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Fly Genetics (fall 2012) Pat O’Farrell [email protected] - 6-4707 Lecture 4 Pseudoalles – Noncoding “genes” – homeotic transformation and regulation – genetic memory and the polycomb system. Sensitized genetics – a powerful screen for modifiers Psuedoalleles Homeotics and the special heritage we have in fly genetics Polycomb and trithorax class mutations, the genetics of chromatin states Pairing and transvection Biological/Developmental/Genomic issue Greater than 90% of our DNA does not code for gene products and appears to have regulatory roles. We do not know how it works. We do not know the logic of its evolutionary conservation. We suspect that differences in these sequences distinguish species. Can genetics lead us to answers? General reading: New: Two nice papers from Ian Duncan summarize the seminal contributions of Ed Lewis to genetics and biology. They can found at http://www.genetics.org/cgi/content/full/160/4/1265 http://www.genetics.org/cgi/content/full/161/1/1 I’ve referenced it before, but in case you didn't get what I said or want a repeat with a different perspective, try this. It is a review that beautifully represents many of the things that I have tried to teach. St Johnsoton, D. (2002) The art and design of genetic screens: Drosophila melanogaster. Nature Rev. Genet. 3, 176 -188 Sensitized Genetics A new a powerful way for screening for genes that contribute to particular biological function. This approach was first developed and used by Mike Simon, an ex-student of our department, when a post-doc in Gerry Rubin's lab. Simon MA, Bowtell DD, Dodson GS, Laverty TR, Rubin GM (1991) Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell 67, 701-16 Idea based on several facts. 1. One good copy of almost any region in the genome is sufficient for wt function (demonstrated by small deletions genome wide Lindsley et al., 1972) (haploinsufficiency is rare) 2. Removing one copy of a gene reduces its function in half (Muller et al., 1931) (Surprising fact that has verified repeatedly and is true for the vast majority of genes). 3. When an essential pathway has been compromised by a hypomorphic mutation so that a weak phenotype is seen – the severity of this phenotype is very sensitive to conditions. It was argued that a hypomorphic mutant would reduce the activity of the entire pathway to which it contributed and consequently reducing the dose of another gene in the pathway might further reduce the activity of the pathway and exacerbate the phenotype, while the same heterozygous defect would have no effect in the absence of the sensitizing mutation. The authors studied receptor tyrosine kinases that are required for normal eye development and discovered Ras and Ras regulatory proteins as enhancers of defects in RTK function and suppressors of mild gain-offunction RTK mutant. Their work ended up organizing the previously chaotic field of Ras signaling, and revealed powerful and general way to screen for genes in particular functional pathway. WT eye Ellipse (gain of function RTK) Ellipse, modifier mutant/+ Almost any process in molecular biology, cell biology, or development can be dissected by modifier screens in the eye (note the eye is best but similar things can be done in other tissues). To do this one might express an RNAi in the eye that compromised a particular process, and then cross to introduce mutagenized chromosomes and identify modifiers of the phenotype. Note that these show up as dominants even if they are effectively recessive genes. As a dominant screen, there is no requirement to homozygous the mutation. Individual F1 progeny are screened and the mutants subsequently recovered. Many of the mutants are recessive lethals – explain this to yourself. A special heritage http://www.genetics.org/cgi/content/full/160/4/1265 http://www.genetics.org/cgi/content/full/161/1/1 The work of EB Lewis beginning in the 1940’s gave us an extraordinary view of genetic and of development. The scientific community is still trying to catch up with the implications of some of the things he found. I will mention two genetic phenomena that he described because they are especially meaningful to the genetics of regulation. Homeotic mutations: Cause the development of normal structure in the wrong place! (The normal function of a homeotic gene is to direct the expression of the normal structure in its normal place. Homeosis (the transformation of one structure into another) occurs when a homeotic gene is ectopically expressed in a position where it should not be, as in the neomorphic Antp mutant, or indirectly because of the absence of expression of a homeotic gene in its normal domain. Regulatory interactions (repression hierarchy) among the homeotic genes contributes to their normal patterns of expression, and loss of expression of one gene can result in another gene being derepressed in the formally occupied domain.) Lewis studied a cluster of “genes” specifying the differences between segments, the Bithorax Complex. Fig. Phenotypes of bithorax complex mutations. (A) Dorsal view of a wild-type male. The T2 segment produces the single pair of wings as well as almost all of the dorsal thorax. Dorsally, T3 produces only the halteres, small clubshaped organs located posterior to the wings. (B) The famous fourwinged fly, in which T3 is transformed to T2. This male is hemizygous for the triple-mutant combination abx bx3 pbx. The abx pseudoallele has effects similar to bx mutations, but causes a stronger transformation of the very anterior portion of T3. (C) A Cbx male showing transformation of T2 toward T3. Generally, Cbx transforms only the posterior portion of T2, as seen on the right side of this fly. Occasionally, all of T2 is affected, as occurs on the left side. (D) Ventral view of a wild-type female. Each of the thoracic segments produces a pair of legs. (E) Ventral view of a bxd hemizygous female. An extra pair of legs is present as a result of the transformation of A1 toward T3. (F) Ventral view of an Hab female. The third pair of legs is lost because T3 is transformed toward A2. (B–F courtesy of E. B. Lewis.) Pseudoallelism: Ubx/bx – transformed the anterior half of the haltere to wing Ubx/pbx – Transfromed the posterior part of haltere to wing bx/pbx – wild type Logic of complementation places bx and pbx in the Ubx complementation group yet they complement each other. What is the explanation? Transvection: Ubx/bx – mild phenotype Ubx + R/bx – strong phenotype Ubx + R/bx + R – Mild phenotype R is rearrangement (and inversion) were the break points are far away from the Bithorax Complex. The rearrangement interfered with alignment/pairing of the homologs when it was heterozygous, and this increased the severity of the phenotype. Why? The Bithorax Complex (BxC) The Complex is huge, and very little of sequence is occupied by coding sequence. There are three homeobox transcription factors, and about 100kb of regulatory sequence. Molecular dissection of this and other loci have taught us that there are blocks of regulatory sequence that are about gene size, that guide spatial and temporal aspects of the program of gene expression. Mutations such as bx and pbx appear to be mutations in such regulatory sequences. Now can you explain pseudoallelism? For the future. Can genetics dissect the logic of regulation, the task that the bulk of our sequences are devoted to?