VL 21 Flashcards
Genom Archaes
- usually circular; 0,35 – 16 MBp
- some linear (Streptomycetes, Borrelia)
- compacted by histone-like protein
- mostly haploid
Genom Eukarota
- (usually) larger genomes
- several linear chromosomes/cell
- genes with Exons and Introns
- many non-coding DNA regions
- compacted by histones - mostly diploid
GC Content
= (G+C)/(A+T+G+C)
- konstant für jede Art
- 14-75%
- DNA melting point
GC Skew
(G-C)/(G+C)
Definition of an Open Reading Frame
- An open reading frame (ORF) starts with any start codon and ends with the next stop codon.
- The first start codon of an ORF can be the correct translational start site but it does not have to be.
- ATG is the most common start codon but not the only one possible: Alternatives: GTG, TTG, CTG
- (+) and (-) strands are coding, there is no general preference.
Gene und Genome von Bakterien
- Functional genes with Transcription control signals
- Promotors, Operators, Transcription terminators etc.
- Single genes and Operons
- Protein-encoding genes (≈95 % in bacaterial/archaeal genomes)
- Ribosomal RNA genes (1-10 copies/ bacterial chromosome)
- tRNA genes (32-60)
Intergenic regions von Bakterien
Small Regulatory RNAs
Chromosomen von Bakterien
- zirkular
- Origin of replication und Replication termination region
- Chromosome maps in “Minutes” (classical) or Base pairs (modern)
Origin of replication
DNA Replication (1-3) -Replication start point and copy number control
In vivo Functions of Naturally Occuring Bacterial Plasmids
(1) Genes for autonomous Replication Transfer genes (tra Operon): F-Plasmid, R-Plasmids
(2) Resistence genes against antibiotics (R-Plasmids) Resistence genes against toxic transition metals
(3) Unusual sources of carbon: Hydrocarbon degradation
(4) Pathogenicity / infectivity: Hämolysins, enterotoxins
(5) Tumors (in plants: T-DNA, Agrobacterium tumefaciens)
(6) Genomes of Bacteriophages
Plasmide
- extra-chromosomal genetic elements; circular or linear
- 3 KBp up to >500 KBp in length
- when integrated into chromosome: Episome
Facultatively present genes
Antibiotics resistence genes bla
Metabolic genes lacZ Single strand origin of replication f1-ori
lac-Promoter / Operator p.lac
lacZ a-fragment lacZ a Multiple Cloning Site (=Polylinker) MCS
Blue/ White CloningVector
pBluescript II SK- polylinker (=MCS) with “Gene of Interest” in XhoI und BamHI site
->lacZ a
Gene disrupted, when Insertion present
->no b-Galactosidase activity
Blue/ white-Selection on Agar plates with X-Gal = dye precursor and IPTG = p.lac inducer
Bacterial Plasmids in Molecular Biologie
(1) Cloning/ gene amplification/ sequencing
(2) Heterologous gene expression / over-production of foreign proteins in E. coli or other Bacteria/ Archaea (3) Site-directed mutagenesis of genes/ proteins
(4) Shuttle vectors for plasmid transfer between E. coli and other, non-related ppecies (e.g. Haloferax volcanii)
(5) Shuttle vectors for plasmid transfer between E. coli and Eukarya (e.g. human cell lines)
Mechanisms of Changes in Genes andGenomes
Replication errors -> Point mutation
Nucleotide Damage-> Point mutations
Recombitation -> Inversion,Deltetion,Transposition
DNA uptake -> Insertion
