Microbiology Exam 5

The nucleotide sequence of the genome

Genotype

The physical properties of the organism

Phenotype

A heritable change in the base sequence of the genome

Mutation

The strain isolated from nature

Wild type strain

A strain carrying a change in its nucleotide sequence

Mutant

An altered phenotype relative to the parental strain

Mutant phenotype

The specific mutation is a rare event; mutant of interest will be greatly outnumbered by the parental strain

Problem

The mutant has an advantage under certain environmental conditions and will outcompete and replace the parent

Selectable Mutations

Mutation does not provide an advantage so it will remain rare in the population

Non-selectable mutations

What certain gene is intentionally added to acquire a certain trait for easy screening for mutants?

Selectable marker gene

Examples of selectable marker genes

antibiotic resistant gene, fluorescent gene, nutritional gene, etc.

Replica plating method to screen nutritional mutants

Example of negative selection

Does not affect amino acid sequence

Silent mutation

A codon become a stop codon, short and incomplete polypeptide

Nonsense mutation

An amino acid that has been changed, altered polypeptide

Missense mutation

A type of genetic mutation that occurs when one or more nucleotides are inserted or deleted in a DNA sequence

Frameshift Mutations

What can result in frameshift of +/- 1 to 2 bases?

Insertions or deletions of one or a few nucleotides

In a frameshift mutation, what is mainly changed?

most amino acids

A genetic change that restores a mutated gene to its original, wild-type sequence and function

Back or reversion mutation

Where does a mutation occur in a reverse mutation?

Previously mutated position

Production of genetic mutations

Mutagenesis

Spontaneous mutation rate is very low, so it is important to...

Induce mutation to increase the rate

Simple test to screen chemicals for potential mutagenicity

Ames test

What is the importance of the Ames test?

Screens for an increase in the rate of back mutations in auxotrophic bacteria

What are two ways of transferring genetic material?

Vertical gene transfer and Horizontal gene transfer

What are three mechanisms of genetic exchange?

transformation, transduction, conjugation

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

transformation

A bacterial virus transfers DNA from one cell to another

transduction

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

Conjugation

What does a donor cell contain that a recipient cell does not?

Conjugative plasmid

F- cell

lacks F plasmid; recipient cell

Transfer plasmid DNA from donor to recipient cell; some viruses also use the same mechanism

Rolling circle mechanism

Contains several transposable elements and TRA genes that encode transfer functions

F (fertility) plasmid

Can exist independently or integrate into host chromosome

Episome

EX. F plasmid

Cell with integrated F plasmid

Hfr (high frequency of recombination)

Mobile pieces of DNA that can copy themselves into entirely new areas of the chromosomes

Transposable elements

Where can transposable elements/mobile DNA be found?

All 3 domains of life

What is the process that transposable elements move?

Transposition

What are the two main types of transposable elements in bacteria?

Insertion sequence or transposons

What can move from one location to another within a genome?

insertion sequence (2 inverted repeats + transposase gene)

What is a segment of DNA that can move from one location to the next within a genome?

transposons (2 inverted repeats + transposase gene + other genes)

Recognize specific base sequences within DNA and cut the DNA at specific sites

Restriction enzyme

Where are restriction enzymes found?

Commonly in prokaryotes (Bacteria/Archaea) but rare in eukaryotes

Inverted repeat sequence

palindromes

Sense the methylation at specific base pairs in the recognition sequences

Restriction enzymes

What is a helpful characteristic of restriction enzymes?

it can be used as a defense mechanism against hostile foreign DNA, such as a virus genome

Defense mechanisms against horizontal gene transfer:

physical barriers and restriction enzymes

Cleavage site is far from the recognition sequences

Type I and III restriction enzymes

Cleavage site is located within the recognition sequences

Type II restriction enzyme

What type of ends do restriction enzymes form?

Sticky (cohesive) and blunt

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

What is an acquired immunity in prokaryotes?

CRISPR-Cas9

Contains RNA copies of the phage memories

CRISPR RNAs (crRNAs)

What combines with crRNA to hunt for matching phage DNA?

Cas9

Separates DNA fragments by size and shape

Gel electrophoresis

A bond formed between adjacent nucleotides which consists of a phosphate group that links the sugars of two nucleotides

phosphodiester bonds

Single stranded DNA or RNA can be used to screen for....

homologous sequences by utilizing complementary base pairing

Laboratory technique that detects specific DNA or RNA sequences by using complementary base pairing

Nucleic acid hybridization

What is the procedure for nucleic acid hybridization?

1. Label known nucleic acid with radioactivity or a fluorescent dye

2. Hybridize probes with unknown samples

3. If the probe hybridizes, this indicates a match in the sequence

Molecular biology technique used to identify specific DNA sequences within a complex mixture that was invented by Professor Sir Edwin Southern

Southern blotting

Laboratory technique used to analyze RNA samples, specifically to detect and quantify specific RNA sequences within a complex mixture

Northern Blotting

Makes a DNA copy of RNA -> cDNA

reverse transcriptase

method to amplify DNA; rapidly increases the amount of DNA in a sample

Polymerase chain reaction

What is added to indicate where amplification will begin in PCR?

Primers of known sequence, along with special heat tolerant DNA polymerase and nucleotides

The study of genetic defeats and cancer, forensic, taxonomy, and evolutionary studies

Gene mapping

DNA sequencing method that identifies the order of bases in a DNA segment

Sanger sequencing

How are sequences assembled?

By pairing their overlaps to generate the long continuous sequence

Tracks the expression of thousands of genes and used to identify the devise treatments for diseases based on the genetic profile of the disease

Microarray

Northern blotting vs. Microarray

Microarray involves thousands of genes where northern blotting only involves a few genes at a time

Plasmids as cloning vectors

are small in size and easy to isolate DNA

Plasmids as cloning vectors have a presence of?

selectable markers

Multiple cloning site

Polylinker

Carried out normally by chemical transformation or electroporation

Vector transfer

What are desirable features in a cloning host?

• Fast growth rate

• Can be grown in large quantities using ordinary culture methods

• Nonpathogenic

• Genome that is well delineated

• Maintains foreign genes through multiple generations

Capable of accepting plasmid or bacteriophage vectors; allowing it to accept the host

competence

In a desirable cloning host, will there be a high or low yield of proteins secreted from expressed foreign genes?

high

Replicates a narrow range of host cells, EX. pUC plasmids

simple cloning vector

What are the 4 types of cloning vectors?

Ti plasmid, expression, shuttle, and simple cloning vectors

Replicates in a wide range of host cells (multiple unrelated hosts) and usually requires multiple selection markers for the different hosts

Shuttle vectors

Allows experimenter to control the expression of cloned genes (transcriptional control) and allows high levels of protein expression

Expression vector

The plasmid in plant pathogen is agrobacterium tumefaciens and is used to introduce foreign DNA into plants

Ti plasmid vector

What contains genes that mobilize DNA for transfer to the plant?

Ti plasmid vector

What are the three steps in gene cloning?

1. Isolation of source DNA

2. Inserting DNA fragment into cloning vector

3. Introduction of cloned DNA into host organism

What is often used to get recombinant DNA into host?

Transformation in the 3rd step of gene cloning

Enzyme that joins two DNA molecules

DNA ligase

Fragmentation of source DNA happens by

restriction enzymes

Vector transfer is carried out normally by...

chemical transformation or electroporation

Contains polylinker lacZ gene and ampicillin resistance

plasmids as cloning vectors

What does a blue colony indicate?

no foreign DNA inserted

What does a while colony indicate?

foreign DNA has been inserted and lacZ gene is inactivated

Inactivator lacZ cannot process

x-gal (galactose homolog)

Differences in ... usage between organisms

codon

Presence of ... is in eukaryotes

introns

... to prokaryotic host

toxicity

Adavantages using bacterial system

quick replication and cheap production cost

first human protein made commercially by genetic engineering

Insulin

What is translated as a fusion protein by the bacterial cells?

Insulin

The A&B chains ... assemble to form biologically active insulin

spontaneously

Widely produced growth hormone that allows for reverse transcriptase to generate cDNA

somatotropin

What can be transformed into E.coli?

somatotropin