Quizz
Part I DNA and RNA structure
Name the DNA bases and classify them?
Adenine, Guanine, Thymin, Cytosine. A,G: Purin bases C, T Pyrimidine Bases
What does a nucleotide consist of?
Base, Ribose, phosphate ester
What is the structural formula of AMP?
XXPic
What types of double stranded DNA do you know?
A, B, Z
What is the most common, physilogical type of DNA?
B-DNA
How many bases are there per turn in B-DNA?
10.5
Name at least two ways to denature DNA?
By heating, high pH
What is the major principle underlying Northern Blotting? Explain this principle
Hybridization: Annealing of complementary (similar ) strands. (A little figure would help)
Explain a southern blot?
XXpicture:
DNA is cut and run on a gel, thus separted by size, denatured and transfered to immobilized membrane, incubation with labelled probe, probe hybridizes, unbound probe is washed away, label oif probe is visualized
At which wavelength does DNA absorb UV light? (The absorbance peak)
260nm (This number is so important that you should know it by heart)
What can you observe if you heat double stranded DNA in a photometer monitoring at 260nm? Explain this effect
At some point the absorbance at 260 nm rises drastically. This is because single stranded DNA absorbs proprtionally more UV light. This is dependent on base stacking.
Explain the melting temperature Tm of DNA?
The mid point of transition between double helical and single stranded DNA. Thus in other words half of the DNA is single and half is double stranded.
How could you determine the melting temperarture of a DNA?
Slowly heating and monitoring at 260nm. Plotting A260 versus temperature and determining the point in which the absorbance has risen by half a delta (final, begin) (see lectures slide)
Give at least two things that influence the melting temperature of DNA? (With explanation)
GC content, 3 hydrogen bonds versus 2 for AT pairs, stringer stacking effects
ionic strength, negativ charges of the two DNA backbones repell each other, this can be shielded by cations
What is the tyical topological form plasmid DNA is in? What consequences does this have?
In a closed circle. As the circle is closed, constraints are imposed.
Why can the supercoiling of DNA play an important role in day to day lab routine?
Cicrular DNA runs different if either being relaxed or in supercoiled form.
Describe why Ethidium bromide can be used to visualized DNA?
Ehtididum Bromide intercalates in the DNA. When Ethdium bromide is intercalated in DNA its fluorecence increases dramatically and thus DNA can be detected.
How does Ethidium Bromide effect DNA?
It unwinds the DNA.
Explain the Lk (Linkage) number of DNA?
The number of times one strands wraps around the other
How can the linkage number of circular double stranded DNA be changed?
Only by at least single strand breakage
Which enzymes change the linkage number of DNA? Which classes do you know and what do they do?
Topoisomerases classes I and II. Topoisomerases I breaks one strand, Topoisomerase II breaks both strands. (Extra points for subclassiifcation)
Why is Topoisomerase II of vital importance for organisms?
Dentanglement of DNA after replication
Schematically show how topoisomerases work?
XXpicture
What are the major differences between DNA and RNA?
RNA uses Uracil instead of Thymine. RNA uses Ribose instead of deoxyribose. RNA can form non canonical base pairs. RNA can bild complex strcutures
Part II Packaging of DNA
What is so remarkable about RNA that made some people speculate about an RNA world?
RNA can act as an enzyme and can serve as a store of genetic information.
Why does DNA need to be packaged?
DNA as a molecule is very long. It needs to fit into a cell or into the nucleus
Do bacteria have histones?
No but they have similarly positively charged proteins, like IHF (Integration host factor)
What are the four histones making up the core particle
H2a, H2b, H3, H4
Where does H1 come into play?
As an outer particle, additional packing
How long (in nt) is a strech of DNA around a nucleosme?
~146b
How can one determine the length of DNA around a nucleosome?
micrococal DNAase digestion, the nucleosme protects the DNA, then run the fragments on a gel
If you digest chromosomal DNA with micrococal DNA and run them on a gel you get shortisch fragments of around 170-200bp but also longer and much longer bands, where are these coming from?
Not complete digestion
What histone modifications do you know?
–Acetylation (Ac)
–Ubiquitination (Ub)
–Methylation (Me)
–Phosphorylation (P)
–Sumoylation (Su)
What does HAT stand for?
Histone acetylase.
What is H3K27me3 short for and with what is this histone modification associated?
Histone 3 Lysine 27 triple metylated
It is associated with genes.
Which chemical modification of DNA do you know?
Metyhlation of Cytosines
Sometimes "packed" chromosomal DNA needs to be accessed, how is this problem solved?
By nucleosome remodelling factors. These can change the localization of the nucloesomes with respect to the DNA
What does a bromo- and what does a chromodomain do, in the context of binding to certain structures?
-Bromodomain (binds acetylated lysines)
-Chromodomain (binds methylated lysines)
Part III DNA Replication and Repair
Why is DNA methylation risky?
Methylated cytosines are less chemically stable, so more likely to undergo deamination, which changes the base to thymine
What can CsCl gradients be used for?
For separation, e.g. 14N and 15N DNA
What does semi-conservative DNA replication mean?
Two copies are produced and each contains one original strand
Be able to interpret the Raghuraman et al experiment (if you get similar results and techniques)
XXXPic the Raghuraman experiment
Where does replication begin?
At specific sites: ori
What is the role of DnaA in E.coli.
Its role is in replication initiation. DnaA protein binds to Ori site (DnaA boxes on the DNA) DnaA -ATP multimerize which bends the DNA thus facilitating its unwinding (often at AT rich sites close to the ori)
more background available Bacterial replication initiator DnaA. Rules for DnaA binding and rolesof DnaA in origin unwinding and helicase loading
The DNA helicase DnaB is necessary in DNA replication, can you explain why?
DNA needs to be opened to make it accesible
What is the role of single stranded binding proteins during replication?
To stabilize single stranded DNA
During DNA replication the DNA is unwound by a helicase, which topological problem is induced by this and how is it solved?
The DNA is overwound, this is relaxed by topoisomerases
How is the great fidelity of DNA replicative polymerase explained?
mismatches have a different shape, in addition the polymerase has a proofreading activity
Which complex helps DNA polymerase to become processive as opposed to falling off and jumping on all the time?
The sliding clamp which is loaded by the sliding clamp loader
What does DNA polymerase need to start synthesis (apart from a template, dNTPs, etc)?
It needs a primer, this is synthesized by Primase
What are Okazaki fragments and why do they occur?
They are a result of DNA synthesis on the lagging strand, where DNA is made in short discontinous stretches, as the fork moves.
What is the difference in DNA synthesis primer removal by pro- and eukaryotes?
Procaryotes can directly remove the primer with DNA polymerase I, wherease in eukaryotes the RNA primer + a bit of strand are displaced and then cut off by e.g. Fen1
What has to be taken into account w.r.t. histones when DNA is newly synthesized?
The histones code has to be preserved.
DnaA occurs in a ATP and ADP bound form, why?
DnaA can not initiate origin firing, this is one of the safeguard mechanisms against overstimulation of replication initiation
Dam methylase methylates the A in the sequence GATC, what is the role in the context of DNA replication?
GATC overlaps oriC after replication these sites are only hemimethylated a secific protein bins these sites (SeqA) and thus blocks the ori
DNA repair
What is the role of telomerase?
it solved the "end" problem by prolonging special structures "telomeres" at chromosome ends.
What is a transition?
Purin versus Purin exchange
What is a transversion?
Purin versus Pyrimidin exchange, or vice versa
Name some sources of mistakes which could eventually lead to mutations:
tuatomeric structures of DNA bases
cell internal metabolies, e.g. reactive oxygen species
external chemicals e.g. alkylation agents
radiation e.g. UV or X-ray
deamination
What is a simple mechanism to repair UV induced tymine dimerization?
Direct reversal of the damage by photolyases
Ada is involved in alkylation repaair, what was special about this repair
The protein transfers the alkyl group onto itself, as this is irreversible the protein is no enzymes and is thus "used up" 3/14
What is the role of Mut SLH?
Its role is in mismatch repair MutS recognizes a mismatch, MutH a hemimethylated strand and nicks the strand 3/15
Explain Base excision repair
damaged bases are removed by glycosylase (there are many different ones) AP site is recognized by an endonuclease, it cleaves the backbone and the hole is filled 3/17
When is nucleotide excision repair being used?
For bulky lesions 3/18
What are TLS polymerase
Translesion polymerases, they are able to polymerize in a non-templated fashion when damage has occurred 3/19
Why are TLS polymerase a last resort?
The TLs polymerase are error prone and just enusure that DNa can be replicated, this often leads to mutations. It can sageguard replication though 3/19ff
Recombination
Explain the LexA-RecA system.
Normally LexA is blocking the syntehsis of SOS genes by binding to a DNA operator. If RecA bind ssDNA it is activated and cleaves LexA. Cleaved LexA can't bind to the operator anymore, SOS genes are read. 3/27
What is NHEJ
Non homologous end joining, joining DNA ends 4/10 ff
When resection occurs during NHEJ, what happens?
DNA ends are resected, regions of microhomology are found, DNA pairs, gaps are filled and overhangs trimmed 4/13
Briefly depict the process of homology directed repair with synthesis dependte strand annealing (no protein names are necessary, but give the neccessary terms for the DNA structures)?
Resection occurs to generate 3' overhangs, one of the 3' overhangs invades an intact duplex, generating a heteroduplex and a displacement loop,
new DNA is synthesized, until this can pair with the other 3' overhang. Then repair replication and synthesis 4/17-18 (Probably helpful if you could draw a diagrm here)
Genes involved in repair and recombination are often implicated in some diseases as well, name two example for such a disease
Werner Syndrome
Bloom Syndrome
Rothmund Thomson Syndrome
What is the role of RecA (RAd51) in homology directed repair, where else does it occur?
It loads onto the single stranded DNA and helps in strand exchange 4/30 it also plays a role in homolgous recombination
What is the consequence of using homologous chromosomes as templates for homology-directed repair?
DNa is changed and is afterwards a copy of the homologous chromosome. This can lead to a loss of heterozygousity 4/37
Does the cleavage of a holiday junction always lead to large areas of recombinant product?
No this depends on how the junction is cleaved 4/44
Where is RuvAB involved in the broad complex of recombination?
Movement of the holiday junctions
Depict the first steps in homologous recombination
After a double strand break, DNA is resected and bound by proteins which help in inavding the other duplex. DNA
is synthesiszed from the invading strand. The displaced strand of the D-loop is captured by the remaining 3' end.
DNA synthesis occurs and holiday junctions form 4/51
What is the relationship between homology directed repair and homologous recombination?
They are similar and the in initial processes are almost the same 4/52
How can damaged replication forks (specifically nicks) be repaired with a principle remeniscent of recombination?
single strand nick leads to a ds break after strand separtion. One strand of the "broken off" end invades the
"whole" part a D-loop structure is formed, DNA synthesis occurs, the displaces strand is captured by the lagging strand
lagging strand synthesis is restarted and after clevaged the fork is restored see 4/54
Describe Break Indcued replication
Resection occurs to generate 3' overhangs, one of the 3' overhangs invades an intact duplex, generating a heteroduplex and a displacement loop,
new DNA is synthesized. The displaced strand from the D-loop is not captured. Here lagging strand synthesis starts occuring
What are some consequences of Break Indcued replication?
Potentially loss of heterozygousity
Potenntially this Replication is not as precise as normal replication 4/58f
Mobile Elements
Name at least two problems that can occur with recombinationndue to sequence similarity regions?
Recombination on the same chromosome
Unequal crossover between chromatids or between homologs can lead to deletions and duplications
Crossing over between different chromosomes can lead to DNA translocation 4/61
Be reads to explain a C0t curve
XXPic C0t Curve 5a/2
What did Barbara McClintock discover
transposition / transposable elements 5a/3
Explain autonmous and non-autonomous elements
Autonomous elements encode the enzmes needed to move. Non-autonomous elements lack these and rely on enzymes of Autonomous elements 5a/4
Would you rate the number of transposable elements in the humann genome as high or low?D
High 5a/4
What is the state of many transposons in the human genome?
They are inactive e.g. due to mutation 5a/9
If a mobile elements jumps into a functional gene, what can be the consequence for gene function?
The gene function or regulation can be affected potentially resulting in disease 5a/5
Does transposition necessarily require homology?
No in general not, but be aware of the special cases 5a/10 and e.g. 5b/64
What are Class I and Class II elements?
Class I elements are retrotransposons that move via an RNA intermediate, Class II elements are DNA only transposons 5a/11
What is the general structure of a Class II element?
It has terminal inverted repeats and a transposase gene. 5a/15
What are the two major classes of Class I elments?
LTR and non LTR 5a/15
What is remarkable for Class II elements when it comes to the species barrier?
Class II elements can likely cross the species barrier 5a/19
What is the predominant movement mechanism for DNA elements?
Cut and Paste 5a/20
What is the less common movement mechanism for DNA elements?
Nick and Paste 5a/20
When it comes to bacterial transposons, which other genes apart from the transposase do they often carry?
E.g. pathogenicity factors and antibiotica resistance 5a/21
What is an IS Element?
A bacterial cut and paste DNA element only encoding its own transposase 5a/22
What is a compound transposon?
Two simple transpons (IS elements) flanking another gene(s) 5a/22
For a DNA cut and paste transposon, how many transposase proteins would you need in general to mobilize one transposon?
Two 5a/25
How is it ensured that transposon ends are brought together before cutting for cut and paste DNA elements?
The transposase cuts in trans 5a/26
In the case of DNA cut and paste elements: When such an element inserts into the sequence CGAT. How will the sequence look after insertion.
GCATxxxxTransposonxxxxGCAT 5a/27
After a DNA cut and paste transposon leaves a certain site a footprint remains. Explain what this is.
Upon insertion a piece of DNA is duplicated, when the transposon leaves, this duplicated DNA can remain -> a footprint 5a/30
After a DNA cut and paste transposon leaves a certain site a precise excision can occur. Explain what this is and name conditions.
When the transposon leaves and the resulting gap can be repaired by homolgy directed repair from a transposon free site, the original state can be resoted and no footprint remains 5a/32
Explain in broad, superficial terms where VDJ recombination occurs?
Antibodies joining of different pieces, more possible antibodies, several possible fragments for each V D J pieces 5a/34ff
Mobible Elements
What is the relationship between VDJ recombination and transposons?
They could be related as there are certain similarites both in the enzymes as well as in the signal DNa sequences 5a/38
Mobile Elements
Name an application in the lab for the Drosophile P element?
Using microinjection this is a way to make transgeic flies. 5a/42
Mobile Elements
What is sleeping beauty and how was "she" awakened from "her" sleep?
This is a fish transpon which was no longer active. Using the consensus sequence the ancestral working state could be restored 5a/43
What is sleeping beauty and how did "she" fall asleep in the first place?
This is a fish transpon which was no longer active. Due to mutations the inserted transposons became "dormant" 5a/43f
Name the two clasess of RNA retrotransposons
LTR and non-LTR elements 5b/51
Mobile elements
What does LTR stand for in LTR (RNA) elements?
long terminal repeat 5b/51
Mobile Elements
Within a LTR Retroelement you usually find coding regions for 4 proteins, anme these?
INtegrase, GAG, PROtease, RT-RNase reverse transcriptase+ RNAse activity 5b/54
You have a working LTR retroelement, when you compare that to a retrovirus which protein coding regions is missing?
ENV for extracellular stage 5b/55
Mobile Elements
Describe how a LTR element multiplies and integrates into new regions?
What can happen when you have two LTR elements spaced a bit apart from each other with the intervening DNA?
It can get lost due to hmologous recombination bewteen the elements 5b/59
Do you rate the non-LTR element occurrence in mamamalian genomes as absent/low/medium/high/very high?
very high 5b/61
Why are RNA elements very often mutated?
Reverse Transcription is error prone. reverse transcription is part of the life cycle. 5b/61
What are the typical elements for a LINE element
ORF1/ORF2 and polyA 5b/61
What is the sequence requirement for an insertion of some non-LTR elements (e.g. LINEs) and why?
a stretch of Ts that pair with the polyA tail 5b/64
How does DNA methylation affect IS10 transposition?
DNA methylation both prevents transposase expresison and transposon end activity 5b/76
In which cells is the Drosophila P element active and in which one is it not active?
It is active in germ line but not somatic cells. 5b/77
Ty5 is a relatively "benigin" element when it comes to gene mutations why?
It preferabley inserts into heterochromatin 5b/80
Explain what local hopping means for certain transposons?
If transposons preferably insert in a location close to the donor site. 5b/80
What does CSSR stand for?
Conservaive site-specific recombination 5c/88
When it comes to CSSR recombinases which enzyme class do they fall into?
They are topoisomerases 5c/90
Which two major classes of CSSR recombinases exist (hint name the AA)?
Serine and Tyrosine type 5c/90
What is the difference between Serine and Tyrosine Recombinases when it comes to the intermedieate form, describe both forms?
Tyrosine recombinases break DNA and form DNA-3P-tyrosine linkage, Serine recombinases break DNA and form a DNA 5P-serine linkage 5c/90
What is the Cre enzyme and what does it do?
a (tyrosine) recombinase that recognizes lox sites? 5c/100
For what can the cre lox system be used for?
To delete DNA between two lox sites, this is often used as a tool where Cre is expressed under a specfic promoter. After Cre expression, a piece of DNA is removed and for example a tissue specific knockout is made.
The bacteriophage lambda system has four sites that are named on DNA (hint two sites get converted into two other sites), name the sites?
attP, attB -> attL, attR 5c/121
What is the Invitrogen Getway system system building on (which molecular mechanism from which organisms)?
phage lambda recombination 5c/121
Transcription
What are the major two differences between RNA and DNA?
RNA has U instead of T aand ribose instead of deoxyribose. 6a/2
Name the three major phases in transcription
Initiation, Elongation, Termination 6a/3
Name the three major eukaryotic RNA polymerases and their primary role
RNA polymerase I (large ribosomal RNA), II (mRNA), III (tRNA and 5S rRNA)
What is special for plant RNA polymerases?
They have an additional polymerase (apart form I, II , III) to transcribe regulatory RNAs
When it comes to how much work RNA polymerases have how would you rate the transcription of RNA Polymerase I+III 0/4< x <1/4< x <2/4< x <3/4< x <4/4 ?
3/4 <x< 4/4 or the main load 6a/5
What can happen to the C-terminal domain of eukaryotic RNA polymerase II?
It can become phosphorylated 6a/8
What does the bacterial sigma factor do?
It contacts the prmooter 6a/10
What are the major two bacterial promoter elements (that are close to the TSS) (hint name them by position)?
-35 and -10 element
In which bacterial promoters do you find the UP element? (somewhat active, medium active, very active)?
very active 6a/11
Do you know the consensensus sequence of the -10 sigma70 E.coli element?
TATAAT 6a/11
What is an anti-sigma factor?
Proteins beinfind to sigma factors and inhibiting their function 6a/13
In S. typhimurium flagelum assembly an anti sigma factor is involved, can you say how this factor FlgM is later loosing its influence
It is exported through the incomplete flagellum apparatus 6a/14
Eukaryotic promoers have a box very similar in sequence to the bacterial -10 element, how is this box called?
TATA box 6a/16
Which protein do all thee eukaryotic RNA polymerases need for establishing the pre-initiaion complex?
TATA binding protein TBP 6a/16
When it comes to transcription what is the hallmark of a closed complex?
RNA polymerase is in position and the DNA is not yet opened, then the complex of polymerase and promoter is called closed complex 6a/21
When it comes to transcription what is the hallmark of an open complex?
RNA polymerase has opned up the transcription bubble
Interestingly, when RNA polymerase starts making RNA, a strange phenomenen occurs resulting in short pieces of RNA, explain?
RNA polymerase is not immedetealy working fully instead it enters cycles of abortive inititaiion reuslting in short pieces of RNA being released. This is likely due to a loop of eukaryotic TFIIB and bacterial sigma factors extending into the polymerase 6a/24ff
What is promoter clearance?
When the loop of TFIIB/sigma inserted into the RNA polymerase is displaced and the polymerase breaks away from the promoter 6a/26
What happens to the CTD when eukaryotic RNA polymerase is conevrted into the elognating complex after promoter clearance=
It becomes phosphorylated 6a/26
Describe a simple! experiment to show that abortive initiation takes place in vitro
Incube RNA polymerase holoenzyme with radioactive nucleotides. Run products on gel and visualize with Xray film 6a/27
To show that abortive initiation takes place in vivo special kinds of nucleic acids were used, name them and idicate why these were used?
locked nucleic acids (these are locked in a conformation more favourable for hybridization)
What is the approximate speed of RNA polymerase 0<x <10 x <100< x <1000< x <10000< x <100000 nucleotides per second?
10< x <100 (20-50 nt/s) 6a/31
What is promoter proximal pausing?
When thete is some pausing after only 35-5ß bp have been synthezied by RNA polymerase 6a/32
What are potential reasons for transcriptional pausing?
Short complementary regions in the nascent transcrip forming hairpins or weak DNA-RNA hybrids 6a/32
Which factors can relieve transcriptional pausing?
Elongation factors 6a/32
Transcription
Explain how transcriptional pausing may be relieved?
RNA polymerases reverses direction, mots recently made RNA sepeartes of from DNA this protruding end can be chopped off 6a/33
What is one of the first maturation steps that occurs to eukaryotic mRNA?
This is often capping happening while RNA is still being made 6a/35
What happens after capping to the eu. RNA polymerase II CTD?
It is phosphorlyated at additional serines 6a/36
What is the role of the 5' cap for the mRNA?
It helps in nuclease degradation, elongation termination of transcript, mRNA preocessing, export from the nucleus and directiong translaation 6a/37
What is the minimal structure incorporated into a cap?
7-methylguanine is lined via a 5'-5' triphsphate to the 5' end of the RNA. (In more complex eurkaryotes 2' O of ribose in the second and third base gt methylated) 6a/37
Describe schematically how capping proceeds?
removal of 5' phosphate (PPP-RNA -> PP-RNA), addition of GMP (GPPP-RNA), methlyation of guaanine m7GPPP-RNA)6a/38
What taks care of the nucleosome problem during transcription?
histone chpaperons 6a/39
What topological problem can transcription cause?
Changes in supercoiling 6a/40
What is a (bacterial) intrinsic terminator?
A site where bacterial RNA polymerase terminates transcription without any aadditional factors 6a/42
What are the two main features of a bacterial (non enzymic) terminator?
An inverted repeat that forms a stem loop in the RNA, and a polyA (DNA) region, that leads to less stable AU bse pairs 6a/43
What is the bacterial Rho protein (field transcription)?
An enymatic terminator 6a/44
What is the difference between intrinsic and Rho dependent genes in terms of termination?
The former make a hairpin and have a poly A region the latter (Rgho) don't 6a/44
Which eucaryotic RNA polymerase recognizes intrinsic terminators?
RNA Polymerase III 6a/46
Transcription
When you compare bacterial intrinsic terminators to eu. RNA polymerase III terminators what does the latter not need?
RNA pol III doesn't need the RNA hairpinn 6a/46
Transcription
What is eu. RNA polymerase II termination coupled to?
It is coupled to 3' end processing 6a/47
What happens to most eukaryotic 3' mRNAs?
They get polyadenylated 6a/47
Is the polyA tail of eu. mRNA added to the last based made by RNA polymerse II?
No the nascent transcript is cleaved first and then the polyA tail is added. 6a/48
Give the names of the two major model for eu. RNA polymerase II termination?
torpedo and allosteric model 6a/48
What must a RNA transcription regulatory protein do?
It must specifically recognize its right regulatory sequence e.g. through a protein domain 6b/48
Why can it be favourable to contact the major grove of DNA in terms of sepcificity?
In the major grove all bases can potentially be distinguished this is not nnecessarily the case for the minor grove 6a/60
Which kind of amino acids would you expect to find in proteins that contact DNA and why?
positively charged amino acids, as the DNA is negatively charged 6a/62
Give two examples of domains or motifs found in transcription factors?
helix-turn-helix, homeodomain, zinc finger.... 6b/63ff
Describe the procaryotic trp repressor system?
6b/73
The lac system depends on the input of both glucose and lactose availabilty. Name the promoter states (off/weakly on/strongly on) in dependence of glucose and lactose conentration?
low glucose, lactose available -> strogly on
low glucose, lactose not available -> off
high glucose, lactose available -> weakly on
high glucose, lactose not available -> off
A colleague of yours proposes that bacteria don't phosphrylate protein. Can you proove him wrong by naming a singal transduction pathway?
The two component signal transduction pathway features a histidine kinase.
CAn transcriptional reguklation only occur in the initiaion phase?
No it can also occur during elongation and termination 6b/98
What is anti-termination?
When transcription termination is actively prevented 6b/98
Which famous virsus uses anti-termination?
HIV
What are riboswitches
Riboswitches are RNA pregions that can directly bind a small molecule that controls the RNA secondary structure, regulating transcription or translation 6a/104
Explain how translation is coupled to transcription for the bacterial trp operon?
see 6b/103 (you would need to explain that there are 4 blocks, where 3+4 form a terminator but there is a small ORF with lots of Trp causing eventual staalling making 2 pair with 3) ...
Explain attenuation using the trp operon as example
6b/103 (you would need to explain that there are 4 blocks, where 3+4 form a terminator but there is a small ORF with lots of Trp causing eventual staalling making 2 pair with 3) ...
What does nuclease S1 cut?
single stranded DNA and RNA 6b/108
Small RNA
What does Dicer (and Dicer Like) do to double stranded RNA?
It cleavs it into short pieces 7/5
How can plants defend themselves against plant viruses?
siRNA -> silencing 7/6-20 (Give some details about AGO, Dicer etc)
What is cosupression?
When a construct is brought into a genome in normal sense orientation and both the introduced gene and the endogenous gene are silenced 7/29
You want to suppress a gene in C. elegans, which RNA would you introduce a) sense -----> b) antisense <-------- c) double stranded <-====->
double stranded 7/31
Can small RNAs effect DNA?
Yes siRNAs for example can target regions for Cytosine methylation or histone modifications 7/37
How many RNA polymerase classes would you find the genome of an angiosperm plant?
five 7/38
What is the role of RNA Pol IV in plants?
Production of siRNA 7/38
Describe the core components of how miRNA are processed?
7/47 is enough (If you know about Drosha and others all the better) but core components
What is a *-strand?
The strand of a miRNA that is usually (or probably better: more often) degraded
Which gene class is often targeted by conserved miRNAs?
Transcription factors 7/49
What does tasiRNA stand for?
trans-acting siRNAS
When you compare tasiRNA and miRNAs what is different in respect with how double stranded RNA is formed?
miRNAs can fold back on themsleves, trans-acting siRNAs are processed by RNA dependent RNA polymerase
What does Nat-siRNAs stand for, and how are these usually formed?
Natural cis-acting siRNAs, these are derived from transcripts that overlap in such a way that double stranded RNA can form.
modern Transcriptomics
How could you make your own microarray in the lab featuring several hundred genes? (Or in other words which kind of microarry has the simplest technique)
One could use a nylon microarray where one would spot cDNAs from the genes one is interested in. Extracted RNA from the organism would be labelled with radioisotopes
What is the typical number of probes found on a two color glass side microarray, where cDNAs are spotted [10s 100s 1000s 10,000s 100,000s]?
1000s
What is the typical number of probes spotted on a short oligo one color glass side microarray 10s 100s 1000s 10,000s 100,000s?
100,000s
What is a main advantage of two color microarrays, if you were to compare a mutant versus a wildtype in one condition?
One would label RNA from the mutant with one color and from the wild type with the other color and hybridize the pooled samples onto the microarray. Washing, batch and other artifcats etc. would thus effect both samples the same way, and would hopefully mostly cancel out.
What is a major advantage of two color arrays for poor labs?
They are cheap
What are PM and MM probes on an Affymetrix chip?
These are "p"erfect match and "m"ismatch probes. Both are usually 25nt long. However whilst PM probes should hybridize perfectly, MM probes have a nt exhange in the 13th nucleotide.
What is a major advantage of having many probes per gene on the Affymetrix chip?
One can hope to remove spatial artifacts, and generally having more probes per gene helps in getting a robust estimate. This is the same as in the lab where you would do replicates. In addition one can order the probes 5' to 3' to monitor degradation.
What can be a disadvatage of Affymetrix chips?
The price. In addition gene models might get revised, new genes be found and this would not be represented (a problem of all microarrays)
Name some general advantages of microarays?
Rapid, Method and data analysis well described and supported, not too expensive
Name some general disadvantages of microarays?
Closed system approach (what's on is on and what isn't can't be measured), difficult to correlate with absolute transcript number, problems to detect alternative splice forms (but tiling arrays can help here), some sensitivity problems with lowly expressed genes
Name some (next generation) platforms that could be used for sequencing (at least three)?
Solid, Iontorrent, 454, Illumina, PacBio
Please give an estimate about the output of a sequencing run for 454/solid/illumina 100K bases 1M base 10 Mbases 100Mbases 1Gbase 10 Gbase 100 Gbase 1Tbase?
454~1Gbase, Illumina and Solid 100s of Gbases
Estimate the sequence read length of a contigous read (if forward and rverse reads can be obtained choose the longer read e.g. 15+75 => 75) per platform for 454/illumina/solid 10-49, 50-99, 100-499, 500-1000, >1000?
454 500-1000, solid 75 forward and 35 reverse-> 50-99, Illumina 100 forward and reverse -> 100-499
What is the principle behind illumina sequncing (Explain the method)?
All dNTPs are blocked and labelled with a dye (4 different dyes for 4 dNTP derivatives). Thus only one dnucleotide can be incorporated at each time. After incorporation, these area imaged and then the dye and the block is cleaved off-> a new cylce can be performed.
When you use RNA sequencing to determine expression, what are the major disadvantages?
expensive, computationally more difficult, having a reference is very important
When you use RNA sequencing to determine expression, what are the major advantages?
It is an open platform you sequence all that is in there (except probably rRNA which is usually filtered out), you can get single base reolution, you can sequence as deep as you want (limited only by sample amounts), you might be able to get quantitaive and qualitative information
When you use RNA sequencing to determine expression, what are the major advantages?
It is an open platform you sequence all that is in there (except probably rRNA which is usually filtered out), you can get single base reolution, you can sequence as deep as you want (limited only by sample amounts), you might be able to get quantitaive and qualitative information