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MCB 250 Final Exam Review Set
Terms in this set (161)
What is the DNA region that transposition involves?
What are the two characteristics of strongly polar mutations?
- Not revertible
- Transmissible, where mutations can spread to other gene systems
What are the four properties of transposable elements in prokaryotes?
- Double-stranded DNA
- Flanked by inverted repeats
- Contain one or more genes
- When transposes it produces a target-site duplication
How can you demonstrate that transposable elements have inverted repeats?
1. Denature the double-stranded DNA.
2. Cool down to reanneal
3. See intra-strand annealing between the inverted repeats
What are the two types of transposable elements in prokaryotes?
- Insertion sequences
What are the six properties of insertion sequences?
- Usually very small
- Flanked by IRs
- Encode at least one gene
- Provide their own transposition functions
- Do not code for noticeable traits
- Can cause mutations by inserting into genes
What are the two properties of Class I transposons? What is the alternative name for Class I transposons?
- A segment of DNA flanked by two copies of an IS element
- Each IS element itself flanked by IRs
What is an example of Class I transposon and its function?
Tn5, kanamycin resistance gene
What are the two properties of Class II transposons?
- Contains IR elements at each end
- The IRs at each end are not part of IS elements
What is an example of Class II transposon and its function?
Tn3, penicillin resistance gene
What is the characteristic of insertion sites for transposable elements?
Usually very random
What are the DNA sites involved in the moving of transposable elements?
IR elements and target site
What are the proteins involving in the moving of transposable elements?
Transposases that are usually encoded by the transposable element
What does the moving of a transposable element produce?
A target-site duplication
What does target-site duplication imply about the moving of transposable elements?
Asymmetric cutting of DNA by enzymes
What are the two properties of transposases?
- DNA Recombinases
- Nicks and rejoins dsDNA
How is the mechanism of transposases different from site-specific recombinases in four ways?
- Does not form a covalent bond with the nicked DNA
- the OH group of water used as a nucleophile
- contains an invariant triad of residues, DDE (aspartic acid, aspartic acid, glutamic acid), at the active site
- Contains a bound Mg++ at the active site
What are the two major domains in transposases?
- Contains a DNA binding domain that recognizes a specific sequence
- Contains a protein-protein interaction domain
What is the order of domains from N terminus to C terminus?
DNA binding -> DDE -> protein-protein contact
How does the transposes interact with the DNA?
1. The DNA binding domain of the transposase interacts with the inverted repeat
2. The protein-protein contact domain of the two tranposases interact with each other bringing the two inverted repeats together
What is the difference between replicative transposition and nonreplicative transposition?
In replicative transposition the transposable element is found in the donor and recipient DNA while in nonreplicative transposition the element is only found in the recipient DNA
What is the first step of replicative transposition?
Involves a fusion of the two replicons during tranposition and formation of a cointegrate
What are the three steps of the formation of the cointegrate?
1. Nicking DNA
2. Joining free ends
3. Replication of transposon
What happens after cointegration and what proteins are involved and what are the properties of these proteins?
Recombination between res sites
Is a cointegrate formed during nonreplicative transposition?
Is resolvase involved in nonreplicative transposition?
What is the frequency of transposition?
10 ^ -6 to 10 ^ -7
What is the frequency of transposition when genetic material is introduced without a copy of transposable element?
10 ^ -3 to 10 ^ -4
What is the frequency of transposition when the genetic material already contains a copy of the transposable element?
10 ^ -6 to 10 ^ -7
What are the four properties of Inhibitor or Inh that inhibits tranposition?
- Synthesized in the same ORF as tnpA
- but starts translation at Met codon position 56
- missing 55 amino-terminal amino acids
- binds transposase and inhibits its activity
How does Inh inhibit transposition?
The incomplete sequence has a protein binding domain that dimerizes with the transposase causing an incomplete transposition complex prevent transposition
What are four gene types that are usually contained within transposable elements?
- Antibiotic resistance genes
- Resistance to toxic agents
- Toxin production and other pathogenicity determinants
- Catabolic systems
How can tranposition cause a loss of function?
Insertion of transpositional element into gene
How can transposition cause a gain of function?
Insertion of transposition element that contains a promoter before a gene region
What cause resolution of a direct repeat indicate? An inverted repeat?
What are the two types of transposons in eukaryotes?
- Classical DNA transposons
What are the five characteristics of classical DNA transposons?
- Prokaryotic-like transposons
- Flanked by terminal inverted repeat sequences
- Target-site duplications
- Autonomous or non-autonomous
What are two examples of classical DNA transposons?
- Ac/Ds system in maize
- P Elements in Drosophila
What are the Ac and Ds gene regions?
Ac is an autonomous transposon or activator while Ds is a set of related, non-autonomous transposons or dissociator
What is the ordering of lengths of transposases among Ds-a, Ds-b, and Ds-c from greatest to least?
Ds-a > Ds-b > Ds-c
What gene in maize controls the color?
Gene c controls pigment production
How does the insertion of a mutation into gene c work and what is the outcome?
Ac transposes Ds into gene c causing a lack of pigment
How does a spotted kernal form?
Excision of mutation of Ds out of gene c
What is the phenomenon when a male fruit fly with a P element mates with a female fruit fly without P elements?
Why does P element transposition not occur if the female fruit fly has P elements but does occur if the male fruit fly has P elements?
The female egg is large so a large portion of repressor is produced that represses transposition while the male sperm is small meaning not a lot of repressor is produced causing more transposition
What are the four properties of retrotransposons?
- replicative transposition
- transpose via an RNA intermediate
- reverse transcriptase (RNA to DNA)
- autonomous or non-autonomous
What are the two types of replicative transposons?
- long-term repeats
- non long-term repeats
What are the six properties of LTR retrotransposons?
- Also called viral-like retrotransposons
- Long terminal repeats
- IR embedded in LTR
- target-site duplications
- reverse transcriptase (RT), integrase
- viral coat-like proteins
What are the four steps of the LTR retrotransposon lifecycle?
1. Transcription of retrotransposon
2. Translation of mRNA to make RT, Int, viral-like coat proteins in cytoplasm.
3. Reverse transcriptase produces dsDNA that is the orignal retrotransposon region
4. Integrase integrates retrotransposon into new site
Where do Ty elements usually transpose?
Upstream of RNA pol III promoters
What are the seven properties of non-LTR retrotransposons?
- Also called poly-A retrotransposons
- No LTRs
- No IR
- Target-site duplications
- 5' and 3' untranslated regions (UTR)
- May have ORFs that encode an RNA binding enzyme and an enzyme with reverse transcriptase/endonuclease activity
- poly-A tail
What are the seven steps in the lifecycle of Non-LTR retrotransposition?
2. Export of mRNA out of nucleus
3. Translation and protein association
4. RNA binding by RT/Endonuclease
5. Import into nucleus
6. Reverse transcription of RNA to dsDNA
What are the specific steps in transposition of mRNA sequence into DNA?
1. Transcription of transposable element.
2. Translation of mRNA producing ORF1 and ORF2 proteins that bind to mRNA near the poly-A site
3. Binding of mRNA to target DNA
4. Endonuclease cleaves target site forming RNA-DNA hybrid
5. Synthesis of cDNA strand
6. RNA degradation and second strand synthesis
7. Joining and repairing completing transposition
What are examples of Non-LTR containing retrotransposons?
Long Interspersed Nuclear Element (LINE)
Short Interspersed Nuclear Element (SINE)
What is the major property of LINE transposons?
Most are non-autonomous and inactive
What is the major property of SINE transposons?
All are non-autonomous, do not encode functional proteins
What are examples of retroviruses?
- Rous Sarcoma Virus (RSV)
- Human Immunodeficiency Virus (HIV)
What is the name of a DNA copy integrated into the host genome?
What are the two sites of gene regulation in eukaryotes?
- Transcriptional control
- Post transcriptional control
What are the three types of RNAP in eukaryotes and what do they transcribe?
- RNAP I for rRNA
- RNAP II for mRNA
- RNAP III for tRNA, 5S rRNA, and others
What is most of the control in eukaryotes?
What are the two fundamental mechanisms of gene regulation in eukaryotes?
- Nucleosome remodeling
- Recruitment of RNA polymerase
What type of DNA produces little transcription and what type produces lots of transcription?
What are the steps for nucleosome remodeling?
1. Activator binds enhancer
2. Histone acetylase binds activator
3. Histone acetylase acetylates nucleosome exposing promoter increasing transcription
How does chromatin remodeler work?
1. Activator binds enhancer
2. Chromatin remodeling complex binds activator
3. Nucleosome is remodeled exposing promoter causing more transcription
What are two mechanisms SWI/SNF in chromatin remodeling?
- Sliding where DNA slides along the nucleosome exposing the transcribable region
- Transfer where the transcribable region is placed on a free exposed DNA segment
How does recruitment of RNAP work?
1. Activator binds to enhancement region
2. Activator attracts mediator that is bound to RNAP or
TFIID-TBP that attracts RNAP down the line
What is the mediator complex consisted of?
Over 20 subunits
What is the activator of gal genes for Saccharomyces cerevisiae?
What are the three domains in Gal4?
- DNA binding domain
- Acid Activation Domain
- Dimerization Domain
What is the site that Gal4 binds to?
Upstream Activating Sequence
What is required for Gal4 to be able to bind to the UAS sites and what is the function of this complex?
RSC complex that engages in nucleosome remodelling allowing the region to be exposed for Gal4 to bind to
What does Gal4 interact with to allow greater transcription of gal genes?
SWI/SNF chromatin remodelers
What two things does Gal4 do to enhance transcription of Gal regions?
- Recruits RNAP laborers
- Recruits chromatin remodelers
What three transcriptional regulators are involved in sensing galactose and what are their roles?
- Gal4: transcriptional activator
- Gal80: anti-activator which binds Gal4
- Gal3: an anti-anti-activator which binds Gal80 and senses galactose
How does high concentrations of galactose stimulate the transcription of gal genes?
Gal3-galactose binds to Gal80 and causes Gal80 to be released from Gal4
What residues of proteins can be phosphorylated?
What does CREB stand for?
cAMP response element binding protein
How does CREB get activated?
1. cAMP activates Protein Kinase A
2. Activated Protein Kinase A phosphorylates CREB
3. Phosphorylated CREB interacts with and helps recruit RNAP II
What are the five steps of the JAK-STAT pathway?
1. Ligand binding
2. Receptor dimerization
3. Receptor autophosphorylation by JAK kinase
4. Binding and phosphorylation of STAT
5. STAT dimerization and activation of transcription in nucleus
What are the four steps of the RAS and MAP Kinase (MAPK) pathway?
- Ligand binding, receptor dimerization, auto phosphorylation
- Binding and activation of RAS by Grb2 and SOS
- Cascade of kinase activities
- Activation and transcription in the nucleus
What are the two components of hormone-response systems and their functions?
- Hormone receptor: transcriptional activator
- Hormone response element - a DNA binding site
What is an example of an hormone receptor activation in the cytoplasm and how does it work?
1. Glucocorticoid hormone enters the cell and kicks off Hsp90 (heat shock protein) from glucocorticoid receptor
2. Glucocorticoid receptor (GR) binds GRE and stimulates transcription
What is an example of an hormone receptor activation in the nucleus and how does it work?
1. Hormone receptor binds to response element but is inactive
2. Thyroid hormone binds to hormone receptor allowing recruitment of RNAP
What is the process of occlusion and what types of organisms engage in this gene regulatory process?
Repressor protein can directly block RNAP binding, prokaryotes
What are the four types of repression mechanisms in eukaryotes?
- Direct Repression
- Indirect Repression
How does competitive repression work?
Protein binds to repressor binding site which overlaps activator binding site causing the activator protein to be unable to bind
How does inhibition repression work?
Both the activator and repressor protein can bind their sites but the repressor protein interacts with the activator protein that does not allow it to recruit RNAP
How does direct repression work?
The repressor binds the site and interacts with the transcription laborers preventing transcription
How does indirect repression work?
Histone deacetylase converts acetyl groups to amine groups causing the promoter to be hidden in a web of nucleosomes
What are the steps taken when glucose is present to prevent transcription of gal1?
1. Glucose promotes Mig1 to bind to the Mig1 site
2. Mig1 recruits Tup1 to the site
3. Tup1 either deacetylates sites around the promoter making it less accessible or may directly interact with the transcription laborers to inhibit initiation
What prevents a enhancer protein from promoting the transcription at any given promoter? What is an example of such protein?
Where are insulators located?
At the beginnings of a DNA loop that maintains the structure
Where is interaction between enhancer and promoter permitted and where is it disallowed?
Interaction prohibited between DNA loops
Interaction permitted within DNA loops
What are two roles of DNA methylation in eukaryotes?
- Regulate chromatin structure
- Silence gene transcription
Where does DNA methyltransferase add the methyl group to cytosine?
Can methylcytosine base pair with guanine?
What is the main target for DNA methylation?
How much of the CpG sites are methylated in human adults?
Where are the majority of CpG sites clustered together and what is the term for this?
promoter linked cis-regulatory regions of protein-coding genes
What are the two manners in which DNA methylation silences gene expression?
- May directly disrupt binding of the transcription machinery or activators
- Helps recruit proteins involved in chromatin remodeling
How is the DNA methylation pattern passed down to daughter cells in two steps?
1. DNA is replicated creating a hemi-methylated CpG
2. An enzyme called maintenance methyltransferase follows the replication fork restoring the fully methylated CpGs
Do females express genes in both X chromosomes at high levels?
No, one chromosome is epigenetically silenced
What two actions are done to inactivate female X chromosomes?
- Hypermethylation of its DNA
- Hypoacetylation of its histones
What is the heavily condensed DNA of the inactive female X chromosome called?
When does inactivation of the X chromosome occur?
32-64 cells after fertilization
Does inactivation occur randomly?
What is combinatorial control?
More than one signal controls expression of a given gene where the more bound the better the transcription rate
What is cooperative activation?
All proteins must bind for transcription to occur
What are the progression of steps in splicing?
1. U1 binds 5' splice site
2. U2 binds to branchpoint recognition sequence
3. U4-U5-U6 complex binds to 5' splice site
4. U1 is displaced by U6
5. U6 binds to U2 bringing 5'-splice site near the branchpoint
6. U5 binds exon sequences and intron is removed as lariat
What are the four alternative splicing patterns?
- Alternative selection of promoters
- Alternative selection of cleavage/polyadenylation sites
- Intron retaining mode
- Exon cassette mode
What is the alternative selection of promoters?
Alternative promoter used and splicing result in proteins with different N-terminal sequences
What is the alternative selection of cleavage/polyadenylation sites?
Alternative polyadenylation sites recognized and splicing result in proteins with different C-terminal sequences
What are the two regulations of splicing of mRNA?
- Splicing activators
- Splicing repressors
What is an example of a splicing activator? What is it rich in?
SR protein, serine and arginine
What are the two major domains of the SR protein?
- Binds the RNA
- Binds the splicing laborers
Where does SR protein bind to?
Exonic splicing enhancers (ESEs)
How does differences in chromosomes affect expression of genes in fruit flies?
In XX fruit flies there is twice the number of expression for transcriptional activators SisA and SisB meaning far greater amounts than the Deadpan transcriptional repressor encoded by autosomal chromosomes. Increase in transcriptional activators leads to development of sex lethal protein (SxI) in XX but not in XY
What are the three genes involved in sex determination in fruit flies and their functions?
- sex lethal: splicing repressor
- transformer: splicing activator
- double sex (transcriptional factor)
What is the progression of steps that cause sex differentiation in fruit flies?
1. sxI gene is expressed in females causing exons 2 and 3 in sxI operon to be inhibited from splicing leading to 2 and 4 exons to be joined together to form functional sxI protein
2. SxI protein inhibits splicing between exons 1 and 2 in tra operon causing 1 and 3 exons to join together creating functional Tra
3. Tra binds to Dsx at exon 4 causing early termination and expression of first four exons instead of the six exons that would be present in males
What is the function of the Dsx protein in females? In males?
- Repress male genes and activate female genes
- Repress female genes
How is iron brought into the cell?
Transferrin binds iron and transferrin receptor intakes the complex so iron can enter the cell
What is the relationship between transferrin receptor and iron concentration?
How is Tfr mRNA stability influenced by iron concentration?
mRNA is stable when iron concentrations are low and unstable when iron concentrations are high
What binds to mRNA under low iron conditions?
Aconitase or iron response protein (IRP)
Where is aconitase also found?
What does aconitase do under high iron concentrations?
Binds iron, ditches mRNA for Tfr, and is used in the TCA cycle
Is apoaconitase able to be used for the TCA cycle?
What are the two major processes for RNAi?
- Protecting cells from dsRNA viruses
- Regulating gene expression
What are the steps of RNAi mechanism?
1. Dicer cleaves dsRNA creating siRNA
2. Specific proteins associate with siRNA forming an RNA-induced silencing complex (RISC)
3. RISC binds by base pair complementarity
4. mRNA either is degraded to repressed
What part of Dicer binds to the 3' end of RNA?
Which outcome degrades mRNA and which outcome represses translation while keeping mRNA intact?
Perfect base-pairing causes degradation
Imperfect base-pairing causes translational repression
How does RNAi help with protecting against viruses?
Viruses that embed their genetic material into the genome will transcribe their own mRNA and when a dsRNA is injected by a virus it gets cleaved into siRNA and binds to the viral mRNA transcript preventing translation
What is microRNA cleaved by?
What does miRNA form when exiting the nucleus and entering the cytoplasm?
What cleaves miRNA to form what?
Dicer to form RISC
Is there perfect base-pairing between mRNA and cleaved miRNA?
Where are three places dsRNA come from?
- dsRNA virus
- microRNA genes
What are the two types of transgenes?
- planned, antisense RNA
What is antisense RNA?
An RNA complementary to an mRNA
What are antisense annealing to mRNA trigger?
Dicer mediated cleavage, RISC formation and RNAi
What are six possible antigens?
- nucleic acids
- small molecules
- large molecules
What part of the antigen does the antibody bind to?
What are the two domains of antibodies?
- Variable domains
- Constant domains
Does the light chain or heavy chain bind the antibody?
What are the five classes or isotypes of antibodies?
IgG, IgM, IgA, IgE, IgD
What determines the class or isotypes of antibodies?
How are multiple combination of antibodies developed?
Which cells begin making antibodies?
How many variable gene segments, diversity gene segments, joining gene segments, and constant domain gene segments are there in what type of cell and what part of the antibody does this code for?
- 100 variable gene segments
- 12 diversity gene segments
- 4 joining gene segments
- 5 constant gene segments
How many variable gene segments, joining gene segments, and constant domain gene segments are there in what type of cell and what part of the antibody does this code for?
- 300 variable gene segments
- 4 joining gene segments
- 2 constant domain gene segments
Where is somatic recombination imprecise?
H and L chain
How does clonal selection work?
- Ag binding to right B cell
- Activation occurs
- Clonal expansion and proliferation
- Differentiation into Memory B cells or plasma cells to fight infection
What are the three dna sites involved in somatic recombination?
- recombination signal sequences
- found next to V, D, and J gene segments
- bind recombinase
What are the protein factors involved in somatic recombination?
- RAG-1/RAG-2 (recombination activating gene), a site-specific recombinase
- HMG, a DNA bending protein
What is the makeup of the recombination signal sequences?
- 12 bp spacer or 23 bp spacer
Where are the 12 and 23 bp spacer in the V, D, and J regions?
23 bp spacer by the V and J segments and 12 bp spacer by the D segment
Where are the 12 and 23 bp spacer in the light chain?
23 bp spacer and one end and 12 bp spacer on the other end
Do the spacers have to be unlike for recombinases to join the ends together?
What are the first two steps of the mechanism of somatic recombination?
1. Single-stranded cleavage
2. Hairpin formation
What are the two possible outcomes after the first two steps?
- Signal joint formation that is discarded
- Hairpin opening and fusion of coding segment
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