L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 Lecture 5. GENETICS OF PROKARYOTES 1. Basic concepts 2. The prokaryotic genome 3. The pan-genome 4. Genetic interchange and recombination 4.1. Recombination 4.2. Transformation 4.3. Conjugation 4.4. Transduction 5. Transposable elements 6. Genetic manipulation of microorganisms (“Genetic engineering”)
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Lecture 5. GENETICS OF PROKARYOTES · L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014 . 1. BASIC CONCEPTS. Genome: molecule/s storing the genetic information (DNA in all cells;
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L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
Lecture 5. GENETICS OF PROKARYOTES
1. Basic concepts
2. The prokaryotic genome
3. The pan-genome
4. Genetic interchange and recombination
4.1. Recombination
4.2. Transformation
4.3. Conjugation
4.4. Transduction
5. Transposable elements
6. Genetic manipulation of microorganisms
(“Genetic engineering”)
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
1. BASIC CONCEPTS
Genome: molecule/s storing the genetic information (DNA in all cells; DNA and/or RNA in viruses) Gene: the basic unit of genetic information. A fragment of DNA or RNA, including regulatory sequences, coding for a protein or RNA.
Expression: transcription (when the gene product is a rRNA or tRNA) or transcription and translation (when the gene product is a protein; implies mRNA)
Coding sequence (ORF: open reading frame)
5’ 3’
Regulatory sequences
DNA RNA PROTEIN Translation
GENETIC CODE
Transcription
Reverse transcription
Replication
Replication
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
2. THE PROKARYOTIC GENOME
Chromosome: carries genes essential for survival Plasmid/s: non essential* genes. Selectve advantages
Chromosome
Plasmids
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
2. THE PROKARYOTIC GENOME 2.1. CHROMOSOME
Chromosome
Plasmid Number: normally, only one Copy number: 1 – 100 Size: 0.5 Mb – 10 Mb Structure: cccDNA (with some exceptions) supercoiled Packaging: basic proteins, cations, etc.
Protein coding sequences (87%)
RNA coding sequences (0.8%) Regulatory non-coding
sequences (11%)
Non-coding sequences (0.7%)
Escherichia coli chromosome
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
2. THE PROKARYOTIC GENOME 2.1. CHROMOSOME
OPERON REGULON
Only one promoter. Co-transcription of several genes. Polycistronic RNA
One regulatory molecule Co-expression of several operons
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
2. THE PROKARYOTIC GENOME 2.2. PLASMIDS
Circular (normally) DNA molecules Chromosome-independent replication
Genes non-essential* for growths Size range from 1 Kb to 1 Mb (megaplasmids)
Chromosome
Plasmid High/low copy number Incompatibility groups Curation: plasmid loss (induced or spontaneous)
Plasmid types:
Cryptic
Conjugative
Resistance
Metabolic
Virulence
Episome
“Engineered”
Plasmids R
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
2. THE PROKARYOTIC GENOME 2.2. PLASMIDS
ROLLING CIRCLE REPLICATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
Core genome
vs.
Accessory genome
(strain and environmental
sequences)
3. THE PAN-GENOME
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
4. GENETIC INTERCHANGE AND RECOMBINATION
GENETIC VARIABILITY
GENETIC VARIABILITY Eukaryotes: Individual level (mutation and recombination) Population level (sexual reproduction) Prokaryotes: Individual level (mutation and recombination) Population level (HGT: horizontal gene transfer or LGT: lateral GT)
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
These mechanisms transfer DNA to receptor cells. This DNA will “stay” if it recombines with the receptor genome
4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
4.1. RECOMBINATION
Together with point mutations, this is a mechanisms of generating genetic diversity
Transfer of DNA between different molecules. Homologous recombination requieres large straches of homologous sequences (>100pb)
4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
Barrier to the recombination: restriction-modification systems
CH3
CH3
Methylated Chromosomic DNA
Methylase
Restriction enzyme
Exogenous DNA
4.1. RECOMBINATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
RESTRICTION ENZYMES
4.1. RECOMBINATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
4.1. TRANSFORMATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
Definition? Competent cells
Viral DNA: transfection
DNA binding proteins Autolysines Nucleases
DNA carrier proteins
4.1. TRANSFORMATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
Natural or recombinant plasmid
(Genetic Engineering) 4.1. TRANSFORMATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
DNasa
4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
Conjugative plasmids [e.g.: plasmid F (factor F)]
F+
F-
F+
F+
4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
Integration into the genome (episomes)
bbe
Plasmid F
Chromosome
INtegrated plasmid F
Plasmid F‘
4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
From Hfr to F- From F ‘ to F-
2 cells with F ‘ 2 recombinat cells
4.1. CONJUGATION 4. GENETIC INTERCHANGE AND RECOMBINATION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
DNase
4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANDSUCTION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
LYTIC vs LYSOGENIC CYCLES
Virulent phages Always lysis
Temperate phages Integration/Lysis
4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANDSUCTION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
GENERALIZED TRANSDUCTION
Defective phage
Transduced cell
4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANSDUCTION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
SPECIALIZED TRANSDUCTION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
Gene Transfer Agents (GTAs)
4. GENETIC INTERCHANGE AND RECOMBINATION 4.4. TRANSDUCTION
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
5. TRANSPOSABLE ELEMENTS
DNA fragments that can move and integrate in a new genomic region (transposition)
Insertion sequences
Transposons
Replicative transposons
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
5. TRANSPOSABLE ELEMENTS
CUT AND PASTE
Genomic region B (without a mobile element) Genomic region A (with a mobile element)
Genomic region B (with a mobile element) Genomic region A (without a mobile element)
COPY AND PASTE
Genomic region B (without a mobile element) Genomic region A (with a mobile element)
Genomic region A (with a mobile element) Genomic region B (with a mobile element)
Transposable element Transposable element
Transposable element
Transposable element
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
5. TRANSPOSABLE ELEMENTS
5’ 3’
3’ 5’ P
Gene expression inactivation
Over-expression
5’ 3’ No effect
5’ 3’
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
6. GENETIC MANIPULATION OF MICROORGANISMS 6.1. GENE CLONING
Restriction enzymes / Taq polimerase
Restriction enzymes
DNA ligases
Transformation
bb
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
6. GENETIC MANIPULATION OF MICROORGANISMS 6.2. CLONING VECTORS
BBE
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
6. GENETIC MANIPULATION OF MICROORGANISMS 6.2. CLONING VECTORS
L. 5: Prokaryotic Genetics. 2nd Biology ARA 2013-2014
6. GENETIC MANIPULATION OF MICROORGANISMS 6.3. METAGENOMICS