1. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 1. One-to-many relationship of phenotypes to genes This concept is based on the observation that many different genes can affect a single phenotype. This is easy to understand in terms of a character such as eye color, in which there are complex metabolic pathways with numerous enzymatic steps, each encoded by one or more gene products. Genetic heterogeneity is the term used to refer to a given condition that may be caused by different genes. This concept is based on the observation that many different genes can affect a single phenotype. This is easy to understand in terms of a character such as eye color, in which there are complex metabolic pathways with numerous enzymatic steps, each encoded by one or more gene products. Genetic heterogeneity is the term used to refer to a given condition that may be caused by different genes. One goal of genetic analysis is to identify all the genes that affect a specific phenotype and to understand their genetic, cellular, developmental, and molecular roles. To do this, we need ways of sorting mutations and genes. One goal of genetic analysis is to identify all the genes that affect a specific phenotype and to understand their genetic, cellular, developmental, and molecular roles. To do this, we need ways of sorting mutations and genes.  We first will consider how we can use genetic analysis to determine if two mutants are caused by mutational hits in the same gene (that is, they are alleles) or in different genes.  Later, we will consider how genetic analysis can be used to make inferences about gene interactions in developmental and biochemical pathways.

2. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 2. The complementation test The allelism test that finds widest application is the complementation test, which is illustrated in the following example. The allelism test that finds widest application is the complementation test, which is illustrated in the following example.  Consider a species of flower in which the wild-type color is blue. We have induced three white-petaled mutants and have obtained pure-breeding strains (all homozygous). We can call the mutant strains $, £, and ¥, using currency symbols to avoid prejudicing our thinking concerning dominance. In each case the results show that the mutant condition is determined by the recessive allele of a single gene. However, are they three alleles of one gene, or of two or three genes? The question can be answered by asking if the mutants complement each other. Complementation is the production of a wild-type phenotype when two recessive mutant alleles are united in the same cell. Complementation is the production of a wild-type phenotype when two recessive mutant alleles are united in the same cell.

3. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 3. Performing the complementation test In a diploid organism the complementation test is performed by intercrossing homozygous recessive mutants two at a time and observing whether or not the progeny have wild-type phenotype. If recessive mutations represent alleles of the same gene, then obviously they will not complement because they both represent lost gene function. Such alleles can be thought of generally as a’ and a", using primes to distinguish between two different mutant alleles of a gene whose wild- type allele is a +. These alleles could have different mutant sites but would be functionally identical. The heterozygote a’/a" would be In a diploid organism the complementation test is performed by intercrossing homozygous recessive mutants two at a time and observing whether or not the progeny have wild-type phenotype. If recessive mutations represent alleles of the same gene, then obviously they will not complement because they both represent lost gene function. Such alleles can be thought of generally as a’ and a", using primes to distinguish between two different mutant alleles of a gene whose wild- type allele is a +. These alleles could have different mutant sites but would be functionally identical. The heterozygote a’/a" would be However, two recessive mutations in different genes would have wild-type function provided by the respective wild-type alleles. Here we can name the genes a1 and a2, after their mutant alleles. Heterozygotes would be a1/+ ; +/a2 (unlinked genes) or a1+/+a2 (linked genes), and we can diagram them as follows: However, two recessive mutations in different genes would have wild-type function provided by the respective wild-type alleles. Here we can name the genes a1 and a2, after their mutant alleles. Heterozygotes would be a1/+ ; +/a2 (unlinked genes) or a1+/+a2 (linked genes), and we can diagram them as follows:

4. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 4. Mutants that complement We now return to the flower example and intercross the mutant strains to test for complementation. Assume the results of intercrossing mutants $, £, and ¥ are as follows: We now return to the flower example and intercross the mutant strains to test for complementation. Assume the results of intercrossing mutants $, £, and ¥ are as follows: From this set of results we would conclude that mutants $ and £ must be caused by alleles of one gene (say w1) because they do not complement; but ¥ must be caused by a mutant allele of another gene (w2). From this set of results we would conclude that mutants $ and £ must be caused by alleles of one gene (say w1) because they do not complement; but ¥ must be caused by a mutant allele of another gene (w2). The molecular explanation of such results is often in terms of biochemical pathways in the cell. How does complementation work at the molecular level? Although it is conventional to say that it is mutants that complement, in fact the active agents in complementation are the proteins produced by the wild-type alleles. The molecular explanation of such results is often in terms of biochemical pathways in the cell. How does complementation work at the molecular level? Although it is conventional to say that it is mutants that complement, in fact the active agents in complementation are the proteins produced by the wild-type alleles.

5. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 5. The biochemical explanation The normal blue color of the flower is caused by a blue pigment called anthocyanin. Pigments are chemicals that absorb certain parts of the visible spectrum; in the case of the harebell the anthocyanin absorbs all wavelengths except blue, which is reflected into the eye of the observer. However, this anthocyanin is made from chemical precursors that are not pigments; that is, they do not absorb light of any specific wavelength and simply reflect back the white light of the sun to the observer, giving a white appearance. The blue pigment is the end product of a series of biochemical conversions of nonpigments. Each step is catalyzed by a specific enzyme coded by a specific gene. We can accommodate the results with a pathway as follows: The normal blue color of the flower is caused by a blue pigment called anthocyanin. Pigments are chemicals that absorb certain parts of the visible spectrum; in the case of the harebell the anthocyanin absorbs all wavelengths except blue, which is reflected into the eye of the observer. However, this anthocyanin is made from chemical precursors that are not pigments; that is, they do not absorb light of any specific wavelength and simply reflect back the white light of the sun to the observer, giving a white appearance. The blue pigment is the end product of a series of biochemical conversions of nonpigments. Each step is catalyzed by a specific enzyme coded by a specific gene. We can accommodate the results with a pathway as follows: A mutation in either of the genes in homozygous condition will lead to the accumulation of a precursor, which will simply make the plant white. Now, the mutant designations can be written as follows: A mutation in either of the genes in homozygous condition will lead to the accumulation of a precursor, which will simply make the plant white. Now, the mutant designations can be written as follows:

6. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 6. Complementation Three phenotypically identical white mutants, $, £, and ¥, are intercrossed to form heterozygotes whose phenotypes reveal whether or not the mutations complement each other. (Only two of the three possible crosses are shown here.) If two mutations are in different genes (such as £ and ¥), then complementation results in the completion of the biochemical pathway (the end product is a blue pigment in this example). If mutations are in the same gene (such as $ and £), no complementation occurs because the biochemical pathway is blocked at the step controlled by that gene, and the intermediates in the pathway are colorless (white).

7. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 7. Complementation in human genetic diseases Profound congenital hearing loss is often genetic, and when genetic it is usually autosomal recessive. However, when two people with autosomal recessive profound hearing loss marry, as they often do, the children usually have normal hearing. Profound congenital hearing loss is often genetic, and when genetic it is usually autosomal recessive. However, when two people with autosomal recessive profound hearing loss marry, as they often do, the children usually have normal hearing. This is an example of complementation. The children will have normal hearing whenever the parents carry mutations in different genes. Diseases and developmental defects represent the failure of a pathway. It is easy to see that many different genes would be needed to construct so exquisite a machine as the cochlear hair cell, and a defect in any of those genes could lead to deafness. Such locus heterogeneity is only to be expected in conditions like deafness, blindness or mental retardation, where a rather general pathway has failed; but even with more specific pathologies, multiple loci are very frequent. This is an example of complementation. The children will have normal hearing whenever the parents carry mutations in different genes. Diseases and developmental defects represent the failure of a pathway. It is easy to see that many different genes would be needed to construct so exquisite a machine as the cochlear hair cell, and a defect in any of those genes could lead to deafness. Such locus heterogeneity is only to be expected in conditions like deafness, blindness or mental retardation, where a rather general pathway has failed; but even with more specific pathologies, multiple loci are very frequent. A striking example is Usher syndrome, an autosomal recessive combination of hearing loss and retinitis pigmentosa, which can be caused by mutations at eight or more unlinked loci A striking example is Usher syndrome, an autosomal recessive combination of hearing loss and retinitis pigmentosa, which can be caused by mutations at eight or more unlinked loci

8. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 8. Complementation test for rare human genetic diseases Genetic complementation for rare and invalidating genetic diseases cannot be observed in human pedigree, because affected people do not marry Genetic complementation for rare and invalidating genetic diseases cannot be observed in human pedigree, because affected people do not marry However, some mammalian somatic cells can be cultured in a well-defined medium. In addition, cultured cells can be fused to produce somatic hybrids; although cell fusion occurs spontaneously at very low rate, it can be increased in the presence of certain viruses that have a lipoprotein envelope similar to the plasma membrane of animal cells. A mutant viral glycoprotein in the envelope promotes cell fusion. Cell fusion is also promoted by polyethylene glycol, which causes the plasma membranes of adjacent cells to adhere to each other and to fuse.

9. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 9. An early demonstration of complementation in fused human cells As most fused animal cells undergo cell division, the nuclei eventually fuse, producing viable cells with a single nucleus that contains chromosomes from both “parents.” It is even possible to fuse cells from different species

10. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 9. Xeroderma pigmentosum Cell fusion is the basis for the complementation test in human; if a genetic defect is assayable in cultured cells, complementation analysis by cell fusion can be undertaken. Cell fusion is the basis for the complementation test in human; if a genetic defect is assayable in cultured cells, complementation analysis by cell fusion can be undertaken. For example, the autosomal recessive disease xeroderma pigmentosum (XP) involves defects in repair of UV-induced damage in DNA. Patients are abnormally sensitive to sunlight, developing skin cancer after relatively brief exposure. For example, the autosomal recessive disease xeroderma pigmentosum (XP) involves defects in repair of UV-induced damage in DNA. Patients are abnormally sensitive to sunlight, developing skin cancer after relatively brief exposure. Multiple basocellular carcinomas on the face of an XP patient. Thick arrow points to a recent lesion, and thin arrow to a scar of an old lesion

11. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 10. First demonstration of complementation groups in XP By fusing fibroblasts from various patients with XP, seven main complementation groups have been defined By fusing fibroblasts from various patients with XP, seven main complementation groups have been defined

12. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 11. Different sensitivity to UV radiation of cells from different complementation groups Hypersensitivity to UV radiation of XP cells in culture. Here the cells from a number of complementation groups are shown. There is a variation between complementation groups, but all are more sensitive to UV radiation than are normal cells. The difference in UV photosensitivity between normal and diseased cells is evident from the survival curves of cultured cells treated with UV light Hypersensitivity to UV radiation of XP cells in culture. Here the cells from a number of complementation groups are shown. There is a variation between complementation groups, but all are more sensitive to UV radiation than are normal cells. The difference in UV photosensitivity between normal and diseased cells is evident from the survival curves of cultured cells treated with UV light