Griffith The Experiment
What is a gene?
The work of Sutton and Morgan established that genes reside on chromosomes But chromosomes contain proteins and DNA So which one is the hereditary material
Several experiments ultimately revealed the nature of the genetic material
The Griffith Experiment
In 1928, Frederick Griffith discovered transformation while working on Streptococcus pneumoniae The bacterium exists in two strains S
Forms smooth colonies in a culture dish Cells produce a polysaccharide coat and can cause disease
R
Forms rough colonies in a culture dish Cells do not produce a polysaccharide coat and are therefore harmless
How Griffith discovered transformation
Thus, the dead S bacteria somehow "transformed" the live R bacteria into live S bacteria
The Avery and Hershey-Chase Experiments
Two key experiments that demonstrated conclusively that DNA, and not protein, is the hereditary material Oswald Avery and his coworkers Colin MacLeod and Maclyn McCarty published their results in 1944 Alfred Hershey and Martha Chase published their results in 1952
The Avery Experiments
Avery and his colleagues prepared the same mixture of dead S and live R bacteria as Griffith did They then subjected it to various experiments
All of the experiments revealed that the properties of the transforming principle resembled those of DNA
1. 2. 3. 4. Same chemistry and physical properties as DNA Not affected by lipid and protein extraction Not destroyed by protein- or RNA-digesting enzymes Destroyed by DNA-digesting enzymes
The Hershey-Chase Experiment
Viruses that infect bacteria have a simple structure DNA core surrounded by a protein coat Hershey and Chase used two different radioactive isotopes to label the protein and DNA Incubation of the labeled viruses with host bacteria revealed that only the DNA entered the cell Therefore, DNA is the genetic material
The HersheyChase Experiment
Thus, viral DNA directs the production of new viruses
Discovering the Structure of DNA
DNA is made up of nucleotides Each nucleotide has a central sugar, a phosphate group and an organic base
The bases are of two main types Purines Large bases Adenine (A) and Guanine (G) Pyrimidines Small bases Cytosine (C) and Thymine (T)
The four nucleotide subunits that make up DNA
Nitrogenous base
5-C sugar
Erwin Chargaff made key DNA observations that became known as Chargaff's rule
Purines = Pyrimidines A = T and C = G
Rosalind Franklin's X-ray diffraction experiments revealed that DNA had the shape of a coiled spring or helix
Rosalind Franklin (1920-1958)
In 1953, James Watson and Francis Crick deduced that DNA was a double helix They came to their conclusion using Tinkertoy models and the research of Chargaff and Franklin
James Watson (1928)
Francis Crick (1916-2004)
The DNA double helix
Dimensions suggested by X-ray diffraction
The two possible basepairs
How the DNA Molecule Replicates
The two DNA strands are held together by weak hydrogen bonds between complementary base pairs A and T C and G
If the sequence on one strand is The other's sequence must be
ATACGCAT TATGCGTA
Each chain is a complementary mirror image of the other
So either can be used as template to reconstruct the other
There are 3 possible methods for DNA replication
Daughter DNAs contain one old and one new strand
Original DNA molecule is preserved
Old and new DNA are dispersed in daughter molecules
These three mechanisms were tested in 1958 by Matthew Meselson and Franklin Stahl
Thus, DNA replication is semi-conservative
How DNA Copies Itself
The process of DNA replication can be summarized as such The enzyme helicase first unwinds the double helix The enzyme primase puts down a short piece of RNA termed the primer
DNA polymerase reads along each naked single strand adding the complementary nucleotide
How nucleotides are added in DNA replication
Template strand
HO 3'
New strand
5'
Template strand
HO 3'
New strand
5'
C
Sugarphosphate backbone
O P
G
O
P O P
C
G
O
P
T
O P
A
O
P O P
T
A
O
P
A
O
P
T
O
P
A
DNA polymerase
O P
T
O
P
C
O
G
O
P O P OH O
C
G
O
P
P
A
O P
3'
A
P P
P P
T
O
T
A
O O
P Pyrophosphate
OH
A
OH P
3'
P 5'
O 5'
DNA polymerase can only build a strand of DNA in one direction
The leading strand is made continuously from one primer The lagging strand is assembled in segments created from many primers
RNA primers are removed and replaced with DNA Ligase joins the ends of newly-synthesized DNA
Mechanisms exist for DNA proofreading and repair
Transcription
The path of genetic information is often called the central dogma
DNA
RNA
Protein
A cell uses three kinds of RNA to make proteins Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA)
Transcription
Gene expression is the use of information in DNA to direct the production of proteins It occurs in two stages
Transcription
The transcriber is RNA polymerase
It binds to one DNA strand at a site called the promoter It then moves along the DNA pairing complementary nucleotides It disengages at a stop signal
Translation
Translation converts the order of the nucleotides of a gene into the order of amino acids in a protein The rules that govern translation are called the genetic code
mRNAs are the "blueprint" copies of nuclear genes mRNAs are "read" by a ribosome in threenucleotide units, termed codons Each three-nucleotide sequence codes for an amino acid or stop signal
The genetic code is (almost) universal Only a few exceptions have been found
Ribosomes
The protein-making factories of cells They use mRNA to direct the assembly of a protein
A ribosome is made up of two subunits Each of which is composed of proteins and rRNA
Sites play key roles in translation
Transfer RNA
tRNAs bring amino acids to the ribosome They have two business ends Anticodon which is complementary to the codon on mRNA end 3'OH to which the amino acid attaches
Hydrogen bonding causes hairpin loops
3-D shape
Making the Protein
mRNA binds to the small ribosomal subunit The large subunit joins the complex, forming the complete ribosome mRNA threads through the ribosome producing the polypeptide
How translation works
The process continues until a stop codon enters the A site The ribosome complex falls apart and the protein is released
Architecture of the Gene
In eukaryotes, genes are fragmented They are composed of Exons Sequences that code for amino acids Introns Sequences that don't
Eukaryotic cells transcribe the entire gene, producing a primary RNA transcript This transcript is then heavily processed to produce the mature mRNA transcript This leaves the nucleus for the cytoplasm
Processing eukaryotic mRNA
Protect from degradation and facilitate translation
Different combinations of exons can generate different polypeptides via alternative splicing
7. Phosphorylation or other chemical modifications can alter the activity of a protein after it is translated.
6. The polypeptide chain grows until the protetin is completed.
Amino acid
Completed polypeptide
tRNA
5' Ribosome moves toward 3' end
How protein synthesis works in eukaryotes
Cytoplasm
5. tRNAs bring their amino acids in at the A site of the ribosome. Peptide bonds form between amino acids at the P site, and tRNAs exit the ribosome from the E site.
Ribosome
DNA Nuclear membrane 3' 5' 5' 3' Primary RNA transcript 5' Cap mRNA Poly-A tail mRNA 2. Introns are excised from the RNA transcript, and the remaining exons are spliced together, producing mRNA
4. tRNA molecules become attached to specific amino acids with the help of activating enzymes. Amino acids are brought to the ribosome in the order dictated by the mRNA.
3' RNA polymerase 1. In the cell nucleus, RNA polymerase transcribes RNA from DNA
Cap
Small ribosomal subunit
Nuclear pore Large ribosomal subunit
5'
3' Poly-A tail
Introns 3'
Exons
3. mRNA is transported out of the nucleus. In the cytoplasm, ribosomal subunits bind to the mRNA
Architecture of the Gene
Most eukaryotic genes exist in multiple copies Clusters of almost identical sequences called multigene families As few as three and as many as several hundred genes
Transposable sequences or transposons are DNA sequences that can move about in the genome They are repeated thousands of times, scattered randomly about the chromosomes
Turning Genes Off and On
Genes are typically controlled at the level of transcription In prokaryotes, proteins either block or allow the RNA polymerase access to the promoter Repressors block the promoter Activators make the promoter more accessible Most genes are turned off except when needed
The lac Operon
An operon is a segment of DNA that contains a cluster of genes that are transcribed as a unit The lac operon contains Three structural genes
Encode enzymes involved in lactose metabolism
Two adjacent DNA elements Promoter
Site where RNA polymerase binds
Operator
Site where the lac repressor binds
The lac Operon
In the absence of lactose, the lac repressor binds to the operator RNA polymerase cannot access the promoter Therefore, the lac operon is shut down
The lac Operon
In the presence of lactose, a metabolite of lactose called allolactose binds to the repressor This induces a change in the shape of the repressor which makes it fall off the operator RNA polymerase can now bind to the promoter
Transcription of the lac operon is ON
The lac Operon
What if the cell encounters lactose, and it already has glucose? The bacterial cell actually prefers glucose! The lac operon is also regulated by an activator The activator is a protein called CAP It binds to the CAP-binding site and gives the RNA polymerase more access to the promoter However, a "low glucose" signal molecule has to bind to CAP before CAP can bind to the DNA
Activators and repressors of the lac operon
Enhancers
DNA sequences that make the promoters of genes more accessible to many regulatory proteins at the same time
Usually located far away from the gene they regulate Common in eukaryotes; rare in prokaryotes
Mutation
The genetic material can be altered in two ways Recombination Change in the positioning of the genetic material Mutation Change in the content of the genetic material
Bithorax mutant
Mutation
Mutation and recombination provide the raw material for evolution
Evolution can be viewed as the selection of particular combinations of alleles from a pool of alternatives The rate of evolution is ultimately limited by the rate at which these alternatives are generated Mutations in germ-line tissues can be inherited Mutations in somatic tissues are not inherited They can be passed from one cell to all its descendants
Kinds of Mutation
Mutations are caused in one of two ways
Errors in DNA replication Mispairing of bases by DNA polymerase
Mutagens Agents that damage DNA
Kinds of Mutation
The sequence of DNA can be altered in one of two main ways Point mutations Alteration of one or a few bases Base substitutions, insertion or deletion Frame-shift mutations Insertions or deletions that throw off the reading frame
Kinds of Mutation
The position of genes can be altered in one of two main ways Transposition Movement of genes from one part of the genome to another Occurs in both eukaryotes and prokaryotes Chromosomal rearrangements Changes in position and/or number of large segments of chromosomes in eukaryotes
Mutation, Smoking and Lung Cancer
Agents that cause cancer are called carcinogens These are typically mutagens
The hypothesis that chemicals cause cancer was first advanced in the 18th century Many investigations since then have determined that chemicals can cause cancer in both animals and humans For example, tars and other chemicals in cigarette smoke can cause cancer of the lung