Mosaic AnalysisReading: Chapter 5, pp140-141; Reference chapter D, pp820-823Problem set FTwin spots in DrosophilaAlthough segregation and recombination in mitosis do not occur atthe same frequency as in meiosis, under the proper conditions,crossing over can be observed in mitosis. Curt Stern was the first todescribe mitotic recombination. In females heterozygous for therecessive X-linked yellow body (y) and the singed bristle (sn) alleles(y+ sn/ y sn+), Stern noted that most females are wild type with greybodies and normal bristles. However, some females had patches ofyellow or singed (short twisted bristles). Other females had twinspots, or two adjacent regions, one exhibiting a yellow phenotypeand the other exhibiting a singed phenotype. Since the two spotswere adjacent, Stern reasoned that the twin spots were reciprocalproducts of the same event, the product of mitotic crossing over.Since the order on the X chromosome is centromere-sn-y, a crossoverbetween the centromere and the sn gene will result in a twin spot. Asingle singed patch can be explained by mitotic nondisjunction anda yellow patch can be explained by mitotic nondisjunction or by amitotic crossover between the sn and y genes. Individuals that arecomposed of cells of more than one genotype are referred to asgenetic mosaics.The use of genetic mosaics to determine the site of gene functionIt turns out that the Sevenless protein is expressed in many cells ofthe developing ommatidium. How then can we determine in whichcells sevenless functions? Drosophila geneticists can use mosaicanimals to address this question. Let’s consider the experiments todetermine the site of Sevenless function first, and then we will turnto Boss.The basic approach is to make mosaic animals that havesome cells in the eye that are functionally wild type for Sevenlessand others that are mutant (Figure 1). X rays stimulate mitoticrecombination, causing frequent crossing over between homologsduring mitosis; it is thought that induced chromosomal breaksstimulate mitotic recombination. When mitotic recombination isstimulated in animals that are heterozygous for the sev mutation,clones of homozygous mutant cells are produced. We also need toknow which cells are mutant, and the chromosome bearing the sevmutation also contains a white mutation. Mosaic eyes will havepatches of white tissue. These eyes are sectioned and the cells thatare wild type for white, and hence wild type for sev, contain pigmentgranules, while cells that are mutant for white, and hence mutant forsev, don’t contain pigment granules. The white gene is necessary forthe transport of pigment granules.Figure 1. X-ray induction of a mitotic recombination event that willlead to a mosaic clone. Note that the alternate orientation of thehomologous chromosomes would result in a segregation patternthat would not lead to mosaic clones.In the mosaic eyes, some ommatidia will consist of mixturesof wild-type and mutant cells. There were no ommatidia in whichthe R7 cell was white. Moreover, as long as the R7 cell was wild-typefor white, any or all of the cells could be mutant (Figure 2). Theseresults indicate that the requirement for the wild-type sev geneproduct is in the R7 cell, and we say that the requirement for sev iscell autonomous. In other words, to develop into an R7 cell (and nota cone cell) a presumptive R7 cell must make the Sev protein, and itdoesn’t matter whether other cells of the same ommatidium makeSev protein.Figure 2. Examples of sev mosaic ommatidia where the R7 cell isproduced. Only the photoreceptors cells are shown in the diagram.1 is R1, 2 is R2, etc.When researchers performed mosaic analysis for the boss gene, theresults were different. An R7 cell develops properly only if the R8cell is wild type for boss. All of the other cells of the can be mutant forboss, including R7, and R7 will develop normally as long as R8 iswild type. Conversely, if R8 is mutant, even when all of the othercells of the ommatidium are wild type, the presumptive R7 cell willdevelop into a cone cell. These results indicated that the wild typeboss gene functioned in the R8 cell to specify the R7 fate, and we saythat the requirement for the gene is cell nonautonomous. This isconsistent with the view that Boss is the Sev ligand.Figure 3. Examples of ommatidia mosaic for boss. Note that thereis a correlation between R8 being wild type for boss and thepresence of R7.Problem set F1. Although the mechanism for generating mosaic animals in C.elegans and Drosophila is different, the principle is the same: a mosaicanimal is generated, and a cell autonomous marker (like white in thefly eye), is used to determine which cells are wild-type and whichcells are mutant for the gene being studied. Acetylcholine is theneurotransmitter that is released from motor neurons in C. elegansand stimulates muscles that control movement to contract by bindingto acetylcholine receptors on the muscle cells. Acetylcholinesterase isthe enzyme that breaks down acetylcholine in the synapse to ensurethat the neurotransmitter acetylcholine does not accumulate. ace-1 isthe gene that encodes the acetylcholinesterase enzyme and recessivemutations in the gene lead to animals that move abnormally. Thisabnormal or uncoordinated movement is caused by the buildup ofacetylcholine in the neuromuscular synapse. You are interested indetermining whether ace-1 gene function is required in motorneurons, in muscles, or in both motor neurons and muscles. Yougenerate mosaic animals that have the recessive ncl-1 mutation andthe ace-1 mutation linked, so that when cells loose the wild-type ncl-1gene, they also loose the wild-type ace-1 gene. The loss of wild-typencl-1 function is cell autonomous and leads to cells with largenucleoli. Thus, ncl-1 can be used as a cell autonomous marker in thisstrain for the loss of ace-1 in specific cells.The division of the C. elegans zygote produces the AB and P1 cells. Allof the neurons in C. elegans are derived from the AB cell and all of themuscles are derived from the P1 cell. Describe the results from yourmosaic analysis if ace-1 acetylcholinesterase is produced by motorneurons; by muscle; by both neurons and muscles. Be sure to includewhich cells contain normal nucleoli (Ncl+) and which cells containenlarged nucleoli (Ncl-) in the different mosaic animals and whichanimals are uncoordinated (Unc).2. In a screen, you identify several new recessive mutations that leadto a loss of R7. You are
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