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Biochem Final
Final Exam
206
Biochemistry
Graduate
05/03/2011

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Term
Gene
Definition
DNA sequence that can be transcribed; includes genes for rRNAs, tRNAs, snRNAs, proteins, etc.
Term
Constitutive Genes

example?
Definition
transcribed at constant level regardless of changes in environment.

Glycolysis Proteins
Term
Inducable genes
Definition
Transcription changes in response to environment
Term
What is the basis of all geneic information flow in cells?
Definition
Complimentary base pairing
Term
rRNA
Synthetic Capacity, Rate of Degredation, Steady State Level
Definition
58%
Slower than synthesis
83% stable
Term
tRNA
Synthetic Capacity
Rate of Degredation
Steady State Level
Definition
Both are about the same
Term
mRNA
Synthetic Capacity
Rate of Degredation
Steady State Level
Definition
32%
Degraded faster than it is made
3%
Term
RNA pol in prokaryotes?
Definition
1 RNA pol in prokaryotes
Term
RNA pol in eukaryotes?
What are they used to make
Definition
multiple RNA pols.
RNA Pol 1: pre-rRNA
RNA Pol 2: pre-mRNA
RNA Pol 3: pre-tRNA
Term
What subunit is important in initiating transcription in prokaryotes? What does it do?
Definition
Sigma factor
Gets the polymerase to the right part to identify where to begin synthesis
Term
RNA polymerase mechanism
Where does energy come from?
Definition
Energy comes from Ribonucleoside Tripohs.
-Synthesis only 5' to 3'
-Initiates on dsDNA template to form ssRNA product
Term
Can RNA polymerase proofread?
Definition
NO
Term
What strand is the coding strand, anticoding?
Definition
Anticoding=template 3' to 5' (make RNA from this)
Coding= 5' to 3'. It should be identical to synthesized RNA except U,T
Term
Where does transcription begin?
Definition
+1 site
Term
Prokaryoteic Transcription
Definition
-Prokaryotic promotor("-10" and "-35") upstream of +1 and bind to RNA pol sigma factor.
Term
Consensus sequence
Definition
The nucleotide that occurs most often at that given position.
Term
The concensus sequence -35 and -10 of many gense. There is another gene were none of the nucleotides are equal. We are told that they are transcribed efficiently. What might be going on?
Definition
It is using a different consensus sequence and recognized by a different sigma factor.
Term
Transcription elongation in prokaryotes:
Definition
-RNA pol unwinds the DNA and a transcription bubble formed
-This opens the complex and RNA is slowly formed
-The sigma factors resists RNA pol resists RNA pol moving
-After short RNA is made, sigma dissociates and RNA pol clears from promoter and makes RNA faster.
Term
Transcription Termination (pro)
Definition
Usually occur at specific 'pause sites' that are often DNA, G:C rich.
Term
Where do eukaryotic RNA pols bind?
What do they use?
Definition
NOT to DNA on their own. They use Transcription factors (TF) to recognize DNA sequences.
Term
Eukaryotic transcription
Definition
Complex of proteins forms from binding of TF's
Additional TF's are recruited.
Finally RNA pol 2 initiates and transcripts at +1
Term
3 ways to weaken Histone-DNA binding
What does this do?
Definition
Makes RNA pol/TF more assessable to DNA
-Histone Acetyl Transferases (HATS)
-Histone methylases (methylated)
-Histone Kinases phosphorylate (serine)
Lys and arg for the first 2 are the molecules acetylated and methylated
Term
3 ways to increase Histone-DNA binding.
What does this do?
Definition
Lessens the space for Pol/TF to access DNA
-Histone deacetylase (HDAC)
Histone demethylase
Histone phosphatases
Term
Where can gene expression be regulated?
Definition
Many levels:
-Transcription
-mRNA processing/transport
-Translation
-Post translation
Term
Repressors:
Definition
Inhibit transcription when bound to DNA
Term
Activators:
Definition
Stimulate transcription when bound to DNA
Term
Ligands:
Definition
Modulate repressors and activators binding DNA
Small molecules that bind proteins
Term
Names/Fxn of Ligands
Definition
Inducers (inactivate repressors)
Corepressors (activate repressors)
Term
Regulation by Lac operon:
Definition
Repression and Activation
Term
What is the lac operon?
Definition
Contains genes from metabolizing lactorse. Transcribed ONLY when lactose is abunant and glucose is limiting.
Term
Polycistronic mRNA
Definition
Long piece of mRNA that have different regions that code for different proteins.

Only in prokaryotes
Term
What is usually bound to either lactose or operator in the Lac operon?
Definition
Repressor
Term
Is lac operon constitutive or inducable?
Definition
Constitutive. Always synthesized at a low level in prokaryotes.
Term
Lac Operon: High Lactose
Definition
Repressor binds lactose. Operator is free. RNA pol binds the promotor. More transcription and polysictronic mRNA
Term
Lac Operon: Low lactose
Definition
Repressor binds to operator. Less RNA pol can bind to the promotor. Low transcription.
Term
What does the polycistronic mRNA in lac operon eventually produce?
Definition
B-galactosidase
Permease
Transacetylase
Term
Camp Regulatory Protein
Definition
(Also known as catabolite activator protein CAP)
Active when CAMP is abundant.
When this complex CRP*CAMP binds to DNA increase affinity for RNA [pol. greater transcription.
Term
Low Glucose, High Glucose and CAMP
Definition
Low glucose= High Camp. High transcription
High glucose=Low CAMP. Low transcription.
Term
Catabolite repression
Definition
Ability of glucose to reduce cAMP and reduce transcription of multiple genes
Term
If there is a gene____ and its transcription is reduce when glucose levels are high in prokaryotic cell and transcription is elevated when glucose levels are low, what is likey to be regulating the gene?
Definition
CRP-cAMP colmplex. Complex binds near the promoter of then gene when glucose levels are low.
Term
When targeting enzymes and pathways for therapeutic purposes, what kind of compound does one want?
Definition
One that inhibits an essential step unique to the offending organism/cell with minimal side effects on human host.
Term
Mechanisms of action for therapeutic destruction of enzymes
Definition
1. Competative inhibition
2. Suicide inactivation
3. Modified substrates that halt many steps in a multi-step process (ex. DNA rep)
Term
Antiviral Drugs Discussed:
Definition
Acyclovinr (acycloguanosine)
AZT
Term
Acyclovir is what kind of analog?
What is it missing?
Definition
Purine analog of guanine
3' OH is missing
Term
First step in in bioactivation of acyclovir? What does this do for the drug?
Definition
Acyclovir monophosphate by HSV kinase (phosphorylated). This gives the drug specificity to infect only cells infected with the virus.
Term
How is bioactivation of acyclovir completed?
Definition
Forms acyclovir triphosphate via host cell enzymes.
Term
What is acyclovir PPP used for?
Definition
An alternate substrate for HSV-DNA polymerase, substituting for GTP.
When it is incorported into growing DNA chain termination occurs b/c lack of 3' OH
Term
Can acyclovir get into normal cells?
Definition
Yes, but its not activated unless it has been activated by HSV kinase.
Term
AZT is used for??
Definition
Combating HIV infection.
Term
What is the target of AZT?
Definition
HIV DNA polymerase (reverse transcriptase)
Term
What type of analog is AZT?
Definition
Pyrmidine analog with azido group instead of 3' OH group
Term
Bioactivation of AZT?
Definition
AZT triphosphate by host cell enzymes.
No specificity for HIV enzyme in this step
Term
How does AZT work?
Definition
It is used as an alternate substrate by HIV DNA Pol known more commonly as RT. When used with causes HIV DNA chain termination. No HIV DNA is made from teh HIV RNA to integrate into the genome, so infection ends.
Term
Reasons AZT selects HIV RT DNA synthesis even though host cell enzymes are used?
Definition
-RT has much lower substrate specificity than host DNA polymerases.
-RT more readily incorporates errors into viral DNA.
-HIV RT lacks proofreasing,mistakes remain
Result in a lower fidelity for DNA synthesis than the good host.
Term
What often happens with AZT to decrease its efficacy.
Definition
Development of resistance to AZT and other anti-HIV drugs is common due to the high mutation rate of HIV RT when DNA is made. This is why drug combos are used.
Term
Anti-cancer drugs
Definition
Methotrexate, 5- flourouracil
Term
Thymidylate Synthase
Definition
Key synthetic step with cofactor recycling in DNA dTMP biosynthesis.
Targeting dTMP focuses on DNA, not RNA
Term
What kind of inhibitor is methotrexate?
Definition
Competative inhibitor of dihydrofolate redcutase
Term
What is methotrexate an analog of?
Definition
Dihydrofolate.
Term
Essentially, what does methotrexate do?
Definition
Slows the recycling of the esential methylene tetrahydrate cofactor for thymidallate synthase, decreasing deoxy TMP synthesis. This also decreases DNA replication.
Term
What is 5-flourouracil an analog of?
Definition
Pyrimidine analog
Term
What kind of inhibitor is 5-flourouracil?
Definition
suicide inactivator of thymidylate synthase.
Term
What cofactor does 5-flourouracil require?
Definition
Co-factor thymidylate synthase methylaene-THF (CH2-THF)
Term
What is 5-flourouracil bioactivated to?
Definition
F-DUMP after enters the cell using host cell enzymes.
This is an example of the salvage pathway.
Term
Side effects of chemo, why?
Definition
Hair loss, Intestinal upset (mucosa), Anemia, decreased immunity, tired (bone marrow); on chemo/then off, then on (etc.)

THis is because all of these cells (hair follicles, intestinal mucosa, bone marrow are rapidly dividing.
Term
By the end of 2009, what was the estimation of the total number of HIV infections world-wide, and what area of the world had by far the most?
Definition
33.3 million
Subsaharan Africa
Term
• Name the three key viral enzymes important in HIV viral replication and maturation. What is the major target cell that the virus infects?
Definition
Reverse Transcriptase, Integrase, Protease
Major target cell are those that contain CD4+ on the surface (macrophage, B, T cells)
Term
• Explain the mechanism by which an HIV particle binds and attaches to a host cell just prior to membrane fusion and cell entering. Include discussion of gp160 components and their roles. What are CD4 and CXCR4, and how do they function in this process?
Definition
• The HIV particle bind with the CD4 particle on the T cell. This happens by a complex known as the gp160 complex. This complex contains a gp 120 and which directly attaches to the CD4 complex, and a gp41 complex which is eventually inserted into the cell membrane. First, the gp120 attaches to the cd4 complex. Next, the complex folds and binds to the chemochine receptor CXCR4 (which is part of the host cell). Next, the gp-41 parti cle is now exposed and inserts HIV through the cell membrane and attaches through the outer surface.
Term
• Review the steps in the process of viral replication starting with the HIV RNA that has entered a CD4+ T cell host. Cite the key enzymes involved from Question 3 above.
Definition
The HIV RNA has entered the cell and reverse transcriptase first acts on it to form cDNA. Next,integrase acts on the cDNA to integrate the viral DNA into the host cell genome. Now, a provirus has ben formed, this remains silent until this T cell becomes activated. Next, the provirus is transcripted and then spliced and translated. Viral RNA is transported into the cytoplasm where proteins are translated and assembled into virus particles that bud from the cell. This is the immature virus. The mature virus is formed by protease action.
Term
Slide 10: What makes HIV unique among viruses, and what is the reason for this?
Definition
Our bodies initially produce an immune response to the HIV virus. However, this response does not last.
Term
Four main stages of HIV progression
Definition
Acute Phase
Asymptomatic Phase
Symptomatic Phase
AIDS
Term
Acute phase of HIV
Definition
2-6 weeks
Flu like symptoms
Rapid decrease in immunity
Term
Asymtomatic phase of HIV
Definition
Average about 10 years
CD4 numbers remain > 500
Reduce gradually
Term
Symptomatic Phase of HIV
Definition
2-3 years
CD4<500
Begin to show more symptoms (increase in amount of virus, decreases immunity)
Term
AIDS Phase of HIV
Definition
0-1 Years
CD4<200
Death
Term
What actually causes the death of an aids patient?
Definition
Not the virus itself.
The patient has a very low immune system and many opportunistic virus' and bacteria infections cause actual death
Term
Three cheif characteristics of AIDS
Definition
-Decrease level of CD4 (below 200)
-Increase of aggressive form of Kaposi’s sarcoma or B cell lymphoma
-Increase in susceptibility to infection with opportunistic pathogens.
Term
AIDS categories A-C
Definition
A- Asymptomatic infection
B-Symtomatic condition – start to treat symptoms here
C- AIDS (CD4<200 or <14%)
Term
Which AIDS symptom usually ends the life of a person with this disease?
Definition
Aids Wasting Syndrome
Term
HAART Acryonym
Definition
Highly Active Antiviral Reactive Therapy
Term
What do HAART drugs contain?
Definition
Combination of drugs with RT inhibitors and protease inhibitors
Term
How do HAART drugs work?
Definition
Decreasing viral load.
Increasing CD4
Term
Major problem with HAART?
Definition
Difficulty with patient compliance
Term
What question cannot be answered about HAART at this time?
Definition
Whether the patient can every safely terminate treatment.
Term
What are the access to treatment issues of HAART?
Definition
-Access in third world countries
-Access in poor inner cities and rural America
-Too expensive, esp in third world countires.
Term
Over time, what does HAART lead to? How is this combated?
Definition
Drug resistance. This is combated by exchanging the drugs used for different ones.
Term
What happens after about 3 months of being treated with HAART agents?
Definition
Over time, the amount of virus in plasma decrease and is usually at 10 at 3 months treatment. (limit of detection is decreased)
Term
All procaryotic and eukaryotic RNAs are modified and processed except what?
Definition
Prokaryotic mRNA, it immediately is acted upon by the ribosome.
Term
3 general types of processing?
Definition
1. Removal of nucleotides
2. Covalent modification of nucleotides
3. Addition of nucleotides not encoded in DNA
Term
How does tRNA processing occur?
Definition
By the 3 main ways of processing.
They are highly processed
-Remove excess nucleotides (endo/exonucleases)
-Addition of nucleotides (all tRNA's have CCA on 3' end)
-Extensive modiications
Term
What do ALL tRNA's have?
Definition
CCA added on the 3' end.
Term
How does rRNA processing occur?
Definition
rRNA's are a part of a large transcript.
Many RNases remove excess RNAs->rRNAs
Term
Genes for rRNA.
How many.
Euk/Pro?
Definition
Many genes in eukaryotes and prokaryotes
Term
Where is rRNA made in eukaryotes?
Definition
Nucleolus of eukaryotes
Term
Where do ribosomal proteins bind, what does this do?
Definition
They bind to the primary transcript and aid processing and prevent degredation.
Term
What is added to eukaryotic mRNA as they undergo extensive processing?
Definition
-CAP @5' END
.N-7 methyl guanine attached to +1 by PDE bond
.2 OH methylation
3' Poly A tail
.
Term
Functions of the mRNA cap
Definition
-Increase stability
-Recognized by splicosomes
-Recognized by ribosomes
Term
What RNA pol is capping associated with?
Definition
RNA pol 2, b/c form on mRNA (which is RNA pol2)
Term
What is polyadenylation?
Definition
Adding Poly A tail!!
Term
CPSF
Definition
Cleavage and polyadenylation specificity factor
-Binds to consensus sequence and forms a complex containing RNA and endonuclease.
-Endonuclease catalyses cleavage of the transcript downstream of the polyadenylation forming a new 3' end. -Poly A polymerase binds and makes tail!
Term
What is template independent RNA polymerase?
Definition
Poly A polymerase
Term
Functions of Poly A tail:
Definition
Increase stability
mRNA transport from nucleus->Cytoplasm
Term
What does splicing remove?
Definition
Introns
Term
What does mature mRNA contain?
Definition
5' CAP
exons
Poly A tail
Term
What happens once mature mRNA is formed?
Definition
translation
Term
Where does coupled trancription/translation occur?
Definition
Prokaryotes.
Term
Splisosomes
What do they do?
What are they made of?
Definition
Splicing
Somplexes of snRNPS
Term
What are snRNPS?
Definition
Small RNA's with proteins.
Term
How do snRNPs assemple? What does this form.
Definition
Sequentially @ each intron, from a sliceosome.
Term
Alternate or differential hnRNA splicing?
Definition
Joins different exons
Form different mRNA
Form different proteins.
(from same inital transcript)
Initally believed rare, now not.
Term
Summary of Euk mRNA processing:
Definition
1. RNA pol 2
2. Cap added
3. Splicing
4. Poly A tail added
Term
What is translation
Definition
Taking nucleic acids and forming the correct amino acid structure->proteins.
Term
Genetic code
Definition
Specific nucleotide sequence in mRNA or DNA designate specific AA
Term
What are adapter molecules in translation?
Definition
tRNAs
Term
Genetic code properties
Definition
-Non overlapping codons
-Proper reading frame
-Univeral
-Unambiguous
-Degenerate or synomous codons (many codons=same AA)
Term
How many Stop codons?
How many initiation codons?
Definition
Stop-3
Start-1 (AUG or methionine)
Term
What is the role of tRNA's in translation?
Definition
convert/adapt mRNA sequence to AA sequence
Term
Characteristics tRNA's use to adapt?
Definition
-Size/Shape (all similar)
-Anticodon
-Acceptor stem
Term
What do amino acyl tRNA's do?
Definition
Translate mRNA codon sequences to an AA sequence.
Term
Wobble.
Definition
Some tRNAs recognize multip codons via imperfect base pairing.
Term
What do we need to know for wobble?
Definition
U is messed up
Binds A or G
Term
What do amino acyl TRNA synthetases do?
Definition
Join specific AA to appropraite tRNA's to form aminoacyl tRNA
Term
what is special about the amino acid-trna bond?
Definition
It is a high energy bond that forms the peptide bond during translation.
Term
Can aminoacyl-tRNA's proofread?
Definition
YES!
Term
Steps in translation (simple)
Definition
-Activation
-Initiation
-Elongation
-Termination
Term
Activation of translation
Definition
aminoacyl-tRNA synthetases join proper AA with tRNA (covalently bound forms a high energy bond)
Term
Ribosomes
-Subunits
Definition
Large complex of rRNA and proteins that coordinate translation
2 subunits
-small (1rRNA) +proteins (cleft where large sits)
-large (2,3 RNA) + proteins (tunnel to accomodate growing protein)
Term
Sites of ribsome
Definition
A-Aminoacyl tRNA
P-Peptidyl tRNA
E-Empty tRNA
Term
What does the ribosome accomodate?
Definition
mRNA, aminoacyl tRNA, peptidyl tRNA, and growing protein
Term
Met formyl vs regular
Definition
N-formyl methionyl tRNA is only in prokaryotic initiation.
Formed by prokaryotic methionyl synthetase and formyltransferases.
Term
Prokaryotic Translation initiation:
Definition
tRNA i/f met is used.
Shine dalgarco sequence in mRNA precedes AUG to determine proper AUG
Term
Prokaryotic initiation factors 1,2,3
Definition
1 and 3 binds to the small ribosome and prevent the large ribosome from prematurely adding.
2 + GTP binds with the formyl AUG. This interacts and recognizes the S.D.
Large subunit adds. (1,3 released)
Term
Eukaryotic Initiation of translation:
Definition
-eIF-4 binds with the cap and begins formation of the initiation process
-Complex moves along mRNA in the 5 to 3 direction until reaches the AUG. Here eIF-2 brings to small ribosomal subunit.No SD.
-Preinitiation complex pauses and large ribosomal subunit joins with Met-tRNA (this is positioned in ribosome P site)
then the eIF's dissociate.
Term
What is the role of EF-TU GTP in the elongation phase of translation?
Definition
Binds aminoacyl-trna and brings to the A site.
They bind to the acceptor stem of amino-acyl trnas.
Term
Why do EF-Tu NOT bind f-Met-trna?
Definition
These are for initation. They have a unique acceptor them that prevents them from binding to Ef-TU
Term
Where do the EF-TU GTP aminoacyl tRNA complexes go once formed?
Definition
A site
Term
What happens in the elongation phase once the EF-TU GTP touches the ribosome?
Definition
GTP goes to GDP (high energy bond) and the aminoacyl trana is released into the A site and EF*TU*GDP dissociates
Term
Peptidyl transferase?
What. Where. When.
Definition
Forms the peptide bond in the large ribosomal subunit after dissociation of EF-TU*GDP.
Term
Why peptidyl transferase activity happen?
Definition
Due to rRNA and ribosomal proteins b/c rRNA has catalytic activity.
Term
What is translocation?
Definition
EF-G*GTP binds ribosome. Moves to put next codon in the open A site; uncharged tRNA in E dissociated (GTP->GDP)

Basically just moving the product from A to P and E...
Term
Termination of translation
Definition
Release factors 1,2,3
Stop codon in the A site.
Ribosome stalls and RF-1 or 2 binds with RF3*GTP which bind the stop codon.

This results in hydrolysis of peptidyl tRNA. Protein released.
Term
Coupled transcription and translation.
Process.
What cells?
Definition
ONLY prokaryotes
Prokaryotic mRNA emerges from RNA pol (no processing), ribosomes form initiation complexes. Translation begins before transcription complete.

ONly in pro b/c euk are in different compartments.
Term
Polysomes
Definition
Many ribosomes translating proteins on a single mRNA both in prokaryotes and eukaryotes.
Term
Streptomycin:
Definition
Binds small ribosomal protein
Inhibits initiation
Term
Chloramphenmicol
Definition
Inhibits peptidyl transferase in large subunit.
Peptide bond formation
Term
Erythromycin
Definition
Inhibits translocation
Term
Tetracycline
Definition
Binds small ribosomal subunit, preventing binding of aminoacyl trna into A site.
Term
Where is a not wiedely used place to regulate gene expression?
Definition
Translation
Term
What regulates globin?
Definition
Availability of heme
Term
What does heme do, indirectly?
Definition
Regulates activity of eIF-2
Term
HCI
What does it do. What is the result
Definition
Heme controlled inhibitor.
Phosphorylates eIF2
Global inhibition of translation
Term
When is HCI active?
Definition
When heme is low
Term
What regulates the trp operon?
Definition
Repression and attenuation
Term
What is attenuation. Where does it occur?
Definition
ONLY in prokaryotes. For coupled transcription and translation.
-Independent mechanism of sensing trp level.
Term
TRP operon
Definition
codes 5 protesin needed to make trp with is required for translation
Term
If TRP is high, what is the activity of the trp operon (repression)
Definition
THe trp operon is low, repressed.
TRP is a co-repressor
Term
If TRP is low, what is the effect on the operon?
Definition
TRp does not bind to the repressor. operon makes trp
Term
How does attenuation work in the trp operon?
Definition
Leader region with coupled transcription and translation controls RNA pol moving into trpE gene via 2 trp codoings and 4 GC rich regions.
Term
Formation of leader mRNA in trp operon regulation
Definition
1-2 bound; 3-4 bound
Termination signal
RNA pol leaves before transcribing genes.

2-3 Bound- no pause/termination signal
RNA pol transcribles trp genes.which make proteins that make trp.
Term
Trp is abundant attenuation
Definition
Trp is high. so 1:2 3:4.
RNAP disoccaites. No transcription
Term
Trp is limiting attenutaiton
Definition
2:3 and ribosome remains stalled. Termination signal not formed.Trp eventually made
Term
Trp is limiting attenutaiton
Definition
2:3 and ribosome remains stalled. Termination signal not formed.Trp eventually made
Term
Free ribosomal proteins translate what proteins?
Definition
Cytoplasmic, Nuclear,Mitochondrial
Term
RER ribosomes make proteins that go where?
Definition
Secreted proteins, PM proteins, ER, lysosomal proteins
Term
Process of RER proteins
Definition
Attached to ER
Enter ER lumen
Transfer vesicles
Golgi (->lysosome or)
Secretory vesicle
PM or secreted
Term
What type of cells do signal hypothesis occur in?
Definition
Eukaryotes
Term
What is a signal peptide?
What type of proteins are they found in?
Definition
15-30 N terminal AA righ in hydrophobic AA that cause ribosomes to go into ERE; inly present in secreted, PM, lysosome or peroxisome proteins
Term
When is the signal peptide removed?
Definition
ER lumen
Term
What is SRP
Definition
Signal recognition particle. Recognized and binds signal peptide as it emerges from ribosome.
Term
What binds the signal peptide? What does it do?
Definition
Signal recognition particle binds the signal peptide
Translation now stops
Term
What happens to the SRP-ribosome complex in in the signal hypothesis?
Definition
Bound to the ER membrane.
Term
In the signal hypothesis how is the ribosome anchored, and where is it anchored to? What happens next?
Definition
Anchored to the Er pore by robophorin/translocon.
Next, SRP is released and signal peptide inserted into pore.
Term
Role of signal peptidase in signal hypothesis
Definition
Cleaves signal peptide
Term
Role of signal peptidase in signal hypothesis
Definition
Cleaves signal peptide
Term
How are proteins modified within the ER?
Definition
Transport vesicles, golgi, secretory vesicles.
Term
What proteins are normally glycosylated?
Definition
Most secreted and membrane bound proteins
Term
What is dolichol phosphate? Found? What cells? What does it do?
Definition
Sugar.
Found in ER membrane of eukaryotes
Differnet carb bodies add to dolichol phosphate
Term
Glycosyl Transferase
Definition
Removes pre formed block of sugars
Term
Proteins that end up in Lysosomes are modified in ER or Golgi by addition of ?
Definition
mannose 6 phosphate.
Term
Nuclear location signal
Definition
Importin complexes. Protein complex enters. Like Yon!
Term
Recombinant DNA
Definition
combining DNA from different sources
Term
Steps in recombinant DNA formtion
Definition
1. Isolatte and purify vector and target DNA
2. Cleave vector and target DNA @ specific sequences via restriction endonucleses
3. Ligate target DNAs into vector recombinant DNA
4. Introduce recombinant DNA into appropriate host (Bacteria yeast, mammal, embryo)
5.Replication/Expression of recmobinant DNA in host cells
6. Identify host cells that specifically contain DNA of interest via screening or selection.
Term
What is a vector?
Definition
Carrier DNA into which target DNA is inserted: often plamids
Term
Most common vector?
Definition
Plasmid
Term
What is the purpose of the origin of replicaiton?
Definition
ORI present so it can be replicated in the host.
Term
What is a shuttle vector?
Definition
Ability to be used in both bacteria hosts and eukaryotic cell hosts.
Term
What do restriction endonucleases do?
Definition
Generate sticky complimentary ends on target DNA and on vectors, allowing insertion of target into a specific site of vector.
Term
LacZ gene
Definition
Plasmids constructed to contain this. Forms beta galactosidase
B galactosidase hydrolyzes X gal to blue
Term
What do PVU I and PSTI do to Lac z?
Definition
Make it from white colongies
Term
Genomic Libraries
Definition
Collection of host cells with recombinant vectors that contain inserts that gother repesent all of the DNA in an organisms genome.
Term
cDNA libraries.
Definition
Recombinant DNA libraries that represent all of the mRNAs transcribed in a cell, tissue or organism under certain conditions. cDNA = complimentary, dS dnA
Term
How to make cDNA:
Definition
-Isolate eukaryotic mRNA on oligo dT columns via poly A tail
-mix oligo dT and bind poly A and acts as a primer for RT
-RT makes cDNA from mRNA template
-RNase H partially degrades mRNA ->multiple RNA primers fro DNA pol 1 synthesizes complimentary DNA as degrades RNA primers
-DNA ligase joins DNA fragments to dsDNA
Term
Screening libraries
Definition
-Scren or proble clones to determine which contains insert of intrest
-Probes are molecules that specifically recognize DNA of intrest
Term
Process of screening libraries
Definition
Spread cells from library on plate
Blot some cells in each colong onto filters
Denature DNA from each colony on filter
Reach with probe containing DNA sequence complimentary to DNA of interest
Probe specifically hybridizes with DNA of intrest
Term
Prokaryotic expression vectors:
Definition
Addition to marker gene and ori
-multiple cloning site preceded by strong promoter
-Target DNA inserted in site, after ATG, which is preceded by Shine delgarno sequence
-Transcription Translation Signal
-Protein has a few N terminal amino acids not present on native protein
Human: growth hormone, insulin, blood coagulation factors,
Term
Where are many eukaryotic proteins also expressed?
Definition
Prokaryotes. They aren't active b.c prokaryotes lack post translational modification enzymes.
Term
Hosts for eukaryote expression?
What can they do?
Definition
Yeast, cultured cells.
Can splice introns
Insert genomic or cDNA
Term
Transgenic animals
Definition
Recombinant DNA introduced into zygote or early embryo. All cells in offspring contain recombinant DNA
Term
Applications of recombinant technology in prokaryotes?
Definition
Genomes of many bacteria have been sequenced. Better understanding of microbial bioogy and virulence factors; may aid development of new drugs to treat/prevent human diseases.
--especially important now bacterial resistance
Term
Applications of human treatment in recombinant technology?
Definition
Human proteins. Various diseases.
Proteins from bacteria.
Human gene therapy
STEM CELL RESEARCH. restrictions
Term
Diagnosis of human diseases using recombinant DNA technology?
Definition
Genetic diseases. Coagulation factors. Lesch Nyhan Syndrome. Sickle Cell. Thalassemias.
RFLP
Term
What is RFLP?
Definition
Restriction fragment lenght polymosphism
Follow patterns of disease inheritance as a family, and diganose and counsel prospective patients.
Also, differentiate different people from milions of others.
Term
Polymerase Chain Rxn (PCR)
Definition
Procedure quickly amplifies select DNA sequences from very small amount of DNA sample.
Term
What must you know in PCR? Why?
Definition
Sequence of DNA bounding region of interest to construct approprate primers
Term
What do you do with the DNA in PCR?
Definition
Place in tube with primers, heat stable DNA pol, and 4DNTPs.

Tube sealed and placed in a thermocycler programmed to heat for a specific time, cool, repeat.
Term
PCR applications
Definition
Amplify genes w/o cloning
test or diagnose genetic diseases amniotic
Find bacterial diseases before widesread
Monitor cancer chemotherapy
Forensics
Moleculer evolution
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