Experience with the Applegate-Nelson-Metzenberg method of mutant enrichment in high sorbose medium

Experience with the Applegate-Nelson-Metzenberg method of mutant enrichment in high sorbose medium 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/vol26/iss1/21 The experiments reported here utilized I5 25% continuous linear sucrose (Beckman, ribonucleose-free) gradients mode in 50mM Tris-HCI, pH 7.8 buffer containing 500mM KCI, 5mM MgC12 ated for 60 minutes at IOOC to stabilize them prior to centrifugation. and I mM dithiothreitol (DTT). The gradients were refrigerThe sample, ribacmes isolated from wild type Neurospora crossa and stored at -7O’C (S.C. Schlitt and P.J. Russell 1974 J. Bacterial. 120: 66&671), was thawed and immediately layered upon the gradient. Gradients were centrifuged in the TV850 rotor for 95~ minutes, 47,OOOrpm ot 4OC. After centrifugation, grodients were displaced upwards through o flow cell to monitor nucleic acid obsorbancy at 26Onm. This method of separation results in significantly greater resolution of the 605 and 375 ribosomol subunits (Fig. IA) when compared with that achieved with the usual separation technique in which gradients ore centrifuged in a Beckman SW27. I swinging bucket rotor for 21 hours, 24,OOOrpmat 4’C (Fig. IB). Another advantage of this technique is that it cwerccmes the frustrating problem of partial degradation of the 375 subunit during centrifugotion of gradients in the SW27.1 rotor. Quontificotion of this 375 degradation, using 605:37S peak amplitude ratios (PAR) indicates that TV850 gradients result in a mean PAR of I .5:l ond a substantially incrosed resolution of the 60Sand 375 subunits when compared to SW27. I gradients which result in a mean PAR of 3:1 for the two subunits.


method of mutant enrichment in high sorbore medium.
The high-sorbore, filtration concentration method of mutent enrichment repated by Applegate et al. (Neurospaa --Newl. 25: 17, 1978) was modified and evaluated for ih efficiency of mutant selection. Conidia were suspended in water, filtered through four la justed to 2x 106-2~10 J en of cheesecloth (20 mesh/inch), ad-/ml, ond IOml of the suspension was placed in o 1Ocm diameter glass petri dish for o one min exposure to W-light (4Bergs/sec/mm2) that resulted in 70 to 90% kill.
The suspension wets transferred to o 5OOml flask containing 250ml Vogel's minimal salts plus 6% rorbore, 0.5% glucose, and 0.5% fructose. The flask was incubated in a waterbath rhoker ot 100 reciprocotionq'min; restriction of growth was greater at 37oC than at 25'C.
At 12 hr intervals (at either temperature), the suspension was parsed through a single layer of Nitex #53 nylon monofilament screen with 35pM openings, (Baylis eoj. Neurospora Newsl. 7: 7, 1965, Twtox 73-511-4); four loyerr of cheerecloth also filtered effectively but were less convenient to handle. When the filtration was complete, nongerminated conidio were collected by one of two methods. 1) If the filtration medium contained no inhibitor, the suspension was mixed with an equal volume of warm (6O'C) complete ogor medium, or appropriately supplemented minimal, and poured into petri dishes (15 to ZOml/dirh). 2) To worh conidia, a dense suspension of '"carrier conidia" (killed by 12h r incubation ot 60°C) was added, and the conidio were pelleted by centrifugotion and resuspended in 15 to 20ml water (additional washing by centrifugotion was sometimes necersory).

Carrier conidio aided in locating the pellet and reduced the likelihood that living conidia would stick to each other during centrifugation or would be lost in the supernotont. (Carrier conidio hove previously been used in this way D.E.A. Catch&de.) Aliquots (I ml)of resuspended conidio were placed in Petri dishes and mixed with warm ogar medium.
After a 60hr filtration period ot 37oC, there were 500 to 600 survivors, of which about 30% were ouxotrophr; after 84 hr there were less than 100 survivors ond up to 92 % ouxotrophs. Averages from several experiments show that among oil wrvivors 11% were temperature sensitive, 57% were ouxotrophic, ond about 30% produced white oscorporer indicative of chromorome aberrations, in tests carried out by D.D. Perkins. Specific releition for guanine ouxotrophs or for caffeine sensitivity yielded 2% in each care.
The method is thus extremely efficient, in addition to being convenient. A modification of the method hos been useful with another oscomycete, Cochliobolus heterortrophus. By replacing 1.5% glucose in the filtration medium with 2% rorbore and 0.2% glucose and adjusting procedures slightly, the rate of ouxotroph selection was increased from about 0.1% to 1 to 2%. ---Department of Plant Pathology, Cornell University, Ithaca, NY 14853.