1 Flashcards
(438 cards)
1
Q
define Mendel’s first law or The law of segregation
A
- the 2 alleles for a trait will separate during gamete formation and each allele will have an equal probability of inclusion in a gamete
- the random union of gametes, one from each parent at fertilisation, will produce progeny in ratios that are determined by chance.
2
Q
what is testcross
A
a cross of any genotype, often unknown, with the homozygous recessive. this cross can determine the genotype of unknown.
3
Q
what is the product rule
A
the probability of 2 or more independent events occurring together is the product of their individual probabilities
4
Q
what is the sum rule
A
the probability of the occurrence of either 2 or more mutually exclusive events is the sum of their individual probabilities
5
Q
what is monohybrid cross
A
crosses between individuals differing in 1 character e.g. purple vs white
6
Q
what is dihybrid cross
A
cross between individuals differing in 2 characters
7
Q
what is Mendel’s second law or the law of independent assortment
A
during gamete formation segregation of alleles of one gene is independent of the segregation of the alleles of another gene
8
Q
how do you do a hypothesis test with chi-square
A
- first need null hypothesis e.g. 9:3:3:1 (F2 ratio), F1 is a cross between heterozygous . P is homozygous
- chi-square = the sum of [ (O-E)^2/E]
9
Q
what is probability less than 5% mean
A
2 std from the mean is consider as significant
10
Q
what does more std from the mean suggest
A
less confident and less fit on 9:3:3:1
11
Q
how do you interpret a result that has p>0.05
A
do not reject, retain. not significant different from expected, therefore accept null hypothesis
12
Q
what r the factors affecting observed phenotype and genotype
A
- Pleiotropy
- Penetrance and expressivity
- lethal alleles.
- interactions among genes (epistasis)
- sex linked inheritance.
13
Q
what is pleiotropy and give example
A
A single gene affects 2 or more characters result in multiple phenotypes.
e. g A^Y in mice affects coat colour (yellow) and weight (obese)
e. g. W white spotting gene in mice affects migration of 1.melanocytes (white spotting).2.germ cells (sterility)3. blood precursor cells (anemia)
14
Q
many genes can influence 1 single phenotype, give example
A
- coat colour
gene 1: agouti: distribution of colour on each hair
gene 2: dark colour of hair black or brown
gene 3: albino or pigmented
15
Q
what is null alleles
A
mutation of gene r produce no units of R
16
Q
what is haplosufficient
A
single copy of wild type gene, enough product to carry out.this is happened when the WT is dominant
17
Q
what is haploinsufficient
A
when the WT is recessive and mutation is dominant
- a dominant mutation may occur when one dose of the WT allele is not sufficient to produce enough functional product for normal activity. the mutant allele is dominant. the WT allele is haploinsufficient
18
Q
what does loss- of function mutation result in
A
decrease or complete loss of the functional activity of a gene product . these gene are normally recessive
19
Q
what do u call the mutation of complete loss of gene function
A
null mutation or amorphic mutation
20
Q
what do u call the mutation of partial loss of gene function
A
leaky mutation or hypomorphic mutation
21
Q
what do u call the mutation of altered products of a gene
A
dominant negative mutations
22
Q
give an example of loss of function mutation
A
osteogensis imperfecta
- dominant negative mutations in a collagen gene
- no long functioning causing brittle bone
23
Q
what does gain of function mutations result
A
result in a new function or have increased levels of expression. these mutation are often dominant
24
Q
what do u call mutation that caused increased gene activity
A
hypermorphic mutation
these may be semi-dominant where the homozygous is more severe than heterozygous
25
what do u call the mutation that cause novel function
neomorphic mutation
26
give an e.g. of dominant gain of function mutation
e. g. 1 Drosophila Antennapaedia (Antp) mutant has legs in place of antennae
e. g. 2. maize leision mimic mutants (Les) have spontaneous necrotic spots in the absence of pathogen
- dominant mutations with expression of a gene in the wrong place or at the wrong time
- genetically dominant because the gain of function cannot be compensated by the WT allele
27
give an example of incomplete dominance
4 o'clock plants
A1A1 red
A2A2 white
A1A2 pink
28
give an example of codominance
ABO blood type
29
wht r the factors affecting observed phenotype and genotype
1. pleiotropy
2. penetrance and expressivity
3. lethal alleles
4. interactions among species (epistasis)
5. sex-linked inheritance
30
what is incomplete penetrance and variable expressivity
incomplete penetrance and variable expressivity can result in modification of segregation ratios or apparent inheritance of an allele, or the phenotype will vary between individuals
31
what is penetrance
penetrance is the proportion of individuals of a specific genotype that exhibit the corresponding phenotype
32
what is the penetrance of aa->100% phenotype
| receissive mutation a
1.0
33
what is the penetrance of dd-> 80% phenotype ->20% normal recessive mutation d
penetrance 0.8
34
give an e.g. of incomplete penetrance
polydactyl
- single dominant gene with incomplete penetrance
- at leat 1 in 4 people with mutation have 5 digits
35
what is variable expressivity
is the extent to which a phenotype is expressed
36
give an e.g. of variable expressivity
Piebald spotting beagles
| - spotting is due to dominant gene S^p with variable expressivity
37
what'd the difference between pleiotroph, penetrance, expressivity
Pleiotrophy: Gene X cause phenotype 1,2,3
Penetrance: all individual have the mutation but not all individual show phenotype
Expressivity: all individuals have the mutation, all shown phenotype but differ in severity
38
what is gene interactions
no gene operates alone to produce a phenotype
- networks of genes collaborate to make up biosynthetic signal transduction or developmental pathways
- when 2 genes affect the same trait genetic interactions often occur and can sometimes lead to altered Mendelian ratios in F2
39
what id gene interaction due to
epistasis
40
what id epistasis
an allele of 1 gene modifies or prevent the expression of alleles of another gene
41
what does epistasis result in dihybrid cross
- epistasis results in fewer than 4 phenotypes from a dihybrid cross
42
what r the gene interactions lead to modifies F2 Mendelian ratios
1. complementary gene interaction (9:7)
2. duplicate gene interaction (15:1)
3. receissive epistasis (9:3:4)
43
what is complementary gene interaction
recessive mutation in 2 different genes, acting in the same pathway, produce the same phenotype
9:7
44
what is duplicate gene interaction
either of 2 gene products perform the same function
| 15:1
45
what is recessive epistasis
a recessive allele masks the expression of other genes
| 9:3:4
46
how do you perform functional allelism for recessive genes?
unknown cross with mutants to produce F1
- if genes allelic (mutation of same gene) -> F1 mutant phenotype; failure to complement
- if non-allelic (the mutation is not on the same gene) -> F1 WT phenotype, complementation
if non allelic and intercross F1 AaBb X AaBb can get gene interaction with a modified F2 ratio
47
what does 9:3:3:1 tell you
the genes are unlinked, and the traits are on different genes
48
whats the ratio for complementary gene interaction
9:7
9 WT, 7 homozygous for aa or bb -> albino
mutation of aa or bb will be albinism
49
whats the ratio for duplicate gene interaction
15:1 segregatio ratio; 15 purple and 1 white
| 2 gene R or P gene are responsible for conversion to purple, result in genetic redundancy
50
whats the ratio for recessive epistasis
9:3:4
9 black, 3 brown, 4 albino
cc epistatic over B, the cc genotype masks expression of other coat colour genes. e.g B-cc will be albino
e.g. cross BBcc(albino) X bbCC(brown)
F1 is BbCc (black) and F2 will have a modified ratio
51
whats the explanation in terms of metabolic pathways
Precursor --C--> intermediate ---B/b---> Black pigment/Brown pigment
52
what is epistasis
epistasis is the interaction b/t different genes. if 1 allele or allelic pair masks the expression of an allele at a second gene, that allele or allelic pair is epistasis to the second gene
53
give example of recessive epistasis
e.g. B and E genes control coat colour
B controls melanin distribution
E control eumelanin synthesis
Precursor->eumelanin
- B-lots of eumelanin =black
- bb less eumelanin =brown
precursor->pheomelanin
ee=no eumelanin synthesis =yellow
```
bbEe chocolate
B-ee yellow
B-E- black
bbEE (chocolate) x BBee(yellow)
F1 BbEe black
F2 9 black, 3 chocolate, 4 yellow
```
54
what derives the X and Y chromosomes
autosomes
55
describe the X chromosome
- 165 megabases
- ~1000 genes
- highly conserved in placental mammals
56
describe the Y chromosome
- ~58 Megabases, 156 transcribed units, 78 protein-coding genes, 27 unique genes
- specialized male and fertility genes
- many repetitive sequences and palindromes
57
males are homozygous for X-linked genes: male cannot be homozygous or heterozygous
T
58
what cross can you do to determine sex-linkage
- inheritance of X-linked trays produces different outcomes in reciprocal crosses
- reciprocal cross have the same ratio-not sex linked, different ratio-sex linked
59
males inherit X-chromosomes from their mothers and transmit X chromosomes to their daughter
T
60
the differences between reciprocal crosses observed by Morgan are not anticipated by Mendel's law of heredity
T
61
what is dosage compensation of sex chromosome
- in mammals females have 2 copies of each X-linked gene and males have 1 copy of each X-linked gene
- many X-linked genes are expressed at the same level in males and females through dosage compensation
- DOSAGE COMPENSATION: any mechanism that compensates for the differences in the number of copies of genes due to the different chromosome constitutions of males and females
62
give an e.g. of dosage compensation mechanism and what does this require
random X-chromosome inactivation in placental mammals. this requires Xist gene
63
how does Xist gene work in X-chromosome inactivation
1. random X chromosome inactivation requires the X-inactivation-specific-transcript (Xist) gene
2. Xist is transcribed to produce a large (~17kb) non-coding Xist RNA
3. Xist coats the X-chromosome in cis (on the same chromosome)
4. Xist recruits proteins that inactivate or silence gene expression
64
random X-chromosome inactivation in placental mammals
1. in female one X-chromosome i inactivated
2. the inactivated chromosome may be from either the male parent or the female parent
3. the inactivation is permanent so that all females have clones of 2 types of somatic cells: half with the maternal X inactivated and half with paternal X-inactivated
65
give and e.g that due to dosage compensation and random X-chromosome inactivation
Tortoiseshell cats
- all tortoiseshell cats are female and heterozygous X(B)X(b)
- some of the cell inactivate the B, some of the cell inactivate the b, result in different patterns.
66
what is the characteristic of human X-linked recessive trait and give e.g.
what r some e.g. of human X-linked recessive traits
- present more in males than females
| e. g. Haemophilia A, red-green colour blindness
67
what is the characteristic of human X-linked dominant trait and give e.g.
- trait occurs with ~ equal frequency in males and females
| e. g. fragile X syndrome
68
To construct a linkage map, perform the following steps in this order:
Determine which offspring have parental genotypes and which have recombinant genotypes.
Determine which offspring are recombinants between genes m and d. Remember that identifying the original linkage in the trihybrid parent (MmDdPp) is key to determining this. (See Hints 2 and 3.)
Determine which offspring are recombinants between genes m and p. And finally, determine which offspring are recombinants between genes d and p.
Calculate the recombination frequency between each pair of genes. Do so by adding up the number of recombinants for a given pair and dividing that number by the total number of offspring (1000). Multiply by 100 to get the recombination frequency.
Recombination frequency=Number of recombinantsTotal number of offspring x 100
Use the recombination frequencies to construct your linkage map. Remember that a 1% recombination frequency is equal to one map unit, or one centimorgan (cM).
Once you determine the arrangement of the genes along the chromosome, remember to take into account double crossover events in calculating the distance between the two genes that are farthest apart.
69
recombination frequency between m and p is slightly less than that sum. This is because of double crossover events--the times that crossovers occur both between m and d and between d and p. In a double-crossover event
This is because of double crossover events--the times that crossovers occur both between m and d and between d and p. In a double-crossover event, the second crossover effectively “cancels out” the first, reducing the number of recombinants between m and p that are observed, while contributing to the number of recombinants between each of the other two pairs of genes. Therefore, adding the smaller map distances to calculate larger distances avoids inaccuracies due to double-crossover events that produce non-recombinant genotypes.
70
what does linked alleles do
tend to be inherited together
71
where is the site of crossing over
chiasmata
| crossing over is between chromatids not chromosomes
72
what happen to unlinked genes
independent assortment
73
how does recombination occur in meiosis
recombination in meiosis holds the chromosomes in place reducing non-disjunction (there is always 1 cross over between homologues)
74
what is test-cross
unknown X double recessive
| - the double recessive gametes will not contribute to the phenotype of the offspring
75
what happen to the distant apart gene on 1 chromosome
more cross over, thus higher recombinant frequencies
76
what is 1 map unit
the distance between genes for which 1 product of meiosis out of 100 is a recombinant
recombination frequency of 0.01 = 1 map unit (m.u)
77
what is Cis and Trans in mapping
- Cis (coupling): AB/ab
| - Trans (repulsion): Ab/aB
78
what is interference
a cross over event at one point in a chromosome alters the probability of another crossover in an adjacent region
I = 1-c
79
what is coefficient of coincidence
c=Observed freq double recombinants/expected freq double recombinants
80
expected greg droble cross over
assume crossing over in adjacent chromosomal regions is independent e.g. abc
= rec.freq ab x rec.freq bc
81
what does -ve interference mean
a cross over in 1 region promotes crossing over in the adjacent region
82
what does +ve interference mean
a cross over in 1 region interferes with crossing over in the adjacent region
83
Crossing over during meiosis occurs between alleles on nonsister chromatids.
T
84
Detecting SNPs
• If the SNP is within a restriction site then it may be useful as an RFLP (next slides) but SNPs can also be identified by high resolution melting analysis, microarray analysis or sequencing (“next generation sequencing” is cheap and quick)
85
mapping with
| molecular markers
RFLPs - Restriction Fragment Length Polymorphisms.
| EcoRI recognition sequence: GAATTC
86
Detecting SNPs
• If the SNP is within a restriction site then it may be useful as an RFLP (next slides) but SNPs can also be identified by high resolution melting analysis, microarray analysis or sequencing (“next generation sequencing” is cheap and quick)
87
mapping with
| molecular markers
RFLPs - Restriction Fragment Length Polymorphisms.
| EcoRI recognition sequence: GAATTC
88
what is hapmap
Hapmap :a catalog of common genetic variants that occur in human beings.
89
Haplotype - a genotype including two or more tightly linked loci. Using haplotypes to deduce gene position
T
90
what is Euploidy
Euploidy: multiples of the basic chromosome set
| e.g. n,2n,3n,4n
91
what is aneuploidy
Aneuploidy: gain or loss of a single chromosome
| e.g. monosomic 2n-1 or trisomic 2n+1
92
what is karyotype
The karyotype - the chromosome set as seen during mitotic metaphase.
93
what is G-band
G-banded human karyotype, 44A, XX produced with Giemsa stain
Detail of Chr 13 p = short arm
q = long arm
94
• Example of a euploid species?
watermelon->3n
| strawberry -> 8n auctaploid
95
• Example of a phenotype caused by aneuploidy?
down syndrome -> extra copy of chromosome 21
96
what is karyotype
The karyotype - the chromosome set as seen during mitotic metaphase.
97
Triploids are usually sterile - failure of chromosome pairing in meiosis. Most gametes are an unbalanced set - aneuploid - and are usually sterile.
2 pairing possibilities of one set of chromosomes in a triploid.-> all unbalanced gametes
- trivalent or Bivalent+univalent
98
• Example of a euploid species?
watermelon->3n
| strawberry -> 8n auctaploid
99
• Example of a phenotype caused by aneuploidy?
down syndrome -> extra copy of chromosome 21
100
Sex-determination in Drosophila melanogaster relies on correct gene dosage
```
Female
XX : AA = 1
-active dimer- Sxl on, therefore female slicing of transcript
Male
X : AA = 0.5
```
101
autopolyploids
Autopolyploids: multiple chromosomes sets from within one
species, eg 2n = AAAA Autotetraploid
Origin: Spindle failure in mitosis or meiosis; environmental, colchicine.
102
what is the origin of autotetraploid
Metaphase of mitosis in a diploid, 2n = 4, cell
| If tetraploid cell goes through meiosis it will produce 2n gametes
103
Triploids are usually sterile - failure of chromosome pairing in meiosis. Most gametes are an unbalanced set - aneuploid - and are usually sterile.
2 pairing possibilities of one set of chromosomes in a triploid.
- trivalent or Bivalent+univalent
104
Autotetraploids Double 2n ➞ 4n
Can have a regular meiosis - depends on chromosomes
pairing as bivalents or quadrivalents.
two bivalent or one quadrivalent gametes are all balanced
| but univalent + trivalent is unbalanced
105
Why do aneuploids have reduced viability?
Inviability can result from:
1. Imbalanceofthegeneticmaterial
AAA BBB CCC Gene doses on ABC balanced: 1:1
AA BBB CC Genes doses on A/C and B ratio of 2:3 or 1:1.5
2. Expression of deleterious recessives (monosomics)
Ll (diploid) versus l (monoploid) where l is a recessive lethal allele
106
Monoploids (n)
Usually inviable because (a) lethal genes expressed.
(b) If viable, germ cells cannot proceed through meiosis because chromosomes have no pairing partner.
But
Hymenoptera: Male bees, wasps and ants are haploid. Develop from unfertilised eggs (parthenogenesis). Produce gametes from modified meiosis.
Females (queens and workers) are diploid. Develop from fertilized eggs.
107
autopolyploids
Autopolyploids: multiple chromosomes sets from within one
| species, eg 2n = AAAA Autotetraploid
108
what is the origin of autotetraploid
Metaphase of mitosis in a diploid, 2n = 4, cell
109
Origin of triploids
Meiosis in tetraploid 4n-> 2n gametes
Meiosis in diploid 2n-> n gametes
Fertilization 2n + n gametes-> triploid 3n zygote
Examples: seedless watermelon, bananas; oysters that don’t spawn
110
Genetic Ratios in an autotetraploid
What are the genetic ratios in progeny of an autotetraploid with genotype AAaa that is selfed? Assume all the chromosomes pair as bivalents.
How does this compare to the ratios of progeny for an Aa diploid individual that is selfed?
111
Mutations Are Rare and Occur at Random
• Every organism carries mutant alleles, whether they manifest in distinct phenotypes or not
• Mutations occur in every generation
• Humans acquire one to four new mutations each
generation
112
Gene Mutations Modify DNA Sequence
* Gene mutations substitute, add, or delete one or more DNA base pairs
* Localized mutations, or point mutations, occur at a specific, identifiable position in a gene
* Such mutations have varied consequences depending on the type of sequence change and the location of the affected part of the gene
113
what is the consequence of silent mutation
no change to a.a. sequence
114
what is the consequence of missense mutation
changes one a.a. of the polypeptides
115
what is the consequence of non-sense mutation
create stop codon and prematurely terminates translation
116
what is the consequence of frameshift mutation
result in wrong sequence of a.a.
117
what is the consequence of promoter mutation
changes timing or amount of gene transcription
118
what is the consequence of polyadenylation mutation
alters sequence of mRNA, may affect mRNA stability
119
what is the consequence of splice site mutation
may retain intron sequence in or exclude exon sequence from mature mRNA
120
what is the consequence of DNA replication mutation: triplet-repeat expansion
increase number of repeats of DNA triplets, causing instability of mRNA or incorrect number of repeating a.a. in a protein
121
Base-Pair Substitution Mutations
* Base-pair substitution mutation: the replacement of one nucleotide base pair by another
* Transition mutations: one purine (A, G) replaces another, or one pyrimidine (C, T) replaces another
* Transversion mutations: a pyrimidine is replaced by a purine or vice versa
122
• Base-pair substitution mutation
• Base-pair substitution mutation: the replacement of one nucleotide base pair by another
123
Transition mutations
Transition mutations: one purine (A, G) replaces another, or one pyrimidine (C, T) replaces another
124
Transversion mutations
Transversion mutations: a pyrimidine is replaced by a purine or vice versa
125
Three Types of Base-Pair Substitution Mutations
* Silent mutation: a base-pair change, no amino acid sequence change
* Missense mutation: a base-pair change, amino acid change
* Nonsense mutation: a base-pair change, early stop codon
126
Frameshift Mutations
* Insertion or deletion of one or more base pairs
* addition or deletion of mRNA nucleotides
* alters the reading frame
* The wrong amino acid sequence and premature stop codons are produced downstream of the altered nucleotides
127
Regulatory Mutations
* Mutations that alter the amount of protein product
* No amino acid sequence change
* Can be in promoters, introns, 5!-UTR, 3!-UTR
128
Cryptic Splice Sites
• These mutations produce new splice sites
• Replace or compete with authentic splice sites during
mRNA processing
• These are called cryptic splice sites
129
Forward Mutation and Reversion
* Forward mutation: converts a wild-type allele to a mutant allele
* Reverse mutations or reversions: convert mutant alleles to wild-type or near wild-type
* True reversion: wild-type DNA sequence is restored by a second mutation within the same codon
130
Types of Reversion
* Intragenic reversion: mutation elsewhere in the same gene
| * Second-site reversion: mutation in a different gene and together the two mutations restore the organism to wild-type
131
Gene Mutations May Arise from Spontaneous Events
• Spontaneous mutations arise in cells without exposure to agents capable of inducing mutation
• Errors in DNA replication
- Strand slippage during DNA replication
• Spontaneous changes in the chemical structure of a nucleotide base
-Tautomers
-Depurination
- Deamination
132
what is tautomer mutation
changing the component of molecules arrangement.
WT AT have 2 H bonds, CG 3 H bonds
rate tautomer: TG 3 H bond, AC 2H bonds.
e.g. Tautomertic shift of the common T (keto) form to the rate (enrol) form, the T(enrol) tautomer mispairs with G (keto), tautomeric shift of T (enrol) back to T (keto), transition mutation A-T ->G-C
133
what is deprivation
depurination remove G from the nucleotide leaving an Apurinic site (empty), during DNA replication cycle, adenine is most commonly incorporated leading to a transition mutation.
134
what is deamination
the lost of amine group, Oxygen group replace
| e.g. cytosine deamination become uracil
135
Mutations May Be Induced by Chemicals or Ionizing Radiation
* Induced mutations are produced by interactions between DNA and physical, chemical, or biological agents that generate mutations
* Agents that cause DNA damage leading to mutations are called mutagens
* Mutagens interact with DNA in specific ways to cause particular types of sequence changes
136
DNA Intercalating Agents
• Some molecules are able to fit between DNA base pairs
• These intercalating agents distort the DNA duplex; some can also form bulky adducts that contribute to DNA distortion
• The distortion leads to DNA nicking that is not efficiently repaired, resulting in added or lost nucleotides
e.g. antiseptic proflavin, smoke Benzopyrene (BaP)
137
Nucleotide Base Analogs
* A base analog is a chemical compound with a structure similar to a DNA nucleotide
* These can pair with normal nucleotides—DNA polymerases cannot distinguish the analogs from normal nucleotides
* For example, 5-bromodeoxyuridine acts as an analog of thymine, mispair with Adenine
138
Deaminating Agents
* A deaminating agent removes amino groups from nucleotide bases
* Deamination of 5-methylcytosine leads to a G-C to A-T substitution
* Deamination of adenine produces hypoxanthine, which can mispair and lead to A-T to G-C base-pair substitutions
139
Alkylating Agents
• Alkylating agents added bulky side groups such as methyl (CH3) and ethyl (CH3-CH2) groups to bases
• These added groups are called bulky adducts
• Ethyl methanesulfonate (EMS) is a powerful
alkylating agent
• Bulky adducts interfere with DNA base pairing and distort the DNA double helix
140
• Hydroxylating agents
* Hydroxylating agents add hydroxyl groups to a recipient compound
* Hydroxylamine adds a hydroxyl group to cytosine, creating hydroxylaminocytosine
* This can mispair with adenine, creating a C-G to T-A transition mutation
141
Oxidative Reactions
* Oxidation is a chemical process of electron transfer by addition of an oxygen atom or removal of a hydrogen atom
* Compounds that contain reactive forms of oxygen (oxygen radicals) react with DNA bases
* For example, oxidized guanine can mispair with adenine, leading to a tranversion mutation (G-C to T-A)
142
DNA Intercalating Agents
* Some molecules are able to fit between DNA base pairs
* These intercalating agents distort the DNA duplex; some can also form bulky adducts that contribute to DNA distortion
* The distortion leads to DNA nicking that is not efficiently repaired, resulting in added or lost nucleotides
143
Photoproducts
• Pyrimidine dimers, covalent bonds between adjacent pyrimidine nucleotides
• Two examples
• Thymine dimer, formed between the 5 and 6 carbons of
adjacent thymines
• 6-4 photoproduct formed by a bond between carbon 6 on one thymine and carbon 4 on the other
144
Photoproducts
• Pyrimidine dimers, covalent bonds between adjacent pyrimidine nucleotides
• Two examples
• Thymine dimer, formed between the 5 and 6 carbons of
adjacent thymines
• 6-4 photoproduct formed by a bond between carbon 6 on one thymine and carbon 4 on the other
145
Other Types of Radiation
* High energy irradiation, X-rays and radioactive materials
| * Single-stranded or double-stranded breaks in DNA • Block DNA replication
146
The Ames Test
* The Ames test tests chemicals and their breakdown products for mutagenic potential
* Uses bacteria with frameshift and base substitution mutations
* Cannot synthesize histidine, his-
* Bacteria + liver enzymes exposed to compounds
* Plated on medium lacking histidine
147
Ames Test Results
* The number of revertants from his- to his+ are assayed for each treatment or control
* The suggestion that a compound is mutagenic is shown by a significant increase in the reversion rate in treated strains relative to controls
* This includes strains with one type of mutation (base-substitution) compared to those with another (frameshift), and untreated strains
148
Repair Systems Correct Some DNA Damage
• Repair of UV-induced photoproducts • Nucleotide base excision repair
149
Repair of UV-Induced Photoproducts
* Pyrimidine dimers can be directly repaired by photoreactive repair, in bacteria, single-celled eukaryotes, plants, and some animals (not humans)
* The enzyme photolyase uses energy from visible light to break the bonds between pyrimidine dimers
* Photolyase is encoded by the E. coli phr (photoreactive repair) gene
150
Nucleotide Base Excision Repair
* Nucleotide base excision repair is a multistep process
* DNA glycosylases recognize and remove modified purine bases, leaving an apurinic site
* AP nuclease then removes the remainder of the nucleotide and DNA polymerase and DNA ligase replace the gap with the appropriate nucleotide
151
Which type of DNA damage is repaired by the enzyme photolyase?
thymine dimer
152
Thymine dimers are most commonly caused by which of the following?
U-V irradiation
153
Features That Make Bacteria Useful to Geneticists
* Few genes
* One copy of each gene
* Rapid generation times
* Many progeny
* Ease of propagation
* Numerous heritable differences
154
Genetic Transfer Occurs by Three Processes
• Conjugation is the transfer of replicated DNA from a donor to a recipient
• Transformation is the uptake of DNA from the environment
• Transduction is the transfer of DNA from one bacterium to another by a viral vector
155
Genetic Transfer
* One-way transfer of genetic material from a donor bacterial cell to a recipient cell
* Extrachromosomal plasmid or a portion of the bacterial chromosome
156
Characteristics of Bacterial Genomes
* One covalently closed circle
* ds DNA
* A few thousand to a few million base pairs • Essential genes
157
Plasmids
* Covalently closed circle • Multiple copies
* ds DNA
* Small
* Non-essential genes
158
Types of Plasmids
* F (fertility) plasmid contains transfer genes
* R (resistance) plasmid carries antibiotic resistance genes • Plasmids are easily modified
* Used in recombinant DNA
* High copy number plasmids
* 50 copies per cell
* replicate independently from the chromosome • Low copy number plasmids
* 1or2percell
* Replicate with the chromosomes
159
Transfer of the F Factor
* One-way transfer of genetic information • Donors to recipients
* Donor ability via F factor, fertility factor • F+ cells have F plasmid
* F- cells lack F plasmid
160
Donor and Recipient Cells in Bacterial Crosses
* F+ cells have F plasmid
* F- cells lack F plasmid
* 100 kb in length 40 genes that control conjugation
* Donor cells transfer genetic information to a recipient cell
* The exconjugant cell is the recipient cell with its genetic information modified by receiving DNA from the donor cell
161
IS Sequences on plasmid
* F factor includes four insertion sequences (IS)
* Role in bacterial gene transfer
* F factor can integrate into the bacterial chromosome, to form an episome
162
Hfr Gene Transfer
* Gene transfer by rolling circle replication
* High frequency of transfer (Hfr)
* Incomplete transfer of chromosome
* The segment of T strand DNA enters the recipient and is used to generate a double-stranded linear fragment
163
Key Outcomes of Hfr × F- Mating
* Transfer of one or more donor alleles into the recipient chromosome
* Occurs by homologous recombination
* Forms an exconjugant chromosome
* The F factor is not fully transferred during the mating
* Therefore, the recipient cell is not converted into a donor cell
164
Interrupted Mating Analysis Produces Time-of-Entry Maps
• Breaking the conjugation tube
• Stops mating before the Hfr chromosome can be
completely transferred
• Time-of-entry mapping
• Transfer genes in a specific order
• Distance of the gene from the origin of transfer (oriT) is related to time of transfer
• Genes closest to oriT will transfer earlier
165
Consolidation of Hfr Maps
* Multiple Hfr strains are used to construct a time-of- entry map
* The maps are consolidated by identifying overlap regions
* Circular map of the donor chromosome
166
Construct Overlapping Linear Maps
Data from each Hfr strain is used to create partial overlapping maps
167
Construction of the Circular Map
Linear maps are used to create an overall circular map
168
Conjugation with Fʹ′ Strains Produces Partial Diploids
6.3 Ces Partial Diploids
• Imperfect excision of the F factor from an Hfr chromosome produces F’ factor
• A functional F factor
• Fʹ′ factor contains all its own DNA plus a segment of
the bacterial chromosome
169
Fʹ′ Factor Mating
* Exconjugants that contain a complete Fʹ′ factor are called partial diploids because they contain two copies of the bacterial chromosome genes found on the Fʹ′ factor
* The partial diploidy is retained as a characteristic of the exconjugants and their descendants
* Partial diploids can be used to examine the mode of action of bacterial genes, and their regulation
170
Transposable Genetic Elements Move Throughout the Genome
* Transposable genetic elements DNA sequences • Move via transposition
* Many forms, lengths, copy numbers
* Can cause a mutation via insertional inactivation
171
Discovery of Transposition
* Barbara McClintock discovered transposition in maize
* C = purple kernels, c1 = colourless; Sh = plump kernels, sh = shrunken; Wx = shiny kernels, wx = waxy
* C Sh Wx/c1 sh w, mostly purple kernals
* Sectors that lacked colour
* Colourless sectors were all shrunken and waxy
172
McClintock’s Results
* Colourless sectors had terminal deletion of one chromosome 9 homologue
* Purple sectors, intact chromosome 9
173
Interpretation of McClintock’s Results
* Dissociation (Ds) element, at the site of chromosome breakage
* Second element, activator (Ac) element, required for chromosome breakage
174
Unstable Mutants
* Colourless kernels with varied patterns of purple spotting
* Unstable mutant alleles, insertion of Ds into the C locus to produce a kernel lacking pigmentation (the mutation is called c1DS)
* Rare transposition of Ds out of the gene
* Reversion to wild type
* Array of purple spots on the kernels
175
Transposable Elements
• Transposition requires the enzyme transposase
• Gene encoding transposase is carried by some
transposable elements
• Some transposable elements also carry other genes
• Some elements contain only repetitive sequences
176
Types of Transposable Genetic Elements
1. Autonomous transposable elements
• transposase gene
• all DNA sequences needed to carry out transposition (e.g., Ac)
2. Non-autonomous transposable elements
• no transposase gene
• may lack the sequences needed for transposition (e.g., Ds)
• unable to move unless transposase is provided by an autonomous element elsewhere in the genome
177
Transposition Modifies Bacterial Genomes
• Two categories of transposable elements • IS (insertion sequence) elements
• simple transposable elements
• containing only the genes and sequences needed for
autonomous transposition
• Two types of transposons, that carry multiple genes and confer new traits on bacteria
178
Insertion Sequences
• About 1000 bp of DNA
• Transposase gene, bracketed by a short, inverted
repeat (IR) sequence (needed for transposition)
• Different IS elements have different IR sequences
179
Insertion Sequence Transposition
• Begins with a cleavage at the ends of IS elements
• Transposase recognizes and cleaves a sequence at
the new integration site
• IS element is ligated into the new site
• Gaps filled by DNA replication
• Pair of direct repeats is produced upon integration17
180
Transposons
• Two types of transposons (Tn), composite and simple
• Different types of sequences flanking the transposon • The genes most commonly carried by transposons
confer antibiotic-resistance
181
Composite Transposons
* Several kb
* One or more functional genes
* Complete IS elements at each end, inverted repeats
* At least one of these contains transposase gene
* The transposon Tn10 has a typical composite transposon structure
182
Simple Transposons
• Flanked by very short IR sequences
183
Transposition Mechanisms
1. Duplication of the target site (direct repeats to either side of the inserted element)
2. There are two mechanisms of transposition that lead to target site duplication
• conservative transposition
• replicative transposition
184
Conservative Transposition
* Excises a transposable element from one position and inserts it into a new location
* Cut-and-paste
* Moves transposable elements around the genome
* No increase in the number of transposable elements
185
Replicative Transposition
• Copying of the transposable element
• Increase in the number of elements per genome
• Initiation of replication of the element in a plasmid
• Transposase facilitates formation of a cointegrate –
a temporary fusion of the plasmids
• A recombination-like process resolves the cointegrate
• Both plasmids have a copy of the element
186
Transposition Modifies Eukaryotic Genomes
1. Two groups of eukaryotic transposable elements
2. DNA transposons
• transposed through conservative or replicative transposition
3. Retrotransposons
• transcribed
• reverse transcriptase produces a ds DNA copy • inserted into the genome
187
Drosophila P Elements
```
• Drosophila melanogaster
• Transposable element
• 20 – 50 copies per genome
• Cross-species transfer in 1960s
• Today, all wild-caught D. melanogaster contain P
elements
• Rapid evolution
```
188
P Element Structure
* About 2900 bp long
* Gene encoding transposase • 31-bp inverted repeats
* Nonfunctional elements with no transposase
189
Hybrid Dysgenesis
• P elements produce hybrid dysgenesis
• Sterility in the F1 progeny of certain crosses
• Occurs in cross:
-M cytotype females have no P elements (lab strain)
- P strain males contain about 30 P elements (wild strain)
- Hybrid dysgenesis is observed in the offspring
• Sterility via widespread transposition of multiple P elements
• P elements are biological mutagens
190
Retroviruses
• Retroviruses infect eukaryotic cells
• Genomes are single-stranded RNA
• Flanked by long terminal repeats (LTRs)
• RNA is transcribed into ds DNA by reverse transcriptase
• DNA integrates into host genome
• Viral genes gag and env, produce new retroviral
particles;
• pol encodes reverse transcriptase
191
Retrotransposons Are Related to Retroviruses
```
1. Retroviruses
• Carry pol, gag, env
• Produce viral particles
2. Retrotransposons
• No capsid formation • No viral particles
• Dependent on host
```
192
LINE and SINE Elements of Humans
• 45% of the human genome from former transposable elements
| • No longer move • Still functional
193
LINE
* 600,000 per genome • 6.5–8.0kb
| * Cause mutation
194
SINE
• 1.2 million per genome • 100–300bp
195
what does Activator element do
ac-activated chromosome breakage at Dc(dissociation elelment)
196
McClintock's transposable genetic element hypothesis was that the unstable mutant phenotype resulted when a transposable element (Ds) created a mutation by its insertion into the C allele and led to reversion when the expression of Ac led to its removal.
t
197
What is the main enzyme responsible for excising and copying transposable genetic elements from chromosomes and inserting them into new locations?
transposase
198
What does the model of hybrid dysgenesis predict for the F1 and F2 generations when an M-cytotype male is crossed to a P-cytotype female?
F1 and F2 will be WT and have normal fertility
199
how do you determine hybrid dysgenesis for F1 and F2
look at female. If the female is M cytotope, no P element. the F1 will be sterile WT.F2 no progeny due to F1 fertility caused by hybrid dysgenesis.
if the female is p cytotype. the F1 and F2 is WT and have normal fertility
200
In what way do retrotransposons generally differ from typical DNA transposons?
They carry a pol gene that encodes reverse transcriptase.
201
P elements cause which phenomenon responsible for sterility in Drosophila?
hybrid dysgenesis
202
auxotrophic
need additional nutrient to grow
203
prototrophic
can grown in minimal medium
204
exconjugant
• The exconjugant cell is the recipient cell with its genetic information modified by receiving DNA from the donor cell
205
genes important in F factor transfer in plasmid
```
OriT: origin of transfer
tra1 encode for relaxase
traD encode for coupling proteins.
traK, traB and traP encode for exporter proteins
traA encode for pilin
```
206
IS element on F plasmid
- contain IS element and transposon that is antibiotic resistant gene
207
episome
• F factor can integrate into the bacterial chromosome, to form an episome
208
conjugation of F+ and F- cells. not exammable
- a mechanism of transfer and copying process
1. the donor cell (F+) assembles a conjugation pilus to contact the recipient cell (F-)
2. the relaxosome complex binds the F factor at oriT and cleaves the T stand of the DNA
3. the relaxosome partially degrades, leaving relaxase bound at the 5' end of T stand. the relaxase - T strand complex binds to a coupling factor to prepare for export. rolling circle DNA replication begins in the donor
4. the exporter moves the relaxes-T stand complex into the recipient cell. rolling circle replication in the donor spools the T stand tp the recipient, where it is a template for DNA replication
5. the completion os replication in both cells leaves the do not (F+) unchanged and converts the recipient cell to a F+ do not state
209
F+ X F- OUTCOME
exconjugant converted to donor state. F- become F+. the donor bacterial gene did not transfer to exconjugant, only F plasmid
210
Hfr X F-
exconjugant did not converted to donor state, the exconjugant remain F-. the donor bacterial gene did transfer to exconjugant
211
Bacteriophage Life Cycles
• Bacteriophage particles
212
Bacterial Transduction Is Mediated by Bacteriophages
• Genetic material, donor -> recipient cell via bacteriophage
• Bacteriophage infects a donor cell
• Donor DNA can be included in the phage
chromosome
• Progeny phage then infect a recipient cell, inject the donor DNA
• Use bacterial proteins as a means of entry
• Eg. maltose binding protein of E. coli
213
Steps of the Lytic Cycle
• The lytic cycle is a six-step process that leads to lysis of the host cell
1. Attachment of the phage to the host cell
2. Injection of the phage chromosome into the host, followed by circularization of the phage chromosome
3. Replication of phage DNA using host proteins and enzymes
Remaining Steps of the Lytic Cycle
4. Transcription and translation of phage genes, and subsequent production of heads, sheaths, and tail fibers for assembly of progeny phage
5. Packaging of phage chromosomes into phage heads
6. Lysis of the host cell, and release of progeny phage particles
214
The Lysogenic Cycle
• Temperate phages have an alternate, temporary life cycle
• Called the lysogenic cycle
• Integration of the phage chromosome into the
bacterial chromosome, called lysogeny
• Lysogeny can persist for many bacterial cell cycles,
but eventually comes to an end
• Lytic cycle is triggered
215
Steps of the Lysogenic Cycle
1. Attachmentofthephageparticletothehost cell
2. InjectionofthephageDNAintothehost, followed by phage-chromosome circularization
• Same as the lytic cycle
3. Integrationofthephagechromosomeintothe host chromosome, via recombination, at specific
sequence in both chromosomes
Additional Steps of the Lysogenic Cycle
• Once integrated into the host chromosome, called the prophage
4. Excision of the prophage • environmental signal
• site-specific recombination
5. Resumption of the lytic cycle, beginning with phage-chromosome replication
216
Discovery of Transduction
* The transfer of genetic material from a donor bacterium to a recipient via a phage
* Due to rare errors in packaging of phage DNA during the lytic cycle
217
2 types of transduction
```
1.Generalized transduction
• transfer of any bacterial genes
2.Specialized transduction
• only by temperate phages
• only genes transferred are those close to the integration site
```
218
Generalized Transduction
* Package a random piece of bacterial DNA into progeny phage heads
* An error
* DNA to be packaged by size
* By bacteriophages that cannot distinguish between phage and bacterial DNA
* The phage P1 infects E. coli, and has a genome of approximately 100 kb
219
Steps of Generalized Transduction
1. A normal P1 phage attaches to a donor bacterium and injects its DNA into the cell
2. Replication of the phage chromosome is followed by transcription and translation to produce phage proteins
3. Progeny phage are assembled normally, but some receive a host DNA fragment instead of phage DNA
4. Host cell lysis releases all the progeny phages
Additional Steps of Generalized Transduction
5. Generalize transducing phage (incorrectly packaged) attach to new recipient cells and inject their DNA
6. In each recipient, homologous recombination occurs between the donor fragment and the recipient chromosome
7. A stable transductant strain results
220
cotransduction
• Close genes are transferred together frequently
• Distant genes are rarely, or never, cotransduced
• Cotransduction frequency depends on the distance between two genes
• Can be used for mapping
• Due to
- the likelihood of two genes being packaged together into a single phage head
- chances of separation by a crossover event in the recipient
221
A Two-Step Strategy
* A two-step strategy reduces the number of colonies that must be tested in a cotransduction experiment
* Selected marker screen, colonies are selected for acquisition of one of the donor alleles
* Then the colonies are tested for the acquisition of the second allele in an unselected marker screen
222
Experiments of Charles Yanofsky
* In 1959, Charles Yanofsky obtained cotransformation data for genes that are part of the tryptophan operon
* Selected for cys+, a gene outside the operon, then tested the colonies for acquisition of one of four trp operon genes
* In four separate experiments
* He proposed a gene order of trpE-trpC-trpB-trpA
223
Specialized Transduction
• Temperate phages, such as λ phage, integrate into host chromosomes at specific sites
• Integration site
- att site
- 15-bp sequence, called attP in lambda phage, and attB in the E. coli host
- excision of the prophage is usually the exact reversal of the integration
224
Errors in Excision
• Excision of the prophage may be inaccurate
• Part of the prophage and part of the bacterial DNA
is removed
• Produces a specialized transducing phage
• In E. coli, the genes galK, and bioA, are found to either side of the attB site
225
Two Types of Specialized Transducing Lambda Phage
• Genes next to the attB site are captured by aberrant excision of lambda prophages
• The λdgal+ specialized transducing phage
- galK gene
- can infect host cells but lacks genes needed to complete either lysis or lysogeny
• The λdbio+ specialized transducing phage
- bioA gene
- can induce lysis but lacks the genes needed for lysogeny
226
F' X F- outcome
exconjugant converted to donor state, F- to F'. donor bacterial genes can be transferred to exconjugant
227
Benzer Studied the rII Locus of the T4 Bacteriophage
* The rII locus determines whether and how the phage will lyse its E. coli host
* Benzer showed that two genes, rIIA and rIIB, control the ability of T4 phages to lyse their hosts, with the wild type allowing lysis of multiple E. coli strains
* Mutants of either rIIA or rIIB limit the phage to just the B strain of E. coli
228
transformation step
1. double stranded donor DNA binds at the receptor site. 1 strand is degraded as it enters the recipient cell.
2. the transforming strand pairs with the homologous region of the recipient chromosome
3. the transforming strand displaces a recipient strand, forming complementary heteroduplex DNA (a-/a+). the excess strand degraded
4. DNA replication and cell division produce 1 transformant and 1 nontranformant.
229
Which of the following mechanisms of gene transfer in bacteria can produce recombinants for only a specific region (a few genes) of the bacterial chromosome?
• Specialized transduction
230
Some bacteriophages can be either lytic or lysogenic. These are called ________ phages.
temperate
231
whats population genetics
* Study of allele frequencies in populations
* Interpretation of allele frequencies across populations to explain natural selection and evolution
* Variance in allele frequencies in response to environmental gradients
* Epidemiology
232
How can we use population genetics?
* Forensic genetics
* Conservation genetics
* Management of pesticide and antibiotic resistance
* Origins of people and populations of animals
233
Conservation genetics
* Design of captive breeding programs
* Mating structures in endangered populations • Resolving taxonomic uncertainties
* Source populations for recovery programs
* Paternity testing
234
Origins of genetic variation
• Evolution proceeds by the differential reproduction of genotypes
• Expect variation to be eliminated by selection
• But high levels of genetic variation observed in
most populations
235
Mutation
```
• Original variation from mutation
• Slow process
Let the mutation rate from a to A be μ.
pn = p0e−nμ
pn = frequency of allele A at time n
p0 = frequency of allele A at time 0
```
236
Variation from recombination
Sex generates variability
Sexual species
• Crossing over and sexual reproduction produces high heterozygosity and shuffling of bases
237
Asexual species
Aseual species
• Variation arises only through mutation
• Amount of variation and the rate of evolution is lower in asexual species
238
Variation from migration
• Gene flow introduces new genetic variation
239
Origin of new functions
* Natural selection can only act on existing DNA sequences
| * Gene duplication permits evolutionary change
240
Repeated sequences can
* Retain original function
* Acquire new functions
* Lose function in some duplicates
241
Gene families
• Similar sequences found together
• Usually from duplication from a common ancestor
• May act in concert to produce gene products
• Various versions switched on in different
tissues
• 1000 gene families known in humans
242
Balanced polymorphisms
* Why are polymorphisms maintained?
| * Why are deleterious alleles maintained in populations?
243
How can polymorphisms be maintained?
* Frequency dependent selection • Neutral fitness
* Variable environments
* Heterozygous advantage
244
Balanced polymorphisms
• When polymorphisms are maintained by heterozygote advantage this is known as a balanced polymorphism.
245
A synthesis of forces maintaining heterozygosity
1. genetic drift (allele frequency bounce around)
2. a tiny mutation happened
3. balanced polymorphism push allele frequency to the centre, maintain heterozogosity
4. selection push to the edge, push to the fittest allele
246
what maintains all the genetic variation? mutation is the origin of the genetic variation
1. heterogenous selection.
2. sex (random mating)
3. migration
247
pseudogenes
duplication, 1 useless and 1 train function
248
cline EXAM
selection maintaining a gradient of allele frequency
249
Hardy-Weinberg law.
"In a large population and in the absence of mutation, selection or migration, allele and genotype frequencies remain constant from one generation to the next".
250
What causes perturbations?
1. migration
2. non-random mating (e.g. inbreeding, small population)
3. small population
4. selection
251
WAHLUND EFFECT
* 2 populations different allele frequencies mix.
* Causes a deviation of expected genotype frequencies from that predicted.
* Detected as a deficit of heterozygotes.
252
Causes of change in allele frequency
* Mutation
* Selection
* Immigration
* Assortative mating
* Drift
253
SELECTION AGAINST A HOMOZYGOTE
* Selection against homozygote reduces genetic variation.
* Variance can be maintained by frequency dependent selection
* Environmental gradients ==> clines
* Selection is inefficient against rare alleles because most are present as heterozygotes
254
Migration (gene flow)
• Migration is a major means of changing allele frequency.
• Migrants introduce new genes to populations. If advantageous, will spread (e.g. pesticide resistance genes)
Example:
Major source of wild-type blow fly populations is garbage tips. Wild-type genes rapidly spread after pesticides are withdrawn from use.
255
Genetic drift
Genetic drift leads to:
(1) Loss of heterozygosity (2) Eventual fixation
• Founder affect
• Low heterozygosity in a population suggests recent founder affect or local inbreeding.
• Small populations will often lose alleles by random chance.
• Problems for zoo populations: desirable alleles are lost.
256
Effects of inbreeding are:
```
• Random gene drif. Allele frequencies become erra9c
• Causes differentiation between
subpopulations
• Uniformity within subpopulations
• Increase in homozygosity
```
257
Inbreeding
• Defined as the mating of relatives
• Can occur by:
1. small population size
2. assortative mating
3. self-fertilization
258
Uses of inbreeding
1. fix 'desirable' traits in domestic animals and plants
| 2. measure the coefficient of inbreeding (F) , tell whether the population is subdivided.
259
Undesirable effects of inbreeding
1. loss of genetic variation
2. fix bad allele in small population
3. reduce fitness
260
Coefficient of inbreeding
• F = probability that two alleles in one individual are identical by descent.
261
Rule for calculating F
n = number of xINDIVIDUALS from one parent to the other
262
self-fertilization
- common in plants
| - heterozygosity is halved each generation
263
Selfing splits a popula9on into a series of homozygous lines
* Selfing does no alter original allele frequency
| * Heterozygosity is halved each genera9on
264
Relatedness
* Coefficient of inbreeding = probability that two alleles are iden9cal by descent in ONE individual
* Coefficient of relatedness, r, = probability that two alleles are iden9cal by descent in TWO DIFFERENT individuals
265
Rule for calculating r
n = number of STEPS (arrowheads) from one individual to the other
266
The inbreeding coefficient is half the relatedness of the parents
T
267
relatedness bro and sis
half
268
relatedness between half sib
1/4
269
relatedness of cousin
1/8
270
Kin selection
* Inclusive fitness = personal fitness + 1/r(fitness of relatives).
* Genes that will reduce personal fitness can spread if they greatly increase the fitness of relatives.
271
Social insects
• Most social insects are haplo-‐diploid
| - relatedness of 2 workers = 3/4 (1+1/2 /2=3/4)
272
inbreeding depression
• Inbreeding depression = decrease in growth, fer9lity and survival following inbreeding and an increase in homozygosity
273
hybrid vigour
• Hybrid vigour = heterosis = increase in vigour, usually after crossing inbred lines
274
Two theories of heterosis
• Davenport (1908) Dominance of linked factors – Favourable alleles are dominant
– Crossing inbred lines produces more heterozygous
loci
• East (1936) Overdominance
– Heterozygote is superior
275
Inbreeding depression
• Inbreeding depression is of major concern in domestic animals, humans, endangered species
• Inbreeding reduces survival of infants: – Sumatran tiger 0.03%. already of thru the genetic bottom neck, is already worth
– Brown lemur 90%
276
Conservation genetics
| Importance of maintaining heterozygosity
* Reduced heterozygosity indicates inbreeding-> population viability
* Assumption is probably correct. Heterozygosity is low in many species with low population size
277
Minimum effective popula9on size
nimum effective population size
• If one or two individuals dominate reproduction, the ‘effective population size’, Ne is lower than the actual population size.
278
• Ne can be increased by:
– Avoiding sib matings
| – All individuals contribute to the nest generation – Reduce variance in population size
279
Ne
Ne = the average number individuals that contribute genes to succeeding generations
280
Quantitative traits
* Controlled by many genes and alleles of small effect
* Environmental factors likely to contribute to phenotype
* Crossing experiments will not reveal simple allelic segregations
281
Qualitative trait
Qualitative traits (Mendelian)
• Controlled by a few genes of large effect
• Phenotype can be determined by simple crossing experiments
• Examples
-tongue rolling
282
East demonstrated that:
* Corolla length controlled by about 5 genes
| * Environmental factors contribute to corolla length
283
5 loci affecting corolla length
* Assume each + locus adds 1 cm
| * Here are 35 = 243 different genotypes • Some examples
284
Analysis of quantitative genetic variation
• Traditional methods: “Mate the best with the best and hope for the best”
285
Biometrical genetics
* Describe genetic variance (means and variances)
* Par--on phenotypic variance into contribution from genes and environment
* Predict response to selection
286
Broad heritability
• Proportion of total phenotypic variance due to genetic effects
• Recall that (ignoring genotype by environment
interaction):
287
Additive effects of genes
• A allele + 10 kg
• a allele -‐10 kg Addi-ve effect is 20 kg.
If Aa = 0 kg, then the alleles are addi-ve
If Aa ≠ 0 kg, then the alleles are non additive (i.e. there is dominance).
288
Because not all genes are additive genetic variance can be partioned into an additive and a non-‐additive component.
T
289
Narrow heritability
* Selection can only change allele frequencies
* Cannot change or accumulate non-‐ additive effects
* To predict response to selec-on we must only consider the additive effects of genes.
290
Narrow heritability
* Heritability not fixed for a character
* Refers only to a particular popula-on and environment
* Cannot extrapolate high heritability to other environments
291
Take home message
• The effect of selec-on depends only on addi-ve genetic variance, not non-‐additive genetic variance
• Narrow heritability, h2, not broad heritability, H2, predicts response to selec-on
ok
292
Predic-ng response to selection
```
R = h2 s
R = response to selec-on
h2 = narrow heritability
s = selection differen-al (≈ the proportion of individuals selected)
```
293
IQ
• h2 of IQ is quite high (0.3-‐0.6)
Some have claimed that:
• Liple can be done to improve IQ and social achievement
• Improving the scholas-c and social environment would have no effect on IQ because it is so highly heritable (gene-cally determined)
294
how to estimate genetic variance Sg2
grow the population in a high uniform environment
295
how to estimate environmental variance Se2
genetically uniform (clone) across many environment.
296
no dominance
d=0
297
A1 dominance to A2
d is positive
298
A2 is dominant to A1
d is negative
299
A allele + 10 kg
a allele -10 kg
additive effect is ?
20kg
| 10- -10=20
300
response to selection
you get the parental gene ration, if it is heritable, the mean is different (move L or R), if it is non-heritable, same mean
301
low narrow heritability for things related for reproductive and fitness.
high heritability is related to unimportant things which give the genetic variance
t
302
what does h^2 =0.5 mean
50% is due to additive genetic effect
303
will mating 2 high people give you high child?
if the narrow heritability is high, yes we can
| if it is low, we can't
304
What is a Genome?
• the ‘manual of life’, with 4-letter alphabet
• chromosomes = chapters, with many paragraphs • some paragraphs easy to understand, others not
What is a Genome?
• ‘program’ for making an organism • produces variety of cells, organizes
complex interactions in space + time
• determines much of who we are (though obviously not all)
305
What is Genomics?
* Systematic study at molecular level of entire genome + products
* Description of how genome contributes to unfolding of cell, organism, population, ecosystem
* Molecular biology + genetics on large scale: larger, more complex data sets
* Relies heavily on automated data acquisition, computer-based data analyses
306
Significance of genomics
• Allows detection of genetic variation that increases disease risk
• Allows detection of cancer mutations, aids search for cure/treatment
• For pathogens, genomics aids identification of targets for antibiotics
• promotes consistency, predictability in breeding, enhancing quality + quantity
• non-model organisms can be studied, reverse genetics can be applied
• helps to answer questions: what are we?
where did we come from? what lies in our future?
307
• structural genomics:
sequencing + analysis of genome information
308
• comparative genomics:
infer seq. function, understand genome evolution
309
functional genomics:
what genes do, how they form networks
310
Structural Genomics: provides gene catalog
• Genomes vary in size: single or multiple chromosomes
• organismal complexity increases, gene number increases (loose correlation only)
noncoding sequence amounts vary...
• How does one go about sequencing a genome?
311
Approaches to Sequencing Entire Genomes
* break genome into fragments, sequence in parallel
* whole-genome shotgun (WGS) sequencing: break into lots of smaller pieces, clone or add adaptors to the smaller pieces, sequence each piece from both ends
312
Whole-Genome Shotgun Sequencing
- Break entire genome into fragments, clone and sequence both ends (paired ends), then assemble into contigs based on sequence overlap
• Libraries can be different sizes: e.g. 2-3 Kb, 8-10 Kb, 20-30 Kb
• Sequence 10-12 times total genome size to produce enough overlapping sequence
313
WGS sequencing problem
Problem of repetitive elements
| e.g. AGAGAGAGAG...(length 10 kb) is dealt with by clones from which library?
314
Whole-Genome Shotgun Sequencing
* Sequence generated from both ends of DNA fragments of known size
* Paired-end sequences useful because they flank repetitive elements, can be assigned to same scaffold
* Scaffold: long segment of genome containing contigs + some missing data, but all fragments join up (PESs)
* Can’t be used when repetitive elements are longer than largest clones
315
WGS Sequencing of a Bacterial Chromosome
* Haemophilus influenzae: first genome to be sequenced by paired-end WGS sequencing
* 1.8 × 106 bp, few dispersed repetitive elements
* Three genomic libraries assembled into 140 contigs of unknown relative order and orientation; the sequence thus had gaps between each contig
316
Sequence Gaps
• The 140 gaps consisted of two types
• 98 sequence gaps, for which clones were available
to provide missing sequence (close the gap)
• 42 physical gaps, meaning sequence for which there were no clones available
317
Closing the Physical Gaps
* Lambda genomic libraries probed with sequences from scaffold ends to identify additional clones
* PCR, using primers specific to scaffold ends used to amplify the missing sequence
* Eventually the sequence was assembled into a single contig
318
WGS Sequencing of a Eukaryotic Genome
* Drosophila melanogaster; first large eukaryote with significant repeat elements sequenced using WGS
* Genome ~180 Mb; 120 Mb euchromatin; 60 Mb heterochromatin
* Only euchromatic portion was sequenced, due to difficulties with efficient cloning of heterochromatin
319
WGS Sequencing of the Drosophila Genome
* Three genomic libraries were used with clones of 2 kb, 10 kb, and 130 kb
* Sequence assembled into 50 scaffolds, totaling 115 Mb, with ∼ 800 additional small scaffolds representing about 5 Mb
* Genetic and physical maps used to assign 50 large and 84 of the small scaffolds to locations on four chromosomes
320
Metagenomics
* Microbial populations constitute the majority of life on Earth
* But only a small fraction of these can be cultured in the lab for study
* WGS sequencing of DNA isolated from natural communities composed of a range of organisms is called metagenomics; the data derived from such projects is called a metagenome
321
Why metagenomics?
* Discovery of novel genes, enzymes, metabolic pathways: important in pharmaceutical and agrochemical industries
* Human health: helps to understand role of non- pathogenic bacteria associated with humans. Uncultivatable microbiota of other organisms too...
* Improve strategies for cleaning up polluted and contaminated sites: how do bacteria evolve in such environments?
322
Approaches to find the genes
* Expression profiling (e.g. microarray, RNA sequencing)
* Quantitative Trait Loci (QTL) mapping
* Candidate genes
* SNP association studies
323
QTL mapping requirements
* Two strains that differ strongly for the trait of interest
| * A saturated genetic map
324
The problem of multiple comparisons
* If we analyse 350 markers with a 5% level of significance, we expect 0.05*350 = 17.5 significant associations
* Set significance threshold very low (
325
Interval mapping
Intervl mapping
• Uses information from linkage map.
• Seeks pairs of linked markers that show evidence of a QTL between them.
326
From QTL to gene
• For many model species there is a complete genome sequence.
327
Candidate genes
• List of:
– Obesity genes from different species – Genes related to lipid metabolism
– Etc.
• Design PCR primers for these genes within the target species.
• Compare gene expression with RT-PCR
328
Recombination is rare over short distances. Haplotypes preserved for many generations
T
329
Annotation to Describe Genes
* Annotation: attaching biological functions to the DNA sequences, based on experimental evidence (preferably) or computational analysis
* Genome annotation: identifying location of genes + functional sequences within genome
* Gene annotation defines biochemical, cellular, and biological function of each gene product
330
Experimental Approaches to Annotation
• cDNAs and ESTs* are compared to genome to identify transcribed sequences of a genome
• Complete cDNA clone set from an organism would allow complete annotation of protein coding genes
*EST = expressed sequence tag = short cDNA fragment, 5' or 3’
331
Computational Approaches to Annotation
* Eukaryotic genomes may contain 1000s of genes for which little/no experimental data available
* The use of computational approaches instead is called bioinformatics
* Algorithms predict gene structure based on recognition of open reading frames (ORFs), sequences that could encode polypeptides
* Most algorithms search for ORFs larger than a minimum size, such as 50 amino acids
* Predictions not infallible: single small exons may be separated by very large introns
* Algorithms are 50–90% accurate in predicting exons, but provide information to assist in design of experimental approaches for confirmation
332
Non-Protein-Coding Genes
* Genes encoding functional RNAs much more difficult to predict computationally (don’t contain ORFs)
* Experimental or comparative approaches are usually required for annotation of such genes
333
Annotation to Describe Biological Functions
• Some genes, e.g. lacI, are well studied: annotation can be quite detailed.
• For genes that are not well-studied, other approaches are needed
• Genes with similar sequence are assumed to encode products with similar biochemical functions
334
BLAST (Basic Local Alignment Search Tool)
* cut query + database entries into “words” e.g. 11 bp long
* if exact match found, comparison extended in both directions
* some mismatches OK, but must have minimum score (based on identical and non-identical bases)
335
Assigning Biological Functions to Genes
* Initial annotation of eukaryotic genomes categorizes genes by presumed biological or cellular functions
* About half of the predictions based on sequence similarity to known proteins
* Predicted biological function should be confirmed experimentally, often by examination of mutant phenotypes
336
Identification of Gene Families
* Genomes contain gene families: groups of genes that are evolutionarily related (how?)
* For example, the 23,000 genes of the human genome can be placed into about 10,000 gene families
* Gene family expansion depends on importance to organism; e.g., the olfactory receptor gene family larger in mice (900 genes) than humans (339 genes)
337
Protein Domains
* Sequence conservation does not necessarily extend to entire gene: but may include functionally important parts (protein domains)
* Eukaryotic proteins often modular, consist of distinct domains joined together; some domains found in numerous genes
* Knowledge of conserved protein domains may provide insight into biochemical activities (requires mutant analysis for confirmation)
338
Variation in Genome Organization Among Species
* Hundreds of genome sequence for bacteria, archaea, eukaryotes allow understanding of genome organization
* Bacteria and archaea have fewer genes and higher gene density than eukaryotes, due to lack of introns, compact gene regulatory sequences, and overall lower complexity of protein structure
339
Comparative / Evolutionary genomics:
the comparative study of genomes
340
Interspecific comparisons
identify sequences conserved over evolutionary time
341
Intraspecific comparisons
identify sequence polymorphisms responsible for genetic differences between individuals
342
The Tree of Life
* Large amount of DNA sequence information has helped resolve the tree of life
* Genes encoding rRNAs provide a universal sequence for comparison due to their ubiquity and high degree of conservation
* Woese and colleagues used these to show (late 1970s) that all forms of life fell into three domains: Bacteria, Archaea, and Eukarya
343
Homologous Nucleotides
• Alignment of homologous nucleotides is used to ascertain phylogenetic relationships
344
• Homologous nucleotides:
* Homologous nucleotides: descended from the same nucleotide in the common ancestor of species being compared
* Highly conserved protein-coding DNA sequences used to resolve ancient divergences; noncoding sequence may clarify very recent nodes
345
Interspecific Genome Comparisons
* Two closely related species may share almost their entire genome
* The genomic differences between sister taxa define the differences between two species
* For example, in four closely related Saccharomyces species, the number of species-specific genes constitute just one for every 500,000 years of evolutionary distance
346
The Births and Deaths of Genes
• By comparing genome sequences, geneticists understand how new genes arise:
1. Gene duplication by duplication of genomic DNA 2. Gene duplication by unequal crossover
3. Exon shuffling
4. Reverse transcription
5. Derivation of exons from transposons 6. Lateral (horizontal) gene transfer
7. Gene fusion and gene fission
8. De novo derivation
347
The Births and Deaths of Genes, continued
* Comparison of genomes of Drosophila spp. show ~80% of new genes arise by duplication
* The duplicates were either tandem or dispersed
* 10% were derived from retrotransposition events
* 12% arose de novo, from previously noncoding sequences
348
Gene Duplication
• Most genomes contain a mosaic of gene families derived from ancient or recent duplication events
• Duplication rate is ~ 0.01 genes per million years
• For an average eukaryotic genome of 10,000–30,000
genes: one gene duplication per 3000-10,000 years
349
Fate of Duplicated Genes
* Depends on the molecular basis of the duplication..
* If entire gene duplicated, both copies can produce functional protein product; genes thus redundant and can evolve new functions
* However, fully redundant genes (i.e. with identical function) are not for maintained long...
350
Fully Redundant Genes Are Not Maintained
* They may degenerate due to lack of positive selection, accumulate mutations, become pseudogenes
* Mutations may produce two different genes with complementary functions: “subfunctionalization”
* Mutation in one copy provides new function not provided by other: “neofunctionalization”
351
• Paralogs (paralogous genes):
• Paralogs (paralogous genes): genes originated by duplication event; functions usually biologically distinct, but biochemically related
352
• Orthologs (orthologous genes):
• Orthologs (orthologous genes): genes in different species derived from a single ancestral gene. Usually have equivalent functions in diff. species
353
Lateral Gene Transfer
Lateral Gene Transfer
• the transfer of genetic material between two species
• e.g. sharing of plasmids among bacterial species
• in bacteria and archaea: 1.5 to 14.5% of genes from LGT
354
Lateral Gene Transfer Is Rare in Eukaryotes
• LGT between prokaryotes and eukaryotes is rare
• Why? Different transcriptional and translational
control mechanisms between them
• One prominent exception: transfer of DNA from endosymbionts to their hosts, e.g. chloroplast and mitochondrial genes, now resident in eukaryotic nuclei
355
Interspecific Genome Comparisons: Genome Annotation
* By comparing genomes of closely related species, researchers can refine annotations of predicted genes (i.e. those not experimentally confirmed)
* Sequences conserved among species more likely to be functional than nonconserved sequences
* Can be confirmed by experimental examination
356
contigue
contiguous sequence
357
scaffold
orientation of contains, but don't know the sequence for the repeat sequence in between
358
Conserved Noncoding Sequences
* At least some conserved noncoding sequences (CNSs) are functional
* Centromeric + telomeric sequences, gene regulatory sequences, and genes that produce functional RNAs
* Can be identified by comparisons of distantly related species. E.g. comparisons complement promoter analysis experiments, help predict regulatory sequences
359
Interspecific Genome Comparisons: Gene Order
* Synteny: conserved order of consecutive genes along chromosome
* Microsynteny, synteny at the level of just a few genes, may be detected in organisms in which chromosomal synteny is not preserved
360
Interspecific Genome Comparisons: Gene Order, continued
* Striking feature of eukaryotic genomes: evidence of past whole-genome duplications or smaller duplications involving parts of chromosomes
* Whole-genome duplications are particularly abundant in plants, though not limited to plants
361
computational appraches to annotation
1. identify the 3 reading frames in the forward direction and in the complementary strand
2. highlight all potential start codon
3. highlight all potential stop codon that r in the same reading frame
4. identify open reading frames and corresponding amino acid sequences.
362
Synteny
Synteny: conserved order of consecutive genes along chromosome
363
Microsynteny,
Microsynteny, synteny at the level of just a few genes, may be detected in organisms in which chromosomal synteny is not preserved
364
Interspecific Genome Comparisons: Gene Order, continued
* Striking feature of eukaryotic genomes: evidence of past whole-genome duplications or smaller duplications involving parts of chromosomes
* Whole-genome duplications are particularly abundant in plants, though not limited to plants
365
Chimp (Pan troglodytes) vs human genome
• 3.1 Gb over 48 chromosomes • Chimp chromosomes 2A + 2B
= human chromosome 2 (fusion event)
• Very high level of synteny (shared gene order)
366
What It Means to be Human
* ∼ 4.0 % of the euchromatic sequence in each species (humans and chimpanzees) is lineage specific
* Single nucleotide substitutions number about 30 million (1 every 100 bp, or 1%)
* the vast majority of SNPs are in noncoding DNA
367
Comparison of Human and Chimpanzee Genomes
* 29% of orthologous proteins identical in sequence, average gene encodes only 2 amino acid differences
* Indels: 5 million, account for 3% of the genomic differences
* Genomes differ by several hundred genes, due to gene gains and losses
368
Comparison of Human and Chimpanzee Genomes: Transposable Elements
* Many indel differences are due to activity of lineage- specific transposable elements (e.g. Alu-based recombination)
* SINEs (short interspersed elements) are more active in humans than chimpanzees
* Chimpanzee genome contains two retroviral elements not found in humans
369
Which Genomic Changes Are Functionally Important in Human Evolution?
* It is useful to determine whether observed differences occurred in chimp or human lineage
* To do this, need more distantly related species–Gorilla
* Gorilla helps show which features are ancestral (pre- existing in a common ancestor) and which are derived (specific to one of the lineages)
370
Can we re-create our last common ancestor?
infer ancestral genome (chuman), then infer intermediate of chuman and human (Australopithecus) then implant in human?
371
Intraspecific Genome Comparisons
* Allelic differences (polymorphisms in DNA) cause phenotypic differences between individuals of a species
* Evolutionary history of a species is reflected in distribution of polymorphic alleles among populations
* Genome of haploid or inbred diploid individual used as reference genome sequence, for comparison to others
372
Intraspecific Genome Comparisons
Phylogenetic tree showing distances between human populations (microsatellites, Indels, SNPs)
373
Human Genetic Diversity
* The first two human genome sequencing projects identified a limited set of polymorphisms because multiple donors were used
* DNA sequenced by Celera was isolated from a single individual, with a maximum of two possible alleles at any locus
* By the end of 2010, entire sequences for hundreds of individuals were available, representing considerable diversity, millions of polymorphisms are now known
374
SNPs and Indels
* A sampling of SNP (single nucleotide polymorphism) between two humans reveals differences at about 1 in 1000 bases, or approximately 3 million polymorphic sites
* SNP variation accumulates due to mutation at the rate of about 30 SNPs in each individual’s germ cells per generation
* Analyses of multiple human genomes has also revealed a high frequency of insertion or deletion (indel) and small inversion variants
375
phylogenetic shadowing
identification of sequence conserved among ALL species of a related group
376
phylogenetic footprint
comparison between distantly related organisms to find conserved areas
377
TATA box
TATAAA
378
start codon
AUG
379
Functional Genomics
• What makes two cells different?
• They have had a different developmental
pathway and been exposed to different signals
• Different genes expressed in each
• means there is a different protein complement • Means their metabolism is different
• Functional genomics can describe these differences (and others) and use them to understand the processes going on in the cell and organism
380
Functional genomics
unctional genomics
• Aims to describe the functions of all genes in the genome
• Expression
• Control of expression • Products of expression
• High-throughput methods! • Deals with global data sets • Requires good bioinformatics
381
Transcriptome
Trnscriptome – set of all RNA molecules expressed in particular cell types or under particular conditions
382
Proteome
– set of all the proteins in particular cell types or under particular conditions
383
• Metabolome -
set of all metabolites in particular cell types or under particular conditions
384
Interactome
– set of all physical interactions between DNA, RNA and protein molecules
385
• Phenome
– set of all phenotypes expressed by a cell, tissue or organism
386
Variation via alternative splicing
Alternative splicing can produce a large number of transcripts from a single gene resulting in many different proteins that vary in their function
Alternative splicing produces 2 receptors that bind different ligands
387
Post-translational events
• Phosphorylation
• Glycosylation
• Cleavage of signal peptides

388
Expression Profiling – mRNA
* A set of procedures that relies on the fact that DNA is transcribed into mRNA before that is translated
* Genome expression can be measured qualitatively and quantitatively
* Using mRNA, expression profiling requires • Synchronization of the cell cycle
* Methods to preserve mRNA information
* Methods to assay gene expression
* Microarrays of oligonucleotides or cDNA
* RNA-seq to sequence transcripts (next-gen sequencing)
* Methods of data analysis (e.g. bioinformatics, statistics)
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Using Microarrays to assay gene expression
• cDNA microarray
• cDNA is spotted onto a microscope slide (10-50
mm–2)
• Oligonucleotide microarray
• Synthetic sequences (25 bp) are placed on a slide
• Whole genome tiling (oligonucleotide) arrays are available
• Shows parts of the genome that are expressed • Identified many small RNAs
• Allows comparison across tissues and
treatments
390
cDNA synthesis
Important notes:
Because the poly-T primer targets all poly-A sequences, including those of mRNA molecules, this procedure allows us to construct a cDNA library of all the genes that are expressed by a genome at any point in time. Can also use random primers to prime cDNA synthesis
Like the genome a cDNA library can be considered a ‘global data set’
391
Synthesising labelled cDNA
* Add a labelled or modified nucleotide into the reaction
* Eg. cy3-labelled dCTP
* Eg. aminoallyl-modified dUTP
* This can then be labelled afterwards with a fluorescent label
392
Microarrays- advantages
* If you have a genome sequence you can make oligonucleotides for all predicted ORFs
* Can easily and relatively cheaply assay gene expression
393
Microarrays- disadvantages
• Can’t detect novel transcripts (need genome sequence)
• Indirect method- transcript abundance inferred from
hybridization intensity
• Low signal to noise ratio- hard to detect transcripts
expressed at a low level (often transcripts of transcription
factors)
• limited dynamic range of detection owing to both background
and saturation of signals.
• Signal becomes saturated so high expression may not be
accurately quantified
• Reproducibility not great
• Cross comparison difficult and relies on complicated normalisation methods
394
“Next-generation” sequencing to study the transcriptome (RNA-seq)
* “deep sequencing”
* Methods that make sequencing quicker (in the order of 10s of Mb/hour) and cheaper (10000 times)
* Sequencing by synthesis (with or without amplification of template)
* Sequencing by ligation
* Can determine mRNA and small RNA components in different tissue, cell type or treatment
* No cloning step so preparation is quick
395
RNA-seq method
```
• Can convert RNA to cDNA or leave as RNA
• Fragment cDNA or RNA
• Add adaptors
• Use next gene methods
to sequence
• Align to genome
Count reads
• Analyse differences
that suggest alternative splicing
```
396
RNA-seq- disadvantages
• Sequencing is more error prone than Sanger sequencing
• The cleavage of cDNA or mRNA may bias results
• Some methods use PCR to amplify cDNA which may
introduce artifacts
• Mostly info is not strand specific
• Huge data sets require lots of computing space and
bioinformatic analysis
397
RNA-seq - advantages
• Does not require knowledge of genome (useful for non-model organisms)
• Reveals sequence variation (alternative splicing, polyA addition)
• Low background
• High dynamic range (>9000 fold vs 100s fold for microarray)
(depends on number of reads done)
• Accuracy comparable to real-time quantitative PCR
• Good reproducibility
• Uses less RNA than microarray
• Much cheaper than other methods (10000 times less)
• Fast (13-83 Mb/h cf 0.03-0.07 Mb/h Sanger)
398
2-D Polyacrylamide Gel
| Electrophoresis
* Separation based on charge
* Separation based on molecular weight
* can have denaturing or native gels in 2nd dimension
* Native gels identify protein complexes
399
Expression Profiling – Proteins
• mRNA studies can’t confirm how much protein is produced, and if/where proteins are modified
• Many more proteins than genes so more complex than genome
• 40% correlation estimated between transcript and protein levels
• Development of proteome can look at localisation
by studying organelles, compare cells, help determine what metabolism is occurring
• Expression profiling of proteins requires • Separation of the different proteins/peptides
• Gel-based
• Liquid chromatography
• Characterization of proteins of interest
400
Proteomics
cleavage of protein with protease e.g. trpsin
Separation of peptides by liquid chromatography
mass spectrometry
fragmentation of selected peptides and analysis of the resulting MS/MS spectra
data analysis, including identification and quantification of proteins from several detected peptides, as well as downstream bioinformatic analysis depending on the specific application
401
Proteomics
* Different methods allow post-translational modifications to be identified
* Phosphoproteome • Glycoproteome
* Basic methods tell you what is there but are not quantitative
* Comparison of different samples by
* 2D-DIGE (2D-difference gel electrophoresis)
* iTRAQ (Isobaric tags for relative and absolute quantification)
* Label free quantitative proteomics
402
Studying gene function (Phenome)
* Various methods can be used to reduce gene expression so the role of the gene can be determined
* RNAi uses hairpin constructs to generate siRNA that silence genes
* CRISPR-Cas can be used for gene knockdown and modulation of transcription (up or down)
* Methods for high-throughput analysis developed
403
And after all this?
And after all this?
• We need to put all the data together to gain insights of how cells and organisms work and interact (systems biology)
• Use the data to answer our question looking at data for transcript, protein, activity, interaction etc
• These techniques may have a big impact on medicine
• Tailor treatments based on the protein or transcript expression in a disease
• Reduce costs by reducing use of ineffective treatments
404
transcription factor
Transcription factors - proteins that bind DNA and regulate gene expression. Can activate or repress gene expression.
Transcription factors are classified by the type of DNA binding domain eg homeodomain, helix-loop-helix (sex determination in Drosophila)
405
The Study of Development
The Study of Development
What makes two cells different? • Different genes expressed
• means there is a different protein complement
• allows cells to differentiate into different cell types • Sets of genes controlled by particular transcription
factors
• So how does one cell develop to produce daughter cells that express different transcription factors even though each cell has the same genes
• Position of the cell is important
• Cell lineage can be important
• But initially one cell produces the whole organism
406
The Study of Development
How are cells organised into a coherent body plan during development?
• Body plan is the specific pattern of body parts eg tetrapods have four limbs, insects have six.
• Developmental genetics uses the tools of genetics to dissect the role of genes in developmental pathways.
407
The study of development - techniques
A. Mutant genes
Phenotype - what tissue is affected? What stage of development?
What is the protein product? - Clone and sequence the gene.
Christiane Nüsslein-Volhard and Eric Weischaus, Nobel Prize in Medicine in 1995.
•P erformed mutational screens to isolate genes controlling pattern formation during the early development of D. melanogaster.
Maternal-effect genes: the phenotype of the offspring reflects the genotype of the mother
408
B. Analysis of gene function
(a) RNA in situ hybridization- where and when is a gene expressed? • Shows the location of the mRNA of a particular gene
• Uses histochemically or radioactively tagged DNA or RNA probe complementary to the mRNA
(b) Immunohistochemistry
• Shows the location of the protein product
• Uses histochemically tagged antibodies that bind to the protein (c) Transgenesis
• P element mediated gene transfer in Drosophila
• Gene microinjection or knockouts in mice
(d) RNA interference - reduces or knocks out gene expression
409
Oogenesis   The egg chamber consists of:
(a) germ-line = oocyte + 15 nurse cells
• arises by mitosis from a single oogonium
• shares a common cytoplasm, connected by ring canals
• nurse cells provide most of the contents of the oocyte cytoplasm
(b) somatic cells = follicle cells
• make the egg shell (chorion proteins)
• interact with the oocyte to help establish axes of the body of the fly
410
Embryonic development
(i) Fertilized egg
ii) Syncytial blastoderm • Nuclei divide but the egg does not
•S oluble molecules can diffuse
throughout the common egg cytoplasm
(iii) Nuclear migration
• Nuclei migrate toward the periphery of the egg
(iv) Cellular blastoderm
•P lasma membrane invaginates around each nucleus
• Contents of each cell isolated from other cells
411
gastrulation
Gastrulation
| Movement of cells to the interior forms three cell layers from one. Cells move through the cephalic furrow.
412
segmentation
```
Segmentation visible by 10 hrs segments:
3 head
3 thoracic 8 abdominal
Larva hatches at 24 hrs.
Denticle bands
characteristic for each
segment
```
413
How do genes specify the Anterior - Posterior axis of the embryo?
bicoid and nanos are maternal-effect genes in Drosophila bcd/bcd female larvae without head or thoracic segments nos/nos female larvae without abdomens
Localization of bicoid and nanos mRNA
•b icoid mRNA is synthesized by nurse cells • mRNA passes through ring canals into the oocyte • Anchored to the cytoskeleton at the anterior
•T ranslation begins after the egg is laid
•n anos mRNA is anchored to the posterior
414
hb-m mRNA is uniformly distributed.
| NOS - an RNA binding protein, inhibits translation of hb- m mRNA. Gradient of HB-M.
T
415
Morphogens:
regulatory molecules that control cell fate in a concentration dependent fashion
bicoid and hunchback-m
Maternal effect genes
Protein products (BCD and HB-M) are transcription factors
Form a concentration gradient in the egg
416
The hierarchical cascade of gene expression leading to A-P pattern in Drosophila
T
417
What is the evidence that bicoid contributes A-P positional information?
1. Thephenotypeofthenull mutant: Larvae from bcd/bcd mothers have no head or thorax
2. BCD protein is expressed in the appropriate position in the embryo
3. Increasing dosage of bcd+ gene pushes the cephalic furrow to a more posterior position.
4. Rescue of bcd embryos to wild-type by anterior
cytoplasm or bcd+ mRNA
418
One way to define positional information is to localise
| 
• In the Drosophila embryo it can
be used because the system
starts with a single cell and division do not result in separate cells.
•More generally it can be used to distribute local determinants to progeny cells through asymmetric division
419
A different type of positional information- Induction
Used in defining the dorsal ventral axis
| Formation of a concentration gradient of a diffusable molecule is a common way of determining position
420
Homeotic mutations -
Homeotic mutations - mutations that lead to the replacement of one body part by another
(mutations are in hox genes) (a) Wild-type
(b) Ultrabithorax (Ubx) Loss-of- function results in development of forewings in place of hind wings
(c) Antennapaedia (Antp) in Drosophila is a gain-of-function mutation causing expression of the gene product in the head as well as thoracic segments resulting in legs in the place of antennae.
421
Hox genes are expressed in structures affected by Hox-gene mutations
Ubx promotes hindwing formation and represses forewing development. It is not expressed in the forewing but is in the hindwing (to stop it becoming a forewing).
422
Hox proteins repress appendage formation in the abdomen
DII: Distal-less (red) (Hox gene target)
Ubx: ultrabithorax (pink)
En: engrailed (blue) (not Hox gene, a segment polarity gene)
abd-A: abdominal A-
423
In humans, a large number of genes associated with immune function share sequence similarity and are evolutionarily related. These would be known as what kind of group?
a gene family
424
why do aneuploids have reduced viability
1. imbalance of the genetic material
| 2. expression of deleterious recessives
425
* Loss of chromosome segment - deletion
| * Duplication chrom. segment - duplication
t
426
Rearrangement of gene order
Within a chromosome - inversion
Between chromosomes - translocation
t
427
2. Deletions and duplications
Deletions
Loss of chromosome segment -small intragenic deletions or larger multigenic deletions
(a) Cytology - Pairing loop in heterozygotes in meiosis (pachytene) and in
polytene chromosomes - loss of bands from deletion chromosome (b) Drosophila deletion heterozygote
428
Deletions
(b) Phenotypic effect
Homozygous deletion - usually lethal.
Heterozygote - may be lethal - loss of normal gene balance or uncovering of deleterious recessives.
Pseudominance - unmasking of recessive alleles on the complete chromosome. Loss - of - heterozygosity.
429
Deletion mapping -
breed flies to pair each deletion with each mutation. fa phenotype observed with all deletions except 258-11 and 258-14 Therefore fa maps to band 3C-7
430
1. Cri-du-chat syndrome
```
loss of
the tip of chromosome 5.
• Funny crying as babies
• Some mental retardation
• Live to adulthood
```
431
2. Williams syndrome:
Elfin facial features, unusual development of nervous system, overly friendly, musical ability, cardiovascular problems.
Deletion of 20 genes from the region q11.23 of chromosome 7
432
Red/green colour- blindness
Red/green colour- blindness results from gene deletions due to unequal crossing over.
• Red and green visual pigment (opsin) genes are closely linked on the X chromosome
• 1 red opsin gene, 1 - 5 green opsin genes
• red-green gene sequences 98% identical
• green gene repeats 99.9% identical including intergenic DNA
433
4. Schizophrenia
* 22q11.2, 1q21.1, 15q13.3 deletions implicated
* Lupski Nature 455:178 (comment)
* International Schizophrenia Consortium Nature 455:237- 241
434
Duplications
(a) Tandem duplications - unequal crossing over
435
Gene duplication is a powerful force in evolution
• Whole gene duplication - duplicated gene can mutate and evolve new function while original gene maintains old function.
eg globin genes. Haemoglobin - 2 α chains + 2 β chains
α genes on chromosome 16, β genes on chromosome 11,
Ψ non-functional pseudo-genes
Genes expressed at different developmental stages. Different chains have different oxygen binding affinities.
436
Duplications
Dupliations
b. Insertional duplications - extra copies elsewhere in the genome
• Can occur through incorporation of a “retrogene” • Short leg dogs have a retrogene duplication
active in fetal humurol bones • Fibroblast growth factor 4
• Inapproriate expression in humans causes dwarfism
437
3. Inversions
Inversions
Paracentric - does not include centromere, 2 breaks in one arm
• Balanced genome - no loss or gain of genetic material
• If break point is within an essential gene lethality
438
Cytological effects of inversions
(i) Pairing to form inversion loop
| (ii) Changed chromosome arm ratios
