What are some differences between bacterial genes and eukaryotic genes?
- size: bacterial gene size is small with fewer genes compared to eukaryotic genes which are large with more genes
- packing: bacterial genes have histone proteins and are supercoiled, eukaryotic genes have histone octamers and are coiled into chromatin
- bacterial DNA is circular, eukaryotic DNA is linear
- Introns: bacterial genome has none, eukaryotic genome has them within genes
- chromosomes: bacteria - often 1, eukaryotes - multiple
- mitochondria: bacteria - none, eukaryotes - present
Plasmids
Circular “extra chromosomal” DNA in bacteria
F plasmids
Fertility plasmids –> assist with transfer
R plasmids
Resistance plasmids -> give resistance
How lateral/horizontal gene transfer happens
What are the 3 types of lateral gene transfer?
- conjugation
- transformation
- transduction
What is conjugation?
The transfer of replicated DNA from a donor cell to a recipient cell through temporary contact
The donated genetic material reforms as a plasmid or as part of the homologous region of the host
What is transformation?
uptake of DNA from the environment
What is transduction?
transfer of DNA from one bacterium to another via a viral vector (like a bacteriophage)
What is the evidence for the endosymbiotic origin of mitochrondria and chloroplasts?
- Double membrane on the organelles is derived from a similar bacterial membrane
- similar in size to extant bacteria
- Organellular DNA is packaged in a similar way to bacteria and dissimilar from the way that nuclear DNA is packaged
- Transcriptional and translational techniques are similar to bacteria
- protein-coding sequences are similar to bacteria than to eukaryotes or archaea
What is antibiotic resistance?
an inherited trait of a microbe that allows it to grow in the prescence of an antibiotic (kills or prevents bacteria growth)
How was the semi-conservative replication of DNA discovered?
Meselson-Stahl Experiment: DNA was grown in N-15 (heavy), then transferred to N-14 to grow. 1st gen (N-15 solution) were all heavy, 2nd gen (N-14) were all hybrid, 3rd gen were half-light, half-hybrid, 4th gen were 75% light, 25% hybrid, and so on.
If it had been conservative, there would be no hybrids. If it was dispersive, there would have been no light (all hybrid).
What proteins are involved in DNA replication?
7 proteins - part of the replisome:
1. DNA topoisomerase (relaxes supercoiling)
2. Helicase (unwinds double helix)
3. SSB (prevents reannealing of separated strands by covering them)
4. Primase (synthesizes RNA primers)
5. DNA pol III (synthesizes DNA)
6. DNA pol I (removes and replaces RNA primer with DNA)
7. DNA ligase (joins DNA segments/Okazaki fragments)
replisome: the complex of proteins at each replication fork
What happens at the end of a linear chromosome?
Replication is unable to replicate the very end of linear chromosomes due to a requirement of an RNA primer. Chromosomes become slightly shorter, so telomeres (repetitive noncoding sequences) are at the end to prevent consequences.
Hayflick limit: # of times that a normal differentiated somatic cell can undergo cell division before it undergoes apoptosis. Usually ~50-70 cell divisions.
What is a ribonucleoprotein and how does it provide evidence for an “RNA world”?
Ribonucleoprotein - an enzymatic complex that includes protein and ribonucleic acid
How are the leading and lagging strands elongated?
Leading strand is elongated continuously while the lagging strand is elongated discontinuously in Okazaki fragments.
Elongated 5’ to 3’
Its like tangent (trig), CAST – when tan is positive in the replication bubble, the strand is leading!
What is Sanger Sequencing?
A method that uses DNA primers and DNA replication reactions to create DNA fragments
What is the role of Taq polymerase in PCR?
Replaces DNA polymerase becaue it is heat-stable
How do we design forward and reverse DNA primers in a PCR reaction?
DNA polymerase can only extend from 3’ OH, so each primer’s 3’ end must point into the region you want to copy, and the primers must match the sequences
What are the main “ingredients” in Sanger sequencing reactions?
- DNA strand to be sequenced
- Single stranded DNA primer
- DNA polymerase,
- Large amounts of each of the four deoxynucleotides
- Small amount of one dideoxy- nucleotide (A, C, T or G)
Why are four different reactions
required?
Because the reaction needs to completed for A, C, T and G terminating nucleotides separately
What is PCR amplification?
In vitro DNA replication, requires a DNA template, a supply of the 4 DNA nucleotides (dATP, dGTP, dCPT, dTTP), Taq polymerase, 2 different DNA primers, and a buffer solution
You need to know the sequences you want to amplify in order to design primers, and PCR only works for small fragments
What is the role of Taq polymerase in PCR?
It acts as DNA polymerase, but it can withstand the intense heat used to denature the DNA.
How do we design forward and reverse DNA primers in a PCR reaction?
Usually >18 bp, and need to match the sequences in the proper orentation. DNA polymerase can only extend from 3’ OH, so each primer’s 3’ end must point to the region you want to copy.
