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Basics of Molecular Genetics – the Genetic Material –

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Basics of Molecular Genetics – the Genetic Material – Basics of molecular genetics – The genetic material – • 1944: Avery, MacLeod & McCarty – DNA is the genetic material • 1953: Watson & Crick – molecular model of DNA structure --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – The genetic material – • 1977: Maxam & Gilbert as well as Sanger et al. describe lab methods for DNA sequencing • 1978: Maniatis et al. develop a procedure for gene isolation (construction and screening of cloned libraries) • 1983: Mullis invents the technique known as the polymerase chain reaction (PCR) • 2001: Draft sequences of the human genomes are published (Lander et al., Venter et al.) --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – Some definitions – • The phenotype is the sum of the observable physical or behavioral traits of a cell or organism and it is determined jointly by the organism’s genotype and environment • The genotype consists of the genes that control the trait of interest • A gene is a segment of a DNA molecule (or RNA in some viruses) corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and/or other functional sequence regions • The genome of an organism is • the sum of all of the DNA in one set of chromosomes (broad sense) • the sum of all of the genes in one set of chromosomes (narrow sense) --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – The DNA – • DNA is a macromolecule • In living organisms it is usually existing as in the shape of a double helix • The backbone of the DNA strands is made of sugars (deoxyribose) and phosphate groups • The simple units of the DNA polymer are called nucleotides • There are four different kinds of nucleotides in the DNA: dATP dCTP dGTP dTTP --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – The Eukaryote genome and DNA – • The eukaryote genome has a highly organised, complex structure • A small piece of the genome encodes gene products: this coding region is ca. 2% of the total genome in humans • The human genome contains ca. 3.3 billions base pairs but ca. 20500 genes only Genome in nucleus 30 % 70 % Genes and Regions between related sequences genes 10 % 90 % coding non-coding (information) (introns, pseudogenes) --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – The Eukaryote genome and DNA – --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – The structure of eukaryote genes – promoter structure gene termination signal 5´---- ---- 3´ 3´---- ---- 5´ • Promoter : recognition and binding site for the polymerase • Structure gene : contains the coding sequence • Termination signal : responsible for the termination of the transcription --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – From genes to proteins – • DNA/RNA is able to encode proteins based on the genetic code • a single amino acid is encoded by three consecutive nucleotides (triplets vs. codons) • slight variations on the standard code are existing (e.g. vertebrate mitochondrion) • the genetic code is redundant , degenerated but unambiguous • the process from genes to proteins is called gene expression and it includes transcription (DNA to mRNA) and translation (mRNA to protein) --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – Gene expression in prokaryotes vs. eukaryotes – • Transcription and translation are • Prior to its transport to the cytoplasm, there is a running side by side maturation process of mRNA (cap, polyA tail, splicing) • Genes are continuous DNA fragments • In the genes, there are coding (exons) and non-coding (introns) DNA regions • All RNA types are synthesized by one • There are three different types of RNA-polymerases RNA-polymerase --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – Genomes in Eukaryotes – • In general, three types of Eukaryote genomes are known: • nuclear genomes – ncDNA • mitochondrial genomes – mtDNA • chloroplast genomes – cpDNA – PLANTS • some lower Eukaryotes (fungi) and plants may have plasmids containing DNA as well • mt and cp genomes are existing in a number of copies in the cells • there is a higher chance to extract extranuclear DNA from degraded material • multilayer membranes also help to avoid degradation --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – Genomes in Eukaryotes – • The inheritance of the extranuclear genomes is mainly independent from the nuclear genome • extranuclear genomes tell an independent evolutionary story • combined analysis of genetic markers of different (genomic) origin may lead to more robust phylogeny • Maternal inheritance is widespread, but also paternal (e.g. cpDNA of conifers) or biparental inheritance (mtDNA of yeasts) are possible • Gene transfers are possible between the different types of genomes (evolutionary significance!) – Mitochondrial genomes – • the (circular) mitochondrial genome of vertebrates is much smaller than that of the plants, yeasts etc. • the mitochondrial genes of plants/yeasts do contain introns, while mitochondrial genes of vertebrates do not • tRNA genes are marked in red • mitochondrial genes of vertebrates (markers frequently used for molecular phylogenetic analyses in bold): • 12S rRNA , 16S rRNA • NADH dehydrogenase subunits 1, 2, 3, 4L, 4, 5, 6 • Cytochrome c oxidase subunits I, II, III • ATP synthase subunits 6, 8 • Cytochrome b • other DNA fragments in vertebrate mitochondria: tRNAs, D-loop --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – Chloroplast genomes – • cpDNA of plants (circular) includes genes playing a role in transcription, translation, photosynthesis, electron-transport etc. • the genome size is ca. 120-200 kb • some markers used in molecular phylogenetics and/or possible „barcoding“ candidates are: • rbcL • rpoB, rpoC1 IR: inverted repeats LSC: large single-copy region SSC: small single-copy region --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – Variability of the genetic information – • Molecular phylogeny is based on the idea that there is a multi-level variation in the genetic information • This variation could be detected by using molecular genetic tools • The source of these variations are: • Gene mutations • substitution (point mutation): transition, transversion (SNPs) • insertion • deletion • inversion • Chromosome mutations • structural mutations • numeric mutations • Recombinations (during meiosis) • Transposons (mobile genetic elements) --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – Gene mutations – • Some reasons of mutations: • replication errors (although DNA replication is almost error-free) • transitions (change of a purine-pyrimidine basepair against another purine-pyrimidine basepair) • transversions (change of a purine-pyrimidine basepair against a pyrimidine-purine basepair) • short insertion , deletion or inversion • spontaneous changes of the bases (e.g. depurination) • errors during crossing-over (recombination errors) – can lead to deletions , inversions or duplications • changes induced by irradiation (e.g. UV- or X-rays, radioactive radiation) could lead to thymine-dimers • transposons --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – Gene mutations – • Some consequences of gene mutations on protein-level: • neutral and missense mutation : exchange of the encoded amino acid • frameshift mutation : the reading frame will be shifted • nonsense mutation : change to stop codon • chain elongation : stop codon changes to amino acid • silent mutation : no change in amino acid (synonymous codon) • Molecular phylogenetic hypotheses suppose that closely related organisms show high similarity in their genetic material (i.e. relatively few mutations occured) while distantly related organisms show bigger differences in their DNA --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – Chromosome mutations – • Chromosome mutations could have evolutionary singnificant effects but also could lead to individual defects • Structural mutations of chromosomes • Duplication • Deletion (deficiency) • Inversion • Translocation • Transposition •Numeric mutations of chromosomes • Fusion of chromosomes – the number of chromosomes decreases • Fission of chromosomes – the number of chromosomes increases • Ploidisation (e.g. polyploidy – very common in plants, but rare in animals!) --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – Paleopolyploidy – • Polyploidy is the condition of some organisms and cells manifested by the presence of more than two homologous sets of chromosomes (genomes) • Some examples: triploid (3x): apple, banana tetraploid (4x): tobacco, cotton hexaploid (6x): bread wheat octaploid (8x): sugar cane • The diagram
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