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Microbiology Unit 7


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Study of science of heredity
Structures containing DNA
Segments of DNA
One long double helix w/associated proteins
Nucleotides base
adenine, thymine, cytosine, guanine
Nucleotide dexoxyrybose
pentose sugar
Base pairs
Adenine w/thymine

Cytosine w/ guanine
Gene expression
DNA transcribed to produce RNA; mRNA then transferred to proteins
Genetic composition of organism - entire DNA
Expression of the genes
Bacteria DNA
Circular, 1000 times longer than cell
DNA Replication
1. Two strands of parental DNA unwind and separate in one small segment after another

2. Free nucleotides match up to exbosed bases

3. Newly added nucleotide is joined to growing DNA strand by DNA polymerase

4. Parental unwound a little more to allow addition of next nucleotides

Only occurs in ONE direction around chromosome
Replication fork
Point at which replication occurs
Semiconservative replication process
Each double-stranded DNA molecule contains one original and one new DNA strand
DNA polymerase
Makes a molecule of DNA from a DNA template
Conversion of a DNA message (gene) to an RNA strand
Pairs of DNA bases and mRNA
G in DNA / C in mRNA
C in DNA / G in mRNA
T in DNA / A in mRNA
A in DNA / U in mRNA
Steps of transcription
1. RNA polymerase binds to DNA at promoter (site)

2. RNA polymerase assembles free nucleotides into new chain (base pairs)

3. As new RNA chain grows, RNA polymerase moves along DNA

4. RNA synthesis continues until terminator site

5. RNA polymerase and newly formed, single-stranded mRNA are released from DNA
Promoter site
Starting point for transcription where RNA polymerase binds to DNA
Terminator site
Region of DNA that is endpoint for transcription
Conversion of mRNA to a protein
Genetic code
Relationship among nucleo-base sequence of DNA, corresponding codons of mRNA, and amino acids for which codons code

Code is degenerate- most amino acids are coded for by more than 1 codon
Sense codons
61 codons code for amino acids
Nonsense codons
Stop codons signal the end of the protein molecule's synthesis
Start codon
AUG (methionine)
Found on the tRNA and matches the codon
Steps of translation
1. Two ribosomes subunits, tRNA wi/anticodon UAC, and mRNA molecule assemble.

2. First tRNA binds to start codon

3. First amino acid is transferred by the ribosome

4. After ribosome joins two amino acids w/peptide bond, first tRNA leaves the ribosom

5. Ribsome then moves along mRNA

6. Peptide bonds are formed between amino acids

7. Ends when one of three nonsense codons is reached
Procaryote tranlsation
Can begin before transcription is completed
Three-base sements of mRNA that specify amino acids. mRNA read in 5'--3' direction
Change in nitrogenuous base sequence of DNA - causes a change in product coded for

Some are neutral, some disadvantageous, other beneficial
Base-pair mutation
One base pair in DNA is replaced w/a different base pair
Missense mutations
Base substitution results in amino acid substitution in the synthesized protein
Nonsense mutation
Base substitution results in nonsense codon
Frameshift mutation
A mutagen that causes insertions
Agents in the environment that cause permanent changes in DNA
Spontaneous mutation
Occur in absence of any mutation-causing agents.
Chemical mutagens
Base-pair mutagen (nitrous acid), Base analogs, frameshift mutagen
Ionizing radiation
A mutagen that causes the formation of highly reactive ions
Nonionizing radiation
UV radiation

A mutagen that causes the formation of pyrimidine dimers
UV Damage
Can be repaired by enzymes that cut out and replace damaged portion of DNA
Mutation rate
Probability that gene will mutate when cell divides, expressed as 10 to negative power
Genetic recombination
Rearrangement of genes to form new combinations
Crossing over
Genes from two different chromosomes are recombined into one chromosome
Some of donor's DNA integrated into recepient's DNA
The transfer of DNA from a donor to a recipient as naked DNA in solution

Discovered first by Frederick Griffith w/Streptococcus pneumoniae
Contact between living cells

Transfer of a plasmid from a donor cell to a recipient cell
Circular piece of DNA that self-replicates outside of the chromosome
The transfer of DNA from a donor to a recipient cell by a bacteriophage
Steps of transduction
2. Phage attaches to the donor bacterial cell wall and injects DNA into bacterium

2. Phage DNA acts as template for synthesis of new phage DNA. Bacterial chromosome is broken apart by phage enzymes.

3. Some pieces of bacterial DNA mistakenly packaged inside phage protein coats.

4. When released phage particles later infect new bacteria, bacterial genes are transferred to newly infected recipient cells
F Factor plasmid
Conjugative plasmid carries genes for sex pili and for the transfer of plasmid to another cell
Dissimilation plasmid
Code for ezymes that trigger the catabolism of certain unusual sugars and hydrocarbons
Resistance factor
Plasmids that have significant medical importance
Small segments of DNA that can move from one region of a DNA molecule to another

Frequent movement can wreak havoc inside cell

Found in main chromosome of organism

Vary from simple to complex
Complex transposons
Carry other genes not connected with the transposition process.
Genetic engineering
Genes can be transferred between unrelated species via laboratory manipulations
Recombinant DNA
DNA that has been artificially manipulated to combine genes from two different sources
All industrial applications of microorganisms as well as industrial uses of genetically engineered cells
Self-replicating DNA used to carry a gene from one organism to another
Recombinant DNA Procedure
1. Desired gene inserted into DNA vector such as plasmid or viral genome

2. Vector inserts the DNA into new cell, which is grown to form clone

3. Large quantities of gene can be harvested from the clone
Sticky Ends
Short stretches of single-stranded DNA at ends of DNA fragments

Fragments of DNA produced by same enzyme will spontaneously join by hydrogen bonding
Shuttle vectors
Plasmids that can exist in several different species
Viral vector
Can accept much larger pieces of foreign DNA

Can be inserted into cell by transduction
Can be used to increase amounts of DNA in samples to detectable levels
An electrical current form microscopic pores in membranes of cells. DNA enters cells through pores.
Protoplast fusion
"Hit and miss"

Does not add a specific gene to a cell
Use of a fine glass micropipette to inject DNA
Gene libraries
Can be made by cutting up an entire genome with restriction enzymes and inserting fragments into plasmids or phages
Stretches of DNA that code for protein
Stretches of DNA that do not code for protein
cDNA made from mRNA by reverse transcription

Segment of eukaryotic DNA that lacks introns
Synthetic DNA
Can be made in vitro by a DNA synthesis machine
Easily grown

Genetics are well understood

Produces endotoxins

Must be lysed or gene must be linked to gene that produces a naturally secreted protein
Baker's yeast/Saccharomyces cervisiae
Four times larger than E.coli

Best understood eukaryotic genome

May carry plasmids which can be transferred

More likely to continuously secrete product
Mammalian cells
Can be engineered to produce proteins such as hormones for medical use
Plant cells
Used to produce plants with new properties
Population of cells carrying a specific gene
Human insulin
Synthetic genes linked to beta-galactosidase inserted into E. coli, allowing E.coli to produce and secrete the two polypeptides
Subunit vaccine
Protein portion of a pathogen from genetically engineered yeasts
Animal viruses
Can be engineered to carry gene for pathogen's surface protein.

When used as vaccine, host develops immunity
Southern Blotting
DNA fragments are separated then exposed to radioactive probe

Locates gene in a cell
Genetic Screening
Uses Southern blotting to look for mutations responsible for inherited disease in humans

Screen available for more than 200 diseases
DNA fingerprinting
Restriction fragments
DNA probes
Used to identify bacteria carrying a specific gene
Ti plasmid vector
Plant cells can be engineered using this vector
Enhances nitrogen fixation
Restriction enzyme
Protect a bacterial cell by hydrolyzing phage DNA
Reverse transcriptase
Enzyme that provides copy of mRNA to DNA
Antibiotic resistance
Can be acquired by bacteria by:

Insertion of transposons
Acquiring plasmids

Use on plasmid makes direct selection possible
Nucleoside Analog
A mutagen that is incorporated into DNA in place of a normal base
Composed of rRNA and proteins

Performs translation
Carries the specific amino acid to the ribosome
Carries the message for a polypeptide, translated by the ribosome (translation)
RNA Polymerase
Makes of molecule of RNA from a DNA template
DNA ligase
Joins segments of DNA
Insertion of DNA segments into DNA
Removal of introns
Gene Library
Pieces of DNA stored in yeast cells
Making cDNA
1. Gene composed of introns and exons is transcribed to RNA

2. RNA processing to remove introns

3. Isolate mRNA from cell and add reverse transcriptase

4. First strand of DNA is synthesized

5. mRNA digested by reverse transcriptase

6. Add DNA polymerase to synthesize second strand of DNA

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