Study of a Biochemical Pathway in Neurospora
In this lab you will investigate the biochemical pathway by which Neurospora, a common bread mold, synthesizes the amino acid tryptophan and the pathway leading from tryptophan to the vitamin nicotinamide. You will be given the pathways and seven mutant strains of Neurospora which require an external source of tryptophan (or for some of the mutants, nicotinamide) to grow. They cannot make their own because in each strain, a mutation has altered the gene coding for one of the enzymes in the pathway. By using various techniques, such as plating the mutant strains on medium containing intermediates from the pathway, detecting compounds accumulated by the mutants, and observing the behavior of strains containing two mutations, you will attempt to figure out the step in the pathway at which each mutant is blocked. Solving this puzzle requires putting together many separate pieces of evidence.
What We Expect You To Get Out of This Lab
The biosynthesis of even relatively simple molecules requires many steps, each controlled by a separate enzyme.
Having a series of strains with mutations in different genes but the same phenotype (in this case the inability to grow in the absence of tryptophan, or in some cases, nicotinamide) allows one to dissect a complicated pathway.
Crossfeeding depends on-the ability of one mutant to accumulate a compound which can support the growth of another mutant.
A strain carrying mutations in two different genes affecting the same pathway accumulates compounds like one carrying a single mutation in the gene coding for the enzyme which comes earlier in the pathway.
In chromatography, the distance a particular compound moves depends on its relative affinity for the moving phase and the stationary phase.
Tryptophan is needed by the cell for protein synthesis as well as for nicotinamide synthesis.
Spotting Neurospora conidia (asexual spores) on plates where it grows in restricted form
Testing mutants for nutritional requirements
Detecting fluorescent and UV- (Ultraviolet) absorbing compounds on a chromatogram
Further understanding of the importance of including and analyzing control tubes before drawing conclusions
Interpreting chromatograms and drawing conclusions about compounds accumulated by mutants
Bringing together many pieces of evidence to reach a conclusion
Deciding upon the narrowest possible range of possibilities when the exact step cannot be determined
Terms you should understand
ascus, bioassay, conidia, crossfeeding, double mutants, hyphae, spent medium
A. Biosynthetic Pathways
We are used to the idea that such traits as eye color, stature, and blood types are inherited, but we know little about the biochemical processes by which these traits are expressed. In this experiment, we will see that nutritional requirements are also under genetic control, and that mutants with altered nutritional requirements can be used to dissect a complicated biochemical pathway. The principle is quite simple. Imagine a pathway in which a compound, A, is converted to B by an enzyme, alpha, and B is converted to C by an enzyme, Beta, etc., until finally Z, a required building block, is formed:
alpha beta gamma delta epsilon A <=====> B ----> C ----> D ----> E ----> ----> ----> Z | B'
The information the cell uses to make each of the enzymes represented by Greek letters is found in a particular gene. Now, if the gene that codes for beta is mutated so that beta is absent or non-functional, the resulting mutant organism will require an outside source of Z to grow since it cannot make its own Z. It will also be able to use a variety of compounds in place of Z, such as C, D, E, since enzymes gamma, delta, etc. are present and functional, but it will not be able to use A or B. By making educated guesses about what sort of compounds might be involved in the pathway, one can test a large number of compounds as a substitute for Z.
B. Accumulation of Intermediates
An even more powerful technique can be used to eliminate much of the guesswork. If enzyme beta is missing, compound B will tend to accumulate, since the enzyme that normally would convert it to C is missing. Often B will accumulate in the organism or in its growth medium to concentrations that are many thousands of times higher than those that would be present in the wild type organism. B can often be detected in the growth medium by its ability to support the growth of a mutant that is missing enzyme alpha (This technique is called "crossfeeding.") Thus, a mutant organism missing enzyme a can be used as an assay organism during the purification of B from the medium of the mutant that is missing enzyme beta.
Once B has been isolated, its structure can be determined by standard techniques of organic chemistry.
This general strategy has been extremely successful in identifying the intermediates in a large number of biochemical pathways. Nevertheless, there are a number of possible difficulties that can arise, so that the approach is not quite as easy as it sounds. Sometimes a compound that is accumulated, such as B, is in facile equilibrium with its precursor, A, so that both are accumulated; or, depending on the equilibrium constant, there may even be much more A accumulated than B. Sometimes there is an enzyme from another pathway that converts B to B', a compound which may occur in small amounts in wild type cells which contain little B, but may become a major compound in a mutant that accumulates a great deal of B. Sometimes B will be unstable, or will be a large or highly-charged molecule that cannot get across the cell membrane, so that "crossfeeding" is impossible. Most of these complications can be gotten around one way or another.
C. Double Mutants
A very useful technique for placing mutants in order in a pathway involves the use of double mutants. In the pathway shown previously, a mutant lacking enzyme beta will accumulate compound B. Let us assume we can recognize this by its color, fluorescence, biological activity, odor, or some other characteristic. A mutant lacking enzyme gamma will accumulate compound C, and possibly B as well, and let us assume that we can also detect C if it is present in quantity. If the crossfeeding experiment does not work, how do we decide which mutant is blocked earlier in the pathway? (Remember that in the real world, nobody has put the compounds in alphabetical order for us!) If we make a cross of the mutant blocked at beta with the one blocked at gamma, we can isolate the double mutant that possesses both mutations and is blocked in both steps (beta and gamma). If the order of the steps is as we have shown it above, this double mutant will behave in one respect exactly like the single mutant blocked in beta, that is, it will accumulate compound B, but not C. This is very general: If any "early" enzyme is knocked out, knocking out a "later" one will not have any additional effect. We will use double mutants in conjunction with paper chromatography and nutritional testing to provide evidence in determining where some mutant strains are blocked in a pathway. Notice that in another respect, the double mutant will resemble the single mutant blocked at gamma: it will grow on D, but not on C.
D. The Use of Neurospora to dissect a Pathway
In this experiment you will examine the pathway by which the necessary amino acid tryptophan is synthesized. Tryptophan is needed by the cell for both protein synthesis and as a precursor in the synthesis of nicotinamide, a necessary vitamin. The organism you will use for this study is the orange colored bread mold Neurospora crassa. It has the advantages of being very easy to grow, being completely non-pathogenic, and having a sexual as well as a vegetative cycle so that genetic analysis can be done. Wild type Neurospora has all the enzymes necessary to make both tryptophan and nicotinamide. It will grow on a simple salts medium containing sugar (sucrose) and a single vitamin (biotin) as the only organic constituents. (We call this medium "minimal.") You will investigate the behavior of seven different mutants which are blocked in different steps in the pathway from sugar to tryptophan and nicotinamide and try to determine where in the pathway each mutant is blocked. We know this pathway quite well as a result of the sort of experiment you will be doing. The pathway is shown on page 2.
Work in groups of two.
Parts A-C below can be done in any order. Sign up for a time in the darkroom for part C so that there is not a big crowd there at the end of the period.
A. Nutritional Testing
Please refer to the diagram on page 2 when reading the description which follows on testing possible intermediates.
Unfortunately, not all compounds that are intermediates in this pathway can be tested. "PRA," "CDRP," and "INGP" do not get across the cell membrane and also are not commercially available. Quinolinic acid is charged and may or may not get across the membrane. Indole is not a true intermediate, but it equilibrates easily with INGP in these strains and will be tried. Anthranilic acid, hydroxy kynurenine, and hydroxy anthranilic acid are true intermediates and will be tested.
You will be given eight agar plates containing eight different media. Each contains salts and a special mixture of sugars (sorbose, glucose, and fructose) on which Neurospora grows in a restricted "colonial" form, rather than rapidly spreading over the whole plate as it would on most media. This will allow you to test all eight strains on each of the eight plates.
One of the eight plates has minimal medium (M) which contains just the salts and sugars. The others contain anthranilic acid, indole, tryptophan, hydroxy kynurenine, hydroxy anthranilic acid, quinolinic acid, or nicotinamide in addition to the salts and sugars. The minimal and supplemented plates are labeled M, A, I, T, HK, HA, Q, and N, respectively. Divide the bottom of each plate into eight pie-shaped sectors with a marking pen and mark each sector for spotting with a particular strain: wild type; trp-1, 2, 3, and 4: and nic-1, 2, and 3.
Each pair of students will be given eight vials containing suspensions of conidia (asexual
spores) of these strains.
Flame a loop and dip it into a suspension of wild type conidia and put a small spot onto the appropriate sector of each plate. Be sure the conidia are well-suspended- they settle out quite quickly; also be sure to get a fresh loopful from the vial for each spotting so that each plate gets about the same inoculum. (The staff will demonstrate good sterile techniques for this.) Then spot the seven mutants. Put all the plates at about 25 C and examine them a few days to a week later. Record the results.
We will try to determine whether trp-1 or trp-2 is blocked earlier in the tryptophan pathway by doing a crossfeeding experiment. The laboratory staff has grown wild type, trp-1, and trp-2 in flasks of minimal medium supplemented with 1 x 10-4 M tryptophan (0.lmM). This amount of tryptophan will be completely used up during growth of these strains, including wild type, which does not need an external source of tryptophan. The mutants will accumulate the compound that comes before the missing enzyme.
Prepare twelve small tubes, each containing a final volume of 1.0 ml (0.5 ml of sterile minimal medium at 2X its normal concentration and 0.5 ml of the medium in which various mutants or wild type cells have been grown, referred to as "spent" medium). After adding spent media or tryptophan (a positive control) to the 2X minimal medium, put the rack of tubes in a boiling water bath for five minutes to kill any Neurospora spores that may have been in the spent media. Cool the rack in cold water for a minute or two, and then inoculate the tubes with suspensions of the strains listed below, using a sterile loop. After the cultures have been allowed to grow for a few days or a week at 25'C, check them qualitatively for growth and record the results.
||Medium to be tested
||After sterilizing, inoculate with|
|trp-1 spent medium
trp-1 spent medium
trp-1 spent medium
|trp-2 spent medium
trp-2 spent medium
trp-2 spent medium
|wild spent medium
wild spent medium
wild spent medium
C. Accumulation of Fluorescent Intermediates by Mutants
One strategy for identifying accumulated intermediates is to separate them by paper chromatography and to compare their rate of migration in one or more solvent systems with that of some known compounds which are suspected as possible intermediates. In this part of the laboratory, you will attempt to do this by analyzing a chromatogram that the staff has prepared.
Flasks of various tryptophan and nicotinamide mutants were grown and the spent growth media concentrated 20-fold by evaporation. The media were then spotted about 2 cm from the bottom of a dry sheet of filter paper. The paper was rolled into a cylinder, stapled so that it would stand up by itself, and placed in a jar containing a small amount of solvent -enough to wet the bottom 1 cm of the paper. The solvent, in this case, was three parts n-propyl alcohol and one part 1M NH40H. As the solvent slowly migrated up the paper by capillary action, it carried the various compounds in the spent media various distances up the paper, so that the complicated mixtures were separated into numerous chemical components. The theory behind this simple technique is as follows: A "dry" piece of paper really has a layer of tightly-bound water on its surface which does not move around. A mixture of compounds spotted onto paper can be thought of as being dissolved in this stationary water phase. When an organic solvent creeps up the paper by capillarity, it forms a moving phase distinct from the stationary water phase. A compound that has a high affinity for the moving solvent phase will spend more time in the solvent phase and less time in the water phase and will move most of the way to the top of the paper by the time the solvent reaches the top. On the other hand, a compound that has a high affinity for the stationary water phase will stay near the bottom. Since every molecule must make the decision which phase to be in over and over again, the mobility of a particular compound will reflect its relative affinity for the two phases.
The chromatogram was dried after the solvent reached the top and has been hung in the darkroom for your inspection. By pointing an ultraviolet light at it, you will see a large number of fluorescent compounds.
(Important note: ultraviolet light can damage your eyes if they are not suitably protected. Do not use the ultraviolet lamp without wearing plastic goggles or ordinary glasses!) If you also put the lamp around behind the chromatogram, you will also see a few ultraviolet-absorbing (dark) spots, if you are using a 'short wavelength' UV lamp. The spots are those from the media of trp-1, 2, 3, and 4, nic-1, 2 and 3, and the double mutants (nic-1, nic-2), (nic-2, nic-3) and, (nic-1, nic-3). In addition, there are a number of more or less pure reference compounds obtained from chemical companies. From the color and approximate mobility of the spots from media of various mutants, you should be able to identify a few of them with the reference compounds. In addition, you should be able to place the nic mutants in order in the pathway just by the pattern of accumulations of the double mutants (as explained in the Introduction).
Please do not mark the chromatogram or touch it with wet or greasy hands. It must last for a number of classes and stray marks make it difficult for others to see the compounds.
D. Analyzing Your Data
From the information that you will obtain from nutritional testing, crossfeeding, and examination of the chromatogram, try to figure out where in the pathway each mutant is blocked. Remember that more than one intermediate may be accumulated behind a block point. This is a challenging puzzle. You will not be able to determine the exact blockage point for some of the mutants. For those where you cannot, decide on the narrowest possible range of possibilities.
III. Further Comments
Bioassays can be used quantitatively to measure a great variety of different vitamins, amino acids, etc. Mutants that require various B-complex vitamins are available in such organisms as Neurospora and E. coli. Some bioassays employ "natural" mutants rather than laboratory-produced mutants. For example, some of the bacteria that cause milk to turn sour require many different amino acids for growth. One can make up a medium containing an excess of all of the required amino acids except one, which is omitted. That amino acid can then be assayed in an unknown food product or biological fluid by adding known volumes of the unknown and comparing the amount of growth of the bacteria with the growth given by known amounts of the pure amino acid.
Bioassays are usually not as quick or easy as chemical assays, but they are often much more specific and sensitive. As little as 10-9g of some vitamins can easily be measured by bioassay-an amount that would be difficult or impossible to measure chemically in a complex mixture.
B. Use of this Approach in Higher Organisms
The general approach of getting mutants and putting them in order in a pathway is not restricted to exploration of intermediary metabolism in microorganisms. The same approach to pathways works in all sorts of organisms, though it is technically more difficult in some. The principle has even been used to explore the genetic control of behavior in organisms such as Drosophila, E. coli, Paramecium, and, more recently, mice.
C. The Relationship of This Pathway to the Deficiency Disease Pellagra
Pellagra is a disease that is primarily due to deficiency of nicotinamide in the diet. It used to be prominent in the southeastern part of the U.S. among people who subsisted mostly on corn. Both corn and milk are poor sources of nicotinamide, but people on a primarily milk diet (e.g., babies) do not get pellagra. The explanation of paradox is that milk is an excellent source of tryptophan, and given enough tryptophan, humans can make nicotinamide. Corn, unlike milk, is a very poor source of tryptophan as well as of nicotinamide.