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Biochemistry 6: Model Organisms Ulrike Gaul Boris Pfander Eckhard Wolf Barbara Conradt Nina Uhlenhaut Lucas Jae Topics and schedule of the course 1 25.4. History and methods of genetics UG 2 2.5. Yeast I BP 3 9.5. Yeast II BP 4 16.5. Fly I: embryonic axis formation UG 5 23.5. FlyII: imaginal development UG 6 30.5. Mammals- early development EW 7 6.6. Pentecoste 8 13.6. Mammals- metabolic control EW 9 20.6. Fly III - Glial function in NS UG 10 27.6. Worm - Cell death BC 11 4.7. CRISPR/Cas9 NU 12 11.7. haploid screens LJ 13 18.7. Q&A SB 14 4.8. Exam 10:00 Lynen auditorium SB Introduction to genetics – essentials Transmission genetics is the general process by which traits controlled by factors (genes) are transmitted through gametes from generation to generation. Its fundamental principles were first put forward by Gregor Mendel in the midnineteenth century. Later work by others showed that genes are on chromosomes and that mutant strains can be used to map genes on chromosomes. The recognition that DNA encodes genetic information, the discovery of DNA’s structure, and elucidation of the mechanism of gene expression form the foundation of molecular genetics. Recombinant DNA technology, which allows scientists to prepare large quantities of specific DNA sequences, has revolutionized genetics. Some of the model organisms used in genetics research since the early part of the twentieth century are now used in combination with recombinant DNA technology to study human diseases. From genotype to phenotype The central dogma of molecular biology – DNA makes RNA makes protein – explains how genes control phenotypes Chromosomes All somatic cells of a given species contain an identical number of chromosomes. The number of chromosomes varies between species: Yeast (16) – Garden pea (7) – Worm (6) – Drosophila (4) Mouse (20) – Cattle (30) – Human (23) Chromosomes Some eukaryotic species live as haploids (yeast), most live as diploids. In diploids, nearly all chromosomes occur in pairs. The members of each pair are called homologous chromosomes. One copy comes from the mother, the other from the father (biparental heritance). Chromosomes Chromosomes are best visualized during mitosis. They differ in length, shape and position of their centromeres. In many species, the sex-determining chromosomes (X and Y) are not similar in shape and size, but behave as homologs during meiosis. Cell cycle Interphase (G/0/1 – S – G2) – Mitosis 2n chromosomes are duplicated 4n, then distributed between two daughter cells each 2n Mitosis Prophase: Chromosome condensation, centrioles divide and move apart Metaphase: Chromosomes align on metaphase plate, perpendicular to axis of spindle fibers Anaphase: Sister chromatids of each chromosome disjoin/centromeres split; daughter chromosomes migrate to opposite poles Telophase: Two complete sets of chromosomes, one set arriving at each pole; cytokinesis divides the cytoplasm into two halves. Mitosis Prophase: Chromosome condensation, centrioles divide and move apart Metaphase: Chromosomes align on metaphase plate, perpendicular to axis of spindle fibers Anaphase: Sister chromatids of each chromosome disjoin/centromeres split; daughter chromosomes migrate to opposite poles Telophase: Two complete sets of chromosomes, one set arriving at each pole; cytokinesis divides the cytoplasm into two halves. Life cycle of diploids – the need for meiosis Meiosis converts a diploid cell into a haploid gamete or spore, making sexual reproduction possible. During sexual reproduction, gametes then combine at fertilization to reconstitute the diploid complement found in the parents. In addition to reducing the chromosome set (2n n), genetic exchange occurs between the maternal and paternal homologs during ‘crossing over’, which results in mosaic chromosomes. Meiosis is the major form of genetic recombination. Overview of meiosis As in mitosis, cells entering meiosis have duplicated their chromosomes in the preceding S-phase and start out with 4n. Therefore, two meiotic divisions are necessary to reach 1n per gamete. First meiotic division Prophase I: chromosomes condense, each homologous pair undergoes synapsis, and crossing over occurs between synapsed homologs. Metaphase I: tetrads are aligned at metaphase plate; half of each tetrad is pulled randomly to one or the other pole; sister chromatids stay together. Telophase I: separation of cells. Overview of meiosis Second meiotic division Each gamete receives only one chromatid from the original tetrad. Prophase II: Each dyad is composed of one pair of sister chromatids attached by a common centromere Metaphase II: Centromeres are positioned on the metaphase plate Anaphase II: Sister chromatids are pulled to opposite poles. Telophase II: 1n set of chromosomes. End result: 4 haploid gametes, with each monad a combination of maternal and paternal genetic information Development of gametes differs between spermatogenesis and oogenesis and varies between species. Overview of meiosis As in mitosis, cells entering meiosis have duplicated their chromosomes in the preceding S-phase and start out with 4n. Therefore, two meiotic divisions are necessary to reach 1n per gamete. First meiotic division Prophase I: chromosomes condense, each homologous pair undergoes synapsis, and crossing over occurs between synapsed homologs. Metaphase I: tetrads are aligned at metaphase plate; half of each tetrad is pulled randomly to one or the other pole; sister chromatids stay together. Telophase I: separation of cells. Overview of meiosis Second meiotic division Each gamete receives only one chromatid from the original tetrad. Prophase II: Each dyad is composed of one pair of sister chromatids attached by a common centromere Metaphase II: Centromeres are positioned on the metaphase plate Anaphase II: Sister chromatids are pulled to opposite poles. Telophase II: 1n set of chromosomes. End result : 4 haploid gametes, with each monad a combination of maternal and paternal genetic information Development of gametes differs between spermatogenesis and oogenesis and varies between species. Transmission Genetics I – monohybrid cross Three postulates are necessary to explain the data: 1) Genetic traits are controlled by ‘unit factors’ (genes on chromosomes) that exist in pairs in individual organisms 2) Unit factors can be dominant or recessive 3) Unit factors segregate randomly Transmission Genetics I – monohybrid cross Transmission Genetics I – monohybrid cross Transmission Genetics II – dihybrid (two-factor) cross Mendel’s 9:3:3:1 dihybrid ratio One postulate is necessary to explain the data: The two traits can assort independently Transmission Genetics II – dihybrid (two-factor) cross Unit factors, genes and homologous chromosomes Independent assortment leads to extensive genetic variation Number of possible gametes is 2n (n=haploid number) Fly (4) Human (23) 16 gamete combinations 8x106 gamete combinations Modification of Mendelian ratios While alleles are transmitted from parent to offspring according to Mendelian principles, they sometimes fail to display the clear-cut dominant/recessive relationship observed by Mendel. In many cases, in contrast to Mendelian genetics, two or more genes are known to influence the phenotype of a single characteristic. Another exception to Mendelian inheritance is the presence of genes on X chromosomes, whereby one of the sexes contains only a single member of that chromosome. Phenotypes are often the combined result of both genetics and the environment within which genes are expressed. The result of the various exceptions to Mendelian principles is the occurrence of phenotypic ratios that differ from those resulting from standard monohybrid, dihybrid and trihybrid crosses. Extranuclear inheritance, resulting from the expression of genes present in the DNA found in mitochondria and chloroplasts, modifies Mendelian inheritance patterns. Such genes are often transmitted through the female gamete. Alleles alter phenotypes in different ways Alleles are alternative forms of the same gene. The allele most frequently occurring in a population is called the wild-type allele. This is often, but not always, dominant. Different alleles of genes are created by mutations. Loss-of-function mutations (often recessive) Partial loss-of function = hypomorphic allele Complete loss-of-function = null allele Gain-of-function mutations (often dominant) Overexpression = hypermorph Ectopic expression = neomorph Codominance of alleles Codominant inheritance is characterized by distinct expression of the gene products of both alleles. Example: ABO blood groups in humans with 4 phenotypes Complementation analysis Test whether two independently isolated mutations, which cause a similar phenotype, are alleles of the same gene or of two different genes X-linkage describes genes on the X chromosome Unlike the outcome of typical monohybrid cross, reciprocal crosses of X-linked mutants do not yield identical results. Fly/Human: male is XY, female is XX. X-linked genes: Fly: Eye color gene white Human: Color blindness (deutan type, protan type); Hemophilia (A and B) X-linkage describes genes on the X chromosome Phenotypic traits controlled by recessive X-linked genes are passed from homozygous mothers to all sons, since sons receive their (one and only) X chromosome from the mother and the Y chromosome from the father. Genetic background and the environment can affect phenotypic expression Penetrance – percentage of individuals that show at least some degree of expression of a mutant genotype. E.g. if 15% show wild-type appearance, the gene is said to have a penetrance of 85%. Expressivity – range of expression of the mutant genotype (e.g. eyeless) In the case of the eyeless phenotype, genetic background and environmental factors influence its expression. Extranuclear inheritance modifies Mendelian patterns Organelle heredity: chloroplasts and mitochondria contain DNA inheritance is typically cytoplasmic/maternal mutants show deficiency in organelle function Examples: poky in Neurospora, petite in Saccharomyces Maternal effect: Phenotype for a particular trait is under the control of maternal gene products (mRNA, proteins) present in the egg. This is in contrast to biparental inheritance, where both parents transmit information to the offspring (more later …). Linkage and chromosome mapping in eukaryotes Chromosomes in eukaryotes contain many genes whose locations are fixed along the length of the chromosomes. Unless separated by crossing over, alleles present on a chromosome segregate as a unit during gamete formation. Crossing over between homologs during meiosis creates recombinant gametes with different combinations of alleles that enhance genetic variation. Crossing over between homologs serves as the basis for the construction of chromosome maps. Genetic maps depict the relative locations of genes on chromosomes in a species. Sturtevant and mapping Summary Cells are the fundamental units of life. All present-day cells are believed to have evolved from an ancestral cell that existed more than 3 billion years ago. All cells grow, convert energy from one form to another, sense and respond to their environment, and reproduce themselves. All cells are enclosed by a plasma membrane that separates the inside of the cell from the environment. All cells contain DNA as a store of genetic information and use it to guide the synthesis of RNA molecules and of proteins. The simplest of present-day living organisms are prokaryotes: although they contain DNA, they lack a nucleus and other organelles and probably resemble most closely the ancestral cell. Different species of prokaryotes are diverse in their chemical capabilities and inhabit an amazingly wide range of habitats. Two fundamental evolutionary subdivisions are found: bacteria and archaea. Eukaryotic cells possess a nucleus and other organelles not found in prokaryotes. They probably evolved in a series of stages. An important step was the acquisition of mitochondria, which are thought to have originated from bacteria engulfed by an ancestral eukaryotic cell. Textbooks Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Roberts, Walter Essential Cell Biology 3rd edition, 2010, Garland Science Alberts, Johnson, Lewis, Raff, Roberts, Walter Molecular Biology of the Cell 5th edition, 2008, Garland Science Pollard and Earnshaw Cell Biology - Das Original mit Übersetzungshilfen 2nd edition, 2008, Spektrum Akademischer Verlag