Basics of molecular genetics The genetic material 1944: Avery, MacLeod & McCarty DNA is the genetic material 1953: Watson & Crick molecular model of DNA structure
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.)
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)
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
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 related sequences Regions between genes 10 % 90 % coding (information) non-coding (introns, pseudogenes)
The Eukaryote genome and DNA
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
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)
Gene expression in prokaryotes vs. eukaryotes Transcription and translation are running side by side Genes are continuous DNA fragments All RNA types are synthesized by one RNA-polymerase Prior to its transport to the cytoplasm, there is a maturation process of mrna (cap, polya tail, splicing) In the genes, there are coding (exons) and non-coding (introns) DNA regions There are three different types of RNA-polymerases
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
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!)
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 Mitochondrial genomes 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
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 Chloroplast genomes IR: inverted repeats LSC: large single-copy region SSC: small single-copy region
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)
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
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
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!)
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 summarizes all well-known polyploidization events
Genetic recombination Genetic recombination is the most important mechanism for maintaining genetic variation in many organisms Recombination is the exchange of homologous DNA sequences in general Homologous recombination occurs during meiosis (Prophase I - pachytene) Meiosis occurs in all eukaryotic life cycles involving sexual reproduction Mistakes during crossing over further increase the variability Recombination (to a certain extent) is also possible during mitosis Site-specific recombination is typical for viruses when they are integrating into the host cells Transpositional recombination (caused by transposons) does not need sequence homology
Genetic markers In general, it is not possible and also not necessary to investigate the whole genome of an organism in order to answer questions concerning its evolution Instead of this, we are using so called molecular or genetic markers Molecular markers should be identified by a simple assay non-dna analyses (e.g. allozyme analyses) DNA sequencing fragment analyses RFLP (Restriction Fragment Length Polymorphism) AFLP (Amplified Fragment Length Polymorphism) microsatellite analysis RAPD (Random Amplified Polymorphic DNA) ISSR-PCR (Inter Simple Sequence Repeats) etc. SNP arrays etc. The selection of the genetic marker depends on the question of interest which type of organisms you are working on animals, plants, fungi which level of evolutionary changes should be detected population genetics, phylogeography, phylogeny
Genetic markers Types of genetic markers SNPs (Single Nucleotide Polymorphisms) nowadays, for detecting SNPs, no DNA sequencing is needed Sequences of relatively short DNA segments single-copy protein-encoding genes ribosomal DNA (nuclear and mitochondrial rrnas) introns Repetitive DNA minisatellites or VNTRs (Variable Number of Tandem Repeats) STRs (Short Tandem Repeats)/ microsatellites (commonly used for population genetic analyses) SINEs and LINEs (Short and Long Interspersed Elements) telomere sequences (telomeric repeats are fairly conserved)
Genetic markers Considerations for the selection of molecular markers DNA sequences Nuclear DNA Mitochondrial DNA Chloroplast DNA ANIMALS slow relatively fast fast Evolutionary tempo ANIMALS Frequently used markers 5.8S, 18S, 28S rrna ITS 1, ITS 2 RAG1, RAG2, c-mos β-fibrinogen, myoglobin 12S rrna, 16S rrna (rel. fast) COI, NDx, cyt b (fast) D-loop (very fast) elongation factor 1, 2 rhodopsin, RNA polymerase II PLANTS relatively fast fast (very) slow slow variable Evolutionary tempo PLANTS Frequently used markers 18S rrna ITS 1, ITS 2 Not really used rbcl, atpb, trnk/matk, ndh
From the idea to results using molecular tools PLANNING PHASE DATA COLLECTION Do sampling Formulate a phylogenetic hypothesis DNA sequencing Choose appropriate molecular genetic methods DNA isolation PCR Fragment analyses (microsatellites, ISSR) EVALUATION PHASE Sequence alignment Dendrogramm construction based on distance, MP, ML, BI criteria Evaluation depending on methods Evaluation of results based on the original hypothesis Testing of alternative hypotheses Comparison with other results Final evaluation and interpretation
Further reading Lodish et al.: Molecular Cell Biology (2007), 6 th edition. Hartwell et al.: Genetics: From Genes to Genomes (2006), 3 rd edition. Wink (ed.): An Introduction to Molecular Biotechnology: Molecular Fundamentals, Methods and Applications in Modern Biotechnology (2006), 1 st edition. Avise: Molecular Markers, Natural History, and Evolution (2004), 2 nd edition.