Week 5 Flashcards
Prokaryotic gene regulation:
What is the example of the inducible operon ?
Lac operon in bacteria
Prokaryotic gene regulation: (inducible operons)
In the absence of the inducer, a ______ is bound to the operator and no transcription occurs.
Repressor
Prokaryotic gene regulation: (Repressible operons)
The operon is transcribed in the absence of the ______. A co-repressor may also be needed to bind and activate the repressor before transcription will be ____.
repressor
inhibited
what 5 methods are present in Availability or amount of DNA level regulation (Eukaryotes) ?
- Chromatin remodeling
- DNA methylation
- Gene Deletion
- Gene amplificaiton
- Gene Rearrangement
What method is present for Transcriptional level of regulation in Eukaryotes ?
Gene specific transcription factors
What are the 5 levels of control in regulation of gene expression in eukaryotes ?
- Availiability or amount of DNA
- Transcriptional control
- Post-transcriptional control
- Translational control
- Post-translational control
What are the methods present for gene regulation in the Post-transcriptional level of control?
- Alternative splicing
2. mRNA editing
What are the methods present for gene regulation in the Translational level of control ?
- Translational initiation
2. mRNA stability or accessibility
What are the two main methods used for gene regulation in the Post-translational level of control?
- Phosphorlyation
2. Proteolytic cleavage
In DNA methylation under gene regulation, what bases can be methylated to reduce levels of gene transcription?
C and G
will make the DNA more Negative so that it binds tighter to Histones
What is the best example of Gene deletion level gene regulation in mammals?
- Development of Erythrocytes
- cells will destroy its nucleus and its entire DNA (during differentiation- cell continues to produce large amounts of globin from pre-sythesized mRNA but cannot produce other proteins
Gene amplification by the cell is used in _____ instances when proteins are needed more rapidly than the chromosomal number would permit.
rarely
What is the example of gene rearrangement used for gene regulation (DNA level)
Mammalian immune system is capable of generating diverse antibodies capable of recognizing any array of antigens
-Also, Transposons- migratory pieces of DNA that can insert themselves into DNA
At the transcriptional level of control, what are the examples of the gene specific transcription factors ?
- Nuclear hormone superfamily (steroid/thyroid hormones- common domain structure of central DNA binding domain and C-terminal hormone binding domain
- Super-family recognizes the hormone response element (HRE) to activate the expression of genes containing it
some pre-mRNA transcripts give rise to multiple mature mRNAs. They can be processed in more than one way, this is known as _____ ______.
alternative splicing
Use of alternative splice-sites in the same pre-mRNA allows the synthesis of different proteins from _______.
the same gene
Where is alternative splicing know to occur?
Tissue-specific or developmental fashions
mRNA editing is a ____ type of RNA processing event that alters the sequence of RNA ______.
rare
after it is formed
RNA editing refers to an alteration in the nucleotide sequence of RNA, the sequence of the mRNA differs from the ______ encoding it.
exons
What is the example of mRNA editing in mammals?
Apolipoprotein- in apoB transcripts in the small intestine, a cytidine in the coding region is converted to uridine generating a premature stop codon (otherwise in the liver it is normal and longer protein)
in the translational level of control, translation initiation has for an example of gene expression control ….?
Phosphorylation of translation initiator protein eIF2 results in decreased translation
Describe mRNA stability or accessibility at the translational control level of gene expression ?
- approach used by cell to quantitatively regulate output of a gene
- mRNAs vary greatly in their stability, with half-lives ranging from a few minutes to more than a day
- Example is Ferritin translation (accessibility)
The operon contains ?
Promotor and then all of the structural genes (can have multiple genes for one promoter region on Prokaryote DNA)
What happens when an operon is regulated on or off?
All of the genes within the operon are either turned on or off, can not seperate them
A metabolite of lactose called _____ serves as an inducer, binding to the repressor and inactivating it. RNA polymerase can then bind and transcribe structural genes of ____ operon.
allolactose
lac
When is the lac operon turned on ?
in the presence of Lactose (allolactose is the inducer - metabolite)
-Glucose must be low (cAMP will be high)
What is needed to turn on the Lac operon where RNA polymerase will bind to it?
- low levels of glucose
- high levels of cAMP to turn on CRP (cAMP receptor protein)
- Presence of Lactose
remodeling of chromatin generally refers to displacement of the nucleosome from specific DNA sequences so taht transcription of the genes in that sequence an be _____.
Initiated
What are the two mechanisms that chromatin remodeling occurs by?
- Adenosine triphosphate (ATP) driven remodeling complex (uses hydrolysis to unwind certain sections of DNA)
- Covalent modification of the histone tails through acetylation (makes less positive)
What does Histone acetyltransferase (HAT) do ?
transfer an acetyl group from acetyl coenzyme A to Lysine residues in the tail (amino terminal ends)
What removes the acetyl group from the _____ on histones?
Lysine
-Histone deacetylases (HDAC) will remove acetyl groups, making DNA binding tighter again -can have several lysines for multiple acetylation sites
______ residues in DNA can be methylated to produce ______.
Cytosine
5-methylcytosine
Where are the methylated sequences of Cytosine found on DNA?
near CG-rich sequences (called CpG islands) - often near promoter region of a gene
What is an example of methylation effecting gene transcription?
Globin genes are more heavily methylated in non-erythroid cells (non-RBC lineage)
-mechanism for regulating cell differentiation particularly in fetal development
What does an acetyl group look like ?
O=C-CH3
-linked to another group by middle Carbon
what level do microRNAs (miRNA) regulate gene expression at ?
Post-transcriptional level
What do microRNAs do to regulate at the post-transcriptional level ?
- induce the degradation of a target mRNA
- block translation of the target mRNA
(reduced expression overall)
What is a microRNA?
small RNA molecules that are non coding for proteins and regulate mRNA
True or false:
miRNA can be found in the introns (removed parts) of the genes they regulate?
true
True or false:
One miRNA type can regulate multiple mRNA targets
True
True or false:
one mRNA can only have one miRNA that regulates it
False:
mRNA can have multiple different miRNA that regulates it, and one miRNA can regulate several different types of mRNA
What RNA polymerase transcribes miRNA ?
RNA polymerase II
What happens in the presence of heme to eIF2alpha
the eIF2 will not be phosphorlylated (and inactivated) so it can continue translation
Heme _____ the heme-regulated inhibitor kinase (for eIF2-alpha)
inactivates
What is mRNA editing?
hnRNA and the coding gene are the same, but in different tissues, bases are altered or nucleotides are added or deleted so the mature mRNA differes in different tissues
In Ferritin synthesis, what happens when iron level are low ?
IRE-BP has high affinity for IRE section of mRNA and stays bound to hairpin loop
In Ferritin synthesis, what happens when iron is present?
Iron binds to the IRE-BP and the IRE section of the mRNA is free to translate protein to store more iron in cells (will not store iron that is not there)
what type of control is the transferrin receptor ?
mRNA degradation control of translation
The _____ receptor is a protein located in the cell membranes that permits cells to take up _____, the protein that transports iron in the blood.
Transferrin
The rate of synthesis of the transferrin receptor ______ when intracellular iron levels are low, enabling cells to take up more iron.
increases
What is ferritin involved in ?
a protein involved in storage of iron within cells, synthesized when iron levels increase
What does IRE stand for?
Iron response element that is on the mRNA for ferritin protein production
What does transferrin do ?
a protein that transports iron in the blood
In regulation of degredation of the mRNA for the transferrin receptor, what happens when iron levels are high?
Iron will bind to IRE-BP , then taking it off the mRNA hairpin loop and the mRNA is rapidly degraded preventing translation
What does the poly adenine tail do for mRNA?
protects the mRNA from attack by nucleases , gets shorter as the mRNA ages
The rate of synthesis of the transferrin receptor ______ when intracellular iron levels are low, enabling cells to take up more iron.
Increases
transferrin is in the membrane
What is a restriction enzyme ?
A specific Restriction endonuclease
-recognizes a short sequence of DNA, usually 4 to 6 base pairs and cleaves a phosphodiester bond in both DNA strands (high specificity)
What is a Restriction endonuclease ?
enzyme that enables us to cleave segements of DNA from the genome of various types of cells or to fragment DNA obtained from other sources
What is the utility of restriction endonucleases in recombinant technology ?
- use to identify variations in base sequence in a gene
2. Synthesize recombinant DNA (chimeric DNA)
How does DNA ligase interact with restriction enzyme?
the DNA ligase can fuse the fragmented DNA together again
What is a probe?
A single strand polynucleotide of DNA or RNA that is used to identify a complementary sequence on a larger ssDNA or ssRNA molecule
What is the name for when a probe finds its complementary base pairs?
Annealing or hybridizing (binds to target on ssDNA or RNA)
What is stringency in relation to a probe?
How exact a match the probe must have to the DNA to which it is hybridizing in order for significant hybridization to occur
-low allows for more mismatches to occur
How can stringency be manipulated with a probe?
Raising and lowering temperature
Raising or lowering the salt concentration
(high salt helps lower stringency as it negates the repulsion in the backbone of DNA by mismatched pairs)
What is a single nucleotide polymorphism (SNP)?
A variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population
What is a Restriction fragment length polymorphism(RFLP) ?
A point mutation occurs in a recognition site for a restriction enzyme, so the enzyme can not cut in the same way for individuals with the mutation vs. normal
-usually the mutation will have a larger fragment that binds the probe or smaller than the normal gene
what is the usefulness of Restriction fragment length polymorphisms ?
can be used as a biological marker for genetic diseases in testing
-most RFLP are in the non-coding regions but are regions closely linked to the coding regions for disease
What percentage of DNA human genome codes for actually used genes?
1.5%
What is Polymerase chain reaction ?
In vitro method used for rapid production of large amounts of specific DNA segements
- large quantities of primers added
- DNA is heated to denature, and cooled over and over to stimulate DNA synthesis
In 20 heating and cooling cycles in the Polymerase chain reaction method, (each cycle only a few minutes), how much can a target DNA be amplified ?
over a millionfold
Define Reverse transcriptase ?
mRNA that is isolated and used as template by the enzyme reverse transcriptase , which produces a DNA copy (cDNA) or the RNA
What makes Reverse transcriptase methods different from PCR methods ?
-Reverse transcriptase method makes DNA that only contains exons, no introns (because it came from mRNA) and so missing the regulatory regions of a gene
In gel electrophoresis of DNA, DNA samples are placed into wells at one end of gell (negative charge end) and what defines how they migrate toward the opposite end (positive charge)?
Shorter molecules migrate more rapidly than longer molecules and their negative charge is attracted to the opposite positive charged end
What is the Southern blot method ?
- used to separate DNA molecules by electrophoresis
- the DNA is denatured and hybridized with a probe on nitrocellulose paper (blot)
What is the northern blot method ;?
- used to separate RNA
- already ss so the probe can bind without denaturing needed first
What is the western blot method ?
-used to electrophorese Proteins and probed with specific antibodies
What does VNTR stand for ?
Variable number of tandem repeats
What are the regions called in human genome that has many sequences repeated ?
Highly variable regions - contain a variable number of tandem repeats (VNTR)
Restriction enzymes can digest regions that flank the VNTR region and produces variable sized fragments between individuals, this can be used for ?
“DNA fingerprinting”
-Forensic analysis
Probes used to identify these restriction fragments- the size between the restriction enzymes that flank will vary between all individuals
What is the allele-specific oligonucleotide probe used for ?
probing for mutations that do not sit within restriction enzyme zones, involve general mutations in the coding regions
Ex: Sissy fibrosis
-PCR used to amplify abnormal vs. normal regions
-very specific technique
-one “normal” probe and one “mutant” probe- if heterozygous two dots appear showing both probes bound to DNA
what must occur with Single nucleotide polymorphisms that will qualify it for such a title and not just a random mutation ?
the single change must be present in 1% of the population
- they occur once ever 100 to 300 base pairs
- useful for mapping disease
If a restriction enzyme recognizes a six base sequence, how frequently on average will this enzyme cut a large piece of DNA?
use equation (1/4) to the 6th power (1 in 4,096 bases)
What does the Sanger technique require to work ?
Deoxyribonucleotides and didoxyreibonucleotides, templete DNA
What is Taq polymerase used in ?
PCR
In steroid hormone receptors, what are the two examples of heterodimers that have receptors already bound to the DNA inside nucleus ?
Thyroid hormones
Retinoic Acid receptors
DNA binding proteins are primarily alpha helixes interacting through _______ interactions.
hydrophobic
In DNA binding proteins, _____ _____ bind Zn to generate a small folded domain, 2-6 Zn domains, estrogen and steroid receptors, dimers
Zn finger
What type of DNA binding protein forms a coil through hydrophobic interactions, homo or heterodimers (appearing like an X with the top sections of the X the _____ domains)
Leucine zippers
What type of DNA binding protein is a helix that fits into the major groove, has stable monomers, and regulates homoebox proteins during development?
Helix-turn-helix proteins (2 coils parallel with a third coil perpendicular)
What type of DNA binding protein forms through hydrophobic interactions and is mainly involved in cell differentiation ? (has parallel helix, then loop, then X helix formation)
Helix-loop-helix
When does mRNA editing occur>
Post Splicing modification
Often reversible and tissue specific
Regulatory proteins that turn on and off gene transcription are ________ as one can trigger a cascade of other gene activations through protein synthesis of other regulatory proteins.
Combinatorial
In translational regulation, when levels of Heme are high, what does it prevent ?
phosphorylation of eIF-2Alpha by Heme-regulated inhibitor (HRI) kinase (high levels of Heme inactivate this enzyme)
Why would a cell want translational regulation of Heme and eIF-2Alpha by Heme?
Heme is required for the generation of the holoprotein Hemoglobin (consists of 4 subunits each requiring a heme group)
-If heme is low, the cell can NOT generate functional hemoglobin, if heme is high, it needs to translate globin protiens (make sure eIF-2Alpha stays active by inhibiting the phosphorylation of it)
Which RNA polymerase transcribes miRNA ?
RNA polymerase II
What does miRNA use to target specific mRNA for degredation?
a guide strand - will cleave mRNA or repress translation
In post translational modifications , what does Glycosylation do ?
N or O linked addition of carbohydrate to help protect from proteolysis
In post translational modifications, what is the common place and AA for lipid additions to occur on?
Common for membrane proteins
Common to Cysteine residues
What are 2 main post-translational modifications that impact function ?
Phosphorylation
Acetylation
What is normally the AA that is modified by phosophorylation ?
Serine or Threonine
What is normally modified by Acetylation ?
on Histones to increase DNA accessibility
The generation of multiple protein products from a pre mRNA is done through ?
Alternative splicing
What are three main title ways that Cancer develops at the genetic level ?
- Radiation or chemical carcinogens
- Gene amplification
- Gene rearrangement
In the radiation or chemical carcinogen pathway for Cancer, what are 2 main ways the genes are effected?
- mutation in coding region causes production of hyperactive protein
- mutation in promoter causes excessive expression
What is the main way that Gene amplification causes cancer ?
Expression of multiple copies of the proto-oncogene
What are the 2 main ways that gene rearrangement can cause cancer ?
- Proto-oncogene is now under control of strong promoter or enhancer
- Fusion protein is either overproduced or hyperactive after proto-oncogene is fused with another gene
What are the 3 basic requirements of plasmids ?
- abilify to self replicate
- possession of selection marker
- must be capable of being digested
What is on the plasmid besides the basic 3 requirements ?
Multiple cloning sites for inserting the gene of interest to study
What bond does the restriction endonuclease cleave?
Phosphodiester bond on backbone
What does Cloning combine method wise?
Utility of restriction enzymes with plasmids
-Recombinant DNA can be introduced into cells treated to make them permeable
Where do the oligonucleotide primers in Polymerase Chain reaction anneal to the DNA template on ?
3’ end of DNA templates - act similar to RNA primers in regular DNA replication
In the Southern blot, what is used as a probe on the DNA?
DNA fragment as probe
In the Northern blot, what is used as a probe for total RNA?
total RNA is probed with RNA or DNA fragment
In Western blot, what is used as a probe for total protein ?
probed with antibodies
What does the probe anneal with ?
ssDNA
True or false:
Bacteria can be used as viable hosts for both protein and DNA expression.
True
True or false:
Restriction endonucleases are not required for insertion for you gene of interest into a plasmid.
False
True or false:
PCR is only an avaliable technique if you know the full sequence of the region of interest on a stretch of DNA
False - you only need to know the flanking regions
True or false:
Southern blotting evaluates changes in the size of mRNA
False - it evaluates DNA
What is the difference of a SNP from a RFLP?
the SNP is a single base pair change
the RFLP is a single base pair change that introduces or removes a restriction site
What happens in quantitative reverse transcriptase PCR?
- Isolate RNA
- Use reverse transcriptase to convert mRNA into cDNA (coding DNA)
- Use PCN to amplify cDNA
- Run samples on gel and compare to an internal control
What is Cystic fibrosis mainly caused by ?
mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein which is a chloride channel
- triplet deletion of CFTR gene 508 removing one codon
- leads to pancreatic duct blockage and clogged airways
