Microbiology Exam 5-mine

Wild-type strain

Wild-type strain

strain isolated from nature

Mutant

change in nucleotide sequence

mutant phenotype

altered phenotype relative to parental strain

What is a problem with the isolation of mutants

a specific mutation is a rare event and the mutant of interest will be outnumbered by the parent strain

selectable mutation

mutant has an advantage under certain environmental conditions and will overtake the wild strain

Non-selectable mutation

no growth advantage or disadvantage in any environment. Difficult to isolate

What is a screening strategy for identifying mutants?

selectable marker genes

A mutation during DNA replication will result in what two types of cells?

Wild type and mutant

Name one example of negative selection

Replica plating

Replica plating

method to screen nutritional mutants (leucine auxotrophs)

What are the point mutations?

silent, nonsense, missense

Silent mutation

no change in amino acid sequence

Nonsense mutation

codon becomes stop codon

Missense mutation

amino acid is changed

Transitions

purine to purine and pyrimidine to pyrimidine

Transversion

pyrimidines to purines

Frameshift mutation

one or more nucleotides are added or deleted from DNA

Back mutations

a mutation occurs at a previously mutated position, so it returns to original

Point mutations are typically reversible (True/False)

True

Mutagenesis

induced mutation

Ames Test

• screen chemicals for potential mutagenicity

• screen for increase rate of back mutations in auxotrophic bacteria in presence on chemical disc

Vertical gene transfer

genes passed from parent to offspring

Horizontal gene transfer

movement of genetic material between organisms that are not related by parent and offspring

What are the three types of horizontal gene transfer

Transformation, Transduction, Conjugation

Transformation definition

free DNA is incorporated into a recipient cell and brings about genetic change

Transduction definition

bacterial virus that transfers DNA from one cell to another

Conjugation definition

genetic transfer from a donor cell to a recipient cell requiring cell-to-cell contact

Transformation process

1. free DNA enters cell

2. homologous recombination

Generalized Transduction

• any host gene packaged into virion

• virion lacks viral genome

• requires recombination for stabilization in new host

• lytic or lysogenic

• low frequency

Specialized Tranduction

• only genes near viral integration site are packaged

• virion carries most to all of viral genome

• recombination not needed

• lysogenic

• high frequency

Conjugation process

sex pilus and then the DNA is sent into the other cell and replicated

Rolling circle replication

bacterial conjugation where transfer plasmid DNA from donor to recipient cell and then circular DNA just rolls and replicates

F plasmid

• circular

• ~100k bp

• contains genes that regulate DNA replication

• contains transposable elements

• contains tra genes that encode transfer functions

• episome

Episome

can exist independently or integrate into host chromosome

F-

cell without F plasmid

F+

cell with non-integrated F plasmid

HFr

cells with integrated F plasmid

Transposable elements

mobile DNA

Are transposable elements found in all three domains of life?

yes

What are the two types of transposable elements in bacteria?

Insertion sequences and transposons

Insertion sequences

2 inverted repeats + transposase gene

Transposons

2 inverted repeats + transposase genes + other genes

Restriction enzymes

Recognize specific base sequences within DNA (recognition sequences) and cut the DNA at specific sites

Where are restriction enzymes found

prokaryotes (bacteria and archaea)

Recognition sequences

• inverted repeats

• methylation

• defense mechanism against foreign DNA

Defenses against horizontal gene transfer

physical barriers and restriction enzymes

Type II restriction enzymes

cleavage site locate within the recognition sequences

What are the three types of ends that Type II forms

1. 5’ overhang

2. 3’ overhang

3. Blunt ends

CRSPR

clustered regularly interspaced short palindromic repeats

CRSPR Process

• segments of prokaryotic DNA containing short repetitions of base sequences

• each repetition followed by short segments of spacer DNA from previous exposure to a bacterial virus or plasmid

CRSPR Cas-9

crRNA + tracrRNA + Cas9 endonuclease

How does CRSPR Cas-9 work?

binds to target viral dsDNA and cuts both strands (double stranded cleavage)

CRSPR Cas-9 process

1. virus invades bacterial cell

2. new spacer derived from from virus and integrated into CRSPR sequence

3. CRSPR RNA is formed

4. CRSPR RNA guides molecular machinery to target and destroy viral genome

What is also called magic scissors

CRSPR Cas-9

Gel electrophoresis separates cells by their

shape and size

How does size effect movement in gel electrophoresis

larger fragments move more slowly

How does shape effect movement in gel electrophoresis

compact shapes move faster

How does gel electrophoresis work?

the phosphodiester bond in DNA is negatively charged, so they migrate towards the positive pole. Fragments are then stained

Nucleic Acid hybridization

single stranded DNA or RNA used for screening homologous sequences by utilizing complementary base pairing (hybridization)

Nucleic Acid hybridization procedure

• label know nucleic acid with radioactive/fluorescent dye

• hybridize probes with unknown samples

• if probe hybridizes it is positive

Southern blotting

DNA in the gel labeled with DNA or RNA probe

Northern blotting

RNA in gel labeled with DNA or RNA probe

Western blotting

protein in gel labeled with antibody

Splicing

introns are removed and exons are joined by spliceosome

Spliceosomes contain

small nuclear ribonucleoproteins (snRNPs)

Reverse transcriptase

• makes DNA copy of RNA (cDNA)

• cDNA made from mRNA, tRNA, rRNA

What does reverse transcriptase allow for

a means of synthesizing eukaryotic genes from mRNA transcripts-synthesized gene free of introns

Polymerase Chain Reaction Purpose

rapidly increase the amount of DNA in a sample

For PCR, primers of known sequences are added to indicate where amplification will begin, along with

special heat tolerant DNA polymerase and nucleotides

PCR is repetitively cycled through

denaturation, priming, and extension

Every cycle of PCR increases the amount of DNA by much

double

What is PCR essential for

gene mapping, genetic deficits, cancer, forensics, taxonomy, evolutionary studies

DNA sequencing

1. isolate unknown DNA fragment

2. DNA is denatured to produce single template strand

3. strand labeled with specific primer molecule

4. DNA polymerase and all nucleotides added to a tube. Each tube contains one type of dideoxy nucleotide that stop chain lengthening reaction

5. newly replicated strands are terminated at the point of addition of a dd nucleotide

6. a schematic view illustrating how each fragment will end with a labeled dideoxy nucleotide, after all four tubes are mixed

7. a gel showing the results of a sequencing run for six different strands of DNA. Location and color of the band provide the correct identity and order of the bases

Pyrosequencing

whole-genome shotgun sequencing

Benefits of pyrosequencing

fast and cost effective analysis

pyrosequencing procedure

1. DNA fragmentation

2. separate and sequence small DNA fragments

3. Assemble pieces of DNA sequences together

4. Annotation, finding ORF

Microarray

track the expression of thousands of genes to identify diseases and treatments

Plasmids can be used as

cloning vectors

Why is it easy to isolate DNA in plasmids?

small size

Do plasmids have an independent origin or replication?

yes

Polylinker within plasmid

multiple cloning site, single cut made by restriction enzymes

Vector transfer is normally carried out by

chemical transformation or electroporation

Desirable features in a cloning host

1. fast growth rate

2. grown in large quantities

3. nonpathogenic

4. genome is well delineated

5. capable of accepting plasmid or bacteriophages

6. maintains foreign genes through multiple generations

7. will secrete a high yield or proteins from expressed foreign genes

Types of cloning vectors

1. simple cloning vectors

2. Shuttle vectors

3. Expression vectors

4. TI plasmid vector

simple cloning vectors

replicates in narrow range of host cells

Shuttle vectors

• replicates in wide range of hosts

• easily move genes between multiple hosts

• multiple selection markers

• multiple origins of replication

Expression Vector

• transcriptional control

• multiple cloning sites downstream of promoter region

• high levels of protein expression

• promoter is repressed unless activated by an inducer

• strong transcription terminator sites prevent read-through

TI plasmid vector

used to introduce foreign DNA into plants

Example of TI plasmid vector

Agrobacterium tumefaciens

What DNA is transferred to the plant in TI vectors

T-DNA

Cloning steps

1. Isolation and fragmentation of source DNA

2. Inserting DNA fragment into cloning vector

3. Introduction of cloned DNA into host organism

Where/when is DNA usually inserted

in vitro

DNA ligase

enzyme that joins two DNA molecules. sticky and blunt ends

Introduction of clones DNA into host organism. ___ is often used to get recombinant DNA into host

Transformation

X-gal

colorless substrate used to detect activity of ß-galactose

No vector screening (plasmids as cloning vectors)

• AMP: sensitive

• Lac Z: no gene

• X-gal: no colony on AMP plate

Vector without insert screening (plasmids as cloning vectors)

• AMP: resistant

• Lac Z: intact gene

• X-gal: processed by lac Z protein. Blue colony

Vector with insert

• AMP: resistant

• Lac Z: damaged gene (interrupted by an insert)

• X-gal: not processed (no color) White colony

Expression of mammalian genes in bacteria

• different codon usage

• presence of introns in eukaryotes

• degradation by host intracellular proteases

• toxicity to prokaryotic host

• formation of inclusion bodies

• improper protein folding

• lack of post-translational modification

Advantages using bacterial system

quick replication and cheap production cost