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What is molecular genetics

It is the genetic research with bacteria and phage that led to the discovery of genes, DNA, the molecular basis of inheritance, transcription, translation, the genetic code and biochemical pathways


How were these studies carried out

It started with the isolation of bacterial or phage mutants that are defective for a certain process that is being studied. This leads to the identification of the genes that are required for carrying out that process, which leads to the proteins that the genes encode for and the molecular basis of the phenotype


What is a mutation

A heritable change in genome sequence that can potentially lead to a change in phenotype


What is a phenotype

Observable properties of an organism


What is a mutant

Any organism, cell, or virus differing from the parental strain in genotype


What is a genotype

The nucleotide seqeunce of genome. A mutant contains mutation(s) in its genome


What is WT (wild type)

Typically refers to strains isolated from nature, but depends on context, and often refers to a standard research strain


What are selectable mutations

Mutations that give the mutant organism a growth advantage under certain conditions, such as antibiotic resistance. It allows genetic selection of mutants that can grow and others will die. Ex. antibiotic resistance


What are nonselectable mutations

Those that may not have either an advantage or a disadvantage over the wild-type. Detection of such mutations requires examining a large number of colonies and looking for difference in various ways (screening)


What is screening

Have to look through all the bacteria on your plate and differentiate between them


What is selection

Only the mutants will grow and the wild-types will die


What is replica plating

A method that will facilitate screening. Is useful for identification of cells with a nutritional requirement for growth


What are prototrophs

Bacteria that do not require a specified nutrient for growth, can make everything that it needs by iteslf


What are auxotrophs

Bacteria that require a specified nutrient for growth that is provided in the medium


Describe the replica plating

First you start with a master plate of organisms that have been subjected to mutagensis on growth of complete medium. Then you press the plate onto velveteen. The Velveteen will imprint all of the colonies and you can transfer the imprints to fresh media. Put one imprint on complete medium and one imprint on minimal medium. On the complete medium all of the colonies will grow and on the minimal medium the mutants will not grow (compare)


What are induced mutations

Mutations that are made deliberately or as a result of something in the organism's environment, mutations that can result from exposure to radiation or chemicals


What are spontaneous mutations

Those that occur without external intervention, can result from mistakes in the DNA replication or repair


What are point mutations

Mutations that change only one base pair


What are the 3 different types of point mutations

There can be a missense, nonsense, or silent mutation


What is a missense mutation

A base pair change results in the incorrect amino acid being coded for and results in a faulty protein


What is a nonsense mutation

A base pair change results in a stop codon which results in an incomplete protein


What is a silent mutation

A base pair change does not result in any change of the amino acid being coded for so there is no change in the overall protein


What are frameshift mutations

Deletions or insertions that can cause more dramatic changes. Results in the shift of the reading frame, often results in complete loss of gene function. Every codon after the deletion or insertion will be read incorrectly


What is genetic engineering

It can involve the introduction of specific mutations (site-directed mutagensis)


What can genetic engineering be used for

Can be used to inactivate genes, alter ORF to produce altered function, or change the expression of gene


What is genetic engineering produced with

It will be produced with synthetic DNA. Synthetic primers for PCR, whole synthesis genes, and entire synthetic bacterial genome


What is reversion

Point mutations are typically reversible. A reversion is a mutation in DNA that reverses the effects of a prior mutation


What is a revertant

A strain in which the original phenotype is restored


What are the two types of revertants

Same-site revertant and second-site revertant


Describe same-site revertant

Mutation is at the same site as original mutation, reverts back to the wild type sequence


Describe second-site revertant

Mutation is at a different site in the DNA, but still resotres wild type phenotype. Called a surpressor mutation and compensates for the effect of the original mutation


Describe how a surpressor mutation works

It can replace a amino acid with the same TYPE of amino acid, it won't revert it back to the wild-type genome but this new amino acid can also fold the protein back to the same functional shape


How often do errors occur in DNA replication

10^-6 to 10^-7 per kilobase


How are the error rates of DNA viruses

About 100-1000x greater because RNA repair mechanisms are rare


What are mutagens

Chemical, physical, or biological agents that increase mutation rates


What are the 3 classes of mutagens

Nucleotide base analogs - they resemble nucleotides. Chemicals that induce chemical modifications in DNA, and chemicals that cause frameshift mutations during replication by distorting the DNA structure


What are biological mutagens

Transposons and insertion sequences


What are the 2 main categories of mutagenic radiation

Non-ionizing and Ionizing radition


Describe non-ionizing radition

UV radiation - purines and pyrimidines strongly absorb UV light which results in pyrimidine dimers (thymine dimers)


Describe ionizing radiation

X rays, cosmic rays, and gamma rays - produce free radicals that damage macromolecules in the cell, including DNA


What are the 3 types of DNA repair systems

Direct reversal, repair of single-strand and repair of double-strand damage


Describe direct reversal in DNA repair

Mutated base is recognized by enzymes and repaired without referring to the other strand


Describe repair of single-strand damage in DNA repair

Damaged DNA is removed and repaired using opposite strand as template. This is a method used to repair thymine dimers caused by UV radiation


Describe repair of double-strand damage in DNA repair

A break in the DNA and requires error-prine repair mechanisms, this type of damage is potentially lethal


Why is it important that DNA replication isn't perfect

If DNA replication was perfect then it would prevent evolution. The mutation rate of an organism is a heritable trait. Some organisms have higher mutations rates than others


What kind of bacteria have lower mutation rates than normal

Deinococcus radiodurans is 20-200 times more resistant to radiation than E.coli because of enhanced DNA repair systems.


Describe the Ames test

The Ames test makes practical use of bacterial mutations to detect potentially hazardous chemicals. It can detect an increase in mutations in a bacterial strain treated with a suspected mutagen and relies on the use of an auxotrophic strain. The revertants can grow on the minimal medium and the colonies can be easily counter


Describe the results of the Ames test

It relies on the reversion of an auxotrophic strain on minimal media. There will be a control plate and an experimental plate that contains the mutagen. On the control plate, only a few colonies will form due to spontaneous mutations that will revert them back to prototrophs. On the experimental plate, if the chemical it contains is a mutagen, then there will be many colonies forming because the mutations will revert a lot more back to prototrophs


What is recombination

Physical exchange of DNA between genetic elements (chromosomes, plasmids, viruses, free DNA fragments)


What is homologous recombination

Genetic exchange between homologoues DNA regions. Homologoues DNA regions have a common ancestor and therefore contain similar sequences and contain the same genes


What is a recombinant

An organism that results from gene transfer and recombination


What are the 3 major mechanisms for genetic recombination

Transformation, Transduction, and Conjugation


Describe the process of homologous recombination

First an endonuclease nicks one strand of the donor DNA and SSB proteins bind to the nick to prevent it from re-attaching. Then the RecA protein will facilitate strand invasion where the hanging strand will enter the recipient DNA. The development of the cross-strand exchange occurs and results in the transfer for a section of DNA. Both of the DNA strands will be sections of one another


What are patches.

Single strand transfer


What are splices

Double strand transfer


What is transformation

The genetic transfer process by which "naked" DNA is incorporated into a recipient cell


Who was Fredrick Griffith and describe his experiment

He discovered transformation. He realized that when rats were given live S cells they were killed but when give heat-killed S cells they could live. When given a cell that lacked a capsule that would protect it from the immune system (R cells) the mouse could still live. But if you mixed the dead S-cells with the live R cells, transformation occurs and the mouse dies. Genes from the live R cells are incorporated into the dead S cells and you end up with live S cells


What are competent cells

Cells that are capable of taking up DNA and being transformed


What is transduction

Transfer of DNA from one cell to another by a bacteriophage (virus)


What are the 2 types of transduction

Specialized and Generalized transduction


Define generalized transduction

DNA from any portion of the host genome is packaged inside the virion. A defective risu particle incorporates framents of the cell's chromosome randomly (instead of virus DNA) and the virus can be temperate or virulent


Describe the process of generalized transduction

First a phage enters the cell in the lytic cycle. The phage will use the host cells metabolic activities and make many copies of its own genome. Then when the virus has to repackage itself, one of the virions accidentally packages the host cell DNA (transducing particle). Transduction will occur when this virion infects another host cell and injects the prior host cells DNA instead. Homologous recombination can occur


What is bacterial conjugation

Mating. Mechanism of genetic transfer that involved cell-to-cell contact. Requires a plasmid-encoded mechanism


What is a donor cell

A cell that contains the conjugative plasmid (F+ cell)


What is a recipient cell

A cell that does not contain the conjugative plasmid (F-cell)


What is the F plasmid

The fertility plasmid. It is a short circular DNA molecule. Contains a oriV for replication and oriT for DNA transfer. It contains several transposable elements that allow the plasmid to integrate into the host chromosome and contains tra genes that encode the transfer functions


What is the sex pilus

It is essential for bacterial conjugation and is only produced by the donor cell and is encoded by genes on the F plasmid


What is necessary for DNA transfer by conjugation

DNA synthesis.


How is the DNA synthesized in conjugation

The DNA is synthesized by rolling circle replication starting at the oriT


Describe the process of conjguation

The two bacterial cells (donor and recipient) are connected by a pilus. The F plasmid is nicked in on strand. The transfer of one strand from the F+ cell to the F- cell occurs (rolling circle replication) and the F plasmid is simultaneously replicated in the F+ cell. Then synthesis of the complementary strand begins in the recipient cell. After completion of DNA transfer and synthesis the cells separate. Both are F+ cells in the end


What is an episome

Something that can integrate into the host chromosome (F plasmid)


What are F+ cells

Cells possessing a nonintegrated F plasmid


What are Hfr cells

Cells possessing an integrated F plasmid (high frequency of recombination).


What is the importance of Hfr cells

The integrated F plasmid causes transfer for chomosome, it results in high rates of gene transfer and genetic recombination between genes on the donor chromosome and those of the recipient cell


How does the F plasmid become integrated

Insertion sequences (IS) are mobile DNA elements that are present in both the F plasmid and the E.coli chromosome. Homologous recombination results in integration


Describe the process of how the F plasmid becomes integrated into the chomosome

Both the F plasmid and the chromosome have insertion seqeunces. The insertion sequences will break apart from their respective strand and the F plasmid can integrate itself entire into the chromosome by connecting at the insertion sequences. Results in two copies of the insertion sequence on the chromosome


Describe the transfer of chromosomal DNA by conjugation

Between an Hfr cell and a F- cell. The F plasmid is nicked in one strand and the transfer of F is followed by the chromosomal DNA. This results in part of the chromosomal DNA being brough into the recipient cell.


What is merodiploid

Partial diploid. A bacterial strain that carries two copies of a chromosomal region. DNA region can be introduced into cell by transformation, trasnduction, or conjugation


What is complementation

A functional copy of a gene compensates for a defective copy of the gene in the same cell. Restoring of the wild type phenotype


What are complementation tests used for

They are used to determine if two mutations are in the same gene or different genes. If an introduced DNA fragment compensates for a mutant gene, then it "complements" the mutation and must contain a wilf-type copy of the mutant gene. If the introduced DNA contains a mutation in the same gene, it cannot complement


Describe an example of complementation

Say we have a chromosome with a mutation on the gene that causes the gene to be nonfunctional. (Gene A works but Gene B doesn't). Then you have another organism with the same phenotype but you're not sure if its the same mutation or not. You can bring a stretch of DNA from one organism to another and if both of the organisms have the mutation in the same gene then you will fail to complement. But if the other DNA has (Gene A doesn't work and Gene B works) then both Gene A and Gene B will end up working due to complementation


Describe gene transfer in Archaea

Archaea usually contain a circular chromosome like bacteria and examples of transformation, trasnduction, and conjugation have been found in Archaea.


What are transposable elements

Discrete segments of DNA that move as a unit from one location to another. Can be found in all 3 domains of life They are selfish DNA that are parasitic, only encodes genes for its own propagation and can move from one location to another on a chromosome, to a pahge genome, or to a plasmid


What is transposition

The process of transposable elements moving. Frequency of transposition is usually very low. Low transcription frequency is selected for during evolution to limit international mutations caused by transposons


What are the 2 main types of transposable elements in bacteria

Insertion sequences and Transposons


What do transposable elements carry on their gene

They carry genes encoding for transposase, the enzyme required for hopping around. They also have DNA inverted repeats at their ends and transposons carry additional genes


Describe an insertion sequence (IS)

Are the simplest transposable element. About 1000 nucleotides long and the inverted repeats at the end are about 10-50 base pairs. The only gene carried by the IS is for transposase. The transposase recognizes the inverted repeats on the DNA.


Where are insertion sequences found

They can be found in plasmids and chromosome for Bacteria and Archaea, and also in bacteriophage genomes


What is transposase

The enzyme that catalyzes transposition. allows the DNA to move around


Describe transposons

Are larger than insertion sequences and carry additional DNA between inverted repeats. Most transposons carry antibiotic resistance genes. Transposons allow for rapid exchange of antibiotic resistance genes among bacterial populations


What happens during the insertion of a transposable elements

Insertion of a transposable element (IS or Tn) often generates a duplication of the target seqeunce, its a result of the transposase mechanism