EXAM FOUR NOTES

 

 

 

Molecular Biology

Introduction

Definitions

History

Central Dogma

 

Definitions

Chromosomes

DNA

Gene

Genotype

Phenotype

 

Chromosomes

The structure in cells that carries hereditary information

Composed of DNA and protein

Prokaryotic - circular

Eukaryotic - linear

 

DNA

Nitrogenous base (4 bases - A,T,G & C

Deoxyribose sugar

Phosphate

Double helix structure

 

Genes

Segments of DNA

Functional or regulatory

Complementation

Mutability and variation

 

Genotype

The genetic make-up of an organism; the information that codes for all the characteristics of an organism

Phenotype

The expression or physical manifestation of a gene; how it appears

 

Molecular biology seeks to understand the molecular or chemical  basis of genetics

History of molecular biology is a melding of biochemistry, especially nucleic acid biochemistry and genetics

 

Biochemistry

Meischer

Avery & MacLeod

Hershey & Chase

Watson & Crick

Genetics

Mendel

Morgan

Griffith

Delbruck

Beadle & Tatum

Tatum & Lederberg

 

Genetics

Mendel (1865)

Fluid vs. particulate inheritance

Studied pure breeding pea plants

Law of Segregation

Law of Independent Assortment

Rediscovered by de Vries & others

 

Genetics

Morgan

developed modern science of genetics, especially techniques of mapping genes on chromosomes

used fruit flies because they had a shorter generation time than peas

 

Genetics

Griffith

discovered transformation in 1927

a process by which a nonpathogenic strain is transformed into a pathogenic strain

 

Genetics

Delbruck

developed quantitative methods for analysis of bacteriophage

organized course to teach biologists methods at Cold Spring Harbor

 

Genetics

Beadle & Tatum

developed Neurospora as an experimental organism

established one gene one enzyme hypothesis

complementation testing

generation time is even shorter with Neurospora

 

Genetics

Tatum & Lederburg

discovered conjugation in bacteria

 

Biochemistry

Meischer (1869)

Austrian doctor

isolated a substance called “nuclein” from the nuclei of cells obtained from the pus of surgical bandages

found to contain nitrogenous chemicals, sugar and phosphate

 

Biochemistry

Avery & MacLeod (1944)

isolated Griffith’s transforming factor to a high degree of purity

characterized transforming factor using highly purified enzymes

found transforming factor to be DNA

 

Biochemistry

Hershey & Chase (1952)

used newly developed radioisotopes

S35 for protein

P32 for nucleic acid

labeled bacteriophage (a virus of bacteria)

found P32 went into cells but S35 did not implying that nucleic acid transfer information to cell for new bacteriophages

 

Biochemistry

Watson & Crick (1953)

used X-ray crystallograph to study structure of DNA

by combining chemical data and X-ray data were able to construct a model of DNA

structure inferred function leading to Central Dogma

 

Central Dogma

DNA Structure

Genetic Code

Replication

Transcription

Translation

 

DNA Structure

Sugars

Bases

Phosphates

Double Helix

Anti-parallel

 

Genetic Code

4 bases / 20 amino acids

codons

punctuation

 

Central Dogma states a hypothesis regarding information flow in cell

Replication - the copying of DNA or information for next generation

Transcription - the copying of information for use by the cell

Translation - the conversion of information into useful products -enzymes

 

Replication

replication is semi-conservative

replication occurs at replication fork

replication is discontinuous process

uses DNA polymerase

requires a primer

 

Transcription

RNA polymerase

promoters

produces messenger RNA (mRNA)

requires NO primer

 

Translation

tRNA

amino acid synthase

ribosomes

initiation

termination

 

 

 

Microbial Genetics - DNA Transfer

Review of Information Transfer in Cell

Recombination

Transfer of DNA &Genetic Recombination in Bacteria

Conjugation

Transformation

Transduction

 

Microbial Genetics - DNA Transfer

Recombination - exchange of DNA between two DNA molecules to form new combinations

Crossing over

Occurs when DNA is transfer from one cell to another (transfer from a doner to a recipient)

 

Microbial Genetics - Regulation

Three types of regulation

Constitutive - gene is always turned on

Repression - gene is normally turned on but can be turned off in response to a metabolic signal

Induction - gene is normally turned off but can be turned on in response to a metabolic signal

Microbial Genetics - Regulation

Types of Regulation of mRNA synthesis

Induction - lac operon

Repression - trp operon

Microbial Genetics - Regulation

 

Microbial Genetics - Mutation

Mutation - Introduction

A mutation is a change in the DNA sequence that results in a change in the product protein

Mutations can be neutral, harmful (most) or beneficial

Mutation is the basis of variation within a species and thus provides an important mechanism to evolution

 

Types of Mutation

Base-pair substitution - misssense change in amino acid sequence

Base-pair substitution - nonsense change in DNA sequence - premature stop

Frameshift

Insertion

Deletion

 

Normal gene

DNA transcribed

mRNA translated

No change in gene product

 

Missense mutation

Base substitution

Changes amino acid sequence by one amino acid

May or may not affect activity of protein

 

Nonsense mutation

Mutation changes codon to stop codon

Premature termination of protein

Almost always destroys activity of protein

 

Frameshift mutation

inserts or deletes a base in the DNA

Protein may be of correct size (approximately)

Protein sequence altered significantly

 

Mutatgens

Chemicals that modify DNA

Deaminating agents

Methylating agents

Intercalating agents

Radiation

UV - UV repair system

X-Rays

 

Frequency

Rate at which change occurs usually low

Mostly random although there are “hot spots”

Low rate necessary to provide genetic diversity

Rate can change depending upon what population of cells is exposed to

 

 

 

 

Biotechnology

 

Introduction

            Tools

            Process

            Applications

            Biotechnology

 

Introduction

            Gene is identified and excised from one organism

            Gene is placed in vector (plasmid) and amplified

            Gene is transferred to new organism or used in host organism to make protein product

 

Biotechnology - Tools

 

Restriction endonucleases

            Nucleases cut nucleic acid – at first seem non specific

            Linn & Abner discover that some strains of bacteria are able to resist bacteriophage infection by digesting infecting DNA

            Different bacteria produce different restriction enzymes

 

Restriction Endonucleases

            Cut at specific 4, 6, or 8 base sites

            Site is a “palindrome”

            Racecar

            Madam I’m Adam

            Damit I’m Mad

            Some restriction enzymes generate “sticky ends”

 

Plasmids

            Carry an antibiotic resistance marker

            Carry restriction sites in convenient locations to insert DNA

            Carry characteristics that allow the plasmid to reproduce in several organisms

 

Polymerase Chain Reaction (PCR)

            Allows any segment of DNA to be amplified chemically in minutes

            Uses a thermostable DNA polymerase

            Machine can cycle every 60-90 seconds

 

Agarose Gel Electrophoresis

            Can separate DNA fragments made with restriction enzymes

            Can separate PCR made DNA

            Can be used to sequence DNA

 

Biotechnology - Process

            Basic process worked out by Cohen & Boyer

            Cut plasmid DNA and target DNA with same restriction enzyme

            Mix DNA and allow sticky ends to match up

            Select DNA clones having target gene