of polymerization temperature on the mobility of each nucleic acid species. The data of migration distances of RNAs in gels polymerized at various temperatures were taken from Fig. 2 and replicate data, and analyzed according to Snedcor (16) to obtain the linear regressions.
ferently affects each of the five rRNA species; the heavier the rRNA, the greater the slope; from 0.60 mm/C to 0.85 mm/C (Fig. 4). Peculiarly, the change in mobility of the putative precursor is more similar to light rRNA than to the heavier rRNAs. The mobility of the DNA was hardly affected by the temperature of polymerization of the gel. DISCUSSION
In order to understand the differences in mobility in gels prepared at different temperatures we have to try and visualize the effect of polymerization temperature on the structure of the gels. The gels were prepared under identical concentrations of acrylamide, bis-acrylamide, activator, and catalyst. Thus, the volume occupied by the polyacrylamide should be the same under all conditions. The only variable was the temperature. The gel point in any polymer system is inversely proportional to the molecular weight (length) of the polymer chain. As the polymerization temperature decreases, the rate of initiation decreases and the molecular weight increases, i.e., the gel point at low temperatures is achieved at a much lower degree of conversion of acrylamide. As the polymerization continues to completion, further polymer chains will create more interpenetrating polymer networks decreasing the effec-
90
GRESSEL,
ROSNER
AND
COHEN
tive pore size of the gel. One could thus predict the lower RNA mobility in the gels prepared at lower temperatures (Fig. 4).’ By modifying the temperature of polymerization of the gels, gel systems can be designed to best suit one’s purposes; polymerization at low temperatures for maximal resolution of the rRNA species and polymerization at higher temperatures for best separation of high apparent molecular weight RNAs. The data strongly indicate the idiosyncracies and drawbacks of using rRNA molecular weights to estimate the molecular weights of RNA species with apparently different tertiary conformations from rRNA (see also 8,17). The data also indicate the inherent dangers of constructing processing pathways based on extrapolated molecular weights ( 10). The tertiary structure of rRNA, based on its partly base paired, partly open secondary structure is probably analogous to the classically random coiled proteins, which are used for theoretical studies on gel filtration and electrophoresis. The DNA molecules are probably most similar to rigid rods and putative precursor to open structured single-stranded RNA. It is quite clear from the literature that the relative mobilities of the rRNAs are least affected by changing the parameters of electrophoresis (e.g., 8,15,17). Actually, by changing conditions such as electrophoresis temperature and voltage gradients, Fisher and Dingman (8) were able to demonstrate differences in the absolute mobilities of single and double stranded viral RNAs compared to rRNA standards. Their data brought them to suggest that “the mobilities reflect molecular radii rather than molecular weights,” but this does not completely explain the differential effects. Acrylamide concentration Gas also found to differentially affect the mobility of “single” versus double-stranded RNA (17) but had little effect on the extrapolated molecular weight of putative rRNA precursors (15,17). This supports that what we have is not a change in effective gel concentration, but an effect on the configuration of the fiber maze forming the pores. It is possible that the greater protein mobility found with high running temperatures (6) reflects the higher gel polymerization temperatures and not the running temperatures. The molecular weight of the precursor varied from 2.0 to 2.5 X lo6 with gels prepared from 4 to 35°C (Fig. 1). This is probably due to the fact that one is measuring an extrapolated value and not an intrapolated value, involving a consideration uncertainty. From Fig. 3 one can see that the extrapolation to zero distance of migration at 4°C is 3. I X 1O6 ’ One reviewer offers the following interesting explanation. Shorter polymer chains generated at higher temperatures offer less resistance to the “ballistic” migration of random coiled RNA than longer chains generated at lower temperatures. The effect should be accentuated in direct relation to molecular weight.
GEL
POLYMERIZATION
AND
NA
MOBILITY
91
MW which is not very far from the value of 2.5 X 10” estimated for the precursor in gels polymerized at 35°C. Yet, the extrapolated molecular weight of the origin remains constant despite increases in the duration of electrophoresis. Obviously if we could try a sample of molecular weight 3.1 X lo6 it would move to a certain degree and not stay at zero distance, as we are dealing with an average pore size and not with absolute values. As the relative mobility of the putative precursor varied relative to the five rRNA markers, one can assume that its secondary and tertiary structures differ from that of rRNA. The fact that the mobility of the DNA molecules is unaffected by the pore size [or acrylamide concentration (15)], is probably connected to its rigid structure which enables it to move at an equal rate through decreasingly smaller pores than the open more single stranded RNA. It has been suggested that the DNA aligns parallel to the direction of migration (8). ACKNOWLEDGMENTS We gratefully acknowledge the discussions and cooperation of Yaffa Nevo, who first called our attention to the discrepancies between laboratories. The technical assistance of Ofra Kirsten in part of these experiments helped us immensely. We thank Prof. Moshe Levy of the Plastic Research Department of this institution for his valuable contribution to the part of the discussion of our results concerning the theoretical basis of gel polymerization. An interesting useful correspondence with Dr. A. Chrambach is gratefully acknowledged.
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