Chapter 9

Why did leprosy become less common over time?

Socioeconomic factors and general advancement of life helped to reduce the prevalence of leprosy.

Role of bacteria and viruses is human society

Bacterial function in oceans, agriculture, natural flora, disease, and commercial products.

Advantages of using bacteria and viruses for genetic studies

• Reproduction is rapid

• many progeny produced

• haploid genome allows mutations to be expressed directly

• asexual reproduction simplifies isolation of genetically pure strains

• growth in the laboratory

• genomes are small

• techniques available for isolating and manipulating their genes

• medical importance

• genetically engineered for commercial value.

Characteristics of bacteria

diverse shapes and sizes, some are photosynthetic, replication occurs prior to binary fission

Prototrophic

wild type bacteria, can synthesize all the organic compounds required for its growth.

Auxotrophic

mutant type bacteria, requires the specific nutrients it cannot synthesize through the medium

Minimum media

only required by prototrophic bacteria

Complete media

contains all substances required by all bacteria, including auxotrophic bacteria

Colony

group of genetically identical cells

Purpose of Replica Plating

To identify and isolate prototrophic and auxotrophic colonies

Steps of Replica Plating

Place bacteria on complete medium plate. Replica plate the colonies by pressing a sterile velvet surface to the plate, cells adhere to the velvet. Press the velvet onto a minimum medium and again on a complete medium. A colony that only grows on the complete medium has a mutation in a gene that encodes the synthesis of an essential nutrient.

Shape of bacterial genome

mostly circular, single chromosome/DNA molecule

Plasmids

small circular DNA molecules

Episomes

freely replicating plasmids that are able to integrate into bacterial chromosomes

What are responsible for the spread of antibiotic resistance within bacteria?

plasmids

F factor

fertility factor in E.coli, contains multiple genes that are helpful in insertion to the bacterial chromosome.

Plasmid replication

plasmids replicate independently of its bacterial chromosome, begins at the origin of replication, continues around the circle in both directions.

Conjugation

genetic material passes from one bacterium to another, no reciprocal exchange

F positive cell

donor cells containing F factor

F negative cells

recipient cells lacking F factor

Mechanism of conjugation

• Sex pilus connects the two cells

• One DNA strand of F factor is nicked at origin of transfer and separates

• Replication takes place on F factor, replacing the nicked strand

• 5' end of nicked DNA passes into recipient cell, where single strand is replicated, producing a circular, double-stranded copy of F plasmid

• F negative cell becomes F positive.

Sex pilus

A thin connection between two bacteria through which genetic material passes during conjugation.

Lederberg and Tatum Experiment

• Two auxotrophic strains were combined

• able to grow on minimal media because genetic recombination had taken place

• they could now synthesize necessary nutrients.

Conclusion of the Lederberg and Tatum Experiment

Genetic exchange and recombination took place between the two mutant strains

Davis's U-Tube Experiment

Two auxotrophic strains were separated by a filter that allowed mixing of medium but not bacteria. No prototrophic bacteria were produced.

Conclusion of Davis's U-Tube Experiment

Genetic exchange requires direct contact between bacterial cells

How does antibiotic resistance occur?

Comes from genes located on R plasmids that are transferred naturally between same or different bacteria species

Transformation

• bacteria takes up free DNA from the medium

• can engage in rapid crossing over and recombination, incorporating it into the chromosome.

Competent Cells

cells that can take up DNA through transformation

Transformant Cells

cell that has received genetic material through transformation

Cotransformed Cells

cells that are transformed by two or more genes

How is transformation used to map bacterial genes?

• DNA from a donor cell is fragmented

• Fragments are taken up by the recipient cell

• donor DNA becomes incorporated into bacterial chromosome through crossing over

• Genes close to one another on chromosome are more likely to be present on same DNA fragment and be recombined together

Conclusion of Transformation Gene Mapping Experiment

rate of cotransformation is inversely proportional to distance between genes

Transduction

Bacterial viruses take DNA from one bacterium to another

Horizontal Gene Transfer

genes passed between individual members of different species by nonreproductive mechanisms

Vertical Gene Transfer

genes passed down within a species through generations

Eukaryotes can only use what type of gene transfer?

Vertical gene transfer

Restriction-Modification Systems

Bacteria produce restriction endonucleases that recognize and cleave double-stranded DNA at specific nucleotide sequences.

How do bacteria protect their DNA from their restriction-modification system?

modify the recognition sequences on their DNA by adding methyl groups.

CRISPR-CAS systems

A defense system used by bacteria and archaea. They combine CRISPR RNAs with CAS proteins to provide defense against specific foreign DNA molecules.

CRISPR Array

• Located in the bacterial chromosome

• consists of a series of repeated CRISPR sequences separated by photospacers

• Adjacent to CRISPR array are genes that encode Cas proteins.

Protospacer

The bacteria takes the cut up virus, makes a copy of it and chops the copy up into fragments, known as protospacers.

Three stages of the CRISPR-CAS system

adaptation, expression, and interference

CRISPR-CAS Stage 1: Adaptation

• foreign DNA enters the cell, is identified and cleaved by CAS proteins and made into protospacers

• protospacers processed and incorporated into CRISPR array, where they can trigger a reaction if cell ever encounters foreign DNA again

CRISPR-CAS Stage 2: Expression

• The CRISPR Array is transcribed into a long CRISPR precursor RNA

• cleaved by Cas proteins and processed into crRNAs

• each of which contains a spacer sequence that is homologous to the foreign DNA

• crRNA combines with a Cas protein to form an effector complex.

CRISPR-CAS Stage 3: Interference

• When the same foreign DNA reenters the cell, the effector complex binds to it

• Cas protein cleaves the foreign DNA, degrading it.

PAM Sequences

Protospacer-adjacent-motif. Protects the bacterial DNA from being harmed by the CRISPR system.

Virus

nucleic acid surrounded by a protein coat

Bacteriophage

A virus that infects bacteria

Virulent pathogens

reproduce through the lytic cycle and always kill the host cells

Temperate phages

Phage that enters the lysogenic cycle and remains in the bacteria chromosome as an inactive prophage

Steps of the Lytic Cycle

• Phage binds to bacterium

• phage inserts its DNA/RNA into bacteria

• host DNA is digested

• Phage DNA replicates

• host cell transcribes and translates phage DNA, producing phage proteins

• Assembly of new phages is completed

• phage-encoded enzyme causes cell to lyse.

• New phages are released to start cycle again.

Stages of the Lysogenic Cycle

• phage binds to the bacterium

• Phage DNA/RNA enters host cell

• phage DNA integrates into bacterial chromosome and becomes a prophage

• prophage is replicated as part of bacterial chromosome

• replication can continue through many cell divisions

• prophage may separate from chromosome

• cell will enter lytic cycle.

Generalized Transduction

any gene may be transferred

What can generalized transduction be used for?

Map genes. Check if cotransduced, if so, they must be next to each other in the genome.

Specialized transduction

only a few genes are transferred

Lederberg and Zinder Experiment Purpose

To test if cell-cell contact is required for genetic exchange

Conclusion of the Lederberg and Zinder Experiment

found that cell-cell contact isn't required when bacteriophages are present

Retrovirus

RNA virus that has been integrated into the host genome; uses reverse transcriptase.

Reverse transcriptase

synthesizing DNA from RNA or DNA template

Retrovirus Infection Process

• proteins on capsid allow virus to be identified and allow them access into cell.

• When it binds to the cell at receptor, viral core enters host cell

• Viral RNA uses reverse transcriptase to make complimentary DNA and viral RNA degrades

• Reverse transcriptase synthesizes second DNA strand

• Viral DNA enters nucleus and integrated into host chromosome

• Forms a provirus.

Retrovirus Replication Process

• On activation, proviral DNA is transcribed into viral DNA

• exported to the cytoplasm.

• Viral RNA, proteins, new capsids, and envelopes are assembled.

• An assembled virus buds from the cell membrane.

What cells does HIV attack?

helper T cells

Undetectable HIV

The HIV isn't gone, it could still be hiding in the genome. If the individual gets sick or doesn't take their medicine, the virus could flare up again.

What population of individuals is immune to HIV?

Ashkenazi Jews. They lack the binding receptors on their T cells

What type of virus causes Influenza?

RNA virus

Three main types of influenza

Influenza A, B, and C

What is the most common type of influenza?

Influenza A

How is Influenza A categorized?

It is divided into subtypes based upon expression of hemagglutinin and neuraminidase

How do new strains of influenza appear?

antigenic shift; genetic material from different strains combine

How are new viral genomes created?

through the reassortment of RNA molecules of different strains

Rhinoviruses

small RNA viruses that often cause respiratory infections

Zika virus

RNA virus that has spread rapidly around the world in recent years. It has a mosquito vector, mild to no symptoms, but causes severe neurological symptoms and deformities in infants.