Genomes and Evolution (1-10) Flashcards
(90 cards)
1
Q
What ideas about DNA did Phoebus Levene present?
A
In 1909 - tetranucleotide theory
In 1930 - each building block of DNA is a nucleotide
2
Q
What is the sugar used is DNA?
A
deoxyribose (the 2’ C has loss of O)
3
Q
Which nitrogenous bases are purines?
A
adenine and guanine
4
Q
Which nitrogenous bases are pyrimidines?
A
cytosine, thymine and uracil
5
Q
Where is the glycosidic bond formed in nucleosides?
A
between sugar C-1’ and
N-9 (purines) or
N-1 (pyrimidines)
6
Q
Where is the ester link formed in nucleotides?
A
between sugar C-5’ and the phosphate
7
Q
What is 2’-deoxythymidine-5’ monophosphate
A
thymine nucleotide (dTMP)
8
Q
What is 2’deoxycytidine-5’ triphosphate
A
cytosine nucleotide (dCTP)
9
Q
What is at the 5’ and 3’ end of DNA?
A
5’ - phosphate group
3’ - hydroxyl group
10
Q
Why did the tetranucleotide theory stop scientists believing that DNA was the genetic material?
A
Phoebus Levene proposed that the four nucleotide bases occurred in tetranucleotide blocks with the bases pointed outwards stacked like a pile of pennies, he assumed the base ratio 1:1:1:1 - DNA was therefore simple and repetitive with no differences so couldn’t be the genetic material.
11
Q
How was the tetranucleotide theory disproven?
A
Erwin Chargraff in 1950 found that different organisms have different proportions of bases also he found the base ratios - %T=%A %G=%C
12
Q
What technique was used by Linus Pauling to describe the alpha helix structure of proteins?
A
X-ray crystallography - the crystalline target molecule diffracts x-rays and causes exposed patches (reflections) on films - a cross was formed by the reflections
13
Q
Why did Rosalind Franklin’s photo 51 of DNA produce a cross?
A
She used hydrated DNA and the DNA helices aligned forming a diffraction grid - the cross showed that DNA must be helical
14
Q
How was the height of DNA helical turns measured from photo 51?
A
In X-ray diffraction patterns the closer the spots the larger the actual distance, thus the close horizontal lines corresponded to large features: the helical turns ~3.4nm
15
Q
Why did Linus Pauling’s triple-helix model of DNA fail?
A
the nitrogenous bases faced out so the negative charges on the stacked phosphate groups would repel - the molecule wasn’t stable
16
Q
What are the 6 key features of the Watson-Crick model of DNA?
A
1 - right handed helix
2 - antiparallel strands
3 - bases on the inside, sugar phosphate outside
4 - complementary base pairing
5 - base pair distance 10.5 base pairs per turn, helix turn 3.6nm
6 - major and minor grooves
17
Q
What type of bonding is present between base pairs?
A
Weak hydrogen bonding- if it was strong you couldn’t separate strands A-T: 2 H bonds G-C: 3 H bonds
18
Q
How is bacterial DNA compacted?
A
by looping and supercoiling
19
Q
How is eukaryotic DNA organised?
A
DNA duplex wraps around histone proteins forming nucleosomes, the nucleosomes are coiled - chromatid fibre - coiled, coiled, coiled into metaphase chromatid
20
Q
Why is DNA replication semi-conservative?
A
Each daughter DNA molecules contains one parental strand and one newly synthesised strand. -> DNA strands are complementary so both can act as templates
21
Q
What are the two predictions made from the Watson-Crick model of DNA?
A
- DNA strands are held together by ‘Watson-Crick base pairing’ A-T C-G (consistent with Chargraffs rules) - strands are antiparallel
- Each DNA strand is complementary to the other so both can act as a template for replication - DNA replication is semi-conservative
22
Q
How did Meselson and Stahl test the ‘Watson-Crick’ model of DNA?
A
They used CsCl equilibrium density gradient centrifugation which separates molecules on the basis of density - showed that DNA is semi-conservative
23
Q
What conditions are required for DNA replication?
A
Template DNA, deoxynucleotide triphosphate (dNTPs), co-factor Mg2+, ATP energy source, primers
24
Q
What direction are new strands of DNA synthesised in?
A
5’ → 3’
25
What suggests that Pol I has editing proof-reading abilities?
Pol I has 3' → 5' and 5' → 3' exonuclease activity - can correct mistakes
26
What happens when nucleotides are incorporated into DNA?
pyrophosphate (2 distal phosphates) is removed from dNTP providing energy to incorporate the new dNMP (the sugar proximal phosphate is incorporated)
27
What did Arthur Kornberg use to test if DNA strands are anti-parallel?
radio labelled dNTPs
28
How does DNA polymerase edit DNA?
1 - wrong nucleotide incorporated
2 - unpaired 3'OH end left - points wrong direction blocking elongation
3 - DNA polymerase 3' → 5' exonuclease activity removes mismatch to leave a base paired 3'OH end
→ DNA replication can continue
29
What are the two active sites on DNA polymerase use for?
1. polymerisation
2. editing site of 3'→5' exonuclease activity
30
Why is DNA replication on the lagging strand broken?
DNA replication is built in the 5'→3' direction, which on the lagging strand is away from the replication fork
31
Why do okazaki fragments form?
Okaskai fragments occur on the lagging strand as a consequence of directionality - new DNA is built in the 5'→3' direction
32
Why is there no 3'→5' synthesis of new DNA?
the need for proofreading - 3'→5' synthesis does not allow proofreading as if a nucleotide was removed a 5'phosphate end would be left - an incoming correct nucleotide cannot proceed as no high energy bond cleaved.
33
What is the function of DNA primase?
synthesis RNA primers for DNA replication (its an RNA polymerase which don't require primers)
34
During DNA replication how are RNA primers extended?
DNA Pol III extends RNA primers using dNTPs
35
How does DNA ligase seal gaps between okazaki fragments?
uses ATP as energy source, catalyses the phosphodiester bond between the 3'OH upstream and 5'phosphate downstream the Okazaki fragment
36
What does clamping do to Pol III during DNA replication?
Converts Pol III to have high processivity - can replicate long stretched of DNA ~1000 bases /s
37
What is processivity?
A measure of an enzyme's ability to catalyse consecutive reactions without realising its substrate.
38
Why is the lagging strand unclamped during DNA replication?
So the okazaki fragments can be repaired - why Poll III has low processivity, if it had high processivity it wouldn't let go
39
What happens when the lagging strand primed DNA duplex is captured by Pol III during DNA replication?
Its clamped forming the lagging strand into a loop
40
What is the function of DNA helicase?
To unwind the double helix - by braking hydrogen bonds
41
What is the function of SSB (single stranded DNA binding protein)?
SSB is required for DNA replication to prevent ssDNA fro forming secondary structures - prevents inappropriate base pairing and protects from nuclease
42
What is positive and negative supercoiling?
Positive → results from overwinding of DNA - makes separation difficult
Negative → arises from undermining - easier to replicate so regulated by topoisomerases
43
What is the function of type I and II topoisomerase?
Type II → converts +ve supercoiled DNA to -ve - makes nick in both strands, re-ligates it
Type I → relaxes -ve supercoiled DNA - makes nick in one strand, unwinds and re-ligates it
44
What is the function of topoisomerase IV in bacterial DNA polymerisation?
Bacterial replication is bi-directional - two Pol III complexes bind and proceed in both directions, topoisomerase IV breaks the catenated daughter chromosomes by double stranded break and re-ligation.
45
What is a telomere?
A section of repetitive DNA at the end of our chromosomes that causes DNA Pol to fall off leaving the lagging strand slightly shorter - protects ends from degregation, inbuilt ageing
46
How does Sanger Sequencing use dideoxynucletoides?
Chain terminators - ddNTPs have no 3'-OH group so when incorporated into DNA no more nucleotides can be added.
47
What are the components of Sanger Sequencing?
ss template DNA, a complementary primer, DNA polymerase, normal nucleotides and radio labeled ddNTPs
48
What are the steps in Sanger Sequencing?
1. add template + primer + DNA Pol + dNTPs
2. add appropriate ddNTP
3. separate fragments via size using electrophoresis
4. autoradiograph and read sequence
49
What is PCR?
(polymerase chain reaction) a tool to amplify a specific DNA sequence.
50
What are the components of PCR?
Template dsDNA, two specific oligodeoxynucleotide primers (hybridise at each end of target sequence), dNTPs, buffer, MgCl2, Taq polymerase and a thermocycler.
51
Why is Taq polymerase used in PCR?
due to its thermal stability - it thrives at ~70C and can survive 50C-80C (won't be denatured)
52
What are the steps in PCR?
1. heat to 94C - denatures target DNA, Taq polymerase not denatured
2. cooled to 45C-65C - hybridisation of primers, one to each strand
3. heat to 72C - optimum temp. for extension of the primers by Taq polymerase
4. repeat for 30 cycles - exponential amplification
53
In a phylogenetic tree what does a clade and node represent?
Clade - all the organisms that share a common ancestor
Node - point where lineage splits, speciation event
54
Why is it more difficult to map phylogenetic trees for microorganisms?
Horizontal gene transfer - the transfer of genetic material to another organisms without reproduction
55
What are the 3 types of evidence of phylogeny?
1. Morphology - the study of the size, shape and structure of organisms
2. Fossils - can offer insight into evolutionary timings
3. Molecular - analysing DNA or protein sequences to infer phylogenetic relationships
56
What is a synapomorphy?
Derived form of a trait shared by a group pf related species - between an ancestor and its descendants
57
What are the 3 major domains of life presented by Carl Woese?
1. Bacteria (eubacteria)
2. Archaea (archaebacteria)
3. Eukaryotes
58
What is microevolution?
Changes in allele frequencies in a population of species over time
3 main mechanisms - natural selection, genetic drift, gene flow
59
What is macroevolution?
Change at or above the level of the species
60
What influenced Darwin's theory of evolution?
Economic population arguments - 'invisible hand', population growth and food supply
Naturalistic framework - Isaac Newton: universal laws, geologists: Hutton and Lyle 'deep time' (Earth older than biblical time)
Observations and prior knowledge using logical inference - Lamarck's ideas, his HMS Beage journey, Wallace's letter (same idea)
61
What were Darwins 3 postulates for the theory of evolution by natural selection?
1. variation - for any particular trait individuals within a species are variable
2. heritable - some of this variation is heritable
3. selective - reproduction is not random but selected by nature (most favourable variations reproduce more)
62
Natural selection tends to fix favourable alles ina population, does this happen faster in dominant or recessive advantageous alleles?
Dominant advantageous alleles - with recessive (aa) it takes longer accumulate to fixation takes longer
63
How do frequencies of mutations change in populations if they have no selective value?
Genetic drift - change in gene variant due to random chance: sampling error, not every sperm/egg makes to the next generation - removes genetic variation within demes but leads to differential between demes
64
What is gene flow?
The movement of alleles between previously separate populations, migration of adults leading to subsequent mating - introduces new alleles into demes within a meta population, can lead to genetic homogeneity between demes.
65
What are the 3 main mechanisms of speciation?
1. Gene flow
2. Genetic drift
3. Selection pressure and reproductive isolation
(all 3 together promote population divergence and can lead to speciation)
66
What is a species at the eukaryotic level?
A population of organism that can potentially or actually interbreed to produce fertile offspring - species are reproductively isolated from other groups
67
What is Mendel's first Law of Inheritance: the Law of Segregation?
The two coexisting alleles of an individual for each trait segregate during gamete formation, they fuse randomly so offspring has one allele from each parent, the dominant one is expressed - so there is discrete inheritance rather than blending
68
How did Mendel discover the Law of Segregation?
He bred pure green (yy) and yellow (YY) peas expecting mid yellow-green offspring but they were all yellow (Yy) - he called the yellow colour dominant. He then bred the F1 gen (Yy) with pure green (yy) peas producing a 1:1 green:yellow ratio concluding discrete inheritance,
69
What is the chromosome theory of inheritance?
Chromosomes carry the unit of heredity proposed by Walter Sutton.
70
How did Walter Sutton discover the chromosome theory of inheritance?
He observed grasshopper cells and found that:
female cells = 22 chromosomes XX
male cells = 22 chromosomes X
gametes = 11 chromones +/- X
sex was determined via chromsome-based inheritance, Mendel's 'factors' that give us our phenotype carried on chromosomes
71
What did T.H Morgan propose about eye-colour in fruit flies?
Demonstrated sex-linkage - the X chromosome carries genes other than sex determinants
eye colour gene carried on X chromosome not he Y, so males always inherit mutant as no corresponding allele to mask it.
72
What is the inborn errors of metabolism theory?
Archibald Garrod proposed that in each case an inheritable factor for a metabolic step was defective.
73
What 2 disorders did Archibald Garrod study for his concept of 'the inborn errors of metabolism'?
1. Albinism: lack of pigment - production of pigment defective
2. Alkaptonuria: individuals secrete homogenistic acid (black when oxidised) into their wee - conversion of homegenistic acid to tyrosine defective
74
What is the central dogma theory?
Francis Crick proposed that 'once information has got into a protein it can't get out again' describing the transfer of information from DNA to RNA to protein.
75
What organism was the first to have's genome sequenced?
Haemophilus influenza - in 1995 by J, Craig Venter
76
What are the goals of the human genome project?
1. identify all the genes in human DNA
2. determine the sequence of DNA base pairs
3. store this information in databases
4. improve tools for data analysis
5. transfer related technologies to the private sector
6. address ethical, legal and social issues
77
What were the findings from the human genome project?
1. people expected ~100,000 human genes, only found ~20,000
2. only 1.5% of our genome codes for proteins
78
What are some of the benefits of the human genome project?
Better understanding of our evolutionary history, personalise medicine...
79
Why was bread mould used for the one gene-one enzyme experiments?
It has a fast and convinient life cycle:
- grows haploid so there is only one copy of each gene, if its mutated can't be masked
- fuses for a brief diploid phase
Grows in simple medium, phototroph so makes its own compounds.
80
How did Beadle and Tatumn's 'one gene-one enzyme' experiment demonstrate Archibald Garrod's theory that hereditary diseases are 'inborn errors of metabolism'?
They demonstrated a clear connection between gene and metabolic enzymes by generating mutants with missing or altered steps in a biochemical pathway and growing them in different media.
81
What mutant did Beadle and Tatum generate for the one gene-one enzyme experiment?
Arginine auxotrophs of Neurospora crassa
auxotroph: a mutant that requires a particular additional nutrient
82
In the 'one gene-one enzyme' experiment how did they identify mutated nuclei?
1. They exposed the spores to radiation - some had rare mutated nuclei (arg-)
2. Grew 100s of their descendants in complete medium
3. Transferred these into minimal medium
4. Identified mutant by their failure to grow
83
In the 'one gene-one enzyme' experiment how did they identify nutritional requirement?
They grew each of the mutants on minimal medium + 20 amino acids - if it grew they then tested it in 20 different vials with just 1 aa + minimal medium. If it grew they knew that's the pathway disrupted in the mutant.
84
What did Beadle and Tatum conclude from their 'one gene-one enzyme' experiment?
There are multiple genes coding for a genetic pathway
- mutants of step 1 and step 2 arginine biosynthesis complemented each other
85
What did Friedrich Miescher unknowingly discover in 1869?
nucleic acid - he investigated the nuclei of leukocytes found in pus and discovered a substance called nuclein and proposed that this might be the basis of heredity
86
What did Frederick Griffith use to demonstrate bacterial transformation?
With Streptococcus pneunomiae
R (rough colonies): non-pathogenic
S (smooth colonies): pathogenic
87
How did Frederick Griffith demonstrate the transforming principle of bacterial transformation?
Injeceted a mouse with heat-killed S cells and living R-cells - mouse caught pneumonia
- a transforming principle from the S cell survived heat treatment and altered some of the R cells
88
What the Avery-MacLeod-McCarty experiments show that DNA is the transforming factor?
They added protease, RNase and DNase with heat killed S cells to living R Cells
- they found R + S cells with protease and RNase
- only R with DNase, showing that DNase destroys the transforming factor (no R had become S cells, no DNA was transferred via horizontal gene transfer as destroyed by DNase)
89
How did Martha Chase trace phage protein and DNA to see if protein was the hereditary material?
Labelled protein and DNA radioactively
proteins - sulphur 35s
DNA - phosphorus 32p
90
How did the Hershey-Chase experiments show that DNA was the genetic material?
After 32p labelled phages attached to the E. coli, the 'ghosts' were knocked off - some bacteria were 32p-labelled
