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Bacterial genetics. Chapter 8. 9.1 Mutations and Mutants 9.2 Genetic Recombination 9.3 Genetic Transformation 9.4 Transduction 9.5 Conjugation 9.6 Plasmids 9.7 Transposons and Insertion Sequences 9.8 Comparative Prokaryotic Genomics 9.9 Genetics in Eukaryotic Microorganisms .

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slide1

Bacterial genetics

Chapter 8

9.1 Mutations and Mutants

9.2 Genetic Recombination

9.3 Genetic Transformation

9.4 Transduction

9.5 Conjugation

9.6 Plasmids

9.7 Transposons and Insertion Sequences

9.8 Comparative Prokaryotic Genomics

9.9 Genetics in Eukaryotic Microorganisms

slide2
Microorganisms provide relatively simple systems for studying genetic phenomena and are thus useful tools in attempts to decipher the mechanisms underlying the genetics of all organisms.
  • Microorganisms are used for the isolation and duplication of specific genes from other organisms, a technique called molecular cloning. In molecular cloning, genes are manipulated and placed in a microorganism where they can be induced to increase in number.
slide3

Section 1. Mutation and recombination

Mutation is an inherited change in the base sequence of the nucleic acid comprising the genome of an organism. Mutation usually brings about only a very small amount of genetic change in a cell.

slide4

Genetic recombinationis the process by which genetic elements contained in two separate genomes are brought together in one unit. This mechanism may enable the organism to carry out some new function and result in adaptation to changing environments. Genetic recombination usually involves much larger changes. Entire genes, sets of genes, or even whole chromosomes, are transferred between organisms.

slide5

Section 2.Techniques of bacterial genetics: in vivo

In vivo : manipulate the genetic material within the organism

Genetic Transformation

Transduction

Conjugation

slide6

Section 3. Techniques of bacterial genetics: in vitro

In vitro: operate genetic material in the test tube

Restriction Enzymes

Molecular Cloning

Amplifying DNA: PCR

slide7

WORKING GLOSSARY

Auxotroph an organism that has developed a nutritional requirement through mutation

Cloning vector genetic element into which genes can be recombined and replicated

Conjugationtransfer of genes from one prokaryotic cell to another by a mechanism involving cell-to-cell contact and a plasmid

Diploid a eukaryotic cell or organism containing two sets of chromosomes

Electroporationthe use of an electric pulse to induce cells to take up free DNA

Gene disruptionuse of genetic techniques to inactivate a gene by inserting within it a DNA fragment containing an easily selectable marker. The inserted fragment is called a cassette, and the process of insertion, cassette mutagenesis

slide8

Genetic mapthe arrangement of genes on a chromosome

Genomethe total complement of genes of a cell or a virus

Genotypethe precise genetic makeup of an organism

Hybridizationformation of a duplex nucleic acid molecule with strands derived from different sources by complementary base pairing

Molecular cloning isolation and incorporation of a fragment of DNA into a vector where it can be replicated

Haploida cell or organism that has only one set of chromosomes

Mutagensagents that cause mutation

Mutantan organism whose genome carries a mutation

Mutationan inheritable change in the base sequence of the genome of an organism

slide9

Nucleic acid probea strand of nucleic acid that can be labeled and used to hybridize to a complementary molecule from mixture of other nucleic acids

Phenotypethe observable characteristics of an organism

Plasmidan extrachromosomal genetic element that has no extracellular form

Point mutationa mutation that involves one or only a very few base pairs

Polymerase chain reaction(PCR)a method used to amplify a specific DNA sequence in vitro by repeated cycles of synthesis using specific primers and DNA polymerase

Recombinationthe process by which parts or all of the DNA molecules from two separate sources are exchanged or brought together into a single unit.

slide10

Restriction enzymean enzyme that recognizes and makes double-stranded breaks at specific DNA sequences

Shotgun cloningmaking a gene library by closing random DNA fragments

Site-directed mutagenesis a technique whereby a gene with a specific mutation can be constructed in vitro

Synthetic DNAa DNA molecule made by a chemical process in a laboratory

Transductiontransfer of host genes from one cell to another by a virus

Transformationtransfer of bacterial genes involving free DNA

Transposon a type of transposable element that carries genes in addition to those involved in transposition

slide11

Genetic Recombination

Genetic recombination involves the physical exchange of genetic material between genetic elements.

Homologous recombinationresults in genetic exchange between homologousDNA sequences from two different sources. This type of recombination is extremely important to all organisms. However, it is also very complex. Even in the bacterium Escherichia coli there are at least 25 genes involved.

slide12

A simplified version of one molecular mechanism of recombination. Homologous DNA molecules pair and exchange DNA segments.

The mechanism involves breakage and reunion of paired segments. Two of the proteins involved, a single-stranded binding (SSB) protein and the RecA protein.

Note that there are two possible outcomes, depending on which strands are cut during the resolution process. In one outcome the recombinant molecules have patches, whereas in the other the two parental molecules appear to have been cut and then spliced together.

slide13

Detection of Recombination

In order to detect physical exchange of DNA segments, the cells resulting from recombination must be phenotypically different from the parents.

Strains that lack some selectable characteristic that the recombinants will possess. For instance, the recipient may not be able to grow on a particular medium, and genetic recombinants are selected that can.

Various kinds of selectable and nonselectable markers (such as drug resistance, nutritional requirements, and so on) may be used.

slide14

Complementation

This can be determined by a type of experiment called a complementation test.

Complementation was first used in diploid eukaryotic organisms

slide15

Genetic Transformation

  • In prokaryotes genetic recombination is observed because fragments of homologous DNA from a donor chromosome are transferred to a recipient cell by one of three processes:
  • transformation, which involves donor DNA free in the environment
  • transduction, in which the donor DNA transfer is mediated by a virus
  • conjugation, in which the transfer involves cell-to-cell contact
slide16

Three main processes of genetic recombination in prokaryotes fragments of homologous DNA from a donor chromosome are transferred to a recipient cell

(1) Transformation, which involves donor DNA free in the environment

(2) Transduction, in which the donor DNA transfer is mediated by a virus

(3) Conjugation, in which the transfer involves cell-to-cell contact and a conjugative plasmid in the donor cell

slide17

Transformation

Transduction

Conjugation

slide18

Transformation

Competence

A cell that is able to take up a molecule of DNA and be transformed is said to be competent.

slide19

The introduction of DNA into cells by mixing the DNA and the cell

  • Binding of free DNA by a membrane-bound DNA binding protein.
  • (b) Passage of one of the two strands into the cell while nuclease activity degrades the other strand.
  • (c) The single strand in the cell is bound by specific proteins, and recombination with homologous regions of the bacterial chromosome mediated by RecA protein occurs.

Transformed cell

slide21

Transduction

Concept

Transduction involves transfer of host genes from one bacterium to another by viruses. In generalized transduction, defective virus particles randomly incorporate fragments of the cell's chromosomal DNA; virtually any gene of the donor can be transferred, but the efficiency is low. In specialized transduction, the DNA of a temperate virus excises incorrectly and brings adjacent host genes along with it; only genes close to the integration point of the virus are transferred, but the efficiency may be high.

slide22

In transduction, DNA is transferred from cell to cell through the agency of viruses. Genetic transfer of host genes by viruses can occur in two ways.

Generalized transduction

And

Specialized transduction

slide23

Generalized transduction: host DNA derived from virtually any portion of the host genome becomes a part of the DNA of the mature virus particle in place of the virus genome.

Specialized transduction: occurs only in some temperate viruses; DNA from a specific region of the host chromosome is integrated directly into the virus genome - usually replacing some of the virus genes.

slide24

Generalized transduction

In generalized transduction, virtually any genetic marker can be transferred from donor to recipient

During a lytic infection, the enzymes responsible for packaging viral DNA into the bacteriophage sometimes accidentally package host DNA. This DNA cannot replicate, it can undergo genetic recombination with the DNA of the new host.

slide25

Specialized Transduction

the DNA of lambda is inserted into the host DNA at the site adjacent to the galactose genes

On induction, Under rare conditions, the phage genome is excised incorrectly

A portion of host DNA is exchanged for phage DNA, called lambda dgal ( dgal means "defective galactose“ )

Phage synthesis is completed

Cell lyses and releases defective phage capable of transducing galactose genes

slide28

Genetic Recombination

Genetic recombination involves the physical exchange of genetic material between genetic elements. It results in genetic exchange between homologous DNA sequences from two different sources.

Homologous recombination is extremely important to all organisms. However, it is also very complex .

slide29

conjugation

Bacterial conjugation (mating) is a process of genetic transfer that involves cell-to-cell contact.

Direct contact between two conjugating bacteria is first made via a pilus. The cells are then drawn together for the actual transfer of DNA. Note the F-specific bacteriophages on the pilus

slide30

Conjugation involves a donor cell, which contains a particular type of conjugative plasmid, and a recipient cell, which does not.

The genes that control conjugation are contained in the tra region of the plasmid (see Section 9.8 in your text ). Many genes in the tra region have to do with the synthesis of a surface structure, the sex pilus . Only donor cells have these pili,

The pili make specific contact with a receptor on the recipient and then retract, pulling the two cells together. The contacts between the donor and recipient cells then become stabilized, probably from fusion of the outer membranes, and the DNA is then transferred from one cell to another.

slide31

Mechanism of DNA Transfer DuringConjugation

A mechanism of DNA synthesis in certain bacteriophages, called rolling circle replication, was presented here to explains DNA transfer during conjugation .

if the DNA of the donor is labeled, some labeled DNA is transferred to the recipient but only a single labeled strand is transferred. Therefore, at the end of the process, both donor and recipient possess completely formed plasmids.

slide32

DNA Transfer in Bacteria

transformation

transduction

conjugation

slide34

Three main processes of genetic recombination in prokaryotes fragments of homologous DNA from a donor chromosome are transferred to a recipient cell

(1) Transformation, which involves donor DNA free in the environment

(2) Transduction, in which the donor DNA transfer is mediated by a virus

(3) Conjugation, in which the transfer involves cell-to-cell contact and a conjugative plasmid in the donor cell

slide35

Transformation

Transduction

Conjugation

slide36

Transformation

A number of prokaryotes have been found to be naturally transformable, including certain species of both gram-negative and gram-positive Bacteria and some species of Archaea. However, even within transformable genera, only certain strains or species are transformable

slide37

Competence

A cell that is able to take up a molecule of DNA and be transformed is said to be competent. Competence in most naturally transformable bacteria is regulated, and special proteins play a role in the uptake and processing of DNA. These competence-specific proteins may include a membrane-associated DNA binding protein, a cell wall autolysin, and various nucleases.

Competent cells bind much more DNA than do noncompetent cells as much as 1000 times more

slide38

The introduction of DNA into cells by mixing the DNA and the cell

  • Binding of free DNA by a membrane-bound DNA binding protein.
  • (b) Passage of one of the two strands into the cell while nuclease activity degrades the other strand.
  • (c) The single strand in the cell is bound by specific proteins, and recombination with homologous regions of the bacterial chromosome mediated by RecA protein occurs.

Transformed cell

slide40

Artificially Induced Competence

High efficiency natural transformation is found only in a few bacteria; Azotobacter, Bacillus, Streptococcus,, for example, are easily transformed. Many prokaryotes are transformed only poorly or not at all under natural conditions. Determination of how to induce competence in such bacteria may involve considerable empirical study, with variation in culture medium, temperature, and other factors

when E. coli is treated with high concentrations of calcium ions and then stored in the cold, the transformation by plasmid DNA is relatively efficient.

slide41

DNA Transfer by Electroporation

for artificial induction of competence are being supplanted by a new method termed electroporation.

Small pores are produced in the membranes of cells exposed to pulsed electric fields. When DNA molecules are

present outside the cells during the electric pulse, they can then enter the cells through these pores. This process is called electroporation.

slide42

Transfection

Bacteria can be transformed with DNA extracted from a bacterial virus rather than from another bacterium, a process known as transfection.

slide44

Transduction

In transduction, DNA is transferred from cell to cell through the agency of viruses. Genetic transfer of host genes by viruses can occur in two ways.

Generalized transduction

And

Specialized transduction

slide45

Concept:

Transduction involves transfer of host genes from one bacterium to another by viruses. In generalized transduction, defective virus particles randomly incorporate fragments of the cell's chromosomal DNA; virtually any gene of the donor can be transferred, but the efficiency is low. In specialized transduction, the DNA of a temperate virus excises incorrectly and brings adjacent host genes along with it; only genes close to the integration point of the virus are transferred, but the efficiency may be high.

slide46

Generalized transduction: host DNA derived from virtually any portion of the host genome becomes a part of the DNA of the mature virus particle in place of the virus genome.

Specialized transduction: occurs only in some temperate viruses; DNA from a specific region of the host chromosome is integrated directly into the virus genome - usually replacing some of the virus genes.

slide47

Transduction has been found to occur in a variety of prokaryotes, including certain species of the Bacteria: Desulfovibrio, Escherichia, Pseudomonas, Rhodococcus,Rhodobacter, Salmonella, Staphylococcus, and Xanthobacter, as well as the archaean Methanobacterium thermoautotrophicum.

Not all phages can be transducer and not all bacteria are transducible

slide48

Generalized transduction

In generalized transduction, virtually any genetic marker can be transferred from donor to recipient

During a lytic infection, the enzymes responsible for packaging viral DNA into the bacteriophage sometimes accidentally package host DNA. This DNA cannot replicate, it can undergo genetic recombination with the DNA of the new host.

slide50

Specialized Transduction

the DNA of lambda is inserted into the host DNA at the site adjacent to the galactose genes

On induction, Under rare conditions, the phage genome is excised incorrectly

A portion of host DNA is exchanged for phage DNA, called lambda dgal ( dgal means "defective galactose“ )

Phage synthesis is completed

Cell lyses and releases defective phage capable of transducing galactose genes

slide51

Conjugation

Bacterial conjugation (mating) is a process of genetic transfer that involves cell-to-cell contact.

Direct contact between two conjugating bacteria is first made via a pilus. The cells are then drawn together for the actual transfer of DNA.

slide52

Conjugation involves a donor cell, which contains a particular type of conjugative plasmid, and a recipient cell, which does not.

The genes that control conjugation are contained in the tra region of the plasmid (see Section 9.8 in your text ). Many genes in the tra region have to do with the synthesis of a surface structure, the sex pilus . Only donor cells have these pili,

The pili make specific contact with a receptor on the recipient and then retract, pulling the two cells together. The contacts between the donor and recipient cells then become stabilized, probably from fusion of the outer membranes, and the DNA is then transferred from one cell to another.

slide58

F-

F+

Cells possessing an unintegrated F plasmid are called F', and strains that can act as recipients for F' (or Hfr, see later in this section) are called F-. F- cells lack the F plasmid;

Hrf

F’

slide59

F+ x F-

F-

F+

F’

Hrf

slide60

Mechanism of DNA Transfer DuringConjugation

A mechanism of DNA synthesis in certain bacteriophages, called rolling circle replication, was presented here to explains DNA transfer during conjugation .

if the DNA of the donor is labeled, some labeled DNA is transferred to the recipient but only a single labeled strand is transferred. Therefore, at the end of the process, both donor and recipient possess completely formed plasmids.