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HEREDITY AND DEVELOPMENT: SECOND EDITION 140 6 Genetics—Old and New By the 1930’s, after millions of crosses had been made and billions of off- spring classified, geneticists had the satisfying feeling that the big questions that had been asked for centuries had been answered. When studies were made of inheritance in species not previously investigated, the results con- firmed the rules of Mendel and Morgan. The science of genetics had reached an acceptable level of maturity: it could predict the outcome of experiments. If one were to ask the question, however, just what had been accom- plished, the answer might seem unimpressive. One understood how the genes for eye color were inherited, but there was no understanding of either the molecular structure or the mode of action of the gene. What had been worked out were the rules governing the transmission of genes from parent to off- spring. These rules held for plants, animals, and microorganisms: their uni- versality was impressive. Before we ask the new questions let us summarize the answers to the old. In the following list, the generally established concepts of classical genetics appear in italics; expansions or exceptions to each concept—which for the most part have not been mentioned before—are in roman type. 1. The basic morphology, physiology, and biochemistry of an organism is determined by its inheritance, acting in a definite environment; that is, by a process of reproduction it has originated from other organisms similar to it. 2. Inheritance is the transmission of genes from parents to offspring.

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HEREDITY AND DEVELOPMENT: SECOND EDITION 141 Some of the characteristics of the individual, especially those of the early embryo, are determined solely by the action of the maternal genes during the formation of the ovum in the ovary. Thus the sperm enters an ovum that has some of its characters, such as size, color, and rate at which mitosis will occur, already determined. The paternal genes will not affect these characters but they will affect the ova produced when the individual reaches maturity. The effect of the paternal genes on the early embryo, therefore, may be delayed for a generation. 3. Genes are situated on chromosomes. There are few exceptions to this generalization. A fraction, possibly very small, of inheritance is depen- dent upon non-chromosomal structures such as mitochondria, plastids, and some virus-like bodies. Even the genes of bacteria and viruses are parts of chromosome-like structures. 4. Each gene occupies a particular site, or locus, on a chromosome. In some instances the position of the locus may be changed by inversions and translocations, which shift the position of one or more genes in rela- tion to other genes. 5. Each chromosome has many genes, and these are arranged in a linear order. As an exception to this we should remember that some chromo- somes, such as the Y of Drosophila, have only a few genes. 6. The cells of an animal, except those in the process of ova or sperm for- mation, contain two of each kind of chromosome (diploid condition); that is, all chromosomes are present in homologous pairs, and each gene locus will be represented twice. There are several well-known exceptions: (a) In some species there are differences in chromosome number between different kinds of individuals. In bees, for example, the females (queens and workers) are diploid and the males (drones) are haploid. (b) It is generally believed that an individual will have the same number of chromosomes in all of its cells (except the germ cells) but some exceptions to this are known. In the liver cells of some species of vertebrates, for example, different classes of cells exist. Some have the expected diploid number but others may have twice this number, (c) Sex chromosomes, as in the case of XO males, offer still another exception to chromosomes existing in homologous pairs, 7. For each mitotic cycle every gene is duplicated from the chemical sub- stances in the cell. Cellular reproduction involves a concurrent genic reproduction. 8. Genes are capable of existing in several states (alleles), each having detectable effects. The change from one such state to another is known as mutation. In spite of this ability to change, genes are very stable. On the average, a gene might be expected to duplicate at least a million times before a mutation occurs.

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HEREDITY AND DEVELOPMENT: SECOND EDITION 142 9. Genes can be transferred from one homologous chromosome to another by crossing over. This process is a normal part of meiosis. Crossing over seems not to occur in some instances—for example, in the male of Drosophila. Genes can also be transferred to non-homologous chromo- somes by translocations. 10. Every gamete receives one chromosome of each homologous pair. This distribution of chromosomes to the gametes is a matter of chance. Thus, each type of chromosome in every pair of homologous chromosomes will be distributed to 50 per cent of the gametes. In the case of males with XO sex chromosomes, half of the gametes will receive no sex chromosome at all. This is a complication, though not an exception, to the rule as stated. 11. The distribution to the gametes of the chromosomes of one homologous pair has no effect on the distribution of the chromosomes of the other pairs. There are, however, a few cases known in which the chromo- somes enter the gametes in specific groups. 12. Fertilization consists of the random union of male and female gametes, each with one chromosome of every homologous pair. Therefore, the zygote receives one chromosome of each homologous pair from its father and one from its mother. Once again, some sex chromosomes, as in XO males, introduce a complication to this general rule. 13. When the cells of an organism contain two different alleles of the same gene (heterozygous condition), one allele (the dominant) has a greater phenotypic effect than the other (the recessive). In most cases of this sort the heterozygote appears to be identical with individuals homozygous for the dominant alleles. In a few exceptional cases, the heterozygotes are intermediate in appearance between the homozygous dominant and homozygous recessive types. 14. Genes produce their effects through the production of chemical sub- stances, which in turn control the biochemical reactions of the cell. A fruitful hypothesis, though unproved before 1940, was that each gene controls the production of a specific enzyme, which in turn controls a specific biochemical reaction. From these simple propositions one can deduce most of the phenomena of classical genetics. Once the concepts of transmission genetics had been established, geneti- cists turned to three other major problems. Some who wished to understand better the role of genes in evolution went on to revolutionize evolutionary biology, but their discoveries will not be discussed here. The other major problems were: What is the gene? How does it act? The ‘what is it?’ question will be continued in the next chapters; the

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HEREDITY AND DEVELOPMENT: SECOND EDITION 143 ‘how does it act?’ question will start now and continue in the next chapter. Inborn Errors of Metabolism. In man there is a rare disease—affecting about one in a million—known as alkaptonuria. It makes its presence known very early, since afflicted babies stain their diapers. The urine of these babies contains a substance known as homogentisic acid (or alkapton), which becomes dark red or black when oxidized. The disease is benign, although in later life it may be associated with arthritis. An English physician, Archibald E.Garrod (1857–1936), noticed that the parents of these children were often first cousins and he speculated about the possibility of the disease being inher- ited. He consulted Bateson, who in 1902 suggested that the available data could be understood if one assumed that alkaptonuria is caused by a recessive gene. This was the first Mendelian recessive discovered in man. Garrod recognized alkaptonuria as a genetic disease and spoke of it as an ‘inborn error of metabolism.’ He suggested that individuals with alkap- tonuria lack an enzyme. This enzyme is present in normal individuals where it converts homogentisic acid to simpler substances, which are excreted in normal urine. In the absence of the enzyme, therefore, the urine contains homogentisic acid. Normal individuals, and heterozygotes, are able to pro- duce the enzyme. Thus Garrod hypothesized that one of the things that genes can do is to make enzymes. Neither Garrod nor alkaptonuria is mentioned in any of the books written by the Morgan school in the years of active discovery. Morgan and many other geneticists were adamant in ignoring hypotheses they could not test. As he expressed it: ‘It is the prerogative of science, in comparison with the specu- lative procedures of philosophy and metaphysics, to cherish those theories that can be given an experimental verification and to disregard the rest, not because they are wrong, but because they are useless.’ There was very little that Morgan and his school could cherish in the hypothesis ‘genes control the production of enzymes’—the techniques of biochemistry were too poorly developed. Beadle, Tatum, and Neurospora. By the late 1930’s, George W.Beadle (born 1903) with two associates, first Boris Ephrussi (born 1901) and later Edward L.Tatum (born 1909), thought the time ripe to make a vigorous attempt to discover how genes act. Somehow the genes must produce or con- trol the production of molecules and the interactions of these molecules must result in the phenotypic expression of the genes. It was impossible to think of gene action in any other way; but how could one possibly get at the problem? Every cell has thousands of

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HEREDITY AND DEVELOPMENT: SECOND EDITION 144 genes: how would one investigate the molecules produced by any one of them? First, Beadle and Ephrussi studied the production of eye color in Drosophila. The experiments seemed to show that many substances were involved and that some were probably enzymes. Enough was discovered to suggest the hypothesis: one gene—one enzyme, meaning that the primary function of each gene is to produce a specific enzyme. But the biochemistry of Drosophila proved to be too complex to test the hypothesis adequately and, for the first time, that noble animal let a geneticist down. Some other biological system would have to be used. Metabolic Pathways in Cells. During the 1930’s biochemists had been successful in learning about many of the molecular reactions that occur in cells. Thus, the oxidation of glucose, which is so basic for life and which for generations had been expressed: C6H12O6+6 O2 → 6 H2O+6 CO2 was found to consist of dozens of intermediate reactions, each controlled by a specific enzyme. The speed of these reactions is incredible: from glucose to H2O and CO2 requires far less than a second. Very special methods had to be devised to discover intermediate reactions passed through so quickly. It was found, for example, that a poison may pro- duce its effects by destroying or inhibiting a specific enzyme. When the enzyme is made ineffectual, the sequence of reactions is blocked and its sub- strate (the molecules it should have changed) accumulate in the cell. For example, let us suppose that this sequence of reactions occurs: If enzyme 1 is eliminated, the reaction cannot go beyond the production of molecules of A. Thus, B, C, and D will not be formed but the amount of A may increase to the point where it can be detected. One can check the hypoth- esis that enzyme 1 is not working by supplying the cell with molecules of B. The cell should then be able to make C and D. Similarly, if something goes wrong with enzyme 2, molecules of B will accumulate. Thus, by throwing chemical wrenches into the biochemical gears of the cell, one can learn about the sequence of reactions in normal metabolism. With this in mind, Beadle and Tatum sought to apply the methods of genet- ics and biochemistry to learn if the genes do, in fact, make enzymes. They reasoned that if gene A is responsible for producing enzyme 1, a mutation to an a allele might result in an abnormal and

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HEREDITY AND DEVELOPMENT: SECOND EDITION 145 ineffectual enzyme, or none at all. This was a reasonable hypothesis, and unbeknown to them, proposed long before by Garrod. But how could one test it? If enzyme 1 is necessary for life, all aa individuals would die or possibly never appear at all. Geneticists were rather sure that many mutants did behave this way. They were familiar with a large number of lethal genes which kill homozygous individuals in various unknown ways. Quite possibly these individuals were dying because they lacked some essential enzyme. But which one? Try to imagine how one might test the hypothesis: each enzyme in the cell is produced under the influence of a specific gene. By noticing the stained diapers, Garrod had developed the hypothesis that individuals homozygous for the alkaptonuria gene lack the enzyme that normally degrades homogen- tisic acid. But if the diapers had not been stained in a specific way, how could he have discovered the nature of the disease? One cannot expect that all types of molecules produced by genetic diseases are excreted in the urine, and cer- tainly very few are helpful enough to reveal their presence by turning black in the presence of air. Recall that Mendel’s principal innovation was remarkably simple: he counted the numbers of offspring in each phenotypic class. Beadle and Tatum were to be equally simple and elegant. The standard procedure for a geneticist had been to determine what the genes do: they produce blue eyes, round seeds, or vestigial wings. It was reasonable to assume that these pheno- typic expressions of genes were, themselves, consequences of various bio- chemical reactions. One always started with the gene. Beadle and Tatum reversed this procedure. Instead of beginning with the genes and trying to find the biochemical reactions, they started with the biochemical reactions and searched for the genes that controlled them. Sounds good but, again, what does one do? They had tried to experiment with Drosophila but it proved to be far too complex. Instead they deliberately sought an organism that had the biochemical and genetic properties that would enable them to answer their questions. In 1932, when Morgan had been asked to predict how new discoveries were to be made in genetics he replied, ‘By a search for favorable material’ (Readings, Chapter 5). The favorable material that Bea- dle and Tatum found was the fungus Neurospora crassa. Neurospora crassa. This is the red bread mold. It can be grown on a vari- ety of media in the laboratory but, for reasons that will shortly become clear, it was necessary for Beadle and Tatum to know exactly what sorts of molecules are required for growth. The list was discovered to be surprisingly short: air, water, inorganic salts, sucrose, and a single vitamin—biotin. Thus, sucrose and biotin are the only organic molecules

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HEREDITY AND DEVELOPMENT: SECOND EDITION 146 required. From these simple foods, Neurospora is able to synthesize all of the molecules necessary for its structure and life: amino acids, proteins, vita- mins, nucleic acids, carbohydrates, fats, and so on. Neurospora has a life cycle almost as simple as its nutritional require- ments. The colonies are haploid for most of their life cycle. All look more or less alike but, in reality, there are two mating types, A and a, which corre- spond to the sexes of other organisms. If A and a colonies are grown together, parts will fuse and A nuclei will unite with (fertilize) a nuclei to produce diploid zygotes. The zygote immediately undergoes meiosis to produce four haploid nuclei. These then divide by mitosis to produce eight haploid spores capable of growing into as many new colonies. These eight spores are encased in a spore sac, or ascus, from which they can be removed one by one and allowed to grow into a new colony. Since each colony will be haploid, every gene will be expressed—there are no dominant alleles to suppress the recessives. The amino acid arginine is among the many molecules that Neurospora normally synthesizes. Let us suppose that we wish to learn if the synthesis of arginine is under genetic control. As a working hypothesis, Beadle, Tatum, and their associates assumed that a specific gene supervises the production of a specific enzyme that, in turn, catalyzes the reaction that leads to the forma- tion of arginine from some unknown precursor. Presumably this gene could mutate to an allelic form that would be unable to make the enzyme. Unless such a mutation occurred, one would never suspect the presence of the origi- nal gene. A spore with the mutant allele could never produce a colony: such a mutant allele would be a ‘lethal.’ Thus, to solve the problem, Beadle and Tatum had to devise a method for detecting these lethal mutations and for maintaining them in culture. This sounds impossible, but they did it. First, they used X-rays to produce muta- tions—as Muller had done a decade before. They assumed that the radiations would produce all sorts of mutations but, if they were lucky and looked long enough, they might discover some mutants involved in the synthesis of argi- nine. Spores from the irradiated colonies were placed on the culture medium that contained the minimum variety of molecules necessary for growth. Most of the spores grew, showing that if mutations had occurred they did not pre- vent the Neurospora from synthesizing its constituent molecules from the few simple chemicals of the basic medium. Some of the spores did not germi- nate. Presumably these had genes that had mutated to allelic states than made the production of some essential enzyme impossible. But what enzyme or enzymes? Beadle and Tatum reasoned that if one of the genes necessary for the syn- thesis of arginine had mutated, the defect might be overcome by add-

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HEREDITY AND DEVELOPMENT: SECOND EDITION 147 ing arginine to the medium. So the spores that had not germinated on the min- imal medium were transferred to a minimal medium supplemented with argi- nine. Most of these spores still did not germinate, but others did and from them strains were established that could grow on the minimal medium enriched with arginine. The next step in the analysis was to determine if the inability to grow with- out arginine has a genetic basis. This can be done by crossing the presumed mutant strains to normal Neurospora of the opposite mating type. Spores obtained from such a cross were grown in separate tubes. Half were able to grow on the minimal medium but half required arginine. These results were consistent with the hypothesis that in the wild type Neurospora there is a gene, A, which somehow is necessary for the synthesis of arginine. The X- rays used in the experiment had caused A to mutate to a, which was unable to play some essential role in arginine synthesis. Numerous strains that required arginine were found in Beadle and Tatum’s laboratory. Were all of them the same genetically? There were two main possibilities: 1. All could have originated from mutations at the same locus: A → a. 2. Many different genes might be involved in arginine synthesis: A1, A2, A3, etc. Any one of these might mutate to an allelic state that could no longer function. Thus A1 could mutate to a1, A2, to a2, and so on. Genetic crosses could be made to test the alternatives. Thus, if both strains are a, all of the offspring will be a. Alternatively, if different gene loci are involved, some wild type colonies will appear among the offspring. For example, consider a cross of a1×a2. If a mutation had occurred at only one locus, which is overwhelmingly probable, and not at both A1 and A2, the a1 strain would also have the A2 allele. Similarity, a2 would have A1. The diploid zygotes formed in this cross will be A1 a1 A2 a2. These will produce spores. If the A1 and A2 loci are on different chromosomes, the haploid colonies formed from the diploid zygotes should be: 1/4 A1A2. These are normal and will be able to grow on minimal media. 1/4 A1a2. These will require arginine since the a2 allele will not function. 1/4 a1A. These will require arginine since a1 is not functioning. 1/4 a1a2. These will require arginine since neither a1 nor a2 is able to function.

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HEREDITY AND DEVELOPMENT: SECOND EDITION 148 If the loci are on the same chromosome, the frequency of each class of recombinants will depend on the amount of crossing over. By making these crosses, Beadle and Tatum found seven genetically dif- ferent mutants, each requiring arginine for growth. One can conclude, there- fore, that a minimum of seven genes are required by Neurospora to make arginine. This is interesting but our primary concern is to learn what these genes do. If we assume that they are involved in the formation of enzymes, there are two main possibilities. First, all seven genes could be involved in the formation of a single enzyme that has the function of converting some unknown precursors into arginine. Second, each of the seven genes could be involved in the formation of a different enzyme, each catalyzing a different reaction in the metabolic pathway to arginine (the one gene-one enzyme hypothesis). We could think in terms of this abbreviated model: A is an unknown starting compound that finally becomes arginine after pass- ing through intermediate stages B…G, each step being catalyzed by one of the gene-controlled enzymes E1…E7. The failure of any enzyme would have the same consequence so far as arginine is concerned: there would be none. Beadle and Tatum favored the hypothesis that each of the seven mutants that required arginine for growth had something wrong with a different enzyme, rather than the hypothesis that all the mutants were involved with the same enzyme. But preference hardly makes a hypothesis probable. They would have to learn more about how arginine is made in the cells. Recall that Beadle and Tatum were using biochemical reactions to dis- cover genes rather than genes to discover the reactions. They had chosen to work with arginine because a good deal was already known about its metabolism. In 1932 Hans A.Krebs had discovered that in some vertebrate cells arginine is formed from citrulline, citrulline from ornithine, and ornithine from an unknown precursor. A specific enzyme is required for each transformation. Thus the reaction can be abbreviated: If Neurospora has a similar metabolic pathway, one should be able to see how the seven mutants are involved, since the failure of each specific enzyme would have a different consequence. If something was wrong with enzyme Ex, the reaction would end with precursor X;

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HEREDITY AND DEVELOPMENT: SECOND EDITION 149 arginine would not be formed, but neither would ornithine or citrulline. Simi- larity, if enzyme Eo was absent or ineffectual, ornithine would be formed but neither citrulline nor arginine. It is experimentally possible to test these various alternatives. For exam- ple, let us suppose that a mutant strain requires arginine for growth because enzyme Ex is absent or ineffectual. If this is so, then growth should not depend only on the addition of arginine; either ornithine or citrulline should be equally effective. Similarity, if enzyme Eo is absent, either citrulline or arginine should make growth possible. In this case, the addition of ornithine would not help. If enzyme Ec is absent or defective, only arginine can cure the deficiency. Close study of the seven mutants gave the answer. In the case of four, growth was possible if ornithine, citrulline, or arginine was added. This sug- gested that the sequence of reactions was blocked in the unknown pre- ornithine part of the metabolic pathway. Two of the strains would grow if either citrulline or arginine was added to the basal medium, but ornithine did not help. This suggested that the block was between ornithine and citrulline. The data suggested something else of great interest: since two genetically different mutants both blocked the reaction, it is reasonable to think that ornithine does not change directly into citrulline. Instead ornithine probably changes to some unknown intermediate, in a reaction catalyzed by one enzyme, and then from the unknown intermediate to citrulline under the influence of a second enzyme. Finally, one strain was found that would grow only on arginine: neither citrulline nor ornithine would permit growth. This suggested that enzyme Ec was absent or inactive. We can tentatively conclude that the metabolic pathway ending in arginine has at least seven gene-controlled enzymes and at least seven products of the reactions catalyzed by these enzymes. Three of the products are known. We can, therefore, modify the original model to read as follows: In the experiments described so far, the presence or absence of enzymes had been suggested, not demonstrated. Additional data, obtained after new labora- tory techniques had been perfected, proved that the mutants either lack enzymes or have ones so abnormal that they cannot function. Thus Beadle and Tatum had been able to show that one way genes act is to produce enzymes that control specific biochemical reactions. This hypothe- sis was established as true beyond reasonable doubt. At the time two addi- tional extensions of the hypothesis seemed probable.

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HEREDITY AND DEVELOPMENT: SECOND EDITION 150 1. Every enzyme is dependent on a specific gene for its formation. 2. The primary effect of all genes is the formation of enzymes and other molecules. Much as Sutton had linked cytology and genetics in the early 1900’s, Beadle and Tatum linked genetics and biochemistry in the early 1940’s. The type of experimentation introduced by Beadle and Tatum was vigorously pursued by numerous investigators, using not only Neurospora, but other molds, yeasts, and bacteria as well. While this was going on still another approach to the study of genetics at the molecular level was being made. This was a line of investigation that began in the late 1920’s and eventually led to the identifica- tion of the gene as DNA—the topic of the next chapter. Suggested Readings H.J.Muller’s Pilgrim Trust Lecture of 1945 to the Royal Society of London, entitled ‘The Gene,’ is reprinted in Readings in Heredity and Development. It is a masterful synthesis of what could be induced about the nature of the gene from the data of classical genetics. A more complete bibliography on the nature of the gene and the pioneering work of Beadle and Tatum will be found in the Readings. BEADLE, GEORGE W. 1963. Genetics and Modern Biology. Philadelphia: American Philosophical Society. Memoirs Volume 57. BEADLE, GEORGE and MURIEL. 1967. The Language of Life. An Introduction to the Science of Genetics. Garden City, New York: Doubleday Anchor Books. CARLSON, ELOF AXEL. 1966. The Gene: A Critical History. Philadelphia: W.B. Saun- ders. MORGAN, THOMAS HUNT. 1926. The Theory of the Gene. New Haven: Yale Univer- sity Press. Reprinted 1964 by Hafner, New York. SINGLETON, W.RALPH. 1967. Elementary Genetics. Second Edition. Princeton: D. van Nostrand. Chapter 5. SRB, A.M., and N.H.HOROWITZ. 1944. ‘The ornithine cycle in Neurospora and its genetic control.’ Journal of Biological Chemistry 154:129–139. WAGNER, R.P., and H.K.MITCHELL. 1964. Genetics and Metabolism. Second Edition. New York: Wiley. WHITEHOUSE, H.L.K. 1969. Toward an Understanding of the Mechanism of Heredity. New York: St. Martin’s Press. Questions 1. Considering the data available at the time, what could one have con- cluded about the nature of the gene in 1900, 1905, 1915, 1930, and 1945? 2. Why was it necessary for Beadle and Tatum to know what substances are required for growth by Neurospora? 3. If you wished to use Drosophila to answer the sorts of questions that

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HEREDITY AND DEVELOPMENT: SECOND EDITION 151 Beadle and Tatum asked of Neurospora, outline how you would plan your work. 4. Assume that Neurospora synthesizes molecules of substance A from substrate B. Four genetically different strains are discovered that cannot grow on the minimal medium but can grow if A is added. What can you conclude about the synthesis of A? 5. Two strains of Neurospora are discovered that cannot grow unless the amino acid tryptophan is added to the growth medium. How would you decide whether the strains are genetically the same or different? 6. The meiotic and mitotic divisions that produce the eight spores of Neu- rospora occur in a geometrically exact manner. Thus, if the four chro- matids of a tetrad on the first meiotic spindle are designated, left to right, A A1 B B1, they will occupy predictable positions in the four cells formed by the two meiotic divisions. Thus, A will be in the left-most cell, A1 next, then B, and finally B1 on the extreme right. The mitotic division that follows produces the eight spores, which are arranged in a linear order: A A A1 A1 B B B1 B1. This regular arrangement will occur if there is no crossing over. A strain of Neurospora that can synthesize arginine is crossed with one that cannot. A spore case formed in this cross is opened and the eight spores removed in order and each is placed in a tube with minimal medium. Describe what will happen in each tube. 7. A cross of the above type is made and the spores are removed in order and placed in separate tubes. Growth occurs in tubes 1, 2, 5, and 6 but not in 3, 4, 7, or 8. How would you explain these results?