Exam 1 Flashcards
(44 cards)
Characteristics of hereditary material
- Localized to the nucleus and a component of chromosomes.
- Present in stable form in cells.
- Sufficiently complex to contain information needed for structure, function,
development, and reproduction of an organism. - Able to accurately replicate itself so that daughter cells contain the same
information as parent cells. - Mutable; undergoing a low rate of mutations that introduces genetic variation
and serves as a foundation for evolutionary change.
Avery, McLeod, and McCarty Experiment
Original observation: Heat killed S type (smooth, nonvirulent) was injected into mice along with live R type (rough, nonvirulent). The R type was transformed into S type and killed the mouse.
In order to test what the transforming factor (and therefore likely hereditary material), Avery et al set up assays that eliminated (through enzymes) for RNA, proteins/polysaccharides, DNA, and lipids. Only the assay that removed DNA from the heat killed S type prevented the transformation of R type, therefore indicating that DNA is the hereditary material
Hershey-Chase Experiment
Bacteriophages were grown in mediums that radioactively labelled the DNA and proteins and were then set to infect bacteria. In the final bacterial culture, only the radioactive DNA was found whereas the radioactive proteins remained in the phage ghosts
The 4 nucleotides
A, G (purine/2 rings) and T,C (pyrimidine/ 1 ring)
Phosphodiester bonds
The 3’ OH group will react with the 5’ phosphate group
How did the Meselson-Stahl experiment prove the semiconservative replication model? How would the results have change for either conservative or dispersive?
Heavy parent strand was then 50/50 light heavy, which then separated into two distinct bands
Huberman and Rigg’s Experiment
Pulse-chase experiment in which pulse cells would be darkly visible in regions of active replication. In their results, there were dark/pulses regions on either side of each of the origins of replication, indicating that replication was happening bidirectionally
Replication Initiation in Bacteria
DnaA binds to 9-mer seq. which forces the AT rich 12-mer seq. to unwind. DnaC will help recruit DnaB(helicase) which opens the bubble for replication
Characteristics of DNA polymerase
Only adds nucleotides to the 3’ end
Can not initiate replication de novo (needs RNA primer)
Contains proofreading 3’->5’ exonuclease domain
Replication in bacteria
1) DnaB(helicase) and topoisomerase unwind the DNA
2) Protect the ssDNA
- single-stranded binding proteins
3)DnaG(RNA primase/polymerase) begins synthesis of a new strand utilizing RNA nucleotides
4) DNA polymerase III extends the daughter strands from the RNA primers
5) RNA primers must be removed
-DNA polymerase I or RNase H
6) Ligation of Okazaki fragments
Replisome
topoisomerase, Helicase, SSB, primase, DNA pol III, DNA pol I, DNA ligase
End problem of linear chromosomes
Circular chromosomes can just be nicked and ligated together at the end of replication.
In linear chromosomes, the lagging strand will have a primer on the 5’ end of the new strand that leaves and 3’ overhang after the primer is removed
Telomerase mechanism
1) telomerase uses its associated RNA template to synthesize a complement that extends the 3’ overhang. It shifts and repeats this before primase and DNA pol synthesize the complementary strand and fill in the gap
Why is telomerase activity so tightly regulated
Telomerase being dysfunctional would result in the eventual shortening of coding portions of chromosomes. Additionally, reactivation of telomerase can lead to ageing cells continually replicating (cancer)
PCR
- Denature the double-stranded template DNA into two
single-stranded template strands at 95-100°C
* This is why we need thermal stable DNA
polymerases. - Anneal the primers to the denatured template strands
at 45-68°C (this can vary depending on your primer
sequence). - Extension (72°C): The Taq DNA polymerase extends
from the 3’-OH of the primers generating the fragment
of DNA of interest. - Repeat 30-35 times usually
VNTRs
Variable Number Tandem Repeats
-inherited, detected by PCR
-sequencing flanking the repeats are identical in different people
Sanger DNA sequencing
DNA pol is ised to replicate new DNA from a ss template. Replication is terminated by a ddNTP which lacks a 3’ OH group which prevents the addition of more phosphodiester bonds
Bacterial Promoters
pribnow box, -35 and -10 consensus sequences (the sigma subunits in holoenzymes are specific to these sequences)
Why are eukaryotic promoters more diverse and complex?
There are multiple transcription factors in eukaryotes, which are polymerase specific and therefore bind to different promoters
Bacterial Transcription (intiation)
Due to the intrinsic helicase activity of the holoenzyme, it begins to unwind the DNA after promoter recognition to form the open promoter complex.
Bacterial Transcription (elongation)
RNA polymerase begins transcription and shortly after the sigma subunit disassociates. DNA is unwound ahead of the enzyme to maintain ~18 bp of open DNA
Bacterial Transcription (termination)
Transcription continues until the RNA pol reaches the termination sequence in which it then dissociates from the DNA. Termination can either be rho dependent or intrinsic
Eukaryotic promoters
TATA box, CAAT box, GT rich box
Eukaryotic termination and how it compares to bacterial
The DNA’s CTD has an associated RNase that will cleave ~30 bp downstream from the Poly-A signal. The RNase then goes and eats the rest of the RNA around RNA pol II< facilitating its release
