The detection of irreparable mutants in Neurospor

Kilbey, B. J.

Fungal Genetics Reports

English

Kansas State University

Fungal Genetics Reports Volume 6 Article 15 The detection of irreparable mutants in Neurospora B. J. Kilbey Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Kilbey, B. J. (1964) "The detection of irreparable mutants in Neurospora," Fungal Genetics Reports: Vol. 6, Article 15. https://doi.org/10.4148/1941-4765.2102 This Technical Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. The detection of irreparable mutants in Neurospora Abstract The detection of irreparable mutants in Neurospora This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol6/iss1/15 the use of tests done in 4” test tubes clored with Gxoid Caps. Both are described in Catcheside 1960 Proc. Roy. Sot. London 8153: 179; more detail of the plate tests is given in Ahmad and Cotcheside 1960 Heredity 15:55. For the tests in tubes we use baskets holding 64 tubes in 8 rows of 8 each. Each basket is labelled in a standard fashion with a code number corresponding to the protocol of the matrix to be set up. Drops of conidial suspewion are added to each by means of Pasteur pipettes. The medium contains agar, not sloped, so that the conidial mixture sits on the top and is easily visible on irapection. Daily records are kept on record sheets, for up to IO to 14 days. Beyond this time the medium tends to dry out too much, so concentrating the constituents. The concentration of the medium, OS well as &her factors such as the temperature of incubation, affects the ability to grow. Crossing methods. We use the standard Westergaard ond Mitchell formula in 6” tubes. A piece of folded filter paper is inserted into the medium, which contains agar and is sloped. The female parent is allowed to grow first and when abundant protoperithecio are seen to have been formed, the conidiol parent is added. For conidiation, quite dense suspensions of conidio are made in 2 ml. of sterile distilled water and this suspension is then added to the slopes containing the female parent, after clearing out any excessive conidiol growth that there moy be. The tube is rotated between the hands to distribute the conidia and, after a time, the excess liquid is decanted. - - - Deportment of Genetics, John Curtin School of Medical Research, Australian Notional University, Canberra, A. C. T., Australia. Fonkhauser, D. 8. Grinding of lyophilized mycelial pads. The grinding with mortar and pestle of lyophilized mycelial pads grown in 125 ml flasks (containing 50 ml of medium) has proven very difficult and time con- suming. A much quicker and more effective method is as follow: The Iyophilized pad is placed in an I8 x 150 mm test tube and ‘chopped’ into small pieces with two stainless steel spatulas (a” long with a flat end 2” x l/4” ). With the spatulas still in the tube, and with the top of the tube held firmly, it is placed inside the cup of a Vortex Jr. Mixer and agitated for I5 to 20 seconds, giving (1 semi-fine to fine powder according to the length of agitation. A tube will occasionally chip at the top, but this can be minimized by checking for cracked tubes beforehand, and by holding the tube at the top. 20 x I50 mm i&s should not be used because they break too easily. The tube should not be pressed down with any more force than is necessary to hold it in the cup because contact with the screw ot the cup base will scratch,and eventually break, the bottom of the tube. In three months, we have never had a tube disintegrate. This method will grind up to 400 mg of powder, yielding as fine a powder as desired. The enzymes, tryptaphan synthetase and indole glycerol phosphate synthetose, are extracted as completely from these powders (IS from those prepared by use of mortar and pestle. - - - Deportment of Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio. Kilbey, B. J. The detection of irreparable The heterokaryon system used by Atwood and Mukai mutants in Neurospora. (I953 Proc. Notl. Acad. Sci. U. S. 39:1027)for the detection of irreparable mutants in Neurorpora is open to two main criticisms: first, the hetero- koryotic component in which irreparable mutants ore scored carries the amyceliol ond methioninelers genetic markers, and, second, the tests for reparability are made with medium containing sorbose. Both the genetic background and the plating environment &e probably unfavorable for the detection of reporable mutonk (Horowitz 1963 NN#3:5). In on attempt to obviate these criticisms, on entirely new heterokoryon has been prepared. Both the components of the heterokaryon have been derived from the K3/17 strain of K#lmark (Kblmork and Kilbey 1962 Z&t. fijr Vererbungslehre 93:356). Th is strain carries (1 complex of colonial determinank and re- quires adenine and inositol for growth. The components of the heterokaryon are: A adenineless inositolless albino + 8 adenineless + + lysineless The mutations involved are adenineless (ad-3A 38701 ), inositolless (inos 37401 ), albino (undetermined - but induced with U. V. ) and lysineless (undetermined but induced with X-rays). All the components of the system possess an identical colonial growth pattern. Heterokoryotic colonies develop on Fries’ minimal medium supplemented with adenine. Recessive lethal mutations are scored in the A component of the heterokoryon by the absence of albino colonies when the heterokaryotic colonies ore streaked out on minimal medium supplemented with adenine and inositol. Recess- ive lethals ccln be tested for reparability by their ability to give albino colonies on complete medium. An additional feature of this heterokaryon is the facility for scoring adenine reversions along with recessive lethal mutations. This feature was included in order that certain problems of mutagen specificity might be studied. The advantages of this system can be listed as follows: (I ) S’ lnce the K3/17 strain is associated with high viability, it is unlikely that the reparable mutations will be adversely influenced by the genetic background. (2) The need for sorbose at any stage in the experiment has been eliminated. (3) In contrast to the omycel- ial marker, the albino marker permits scoring to be done without recourse to the microscope. (4) The tech- nique described by Atwood and Mukai can be abbreviated since the punch tube stage in the experiment is eliminated. (5) Reversion ccln also be studied with this heterokaryon. I om greatly indebted to Mrs. M. Griffiths for supplying me with much of the information concerning the performonce of this heterokoryon under experimental conditions. This work was performed at the Medical Research Council’s Mutagenesis Research Unit, West Mains Rood, Edinburgh, Scotland. - - - Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee. Lacy, A. M. Analysis of progeny from interallelic crosses. One of two general methods is usually employed in estimating the viable and total spore populations plated from interollelic crosses. In the first, the total spore population is estimated by counting the number of spores in an aliquot of the suspension to be plated and the viable spare population is estimated by.counting the number of colonies arising when o suitable dilution of the spore suspension is plated on supplemented medium. In the second method, the number of spores counted in an oliquot of spore suspen- sion is used (II o rough estimate for plating purposes and the total and viable spore populations are deter- mined by direct microscopic observation of germinated and ungerminated spores on the plates after sever- al days’ incubation. Experiments with progeny from interollelic td (tryp-3) crosses indicated that different results are ob- tained when the same cross is examined by both%ethods;In general, much lower and less reproducible estimates of viability were obtained by the first method. The first method provides an estimate of only those spores able to give rise to colonies, while the second gives an estimate of all those able to germin- ate. The first class of spores may really be much smaller than the second class and more subject to the effect of external factors, but one must consider the possibility of selection for the wild type. Possibly any wild type spore which can germinate will form a colony while not all mutant spores capable of germ- ination ore able to form colonies on the supplemented plates. In fact, if one partulates that any spore that is wild type will germinate and form a colony, then it may be more realistic to base wild type fre- quencies on the total spore population regardless of the percentage of viable spores. This reasoning is supported by results from interollelic td cresses (Lacy 1959 ph. D. Thesis, Yale University ). Crosses mode at different times between the s&e hvo mutants sometimes give rise to widely differing viable spore percentages. When the wild type frequencies ore based on the viable spore population, they also vary widely. However, when the wild type frequencies we based on the total spore population, they show reassuring similarity. The following crossing and plating method used currently in our laboratory is more a product of 

The detection of irreparable mutants in Neurospora

B. J. Kilbey

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The detection of irreparable mutants in Neurospora

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