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 summarizes all well-known polyploidization events

--- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – 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

--- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – 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 --- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – 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)

--- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – 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 • 5.8S, 18S, 28S rRNA • 12S rRNA, 16S rRNA (rel. fast) Frequently used • ITS 1, ITS 2 • RAG1, RAG2, c-mos • COI , NDx, cyt b (fast) markers • β-fibrinogen, myoglobin • D-loop (very fast) • elongation factor 1, 2 • rhodopsin, RNA polymerase II

PLANTS relatively fast – fast (very) slow slow – variable Evolutionary tempo PLANTS • 18S rRNA Not really used rbcL , atpB, trnK/matK, ndh Frequently used • ITS 1, ITS 2 markers

--- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – From the idea to results using molecular tools –

Formulate a phylogenetic hypothesis

Do sampling Choose appropriate molecular genetic

PLANNING PHASE methods

DNA isolation

PCR

DNA sequencing Fragment analyses DATA COLLECTION (microsatellites, ISSR )

Sequence alignment Evaluation depending on methods

S Dendrogramm construction based on

H distance, MP, ML, BI criteria

I Evaluation of results based on the original hypothesis

E Testing of alternative Comparison with other results hypotheses

Final evaluation and interpretation

--- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA --- – 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), 2nd edition.

--- Introductory seminar on the use of molecular tools in natural history collections - 6-7 November 2007, RMCA ---