Nuclear density determination and the purification of wild type Neurospora nuclei using Percoll gradients

Nuclear density determination and the purification of wild type Neurospora nuclei using Percoll gradients. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This technical note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol27/iss1/20 We compared the sensitivity of this procedure to two other cornnon protease assays using hemoglobin (Kunitz 1947 J. Gen. Physiol. x:291) and casein yellow (Anson 1938 J. Gen. Physiol. z:79). The latter assays are short-term so we modified our procedure to include a reaction tenination step, adding 0.5 ml of 10% Trichloroacetic acid at appropriate time intervals. We found that azocoll is approximately loo-fold nmre sensitive, over a 30 min interval, to hydrolysis by trypsin than are either casein yellow or hemoglobin (Figure 3). To demonstrate further the sensitivity of the azocoll assay, we assayed several commercial proteases over a ten h. period and readily detected one nanogram quantities of trypsin, subtilisin, and thermolysin (Sigma). The solid reaqent disoenser qreatlv reduces the time needed to measure &x011 &r ea& &&ion mixture and makes feasible the use of azocoll for investigations requiring large numbers of protease assays. This method of measuring azocoll is both rapid and accurate (machine error is +4%). The dispenser could readily be used to measure other insoluble substrates. Iloreover, by using teflon rods with suitablv sized holes drilled in them. one could dicpense different amounts of solid substrates. (This research was sponsored jointly by NSF Grant PCM 76.80227 and the Office of Health and Environmental Research, U. S. Dept. Energy Contract W-7405.eng-26 with Union Carbide). Universitv of Tennessee-Oak Ridqe Graduate School of Biomedical Sciences and Biology Di;isian, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830. Figure 3. -Comparison of three short-term protease assays. Hemoglobin, casein yellow, and azocoll are compared as substrates for trypsin over a short time interval. The hemoglobin (0 ) and casein ( l ) reaction mixtures each contained 1% substrate and 0.1% (1.0 mg/ml) trypsin. The azocoll reaction mixtures contained 18 mg of substrate and either 0.01 mg trypsinfml (A ) or 0.033 mg trypsinlml (A ). Reactions were terminated by addition of 10% trichloroacetic acid. Ialbat, K. and P. J. Russell. Nuclear density determination and the purification of wild type Neurospora nuclei using Percoll gradients. The study of precursor ribosomal RNA (pre-i-RNA) maturation in ribosame biosynthesis mutants of N.crassa _~ is facilitated by the isolation of RNA from purified nuclei. Problems have been encountered in attempts to purify nuclei with Ludox gradients. Specifically, Ludox precipitates at low temperatures when exposed to Triton X-100, which is an essential component of the buffer used in the nuclei isolation steps. Therefore, a new gradient medium, Percoll (Pharmacia Fine Chemicals, Piscataway, N.J.) was tested for its applicability. The use of Percoll rather than Ludox eliminated probelms with precipitation. In addition it was possible to determine the buoyant density of the nuclei accurately, since the colloidal silica particles are coated with polyvinylpyrrolidone to which the nuclear membrane is impermeable. Flasks of liquid Vogel's minimal medium inoculated with wild type conidia (2x107 ml-') were incubated for 8 h. at 25'C. Crude nuclear pellets were prepared from these mid-logarithmic phase cultures using a modiy Hautala gal. (1977 J. Bacterial. 130:704). As in the original age. Modifications inculded centrifuge of the fied version of the procedure described b method, a French pressure cell was used for efficient cell break supernatant liquid from the post-Omnimixer homogenized cell suspension at 2,300 9 (rav 8.26cm) rather than 5JO 9 for each centrifugation. For subsequent steps, changes in buffer B were necessary to maintain the correct osmolality for the Percoll gradient step. TO generate a medium having an osmolality of 320 mOs/kg H20, it is necessary to mix Percoll with 2.5 M sucrose in a 9:l ratio. Lower starting densities of Percall can be obtained by adding the appropriate amount of 0.25 sucrose. Since, in the Hautala method, the crude nuclear pellet is s,xpended in buffer B which contains 1 M sucrose (i.e., 50 rrM Tris-HCl, pH 7.5; 5 ti MgC12; 10 mM CaC12; 1 M sxrose; and 1% (v/v) Triton X-100), it was necessary to reduce the sucrose concentration in the experiments reported here from 1.0 to 0.25 M, while keeping the other ingredients the same. Thus, the crude nuclear pellets that were obtained were suspended in 8-10 vol of the modified buffer B and homogenized in 40-ml Potter-Elvehjem tissue grinders. The suspensions were then mixed with the appropriate amount of Percoll (isotonic in 2.5 M sucrose) in Beckman 1.6 x 7.62 cm, 10.4.ml polycarbonate bottle assemblies which were centrifuged at 4 C for 45 min at 58,300 p (raV 6.66 cm) using a DuPont-Sorvall T865.1 rotor in a DuPont-Sorvall OTD-2 ultracentrifuge. Owing to the size heterogeneity among Percoll particles, they sediment (and diffuse) at different rates in a gravitational field, thereby creating a density gradient. The biological material in the gradient, in this case nuclei, bands isopycnically, so that the sample particles reach a position where their densities and that of the surrounding Percoll medium are equal. As is the case with isopycnic separation using cesium chloride gradients, a fixed angle rotor has advantage over a swinqing bucket Potor since with fixed angle rotors reorientation of the tube contents does not occur to alter the final separation of the zones and there is better resolution of the experimental materials since they are banded over a larger cross sectional area. A range of starting densities of Percoll from 1.05 to 1.12 gm ml-l were tested in separate experiments to determine the most useful for banding Neurospora nuclei. After each experiment the tube contents were fractionated into 12 fractions, and their refractive index determined with an A/O Refractometer. The results showed that the centrifugation generated adequate Percoll gradients. The nuclei banded to one region of the gradient but the band was not homogeneous: the upper part was relatively disperse, the center was dense and homogeneous, and the lower part exhibited crane clumps. Based on refractive index measurements, the density of the nuclei was determined to be 1.078 gm mlThe nuclei may be recovered by centrifuging gradient fractions containing nuclei for 2 h at 100,000 2 (rav) in a swinging bucket rotor. Under these conditions, the silica particles pellet and the nuclei remain above the gel formed. The nuclei nay then be pelleted from the supernatant liquid by centrifugation for 20 min at 5,000 g (ray). In conclusion, the results indicate that Percoll is an effective alternative to Ludox for the purification of Neurospora nuclei from crude nuclear preparations. The absence of large osmotic effects such as is observed with other gradient materials has allowed the density of wild type nuclei to be determined. Finally, although RNA extracted from crude nuclei includes high molecular weight species that are presumptive precursors to mature rRNA (K. Talbot 1980 Baccalaureate Thesis, Reed College), studies of pre-rRNA processing in the nucleus will be greatly facilitated now that pure nuclei can be obtained. (Supported by NIGMS, NIH grant GM22488). Biology Department, Reed College, Portland, Oregon 97202.

The study of precursor ribosomal RNA (pre-i-RNA) maturation in ribosame biosynthesis mutants of N.crassa -_ĩ s facilitated by the isolation of RNA from purified nuclei.
Problems have been encountered in attempts to purify nuclei with Ludox gradients.Specifically, Ludox precipitates at low temperatures when exposed to Triton X-100, which is an essential component of the buffer used in the nuclei isolation steps.Therefore, a new gradient medium, Percoll (Pharmacia Fine Chemicals, Piscataway, N.J.) was tested for its applicability.The use of Percoll rather than Ludox eliminated probelms with precipitation.
In addition it was possible to determine the buoyant density of the nuclei accurately, since the colloidal silica particles are coated with polyvinylpyrrolidone to which the nuclear membrane is impermeable.A range of starting densities of Percoll from 1.05 to 1.12 gm ml-l were tested in separate experiments to determine the most useful for banding Neurospora nuclei.

Flasks of liquid
After each experiment the tube contents were fractionated into 12 fractions, and their refractive index determined with an A/O Refractometer.The results showed that the centrifugation generated adequate Percoll gradients.The nuclei banded to one region of the gradient but the band was not homogeneous: the upper part was relatively disperse, the center was dense and homogeneous, and the lower part exhibited crane clumps.Based on refractive index measurements, the density of the nuclei was determined to be 1.078 gm ml-The nuclei may be recovered by centrifuging gradient fractions containing nuclei for 2 h at 100,000 2 (rav) in a swinging bucket rotor.Under these conditions, the silica particles pellet and the nuclei remain above the gel formed.The nuclei nay then be pelleted from the supernatant liquid by centrifugation for 20 min at 5,000 g (ray).

In conclusion, the results indicate that
Vogel's minimal medium inoculated with wild type conidia (2x107 ml-') were incubated for 8 h. at 25'C.Crude nuclear pellets were prepared from these mid-logarithmic phase cultures using a modiy Hautala gal.(1977 J. Bacterial.130:704).As in the original age.Modifications inculded centrifuge of the fied version of the procedure described b method, a French pressure cell was used for efficient cell break supernatant liquid from the post-Omnimixer homogenized cell suspension at 2,300 9 (rav 8.26cm) rather than 5JO 9 for each centrifugation.For subsequent steps, changes in buffer B were necessary to maintain the correct osmolality for the Percoll gradient step.TO generate a medium having an osmolality of 320 mOs/kg H20, it is necessary to mix Percoll with 2.5 M sucrose in a 9:l ratio.Lower starting densities of Percall can be obtained by adding the appropriate amount of 0.25 sucrose.Since, in the Hautala method, the crude nuclear pellet is s,xpended in buffer B which contains 1 M sucrose (i.e., 50 rrM Tris-HCl, pH 7.5; 5 ti MgC12; 10 mM CaC12; 1 M sxrose; and 1% (v/v) Triton X-100), it was necessary to reduce the sucrose concentration in the experiments reported here from 1.0 to 0.25 M, while keeping the other ingredients the same.Thus, the crude nuclear pellets that were obtained were suspended in 8-10 vol of the modified buffer B and homogenized in 40-ml Potter-Elvehjem tissue grinders.The suspensions were then mixed with the appropriate amount of Percoll (isotonic in 2.5 M sucrose) in Beckman 1.6 x 7.62 cm, 10.4.ml polycarbonate bottle assemblies which were centrifuged at 4 C for 45 min at 58,300 p (raV 6.66 cm) using a .Owing to the size heterogeneity among Percoll particles, they sediment (and diffuse) at different rates in a gravitational field, thereby creating a density gradient.The biological material in the gradient, in this case nuclei, bands isopycnically, so that the sample particles reach a position where their densities and that of the surrounding Percoll medium are equal.As is the case with isopycnic separation using cesium chloride gradients, a fixed angle rotor has advantage over a swinqing bucket Potor since with fixed angle rotors reorientation of the tube contents does not occur to alter the final separation of the zones and there is better resolution of the experimental materials since they are banded over a larger cross sectional area.
Percoll is an effective alternative to Ludox for the purification of Neurospora nuclei from crude nuclear preparations.The absence of large osmotic effects such as is observed with other gradient materials has allowed the density of wild type nuclei to be determined.Finally, although RNA extracted from crude nuclei includes high molecular weight species that are presumptive precursors to mature rRNA (K.Talbot 1980 Baccalaureate Thesis, Reed College), studies of pre-rRNA processing in the nucleus will be greatly facilitated now that pure nuclei can be obtained.(Supported by NIGMS, NIH grant GM22488).---Biology Department, Reed College, Portland, Oregon 97202.