MicroBio Mod. 4

Genetics and Application

Replication

Makes possible the flow of genetic information from one generation to the next.

• vertical gene transfer

• the DNA of a cell replicates before cell division so that each offspring cell receives a chromosome identical to the parent’s.

• happens before transcription and translation

Transcription

DNA is transcribed to make RNA (mRNA, tRNA, and rRNA)

• genetic information in DNA is copied, or transcribed, into a complementary base sequence of RNA

• Adenine pairs with uracil in the RNA sequence instead of thymine.

• requires RNA polymerase

• begins when RNA polymerase binds to the DNA at a site called the promoter.

• RNA polymerase unwinds the DNA and begins copying at the start point on only one of the two strands.

• transcription stops at a site called the terminator.

Translation

Involves decoding the “language” of nucleic acids and converting it into the “language” of proteins.

• the ribosome attaches to the mRNA sequence and starts translating it into codons (groups of 3 nucleotides)

• each codon corresponds to one amino acid, but an amino acid can code for more than one codon (64 codons but 24 amino acids)

• the amino acids form into a long chain to make a protein

• start codon: AUG

• stop codons: UAG, UAA, UGA (aka nonsense codons)

Shine Dalgarno sequence

A flag for a ribosome to sit at a specific location and start transcribing

tRNA (transfer RNA)

A small RNA molecule that acts as the physical link between the genetic code in messenger RNA (mRNA) and the amino acids that make up proteins

Mutation

Change in the genetic material

• may be neutral, beneficial, or harmful

• Mutagen: Agent that causes mutations

• Spontaneous mutations: Occur in the absence of a

  mutagen

Mutation: Base Substitution (Point Mutation)

A change in one base

Mutation: Silent Mutation

A change in codon but no change to the amino acid (because each amino acid has multiple codons that code for it)

• neutral

Mutation: Missense Mutation

A type of point mutation

• results in a change in amino acid from a change in nucleotide

• could be positive or negative

Mutation: Nonsense Mutation

Results in a stop codon before there should be

• “nonsense” because the protein hasn’t been fully created - it means nothing

Mutation: Frameshift Mutation

Insertion or deletion of one or more nucleotide pairs

• catastrophic disruption to protein

• results in completely different proteins because there are completely different amino acids in the sequence

Causes of mutations

Ionizing radiation (x-rays and gamma rays) - cause the formation of ions that can react with nucleotides and the deoxyribose phosphate backbone

• disrupts DNA by causing adjacent thymine bases to become crosslinked (thymine dimer) and disrupt normal base pairing

Repairing mutations

Nucleotide expression:

• an enzyme cuts out the damaged DNA and DNA polymerase fills the gap by synthesizing new DNA using the intact strand as a template

• DNA ligase seals the remaining gap by joining the old and new strands

Plasmid

An independent piece of DNA not associated with the bacterial chromosome (circular and smaller)

• carries genes just like bacterial chromosome does

Conjugative Plasmid

Carries genes for s*x pili and transfer of the plasmid

Dissimilation plasmids

Encode enzymes for catabolism of unusual compounds

R factors

Encode antibiotic resistance

Transposon

“Jumping genes”

• segments of DNA that can move from one region of DNA to another

• mediated through an enzyme called transposase

• Targets a sequence of DNA and cuts it, allowing for the incorporation of DNA into a different region

• It reseals the DNA after

Plasmids, Transposons, and antibiotic resistance

Plasmids and Transposons act as the primary delivery system and "cut-and-paste" machinery for antibiotic resistance genes. Together, they allow bacteria to rapidly acquire, accumulate, and share resistance to drugs.

• Transposons physically "cut and paste" antibiotic resistance genes from the main bacterial chromosome and insert them directly into plasmids. Plasmids then transport these genes between different bacteria

Recombination

Exchange of DNA and incorporation of it into the host chromosome/genome

Bacterial DNA structure

• Polymer of nucleotides: adenine, thymine, cytosine, guanine

• Double helix associated with proteins

• "Backbone" is deoxyribose-phosphate

• (sugar-phosphate)

• Strands held together by hydrogen bonds between AT and CG

• Strands are antiparallel

• 5’ and 3’ at opposite ends

Transformation

Cell incorporates a piece of external DNA into the cell.

• movement of “naked” DNA between cells

Requires receptors for DNA incorporation (some bacteria have it)

• called a “competent” bacteria if it has these receptors

• natural setting

Holes in the cell membrane can induce cells to allow DNA in

• laboratory setting through heat, electric shock, chemicals, etc

Conjugation

Requires direct contact between cells

• the cells have to be of opposing mating type (one carries the plasmid and the other doesn’t)

Requires pilis - allows for transfer of material

• coded for by genes on plasmid (small pieces of circular DNA w/ nonessential genes on them)

• plasmid called “F factor” because genes lie on it, meaning it can engage in conjugation

So, cells carrying plasmid are F+ (F positive) and cells w/out plasmid are F- (F minus)

F+ cell

A cell carrying a plasmid for conjugation

F- cell

A cell without a plasmid

• the plasmid is transferred to the recipient F- cell during conjugation, turning it into an F+ cell.

High frequency recombinant (Hfr) cell

Occurs when the F factor (plasmid) becomes integrated into the chromosome

Transduction

Utilizes a virus as the means to transfer DNA between cells

• no cell to cell contact required

• the infection process in a cell is required

Bacteriophage - a virus that targets only bacteria

1. Delivers its own genetic material into the cell

2. Codes for proteins that break the host chromosome down

3. Occasionally during phage assembly, pieces of bacterial DNA are packaged in a phase capsid.

• the donor cell lyses and releases phage particles containing bacterial DNA

4. A phage carrying bacterial DNA infects a new host cell, the recipient cell.

5. Recombination can occur

• produces a recombinant cell w/ genotype different from both the donor and recipient cells.

Can occur naturally or intentionally in a lab.

Ames Test

Identifies chemical carcinogens

Takes mutated salmonella that can’t product amino acid histidine

• mix it w/ mutagen that made it so it could not produce histidine

• put it on a medium w/out amino acid histidine

• growth/colonies suggests the chemical the bacteria was exposed to had mutagenic properties strong enough to undo the mutation and revert it back to its natural state (where it can produce its own histidine)

Used to test how carcinogenic/mutagenic certain chemicals are

Genome

All of the genetic material in a cell

Genotype

Genes of an organism

Allele

Variant of a gene (e.g. eye color)

Phenotype

Expression of the genes

Gene

Segment of DNA that encodes a functional product, usually a protein

Vertical gene transfer

Gene transfer within a cell or between generations of cells

• replication for mitosis

• transcription and translation

Horizontal gene transfer

Gene transfer between cells of the same generation

• recombination

Restriction enzymes

Used to insert foreign genes into a plasmid artificially

• cut specific sequences of DNA

• produces overhanging single stranded DNA ends called sticky ends

Sticky ends:

• complementary

• can pair back up and hydrogen bond

• can take any piece of DNA cut w/ the same enzyme and put them together because they’ll have the same overhang. (ligase will seal them back together)

Destroy bacteriophage DNA in bacterial cells

Cannot digest (host) DNA w/ methylated cytosines

Basically:

• restriction enzyme cuts double-stranded DNA at its particular recognition sites

• produces a DNA fragment w/ two sticky ends

• meets w/ DNA from another source cut by the same restriction enzyme (DNA might be a plasmid)

• they join by base pairing

• form either linear or circular molecules

• DNA ligase unites the backbones, producing a recombinant DNA molecule

Bacterial gene expression

Constitutive enzymes are expressed at a fixed rate

Other enzymes are expressed only as needed

• repressible enzymes - silenced at certain points

• inducible enzymes - turned on when needed to build a protein

Operon

How enzymes are turned on and off.

A group of genes that are transcribed together and controlled by one promoter

• inducible operon, transcription must be turned on

• repressible operon, transcription is usually on and must be turned off.

Consists of the promotor and operator as parts of the control region and structural genes that code for the protein.

• regulated by the product of the regulatory gene.

Promotor

A specific DNA sequence located just upstream of a gene.

• acts as a crucial landing pad for RNA polymerase, directing the enzyme to the exact start site to initiate transcription (the process of copying DNA into RNA

Basically, where the assembly of transcription enzymes happens

• is part of the control region

Operator

The operator acts as the on/off switch of an operon.

• is a specific segment of DNA where regulatory proteins (like repressors) bind.

• By interacting with these proteins, the operator controls whether RNA polymerase can transcribe the operon's genes

Binding site for the regulatory protein/gene