Regulation of Gene Expression in Eukaryotes: Part III

A. Eukaryotic Genes are spliced: multiple introns are clipped of the premRNA and go to the mRNA B. Sometimes, alternative splicing results in the production of isoforms that can differ by the presence of absence of specific functional/regulatory domains C. Rare in plants, frequent in animals D. Very frequent in humans: 70% of genes are alternatively spliced, generating a large number of proteins from a small number of genes 1. Involves a cascade of alternative splicing reactions 2. In females (XX), the proteins encoded by individual genes in the cascade bind to sequences in the mRNA encoded by the next gene in the cascade, altering its splicing 3. In males (XY), alternatively spliced transcripts of these genes encode proteins that don't alter the splicing of the mRNA encoded by the next gene in the transcript E. Classic example of regulation at the level of splicing: Sex Determination in Drosophilia 1. Caused by an expansion of a CUG nucleotide triplet repeat within the transcribed regions of a gene that encode a protein kinase that contributes to muscle development 2. When it reaches a threshold level, the number of repeats increases from generation to generation ? degenerative disorder 3. The DMPK (the protein kinase) mRNA accumulates in foci within the nucleus 4. Several proteins, including Mbnl1, bind these repeats and also accumulate in the same foci, leading to sequestration away from their normal site of action a. Mbnl1 competes with another splicing regulator, CELF, for key splicing regulatory sites in the target RNAs b. Targets for Mbnl1 are RNAs that encode key regulators of muscle development such as a chloride channel (CIC-1), a Ca2+ ATPase, insulin receptors; troponins; ? c. Mutations in CIC-1 result in similar symptoms as DM! 5. Mbnl1 is a key regulator of alternative splicing at critical phases of development, with a key switch early after birth F. Many developmental disorders develop from alternative splicing issues, like Myotonic Dystrophy Type I I. Regulation of Gene Splicing II. Male and Female Development Regulation of Gene Expression in Eukaryotes: Part III (4/18/08) Gene Expression Page 1 II. A. Pathways B. Male and Female Development A. DNA Amplification III. Somatic Changes in the DNA carrying a specific gene or set of genes Gene Expression Page 2 1. Specific increase in the number of copies of a gene whose expression is badly needed at some time / tissue a. In these cells (which do not divide anymore), the chorion gene is specifically replicated b. This leads to a large number of copies, each one of them being highly transcribed leading to large production of chorion protein 2. Example: the chorion gene of Drosophila, which encodes a protein that encapsulates the oocyte, is expressed only during late oogenesis by a group of cells that surround the oocyte. 1. Example: Generation of the genes that code for heavy and light chains of immunoglobulin in B lymphocytes 2. B lymphocytes = white blood cells that contribute to the specific immune response 3. B lymphocytes derive from bone marrow stem cells that mature to eventually give rise to plasma cells (produce antigen-binding antibodies), and to memory B cells (facilitate a more rapid production of antibodies during a secondary infection) 4. AB Structure B. DNA restructuring and site-specific recombination 5. One lymphocyte B leads to one antibody that recognizes one and only one molecule = epitope a. Millions of foreign molecules invade the body b. Antibodies are proteins; therefore, genes encode antibodies c. One needs either millions of genes to encode all the antibodies needed (impossible!), or a mechanism that creates variability, starting from a small pool of genes, known as Intrachromosomal recombination between gene segments during the maturation of B lymphocytes 6. Intrachromosomal Recombination 7. Production of a light-chain Ig 8. VJ-Recombination Gene Expression Page 3 8. VJ-Recombination a. Recombination between V and J involves a site-specific recombinase: RAG1 and RAG2 b. A conserved repeat exists at the end of each V segment and the beginning of each J c. Messy: often occurs over a span of 6 bp, leading to hypervariable recombination a. n this case, however, a larger number of gene segments are involved. b. recombination also involves choosing one of several regions for the conserved domain 9. Similar recombination processes are also involved in the production of active genes that encode heavy immunoglobulin genes during B cell maturation. I a. Therefore, each cell contains two sets of immunoglobin gene fragments (two alleles) b. Only one of these alleles is subject to productive recombination; the other one is not. c. Hence, one B cell produces only one antibody type. d. This phenomenon is called ?allelic exclusion?. 10. Lymphocytes are diploid cells Gene Expression Page 4