Basics of molecular genetics -...

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

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 procedurefor gene isolation (construction andscreening of cloned libraries)

• 1983: Mullis invents the technique knownas the polymerase chain reaction (PCR)

• 2001: Draft sequences of the humangenomes are published (Lander et al., Venter et al.)

• 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 regulatoryregions, 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)

– Some definitions –


– 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:

dATPdCTPdGTPdTTP

– The Eukaryote genome and DNA –


Genome in nucleus

Genes andrelated sequences

Regions betweengenes

coding(information)

non-coding(introns, pseudogenes)

30 % 70 %

10 % 90 %

• The eukaryote genome has a highly organised, complex structure

• A small piece of the genome encodes gene products: this codingregion is ca. 2% of the total genome in humans

• The human genome contains ca. 3.3 billions base pairs but ca. 20500 genes only

– The Eukaryote genome and DNA –


– The structure of eukaryote genes –

3´---- ---- 5´5´---- ---- 3´

promoter structure gene termination signal

• 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 includestranscription (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 oneRNA-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 plasmidscontaining 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!)

– Mitochondrial genomes –• the (circular) mitochondrial genome ofvertebrates is much smaller than that ofthe plants, yeasts etc.

• the mitochondrial genes of plants/yeastsdo contain introns, while mitochondrial genes of vertebrates do not

• tRNA genes are marked in red• mitochondrial genes of vertebrates(markers frequently used for molecularphylogenetic 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


– 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

– Variability of the genetic information –


• Molecular phylogeny is based on the idea that there is a multi-levelvariation 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 relatedorganisms show high similarity in their genetic material (i.e. relatively fewmutations occured) while distantly related organisms show biggerdifferences 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 ofsome organisms and cellsmanifested by the presence ofmore than two homologous sets of chromosomes (genomes)

• Some examples:triploid (3x): apple, bananatetraploid (4x): tobacco, cottonhexaploid (6x): bread wheatoctaploid (8x): sugar cane

• The diagram summarizes allwell-known polyploidizationevents

– Genetic recombination –


• Genetic recombination is the most important mechanism for maintaininggenetic 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 sexualreproduction• 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 intothe host cells

• Transpositional recombination (caused by transposons) does not needsequence homology

– Genetic markers –


• In general, it is not possible – and also not necessary – to investigate the wholegenome 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



• 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)



• Considerations for the selection of molecular markers – DNA sequences

rbcL, atpB, trnK/matK, ndh

Not really used• 18S rRNA• ITS 1, ITS 2

PLANTS

Frequently usedmarkers

slow – variable(very) slowrelatively fast – fastPLANTSEvolutionary tempo

• 12S rRNA, 16S rRNA(rel. fast)

• COI, NDx, cyt b (fast)• D-loop (very fast)

• 5.8S, 18S, 28S rRNA• ITS 1, ITS 2

• RAG1, RAG2, c-mos• β-fibrinogen, myoglobin• elongation factor 1, 2

• rhodopsin, RNA polymerase II

ANIMALS

Frequently usedmarkers

relatively fast – fastslowANIMALS

Evolutionary tempo

ChloroplastDNA

Mitochondrial DNANuclear DNA

Formulate a phylogenetic hypothesis

Do sampling Choose appropriate molecular geneticmethods

DNA isolation

PCR

DNA sequencing Fragment analyses(microsatellites, ISSR)

Sequence alignment

Dendrogramm construction based ondistance, MP, ML, BI criteria

Evaluation depending on methods

Evaluation of results based on the original hypothesis

Testing of alternativehypotheses

Comparison with other results

Final evaluation and interpretation

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– From the idea to results using molecular tools –


– Further reading –


Lodish et al.: Molecular Cell Biology (2007), 6th edition.

Hartwell et al.: Genetics: From Genes to Genomes (2006), 3rd

edition.

Wink (ed.): An Introduction to Molecular Biotechnology: Molecular Fundamentals, Methods and Applications in ModernBiotechnology (2006), 1st edition.

Avise: Molecular Markers, Natural History, and Evolution (2004), 2nd edition.

Basics of molecular genetics -...

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