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

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”)


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 PROTEINTranslation

GENETIC CODE

Transcription

Reverse transcription

Replication

Replication


2. THE PROKARYOTIC GENOME

Chromosome: carries genes essential for survivalPlasmid/s: non essential* genes. Selectve advantages

Chromosome

Plasmids


2. THE PROKARYOTIC GENOME2.1. CHROMOSOME

Chromosome

PlasmidNumber: normally, only oneCopy number: 1 – 100Size: 0.5 Mb – 10 MbStructure: cccDNA (with some exceptions) supercoiledPackaging: 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



2. THE PROKARYOTIC GENOME2.1. CHROMOSOME

OPERON REGULON

Only one promoter. Co-transcription of several genes. Polycistronic RNA

One regulatory moleculeCo-expression of several operons


2. THE PROKARYOTIC GENOME2.2. PLASMIDS

Circular (normally) DNA molecules Chromosome-independent replication

Genes non-essential* for growthsSize range from 1 Kb to 1 Mb (megaplasmids)

Chromosome

PlasmidHigh/low copy numberIncompatibility groupsCuration: plasmid loss (induced or spontaneous)

Plasmid types:

Cryptic

Conjugative

Resistance

Metabolic

Virulence

Episome

“Engineered”

Plasmids R


2. THE PROKARYOTIC GENOME

2.2. PLASMIDS

ROLLING CIRCLE REPLICATION


Core genome

vs.

Accessory genome

(strain and environmental

sequences)

3. THE PAN-GENOME


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)


These mechanisms transfer DNA to receptor cells. This DNA will “stay” if it recombines with the receptor genome



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)



Barrier to the recombination: restriction-modification systems

CH3

CH3

Methylated Chromosomic DNA

Methylase

Restriction enzyme

Exogenous DNA

4.1. RECOMBINATION4. GENETIC INTERCHANGE AND RECOMBINATION


RESTRICTION ENZYMES

4.1. RECOMBINATION4. GENETIC INTERCHANGE AND RECOMBINATION


4.1. TRANSFORMATION4. GENETIC INTERCHANGE AND RECOMBINATION


Definition?Competent cells

Viral DNA: transfection

DNA binding proteinsAutolysinesNucleases

DNA carrier proteins

4.1. TRANSFORMATION4. GENETIC INTERCHANGE AND RECOMBINATION


Natural or recombinant plasmid

(Genetic Engineering)4.1. TRANSFORMATION4. GENETIC INTERCHANGE AND RECOMBINATION


4.1. CONJUGATION4. GENETIC INTERCHANGE AND RECOMBINATION


DNasa



Conjugative plasmids [e.g.: plasmid F (factor F)]

F+

F-

F+

F+



Integration into the genome (episomes)

bbe

Plasmid F

Chromosome

INtegrated plasmid F

Plasmid F‘







From Hfr to F- From F ‘ to F-

2 cells with F ‘2 recombinat cells



DNase

4. GENETIC INTERCHANGE AND RECOMBINATION4.4. TRANDSUCTION


LYTIC vs LYSOGENIC CYCLES

Virulent phagesAlways lysis

Temperate phagesIntegration/Lysis

4. GENETIC INTERCHANGE AND RECOMBINATION4.4. TRANDSUCTION


GENERALIZED TRANSDUCTION

Defective phage

Transduced cell

4. GENETIC INTERCHANGE AND RECOMBINATION4.4. TRANSDUCTION


SPECIALIZED TRANSDUCTION


Gene Transfer Agents(GTAs)

4. GENETIC INTERCHANGE AND RECOMBINATION4.4. TRANSDUCTION


5. TRANSPOSABLE ELEMENTS

DNA fragments that can move and integrate in a new genomic region (transposition)

Insertion sequences

Transposons

Replicative transposons



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



5’ 3’

3’5’P

Gene expression inactivation

Over-expression

5’ 3’No effect

5’ 3’


6. GENETIC MANIPULATION OF MICROORGANISMS6.1. GENE CLONING

Restriction enzymes / Taq polimerase

Restriction enzymes

DNA ligases

Transformation

bb


6. GENETIC MANIPULATION OF MICROORGANISMS6.2. CLONING VECTORS

BBE


6. GENETIC MANIPULATION OF MICROORGANISMS6.2. CLONING VECTORS


6. GENETIC MANIPULATION OF MICROORGANISMS

6.3. METAGENOMICS

Documents

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