Introduction to Genetics
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What is Mendel's first rule? Are there exceptions?
Uniformity rule : If both parents are homozygous the F1 generation is uniform. This is not generally true; be prepared to give an example for an exception
What is Mendel's second rule? Are there exceptions?
segregation rule : If both parents are (in the same way) heterozygous the offspring separates 3:1 phenotypically and 1:2:1 genetically Not generally true; be prepared to give an example for an exception
What is Mendel's third rule? Are there exceptions?
combination rule : If both parents are homozygous for two different traits, these will be inherited independetly and can recombine freely. Not generally true; be prepared to give an example for an excetion
How can you determine the genpotype of an indivudal showing the dominant phenoytpe?
by backcrossing with a homozogot recessive (visible by the recessive phenotype)
Be prepared to predict the outcome of crossing schemes
E.g. Cross Aa with BB where the capital is the dominant allele
Be prepared to explain mitosis and maiosis in broad terms?
Naming/recognizing the phases with/by one hallmark is generally enough
Which problems can arise during meiosis leading to severe genetic problems?
Non disjunction: Too many or too little chrosomes of a kind; most well known Trisomy 21
Why is a family incidence of Trisomy 21 a risk factor fro Trisomy 21?
It could be an indication of a balanced chromosomal translocation, think of scenarios where this can cause problems!
What are the Edwards, Patau and Chri-du-Chat sydromes?
The former are Trisomies and Chri Du chat is a loss of a part of a Chromosome arm (5p)
Describe the XY sex system?
see lecture: key facts: dosis compenstation Y smaller; bar body etc; Y defines male sex; lack of Y defines female sex
What is the Klinefeldter syndrome?
XXY chromosomal abberation
What sex do people with Klinefeldter show?
What is the Turner syndrome?
X0 chromosomal abberation
What sex do people with Turner show?
What sex do people with XXY show?
Explain why there can only be female Calico cats?
pigmentation is X chromosomal; Bar inactivaition inactivates randomly one or the other allele in cats; cats have islands of e.g. orange slide 35
How is dosis compensation achieved in Drosophila?
hyperactivation of single X in males
In the ZZ ZW sex determination systems which is male and female?
ZZ is male ZW is female
What is maternal inheritance?
Inheritance through e.g. mitochondria or chlorplast solely transmitted by the mother; be prepared to give an example how to diagnose maternal inheritance
Define Co-Dominance and incomplete Dominance?
see lecture; be prepared to give an example; (! as some of you figured out the problems of classical definitions you can write this down as well)
What is X linked inheritance and by tranfer Y linked inhritance?
Be prepared to diagnose these special cases of inheritance and be ready to use them as exceptions against Mendel laws (which ones?) and be careful in how you cross (see slide 14/15)
What is genetic linkage (Kopplung) and how was it found ?
It was found in flies by Morgan by crossing lines with two traits which invalidated a Mendel law (which one?). For definition refer to the lecture you don't need to know which flies Morgan used
BE PREPARED TO EVALUTE HUMAN FAMILY TREES TO DIAGNOSE HOW GENTIC DISEASES are TRANSMITTED
Know the symbols and typical tell tale signs; after you came up with a diagnosis make sure it fits all individuals; And be ready to use this to determine the genotype of certain indiviudals in the tree
What is special about red/green blindness
It is X -linked and thus able to invalidate one of Mendel's laws. (Which one?)
One gene affected mutliple different phentotypic effects; be prepared to have an example ready: e.g. PKU phenylalanine accumulates AND neuronal effect
Multiple genes affect one phenotype; be prepared to have an example ready; e.g. chicken comb; (human) skin color; you don't need to know the alleles behind
What is Epistasis
One gene or allele can hide/suppress the output phenotype of another gene. Tpyical examples are biochemical pathways where a precurosor needs to be made by geneproduct A which is then converzed by geneprodut B
What is multiple allely
One can have much more than only two alleles per gene; be prepared to give an example e.g. rabit fur color
What is a maternal effect (not maternal inheritance!)
the cytoplasm of the egg cell contains a gene PRODUCT which influences the phenotype of the offspring
What is pentetrance?
Percentage of indiviudals that indeed express a phenotypic trait; complete penetrance would mean whenever a dominat allele is present one sees the phenotype; incomplete penetrance would mean that even though a dominant allele is present its phenotype is not always expressed; example polydactyly where persons having the allele have a normal amount of fingers
What is expressivity
a trait is expressed but differntly in different indivudals; in the example of polydactylie again one might have six complete fingers (high expressivity) or just a double end segment of one finger (low expressivity); penentrance is a yes/no expressivity a how strong
What is the central dogma of molecular biology, given your knowledge about molecular biology, do you think this is coreect? Why/Why not?
DNA=>RNA=>Protein, no e.g. reverse transcriptases, telomerases (both RNA=>DNA); RNA as genetic material
Describe the Avery MacLeod MacCarthy experiment. What did they want to show and how did they succeed?
See Lecture 1/Slide 8ff
Describe the Hersehy/Chase experiment. What did they want to show and how did they succeed?
See Lecture 1/Slide 10ff
Name at least three hints that DNA is carrying genetic information available some time ago
DNA & protein abundant in nucleaus, only the latter abundant in cytosol; mitochondria and chlorplasts both feature DNA and have genetic function; correlation between ploidy and genetic substance; UV induced mutations 1/14
Name a direct evidence that DNA provided the genetic material available to us now.
Recombinant DNA technology 1/17
What is special about Retroviruses?
They use RNA instead of DNA as genetic material which is (reverse) transcribed into DNA 1/21
Name the four DNA bases and classify them?
Purines: Adenine, Guanine; Pyrimidines: Cytosine, Thymine 1/24
What is the difference in the sugar between DNA and RNA?
RNA uses ribose DNA deoxyribose 1/24
Explain the difference between Nucleosides and Nucleotides:
Then Nucleotides have an additional phosphate group, Nucleosides only a base and pentose 1/26f
How are (deoxy)nucleotides joined in e.g. DNA?
Through a phosphodiester bound between C-3' and C-5' 1/29
If you find a T (U) is this indicative for DNA or RNA?
T=>DNA U=>RNA 1/31
What is the main Chargaff rule which helped elucidating DNA structrue?
amount of A = ca amount of T; amount of G = ca amount of C 1/33
How many base pairs are there per Helix turn in B-DNA?
Give the number of base pairs between A-T and G-C base pairs?
2 and 3; 1/38
What is the most prevelant from of DNA and name some other forms?
Mostly B-DNA; Other forms A, Z ... DNA; 1/41ff
Is RNA rather double or single stranded?
Mostly single stranded but can form hairpins etc and there are exceptions 1/46
Name the three major classes of RNA (full name no abbreviation excecept RNA)?
messenger RNA, ribosomal RNA, transfer RNA 1/47
Summarize the role for the hree major classes of RNA ?
rRNA structural components of the ribosomes; tRNA carry amino acids and recognize mRNA code; mRNA are reads by ribosomes for protein synthesis 1/48
Order m/r/tRNA in terms of abundance?
What does Svedberg stand for?
it is a measurement unit for sedimentation velocity 1/51f
What is Hyperchromicity of DNA what can it be used for?
single stranded DNA absorbs MORE UV-light, so de/re-naturing of DNA can be monitored by UV absorbance 1/53ff
What is the melting temperature of DNA, how can it be determined?
The temperature where half of the DNA is in the form of double and the other in the form of single strands. Melting curce: slowly increase T and monitor OD260; 1/55f
What is hybridization?
The association of complementary strands to form a double stranded DNA molecule (also if one molecule is DNA and one is RNA) 1/57f
How does reassociation of DNa help to study DNA?
Highly repetetive regions find each other faster, single copy ones slower, a figure like the one in the lecture emegers C0t 1/61
In which form do bactrial chromosomes often occur?
In circular form 2/1
How many chromosomes do bacteria often have?
They often only have one chromsome 2/2
What are plasmids?
Extrachromosomal DNA molecules usually carrying non-essential genes. 2/3
How are bacterial chromosomes compacted?
First proteins help, secondly supercoiling 2/6
If you electrophorese a circular DNA without any treatment from an organism what might you see?
Multiple bands due to supercoiling states 2/10
What is the typical supercoiling in bacteria and arachae?
negative supercoiling (opposite to helical twist) 2/16
How is the eukaryotic chormatin compstion?
About half DNA, about half proteins. The proteins being histone and non-hostones in a ~1:1 ratio 2/18
Which Histone proteins do you know?
H1, H2A, H2B, H3, H4 2/19
How long is the strectch of DNA around a histone octamer?
What is the role of Hinstones H2A, H2B, H3, H4 and H1?
H1 is the linker the other ones part of the histone octamer 2/21
Draw a histone octamer
Is the wrapping of DNA around histones the only form of packing?
No, see Lecture 2/23-29
After DNA replication are all nucleosomes newly made?
No a lot use old components 2/30ff
Why can epigenetic modifications be passed on post-replication?
Histone modifications are preserved in retained histones 2/35
If you look at the base sequence of an epigenetic modification what has changed?
Define metacentric, submetacentric, acrocentric and telocentri chromosomes?
What is chromosome banding?
Chromsomes are atained with e.g. Giemsa stain to reveal bands this helps in chromosome and region identification 2/40
Describe hetereo-and euchromatin
Heterochromatin is more condensed, less gene rich; Euchromatin more gene rich less condensed during interphase 2/42
Where could you find constitutive heterochroamtin?
E.g. in the centromers and telomers 2/43
What is special about the centromeric histones?
They contan a special form of Histone H3 called CENP-A 2/46
Explain Position effect variagation
See Lecture 2/53
Explain how Xist helps in X-inactivation
See Lecture 2/63
Explain the Meselson-Stahl experiment in full detail
See Lecture 3/4ff Bear in mind that you need several rounds in Light N14
Where does DNA replication begin?
At the origin of replication 3/9
Does replication proceed in one, two or three directions=
in two it is bidirectional 3/9
What does DNA polymerase require?
A template, a primer, dNTPs 3/12
Describe the process of chain elongation?
See Lecture 3/13
Can a DNA polymerase start replication de-novo?
no it needs a primer 3/14
How is the DNA helix unwound at the start of DNA replication in bacteria?
DnaA binds to the origin and recruits DnaB helicase facilitate by DnaC 3/20
Does unwinding the DNA cost for the start of replication energy?
yes in the form of ATP 3/21
What is an immedeate problem of DNA unwinding and how is it solved?
It introduced supercoiling which is relieved by DNA gyrase 3/22
As DNA polymerase can only elongate, how is the elongation process primed?
by a Primase which starts with an RNA primer 3/23
What are Okazaki fragments, why do they come up and where is this a problem?
What does the DNA polymerase do to ensure faithful replication?
It proofreads the incorporated bases 3/28
How are the two strands called in DNA replication and why?
leading and lagging strand. One can be synthesized in one go one in little pieces 3/24ff
How many replication orgins does a bacterial/eurkayotiy organism often have
one (bacteria) per chromsome versus many in eukaryotes 3/36
Which problem do linear chromosomes pose for DNA replication and how is this problem solved?
Chromosome ends, this is solved by e.g. telomerase, mechanism see 3/42ff
Is telomerase always active?
No in most eukaryotic somatic cells it is not active 3/45
Explain a holiday junction
Explain in detail how genetic recombination takes place at the molcular level
Explain missense, siltent and frameshift mutations
What is a transversion and what a transition
transition: pyrimidine base=>pyrimidine base or purine base=>pruine base; Tranvserion Purine=>pyrimidine or vice versa 4/11
What is a temperature sensitive mutation
the gene product functions at one temperature but not the other temperature 4/14
Give a nice example for a temperature sensitive mutation
Himalaye rabbit 4/16
Explain how tautomeric shifts can lead to mutations
How does deamination effect DNA?
Cytosine looses amino group is converted to Uracil; Adenine to Hypoxanthine. This can be recognizes as errors 4/25
Where does Uracil in the DNA come from?
e.g. deamination of Cytosine 4/26
Why is it good that DNA uses T instead of U?
U is a marker for a problem (probably deamination). This wouldn't wotk if it were a DNA base 4/26
Can transpons lead to genetic changes?
Yes they can cause mutations 4/28
Name some agents that can cause mutations
funghal toxins, cosmix/x-rays, Uv, pollutants, chemicals in tobacco .... 4/30
How can base analogs induce mutations
They can substitute for bases but might increase tautomeric shifts or sensitivy to UV 4/31
How can alkylating agaents induce mutations
They change bases and might change base pairing affinity 4/32
What are intercalating agents?
Agents that wedge themselves in between bases =>distort DNA 4/33
What is the hallmark damage induced by UV?
Pyrimidine dimers 4/36
How is Huntington's disease induced?
trinucleotide repeat sequences that increase likely due to polymerase slippage 4/47 ff
What is the error rate of DNA polymerase
One error in 10^5 to one error in 10^7 bases 4/54
Explain the mutSLH system in E.coli
Explain photoreactivation rpeair
What is Xeroderma pigmentosum
A genetic disorder that arises due to NER defects 4/62
How is Xeroderma pigmentosum treated
By absolutely strict avoidance of UV (sunlight) 4/62
How has Xeroderma pigmentosum been used to study repair
Using heterokaryons to establish complementation groups 4/64
Explain DSB repair
Explain the Ames test
Lecture 4/68 ff
What give researches a clue that RNa could be the intermedeate form for protein synthesis?
studies of RNA compsition after phage infection resembled DNA composition of the respective phages 5/5
What is the template and what the coding strand in transcription?
The template strand is where the RNA polymerase reads from (so complementary and antiparallel to the RNA), the coding strand complementary to the temlate strand thus resembles produced RNA 5/8
What are the four steps in E.coli transcription
Promoter recognition, Transcription initiation, chain elongation & Chain termination 5/9
At which position does transcription start?
at +1; 5/10
In Transcription is there a +-0 position?
No only +1 and -1; 5/10
Where are the major consensus sequences for Transcription located for housekeeping bacterial genes?
at -10 and -35; 5/13
How is the baacterial core promoter recognized?
By the sigma factor 5/14
What are alternative sigma factors good for?
To transcribe from different promoters 5/15
Name three sigma factors and their role
Sigma28: Flaggella and chemotaxis; sigma32 heat shock; sigma54 nitrogen metabolism; sigma70 housekeeping 5/15
Is the sigma subunit associated to the bacterial RNA polymerase all the time?
no only in the beginning once initation has completed sigma disscotaes and elongation is done by the core enzyme alone 5/20
What happens with the sigma subunit of bacterial RNAPolymerade after the incorporation of 8-9 nucleotides?
It dissociates as it is no longer needed 5/20
In which direction is RNA synthesized?
From 5' to 3' 5/22
Decribe the process of intrinsic (bactterial) transcription termination?
The template strand codes for an inverted repeat followed by a polyA tail, this is translated and the inverted repeat folds into a stem loop structure , RNA polymerase slows and the weak A-U bps cause RNA polymerase to release the transcript
Decribe the process of rho dependent(bacterial) transcription termination?
RNA contains rut (rho utilization sites) these are recognized by rho (a hexamer) upon ATP activation rho moves on the RNA towards the polymerase and releases it
How is eukaryotic mRNA usually further changed (matured)?
Introns are spliced out, it also gets a 5' cap and a polyA tail
Which RNA polymerase is responsible for mRNA synthesis in humans
RNA polymerase II 5/34
Which box do you often findin eukaryotic core promoters?
The TATA box
Which General Transcription factor bind to the TATA box in Eukaryotes?
TFIID via TBP
What does the cap consist of?
G. (Interestingly this one is linked via 5'-5' linkage)
Which structures do you find at the 5' and 3' end of ekukaryotic mRNA, do you also find these in prokaryotic mRNA?
5' Cap and 3' polyA tail. These do not occur in prokaryotes
Where is the polyA tail in eukaryotic mRNA coming from?
It is added enzymatically and it is NOT DNA encoded
What is the role of the 5' cap?
Protection from rapid degradation, transport out fo the nucleus, easing intron splicing, improving translation
Which complex is responsible for splining mRNA?
The spliceosome 5/59
Can it happen that final RNA has a different sequence (not considering introns) than the coding strand on the DNA?
Yes due to RNA editing, a posttranscriptional process whereby individual bases are changed
How is the genetic code organized?
Commaless, in tripletts, degenerate and non-overlapping
Describe the triplet binding assay and what it was used for
Considering the genetic code what is interesting about Tryptophane (W)?
There is only one code for it (this is also the case for Met but no othr AAs)
Name the (two major) subunits of a pro/eukaryotic ribosome
The prokaryotic 70S ribosome consists of the small 30S and the large 50S SU. The 80S eukaryotic ribosome consists of a 60S and 40S subunit
Schematically draw the structure of a tRNA?
6/31 left side
What is the typical length of a tRNA
What is interesting about several nucleotides in the tRNA
they contain chemically modified bases (or riboses) such as pseudouridine
Some people say the tRNA is an adapter, can you imagine why?
The tRNA contains an anticodon to recognize the corresponding tripled and its 3' end carries the corresponding amino acid
How are tRNA loaded with amino acids?
This is performed by aminoacyl tRNA synthetase which use ATP
If you were to break translaotion down into seperate steps, what would these be?
Initiation, Elongation. Termination
What do you need for translation initiation in prokaryotes?
The ribosome (small and large SU), GTP, charged initiator tRNA, the initiation factors IF1, IF2, If3, and of course mRNA
What is the role of IF1, IF2, IF3 in prokaryotic initiation?
IF1 stablizes the small rib. SU, IF2 binds charged initiator tRNA to 30S mRNa & binds to GTP,; IF3 binds 30S SU to mrNA
Where does translation start for prokaryotic mRNA?
At the AUG start codon, which is preceeded by the Shine-Dalgarno sequence
What is the Shine Dalgarno sequence?
A special sequence a few bp before the prokaryotic AUG start codon, that base pairs with a region in the 16s rRNA
What do you need for translation elongation in prokaryotes?
The ribosome (small and large SU), GTP, charged tRNA, the elongation factors EF-Tu, EF-Ts EF-G and of course mRNA
What is the role of EF-Tu, EF-Ts and EF-G in prokaryotic initiation?
EF-Tu binds GTP, brings aa-tRNA to the A site in the ribosome; Ef-Ts (re)generates active EF-Tu; EF-G stimulates GTP dependent translocation of the ribosome
What is the typical error rate in prokaryotic translation elongation?
about 1 in 10000
What do you need for translation termination in prokaryotes?
The ribosome (small and large SU), GTP, the release factors RF1 or RF2 and RF3
What is the role of RF1, RF2 and RF3 in prokaryotic translation termination?
RF1 : release of polypetide chaain, disscoiation of complex specific for UAA&UAG; RF2 like RF1 but for codes UGA & UAA; RF3 stimulates RF1 and RF2
What is a major difference in pro- and eukaryotic translation when it comes to the very beginng?
Eukaryotes use Met and not fMet as an initition amino acid; Eukaryotes don't have Shine Dalgarno sequence preceeding the start codon but the Kozak Sequence AROUND the start codon
How can you determine where a certain mutation lies in a metabolic pathway?
Give at least 3 examples of posttranslational modifications of proteins
Removal of N-terminal AA, Phosphorylation, Attaching CHO side chaines, trimming of polypeptide chains,
What is a restriction enzyme?
An enzyme binding to specific (often palindromic sites) in the DNA and cutting the DNA then
What are palindromes?
Sequence that when read from back to front are the same (for DNA keep in mind to reverse complement)
What can you use restriction enzymes and DNa ligase for?
Mobilize DNA fragments and put them into a new target
What is a multiple cloning site in a plasmid?
A site where multiple restriction enzymes cut, so DNA can be cloned into this site.
Describe the principle of blue/white selection after plasmid transformation?
What are BACs?
Bacterial artifical chromosomes, these are usually very low copy but can be used to clone large fragments of DNA tyically ~100kb
How would you transform plant cells?
Using modofied Agrobacterium tumefaciens
How would you prepare a cDNA library (detailed)?
What does reverse transcriptase do?
It rerverse trasnscribes RNA into DNA
how would you screen a cDNA library (explain in detail)
Explain PCR in detail?
Explain Southern Blotting
Explain Sanger DNA Sequencing
Explain Sanger DNA Sequencing