Microbial Genetics Microbiology 2314. Genetics The Study of 1. Heredity 2. What Genes Are 3. How Genes Function 4. How Genes Carry Information 5. How.

Microbial GeneticsMicrobial GeneticsMicrobiology 2314

GeneticsGenetics

• The Study of

1. Heredity

2. What Genes Are

3. How Genes Function

4. How Genes Carry Information

5. How Genetic Information is Expressed

6. How Genes are Replicated and Passed

Genome and DNAGenome and DNA• Genome is the genetic

information of the cell.• Genome is composed of

chromosomes containing genes.

• DNA is a double stranded helix.

• Hydrogen bonds exist between nitrogenous base pairs

1. Nitrogenous Bases

a. Adenine

b. Thymine

c. Cytosine

d. Guanine

2. Deoxyribose Sugar

3. Phosphate

GenesGenes1. Are Short

Sections of DNA

2. Code for Proteins

3. 1000’s of Bases

4. 41000 Possibilities

The Genetic CodeThe Genetic Code

• The sequence of nucleotides in DNA or RNA that determines the specific amino acid sequence in the synthesis of proteins.

• It is the biochemical basis of heredity and nearly universal in all organisms.

• An Operon is a unit made up of linked genes that is thought to regulate other genes responsible for protein synthesis. 

Genetic ExpressionGenetic Expression• DNA Transcribed to RNA

• RNA Translated to Protein

How Does DNA Serve as How Does DNA Serve as Genetic InformationGenetic Information

• DNA is transcribed to RNA

• RNA is translated to Protein

• Proteins can then act as enzymes, structural units, etc to carry on the biochemical processes of life.

• Therefore Protein carries on life. Easy as pie.

Genotype and PhenotypeGenotype and Phenotype

• Genotype

1. Genetic Composition of an Organism

2. Represents the Potential Properties

• Phenotype

1. The Expression of the Genes

2. What You See

ReplicationReplication

• The duplication of DNA which occurs during the S phase of Interphase.

• 1 Strand 2 Complementary Strands

• DNA Polymerase

TranscriptionTranscription• The process by which a molecule of DNA

is copied into a complementary strand of RNA.

• 1 Strand DNA 2 Strands RNA

• RNA Polymerase

TranslationTranslation• The process in which the information in the

nucleotide base sequence of mRNA is used to dictate the amino acid sequence of a protein.

• 1 Strand RNA Amino Acid Chain Protein

RNA and Protein SynthesisRNA and Protein Synthesis

• RNA is a Single Stranded Nucleic Acid

• RNA Acts as a Messenger between DNA and Ribosomes

• Process Takes Amino Acids and Forms Proteins

Why Is It Necessary?Why Is It Necessary?

• DNA / Nucleus• Ribosomes / Cytoplasm

• Need a Messenger

CompositionComposition

• Nitrogenous Basesa. Guanineb. Cytosinec. Adenined. Urasil

• Ribose Sugar• Phosphate

DefinitionsDefinitions

• Codon1. Three-base segment of mRNA that specifies amino acids.2. Sense Codons3. Nonsense Codons (AUG / Methionine)

• Anticodon1. Three-base segment of tRNA that docks with a codon.2. Docking results in deposition of amino acid.

Protein Synthesis in Protein Synthesis in Prokaryotes and EukaryotesProkaryotes and Eukaryotes

• The basic plan of protein synthesis in Eukaryotes and Archaea is similar to that in bacteria.

• The major structural and mechanistic themes recur in all domains of life. However, eukaryotic protein synthesis entails more protein components than does prokaryotic protein synthesis, and some steps are more intricate.

Noteworthy Similarities and Noteworthy Similarities and Differences Between the TwoDifferences Between the Two

• Ribosomes. Eukaryotic ribosomes are larger. The differences between eukaryotic and prokaryotic ribosomes can be exploited for the development of antibiotics

• Initiator tRNA. In eukaryotes, the initiating amino acid is methionine rather than N-formylmethionine.

Noteworthy Similarities and Noteworthy Similarities and Differences Between the TwoDifferences Between the Two

• Initiation. The initiating codon in eukaryotes is always AUG. In almost all cases, eukaryotic mRNA has only one start site and hence is the template for a single protein. . In contrast, a prokaryotic mRNA can have multiple start sites, and serve as a template for several proteins.

• Termination. Termination in eukaryotes is carried out by a single release factor, eRF1, compared with two in prokaryotes.

Duh What?

• Protein synthesis is essentially the same in both types of cells. However, in prokaryotes the ribosomes can attach directly to the mRNA molecule while the mRNA is being synthesized. In eukaryotes, the nuclear membrane separates transcription from translation. This separation allows for the RNA to be processed

Regulation of Bacterial Gene Regulation of Bacterial Gene ExpressionExpression

• Efficient Process

• Performed as Needed

• Determined by Structural Genes

• Example: E. coli / Lactose

Operon Model of Gene Operon Model of Gene ExpressionExpression

• The formation of enzymes is determined by structural genes

1. E. coli digests lactose

2. No lactose / No need for enzymes

3. No Inducer / Repressor binds to DNA

4. Repressor blocks Transcription

5. Inducer Present / Inducer binds to Repressor

Why Does This Occur?Why Does This Occur?

• Bacteria do not make all the proteins that they are capable of making all of the time. Rather, they can adapt to their environment and make only those gene products that are essential for them to survive in a particular environment. For example, bacteria do not synthesize the enzymes needed to make tryptophan when there is an abundant supply of tryptophan in the environment. However, when tryptophan is absent from the environment the enzymes are made

• Similarly, just because a bacterium has a gene for resistance to an antibiotic does not mean that that gene will be expressed.

• The resistance gene may only be expressed when the antibiotic is present in the environment.

A Typical Operon

What is an Operon?

• In genetics, an operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter.

• The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create monocistronic mRNAs that are translated separately

Structure of an OperonStructure of an Operon• This is the general structure of an operon:• Promoter – a nucleotide sequence that enables a

gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. In RNA synthesis, promoters indicate which genes should be used for messenger RNA creation – and, by extension, control which proteins the cell manufactures.

• Operator – a segment of DNA that a regulator binds to. It is classically defined in the lac operon as a segment between the promoter and the genes of the operon. In the case of a repressor, the repressor protein physically obstructs the RNA polymerase from transcribing the genes.

• Structural genes – the genes that are co-regulated by the operon.

Lac Operon ModelLac Operon Model

• In the case of the lac operon, the bacterial cell does not need lactose metabolizing proteins expressed if there is no lactose present to act as a substrate.

• It makes sense, therefore, that the regulatory protein - the repressor - blocks transcription unless lactose is present.

In this situation, the repressor is unable to bind to the operator by itself. Hence, structural genes will be expressed at a level that is determined by the strength of the promoter.

• When, the supply of a molecule is sufficient, or when it builds up to sufficient levels, it can bind to the repressor, alter its conformation, and render it unable to bind to its operator.

• A molecule that acts in this way is called an effector.

Trp Operon ModelTrp Operon Model• The trp operon is

an example of a biosynthetic operon whose expression is regulated by an effector:

Induction and RepressionInduction and Repression

• Induction

The process that initiating transcription with an inducer.

• Repression

The repressing of transcription with a repressor.

• Originally operons were thought to exist solely in prokaryotes, but since the discovery of the first operons in eukaryotes in the early 1990s more evidence has arisen to suggest they are more common than previously assumed.

MutationMutation

• A change in the nitrogenous base sequence of DNA; that change causes a change in the product coded for by the mutated gene.

• Neutral

• Hazardous

• Beneficial

• Base Substitution- One base pair in DNA is replaced with a different base pair

• Deletion- A piece of DNA breaks off and is lost

• Duplication and Translocation- A piece of DNA breaks off and is incorporated into another strand of DNA

• Frameshift- Deletion or Addition results in a shift in the DNA frame

Types of MutationsTypes of Mutations

• Single Base Substitution

- Also called Point Mutation

- Two Kinds

1. Transition

Purine Replaced by a Purine

Pyrimidine Replaced by Pyrimidine

2. Transversion

Purine Replaced by a Pyrimidine

or Vice Versa

• Purines1. Adenine2. Guanine

• Pyrimidines1. Thymine2. Cytosine

• Example Transition?• Example Transversion?

Types of MutationsTypes of Mutations• Missence Mutation

- Codon is Altered Producing Altered Amino Acid- Example: Sickle Cell Disease

GAG GTG

• Nonsense Mutation (Example Thalidomide)- Sense Codon is Altered to Stop Codon- TGG TAA- TGG TAG- TCA TGA

Types of MutationsTypes of Mutations

• Silent Mutation

- Codon Changed but Still Codes for Same

- Serine is TCT and TCG and TCA and TCC

• Deletion (Results in Frameshift)

- GAGCCGCAACTTC Deletion Occurs

- GAGCCGCATTC Altered State Results

• Insertion (Results in Frameshift)

- GAGCCGCAACTTC Insertion Occurs

- ACGAGCCGCAACTTC Altered State Results

Types of MutationsTypes of Mutations• Frameshift

GAG CCG CAA CTT C…

ACGAGCCGCAACTTC

GAG CCG CAA CTT C… ACG AGC CGC AAC TTC

MutagensMutagens

• Tobacco products• Nitrous Acid• Mold Toxins• X-rays• Gamma Rays• UV Radiation• Some Artificial Sweeteners

UV LightUV Light

• Skin cancer is the most commonly occurring cancer in the United States.

Tobacco ProductsTobacco Products

• All forms of tobacco products have been shown to cause cancer.

Frequency of MutationsFrequency of Mutations

• Rare

• Somatic / Germline

• Occurs During S Phase

• Cell Error Checks

• 97% Junk DNA

• Males Contribute to More Mutations

RadiumRadium

• 1900’s

• Watch Faces / Medicine

• FDA Limited Power

• E.M. Byers

Identifying MutantsIdentifying Mutants

• Ames Test

- Inexpensive

- Rapid Test

- Assumes Back Mutation or Reversion

is Possible

- Uses His- Salmonella and Rat Liver

Cells

Genetic Transfer and RecombinationGenetic Transfer and Recombination• Bacteria have no sexual reproduction in the sense that

eukaryotes do. The have no alternation of diploid and haploid generations,

no gametes, and no meiosis.• But the essence of sex is genetic recombination, and

bacteria do have three mechanisms to accomplish that:

1. Transformation

2. Conjugation

3. Transduction

TransformationTransformation• Many bacteria can acquire new genes by

taking up DNA molecules (e.g., a plasmid) from their surroundings

• Genes are Transferred as Naked DNA

• Discovered by Frederick Griffith / England 1928

• Occurs Repeatedly in Nature

• Works best if Cells are Closely Related

ConjugationConjugation• Some bacteria, E. coli is an example, can

transfer a portion of their chromosome to a recipient with which they are in direct contact. As the donor replicates its chromosome, the copy is injected into the recipient. At any time that the donor and recipient become separated, the transfer of genes stops. Those genes that successfully made the trip replace their equivalents in the recipient's chromosome.

ConjugationConjugation

• Requires Direct Contact

• Can only occur between cells of opposite mating types. – The donor (or "male") carries a fertility factor

(F+). – The recipient ("female") does not (F−).

TransductionTransduction

• Bacteriophages are viruses that infect bacteria. In the process of assembling new virus particles, some host DNA may be incorporated in them.

• Utilizes a Donor Cell and a Recipient Cell

• Utilizes a Virus

• 2 Pathways

1. Lytic

2. Lysogenic

PlasmidsPlasmids

• Self-replicating circular pieces of DNA

• Found Primarily in Prokaryotic Cells

• Some Found in Eukaryotic Cells

• Plasmids usually contain between 5 and 100 genes. Plasmids are not essential for normal bacterial growth and bacteria may lose or gain them without harm.

Functions of PlasmidsFunctions of Plasmids

• Plasmids code for synthesis of a few proteins not coded for by the nucleoid. For Example: Some exotoxins, such as the tetanus exotoxin and Escherichia coli enterotoxin are coded for by plasmids.

TransposonsTransposons

• Small pieces of DNA that encode enzymes that transpose the transposon, that is, move it from one DNA location to another.

• 700-4000 genes

Note: Provides a powerful natural mechanism for moving genes from one

chromosome to another.

• Plasmids and conjugative transposons are very important in horizontal gene transfer in bacteria. Horizontal gene transfer, also known as lateral gene transfer, is a process in which an organism transfers genetic material to another cell that is not its offspring.

Horizontal and Horizontal and Vertical Gene Vertical Gene

TransferTransfer

• Vertical gene transfer uses reproduction as a means of gene transfer through generations, whereas horizontal gene transfer uses non-reproductive methods of gene transfer.