Barratt, R. W. Growth of Neurospora.
pg.15
Suskind, S. R. Growth of Neurospora.
pp. 15-16
Aiuto, R . Intergenic
mapping by a sorbose selection technique.
pp. 16-17
Horowitz,
N. H., H. Macleod, J. Maxwell and B. Logan. Neurospora teaching experiments.
pp. 17-19
Henningson, K. W. Neurospora
class experiments.
pp. 19-22
Lacy, A. M.
Neurospora in the freshman biology course.
pp. 22-24
Lacy, A. M. Neurospora
in the genetics course.
pp. 24-26
Dutta,
S. K. and N. Richman. The isolation of DNA from Neurospora crassa.
pp. 26- 27
Haskins,
F. A. Accumulation of anthranilic acid by a mutant strain of Neurospora.
pp. 27-28
Herrmann, R.
L. The role of orotic acid in pyrimidine biosynthesis in Neurospora.
pp. 28-30
Ishikawa, T. Neurospora
as part of an undergraduate genetics course.
pp. 30-32
Woodward, V. W.
The use of Neurospora in teaching.
pp. 32-33
Barratt, R. W.
Neurospora as a laboratory contaminant.
pp. 33-34
Cooke, F. Fungal
genetics course based on Coniochaeta.
pg. 35
Barratt, R. W. Growth of Neurospora.
The exercise described below is a portion of a laboratory period dealing with the growth of cells and cell populations in an introductory biology course for undergraduate students. It has given good results in the two or three years that it has been used at Dartmouth College.
Growth of Neurospora ( students work independently).
Select from the instructor's table a Petri plate containing a nutrient agar. The surface of this agar was inoculated a few hours earlier at the point marked "X" with a small amount of mycelium of a wild type strain of the fungus Neurospora. After an adjustment period the hyphae began growing outward, giving a series of radiating mycelial strands. Hold the plate up to the light and note the area of growth with the aid of a hand lens. Select a large straight filament at the edge of the growing region. Using a grease pencil or other marking device, mark the bottom of the dish under this filament, so that you will be able to find it again. Now place the dish, still covered, upside down on the stage of your microscope, so that you can examine this growing filament by looking through the dish bottom and the layer of agar.
The method used to measure the growth rate of these cells is as follows: Inside the ocular of your microscope has been mounted a piece of film bearing 100 regularly-spaced marks. This "scale" is in the focal plane of the ocular, and thus will be superimposed on any object seen through the microscope. To measure the growth rate of the Neurospora filament, the filament must be brought into focus (low power) and the ocular (holding the scale) must be rotated so that the growing tip of the filament moves along the scale. The growth rate of the filament can then be determined in scale divisions per minute. A stage micrometer can be used to determine the size of the scale divisions in microns. Then the growth rate can be expressed in microns per minute.
After you have aligned a growing filament with the scale as described above, begin recording in the table provided in the manual the exact time, in minutes and seconds, at which the growing tip crosses each successive scale division. Continue these measurements for 10 minutes, or until the tip has grown across 10 scale divisions. (Smallest ocular micrometer division about 15 microns.) Plot these measurements on graph paper, using time on the horizontal axis and position (scale divisions) on the vertical axis. Draw a smooth curve through the points.
Questions: Is the growth rate constant? If so, calculate the average growth rate in divisions per minute and then convert to microns per minute. Enter calculations in the table.
Having measured the normal growth rate of the Neurospora filament, you are now asked to study the effect of one of a number of externally applied substances on the growth rate of this same filament. A list of the available agents will be posted on the board. (Among those substances which have been tried are sucrose and other carbon sources, sorbose, glycerol in various concentrations, urea, toxicants like dithiocarbamates, mercurials of various types, some amino acids, yeast extract, dinitrophenol, copper salts and many others.)
Make sure the filament you have been studying is marked so that it can be found again and then remove the plate from the microscope. Remove the cover and place one drop of the substance to be tested on the marked filament. Replace the cover and center the filament under the microscope as before. As before, record the time the filament crosses each division of the ocular scale. Enter these data in the table. Continue these readings for 10 minutes or until the tip grows across 10 divisions. Plot the growth of the treated filament in the same manner as you did that of the untreated one. Connect the points with a smooth curve.
Questions: Is the growth rate of the treated filament constant? If so, calculate the average growth rate in divisions per minute and microns per minute and enter calculations in the table. If the growth rate of the treated filament is not constant, describe the nature of the growth curve. How would you describe the effect of the substance you used on the growth of a Neurospora hypha? How could you prove that the effects observed were due to the applied substance and not due to the water in which it was dissolved? How could you show that the effects observed were not caused by the brief removal of the cover? If you have time, perform experiments designed to answer these questions.
- - - Department of Biological Sciences, Dartmouth College, Hanover,
New Hampshire 03755.
Suskind, S. R. Growth of Neurospora.
The following exercise is taken from "Principles of genetics Laboratory manual" by Hartman, Suskind and Wright (Wm. C. Brown Co., 1965) and is presented here with the permission of the authors and publisher.
The growth of fungi.
The purpose of this experiment is twofold. First is to examine the morphology of an ascomycete, and second, to become acquainted with the concept and use of nutritional mutants. As you know, biochemical genetic studies have relied heavily on the use of the bread mold,Neurospora crassa. The widespread use of Neurospora in biochemical genetic work is based on its rapid growth and development during versatile life cycles, its well characterized genetics (particularly at meiosis), and its simple nutritional requirements. It can be readily propagated asexually, and large populations of a particular genotype can be easily obtained.
Neurospora (wild type) will grow on a medium containing inorganic salts, a carbon source such as sucrose, and one of the B vitamins, biotin. These substances constitute the "minimal" medium, and from these components, Neurospora can synthesize all of its protoplasmic constituents including vitamins, amino acids, purine, pyrimidine and fats.
It is known that mutation can often be recognized phenotypically by the loss of the capacity of the organism to carry out a particular biochemical reaction. This is frequently accompanied by the appearance of a specific nutritional requirement reflecting the effect of the "genetic block". For example, a mutant strain may require a particular vitamin, amino acid, etc. Such mutants are termed "auxotrophic" strains. In the absence of the specific growth factor required by the mutant, the mutation becomes lethal, i.e., the organism cannot grow.
In today's experiment you will set up a bioassay for vitamin B6 (pyridoxine ) using a pyridoxineless mutant, Y-2329, of Neurospora. Three points will be emphasized in this experiment. (Each pair of students will set up the experiment. )
1. The nutritional independence of a wild type of strain, 5297 (i.e., growth on minimal medium without pyridoxine).
2. The nutritional dependence of mutant Y-2329 (i.e., no growth on minimal medium without pyridoxine).
3. The quantitative growth response to varying concentrations of pyridoxine (i.e., microbiological assay for pyridoxine).
A. Each group of students will be provided with a conidiated slant of wild type strains 5297a and the pyridoxine mutant V-2329. For each slant, transfer a loop-full of conidia to a tube containing 5 ml of sterile distilled water and vigorously mix until a suspension of conidia is obtained. These will be your inocula for the growth experiment.
B. 1. 5297a experiment.
Add 20 ml of minimal medium to each of three flasks ( 125 ml Erlenmeyer), stopped with cotton plugs and autoclave.
2. Y-2329 experiment.
a. Add 20 ml of minimal medium to each of three flasks ( 125 ml Erlenmeyer), stopped with cotton plugs and autoclave.
(Minimal control)
b. Add 20 ml of minimal medium to each of 10 flasks ( 125 ml Erlenmeyer), using a pyridoxine stock solution (l ug/ml), add pyridoxine concentrations ranging from 0.1-1.0 ug pyridoxine/flask. Stopper with cotton plugs and autoclave.
C. Place flasks in cold room for 15 minutes. After the flasks have cooled to room temperature, inoculate flasks from B-1 with 2 drops each of the wild strains 5297a conidial suspension. Gently swirl flask contents. The flasks from B-2a, b are similarly inoculated with the conidial suspension of the pyridoxine mutant, Y-2329. Use sterile 1.0 ml pipettes for the inoculation of the flasks.
D. Incubate the flasks at 30C for 72-96 hours. The flasks can be stored in the cold room until next lab.
E. Harvest and weigh the mycelia. The mycelia are fished out of flasks using a small spatula. Sterile technique is not necessary at this point. Squeeze the pads as dry as you can and place each pad in a numbered depression of a spot plate. Dry in the oven (100C) for 3 hours. Weigh the dried samples on an analytical balance.
F. Plot your data, i.e., a pyridoxine response curve: mg. dry weight per pad vs. pyridoxine concentration.
.
Materials per 2 students:
96-120 hr. slant of 5297a - 1
120 hr. slant of Y-2329 (B6) - 1
test tube with 5 ml sterile distilled water - 2
1.0 ml pipettes - 3
125 ml Erlenmeyer flasks - 16
baskets and trays for carrying flasks
pyridoxine stock solution 1 ug/ml - 5 ml
spatula
porcelain or glass spot plate - 2
non-absorbent cotton
minimal medium (incl. 2% sucrose) - 350 ml
References: Straus 1951 Arch. Biochem. 30:2912; Wagner and Mitchell 1964 Genetics and metabolism. 2nd ed. Wiley; p. 163. Vogel and Bonner 1958 p. 1 In Ruhland (ed.) Encyclopedia of plant physiology. Springer; Fincham and Day 1963 Fungal genetics. Davis.
Medium: Fries minimal medium.
- - - McCollum-Pratt Institute, Johns Hopkins University, Baltimore,
Maryland 21218.
Aiuto, R . Intergenic mapping by a sorbose selection technique.
The following exercise has been found useful in laboratory sections in which adequate numbers of dissecting microscopes are not available for Neurospora ascospore isolation. It is adapted from methods presented by Lavigne (1962 NN# 2:20), Smith (1962 NN# 1:16) and Frost (as described in Fincham and Day 1962 Fungal genetics, 1st ed. Blackwell, Oxford). The technique requires the use of a biochemical mutant linked to the temperature-sensitive colonial mutant cot, crossed to another linkage group IV biochemical mutant, and the use of the growth-inhibiting carbon source, sorbose.
Ascospores from a cross of pdx-1, cot x pan-1 are harvested with a sterile loop and suspended in 5 ml of sterile distilled water. Usually a single loop of ascospores is more than sufficient for mapping purposes. Four differently supplemented flasks of medium N are prepared (2% agar, 1% sorbose, 0.1% sucrose ), each containing 50 ml. One ml of the ascospore suspension is heat-shocked at 60C for 30 minutes in each of the flasks of molten medium, and two plates are poured from each flask. After 60 hours of incubation at 33C, two types of colonies can be recognized: typically sorbose-inhibited colonies three tofour mm. in diameter, and colonies about 1 mm. in diameter. These are cot+ and cot- individuals, respectively.
The two minimal plates reveal gene order. Most of the colonies will be cot- and represent one-half the single crossovers in region 1, while the cot+ colonies, far fewer in number, are one-half the doubles. Colonies scored on pyridoxine-supplemented medium (0.2 mg/ml) as cot+ are one-half the single crossovers in region 2 less the number of cot+ colonies observed on minimal medium. Colonies scored as cot- on plates supplemented with calcium pantothenate (0.1 mg/ml) represent the reciprocal single crossover class of region 2 less the number of cot- colonies observed on minimal medium. Completely supplemented plates, those supplemented with both pyridoxine and caIcium pantothenate, furnish the total number of viable ascospores plated, and the total number in each parental class. This latter calculation is determined by the difference of the recombinant classes (counting the values obtained for singles in region 1 and doubles twice) from the total number of colonies appearing on complete medium.
Other linkage group IV crosses can be used, such as me-1 x his-5, cot. Some mutant combinations, however, e.g., pyr-l, cot, do not lend themselves to clear-cut discrimination of cot+and cot- colonies, nor do all mutant combinations respond satisfactorily at 33C. In such instances, colonies must be isolated to appropriately supplemented non-sorbose tubes, i.e., from minimal plates to minimal tubes, and scoring is then done with the tubes.
In addition to permitting a mapping exercise with Neurospora without
the use of dissecting microscopes, this method introduces the student to
a plating technique, and allows instructor and class to discuss the theoretical
basis for mapping genes with recombinant classes incompletely identified.
The exercise can be shortened by considering only gene order (using only
minimal plates) or extended to a four-point cross by isolating colonies
to individual tubes, allowing these isolates to grow up at 25C on non-sorbose
medium, and testing in liquid medium for the fourth marker. - - - Department
of Biology, Albion College, Albion, Michigan 49224.
Horowitz, N. H., H. Macleod, J. Maxwell and B. Logan. Neurospora teaching experiments.
The following are time-tested Neurospora teaching experiments. All of the strains mentioned are, or soon will be, deposited with the Fungal Genetics Stock Center and should not be requested from the authors ( NN editor).
Experiment 1. Growth tests on Neurospora mutants of the methionine-threonine pathway.
Problem: to locate the point of blocking in a series of mutants.
Solutions needed:
1. Liquid minimal medium containing sucrose (2%) and biotin (5ug per liter).
2. Same, containing 50 mg L-methionine per liter.
3. Same, containing 150 mg DL-homocysteine thiolactone per liter.
4. Same, containing 200 mg DL- + Allo-cystathionine per liter.
5.Same, containing 50 mg L-cysteine-HCl per liter.
6. Same, containing 40 mg Na thiosulfate +.5H20 per liter.
7. Same, containing 50 mg L-threonine per liter.
8. Same, containing 50 mg DL-homoserine per liter.
9. Same, containing 50 mg L-methionine + 50 mg L-threonine per liter.
Strains needed:
Each student pair will be given a set of 8 cultures labelled with isolation numbers. The cultures are listed below.
| Mutant # | Locus | FGSC # | Block |
| 80702 | cys-2 | 125 | SO32------/--->S2O32- |
| 39816 | cys-10 | 427 | S2O32-----/-->Cysteine |
| 26104 | me-3 | 112 | Cysteine + Homoserine
-----/---> Cystathionine |
| H98 | me-2 | 283 | Cystathionine-----/--->
Serine + Homocysteine |
| 38706 | me-1 | 560 | Homocysteine
-----/---> Methionine |
| 51504 | hs | 471 | Aspartic acid
-----/---> Homoserine |
| 35423 | thr-2 | 2 | O-Phosphohomoserine
-----/---> Threonine |
| 25a | wild type | 353 | (any wild type can be used) |
Procedure:
Each strain is to be inoculated into each of the media listed and observed for growth (+or-) after 48 and 72 hours of incubation at 25C. Use 20 ml of medium in the culture flasks provided, plug with cotton, and autoclave at 15 lbs for 10 minutes. At the same time, autoclave 8 tubes containing 2 ml of distilled water to be used for making conidial suspensions for inoculation. After the tubes have cooled, make a conidial suspension of each mutant (visibly turbid) and inoculate the flask with a drop of the suspension.
References:
Horowitz 1950 Adv. Genet. 3:33; Adelberg 1955 p.419 In McElroy and Glass (ed.), A symposium on amino acid metabolism. Johns Hopkins Press, Baltimore. The role of thiosulfate in the pathway of cysteine synthesis is somewhat mysterious. See Leinweber and Monty 1965 J. Biol. Chem. 240:782; and Murray 1965 Genetics 52: 801.
Experiment 2. Demonstration of an enzyme deficiency in a Neurospora mutant.
This experiment consists of two parts: Part A is a growth test in which the phenotypic effect of a mutation that abolishes the synthesis of D-amino acid oxidase is demonstrated. Part B is a test for the enzyme in the mutant and in wild type.
Part A. 18 flasks of supplemented minimal medium will be needed. Nine of the flasks are to be inoculated with mutant #38706, blocked between homocysteine and methionine. These flasks are supplemented as indicated below.
| Flask | L-methionine | Flask | D-methionine |
| 1 | 0 ug/ml | 6 | 4 ug/ml |
| 2 | 4 ug/ml | 7 | 8 ug/ml |
| 3 | 8 ug/ml | 8 | 16 ug/ml |
| 4 | 16 ug/ml | 9 | 32 ug/ml |
| 5 | 32 ug/ml |
The second set of nine flasks is identical with that listed, except that all flasks contain, in addition, 10 ug of inositol per ml. These flasks are to be inoculated with the triple mutant 38706, 89601, oxD(8). This mutant carries, besides the methionine gene, a block in the synthesis of inositol, and a block in the synthesis of the D-amino acid oxidase. (The inositol requirement is irrelevant to this experiment; it is present because the oxD mutation was isolated by the inositolless-death method).
After the flasks have been autoclaved and cooled, inoculate them and incubate at 25C for 72 to 96 hours. Fish out the mycelial mats, press out the excess medium on paper towels, and determine the wet weight of each.
Part B. This experiment will use a wild type strain and mutant oxD (8) which has been freed of the methionine and inositol requirements by crossing. Grow three cultures of each in 20 ml of minimal medium contained in 125 ml Erlenmeyer flasks. Grow the cultures for 5 days at 25C. Decant the medium and wash each mycelial mat individually by adding 20 to 25 ml of water to the flask, swirling, and decanting. Repeat, using phosphate buffer, 0.1 M, pH 7.2. The cultures may be stored in the deep freeze at this point if so desired. Only four of the cultures will be needed for the assay; keep the extra pair as spares.
To one wild type culture and to one mutant culture, add 5 ml of phosphate buffer containing D-methionine at a concentration of 2 mg/ml. To the other two, add buffer without substrate. Incubate the flasks at 37 for one hour with shaking. Filter the solutions through paper and determine the keto-acids by the dinitrophenylhydrazone procedure which follows:
1. Add 1 ml dinitrophenylhydrazine (0.1% in 2N HCl ) to 1 ml filtrate and let stand 5 minutes.
2. Add 2 ml absolute ethanol .
3. Add 5 ml 2.5 N NaOH and shake vigorously to mix. Let stand 5 minutes.
4. Read color in the Klett, using the green filter.
Standard curve: In place of filtrate use 0, 10, 20, 30, 40, and 50 ug of sodium pyruvate.
Calculate the amount of a-keto acid produced from D-methionine by the wild type and mutant strains, assuming that pyruvate is twice as chromogenic, gram for gram, as a-ketomethiobutyrate. What reaction is catalyzed by the D-amino acid oxidase? How can you explain the results obtained in Part A?
Reference: Ohnishi, Macleod and Horowitz 1962 J. Biol. Chem. 237: 138.
Experiment 3. Accumulation of imidozole compounds by a histidine-requiring mutant. Feedback control of biosynthesis.
Into a series of 12 numbered 125 ml Erlenmeyer flasks place 20 ml of minimal medium. To each set of 6 flasks add 0, 0.1, 0.3, 0.5, 1.0 and 2.0 mg, respectively, of L-histidine. Plug the flasks with cotton and sterilize for 10 minutes in the autoclave. Inoculate one set with wild type and the other with histidineless C-84 conidia. Incubate at 25 for 4 days.
Place ½ ml of medium from each flask in which growth has occurred into a series of numbered test tubes. At the some time, set up a series of standards consisting of ½ ml minimal medium plus 0, 10, 25, 50 and 75 ug, respectively, of histidine. Determine imidazole derivatives in the solution by means of the Pauly reaction, as modified by Jorpes (1932). The procedure is explained below. Remove the mycelial mat from the flasks analyzed, press out the moisture on paper towels, and determine the wet weight of each. Record the calculated imidozole concentration for each flask and the amount of imidozole accumulated per gram of mycelium.
Determination of histidine (Jorpes, loc. cit.)
Solutions needed:
1. Sample to be tested.
2. A diazonium solution prepared as follows: to 1.5 ml of a solution containing 0.9 g sulfanilic acid and 9 ml conc. HCl in 100 ml are added 1.5 ml of a 5% sodium nitrite solution. Cool on ice for 5 minutes. Then add 6 ml of the nitrite solution with shaking, cool for 6 minutes, and add water to 50 ml. The diazonium solution should be kept cold. It keeps for 24 hours. Best results are obtained with freshly prepared sodium nitrite solution.
3. 1.1% sodium carbonate solution.
Procedure: To ½ ml of solution 1, add 2 ml of solution 2. After 1-3 hours, add 3 ml of solution 3. Read in a Klett-Summerson colorimeter with a blue filter (400-465mu) 4-8 minutes after addition of solution 3.
References:Haas et al. 1952 Genetics 37: 217; Jorpes 1932 Biochem. J. 26:1507; Ames 1955 p. 357 In McElroy and Glass (ed.), Symposium on amino acid metabolism. Johns Hopkins Press, Baltimore.
Discussion: C-84 (his-1 ) is blocked between imidazole glycerol phosphate (IGP) and imidozole acetol phosphate (IAP) in the pathway of histidine biosynthesis. As the mutant grows, IGP accumulates in the mycelium and soon its dephosphorylated derivative leaks out and is found in the medium. As a control, wild type (25a ) is tested and no imidazole compound is found in the medium. Both mutant and wild type take up the histidine originally added.
The phenomenon of feedback control of biosynthesis is demonstrated when it is noted that the amount of imidozole accumulated per gram of mycelium decreases as the amount of added histidine increases. The end product of a pathway has inhibited an early step in its own synthesis.
Experiment 4. Tyrosinase.
The following experiments demonstrate the thermostability and electrophoretic differences between two allelic tyrosinases of Neurospora. The alleles TL and TPR were chosen for this experiment because they are easily distinguished by both tests. If it is desired to omit the electrophoresis experiment and perform only the thermostability test, then the alleles TS and TLare recommended.
Induction: Place 20 ml of ½ strength Vogel's medium containing ½% sucrose into each of ten 125 ml Erlenmeyer flasks. Plug with cotton and sterilize for 15 minutes. Inoculate 5 of the flasks with the strain 4-137 TL and 5 with 65-1434 TPR. Incubate the flasks at 25C for 3 days. Induce 3 flasks of each strain with 2 mg sterile D-phenylalanine (2 mg/ml) per flask using aseptic technique. Leave 2 flasks of each uninduced. Incubate the flasks for an additional 24 hours at 25C on the shaker. Tyrosinase is formed during this time in those flasks containing inducer.
Remove the excess medium by pressing the mycelia between paper towels. Weigh the pooled induced TPR mycelia, and extract the tyrosinase by grinding with sand in a cold mortar, using 4 ml of 0.1 M sodium phosphate buffer, pH 6, per gram of moist mycelium. Do the same with the TL mycelium, and with the uninduced controls, yielding a total of four extracts. Keep the extracts in an ice bath during waiting periods. Spin the extracts at 10,000 x g for 10 minutes or at the highest speed of the clinical centrifuge for 10 minutes, and decant the supernatant, containing the enzyme.
Assay: Add 0.02-0.1 ml crude extract to sufficient sodium phosphate buffer (0.1M, pH 6) to give a final volume of 4 ml, and equilibrate the solution in a water bath at 30C. To start the reaction, rapidly add 1 ml DL-dopa (4 mg/ml in buffer). Shake the tube and read it in the Klett, using the blue filter. Shake the tube again and replace it in the water bath. Five minutes after the first reading was taken, make a second reading on the tube. The difference between the two readings is proportional to the tyrosinase concentration. Demonstrate this proportionality by setting up a concentration series, using either one of the two preparations you have just made. Have at least 5 points, including a zero control, in the series. The readings should be linear with concentration up to about 150 Klett units/5 minutes. Above 150-200 units, the curve departs from linearity owing to the fact that oxygen becomes limiting for the reaction.
Thermostability test: Into 4 Klett tubes place the calculated volume of TPR enzyme which would give a reading of 100-150 Klett units in 5 minutes; make up the volume to 0.5 ml with buffer. Do the same with the TL preparation. Cover the tubes with foil. Keep one tube of each set in an ice bath as a zero control. Set the other six in a water bath at 59C. Remove one tube of each extract after 10 minutes and chill immediately. Repeat after 20 and 30 minutes. To the chilled samples, add 3.5 ml buffer to each tube and proceed with the assay as above. Estimate the half lives of the two enzymes. (More points may be taken on the curves if desired.)
Electrophoresis: Make up 1 liter of 0.05 M sodium phosphate buffer, pH 6. Dissolve 1 gm of bovine serum albumin in 100 ml of the buffer, and pour the solution into an enamel tray. Saturate 8 numbered paper strips in the solution, allow them to drain for a few seconds, and place on the holder of the Spinco Paper Electrophoresis Cell. Mix the BSA solution with the remainder of the buffer and pour into the reservoirs of the cell . Set the strip holder in position, close the cell, and level the buffer in the reservoirs. The enzyme solutions should contain between 1500 and 3000 Klett units per ml for best results in the electrophoresis. Apply 0.01 ml of each to a paper strip with the wire applicator. (If enough cells are available, use a mixture of the two on a third strip.) Replace the tape on the cell cover, and plug in the cell. Turn on the power supply, and set it for constant current at the rate of 1.25 ma per paper strip. Allow to run for from 16 to 24 hours. Turn off the power supply, unplug the cell, remove the cover and the strip holder. Lay the holder, containing the strips, on a flat surface. Spray the strips with a solution containing either 4 mg/ml of DL-dopa or 0.8 mg/ml of epinephrin. Dopachrome (or adrenochrome) will be formed at the position of the enzyme. Use an atomizer that gives a fine spray; do not flood the strips. Measure the distance migrated.
Reference: Horowitz, et al. 1961 Cold Spring Harbor
Symp. Quant. Biol. 26:233. Biology Division, California Institute of Technology,
Pasadena, California 91109.
Henningson, K. W. Neurospora class experiments.
The following Neurospora class experiments are used in a course in biochemical genetics at the Institute of Genetics, University of Copenhagen.
Experiment 1. Tetrad analysis ( ascospore color).
A cross between a mutant, asco (37402)a, with colorless ascospores (requirement for lysine ) and a wild type strain, 74A, is used for tetrad analysis. Since the color of the ascospore is an autonomous character, it is possible to analyze the segregation of the alternative characters of the ascospores within the intact cell. The cross between strains of opposite mating types can be performed in two different ways: if the two strains are inoculated simultaneously, each strain acts as both female and male parent. If desired, one or the other strain can be used as female, by inoculating it before-hand on the crossing medium (Westergaard and Mitchell 1947 Am. J. Botany 34:573) to induce formation of protoperithecia. Then, mating of protoperithecia can be carried out with conidia from the other strain. Either solid or liquid medium may be used. In the case of liquid substrate, filter paper is used to support hyphae and protoperithecia.
Materials:
First day: Test tube cultures of 37402a and 74A. Three test tubes containing 10 ml liquid crossing medium + 300 mg/l lysine. 0.90% NaCl . Strips of filter paper (sterile).
Second day: 2% sodium hypochlorite. Distilled water. Microscope slides and coverslips. Petri dishes, needles and forceps for dissection.
Experimental procedure:
First day: Harvest conidia from both strains by pipetting about 3 ml of 0.9% NaCl into the test tube culture and shaking gently. Transfer from each strain 0.5 ml of spore suspension to the crossing medium. In each of the cultures a strip of filter paper is oriented so that about 5 cm stands out of the liquid. The cultures are incubated at 25C for 3-4 weeks.
Second day: Analysis of the tetrads. After about 3 weeks, the pyriform perithecia contain asci with maturing ascospores. The filter paper which carries most of the perithecia is removed from the test tube and placed in a petri dish containing 2% sodium hypochlorite. After 3-5 minutes, the conidia have been killed by the hypochlorite and the filter paper can be transferred to another dish and washed with distilled water. A few perithecia then are placed on a microscope slide . Under a dissecting microscope the contents of the perithecia are removed in a drop of distilled water and the perithecial wall is removed from the slide. A coverslip is placed on top and the asci are spread by a light pressure. Under the light microscope all asci containing 4 black and 4 colorless ascospores are analyzed. The number of first and second division segregation ( pre- and post-reduction) tetrads is determined and the distance between the gene for ascospore color and the centromere is calculated from the formula: ½ x percentage of post-reduction tetrads.
Reference: Stadler 1956 Genetics 41:528.
Remarks: As the ascospores approach maturity, some asci containing 8 black spores may appear. If these abnormal asci are excluded from the results, the distance between the gene and the centromere is about 14%.
Preparing cultures: The asco strain (37402)a, FGSC#405, which requires lysine, is kept on Vogel's minimal medium + lysine 300 mg/l. Wild type strain 74A is kept on Vogel's minimal medium N (Vogel 1956 Microbial Genet. Bull. 13:42). Stock cultures are maintained and tested before use as described for arginine mutants.
Experiment 2. Isolation and characterization of biochemical mutants.
A. Isolation of biochemical mutants by filtration technique.
Conidia from a wild type strain of N. crassa are irradiated with X-rays and subjected to differential germination on liquid medium. Passage of the culture through a suitable filter at various intervals removes more and more of the germinated wild type conidia until finally the culture contains only conidia which are unable to germinate on minimal medium. After germination and isolation of these residual conidia on complete medium, many show specific growth-factor requirements.
Experimental procedure:
(1) Harvest conidia from 6 cultures of wild type strains 74A by pouring glass pearls into the flasks and shaking. Then pipette 10 ml 0.9% NaCl into each flask and shake again. Filter suspension through a G-2 glass filter. Centrifuge filtrate at 3000 x g for 3-5 minutes and resuspend pellet in 0.9% NaCl . Repeat 3 times. Determine the conidial concentration of the suspension by counting the spores in a haemocytometer. Adjust the final concentration to 1 x 108 spores/ml . A minimum of 10 ml of the final concentration is needed.
(2) Pipette 3 ml of the conidial suspension into each of 2 alcohol-sterilized plexiglass dishes that will fit into the X-ray machine. Irradiate one dish at 4 x 104 r ( 100 kv, 8 cm. distance, 1.7 mm aluminum filter) for 15 minutes. The second dish is kept as a non-irradiated control .
(3) Transfer the irradiated suspension to a test tube. Rinse the plexiglass dish with 3 ml 0.9% NaCl and add this to the test tube. Pipette 4 ml of this irradiated suspension into a 2-liter flask containing 400 ml Vogel's N-minimal medium + 0.5% sucrose. Prepare a control flask of the non-irradiated sample in the same manner. Place the flasks on a mechanical shaker and shake at low speed at 32C. Record inoculation time as zero time for growth.
(4) To determine the survival and germination percentages, dilute 1 ml of the remaining irradiated and control suspensions to 1,000 and 100 conidia/ml. Transfer 5 samples of 1 ml from each dilution to 5 tubes each containing 15 ml of complete medium with sorbose (melted and kept at 42C ). Shake and pour into sterile petri plates. Incubate at 20-25C for 2-3 days (may be stored at 4C ).
(5) To remove the germinated wild type spores, which have formed hyphae, filter the contents of the two flasks through 4 layers of cheesecloth which have been previously fixed in the necks of 2-liter Erlenmeyer flasks and sterilized in situ . After filtration, the filter is replaced by a sterile cotton plug and the flask is placed on the shaker. Ten filtrations are carried out, at 5, 8, 10, 12, 15, 18, 21, 24, 31, and 42 hours after inoculation, respectively. Platings are made from the 8th, 9th and 10th filtrations.
(6) Following the 8th and 9th filtrations, pipette 25 ml from each culture into a sterile flask and plate 20 aliquots from each sample as described above (if necessary the samples can be stored at 4C and plated the following day). From the last filtration, prepare as many plates as possible. All plates are incubated at 32C for 2 days. (May be stored at 4C until analysis can be carried out.)
(7) Determine the number of colonies and calculate the germination and the survival percentages. The number of viable conidia per ml in the cultures, when started, and following the filtrations, is a rough measure of the effectiveness of the filtration.
(8) Isolate colonies, mainly from the last filtration, in small tubes containing 2 ml of complete medium. For transferring small pieces of colonies, a sharp rigid inoculation needle is used. Incubate the cultures at 32C for about 7 days.
(9) Test the colonies isolated on complete medium for growth-factor requirements by transferring conidia to liquid minimal medium N with a loop-shaped inoculating needle. Keep the two cultures together at 32C for 2 days. Use the isolated colonies which are not able to grow on minimal for further analysis. Calculate the percentage of biochemical mutants.
References: Catcheside 1954 J. gen. Microbiol. 11:34; Fries 1947 Nature 150:199; Lester and Gross 1959 Science 129:572; Pontecorvo, Roper, Hemmons, MacDonald and Bufton 1953 Adv. Genet. 5:141; Woodward, De Zeeuw and Srb 1954 Proc. Natl. Acad. Sci. U.S. 40: 192.
Preparing cultures: Wild type strain 74A is kept on minimal medium N. Samples from stock cultures are spread on plates of medium N. The hyphae are used to inoculate agar slants. Conidia are spread on minimal crossing medium + sorbose and single colonies are used to inoculate agar slants of medium N. Then, conidia are used to inoculate six 100-ml Erlenmeyer flasks, each containing 20 ml of crossing medium + 2.5% glycerol (no sucrose). Incubate for 7 days at 20C.
B. Preliminary test of biochemical mutants.
To determine the type of growth-factor requirements of the biochemical mutants, preliminary tests are carried out on mixtures of growth factors. In this way mutants can be characterized as: a) amino acid-requiring, b) vitamin-requiring, or c) purine and pyrimidine requiring.
Materials:
One petri dish ( 17 cm in diameter) of each of the following media: synthetic crossing medium + sorbose; the same + sorbose + casamino acids; the same + sorbose plus a vitamin mixture; the same + sorbose + hydrolyzed yeast nucleic acids. 10 small tubes with complete medium N. 0.9% NaCl and centrifuge tubes. 5 cultures of the biochemical mutants isolated in the filtration experiment. 5 cultures of biochemical mutants with known requirements.
Experimental procedure:
Prepare a suspension of conidia from each mutant by adding 2 ml of 0.9% NaCl to each tube and stirring briefly. Pipette the suspension into a centrifuge tube and adjust the volume to 10 ml with 0.9% NaCl. Centrifuge at 3000 x g for 3-5 minutes. Decant and resuspend conidia in 10 ml 0.9% NaCl. Repeat 2 times. The final volume should be about 5 ml. Prepare a new culture from each mutant on complete medium N. Using an inoculating loop, place a small drop of the conidial suspension of each mutant on each of the media listed above under materials. Before inoculation, mark all plates in such a way that 8 of the mutants are placed at the periphery and 2 in the center. Incubate the plates at 25C for 2 days (can be stored at 4C). Classify the mutants on the basis of their growth factor requirements.
C. Auxanographic test:
Prepare a suspension of conidia from each of the mutants and inoculate, as described above, plates with individual growth factors. Test the amino acid-requiring mutants on plates of minimal crossing medium + sorbose and supplemented, singly, with 150 mg/ml of the following amino acids: L-arginine HCl, L-leucine, lysine, methionine, phenylalanine and proline. Test the mutants requiring purines or pyrimidines on minimal crossing medium + sorbose +adenine sulfate 150 mg/ml. Test the vitamin-requiring mutants on minimal crossing medium + sorbose supplemented with pyridoxine 50 mg/ml and with Ca-pantothenate 50 mg/ml, separately. On a normal sized petri dish, 4 mutants can be tested. Inoculate close to the periphery of the dishes at previously marked positions. Incubate at 25C for 2 days. Many of the auxotrophic mutants isolated by the filtration technique described can be classified by this auxanographic test.
References: Emerson 1955 Vol. 2, pt . 2, p. 443 In Hoppe-Seyler and Thierfelder (ed.) Handbuch der physiologisch- und pathologisch-chemisches Analyse, 10th ed. Springer, Heidelberg; Horowitz 1950 Adv. Genet. 3:33; Pontecorvo 1949 J. gen. Microbiol. 3:122.
Strains used and preparation of cultures:
The following strains with known requirements are used: pro-1 (21863), FGSC#29; phen-1 (H3791 ), FGSC#504; me-8 (P53), FGSC#98; me-5 (9666), FGSC#140; arg-1 (B369), FGSC#324. Cultures are kept on complete medium. Stock cultures are maintained and tested as described for arginine mutants.
D. Precursor test of arginine mutants.
The final step in the physiological characterization of the biochemical mutants is the precursor test. A group of mutants with known requirements for arginine is used to demonstrate the intermediate steps in the biosynthesis of arginine in Neurospora.
Materials:
Cultures of 7 arginine mutants. Plates of minimal medium supplemented as described below. 0.9% NaCl. Centrifuge tubes.
Experimental procedure:
Prepare a suspension of conidia from each of the mutants by adding 2 ml of 0.9% NaCl to each culture tube. Stir briefly and pipette about 1 ml of the conidial suspension into a centrifuge tube. Adjust the volume to 10 ml with 0.9% NaCl. Centrifuge at 300 x 9 for 3-5 minutes. Decant and resuspend conidia in 10 ml 0.9% NaCl. Repeat 2 times. The final volume should be about 5 ml. With a loop a small drop of conidial suspension is placed on the following media:
minimal crossing medium + sorbose; the same + L-arginine 150 mg/l; the same + L- citrulline 150 mg/l; and the same + L-ornithine 150 mg/l. Mark the plates and inoculate at the indicated spots at the periphery. Incubate at 25C for 2 days.
Determine the block in the biosynthetic chain for each of the mutants from their growth patterns. Establish the sequence of the arginine biosynthetic pathway.
References: Srb, Fincham and Bonner 1950 Am. J. Botany 37: 533; Srb and Horowitz 1944 J. Biol. Chem. 154: 129; Vogel and Bonner 1954 Proc. Natl. Acad. Sci. U. S. 40:688.
Cultures and maintenance:
The following strains of arginine mutants should be used: arg-1 (36703T), FGSC#273; arg-1 (B369), FGSC#324; arg-1 (46004), FGSC#528; arg-5 (27947), FGSC#274; arg-5 (27974), FGSC#480; arg-3 (30300), FGSC#1068; and arg-3 (30300), FGSC#1069. The strains are kept on Vogel's minimal medium N +arginine 150 mg/l. Stock cultures are covered with sterile paraffin oil as soon as conidia are formed and are placed at 4C. Samples from a stock culture are taken with a loop and spread on plates. Hyphae formed on the plates are used to inoculate agar slants. Conidia should be tested on liquid minimal medium N for back mutations. If back mutation has occurred, conidia should be spread on minimal medium N + sorbose + arginine and single colonies re-isolated onto agar slants. Each culture must then be tested on liquid minimal medium. Incubate cultures at 25-32C.
Media:
l) Synthetic crossing medium (Westergaard and Mitchell 1947 Am. J. Botany 34:573). A 4x concentrated stock solution is prepared. After dilution sugar is added and the pH adjusted with NaOH.
2) Vogel's minimal medium N (Vogel 1956 Microbial Genet. Bull. 13:42). A 50x concentrated stock solution is prepared. After dilution sugar is added and the pH is adjusted with NaOH.
3) Complete medium N. To each liter of medium N add: yeast extract 2.5 g., malt extract 5 g., vitamin mixture 10 ml, hydrolyzed nucleic acid 2 ml, and casamino acids 15 ml.
4) Vitamin mixture. Dissolve the following in 1 liter of water: thiamine 100 mg, riboflavin 50 mg, pyridoxine 50 mg, Ca-pantothenate 220 mg, p-aminobenzoic acid 200 mg, nicotinamide 200 mg, choline HCl 129 mg, inositol 400 mg.
5) Hydrolyzed nucleic acid. Dissolve 1% yeast nucleic acid and 0.2% thymus nucleic acid in 1 N NaOH. Hydrolyze for 24 hours at 35C. Adjust to pH 6.5 and store at 4C.
6) "Synthetic casein" (casamino acids). In 687 ml of water, dissolve the following: glycine 20 mg, DL-alanine 180 mg, L-leucine 243 mg, DL-isoleucine 485 mg, DL-aspartic acid 410 mg, L-glutamic acid 1090 mg, DL-serine 580 mg, DL-threonine 390 mg, L-proline 400 mg, L-hydroxyproline 10 mg, L-cystine 15 mg, DL-methionine 360 mg, DL-valine 790 mg, DL-phenylalanine 390 mg, L-tryptophan 110 mg, L-tyrosine 325 mg, L-arginine HCl 305 mg, DL-lysine HCl 310 mg and L-histidine HCl 455 mg.
7) Sorbose-containing media. The ordinary carbon source, sucrose, is replaced by a mixture of sorbose, glucose and fructose to give final concentrations of 1.5%. 0.04% and 0.04%, respectively.
- - -Institute of Genetics, University of Copenhagen, Oster Farimagsgade
2A, Copenhagen K, Denmark.
Lacy, A. M. Neurospora in the freshman biology course.
The following laboratory procedure and description of necessary materials is taken from a new "Labtext" From one cell to many, by Funk and Lacy 1966 Wm. C. Brown Co., Dubuque, with permission of authors and publisher. Some introductory material has been deleted.
This manual is designed for use in a one-semester elementary biology course devoted to the "cell to organism" approach. Cell structure and metabolism, genetics, plant and animal development, and , finally, the structure of a multicellular animal (the dogfish shark) are dealt with in this course.
The students begin the Neurospora experiment during the second half of the course, after they have each completed a four-week independent project using a microorganism. Consequently, they are reasonably proficient in aseptic technique and use of the microscope. This experiment does not depend for its success on extensive first-hand knowledge of Neurospora on the part of the instructor. Moreover, this experiment, in fact this whole manual, is primarily designed for colleges which wish to present a modern course in biology but which lack both a large staff of graduate teaching assistants and an unlimited supply of specialized equipment. The experiment occupies about one hour of one laboratory period (of which the other two hours are devoted to corn genetics, blood typing and blood cell identification), about 15 minutes of a second laboratory period and about five minutes of a third period.
Materials:
(per student) Dissecting microscope with 45-60X magnification
1 glass-handled dissecting needle
1 metal-handled microspatula
1 small flask of 95% alcohol
1 microscope slide
10 ( 10 x 75 mm) tubes of supplemented agar medium (medium N )
10 (10 x 75 mm) tubes of minimal liquid medium (medium N)
(per 8 students) 2 tubes of a Neurospora cross (biochemical mutant x "morphological mutant")
1 petri dish of 4% water agar
1 wire test tube rack for small ( 10 or 12 x 75 mm ) test tubes
(per class) 1 large water bath, set at 60C
Procedure:
Anatomy of Neurospora: Each group of four students will be given a test tube in which two mutant strains of N. crassa have been crossed. Observe the different structures in the tube with the dissecting microscope. Also, remove some of the organism aseptically, place it on a slide in wet mount, and observe under the compound microscope. Identify the vegetative structures (mycelium, hyphae, asexual conidia) and sexual reproductive structures (perithecia, ascospores).
Analyzing progeny from a Neurospora cross: By isolating random ascospores ( haploids) from the cross, allowing them to germinate and grow, and then studying the characteristics of each isolate, it should be possible to determine whether or not the two genes involved are linked together on the same chromosome and, if so, how closely linked they are. For this experiment, each student will isolate and study 10 ascospores. When the ascospores are block and ripe, they are shot out of the perithecia and form small black clumps on the upper side of the test tube. Dip the microspatula into the alcohol, flame, and use it to remove a few spores from the side of the tube. Place them on one end of a 2 cm x 3 cm agar block on a glass slide. Note: agar dries out very rapidly; do not leave this cut piece lying around too long before using. Remember to practice aseptic transfer techniques when dipping into the test tube containing the cross. Aseptic technique is, of course, not possible when working on the agar block.
After flaming your microspatula, cut one side of the agar block into 10 small squares as demonstrated by the instructor. Then, using the glass-handled dissecting needle (DO NOTFLAME), and using the highest power magnification of your dissecting microscope (45-60x), place one black spore on each square of agar. With your flat microspatula, pick up each little agar square individually and place right side up in individual small test tubes of supplemented agar medium. Label each tube with your name, the cross used, and an isolation number.
These small test tubes containing the isolated spores should be placed in a 60C water bath for 40 minutes. This treatment kills contaminants which may have fallen onto the agar during the isolation and all parental mycelia and conidia that may have stuck to the ascospores. It also provides the "heat-shock" necessary for initiation of spore germination. After "heatshocking", the tubes should be incubated at room temperature until the next laboratory period (or for at least 4 days).
During the next laboratory period, the following procedures should be carried out: 1) The germination percentage should be determined. 2) The tubes containing germinated spores should be examined and the morphology of the organism in each tube recorded. The instructor will demonstrate to you the different types of morphology you may expect to observe. If one parent in the cross was an albino mutant, set the tubes in the light for at least one hour before classifying. Carotenoid synthesis in Neurospora is light dependent. 3) A few conidia or small bits of the mycelium from each tube should be aseptically transferred to tubes of minimal liquid medium ( a medium which will support the growth of wild type Neurospora, but not of biochemical mutants).
These inoculated tubes of minimal medium should be incubated at room temperature for two or three days. Then the following points should be determined. 1) Which tubes showed growth and which did not? 2) What does this tell you about the phenotypes of these progeny? Genotypes? 3) What is the correlation between the morphological characteristics of the progeny and the biochemical characteristics (as determined by growth tests on minimal medium)? 4) Is the gene locus determining the morphological character linked to the one determining the ability to synthesize a certain nutrient factor? At the end of the laboratory period, pool your data with that of others in the class studying the same cross, so that you can determine with some statistical accuracy whether or not the two genes are linked.
Before the ascospores are shot out of perithecia, they are contained in sacs called asci (eight spores to each) and are arranged in order of meiotic segregation within these asci . Why do people often isolate spores in order from the asci? If you wish, you may try to do this yourself. Remove a ripe, black perithecium from the crossing tube, squeeze it open with forceps, pull an ascus with eight ripe, b1ack spores to a clean part of the agar, and dissect in order with a microneedle. This requires a steady hand.
References:
Barish 1965 The gene concept, p. 82-86. Reinhold, New York; Beadle 1948 Sci. Am. 179:30; Bonner and Mills 1964 Heredity, p. 18-28. 2nd ed. Prentice-Hall, New York; Sinnott, Dunn and Dobzhansky 1958 Principles of genetics, p. 173-174, 321-322, 333-336. 5th ed. McGraw-Hill, New York; Srb, Owen and Edgar 1965 General genetics, p. 98-102. 2nd ed. Freeman, San Francisco.
Preparation of materials:
The glass-handled dissecting needles are made by inserting 00 Genuine Bohemian Insect Pins (Carolina Biological Supply Co. #A740) into the melted ends of hollow soft glass rods (diam. 5 mm; length about 6 inches) and then flattening the glass onto the pin with forceps. The needles are most easily stored by passing them through a rubber stopper and plugging the stopper into an 18 x 75 mm test tube.
The metal-handled microspatula is made by hammering a 3.5 inch piece of nichrome or chromel wire (24 gauge) until the tip is more or less square, cutting the edges off sharply, and inserting the wire into any fairly short inoculating loop holder.
Test tube racks can be made by buying wire fencing of the appropriate mesh, cutting into two rectangles and a square, and weaving or welding the broken ends together to make a three-tiered rack.
Minimal media for Neurospora are available from Difco (0324-15, 0460-15, 0817-01 ). We have not tried these, but they would probably be convenient for course work. The most commonly used vegetative medium for Neurospora is medium N or Vogel's medium (Vogel 1956 Microbial Genet. Bull. 13: 42). This is generally made up 50x and diluted as needed.
The supplements added to the medium will depend upon the mutants used. 150 ug/ml of L-amino acid is usually sufficient for growth of any amino acid mutant, but for crossing and germination higher concentrations in the range of 200-300 ug/ml are often more satisfactory.
Neurospora strains of all kinds are available free of charge (in limited numbers) from the Fungal Genetics Stock Center, Dartmouth College, Hanover, New Hampshire. Albino is one of the most satisfactory morphological mutants for class use. Almost any amino acid mutant is suitable for the biochemical mutant. We usually use albino and a tryp-3 mutant as parents. Be sure to order parents of different mating types (A and a).
Neurospora requires a special medium for crossing. A synthetic crossing medium(Westergaard and Mitchell 1947 Am. J. Botany 34: 573) is generally used in research, but corn meal agar with dextrose (Difco B114) is probably simpler to use for course work. The best procedure is to inoculate one parent onto a slant of corn meal agar and incubate at 25C for five days. At that time, dust the conidia from the second parent over the mycelium of the first and reincubate at 25C. While two weeks should be sufficient to obtain ripe spores, it is wise to start crosses a month or two before needed. When ripe black ascospores appear in clumps on the upper inside surface of the tube, the cross may be stored in the refrigerator until needed. Refrigerated crosses will retain high viability for as long as a year.
- - - Department of Biological Sciences, Goucher College, Towson, Maryland
21204.
Lacy, A. M. Neurospora in the genetics course.
While other organisms are used (with some reluctance) in my genetics course, much of the laboratory time is devoted to a long project using Neurospora crassa. The following procedure, which will probably be published in more detail as part of a projected Genetics Laboratory Manual, is designed for use in a course taken primarily by junior biology majors and pre-med students. All of these students have completed the elementary biology course, in which Neurospora is introduced, and most of them have also completed a year of organic chemistry before taking the genetics course.
This type of project is probably feasible only where the number of students enrolled in the lab is less than 30, and where Neurospora research is routinely done in the department. While we have limited our study to tryptophan mutants, this is, in part, a reflection of our own research interests. Presumably mutants involving any well-investigated pathway could be used. About 8 weeks of a 10-week term are necessary for completion of the project. By no means all of the laboratory time is devoted to Neurospora, but 8 weeks' total time is necessary to allow growing time for several repeats of various tests, especially the intra-genic complementation tests.
The general approach involves:
1) Induction (by UV or nitrous acid) and selection (by filtration and selective plating) of tryp mutants.
2) Determination of the particular tryp locus altered in each mutant.
3) Assignment of 3 or 4 tryp mutants (at least 2 of these tryp-3 mutants) to each student. If the yield of new tryp-3 mutants is small, the supply is augmented by "unknowns" from our stock collection.
4) Characterization of the tryp mutants in relation to each other and to known tryp-3mutants on which published data are available. This procedure includes tests for temperature sensitivity, indole and/or indole-glycerol accumulation, ability to grow on indole, linkage to fluffy, and intra-genic complementation.
5) Assay of crude extracts of a wild type and of a tryp-3 mutant for reaction 2 activity of tryptophan synthetase. For this purpose one batch of wild type strain and one of a tryp-3 strain are grown and the lyophilized mycelium distributed to small groups of students working together on the assay.
6) Preparation of a report by each student. This report is written in the form of a scientific paper and includes the characteristics of the mutants studied, how the mutants are related to previously known mutants, possible explanations for conflicting results, etc.
By setting up this project in such a manner that students are studying unknown mutants within a partially structured procedure, it is possible to allow for individuality of evaluation and speculation and for some freedom of experimentation without requiring the additional teaching staff and the variety of specialized equipment that would be necessary to allow a completely free project.
To present the procedure in detail would be to publish a whole lab manual in the NN. Moreover, the exact procedure differs somewhat from year-to-year, partly in response to availability of equipment, strains, etc., and partly in response to the instructor's need for variety. If any reader desires an amplification of some aspects of this project, I can sometimes be reached by mail, but more surely by telephone. As an example, the procedure by which the students obtain mutants for use in this course is given below.
Neurospora mutant hunt:
When Neurospora (or any other organism) is exposed to a mutagenic agent (such as ultraviolet light, x-rays, nitrous acid, etc. ), mutations may occur in many different genes in many different cells. If we wish to keep only the mutated types, especially if we wish to collect only certain of the mutated types, an effective selection technique is required. In this course we will use a local modification of the Woodward and Srb filtration method (1954 Proc. Natl. Acad. Sci. U. S. 40: 192). This method is based on the fact that wild type Neurospora can grow in a simple medium containing only minerals, biotin, and sucrose, while nutritional mutants cannot. After treatment with a mutagen, the conidial suspension is incubated in liquid minimal medium. The wild type conidia will germinate, grow, and can be filtered off; the ungerminated mutant conidia will pass through the filter. After repeated cycles of incubation and filtering, the relative proportion of mutant conidia remaining in the medium will increase. The filtrate is then distributed in petri dishes containing minimal medium supplemented with sorbose (which causes Neurospora to grow in a compact, pellet-like form) and with nutrients required by the desired mutant type. In an ideal experiment, a large proportion of the little colonies which appear on the plates will be of the desired type.
The following procedure will be used for induction and selection of tryptophan-requiring mutants from wild type 74A.
1) Each group of students will be given 5 slants of wild type strain 74A (grown for 5 days on minimal agar medium). Vogel's minimal medium N is used throughout these experiments.
2) Pour 5 ml of sterile distilled water into each of the 5 slants. Disperse the conidia, using a sterile microspatula.
3) Pour suspensions aseptically through a sterile glass wool filter apparatus until about 15 ml of suspension has collected under the filter. A calcium chloride drying tube (the top plugged, the stem wrapped in cotton and inserted in a test tube, and the bulb lined with glass wool ) is used for this purpose. Remove the filter and stopper the tube of filtrate with a sterile cotton plug.
4) Pipette, aseptically, one drop of well-shaken suspension onto each side of a haemocytometer. Count the number of conidia in each of 5 "big-squares-containing- 16-little-squares" (demonstration). Average the 5 tallies and calculate the number of conidia/ml of suspension. The most efficient concentration of conidia will differ somewhat with the mutagen to be used. For UV treatment, 107-108 conidia/ml is desirable.
5) The induction of mutations:
A. Chemicals. The appropriate concentration of mutagenic chemical is added directly to the conidial suspension and incubated for a given length of time to obtain the desired ratio of mutated to killed nuclei. The action of the chemical is then stopped, usually by the addition of a second chemical which neutralizes its action or by dilution of the mutagenic chemical.
B. Ultra-violet light. Pipette, aseptically, 11- 12 ml of conidial suspension into an empty sterile "deep-dish" petri dish. Quickly replace glass cover. Warm up UV lamp for about 10 minutes. (Do not look at the lamp bulb - the rays can be very damaging to your eyes.) Place the petri dish under the lamp 10 cm below the bulb. (It is desirable to wear rubber gloves while working under the lamp.) The UV source in this experiment is an 8 watt germicidal UV lamp. When you are ready to irradiate your suspension, remove the top of the petri dish (UV rays will not penetrate glass) and, holding the bottom of the dish between thumb and forefinger, rotate gently to obtain maximum exposure of conidia. Time the irradiation and replace the glass petri dish cover (which has been held face down to maintain asepsis). The class will be divided into 5 groups: members of these groups will irradiate their suspensions for 0, 1, 1.25, 1.5, 2 and 3 minutes, respectively.
6) Pipette 5 ml of treated suspension into each of 2 sterile 1-liter Erlenmeyer flasks with filter tops containing 250 ml of minimal medium. Add 0.25 g of streptomycin sulfate " as aseptically as possible". The filter-top plugs are made by placing a rectangular strip of curity 60 cheesecloth across the mouth of an Erlenmeyer flask, then placing the same sized strip across the mouth at a right angle to the first, and then plugging the flask with cotton so that the cheesecloth is pushed down into the flask but the ends are left above it. This makes it possible to remove the plug easily and yet be left with a layer of cheesecloth for filtering.
7) Label flasks with all pertinent information and incubate flask X at 25C and flask Y at 30C.
8) Now for the filtration part of the procedure. The most efficient way of handling the flasks for rapid and aseptic transfer from a flask of liquid, through a sterile cheesecloth filter, and into a sterile empty flask will be demonstrated. The 0 time suspension should be filtered at approximately 9 hours after inoculation. Both the 0 time suspension and the treated suspensions should be filtered at approximately 8 AM, 2 PM and 9 PM for the next two days. Probably one filtration each is sufficient for the 3rd and 4th days. Note: 24 hours after inoculation, filter into flasks containing 100 ml of sterile minimal medium. All other filtrations should be into empty flasks.
9) The time of filtrate plating (usually after 5 days of filtration or when 12 hours passes with no formation of new mycelia ) will be announced in class. Small aliquots of filtrate (the exact amount depending on the concentration of treated suspension and the length of time treated) will be pipetted, aseptically, into flasks of molten, but fairly cool (approximately 45C ) minimal sorbose agar (1% sorbose, 0. 1% sucrose) containing (since we are selecting
for tryptophan requiring mutants ) about 25ug L-tryptophan/ml. The agar suspension will be distributed over 10 already-layered plates for each X and each Y flask, and the plates will be incubated at 25C and 30C, respectively.
10) The plates should be inspected at least once daily (preferably twice) and any visible spots of growth cut out of the agar (aseptically) with a microspatula, inoculated into small tubes of tryptophan-supplemented agar, and incubated at 30C until good growth is obtained (about 3 days). Draw a crayon circle on the petri dish bottom around the spot picked so that you and your lob partners do not repick the some colony later.
11) Transfer, aseptically, tiny wisps of conidia from each agar slant to appropriately labelled small tubes of minimal liquid medium; make two tubes per presumptive mutant. Incubate one at 25C and the other at 37C. Save presumptive mutant stocks in the refrigerator.
12) Those isolates which DO NOT show significant growth on minimal medium at bothtemperatures after 2 days' incubation can be assumed to be mutated in some gene controlling tryptophan formation. Why? Check with the instructor as to what constitutes "significant growth". The mutant stocks should be numbered, transferred onto 3 large tryptophan-supplemented slants, and incubated at 30C.
13) At this point, the tryptophan mutants obtained by the class will be supplemented by tryptophan mutants obtained by the instructor, so that each student will have 3 unknown tryptophan mutants. Study and characterization of these mutant strains will constitute the central experiment of the term's laboratory work.
- - - Department of Biological Sciences, Goucher College, Towson, Maryland
21204.
Dutta, S. K. and N. Richman. The isolation of DNA from Neurospora crassa.
(Please note, more modern protocols for DNA isolation are available, eg on the FGSC methods page)
The exercise given below is used in the laboratory portion of a general genetics course. Students work singly or in groups, depending upon the facilities available. If desired, the students can be assigned the duty of preparing reagents for this exercise during the preceding laboratory period.
Materials needed:
Equipment: table top centrifuges (capacity of at least 1500 rpm ) and centrifuge tubes incubator or waterbath adjustable to both 37 and 60C
125 ml Erlenmeyer flasks
test tubes and test tube racks
graduated cylinders
glass stirring rods
glass-stoppered bottles
Reagents: 1) 0.1 SCC - A stock solution of l0x SCC (sodium saline citrate) can be prepared by mixing 87.5 g NaCl and 44.1 g Na citrate and diluting to 1 liter.
2) Saline EDTA - Add 8.75 g NaCl to 37.23 g disodium EDTA and dilute to 1 liter. Adjust pH to 8.0 with NaOH.
3) Sodium lauryl sulfate - Dissolve 25 g in 75 ml distilled water. Note: solution solidifies below 25C.
4) Tris buffer - Mix 32.5 ml of 0.1 N HCl with 25 ml of 0.2M solution of tris (hydroxymethyl) amino methane and dilute to final volume of 100 ml.
5) Acetate EDTA - Mix 40.8 g Na acetate with 0.037 g EDTA and dilute to 100 ml.
6) RNAse - Obtain commercial RNAse (Calbiochem) and place water solution of this enzyme (2 mg/ml) into boiling water for 10 minutes to destroy DNAse activity.
7) Phenol reagent - Saturate phenol with saline EDTA and adjust pH to 8.0 with 10N NaOH. Caution should be exercised in the preparation and use of this reagent.
8) Isopropanol.
Neurospora powder: Neurospora powder can be produced by harvesting mycelia grown in liquid medium (for a period of 14-18 hours) and then lyophilizing in a freeze dryer. The dry mycelia may be powdered by passage through a Wiley mill (mesh size 60). If facilities are unavailable for the production of Neurospora powder, it can be purchased. The powder may be stored in the freezer for long periods of time.
Isolation procedure:
1) Mix 1 g lyophilized Neurospora powder with 25 ml saline EDTA and 2 ml sodium lauryl sulfate in a 125 ml Erlenmeyer flask.
2) Stir by hand or by use of magnetic stirrer at low speed.
3) Incubate in 60C incubator for 30 minutes.
4) Remove from incubator and add 1-2 drops of chloroform.
5) Transfer to a stoppered bottle and add an equal volume of phenol reagent. Slowly rotate for 3-5 minutes.
6) Pour into centrifuge tubes and centrifuge at 1200-1500 rpm for 10 minutes.
7) Remove supernatant with pipette attached to aspirating tube.
8) Carefully layer two volumes of ethanol on the surface of the supernatant.
9) Carefully spool out DNA with a glass rod and redissolve in small quantity of 0.1 SCC.
10) Adjust pH to 7.8 with Tris buffer.
11) Add RNAse to a concentration of 50 ug/ml DNA solution and incubate for 30-60 minutes at 37C.
12) Remove from incubator and add 1 ml acetate EDTA.
13) Carefully layer approximately 7 ml isopropanol on the surface and spool out the DNA.
14) Redissolve DNA in 0.1 SCC and purify by deproteinization with phenol as
previously described.
Using these procedures, DNA of high molecular weight and good purity can be obtained. If desired, the identification and quantitation of the isolated substance can be made by means of UV-spectrophotometry or chemical tests, such as the diphenylamine reaction. For a 2-hour laboratory period, the procedure can be carried through step 8. The product at this stage is DNA with adhering protein and RNA. The experiment may be terminated at this point or else the DNA can be dissolved in 0.1 SCC and stored in the refrigerator and the isolation can be continued during the next laboratory period.
- - -Department of Biology, Jarvis Christian College (Texas Christian
University), Hawkins, Texas 75765.
Haskins, F. A. Accumulation of anthranilic acid by a mutant strain of Neurospora.
The laboratory exercise given below has been used successfully for a number of years in a genetics course taken primarily by graduate students, but could readily be used in an undergraduate course, if desired.
Preliminary statement:
Some Neurospora mutants accumulate metabolic intermediates in the mycelium or in the surrounding medium. The indentification of such intermediates provides important clues concerning the nature and the genetic control of certain biosynthetic pathways. The object of this exercise is the demonstration of the accumulation of anthranilic acid by a tryptophanless mutant of Neurospora. Anthranilic acid is an intermediate in the biosynthesis of tryptophan (Wagner and Mitchell 1955 Genetics and metabolism, p. 310. 2nd ed. Wiley, New York).
Strains used:
Strain 10575. This strain is defective at the tryp-1 locus. It grows if supplied indole or tryptophan, but is not able to utilize anthranilic acid for growth.
Strain B1312. Judging by its growth responses, this strain appears to be mutant at the tryp-2 locus. Other tryp-2 mutants (e.g. 75001 or 40008) would probably be equally useful in this experiment. Mutants of the tryp-2 type are able to utilize anthranilic acid, indole, tryptophan, or certain other related compounds for growth.
Both mutant strains are readily cultured on slants of Fries' minimal agar supplemented with L-tryptophan at a concentration of 0.1 mg/ml. Cultures approximately 1 week old should be available for the required inoculations.
Equipment and supplies:
Fries minimal medium. (Other minimal media commonly used for Neurospora would probably be equally satisfactory).
Anthranilic acid and L-tryptophan. At a concentration of 1 mg/ml these compounds dissolve quite readily in water. Warming speeds solution. Dilute anthranilic acid to 0.2 mg/ml for convenience in bioassay.
Sterile water. Add 1 or 2 ml of water to each of several 4-inch test tubes, plug with cotton, and autoclave.
Pasteur pipettes. These are conveniently made by cutting 7-mm soft glass tubing into 15 cm lengths, plugging both ends of each length with cotton, and autoclaving. Shortly before use, heat the center of each length to softness in a Bunsen flame and pull out to form 2 pipettes.
Erlenmeyer flasks, 125-ml. Nine flasks will be needed for each student or pair of students.
Pipettes, 1-ml, graduated. Each student, or pair, needs 2.
Graduated cylinders, 25-ml . Each student, or pair, needs 1 .
Chromatography paper, 6 x 11-inch sheets of Whatman No. 1. Several students may use the same sheet of paper.
Chromatography vessels. One is needed for each sheet. Gallon jars may be used, but standard 6" x 12" chromatography jars are more convenient. Saranwrap is satisfactory for covering the jars.
Solvents for chromatography, about 100 ml per vessel. Two freshly prepared solvents are needed:
n-propyl alcohol - 1% ammonia (2:1, v/v) and
n-propyl alcohol - 1% acetic acid (2:1, v/v)
Glass capillaries. These may be drawn out from 7-mm soft glass tubing.
Ultraviolet lamp or "Blacklight". Peak emission of the lamp should be near 360mu. Autoclave.
Drying oven, 80C.
Analytical balance.
Procedure:
Prepare, and plug with cotton, the following flasks; a) 20 ml minimal medium, b) 20 ml minimal + 0.25 mg anthranilic acid, and c) 20 ml minimal + 0.25 mg L-tryptophan. Autoclave the flasks 20 min. at 15 psi, cool, and inoculate each flask with 1 drop of a suspension made by dispersing a small amount of 10575 conidia in sterile water. Incubate the flasks at room temperature or in a 25C incubator if one is available. The response of strain 10575 to minimal medium, to anthranilic acid, and to tryptophan is shown by the growth or lack of growth occurring in the 3 flasks. The medium in flask c, after growth of strain 10575, will be used in next week's laboratory.
After an incubation period of 7 days (4 to 7 days should be satisfactory, depending on the laboratory schedule), observe the 3 flasks under the ultraviolet lamp. The blue fluorescence of the medium in flask c is produced by a number of substances, perhaps the chief of which is anthranilic acid. Proof of the identity of this compound would require its isolation in pure form from the medium. Such isolation will not be attempted as a part of this exercise, but a partial characterization and assay of the compound will be made on the basis of paper chromatography and biological activity.
A. Paper chromatography. Using a glass capillary, apply a spot of the medium from flask c to a previously marked position on a line 3/4 inch from one of the long edges of a 6 x I 1-inch sheet of Whatman No. 1 filter paper. Sufficient medium should be applied to make the spot approximately 1/4 inch in diameter. Let the spot dry, then repeat the application. Continue this sequence of spotting and drying until 10 applications have been made at the same position. As a control, make one application of the anthranilic acid solution having a concentration of 1 mg/ml . The control spot should be applied along the base line about 1 inch from where the medium was applied. Control spots of additional fluorescent compounds may be used if desired. (Also, the chromatographic separation of the constituents of washable black Skrip ink makes a rather striking demonstration which can be observed while the solvent is traveling up the paper.) After all spots are dry, staple together the ends of the sheet to form a cylinder 6 inches high. Do not permit the ends to overlap each other. Place the cylinder in one of the solvent vessels provided, and cover the vessel. Part of the class will use the n-propyl alcohol-ammonia solvent and the remainder will use the n-propyl alcohol-acetic acid solvent. After approximately 2 ½ hours, remove the chromatograms from the solvents, dry, and examine them under the ultraviolet lamp. Mark the solvent front and any fluorescent spots that are apparent. Measure distances from the base line to the solvent front and to the centers of any fluorescent spots. Calculate the Rf value of each spot as the ratio of the distance traveled by the solute to the distance traveled by the solvent. Each student should observe and record results obtained with both solvents.
B. Bioassay. Prepare and autoclave the following flasks: 1) 20 ml minimal medium, 2) same, + 1 ml medium from flask c, 3) same, + 0.02 mg anthranilic acid, 4) same, + 0.05 mg anthranilic acid, 5) same, + 0. 10 mg anthranilic acid, and 6) same, + 0.20 mg anthranilic acid. Inoculate each of the 6 flasks with 1 drop of a conidial suspension of strain B1312 (or other tryp-2 mutant). Incubate at room temperature (or 25C) for 4 days, then harvest the mycelial pads, squeeze out most of the liquid, dry the pads overnight at 80C, and weigh them to the nearest mg. Plot dry weights against quantity of anthranilic acid, for flasks 3, 4, 5, and 6. Assuming that anthranilic acid is the only material in 10575 culture filtrates with activity for strain B 1312, calculate the quantity of the compound present in flask c. (Students typically obtain values approximating 0.15 mg of anthranilic acid/ml, or a total of about 3 mg in flask c.
- - - Department of Agronomy, University of Nebraska, College of Agriculture,
Lincoln, Nebraska 68503.
Herrmann, R. L. The role of orotic acid in pyrimidine biosynthesis in Neurospora.
The experiment below has been used in an advanced biochemistry course for the past four years with considerable success. Part A normally takes three full laboratory periods, starting on a Friday to allow for growth of cultures over the weekend. Part B was adapted from a similar experiment with E. coli described in Cowgill and Pardee (1957 Experiments in biochemical research technique. Wiley, New York)
Objective:
The experiment provides experience in radioactive tracer methodology, in the use of mutant organisms, and in the isolation and characterization of compounds of biological significance.
Equipment and supplies:
Cultures: N. crassa wild type strain 1A and mutant strain pyr-4 (36601).
Medium: Fries basal medium.
Isotope: 6-14C-orotic acid (New England Nuclear Corporation).
Equipment: Flash evaporator
UV spectrophotometer
Clinical centrifuge
Hood
Autoclave
Incubator-shaker
Planchet-type radiation counter
Fraction collector (optional)
Mineralite UV lamp
Chromatography tank
Procedure:
Part A. Incroporation of 6-14C-orotic acid into ribonucleic acids in Neurospora.
Growth procedure: The mold is grown in 125 ml Erlenmeyer flasks. 1 ml of a stock solution of orotic acid-6-14C containing 2.5 x 106 cpm/ml is added directly to 125 ml of Fries medium by means of a pipettor. Unlabelled orotic acid (40 mg) is added separately to the medium, and 12.5 ml volumes are pipetted (plugged pipettes) into each of ten flasks. The flasks are then stoppered with cotton plugs and autoclaved for 20 minutes. After cooling, the flasks are inoculated with 0.2 ml of a conidial suspension of the mold, made by dispersing two loopfuls of the conidia in 10 ml of sterile distilled water. Incubation is at 25C for 3 days, by which time conidiation is just commencing. The contents of the flasks are then filtered on a fritted glass funnel with suction and the mycelial residue is washed with 5 ml of water and then soaked in 100 ml of acetone for 15 minutes. The acetone is removed and the mycelium washed with dry ether and allowed to dry. A brittle disc is thus obtained which is suitable for grinding.
Isolation of ribonucleotides: The mycelium obtained from 10 flasks of the wild strain of the mold grown for 3 days on orotic acid-6-14C is ground in a mortar with 120 mesh carborundum powder for 15 minutes. The resulting powder is extracted 3 times with 10 ml of cold, 10% (w/v) trichloroacetic acid, transferring to a 12-ml Pyrex centrifuge tube in the process. The solid residue is washed once with 4:1 (v/v ) ethanol-water and then extracted 3 times with boiling 3:1 (v/v) ethanol-ether. The extractions are carried out by suspending the centrifuge tube in a boiling water bath and stirring the contents occasionally. The process is carried out for a half-hour the first time and for 6 minutes the remaining times. The lipid-extracted residue is then washed twice with ether and air dried. Five ml of 1N KOH is then added, and the mixture allowed to stand at room temperature for 24 hours. After centrifugation, the supernatant is transferred to a second 12-ml centrifuge tube, cooled on ice, and acidified to a pH of 3 with concentrated perchloric acid (pH test paper). The resulting precipitate of potassium perchlorate and protein is allowed to coagulate for 10 minutes and then removed by centrifugation. The resulting supernatant of ribonucleotides and suspended protein is filtered through a layer of Celite on a fritted glass filter and brought to a pH of 11 with 1N KOH. The solution of ribonucleotides is thus obtained and is allowed to filter into a 1 x 27 cm column containing Dowex 1 anion-exchange resin (Cl- form) of 200-400 mesh size and 10% cross-linking. The column is developed first with 200 ml of water and then with 200 ml of 2N HCl. The optical density of the latter eluate is determined at 260 mu, and an approximate molar extinction coefficient of 10,000, together with an average molecular weight of 350, is used to estimate the concentration of mixed nucleotides (ca. 5 mg). The solution is then evaporated in vacuo to dryness several times to remove hydrochloric acid, yielding a greyish-white residue.
Hydrolysis of ribonucleotides: The mixed ribonucleotides are taken up in several ml of 0.1N HCl and transferred to a glass-stoppered, 10-ml volumetric flask. The mixture is then blown to dryness by means of a stream of charcoal-filtered air. After careful addition of 0.5 ml of concentrated perchloric acid, the flask is heated on the steam bath behind an explosion shield for 40 minutes. The contents of the flask are then transferred to a 12-ml centrifuge tube with the aid of several small portions of water, and, after centrifugation and transfer of the supernatant to a second graduated centrifuge tube, the precipitate is washed and the washings added to the tube. The solution is then diluted to 5 ml, to provide a perchloric acid concentration of about 1N, preparatory to separation of the mixed bases.
Isolation of the purine and pyrimidine bases: The solution of bases in 1N perchloric acid is allowed to filter into the resin bed of a 1 x 27 cm Dowex 50 column (hydrogen form) 200-400 mesh. Elution with 100 ml of water, collecting 10-mi fractions, serves to remove uracil mixed with perchloric acid. Cytosine usually comes off in an 80-ml volume after ca. 160 ml of 2N HCl has been passed through the column. Guanine is next eluted by 3N HCl in an approximately 120-ml volume after about 60 ml of the eluant has passed through the column, and adenine is then removed by ca. 140 ml of 4N HCl after a forerun with about 60 ml of the acid. Elution should be followed by means of the Beckman spectrophotometer and the OD at 260 mu and at max plotted against the volume collected.
The uracil-perchloric acid solution is carefully taken to 3 ml volume and adjusted to a pH of 11 with 5N KOH. After removal of the potassium perchlorate precipitate, the solution is placed on a 1 x 10 cm Dowex 1 column in chloride form. After the solution has filtered into the resin bed, 100 ml of 0.015N ammonium formate buffer pH 9/1 is passed through, and uracil is then removed in a 75-ml volume after 75 ml of 0.015N ammonium formate buffer of pH 8.0 has filtered through the column. Half-milliliter samples of the separated bases are plated on stainless steel planchets, evaporated dryness and counted. Determine the specific activity of each purine and pyrimidine base. Editor's note: Some of the above procedures involving the use of perchloric acid are dangerous and should be performed by students only under the supervision of an experienced chemist and with the proper safety equipment.
Part B. Isolation of excreted orotic acid from N. crassa strain pyr-4 (36601).
Into a 2-liter Fernbach flask place 700 ml of a medium consisting of Fries basal medium + 0.5 mg/ml yeast extract + 0.1 mg/ml asparagine + 0.05 mg/ml cytidine hemisulfate. Plug the flask and autoclave and, after cooling, inoculate with a conidial suspension of strain 36601. After 3 days' growth, harvest the mycelium, collecting the residual nutrient medium. Store the mycelium at OC. Dilute 0.5 ml of the medium with 2.5 ml of water and read the optical density at 290 mu: the reading should be about 0.2. Assuming that 50% of this absorption is due to orotic acid (e290 = 6.2 x l03; mol. wt. of orotic acid monohydrate = 174), how much orotic acid is in the solution? Concentrate the medium to about 200 ml by flash evaporation. Make the solution 0.1N in KOH. After the solution is chilled overnight, potassium orotate should crystallize out. Separate the precipitate by centrifugation, dissolve most of it in as small a volume of boiling water as possible (less than 10 ml), add a little charcoal if much color is still present, and filter while hot. Bring the hot filtrate to 0.1N with HCl and allow orotic acid to crystallize by cooling the solution to 0C overnight. Filter, wash the crystals with cold water and dry them in vacuo.
Identify the crystals as orotic acid monohydrate by some of the following criteria: a) melting point 323C (decomposes); b) spectra at several pH (5 ); c) equivalent weight and pKa, by titration; d) paper chromatography. For paper chromatography place 5 ul of 0.1% orotic acid neutralized with NaOH on Whatman No. 1 paper, develop the descending chromatogram with a solution containing 50 ml of n-butanol, 15.8 ml of 95% ethanol, 11.4 ml of formic acid and 22.8 ml of water. Orotic acid gives a spot with an Rf of 0.43 that can be seen under ultraviolet light. Run a sample of authentic orotic acid for comparison. Samples obtained in Part A may also be run against known compounds in this solvent system.
On the basis of the results of Parts A and B, what can you conclude
about the role of orotic acid in pyrimidine biosynthesis ?
References:
Aronoff 1956 Techniques of radiobiochemistry. Iowa State College Press, Ames; Kamen 1951 Isotopic tracers in biology. Academic Press, New York; Lieberman and Kornberg 1953 Biochim. Biophys. Acta 12:223; Michelson, Drell and Mitchell 1951 Proc. Natl. Acad. Sci. U.S. 37:396; Mitchell, Houlahan and Nyc 1948 J. Biol. Chem. 172:525; Shugar and Fox 1952
Biochim. Biophys. Acta 9:199; Woodward, Munkres and Suyama 1957 Experientia 13:484.
- - - Department of Biochemistry, Boston University School of Medicine,
Boston, Massachusetts -2118
Ishikawa, T. Neurospora as part of an undergraduate genetics course.
The following contribution presents, in outline form, procedures for experiments with Neurospora that are used in a genetics course at the University of Tokyo. The nature of accompanying laboratory lectures is indicated for each experiment, as well. The students, about 15 per class, are undergraduate majors in biology who meet for 5 hours per week for 12 weeks; they are expected to come to the laboratory outside regular class hours to make observations whenever necessary. Media, cultures and sterile glassware for the experiments are prepared by the students, themselves. Several of these exercises may be run simultaneously, depending upon the class schedule.
Experiment 1. Characterization of mutant strains.
Lecture: Mutation, types of mutants, pathway of adenine (AMP) synthesis.
Strains: ad-3; ad-4; ad-6; ad-8; ad-8; lys-5; ylo-1; asco, lys-5; 74A; 3. la.
Procedure:
1) Prepare fresh cultures of mutants and the wild types given. Use glycerol complete slant medium + adenine 100 ug/ml and lysine 100 ug/ml.
2) Sterilize petri dishes, test tubes and pipettes. Prepare sterile water blanks.
3) Prepare media: a) sucrose agar plating medium with and without supplements; b) liquid minimal medium with and without supplements. Supplements: adenine 100 ug/ml, hypoxanthine 100 ug/ml, lysine 100 ug/ml or adenine + lysine.
4) Pour 20 ml agar medium, autoclaved, into each plate. Dispense 1 ml liquid medium into each 10-cm tube, plug autoclave.
5) Observe morphological characters of fresh slant cultures.
6) Make conidial suspensions of each strain in water and shake well.
7) Inoculate conidial suspensions into both media. Mark the strain numbers on plates and tubes and incubate at 25C.
8) Observe growth after 1-3 days. Record morphological and biochemical characters of the strains.
Experiment 2. Allelism test.
Lecture: Discussion of gene, locus, allelism; cis-trans test.
Strains: ad-8 mutants (E6A; E129A; E32A; E1l1A; E34A; E41A); ad-4A.
Procedure:
1) Prepare fresh cultures of mutant strains given. Use glycerol complete slant medium (+adenine 100 ug/ml).
2) Prepare plates of minimal agar medium and sterile water blanks. Sterilize pipettes.
3) Observe morphological characters of fresh slant cultures. Suspend conidia of each strain in 2 ml water.
4) Mark on the bottom of each minimal agar plate 4 spots on the periphery: one spot for two individual mutants and two spots for the combination of these two mutants. Make all possible mutant combinations.
5) Inoculate the spots marked on the bottoms of the plates with conidial suspensions of the appropriate mutants, using 1-ml pipettes. Incubate at 25C.
6) Observe the results every day for at least 3 days. Consider allelic relationships among the mutant strains given. Construct the complementation map for the allelic mutants.
Experiment 3. Recombination and the genetic map (1).
Lecture: Meiosis, tetrad analysis, recombination, genetic map.
Strains: asco, lys-5; 74A.
Procedure:
1) Prepare fresh cultures of both strains. Use glycerol complete slant medium (+ lysine 100 ug/ml).
2) Prepare slants of crossing medium (+ lysine 100 ug/ml) in 3 x 18-cm tubes.
3) Inoculate conidia of both strains into a slant tube. Incubate at 25C for 2 weeks.
4) Observe at least 100 asci under the microscope and calculate the centromere distance for the asco locus.
Experiment 4. Recombination and the genetic map (2).
Lecture: Linkage, linkage groups, genetic map.
Strains: ad-8, lys-5, ylo-1, a; 74A.
Procedure:
1) Prepare fresh cultures of the mutant and wild type given. Use glycerol complete slant medium (+ adenine 100 ug/ml and lysine 100 ug/ml). Prepare crossing medium, supplemented, in 18-cm tubes.
2) From fresh slant cultures, inoculate mutant and wild conidia onto crossing medium. Incubate at 25C for 3-6 weeks, until ascospores are shot.
3) Prepare plating medium (sorbose minimal agar medium + adenine + lysine), 0.05% agar solution and sterile plates.
4) Prepare ascospore suspension in 0.05% agar solution. Count the number of ascospores in 0.05 ml suspension. Heat shock the ascospores at 60C for 40 minutes in a water bath.
5) Pipette spore suspension (200 spores per plate) into sterile plates. Pour medium (40C) into plates containing ascospores. Mix well. Incubate at 25C for 3 days.
6) Prepare isolation medium (minimal medium + 0.5% agar + adenine + lysine). Melt, pour into 7-cm test tubes, plug and autoclave.
7) Isolate 200 colonies; cut out a piece of colony and transfer it into isolation medium. Incubate at 25C for 5-7 days.
8) Prepare test medium (minimal liquid medium with appropriate supplements poured into 7-cm tubes and sterilized).
9) Observe the characters of each isolate and test for biochemical requirements by inoculating conidia into liquid test medium suitably supplemented.
10) What is the genotype of each isolate? Consider the linkage relationships.
Experiment 5. One gene-one enzyme relationship.
Lecture: Protein synthesis; amylases produced by Neurospora.
Strains: 74A; amylase mutant which shows no amylase activity in the culture filtrate.
Procedure:
1) Prepare fresh cultures of amylase mutant and wild type. Use glycerol complete slant medium.
2) Prepare crossing medium in 18-cm tubes.
3) From fresh slants inoculate mutant and wild type into crossing medium. Incubate at 25C for 4 weeks.
4) Prepare plating medium (sorbose minimal medium).
5) Suspend ascospores in 0.05% agar solution. Count the number of ascospores in 0.05 ml suspension. Heat shock ascospores at 60C for 40 minutes.
6) Plate 200 ascospores per plate. Incubate at 25C for 3 days.
7) Prepare isolation medium (minimal medium in small tubes).
8) Isolate 100 colonies. Incubate at 25C for one week.
9) Prepare: a) minimal liquid medium + 1.5% maltose in 20 18-cm tubes (3 ml/tube), and b) minimal liquid medium + 0.2% starch + 1% sucrose in 100 10-cm tubes (1 ml/tube). Plug and autoclave.
10) Inoculate conidia of each isolate, amylase mutant and wild strain into a and b media. Into a put 20 isolates, and into b put 100 isolates. Procedure b is a convenient simple method to observe the presence of amylase activity in the culture filtrate. Procedure a is used to confirm the b result. Incubate at 25C; a for 2 weeks, b for 5 days.
11) After 5 days add to the b cultures 0.1 ml of I2-KI solution. Identify amylase mutants.
12) After 2 weeks, pipette out 0.4 ml culture filtrate from the a cultures and assay for amylase activity. Identify the amylase mutants and compare with the result from procedure b.
Assay method:
Reaction mixture: to 1.6 ml of 0.11% starch dissolved in 0.05M tris buffer (pH 6.0) add 0.4 ml of culture filtrate. Incubate at 37C for 60 minutes. Stop reaction by adding 0.4 ml 1N HCl.
Starch-iodine reaction: to 1 ml of reaction mixture add 0.5 ml 1N HCl and 1 ml of I2(0.03%)-KI (0.3%) solution. Dilute to 10 ml by adding 7.5 ml water.
Measure transmittance at 660 mu.
Experiment 6. Induction of mutation.
Lecture: Mutagenesis, forward and back mutations.
Strains: ad-8 (E6A).
Procedure:
1) Prepare a fresh culture of the ad-8 mutant strain. Use glycerol complete medium in 16 100-ml flasks supplemented with adenine, 100 ug/ml.
2) Prepare plating media: a) to detect revertants, minimal sorbose medium, 15 plates for each irradiation, b) for survival test, minimal sorbose medium + adenine 100 ug/ml, 15 plates for each irradiation.
3) Prepare a conidial suspension of the mutant in sterile water. Spin down the conidia in centrifuge tubes. Wash 3 times with water by centrifuging. Resuspend in 90 ml water. Estimated number of conidia should be approximately 1 x 108 ml.
4) Pipette 10 ml of conidial suspension into a sterile petri dish and irradiate with a 15W UV-lamp at 30 cm. for between 0 and 30 minutes, taking samples at 5 minute intervals.
5) Dilute irradiated suspension for survival test, using sterile dilution blanks (9 ml of water) and 1 ml pipettes. Make dilution of 10-3-10-6 depending upon the killing rate estimated.
6) Pipette diluted suspensions into plates. Pour in agar medium at 40C and immediately mix well. Plate irradiated diluted suspension onto medium b, using 3 dilutions, 5 plates each. Plate irradiated suspension without dilution onto medium a; 10 plates, 0.8 ml inoculum and 5 plates, 0.1 ml inoculum. Incubate at 25C for 4 days.
7) Count the number of colonies per plate. Draw the survival curve and mutation frequency-UV dose curve.
Experiment 7. Cytoplasmic inheritance.
Lecture: Function of mitochondria, nature of poky mutant, results of reciprocal crosses.
Strains: poky A; poky a; 74A; 3. la.
Procedure:
1) Prepare fresh cultures of mutants and wild type. Use glycerol complete slants.
2) Prepare crossing medium in 18-cm tubes.
3) Inoculate protoperithecial parents: inoculate conidia of all four strains, separately, into tubes of crossing medium. Incubate at 25C for 7 days.
4) Make suspensions of conidial parents in sterile water (all four strains). Pour 0.8 ml of conidial suspension into a tube containing a protoperithecial parent culture. Make all possible reciprocal combinations. Incubate at 25C for 2 weeks.
5) Prepare ascospore suspensions in 0.05% agar solution. Count the number of ascospores in 0.05 ml suspension. Heat shock the ascospores at 60C for 40 minutes.
6) Plate the spore suspensions (500 ascospores per plate) on minimal medium + 1.5% sucrose + 1.5% agar.
7) Observe the size of the colonies after 4 days' incubation at 25C.
- - - Botanical Institute, Faculty of Science, University of Tokyo,
Hongo, Tokyo, Japan.
Graham, J. D. and J. H. Morrison. Studies of the lethal effect and photoreversibility of ultraviolet radiation.
Ultraviolet light is one of the most widely studied mutagenic agents. Its mechanism of action has been demonstrated to be a multiple one, consisting of both the formation of photoreversible thymine dimers within the DNA molecule and other, non-reversible alterations of the molecule. This experiment will be carried out both in the dark, thus avoiding photoreactivation, and in the light. When ultraviolet irradiation is carried out in the presence of white light, a considerable portion of the genetic damage induced is repaired through the photoreactivation process.
The second half of the experiment is carried out in almost complete darkness, so it is essential that the students plan and organize the work in advance. Equipment which will be needed during the blocked-out phase should be laid out on the work table in a logical order so that it will not be misplaced.
The student will be furnished a Neurospora culture which has a heavy conidial growth. Suspend the conidia in sterile water by tapping the side of the tube with the index finger. Filter the suspension through a very thin sterile cotton pad into a centrifuge tube to remove the mycelium. Centrifuge the filtered conidial suspension for 10 minutes. Pour off the supernatant and wash the conidia with sterile water, centrifuge and resuspend in sterile water. Determine the concentration of the conidia with a haemocytometer and adjust with sterile water to 5 x 106cells/ml. Irradiate the cells in a petri dish at 50 cm from a 15-watt germicidal lamp for 5 minutes, agitating the suspension constantly while irradiating. Withdraw aliquots at 0, 30, 60, 90, 120 and 300 seconds, diluting each aliquot l0x with Neurospora minimal broth (Difco). Thus, each sample will have been diluted 10 times upon withdrawal from the dish. The platings of each sample, for scoring of survivors, should be as follows: plate 1 ml of the following dilutions, 1 x l0-4, 5 x l0-3, 1 x 10-3, 5 x 10-2, 1 x 10-2, 1 x 10-2 and 1 x 10-1, respectively for the exposure times given above. Pipette the sample into a sterile petri dish and add 10 ml of Neurospora agar (Difco) which has been supplemented with 8 g of sorbose to induce colony formation. The agar should be liquid but not painfully hot to the touch and should be adequately swirled. Incubate the plates right side up at room temperature.
The procedure above should be followed during the second phase of the experiment, except that the lab should be made completely dark to guard against photoreactivation. A yellow safelight bulb may be used, if necessary and if kept at least five feet from the work area.
Construct a graph of survival rate against length of irradiation for Neurospora in both light and dark. Discuss the photoreactivation effect and the effects of ultraviolet light as a mutagenic agent.
- - - Department of Biological Sciences, Kent State University, Kent,
Ohio 44240.
Woodward, V. W. The use of Neurospora in teaching.
The October 1, 1966, letter "To all Neurosporologists", from the editor, was clear in stating the desired emphasis of NN#10, i.e., " the use of Neurospora in teaching", but I am going to take the liberty of expanding that emphasis, via the catchall "in teaching", to two aspects of laboratory teaching not specified in the letter.
The first point is directed to teachers who may wish to use Neurospora as a laboratory teaching tool but who are at present unfamiliar with it. In my opinion it is unwise to incorporate Neurospora into the student laboratories (a) before the laboratory is properly equipped, and (b) before the teacher has become familiar with, maintained, and worked with the organism. The ill-equipped laboratory coupled with inexperienced hands insures contamination of the cultures and a teacher unfamiliar with the eccentricities of Neurospora may only add to the confusion of interpreting experimental results. If Neurospora is used under these conditions, it would be best to "mark" all the stock cultures (e.g., with albino) so that natural contaminants can be screened . My experience leads me to conclude, however, that Neurospora is not an ideal organism for the amateur who has neither the time, equipment nor inclination to become thoroughly familiar with it, and that the confusion created by contamination and unfamiliarity in beginning laboratories for offsets the slim harvest of insight or intellectual stimulation its use may afford.
The second point is directed to teachers who are familiar with Neurospora and who have teaching laboratories equipped to cope with it. These teachers may use Neurospora as a means for designing questions and problems calculated to stimulate the imagination of the student rather than as gimmicks to keep him busy. A plan that I have followed can be adapted to the personality of the teacher and it has proven a worthwhile incentive for my students. In brief, my suggestion is to present to the student the responsibility of becoming acquainted, through the literature, with specific key experiments (each teacher will make his own list), and with some of the questions being asked today by Neurosporologists. In the meantime, the student will do some simple growth kinetics, transferring, ascospore isolations, etc., to become familiar with basic procedures. Following such orientation the student will propose an experimental design, defend the design, modify it to fit all the "feasibles" (time, space, equipment, etc.), and then attempt to execute the experiment.
The isolated pockets of education which permit student participation both in the design and the execution of experiments are rare, and this extends to many graduate schools. It would appear that a well-equipped laboratory coupled with a teacher familiar with the experimental material may well combine to foil the "busy-work" approach taken, usually out of necessity, by teachers with fewer resources, and at the same time take the more positive approach of encouraging student expression .
During the first year such a laboratory was conducted at Rice, one pair of students undertook the task of recovering a radiation-resistant strain of Neurospora, hopefully to compare "repair mechanisms" with those proposed for bacteria. Through repeated irradiation and asexual transfer, they were able to isolate a strain which showed about 20% survival at a dose of ultraviolet light which killed all of the parent conidia. This mutant also showed markedly increased resistance to gamma irradiation. When crossed with wild type the resistant strain produced both sensitive and resistant progeny in a ratio of 1:1. One of the students, an organic chemist theretofore, was accepted at the University of California, San Diego for graduate work in biology: the other, a married woman, stayed at Rice where she earned the master's degree. Later, another pair of student tried to detect and isolate thymidylate synthetase in Neurospora. Their project failed, in the classical sense, but both students gained insight and momentum; one was accepted for graduate study in the biochemistry department at Stanford, and the other in chemistry at UCLA. It is difficult to plot the "human-element-data" of these "experimental" laboratories, but the correlation seems high between excitement about research and the desire to enter graduate school . The relative merits of the more stereotyped student laboratories compared with the type described here with respect to inciting interest in research cannot be proven; however, my own views should be obvious.
What really matters about laboratories designed to acquaint students
with biology is whether or not the students think about the experiments
and subsequently apply the ability to think to their own work. I contend
that the probability of their thinking about their work increases
proportionately with their investment of sweat and tears into the design
and interpretation of their work, as well as the execution. It is a further
contention that extension of this idea within the teaching community will
be for the ultimate good of students whether or not they become professional
biologists. ( This work is supported by f. e. w. (not to be confused with
HEW)). - - - Biology Department, Rice University, Houston, Texas 77001.
Barratt, R. W. Neurospora as a laboratory contaminant.
It may seem inappropriate in an issue of the Newsletter devoted largely to laboratory experiments using Neurospora to include a note on its control. Yet many biologists have refused even to have Neurospora cultures around the laboratory, to say nothing about permitting students
to work with them. Such an attitude has the weight of the testimony of many investigators who in a moment of weakness let down the bars just once, only to suffer widespread contamination of valuable microbial stocks by that "dirty orange bread mold". (This attitude has even been known to work against the employment of Neurosporologists.) The difficulty arises from the ease with which Neurospora macroconidia are dispersed aerially, their longevity under laboratory conditions, the high rate of mycelial growth (4-5 mm/hr) once established on suitable media, and the fact that the fungus will grow readily on many common laboratory media. When then can be done to circumvent these real drawbacks to the use of Neurospora in class experiments?
Several years ago when I was teaching a course in "fungal genetics of filamentous fungi" with E. Kafer at the Cold Spring Harbor laboratory and simultaneously using Neurospora crassa, with its spreading growth habit, and Aspergillus nidulans , with its restricted growth habit, we were faced with this problem in an acute form. Other than the obvious standard microbiological sanitary procedures, including wiping the laboratory bench down with a disinfectant (Lysol or dilute Chlorox solution) prior to and after use, and spraying the air with propylene glycol to settle conidia, we found that very effective control was accomplished by increasing to between 39 and 40C the temperature of incubators being used for other organisms. Ryan, Beadle and Tatum (1943 Am. J. Botany 30:784) reported that "at a temperature above 40C the rate of growth (of mycelia) progressively slowed down and eventually reached zero." Neurospora has a sharp temperature optimum at 34-35C. They further showed that Neurospora has a pH optimum of 5-6 with a marked decrease in growth rate above 6.5. Their experiments were carried out in growth tubes on Fries minimal medium (which is poorly buffered) which showed a decrease in pH as the mycelial front progressed. Thus, well-buffered medium at pH 7 or above provides a poor environment for the growth of Neurospora, though it may not prevent its growth completely.
More recently, two additional methods have become available which help to avoid the contamination problem. Aspergillus workers frequently add sodium desoxycholate (800 mg/liter) to restrict colony size and to enhance conidiation. Many other fungi (e.g., Penicillia) are also resistant to desoxycholate. Neurospora, on the other hand, is completely inhibited by low concentrations of desoxycholate.
In many experiments and experimental techniques, strains of Neurospora with restricted growth rates (colonials), aconidial strains (fluffy), or microconidiating strains can be substituted for wild type macroconidiating strains. Colonial strains have much lower growth rates and are no more of a laboratory hazard than normal fungal aerial contaminants (Penicillium, Aspergillus, Fusarium, Alternaria). Perkins employs aconidial (fluffy) stocks as mating type testers. Microconidia are short-lived, are not borne on long hyphal filaments and conidiophores, and are thus much less of a source of aerial contamination.
Lastly, and as a general practice, whenever practical, students should be provided with aqueous suspensions of Neurospora conidia rather than making any transfers of dry conidia. Further, any petri plate contaminated by Neurospora should be destroyed by autoclaving immediately upon its discovery, and students should be forewarned of the contamination problem. Each of us has his pet story about Neurospora contamination, but one more note of caution is worth mentioning: never discard agar or other suitable substrata into a wastebasket. Neurospora will find it and conidiate abundantly within 2 days. Paranoid individuals should employ only suitably tagged strains (e.g., albino) to avoid the unjustified wrath of their colleagues.
Other Neurospora workers may have developed methods to reduce the risk
of contaminating a laboratory with Neurospora. If so, I encourage them
to communicate with the Newsletter editor. To end on a note of optimism,
we have never, to the best of our knowledge, cross-contaminated any of
the over 1,200 stocks of Neurospora in the FGSC. So the problem is not
insurmountable.
Cooke, F. Fungal genetics course based on Coniochaeta.
Dr. Fred Cooke, of the Biology Department, Queen's University, Kingston, Ontario reports that he has developed a laboratory course in fungal genetics which is carried out with the heterothallic ascomycete Coniochaeta kellermanii. Coniochaeta is similar to Neurospora but has the advantages of a much more restricted growth and a shorter life cycle. Students have been able to isolate several color mutants as well as a number of auxotrophic mutants. In his opinion, the laboratory course could be modified for use with Neurospora.
Dr. Cooke would be happy to provide information about experiments developed for this course to anyone who is interested.