6. 34d. Biol. (1975) 95, 309-326
etermination of the Molecular Weigh Saccharomyces cerevisiae Nuclear DN GAIL D. LAUERAND LYNNCKLOTZ Departme& of BiocFYemistry and Molecdar Biology Harvard University, Cambridge, Mass. 02138, U.S.A. (Received 7 January 1975) Viscoelastic retardation-time experiments on the DNA released from spheroplasts of the yeast Xaccharomyces cerevisiae yield a molecular weight of 2 x log for the largest DNA, assuming linear unbranched DNA, and of 4.3 x log assuming circular unbranohed DNA. Both log and stationary-phase cells give the same results. Compa.rison of these results with the nuclear DNA content of X. cerewi&e determined by renaturation kinetics suggests that the largest piece of DNA in the yeast nucleus may, at least during part of the cell cycle, consist of from one-fourth to all of the yeast genome.
1. Introduction Dupraw (1970) has suggested that all the DNA in a haploid eucaryotic nucleus may oonsist of only one very long piece of circular DNA which is condensed over various region.s at metaphase to yield condensed chromosomes connected by non-condensed DNA, Light microscope pictures which show fine connectors between metaphase chromosomes during mitosis (Wilson, 1909,1928, pp. 750,753,765), and electron microscope pictures which show the chromosomes of various cells oriented in circles with very thin connectors between the chromosomes (Dupraw, 1970, pp. 194,195; Costello 1961; Hsu et d., 1967), are consistent with this idea, as are the micromanipulation exieriments of Hoskins (1968) m ’ which the chromosomes~may be pulled out of a living cell at metaphase one after another like a chain of paper dolls. Nuclease experiments (Hoskins, 1968) suggest that the connector between chromosomes may be DNA. Diacumakos et al. (1971) have since repeated some of Hoskins’ work witth the same results. Also consistent with the one piece of DNA per genome idea is the work of Kraemer et al. (1971) which shows that the DNA content of heteroploid cells is nearly constant even though the chromosome number may vary wildly. The main objection to the idea of one piece of DNA in a eucaryotic genome is the existence of genetic linkage groups which segregate independently. In addition, Ruth Kavenoff’s measurements, using the viscoelastic technique, of the molecular weights of the DNA in chromosomes of various species of Drosophila (Kavenoff & Zimm, 1973) suggest that each chromosome contains one piece of DNA. However, this result may not be inconsistent with the Dnpraw idea because DNA linkers between chromosomes might be extremely fragile and likely to break with shear. The viscoelastic technique is especially well-suited to test the idea of one piece of DNA per genome because it is capable of measuring molecular weights of very big DNA molecules (up to 5 X lOlo at present), and it is sensitive to the largest DNA 309
G. D. LAUER
L. C. KLOTZ
molecules in a solution of DNA heterogeneous in size (Klotz $ Zimm, 1972a,b). We chose the yeast Saccharomyces cerevisiae as our experimental organism because it is a eucaryote with a membrane-bound nucleus and well-studied genetic linkage groups and, most importantly, because it has a small genome size (Bicknell & Douglas, 1970; Whitney I%Hall, 1974). P&es & Fangman (1972) have measured the molecular weights of the DNA of S. cerevisiae by sucrose gradient centrifugation and have found that the largest pieces of DNA are l-4 x lo9 daltons. In similar experiments, Blamire et al. (1972) have found yeast DNA with molecular weights ranging from 4x lo8 to 6x 108. More recently, Petes et al. (1973) have analyzed yeast DNA by electron microscopy and have seen molecules between l-2 x lo8 and 8.4 x lo8 daltons. The renaturation kinetics of S. cerevisiae nuclear DNA have been carried out by two groups. Bicknell& Douglas (1970) calculated a minimum genome size of 9.2 x 10e daltons. Recently, Whitney & Hall (1974; personal communication) have found S. cerevisiae nuclear DNA to contain 95% single-copy DNA with a kinetic complexity 2.3-times that of Bacillus subtilis and 5% repeated DNA. Thus, according to these last workers S. cerevisiae contains about 4.8~ log daltons of DNA in a haploid nucleus. For reasons to be discussed later, this last determination of nuclear DNA content appears to be more accurate than that of Bicknell & Douglas. We have determined the size of the largest pieces of S. cerevisiae DNA using the viscoelastic technique and have found the molecular weight to be 2 x lo9 assuming linear, unbranched DNA and 4.3 x lo9 assuming circular, unbranched DNA. Taking 5 x 10s daltons as the DNA content of a haploid nucleus, our results suggest it is possible that, at least during part of the cell cycle, the DNA in the nucleus of S. cerevisiae may consist of two Lear pieces or of one circular piece of DNA. Another possible interpretation of our results is that the yeast nucleus contains one very large chromosome equal to half the nuclear DNA and about 16 smaller chromosomes corresponding to the DNA in the other 16 linkage groups. Other less likely possibilities will also be discussed.
2. Materials and Metho& (a) Strains,
media and growth colzditions
8. cerevisiae strain 522% given to us by Tom Fox was used in all experimems. Growth medium was yeast/Peptone/dextrose (1 g yeast extract, 2 g Peptone, 2 g dextrose/100 ml). Cells were grown in liquid culture at 30°C with shaking. Generation time was about 2 h. For experiments on log-phase cells, cells were harvested in mid-log growth phase; stationary-phase cells were harvested 2 h into stationary phase. The Escherichia coli strain used was C600. Cells were grown in media containing 8 g nutrient broth, 3 g brain-heart infusion, and 1.5 g yeast extract/l (Difoo) at 37°C with shaking, and harvested in stationary phase. Purified bacteriophage T4 was given to us by Bob Horowitz, and purified bacteriophage PBS2 was given to us by Steve Clark.
(b) Preparation of spheropbks Cells were spheroplasted by a modification of the procedure of Blamire et al. (1972). In the procedure all centrifugations were done for 8 min at top speed in an International clinical centrifuge. The cells were kept on ice except during the 30°C and 37°C incubations. For each experiment 100 ml media were inoculated. The cells were harvested by centrifugation, washed twice with deionized water, then resuspended in 10 ml thioglycolate buffer (Blamire et al., 1972) and incubated at 30°C for 45 min. After this incubation the
OF S. CEREBISjiz4.E
cells ‘were washed 6 times with deionized water and resuspended in 30 ml spheroplasting buffer (adapted from Petes & Fangman, 1972): 1 M-sorbitol, 0~1 &n-sodium citrate, 0.01 MXa,-EDTA (pH 5-S). A 20-fold dilution of the cells was counted using a Petroff-Causer bacteria counter, and the cells were spun down, resuspended in 10 ml spheroplasting buffer plus 0~2 to 0.3 ml Glusulase (Endo Laboratories, Inc.) for each 5 x log cells, and incubated at 37°C for 45 min. Then the spheroplasts were spun down, resuspended in 10 ml LET medium (Kavenoff & Zimm, 1973) and a 20-fold dilution counted, spun down again, resuspended in an appropriate amount of LET, and counted again. (0) Lysis
(i) Yeast High salt buffers (Kavenoff & Zimm, 1973). 1 ml cells in LET : 0.5 M-Na,-EDTA, 0.01 M-Tris (pH 7) ; I ml HET-proteinase (pH 9.5), containing 2 mg proteinase K/ml; 1.8 ml NDS : 1% N-lauryl-sarcosine
: LET in BET.
(ii) Yeast Low salt buffers (adapted from Klotz, 1971). 2 ml boric acid buffer containing 1% sodium dodeeyl sulfate; 0.95 ml boric acid buffer f 50 ~1 Pronase solution (40 mg/ml in 0.195 M-NaCl); 1 ml cells in boric a.cid buffer containing 0.9 M-sorbitol. (iii) E, coli Urea buffers of Tom Roberts (Roberts & Klotz, unpublished data). l-8 ml 8 *I-urea, 2 M-NaCl; 0.95 ml HET + 50 ~1 proteinase K solution (20 mg/ml 2 M-NaCl) ; 0.1 ml 20% sodium dodecyl sulfate; 1 ml cells suspended in LET medium.
(iv) T$ Flmge 4 ml. of a mixture of equal vol. of NDS and glycerol containing 2 i~-NaCl ; 0.1 ml Pronase solution (20 mg/ml in 2 M-NaCl); 0.03 ml total vol. of T4 suspension plus phage buffer without T4 to obtain desired T4 concentration.
(v) Fhage PBS2 3.3 ml of a mixture containing equal vol. of NDS and glycerol containing 2 x-NaCl; W-1 ml Pronase solution (20 mg/ml in 2 M-NaCl) ; 0.2 ml total vol. of PBS2 suspension plus phage buffer without PBS2 to obtain desired PBS2 concentration. (d) Preparation Lysates cedures.
in the chamber
of the viscoelastometer
by the following
ii) Yeast high salt Zysates W&h the chamber at 5O”C, the NDS and HET-proteinase buffers were pipetted into t,he viscometer chamber and mixed thoroughly by running through a pipette several times. Then the 1 ml of spheroplasts in LET was pipetted forcefully into the chamber in order to distribute the cells well Lysates were incubated overnight at 50°C and then the rotor was placed in the chamber and measurements were made at 50°C. (ii) Yeast loul salt lysates The procedure was exactly the same as that used for the yeast high salt lysates except for the change in lysis buffers-2 ml boric acid buffer containing 1% sodium dodeeyl s&fake in place of l-8 ml of NDS, 0.95 ml boric buffer + 50 ~1 Pronase solution in place of 1 ml of HET-proteinase, and 1 ml cells in boric acid buffer containing 0.9 &I-sorbitol in place of 1 ml cells in LET. (iii) E. coli &sates ‘With the chamber at 50°C the urea, HET-proteinase buffer, and sodium dodecyl sulfate solutions were pipetted into the chamber and mixed thoroughly by pipetting. The cell solution was added and mixed by immediately running the solutions in the chamber
6. D. LAUER
AND L. C. KLOTZ
through a pipette once. Lysates were incubated overnight at 50°C, and then the rotor was placed in the chamber and measurements were made at 50°C. (iv) Phages T4 and PBS2 All solutions except the pha.ge were mixed in a test tube on ice. Then the phage suspension was added and mixed into the lysis solutions by gently rolling the test tube on ice for 15 min. The mixture containing unlysed phage was pipetted into the chamber, which was at 2O”C, and in order to lyse the phage the temperature of the chamber was raised to 60°C overnight for T4 and 50°C for at least 5 h for PBSB. The temperature of the chamber was lowered to 20°C to make readings. In all these lysis procedures, except for the low salt lysates of yeast, the final Na’ concentration is 2 M. In the phage experiments, glycerol and a reduction of the temperature to 20°C were used to lengthen the retardation times until they were easily measurable (Chapman et al., 1969). (e) Determination of retardation times All experiments were carried out on double-stranded DNA. 4 to 6 measurements of retardation time were made on each lysate. In all experiments we were careful to work at sufilciently low shear stresses and DNA concentrations so that dilatency (i.e. anomalous increases in retardation times) did not occur (Massa, 1973; Klotz & Zimm, 19723). For the E. co& and bacteriophage experiments, in which the DNA was expected to be homogeneous in size, retardation times were calculated from the slopes of the linear semilog plots of rotor angle versus time. For the yeast cell lysates, retardation times were calculated as above and by an integral method which does not require linear semilog plots. For the slightly curved semilog plots seen in more than half of our yeast experiments, it was difficult to find an “average” slope in an unbiased way; therefore, a method to obtain retardation times using the area under the raw relaxation curves was used. The area under the relaxation curve from t = 10 s after the relaxation was begun (i.e. after the electromagnets driving the rotor were turned off) to t = co was measured using a planimeter (Keuffel and Esser Co., model no. 620015). Exactly how one calculates a retardation time from this area is described in detail in Muller & Klotz (1975). The only point to be made here is that this method yields a rather complicated retardation-time average, the value of which is close to the retardation time for the la!rgest DNA molecules in the lysate, provided that they comprise 10 to 20% or more of the total number of DNA molecules in the lysate. (f) DeJnition
7 is the symbol for the raw retardation-time values measured from the lysates, uncorrected for buffer viscosities and temperature. TOis 7 extrapolated to zero DNA concentration and is obtained from a 7 versus concentration plot. 7i,50 is TO corrected to the standard oonditions of 50°C and water as the solvent by the formula 0.00549T 0 TO ~w,m= 3237 from Kavenoff & Zimm (1973). In this formula, T is the absolute temperature, solvent viscosity, and 0.00549 is the viscosity of water in poises at 323°K.
(1) 7 is the
3. Results (a) Standardizatiofi
curve for 2 iw-Na+
The volumes occupied by DNA molecules in solution, and therefore the retardation times of DNA solutions, are dependent upon salt concentration (Scruggs C%Ross, 1964 ; Ross & Scruggs, 1968; Rosenberg & Studier, 1969). To help inactivate nucleases we wanted to do the yeast molecular weight experiments in O-5 IVI-EDTA. With this concentration of EDTA, the concentration of Na+ in the lysate is 2 M. However, alI
0 i 0
I oi 0
I 20 used
Fra. 1. Retard&ion times in 2 nn-Na+ plotted against concentration for lgsates of ,!Zy. c&, T4, and PBSZ. (a) E. c& data. Experiments done rtt 5O’C. TO = 100 s. Plotted on tho abscissa wo the optiod densities at 540 nm of the cell suspensions used in the lysstes. (b) T4 data. Experiments done at 20°C. r0 = 6.25 LE.Plotted on the absoissa we the amounts of ihe phage suspension used. The titer of the pbage suspension was 2.3 x 1013 psrtioles/ml. (0) PBS2 date. Experiments done at 20°C. TO = 13.3 s. Plotted on the abscissa are the amounts of the phage suspension used. The titer of the phage suspension wais 2 x 10:s particles/ml. Thus the abscissa values we ail1 proportional to DNA concentration.
G. D. LAUER
the theoretical work on retardation times and the calibration of the linear plot for log 7i,50 verszbslog M, where M is molecular weight, had been done in O-195 M-Na’ (Klotz & Zimm, 1972a,b). Therefore, in order to be able to calculate molecular weights of yeast DNA from retardation times, it was necessary to first generate a relation for log ~c,~” versus log M in 2 M-Na+ by doing retardation-time experiments on organisms for which the molecular weights of their DNAs are already known. In Figure 1 are plots of r versus concentration for the three organisms used to standardize the 2 M-Naf curve. Extrapolations to zero DNA concentration give values for TO for E. coli DNA of 100 seconds, for T4 DNA of 6.25 seconds, and for PBS2 DNA of 13.3 seconds. Although the abscissa in Figure 1 are not DNA concentrations, they are quantities (see Fig. 1) which are directly proportional to DNA concentrations, and therefore T will extrapolate to the same value. Correction of the Q-Ovalues to the standard conditions of 50°C and water as solvent yields 45.8 seconds for E. coli DNA, O-277 second for T4 DNA, and O-433 second for PBS2 DNA. Figure 2 is the plot of log M versus log ~k,~,,. The values used for the molecular weights of the DNA of the known organisms are 2-7 x 10s for E. coli (Klotz & Zimm, 1972b; Cairns, 1963), 1*08x lOa for T4 (Freifelder, 1970), and 1.5~ lo8 for PBS2 (Lauer & Klotz, unpublished data). The solid line is a least-squares fit drawn through the three experimental points. The line has a regression coefficient of 1.00, indicating that the three points fall on a good straight line. The equation relating 7k,50 to M from the line is -T:,~,,= 3+3x lo-l4 M1.6o or M = 2-45x 108(r$,60)o.63.
j A least-squares line drawn FIG. 2. Plot of log M veraus log T&, for 2 M-N&+. ( through the E. coli, PBSB, and T4 points generated by using our retardation-time data and standard molecular weight values for the DNA of the three organisms (see text). (-----) A plot of log M w?w&s log -rt,50 for 2 M-N&+ calculated from Ross & Scruggs’ (1968) viscosity data (see text). The 7:,50 for D. melanogmter is that found by Kavenoff & Zimm (1973). The molecular weight crosslines for D. mekmogaster correspond to 4.3 x 10 lo, the value found by Rudkin (1965a,b) for the molecular weight of the largest chromosome of D. melanogaster and 3.4x 10r”, the molecular weight of the largest chromosome of D. meEanogaster from Rudkin’s (196&b) data on the proportion of the total nuclear DNA which is in the largest chromosome and Laird’s (1971) renaturation kinetics data on the total amount of DNA in a haploid set of chromosomes. (A) Our values for retardation times of S. cerevisiae DNA for both log and stationary-phase cells, each calculated by both the semilog and integral methods (explained in text).
OF S. CEREVISPAE
The dashed line is a plot for 2 M-Na+ calculated from the viscosity data of IlRoaa & ruggs (1968) in the following manner. The largest retardation time, TV, for a DEB. lecule may be expressed in terms of intrinsic viscosity as
where a, = 0449 for linear DNA is evaluated from theory (Klotz $ Zimm, 1972a), 77is the solvent viscosity (7 = 0.00547 poise for water at 50°C), and RT is the gas constant times absolute temperature. In this equation [T] is the intrinsic viscosity for DNA of molecular weight M. Intrinsic viscosity can usually be related to molecular weight by an equation of the form (see, e.g., Tanford, 1961, p. 407) [q] = KMa . We have plotted Ross and Scruggs’ double-stranded DNA in 1 M-Na+ at log Jf to obtain a = 0.606 and K = 25”C, their Figure 3 indicates that it Pinally, substituting equation (3) into
intrinsic viscosity data from their Table I for 25°C (Ross & Scruggs: 1968) as log[q] cera?ns 0.301. Although this data is for 1 M-Na” at is valid for 2 M-Naf at 50°C our conditions. equation (2) yields
for 2 Iti-Na + . Inversion of this equation gives M = 2.75 x lo* (+&$‘~‘=a ,
which is the equation which is plotted as the dashed line in Figure 2. The dashed line from Ross & Scruggs’ data (1968) is close to the line generated by our data. For example, T$,~~of 10 seconds yields M = 1.05 x log using the solid line and H = 1*15x lo9 using the dashed line. It is interesting that when the value of 7i,50 measured by Kavenoff & Zimm (1973) for Drosophila naelasogaster is put on the graph, the measured 7c,50 is too small for the expected molecular weight of D. melanogaster chromosomal DNA. The predicted molecular weight for the largest chromosome of D. melanogaster is 4.3 x lOlo (Rndkini 1965a,b) or 3.4 x lOlo if one uses Rudkin’s data to determine the proportion of t,he total nuclear DNA which is in the largest chromosome and Laird’s (l97I) renaturation kinetics data on the total amount of DNA in a haploid set of chromosomes, However, the measured 7’W,50,when placed on our graph, yields a molecular wei.ght of 2.66 x IWO r l?ossible explanations for this could be that the plot of log ~i,~e verszls log M is not hear for very large DNA or that Mavenoff & Zimm (1973) were seeing slightly smaller than chromosome-sized DNA in their 2 M-Na+ experiments.
(b) ControEs (i) Heat treatment A lysate of yeast spheroplasts was prepared as described above, and were made which yielded retardation times averaging 56.7 seconds. Then was removed, the chamber heated to 95°C for 15 minutes then cooled to rotor replaced, and the retardation time measured. In control experiments
four runs the rotor 5O”C, the the same
G. D. LAUER
procedure was repeated with the heat treatment omitted. In the heated sample the whole viscoelastic effect had disappeared, while in the unheated control no change had occurred in the retardation time, a result which is consistent with heat denaturation of DNA. (ii) M&i and strong shear treatment A lysate of yeast spheroplasts was prepared as described above and four retardation-time measurements were made which yielded an average of 62.6 seconds for 7. Then the rotor was removed and the lysate was vigorously stirred with a 100~~1 pipette for about ten seconds. The rotor was replaced and the retardation time measured. Only a very small intensity of relaxation remained, and the retardation time had decreased so much as to be unmeasurable. The rotor was then removed again, and the solution was pipetted rapidly three times with a Pasteur pipette. The rotor was replaced, measurements were made, and it was observed that all relaxation had disappeared. In control experiments in which the chamber was opened, the rot,or removed and replaced, and measurements made, there was no change in retardation time, a result consistent with shear breakage of DNA. (iii) DNAase treatment We have attempted to do the DNAase control in both low and high salt lysates, using Worthington DNAase I (bovine pancrease, RNAase free, code DPFF) in the low salt lysate and Worthington DNAase II (porcine spleen, code HDAC) in the high salt lysates. So far we have been unable to do this control-precipitation occurs when the DNAase solution is added and we see no DNAase activity as measured in the viscometer. We believe we are looking at DNA because of the shear and heat controls. However, we are continuing work on trying to adjust our lysis solutions so that DNAase will work in the lysates because we wish to use the kinetics of DNAase action to determine the structure of the DNA we are measuring. (iv) Effects of varying thioglycobte and spheropbsting times on size of DNA In order to be certain that our spheroplasting procedure was acceptable, the times that the cells were incubated in the thioglycolate buffer and in the spheroplasting buffer were varied. The following combinations of times were tested: 15 minutes in thioglycolate + 45 minutes in spheroplasting buffer; 60 minutes in thioglycolate + 45 minutes in spheroplasting buffer; 90 minutes in thioglycolate + 45 minutes in spheroplasting buffer; 45 minutes in thioglycolate + 30 minutes in spheroplasting buffer; and 4.5 minutes in thioglycolate + 60 minutes in spheroplasting buffer. In each of the above cases, the retardation time measured was equal to or smaller than that measured with the conditions normally used (45 min in each buffer). Therefore, our spheroplasting procedure seems to be optimized with respect to incubation times in the two buffers. (v) Hydroxywea treatment Since the spheropla,sting procedure takes about four hours, we wondered whether it was possible that DNA replication was completing in the log-phase cells during the spheroplasting procedure and giving us a low value for the size of the DNA in log cells. Hydroxyurea is an inhibitor of DNA synthesis in yeast (Slater, 1973). Therefore, several experiments on log cells were run in which all the water used for washing the
OF 8. CERETISIAE
cells and the buffers used for incubations contained 0.1 M-N-hydroxyurea (K and IX Laboratories, Plainview, N.Y.). The retardation times calculated from these experiments were the same as those calculated from experiments done without the inhibitor. Therefore, whether or not DNA replication was being completed in log cells during our normal spheroplasting procedure without the inhibitor, the effective size of the DRA as seen by viscoelastic measurements is the same in stationary-phase cells and in log-phase cells which have their DNA replication arrested by the inhibitor. This is a result which might be expected from the electron microscope pictures of Xewlon et al. (1973) and of Petes et al. (1973) which show that yeast DNA replication is like that of other eucaryotes in that it ooours by multiple internal replication bubbles, since such bubble structures would have about the same retardation times as the linear structures from which they are derived (Muller & Mlotz, 1975). (vi) Stability of lysates Retardation times on lysates have been measured for periods of up to five and no change has been observed. Thus, the lysates are stable. (vii) iiow salt 1ysaCes In order to test whether our yeast results were dependent on salt concentration, we did two experiments on yeast in low salt (0.195 i?f-Na+) buffers. Using the formula M = 2.2x 108 (+,,,)“.“a from Klotz $ Zimm (19726) for calculating M from ~c 50 for experiments done on 0.195 M-N&+, we found M = 2 X 10Q, the same molecular weight as we measured in the high salt experiments. Therefore, our result is not dependent on salt concentration. This also indicates that aggregation of DNA, either due to DNA entanglements (which should occur more readily in high salt due to decreased repulsions of negati-Tely charged DNA strands) or due to a positively-charged protein-mediated aggregation cf DNA (which should occur more readily in low salt), probably is not occurring. times and molecular weights jar DNA from 8. cerevisiae log and statiomry-phase cells
Figure 3 shows two semilog plots of rotor angle versus time for typical measurements made on S. cerevisiae DNA. In about half the S. cerevisiae cell Iysates the semilog plots were linear as in Figure 3(a), indicating that the bulk of the large DNA was reasonably homogeneous in size. The semilog plots for the remaining lysates were curved as shown in Figure 3(b), indicating heterogeneity in the size of the DNA in the lysates. For these curved plots, a line was drawn as a tangent to the curve going through the part of the curve that approached linearity. The slope of such a, line is somewht arbitrary, so all 7 values were also calculated by the integral method described above. All relaxation curves reached a level baseline within a time less than. 4r, indicating no large aggregates of DNA. Figure 4 contains the plots of r versus DNA concentration for log-phase S. cerevisiae for 7 values calculated by the two methods used. Figure 5 shows the same two plots for stationary-phase cells. In each case there seems to be no concentration dependence ; therefore, TOvalues were calculated as number averages of the Q-values. The average values found for the retardation times for the DNA from S. cerevisicce log and station.. ary-phase ceils in 2 Al-Nit+ are in Table 1.
G. D. LAUER
3. Typical semi-log plots of rotor angle in arbitrary units vt~sz~s time for S. cerevisiae retardation-time experiments done in 2 M-Na+ at 50°C. (a) Linear plot typical of those found in about half the lysates and indicating that the bulk of the large DNA was reasonably homogeneous in size. (b) Plot which curves off, indicating that there may have been some DNA in the lysate larger than that which we measured. FIG.
As expected, the semilog method of calculating T values yields slightly higher values than does the integral method. This is because the semilog method “sees” only the largest molecules while the integral method yields an average which weights the largest molecules very heavily. It is evident from the error bars on Figures 4 and 5 that we were able to measure retardation times very precisely, with the average deviation in the values measured at any given concentration always less than &eight seconds and in more than half the lysates less than &five seconds. The scatter in the data might in part be due to slight variations in spheroplasting and lysis procedures. If this is the case, perhaps further experimentation with lysis procedures may yield larger DNA. The molecular weights corresponding to the 7$,s0 values, assuming the DNA measured is linear, are found by plotting the log -rz,50values on the experimentally derived plot of log M vers’us log 7t,50 in Figure 2 (triangles), yielding the values for M for linear DNA in Table 1. If the large DNA we are measuring is really circular, then the molecular weight for a given $,s,, is 2*1× what it would be for linear DNA (Klotz & Zimm, 1972aJ). Values for M, assuming circularity of the DNA, are also shown in Table 1. It is evident that the values for 7; ,.OOcalculated by the semilog and integral methods give nearly the same molecular weights and that the molecular weights found for the DNA from S. cerevisiae log and stationary-phase cells are, within experimental error, equal.
1 old 0
I.0 Cell concentration
FIN. 4. Retardation times at 50°C in 2 M-N&+ WXYUS concentration for lysates of log-phase 8. cereG.siae. In (a) 7 values were calculated by the semilog method; in (b) 7 values were caloulatstl. by the integral method. Each point is the average of between 4 and 8 retardation-time measurements made on a given lysate. The error bars show the average deviation of the measurements made on a lysate from their average.
IO Cell concentration
FIG. 5. Retardation times at 50°C in 2 M-Na+ VW~‘SZIS concentration for lysates of stationaryphase S. cerewisiae. In (a) 7 values were calculated by the semilog method; in (b) 7 values were calculated by the integral method. Each point is the average of between 4 and 8 retardation-time measurements made on a given lysate. The error bars show the average deviation of the measnremerits made on a lysato from their average.
TABLE 1 Betardatior,
times and molecuhr
weights of 8. cerevisiae
M (x 10-y
&o(s) 2 M-h-a+
Log cells Semilog method Integral method
4.69&0,43 4.03 AO.35
Stationary cells Semilog method Integral method
28.713.7 23.9k4.0 [email protected]
The error estimates in TO values are standard deviations of the average 7 values for each cell concentration from the TO line, which is found by averaging the average values of 7 measured at the different cell concentrations. The values of ~i,~,, are found by dividing the TO values by 1.98, the relative viscosity of the buffer to water. The standard deviations in M are found from the relation between M and ~i,~~, by differentiation. Although we have chosen 2.7 x lo9 for the molecular weight of E. coli DNA, values in the literature range between 2.5 and 2.9 x log. If this variation is added to our values, the standard deviation in the molecular weight of X. cerevisiae DNA becomes about O-3 instead of about 0.2 for linears.
Because of this we have averaged the molecular weights calculated by the different methods to yield 1*98~ 10Q or about 2 x log, assuming linearity of the DNA, and 4.26 x 10” or about 4.3 x log, assuming circles for the size of the largest piece of DNA in the yeast nucleus. Because the log M versus log TO, w 5,, points for the DNA from the three organisms used to generate the 2 M-Na+ graph fall on a straight line, it is unlikely that our yeast molecular weights contain much error due to error in the 2 ix-Na+ relation. Also, because the log .M versa8 log 7i,50 line which we have generated for 2 M-Na’ covers a range including DNA of molecular weight 2 x log, our yeast molecular weight values lack the extrapolation error which would be incurred when working with DNA larger than that used to generate the log M versus log 7i,50 relation. The biggest error in our M values for yeast is probably due to our uncertainties in ~i,~~. These uncertainties in M are expressed as standard deviations in M in Table 1 and are calculated from the standard deviations in 7k,50 also in Table 1.
4. Discussion Our value of 2 x log for the molecular weight of the largest DNA in the nucleus of X. cerevisiae, assuming the DNA is linear, is larger than results obtained previously by other workers. Using sedimentation techniques, Blamire et al. (1972) found molecular weights for yeast nuclear DNA between 4 x 1Oa and 6 x lOa. In similar experiments, Petes & Fangman (1972) measured values between 5x lo7 and 1-4~ log, with zb number-average molecular weight of 6 x lOa.
eceuse of the known strong dependence of sedimentation coefficient on centrifuge rotor speed and on molecular weight (Kavenoff, 1972; Rubenstein & Leighton, 1971; Levin & Hutchinson, 1973), we reasoned that the molecular weights measured by previous workers may have been low because of too high rotor speeds; previous experimenters expected to find DNA in the molecular weight range of about 5 x IO8 to 7 x IO”, not 2 x log, and adjusted their rotor speeds accordingly. In the calculations below, we show that Blamire et al. (1972) and Petes C%Fangman (1972) would no-k have seen DNA in the 2 x log range because of too high rotor speeds. The equation derived by Zimm for the change in observed sedimentation coeEcient with rotor speed (see Kavenoff, 1972) is 1.369 x 1O-48 M4(1 - 0f)4X2(revs/min)4 T2(sO)2$
3 (6) > where B is partial specific volume of DNA, p is solvent density, X is the distance from the center of rotation to the DNA band, revs/mm is the centrifuge speed in revs/min, s is the observed sedimentation coefficient at low centrifuge speed and How concentrations and so is s extrapolated to centrifuge speed of zero. This equation may be rearranged and simplified when (revs/min)4 is sufficiently small such that s is not less than about 80% of so to yield ds = so _ s = KM4(revs/rW4 so
From these two equations, one can then 6nd the following expression for K: K = 1*369xlO-48(1
T27)2 In alI subsequent calculations we use the distance from the center of rotation to the middle of the centrifuge tube, as X. For Kavenoff’s conditions (1972), X = 10.8 cm, T = 1.61 cps (17of 15% sucrose at 20°C), p = 1.059 g/cm3, B = 059, and T = 295°K. Therefore, K = 1.4~ 10-4g. The conditions of Blamire et al. (1972) were almost exactly those of Kavenoff: X = 10-8 em,? = 1.61 cps, p = l-059 g/cm3, B = 0.59, and T = 291°K. Correcting the value for K obtained for Mavenoff’s conditions for the temperature change yields K = 1*44x 10-49. To calculate the error which would result in the measured sedimentation coefYicient for DNA of 2 x lo9 under the conditions of Blamire et al. (1972), we substitute so =: 159 S, the so value found by Kavenoff (1972) for B. szcbtilis DNA, which has a mole.. cular weight of 2~ 10’ (Klotz C%Zimm, 1972b; Wake, 1973), X = 1.44~ 10V4$, and revs/mm = 11,000 into equation (7). This yields the impossible result that ds = 212 S, which implies s is less than zero. The experiment of Blamire et al. (1972) is thus out of the range of Kavenoff’s s vers’us (revs/min)4 plot (Kavenoff, 1972) and Blamire et al. (1972) were working under conditions where DNA of 2 x log molecxlar weight shows incredibly large deviations in s. Substituting M = 5~ lo8 and so = 100 S, the values Blamire eEal. (1972) found for their main band DNA, into equation (7) yields ds = 1.32 S, an insignificant deviation. Thus, the centrifuge speed used was good for DNA of 5 x lOa daltons, the size of DNA the experimenters expected to find. Petes & Fangman’s (1972) conditions were quite different from those of Kavenoff 721, with X = 7.3 cm, y = 2.41 cps, p = 1.09 g/cm3, @= 0.59, and T = 278”K2,
G. D. LAUER
For these conditions K = 2.63 x lo- 50. TO find da for DNA of 2 x lo9 daltons under these conditions, we substitute K = 2-63 x 10m50,so = 159 S, and revs/n& = 10,000 into equation (7) to get As = 265 S. Thus, for DNA of 2 x log daltons, Petes $Fangman would have found an s value 26.5 S too low. When 26.5 S is added to 134 S, the largest s value found by these experimenters for yeast DNA, the sum is 160.5 S, which is almost exactly the so value found by Kavenoff (1972) for DNA of molecular weight 2 x 10s, the size of DNA we find in yeast. It is interesting to compare our value of 2 x lo9 daltons (assuming linear DNA) for the size of the largest DNA in the 8. cerevisiae nucleus with the DNA content of 8. cerevisiae nuclei as determined by both DNA per cell measurements and by renaturation kinetics experiments. Prom DNA per cell experiments, the DNA content of a haploid yeast cell has been found to be 1.2 to 1.3 x lOlo daltons, and the DNA content of a haploid spore has been found to be 8.4 x 10s daltons (reviewed by Hartwell, 1970). However, these results have been obtained by methods in which it is impossible to do the proper control, namely a control for positively interfering substances by doing the experiment on cells minus their DNA. In addition these values should be corrected for 5 to 20% mitoehondrial DNA and 1 to 5% 2 pm DNA (Hartwell, 1974). For these reasons we believe that values for nuclear DNA content obtained from renaturation kinetics are more accurate. Two groups have determined the DNA content of X. cerevisiae nuclei by renatnration kinetics with very different results. Bicknell & Douglas (1970) calculated a minimum genome size of 9.2 x 10s daltons. More recently, Whitney & Hall (1974; personal communication) have found 95% single-copy DNA with a kinetic complexity 2*3-times that of B. mbtilis and 5% repeated DNA; assuming 2 x PO9for the molecular weight of B. sabtilis DNA (Klotz & Zimm, 19723; Wake, 1973), Whitney & Hall’s (1974) result yields 4-8x IO9 daltons for the DNA content of a haploid yeast nuoleus. It is evident that interpretation of our results is very dependent upon the DNA content of the 8. cerevisiae nucleus and upon whether we are measuring linear or circular DNA. Because we do not know the true vaIue for nuclear DNA content and are not certain of the form of the DNA we are measuring, all the various possible interpretations of the data will now be discussed. Taking 4-8 x lo9 daltons as the true DNA content of haploid X. cerevisiae nuclei, our results suggest that the DNA in the nucleus may consist of one or two pieces of DNA, depending on whether the DNA is circular or linear. This is surprising oonsidering the genetic evidence that 8. cerevisiae has 17 linkage groups (Mortimer & Hawthorne, 1973). However, any inconsistency between the existence of genetic linkage groups and the finding that all of the yeast nuclear DNA may be contained in one piece disappears if the yeast nuclear DNA consists of chromosomes connected by non-gene-containing DNA lengths longer than 50 map units. Then genes on two chromosomes connected by a linker would appear to assort randomly because of frequent crossing-over between them, just as “characters due to factors placed far apart on a chromosome assort almost at random due to crossing-over between them” (Whitehouse, 1973, p. 86). At 6rst glance, the idea of one or two pieces of DNA per nucleus, which we arrive at by comparing our results with Whitney & Hall’s (1974), also seems incompatible with the work of Finkelstein et cal. (1972) and Cryer et al. (1973) on the location of genes for ribosomal RNA on chromosome 1. Their work suggests that there are
OLECULAR WEIGHT OF S. CEREVISIAE
&net chromosomes which are distributed according to size in a sucrose gradient. owever, our data does not necessarily contradict these experiments. It is possible that all the DNA in the nucleus is contained in one or two piece(s) of DNA which is (are) condensed for various regions along its (their) length(s) into chromosomes. If this is so, then the pieces of DNA linking the chromosomes may be very sensitive to shear and may break under most handling conditions. Breakage of these weak linkers would yield chromosomes which would be distributed according to size in a sucrose gradient. Very likely, some linkers may be broken while others remain intact so that some very big DNA is found as in Petes & Fangman’s (1972) experiments (see our calctala,tions above). It is possible that the DNA content of the S. cerevisiae nucleus is closer to the value of 9.2 x log daltons found by Bicknell 83;Douglas (1970) than to the lower value found by Whitney $ Hall (1974). If we assume that the amount of DNA in a chromosome is proportional to its map length, then the largest chromosome should contain 13% of the DNA in the nucleus (Mortimer L%Hawthorne, 1973). 13% of 9.2 x IO9 daltons is 1.2 x log daltons. In contrast, 2 x log daltons is 22% of 9.2 x 10g daltons, or almost ii, quarter of the genome. That 1.3 x log daltons is significantly smaller than 2 x [email protected]
’ daltons in our experiments is evident from the relations between 7 and X: Q-cc LW*“~* and M CC;r”.63. From these relations it is evident that a small difference in H values corresponds to a much larger difference in 7 values. (The opposite, of course, is trne for sedimentation. s oc .M”.38 ) so M cc s2.63, and a small error in s leada to a larger error in H.9 It Lay be that the largest chromosome is larger than is indicated by genetics. However, we are measuring DNA which, depending on what value is used for DNA content of the nucleus and on whether the DNA is linear or circular, contains between one-fourth and all of the S. cerevisiae genome. Thus, if we are measuring a single chromosome, it is an unusually large one. It should be emphasized that we are probably getting some DNA breakage in our experimen.ts. Since the viscoelastometer sees only the largest molecules, we would not measure mechanically broken material. It is even possible that the DNA in a yeast nnoleus is larger than 2 x log daltons, the value which corresponds to our measured ,* if we assume linearity of the DNA, or 4.3 x 10g daltons, the value which corresponds to onr measured TO if we are measuring circular DNA (Klotz & Zimm, 1972ii). The fact that over half of our semilog plots were gently curved (Fig. 3) suggests that there may have been bigger DNA in our lysates than we measured. If breakage is significant so tha,t more than about 95% of the DNA molecules are brokea, it is hard to measure the unbroken DNA (Klotz & Zimm, 1972a,b). Even though we attempt to lyse the cells gently and thereafter treat the lysates gently, we may still be breaking most of the DNA. Burgi & Hershey (1961) showed that breakage of DNA at a critical rate of shear produces fragments whose number-average length is about half the length of the unbroken molecules. In other words, DNA tends to break in half and then in half again. In the light of these results, it is interesting that the molecular weights we measure are about one-half or one-quarter of the molecular weight expected for a genome-size piece of DNA, if the DNA is linear. If the DNA in the iysates is circular, Burgi & Hershey (1961) show that we would probably never measure the fulLsize &ears which would result if circles were broken because these full-size pieces of DNA would ‘be so rapidly broken down to half or quarter-size pieces that they would seem l;o break as part of the same kinetic event which broke the circles. Although we do not want to firmly support any interpretation of our results, we 22
G. D. LAUER
feel that Whitney & Hall’s (1974) renaturation kinetics probably yield a value which is closer to the true DNA content of the S. cerevisiae nucleus. Whitney & Hall (personal communication) have done the renaturation kinetics both optically and on hydroxylapatite, and their experiments seem to have been carried out ca,refully. Moreover, Whitney & Hall (1974) used B. subtilis, which has a G+C content very close to that of yeast, for their complexity standard, while Bicknell $ Douglas (1970) used E. coli, which has a G+C content much higher than that of yeast, for their standard and made no attempt to see if the G-!-C content had an effect on the intrinsic renaturation rate. We also believe that we are probably measuring linear DNA because previous work with bacterial DNAs (Klotz & Zimm, 19723) suggests that we would not have circular DNA in the machine under our conditions (if the genome is bacteriallike). It is possible that the largest pieces of DNA that we are measuring are halfmolecules. Yeast is in many ways a typical eucaryote, for it has many euoaryotic features, including mitochondria, a membrane-bound nucleus, and genetic linkage groups. Tamaki (1965) has even published light micrographs showing a number of condensed regions of chromatin at mitosis equal to the number of genetic linkage groups; however, his pictures, although the best micrographs of yeast chromosomes published to date, are not totally convincing. More recently, Peterson & Ris (1974) have studied mitosis in X. cerevisiae with the electron microscope and found it “comparable in its general aspect to that observed in typical eucaryotes”. However, Peterson & Ris found that chromosomes do not condense at mitosis and are not individually visible. This observation suggests that yeast may not be a typical eucaryote. Indeed, in various ways yeast seems to be a very unusual eucaryote. Both haploid and diploid cells undergo mitosis, which is not true of most other eucaryotes (Hartwell et al., 1974). Cell division by budding and nuclear migration are found in yeast but not in most other eucaryotes (Hartwell et al., 1974). Though yeast have mitochondria, they can live very well without respiring and without mitochondrial DNA (Linnane et al., 1972). The ribosomal RNA genes for 18 S, 28 S and 5 S RNA are linked together (Cyer et al., 1973; Rubin & Sulston, 1973) as is found in bacteria (Smith et al., 1968) but not in higher organisms (Wimber & Steffensen, 1971; Pardue et al., 1973; Pardue, 1973). To our knowledge, renaturation kinetics of yeasts are the most similar to those of bacteria of any eucaryote (Whitney & Hall, 1974; Bicknell & Douglas, 1970; Christiansen et al., 1971; B&ten & Kohne, 1968). Thus, although a eucaryote, yeast certainly displays some procaryotic characteristics. We have detected DNA molecules in 1.9.cerevisiae which represent either one-fourth or, if Whitney & Hall’s (1974) results are correct, half or all of the genome. At the present time, we cannot distinguish which of the above possibilities is correct. However, we feel that our data, in conjunction with the unusual properties of yeast mentioned above, demands consideration of and more definitive experiments regarding the possibility of DNA molecules larger than chromosome size.
We wish to thank Bruce Doran for helping to keep the machines in working condition, Dr Patricia Whitney for sending us her data in advance of publication, and Tom Roberts for many helpful discussions on various aspects of this work. This research was supported by grant GB34293 from the U.S. National Science Foundation. One of us (G. D. L.) is a predoctoral fellow of the U.S. National Science Foundation.
OF X. CERE VISIAE
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