GN 311 Module 4

Mutations

Change in the genetic material or DNA

Alkaptonuria

Build up of homogentisic acid in cartilage, etc

Phenylketonuria (PKU)

Accumulation of phenylpyruvic acid in the brain

Albinism

Inability to produce melanin pigment

Beadle and Tatum 1941

One gene makes one enzyme

Auxotroph

Requires some chemical growth not minimal media

Prototroph

Will grow on minimal media

Cystic Fibrosis

Mutation in a structural protein

Sickle Cell Anemia

Defective hemoglobin molecule that has one amino acid substitution

Somatic Mutations

Occurs in nonreproductive cells

Germline Mutations

Occurs in cells that give rise to gametes

Protein Loss of Function

Causes complete or partial absence of protein function; recessive

Protein Gain of Function

Causes the cell to produce a protein that is not normally present; dominant

Transition Mutation

Purine to purine vice versa

Transversion Mutation

Purine to pyrimidine vice versa

Missense

Changes the amino acid

Nonsense

Changes codon to a stop codon

Silent

Codes for the same amino acid

Readthrough

Stop codon is changed to a codon that codes for amino acid resulting in a longer protein

Purines

A/G

Pyrimidines

C/T/U

Depurination

Removes glycosidic bond at A or G

Deamination of Cytosine

Results in uracil

Wobble Base Pairing

Mispairing due to flexibility in helix

Base Analogs

5-bromouracil pairs with A/G; 2-aminopurine pairs with T/C

Mutagens that Alter DNA Bases

Alkylating agents, deamination, environmental agents

Intercalating Agents that Cause Frameshift Mutations

Proflavin, ethidium bromide, acridine orange

Fragile X-Syndrome

Repeated CGG with 50-1500 range

Huntington Disease

Repeated CAG with 37-121 range

Xeroderma Pigmentosum

A human disorder where a repair mechanism is defective; results in tumors; repair of thymine dimers

Transposable Genetic Elements

Can move from one site to another, can move to a different chromosome, can alter phenotypes

Ac (Activator)

Complete transposable element with functional transposase and repeats

Ds (Dissociation)

Lacking functional transposase gene so it requires the AC transposase to move

Arabidopsis

Transposase derived proteins regulate plant genes required for plant growth

Bacteria Direct Repair

Photoreactivation repair of pyrimidine dimers by enzyme photolyase

Methyltransferase Direct Repair

Restores correct form to incorrectly methylated guanine bases

What stalls replication in proofreading?

DNA polymerase

Proofreading Exonuclease

Removes incorrect nucleotide

Mismatch Repair Proteins

Recognize abnormal helical structure and identify the incorrect base

DNA Polymerase

Fills in gaps

DNA Ligase

Seals nick

Nucleotide Excision Repair

Removes bulky lesions (damaged DNA)

Base Excision Repair

Removes modified bases

Glycosylases

Recognize and remove defective bases resulting in AP site

AP Endonuclease

Cleaves the phosphodiester bond next to the missing base

Translesion DNA Polyermases

Specialized that can bypass lesions on the DNA during replication

Structural Genes

Encode proteins that are used in metabolism or play a structural role in the cell

Regulatory Genes

Encode proteins that interact with other sequences and affect the transcription a/o translation of these sequences

Regulatory Elements

DNA sequences that are not transcribed, but play a role in regulating other sequences

DNA Binding Domains

Helix turn helix, zinc fingers, leucine zipper

Rapid ON or OFF

Provides the ability to respond rapidly to sudden changes

Sequential Gene Expression

Cascades of gene expression that turn on in order

Constitutive Expression/Housekeeping Genes

Continuously expressed under normal conditions

Positive Control

Regulatory protein (activator) bind to DNA to stimulate transcription

Negative Control

Regulatory protein (repressors) binds to DNA to prevent transcription

Inducible Control

Transcription is normally off and is turned on when a small molecule binds the regulatory protein

Repressible Control

Transcription is normally on and is turned off when a small molecule binds the regulatory protein

Operon

Defined as segment of DNA containing controlling regions and structural genes controlled by those controlling regions

Regulatory Genes - lacI

Codes for repressor

Promoter - lacP

Binds RNA polymerase to allow transcription

Operator - lacO

Interacts with repressor

Structural Gene - lacZ

Beta galactosidase S

Structural Gene - lacY

Permease

Structural Gene - lacA

Transacetylase

Allosteric Change

Operator binding site is altered when repressor binds allolactose

Cis Acting

Action of an element affects only the genes adjacent to it

Trans Acting

Diffusible product is produced, mutant gene does not have to be adjacent to the other genes to affect them

I+

Normal repressor

I-

Repressor cannot bind operator due to repressor’s bad binding site

Is

Repressor cannot bind allolactose

Oc

Constitutive operator, operator cannot bind to repressor

P-

Promoter cannot bind to RNA polymerase

Catabolite Reperssion

When glucose is preferred so there is no need for the lac operon to function when glucose is present

Lac Operon Positive Inducible

Binding of cAMP-CAP complex enhances binding of RNA polymerase to functional levels

Allosteric Action

A change occurs in the conformation of the repressor when it binds to Trp

R-

Inactive repressor - operon always on

Feedback Inhibition

Excess Trp in cell decreases the production of the enzymes used to make Trp

Attenuator

Located in the leader sequence and responsible for decreasing transcription when Trp is present

Anti-Sense RNA

Small RNA molecules complementary to parts of the mRNAR

Riboswitches

RNA sequences in the mRNA that affect the translation of that mRNA; also regulate level of translation

Changes in Chromatin

Histone modification, DNA methylation, remodeling

DNaseI Hypersensitive Sites

Small regions that are upstream from start of transcription

Methylation of Histone Tails

Can either increase or decrease transcription depending on which amino acids are methylated

Acetylation of Histone Tails

Weakens interactions with DNA and may allow transcription factors to bind DNA

Histone Deacetylation (Lys)

Tightens association between histones and DNA

Histone Acetylation (Lys)

Loosens DNA by neutralizing the positive charge on the histones

Histone Code

The combination of modifications present that help regulate chromatin structure and transcription

Histone Methylation

Can activate or repress expression of a gene

DNA Methylation

DNA methyl transferases tends to cause genes to be turned off

Epigenetics

Heritable changes in gene expression that occur without changing the DNA sequence

Epigenetic Effect

Phenotype is caused not by a change in genotype but an epigenetic change

Initiation of Transcription

TF, TF binding sites, insulators

Core (basal) Promoter

Required for transcription of nearby all RNA poll II genes

Transcription Factors

Bind to DNA at transcription factor binding sites; can be activators or repressors

Insulators

Cis DNA elements that block transcription factors from interacting with the wrong one

Northern Blot

Shows relative amounts of RNA present

RNA Processing and Stability

Splicing, degradation, interference

Master Regulator

SxI (sex lethal)

RNA Interference (RNAi)

Eukaryotes, shuts off gene expression using double stranded RNAs

siRNA

Comes from mRNA, transposons or viral RNA and targets the genes that it comes from