Download nov6_part1_Basics of molecular genetics

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
related sequences
Regions between
genes
10 %
90 %
coding
(information)
non-coding
(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
5´---3´---•
•
•
structure gene
termination signal
---- 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
running side by side
• Prior to its transport to the cytoplasm, there is a
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
RNA-polymerase
• There are three different types of RNA-polymerases
--- 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
ANIMALS
Evolutionary tempo
ANIMALS
Frequently used
markers
PLANTS
Evolutionary tempo
PLANTS
Frequently used
markers
Nuclear DNA
Mitochondrial DNA
Chloroplast
DNA
slow
relatively fast – 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
• 12S rRNA, 16S rRNA
(rel. fast)
• COI, NDx, cyt b (fast)
• D-loop (very fast)
relatively fast – fast
(very) slow
slow – variable
• 18S rRNA
• ITS 1, ITS 2
Not really used
rbcL, atpB,
trnK/matK, ndh
--- 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 –
DATA COLLECTION
PLANNING
PHASE
Formulate a phylogenetic hypothesis
Do sampling
Choose appropriate molecular genetic
methods
DNA isolation
PCR
Fragment analyses
(microsatellites, ISSR)
DNA sequencing
Sequence alignment
EVALUATION PHASE
Evaluation depending on methods
Dendrogramm construction based on
distance, MP, ML, BI criteria
Evaluation of results based on the original hypothesis
Testing of alternative
hypotheses
Comparison with other results
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), 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 Modern
Biotechnology (2006), 1st 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 ---