Exam 2 Flashcards
ENCODE Project
Encyclopedia of DNA Elements.
Identified that 80% of the human genome sequence has biochemical activity, even the noncoding sequences.
Tandem Repeated Genes
Encode rRNA and snRNA.
0.4% of genome.
Monocistronic
Simple Sequence DNA and Where it’s Located on the Chromosome
Actual sequence is very similar among all humans, but different ethnicities will have different numbers of repeats.
Found at centromere and telomere.
This DNA is located on the chromosome by FISH.
Backward Slippage
Backward slippage of daughter strand may result in different lengths of SSDNA.
If the mother strand slips, it can cause a deletion (Cystic Fibrosis).
If the daughter strand slips it can cause an addition (Huntington’s Disease).
If a pro-proliferative gene is amplified it can cause oncogenesis.
Telomerase
Binds to 3’ end of lagging strand template of DNA.
Without telomerase, the lagging strand will be shorter by the length of the primer after each division.
The telomere would continue to get shorter and shorter until the cell dies.
Transposons and Retrotransposons
T: cut and paste.
RT: copy and paste.
Coding regions code for transposase/reverse transcriptase.
In RT, tRNA acts as primer.
LINEs and SINEs
Both Non-LTR RT’s.
L: code for proteins (ORF).
S: do not code for proteins. (Alu sequence for DNA repair).
Euchromatin vs. Heterochromatin
E: thinner/loosely packed, active in transcription, formed through acetylation of histones.
H: thicker/tighter packed, not active in transcription, formed through methylation of histones.
Chromosome Scaffold
Radially attach loops of HC to a central scaffold that is positioned along the chromosome axis.
Non histone.
Condensin
Non Histone.
Anchor portion is able to bind to DNA w/o ATP via electrostatic interaction.
ATP hydrolysis facilitates condensin to make HC loops.
Gene Knock Out
Use PCR to make a destruction construct that contains AB resistance.
Homologous Recombination to insert gene.
In humans: embryonic stem cells.
Loss of Function
Transgenic Technology
Artificially introduced DNA.
Gain of Function.
SiRNA
AKA RNAi.
dsRNA interferes with expression of a particular gene which shares a homologous sequence with dsRNA.
Doesn’t totally abolish expression of the gene (knockdown).
CRISPR-Cas9 System
Guide RNA that is complementary to a piece of target DNA.
Guide RNA carries Cas9 enzyme to target DNA where Cas9 will cut it.
RNAP I, II, and III
I: Pre-rRNA
II: workhorse; mRNA, sn, si, miRNAs.
III: tRNA
TATA Box
Most common promoter.
Located 25-35 bp upstream of start site.
Specifies to other molecules where TC begins.
Intro of a point mutation by site-directed mutagenesis reveals its importance.
Promoter-Proximal Elements and Examples
Located further upstream from the starting site than promoter.
Bind TC activators or inhibitors.
Two common examples: CCAAT box, GC rich box.
Enhancer
Control element located a few kb away (US or DS) from TC start site.
Can be located in intron seq’s.
Can be bound by TC factors to enhance TC of an assoc gene.
Enhanceosome
Cooperative binding of multiple TC activators in an enhancer.
Enhancer is not part of enhanceosome.
HMG1
High mobility group 1.
Capable of inducing sharp bend in DNA.
Rapidly migrated in SDS gel.
C-Terminal Domain (CTD) of RNAP II
Consists of tandem heptapeptide repeats.
Serine-rich; when serine is phosphorylated it activates the catalytic core of RNAP II.
3 methods of determining importance of DNA elements
Site-Directed Mutagenesis
Scrambling/Random Mutation
5’ Deletion Assay
DNase 1 Footprinting
Exposing DNA to DNase 1, which is an endonuclease that will randomly cut DNA.
If a TF is bound, the DNA will be protected, and you can determine the sequence of the undigested DNA.
Assembly of RNAP II TC Initiation Complex
TBP: binds to TATA box and causes bending.
TFIIB: helps recruit RNAP II.
TFIIH: has helicase activity.
3 Steps in Post TC Gene Control
1) 5’ cap; makes it resistant to 5’ exonuclease.
2) 3’ Polyadenylation of pre-mRNA by Poly A Polymerase.
3) Alternative splicing via 2 transesterification reactions, with the help of 5 snRNPs that makeup the spliceosome.
Steps of Nuclear mRNA export
1) Assembly of export competent mRNP complex marked with NXF1 and NXT1
2) mRNP targeting to and translocation through the NPC.
3) Disassembly of mRNP complex by displacing factors.
siRNA and miRNA TC Control and Examples
M: hybridizes imperfectly with target RNA; dicer in embryonic limb development
S: hybridizes perfectly with target RNA; RISC
Post TC Control via Degradation of mRNA
Decapping enzyme
Deadenylase
Endonuclease
3 Examples of Integral Proteins
Glycophorin on RBCs.
Bacteriorhodopsin in PS bacteria.
Porins in mitochondria/chloroplasts and bacteria (B sheets twisted into barrell).
Examples of Lipid Anchored Proteins
Src- acylated
Ras and Rab- prenylation
GPI
3 Types of Interactions Peripheral Proteins and PM
1) Shallow insertion of amphipathic alpha helix parallel to the membrane plane (membrane fusion).
2) Insertion of hydrophobic loop (annexin).
3) Electrostatic ionic interactions with membrane lipids (phospholipase).
FRAP
Fluorescent recovery after photobleaching.
Measures movement of proteins.
Shapes of Phospholipids
Cone: phosphatidic acid, PE
Cylinder: PC, PS, PI
Inverted Cone: lysophosphatidylcholine
Facilitated Diffusion
Passage of materials through biomembrane aided by concentration gradient and a transport protein.
Most are uniporters.
No ATP required.
Glut 1
Uniporter
Transports glucose across CM of RBCs.
Patch Clamp
Can be used to study opening and closing of ion channels by measuring electric charges.
Na+ and K+ Pumps
Na: less abundant, not always active.
K: more abundant, always active; most important in maintaining membrane potential.
Aquaporins
Discovered by expressing mRNA for water channels in frog egg. When exposed to hypotonic solution, the cells swelled.
Electrostatic intx with arginine near pore entrance. Then H bonds with asparagine inside channel.
P Pumps
Get phosphorylated.
Na+/K+ ATPase: maintains intracellular K and Na concentrations by pumping against conc gradients.
Ca2+ ATPase pump in muscle cells.
V Class
Pump protons against concentration gradient to lumens of lysosome, endosome, and vacuoles.
Also pump Cl- to maintain charge separation.
ABC Transporters
Bacterial: transport AA, ions, nutrients.
CFTR: faulty assoc with CF.
MDR: multidrug resistance, remove toxic wastes from the body; transport hydrophobic drugs, which is overexpressed in cancer cells.
Myelination in PNS vs. CNS
PNS: schwann cells.
CNS: oligodendrocytes.
Astrocytes
Star shaped cells that can sense NT levels in synapses and respond by releasing molecules that influence neuronal activity.
Release gliotransmitters (thrombospondin).
Western vs Northern vs Southern Blot
W: proteins
N: RNA sequences
S: DNA sequences