PROKARYOTIC AND EUKARYOTIC GENOMES

Sara Kevorkian

What is a genome?

The haploid set of an organisms hereditary material. Haploid = one set of chromosomes Humans are diploid, we have 46 chromosomes in two sets of 23 Includes both coding and non-coding sequences

Prokaryotes and Eukaryotes

Prokaryotes and Eukaryotes organize their DNA differently Prokaryotes = without a nucleus Eukaryotes = with a nucleus Archaea and Bacteria are prokaryotes Eukaryotes include some unicellular organisms and all multicellular organisms

Differences between Prokaryotic and Eukaryotic Genomes

Prokaryotes

Eukaryotes

DNA all over cell One circular chromosome Very small and compact Related genes clustered together Little non-coding DNA No introns Generally haploid

DNA inside nucleus Multiple linear chromosomes Genes widely spread out Each gene regulated separately Lots of non-coding DNA Lots of introns Generally diploid

Lac Operon (Bacterial Gene Structure)

An operon is a cluster of related genes all transcribed at once LacZ, LacY, LacA all required for lactose metabolism The promoter is where RNA polymerase binds The operator is the regulatory site for the Lac operon The terminator is where transcription ends

Lac mRNA

When the Lac operon is transcribed, all three genes are on the same mRNA Each gene is translated separately The Shine-Dalgarno sequence exists in prokaryotes for the ribosome to find the start codon

Bacterial Sex!!!

Plasmids are small circular pieces of DNA found in prokaryotes They can be transferred between organisms They can contain adaptive genes, such as antibacterial resistance Scientists use them for cloning and making transgenic organisms

Plasmid Compared to Chromosome

Eukaryotic Genomes

Structure of a Eukaryotic Gene

Lets start with Exons and Introns

RNA polymerase binds at the promotor A pre-mRNA is made Introns are spliced out of the pre-mRNA Only exons are left in the mRNA Splice sites mark the boundaries between exons and introns Transcription will continue until RNA polymerase hits the polyadenylation site

mRNA
Exons contain both coding and non-coding regions The coding regions are recognized by the ribosome and translated into proteins The non-coding regions are located at the 5 and 3 ends The 5 UTR contains the ribosome binding site The 3 UTR is important for mRNA stability, localization The polyA tail is important for nuclear export

Does this make sense?

Open Reading Frames

Since codons are read in triplets, there are three possible reading frames on a strand of DNA or RNA 5-ATG|TCG|ATC|CAT|GGC|TGC|TAA-3
Met Ser
Cys

Ile

His
Ser Pro

Gly
Met Trp Ala

Cys
Ala

Stop

5-A|TGT|CGA|TCC|ATG|GCT|GCT|AA-3
Arg Asp

5-AT|GTC|GAT|CCA|TGG|CTG|CTA|A-3
Val Leu Leu

Open Reading Frames

When you add in the fact that DNA is double stranded, for any region a gene is located, there are 6 possible reading frames

5-ATG|TCG|ATC|CAT|GGC|TGC|TAA-3
Met Ser Ile His Gly Cys Stop 3-TAG|AGC|TAG|GTA|CCG|ACG|ATT-5 Stop Ser Stop Val Pro Thr Ile

Now we will move on to regulatory regions

Regulatory regions are made of binding sites for activators or repressors(transcription factors) The right combination of transcription factors will turn a gene on or off

Regulatory Elements

Regulatory elements are regions of DNA where protein binds DNA to turn on or off genes Activators promote RNA polymerase binding the promoter Repressors block RNA polymerase binding the promoter They work similarly to prokaryotes, as in the lac operon example, but are often more complex In some cases multiple proteins may need to bind before a gene is activated or repressed

Regulatory Elements

Regulatory elements in Eukaryotes can be anywhere in the genome When they are close to the promoter they are called proximal control elements Farther from the gene of interest, they are called enhancers. Cis-Regulatory Elements (CRE) contain multiple binding sites for regulatory proteins Regulatory elements can occur within introns

Pax 6 Expression

Pax 6 is important for the development of many structures There are different independently acting CREs important for its expression in different tissues

Pax 6 Expression

By adding the Retina CRE in front of a reporter gene, expression of Pax 6 by that CRE can be detected Lac Z was used as a reporter gene

Any questions?

Mutations!!

At the DNA Level

Point mutations
Substitution
5-ATG

of one base pair for another.

CGT TTA-3 becomes 5-ATG CGA TTA-3

Frameshift mutations
Insertion
5-ATG

or deletion of one or more base pairs

CGT TTA-3 becomes 5-ATG ACG TTT A-3

At the Protein Level

Point mutations affect individual codons

A synonymous mutation does not change the amino acid

Ex) CAA(Gln) to CAG(Gln) still encodes Glutamine

A missense mutation changes an amino acid into a different amino acid

A conservative missesnse mutation is chemically similar Ex) GGU(Gly) to GUU(Val) still encodes hydrophobic amino acids A nonconservative missesnse mutation is chemically dissimilar Ex) UCA (Ser) to CCA (Pro) changes from hydrophobic to hydrophilic

A nonsense mutation changes the amino acid to a stop codon

Ex) UAU(Tyr) to UAA(stop) prematurely ends the amino acid chain

At the Protein Level

Insertions or Deletions alter the reading frame

5-ATG|TCG|ATC|CAT|GGC|TGC|TAA-3

Met

Ser

Ile

His

Gly

Cys

Stop

5-ATG|ATC|GAT|CCA|TGG|CTG|CTA|A-3

Met

Ile

Asp Pro

Trp

Leu

Leu

A deletion will work similarly Insertions or deletions of base pairs in multiples of three will retain the reading frame

Needle in a Hay Stack

This is where you come in!!