Chapter 5 Genetic Analysis in Cell Biology. (textbook: Molecular Cell Biology 6 ed, Lodish section: )
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1 Chapter 5 Genetic Analysis in Cell Biology (textbook: Molecular Cell Biology 6 ed, Lodish section: )
2 Understanding gene function: relating function, location, and structure of gene products Phenotype DNA
3 Mutations: types and causes An organism s genotype is its entire set of genes and may denote whether an individual carries mutations in one or more genes (genotype = basic genetic make-up of an individual organism) An organism s phenotype is its function and physical appearance, and depends on that individual s genotype Normal (wild-type) organisms may develop changes termed mutations in their DNA sequence thereby altering their genotype and perhaps their phenotype Organisms that develop such mutations are termed mutants Mutations may be part of the genotype or occur as acquired (somatic) mutations
4 Haploids and diploids A haploid organism has a single copy of each chromosome and its phenotype is a consequence of that one copy A diploid organism has two copies of each chromosome and thus two copies of each gene The two copies of each gene may be the same or the copies may be different. Different forms of each gene are termed alleles (allele= naturally occuring mutation) Diploids that carry identical alleles are termed homozygous Diploids that carry different alleles are termed heterozygous
5 Cross-over mainly occurs in meiosis (gametogenesis) Cross over allows exchange of genetic information between paired chromosomes
6 Genotype and phenotype in diploids Alleles may be dominant or recessive
7 Segregation of a mutation Segregation of Dominant mut Segregation of Recessive mut Dominant mutations usually lead to a gain of function although occasionally they may lead to a loss of function Recessive mutations lead to loss of function
8 Geno-type linked diseases (non-cancerous) An autosome is a non-sex chromosome. It is an ordinarily paired type of chromosome that is the same in both sexes of a species. For example, in humans, there are 22 pairs of autosomes. The X and Y chromosomes are not autosomal. Non-autosomal chromosomes are usually referred to as sex chromosomes, allosomes or heterosomes.
9 Segregation of geno-type linked diseases
10 Various types of mutations NB: single base mutations leading to know functional coding-change are called sense mutations. Single base mutations frequently occur in nature and are called singel nucleotide polymorphisms (SNP s)
11 Spontaneous mutations may result from errors in DNA replication Spontaneous mutations may also result from chemical instability of purine and pyrimidine bases, or from exposure to UV light or chemical carcinogens -Or from cross-over NB: chapter on repair Cross-over can occur in somatic cells, but most frequently is part of meiosis
12 Induction of point mutations by EMS Scientists may induce mutations in experimental organisms by treatment with chemical mutagens (e.g, EMS) or exposure to ionizing radiation EMS= ethylmethane sulfonate Mutational agents are used to generate libraries of mutant organisms which can then be screened for specific functional changes. Identification of mutated gene then gives information on which genes are involved in this functionality.
13 Role of spontaneous somatic mutations in human genetic diseases Rb is a classical Oncogene Rb (retinoblastoma pocketprotein): normal function: prevents cells from entering S-phase (cell cycle). Inactivation by phosphorylation (cyclins+cdks, see previous lecture) allows progression through S-phase -> mitosis. Mutated Rb = cronic inactivated -> continous cell-cycling (no growth arrest!) = cancer
14 Isolation and analysis of mutants The procedures used to identify and isolate mutants are termed genetic screens The screen used depends on whether the organism is haploid or diploid. If diploid, the screen used also depends on whether the mutation is dominant or recessive. Screens with haploid organisms often involve temperaturesensitive mutants Screens for recessive lethal mutations utilize visible markers
15 Example: mutants in Drosophila Mutations may cause only subtle changes or may produce significant changes in development, cellular function, appearance and/or behavior Drosophila melanogaster (fruitfly) is one of the most favored organisms for genetic studes. It has a short generation time and phenotypes are often easily detected (visible)
16 Temperature-sensitive genetic screen used to identify cell cycle mutants in yeast Yeast (especially bakers yeast; saccharomyces cerevisiae) is a favorite for cell cycle analysis. Eucaryotic single-cell organism with: Fast propagation (generation time 2-4h), easy manipulation (DNA-transfection etc.) and a well charachterized genome. Why is it an advantage to use single cells in culture particulary for cell-cycle studies? ;-)
17 Some chromosome abnormalities can be mapped by banding analysis
18 Molecular cloning of genes defined by mutations The use of recombinant DNA techniques to isolate mutation-defined genes is referred to as positional cloning In higher organisms, the cloning of a mutation-defined gene usually involves four steps
19 Positional cloning: Step 1: identification of a cloned segment of DNA near gene of interest Linkage disequilibrium study In situ hybridization
20 Positional cloning: Step 2, and 3 Step 2: isolation of a contiguous stretch of DNA and construction of a physical map in that region Step 3: correlation of the physical map with the genetic map to locate the position of the gene of interest YAC= yeast artificial chromosome, BAC= bacterial artificial chromosome Function like plasmid vectors but can harbor very large foreign DNA fragments (= you can clone smaller DNA fragments in plasmid vectors, but large fragments in BAC s and very large fragments in YAC vectors) Therefore very good for generating tiling libraries
21 Step 4: detection of alterations in expression of transcripts or changes in DNA sequence between the mutant and wild-type Detection of point mutations:
22 Gene replacement and transgenic animals Some genes are identified through means other than mutant analysis To determine the function of these genes, it is possible to replace an organism s wild type gene with an inactive gene to create a gene knockout It is also possible to introduce additional genes (transgenes) to create a transgenic organism Gene knockout and transgenic techniques can involve mutagenesis of cloned genes prior to transfer into the organism (= in vitro mutagenesis)
23 Creation of mice embryonic stemm cells (ES cells) carrying a knockout mutation Specific targeting of gene of interest (GOI)
24 Gene knockout in mice
25 Cell-type-specific gene knockouts in mice Advantage: Effect on specific tissue / process can be studied Embryonal lethal knock-outs can be studied in tissues where the knock out is not lethal
26 Gene-silencing with small mrna-interfering RNAs (sirna) Dicer is an endogenous enzyme which cleaves specific RNA structures (part of the mirna system) sirnas are designed to basepair with specific mrna, which will then not be translated An alternative to Knock-out. Gene-silencing addtionally allows controlled silencing through regulatable promoter upstream of hairpin construct coding region. Endogenous= naturally ocurring within cells
27 Production of transgenic mice The foreign DNA contains a promoter+goi If the promoter is a constitutive active promoter the GOI will be expressed in all cells If the promoter is regulated by proteins only present in certain cells, a tissuespecific expression can be achieved
28 Example: transgenic mouse (GH+) Promoter function seen in transgenic mice. (A) Recombinant plasmid containing rat growth hormone structural gene, mouse metallothionein regulatory region, and bacterial plasmid pbr322. The plasmid, pmgh, was injected into the mouse oocytes. The dark boxes on the injected plasmid correspond to the exons of the GH gene. The direction of transcription is indicated by an arrow. (B) A mouse derived from the eggs injected with pmgh (left) and a normal littermate (right). (From Palmiter et al. 1982, photograph courtesy of R. L. Brinster.) A plasmid containing this fused gene was grown in bacteria (see Chapter 2), the Mt-1/rGH piece was isolated, and about 600 copies of this fragment were injected into pronuclei of recently fertilized mouse eggs. DNA hybridization showed that many of these newborn mice had incorporated numerous copies of the rat growth hormone gene into their chromosomes. These transgenic mice were then fed a diet supplemented with zinc. The zinc induced large amounts of rat growth hormone to be secreted by the livers of these mice. (The liver is where metallothionein is usually made. Growth hormone is usually secreted from the pituitary gland.) The amount of growth hormone secreted correlated with the size of these mice. The transgenic mice became enormous, up to 80 percent larger than their normal littermates
29 After completion of this lecture you should be able to: Define the terms: genotype, phenotype, mutants, wild-type, somatic mutation, haploid, diploid, allele, homozygous, heterozygous, dominant allele, recessive allele Know that cross-over allows exchange of genetic information between paired chromosomes, and mainly happens in meiosis Explain the genetic reasons underlying potential differences between diploid genotype and diploid phenotype Describe and schematically draw the segregation of dominant and recessive mutations Know the difference between X-linked and autosomal gene-linked diseases and how they segregate Briefly define the following concepts of mutations: missense, nonsense, frameshift, insertion, deletion, translocation, inversion, single nucleotide polymorphism (SNP) Know that induction of pointmutations by chemical stimuli is a valuable tool for genetic analysis. and that pointmutations lead to either missense, nonsense or non-affected (sense) mutants Understand that somatic mutation of cell-cycle regulating genes is the prevalent cause of cancer. Know what a genetic screen is and explain why different methods are applyed for investigating recessive and dominant mutations Know why yeast is an excellent organism for studying cell cycle regulator genes Briefly explain the 4 steps involved in Positional cloning of a mutation-defined gene from a higher organism (phenotype gene) Know the term artificial chromosome (BAC and YAC) and that AC s are vectors that can harbor very large foreign DNA fragments Know that in addition to genetic screens for mutants, specific genes may be investigated through generation of geneknock out and transgene organisms (gene phenotype) Briefly describe the classical strategy for generating knock-out mice Formation of ES cells carrying a knock out mutation Generation of knock-out vector carying disrupted (non-functional) form of GOI and selection markers (ie. antibiotic resistance genes) Facilitated homologous recombiantion Selection of ES cells with targeted disruption of GOI Injection of the ES cells into mouse blastocyst. Insertion of blastocyst into pseudopregnant mouse Selection of full knock out mice which arise from progeny carrying the mutation in their germline cells Know that it is possible to generate not only full body knock out mice but also cell type specific knock outs applying the Cre-Lox system. Know the term sirna, and that it is natures own tool for temporary gene silencing, which biotechnologically can be applied as an alternative to the classical knockout Know that transgene mice can be created by simple injection of DNA vector carrying the GOI into fertilized mice eggs.
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