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2

Lecture 1

Chromosome Structure and Function.

3

Describe how DNA is packaged in the nucleus.

DNA wraps around histone octamer proteins forming nucleoli. H1 linked protein connects nucleosomes. Loops of nucleosomes form coils on SARs/MARs, forming solenoids Solenoids form the chromatin fibre.

4

What are the subunits of histones?

H2A; H2B; H3; H4.

5

What are SARs and MARs

Scaffold/Matrix Attachment Proteins

6

Describe the process of heterochromatinisation.

Under-acetylation of H3/H4 tails by histone deacetylase complexes (HDACs). Trimethylation of H3's lysine 9 (H3K9) causing recruitment of heterochromatin protein (HP1). Trimethylation of H4K40.

7

Describe the process of euchromatinisation.

H3/H4 acetylation by Histone Acetylases (HATs).

8

What is the functional difference between heterochromatin and euchromatin?

Euchromatin is associated with transcription factors which allows gene expression. Heterochromatin blocks TFs.

9

What is Positive Effect Variegation?

Mutation causing the gene to be placed nearer heterochromatin. This would decrease the gene expression of the gene causing a formation of a new, recessive allele.

10

What are CpGs?

5'-CG-3' sites.

11

What are CpG islands?

Regions of high CpG frequency.

12

What effect does methylation of CpGs have?

Turns off gene expression.

13

Draw the diagram of CpG methylation/demethylation.

Should include: Dnmt1; hemimethylase; DNA demethylase; DNA methyltransferase 3a/3b.

14

What are MeCPs? Give an example.

Methyl-CpG-binding Proteins; MeCP2.

15

What is the function of MeCP?

Transcription repression via central domain binding to CpGs. Binds to HDACs causing condensation of chromatin.

16

How does HDAC condense chromatin?

Removes acetyl groups allowing tighter wrapping of DNA around histones.

17

What is Rett Syndrome?

X-dominant de novo Childhood Neurodevelopmental disorder - Loss of speech; Severe retardation.

18

What is distinctive about sufferers?

Almost all female but male lethality disproved. De novo mutations almost always occur on paternal X.

19

What causes Rett Syndrome?

MeCP2 mutation (Xq28) - Decrease in efficiency of gene repression/silencing.

20

What is ICF?

Immunodeficiency Centromeric abnormalities and Facial anomalies. Autosomal Recessive.

21

What causes ICF?

Mutations in Dnmt3-Beta - Causes failure of de novo methylation of pericentromeric CpGs.

22

Compare chromosomal replication of S. cerevisiae and mammals.

S. cerevisiae: Origin Recognition Complex (ORC) binds to Autonomous Replication Sequence (ARS); Mammals: Human Initiation Regions (HIRs) are homologues of ORCs which also bind to the Autonomous Replication Sequence.

23

What is the function centromeres?

Ensures equal distribution of replicated chromatids into daughter cells. Acentric fragments do not get segregated.

24

What happens during dicentric chromosome segregation?

If one of the centromeres is inactive, the chromosome will be segregated by the active one and the inactive is split into 2. If both are active, both are pulled apart forming an anaphase bridge in between them which later breaks to separate the chromatids.

25

Describe the centromeres of S. cerevisiae.

Simple sequence; 3 Domains.

26

Describe the centromeres of Drosophila

Tandem repeats - 10bp AT-rich sequence.

27

Describe the centromeres of Mice.

Tandem repeats - 300-5Kbp. CENP-A histone (Similar to H3). Minor satellite. CENP-B box - binds CENP-B (Centromere protein B) protein.

28

Describe the centromeres of humans.

Alpha-satellite. 171bp repeat. Conserved CENP-B box.

29

What is a satellite?

Sequence of tandem repeats.

30

Is centromere formation coded by the genome? Support claim.

No, dependant on chromatin conformation. Centromeric sequences not conserved between species.

31

What are telomeres?

Natural ends of chromosomes.

32

What is the function of telomeres?

Protection; Compensation for sequence loss; Nuclear organisation.

33

What effect does lack of telomeres have on segregation?

Fusion of chromosome occurs causing formation of anaphase bridge. Chromosome breaks into 4. Chromatids fuse again etc.

34

Describe the conservation of telomeres.

TTAGGG repeat. Conserved in most eukaryotes: Conserved orientation/ss overhang. Variable length.

35

What are the specialised transposons in telomeres of Drosophila?

HeTA; TART (Telomere-Associated Retrotransposons.

36

What is telomerase and what is it's function?

Specialised reverse-transcriptase; Involved in telomere replication.

37

Describe the process of telomere replication.

Elongation - Bases added to get rid of overhang; Translocation of the former overhang up, to create a new overhang.

38

Describe the 4 chromosome morphologies.

Telocentric - Centromere at telomere; Acrocentric - Centromere less than 1/4 from telomere; Submetacentric - Centromere 1/4 from end; Metacentric - Centromere in the middle.

39

What are NORs?

Nucleolus Organiser Regions. Present on Acrocentric chromosomes. Tandem repeats of ribosomal DNA

40

What are the distinct types of bands on chromosomes?

G - Giemsen banding; R-banding; C-banding.

41

What are G-bands?

Rich in AT. Rick in L1 elements (retrotransposons). Few genes.

42

What are R-bands?

Reverse of G-bands. GC rich. Rich in Alu elements. Rich in CpG islands. Rich in genes.

43

What are C-bands?

Centromeric heterochromatin.

44

What is FISH? Describe the process.

Fluorescent in-situ Hybridisation. Hybridisation of designed probes to chromosomes. Probes complimentary to sought sequences. Detected directly if probes labeled with a fluorochrome. Detected indirectly if tagged antibodies are used to amplify the probe signal.

45

Give 2 examples of fluorochromes used in FISH.

Fluorescin. Rhodamine.

46

Give 5 examples of probe types used in FISH.

BACs; YACs; Cosmids; Tandem repeats (Satellites). Chromosome paints.

47

What are chromosome paints?

Flow-sorted chromosome chunks.

48

How is Flow cytometry used for fractioning chromosomes?

Metaphase chromosomes stained with fluorescent dye. Fluorescence measured with a laser/detector. Chromosomes charged and collected after deflection towards oppositely charged plate.

49

Give 4 examples of chromosome imbalance.

Polyploidy - Tetraploidy 92XXXX; Aneuploidy - Monosomy 13; Sex monosomy - XO (Turner); Sex trisomy - XXY (Klinefelter).

50

What are the 3 viable autosomal trisomies?

Trisomy 13 - Patau syndrome; Trisomy 18 - Edward syndrome; Trisomy 21 - Down syndrome.

51

What causes non-disjunction?

Unbalance in genomes caused by errors in segregation. Abnormal distribution of cross-overs.

52

Describe normal chromosome segregation.

Centromeres of chromatids attach to spindles of 1 pole each. Rec8 joins chromatids in bivalents. Anap1: Sgo1 protects Rec8 to keep chromatids together. Anap1 triggered by cleavage of Rec8 by separase; Sgo1 degrades after Anap1; Cleavage od centromeric Rec8 during Anap2 allows separation of chromatids.

53

Lecture 2

Chromosomal Rearrangements.

54

What are the 4 types of chromosomal rearrangements?

Deletions, Inversions, Translocations, Duplications.

55

What are chromosomal deletions?

Gene loci deleted from the chromosome.

56

What are the 2 types of deletions?

Terminal and Interstitial.

57

What are terminal deletions?

Single breaks in the chromosome causing loss of everything from break to telomere. New telomere must be acquired.

58

What are interstitial deletions?

2 breaks in the chromosome. Everything in between is lost.

59

Give 4 examples of disorders caused by chromosomal deletions.

Cri duchat; Wolf-Hischhorn Syndrome; Angelman Syndrome; Prader-Willi Syndrome.

60

What causes Cri Duchat? Describe the properties.

5p-; Mental retardation. 80% of cases paternal. Mostly de novo. 1/37000 - 1/50000.

61

What causes Wolf-Hischhorn Syndrome? Describe the properties.

4p- of WHSC-1 gene; Twice as many female cases as male. 1/20000 - 1/50000.

62

Describe the properties of the WHSC-1 gene.

25 exons. Expressed ubiquitously in early development. Encodes developmental protein which shares domains with 4 others.

63

What causes Angelman Syndrome? Describe the properties.

Maternal 15q- 11-13 (Visible in 50%); Jerky movements/Inappropriate laughter/Severe retardation. 1/20000.

64

What causes Prader-Willi Syndrome? Describe the properties.

Paternal 15q- (Visible in 50%); Obesity. IQ ~50. 1/10000.

65

Draw a diagram of an interstitial deletion.

ABCDEFGH => ABCGH + DEF acentric fragment.

66

What is genomic imprinting?

An epigenetic effect causing genetic expression based on paternal/maternal origin.

67

Describe how deletion mapping is carried out.

Crossing a recessive mutant strain of Gene X, with various strains which carry a deletion in different regions of Gene X. Recombinant offspring which show mutant phenotype indicate that the mutation is in the region of the deletion in the other parent because the dominant allele is missing.

68

What are paracentric inversions?

2 breaks on the same arm of the chromosome.

69

How are paracentric inversions detected?

Change in banding pattern.

70

What is the differential phenotype of paracentric inversions?

Normal phenotype in heterozygote carriers, not homozygote carriers.

71

What are the implications of paracentric inversions on meiosis?

Causes an inversion loop in heterozygote bivalents because of altered homology.

72

Draw the diagram of crossing over between a normal and paracentrically inverted chromosome.

Inversion: oADCBE; Products: ABCDE||ABBE; A||ADCBE + Acentric fragment EDCBE.

73

What are the products crossing over of a paracentrically inverted chromosome?

2 balanced gametes - 1 normal, 1 inverted; 2 unbalanced gametes - Deletions; 1 acentric fragment.

74

What are pericentric inversions?

1 break on each arm of the chromosome.

75

What is the differential phenotype of pericentric inversions?

Normal phenotype in heterozygote carriers, not homozygote carriers.

76

How are pericentric inversions detected?

Change in banding pattern.

77

What are the implications of pericentric inversions on meiosis?

Causes an inversion loop in heterozygote bivalents because of altered homology.

78

Draw the diagram of crossing over between a normal and pericentrically inverted chromosome.

Inversion: ACoBD; Products: ABoCD||ABoCA; DCoBD||ACoBD.

79

What are the products crossing over of a pericentrically inverted chromosome?

2 balanced gametes - 1 normal, 1 inverted; 2 unbalanced gametes - Deletions/duplications.

80

What would happen if crossing over occurred outside the inversion loop?

All gametes would be unbalanced.

81

What are balancer chromosomes?

Chromosomes with multiple inversions to prevent bivalent formation therefore crossing over.

82

What are balancer chromosomes used for?

Screens of recessive lethal mutations. Lethal stock can be kept as balancer lethal stock.

83

What is the cause of genetic duplications?

Error in homologous recombination, Retrotransposition event, Duplication of entire chromosome.

84

Why is the other copy of the gene free from selective pressure?

Mutation of one has no deleterious effect. Mutation rate may increase.

85

How can an error in homologous recombination give rise to a gene duplication?

Depending on the homology of chromosomes' repetitive elements, the chromosomes may misalign causing a duplication in one chromosome and a reciprocal deletion in the other.

86

What are the fates of duplicated genes?

Subfunctionalisation; Neofunctionalisation; Degeneration/loss of function.

87

Give an example of a genetic duplication.

Duplicated digestive gene into an anti-freeze gene in a family of ice fish.

88

What is the DDC theory?

Duplication Degeneration Complementation. Each copy equally likely to accumulate mutations. No effect on phenotype as long as the copies complement each other.

89

How are gene duplications detected?

Genomic microarrays - Array CGH.

90

What is Array CGH?

Array Comparative Genomic Hybridisation.

91

How can gene duplications lead to cancer? Give an example.

Duplications of oncogenes e.g.: FGFR1, causes breast cancer.

92

What are translocations?

Movement of a DNA sequence to a different chromosome.

93

What is the differential phenotype of Reciprocal translocations?

Normal phenotype in heterozygote carriers, not homozygote carriers.

94

How are Reciprocal translocations detected?

Change in banding pattern and morphology. Detected by chromosome paints.

95

Draw the diagram of segregation of a Reciprocally translocated heterozygote bivalent.

3 ways of segregation.

96

What are the genetic effects of reciprocal translocations?

Sterility if Adjacently segregated (50%). 30% of fertilisations give Down syndrome-like phenotype.

97

Lecture 3

Chromosomal Rearrangements Continued.

98

What are Robertsonian Translocations?

Fusion of acrocentric chromosomes' centromeres.

99

Which human chromosomes are susceptible to Robertsonian Translocations?

Group D: 13/14/15; Group G: 21/22.

100

Gow are RobTs detected?

Reduction in chromosome number and change in chromosome morphology; FISH, Chromosome paints/ M-FISH.

101

What are the most common RobTs?

13q/14q; 14q/21q.

102

What is unique about 13q/14q RobT?

Breaks are in similar locations unlike with other RobTs which suggests that they're always triggered by the same mechanism.

103

What is the effect of RobT on chromosome segregation?

Trivalent formation.

104

Draw the diagram of RobT of 14q and 21q.

Products: Alternate segregation - Normal and Balanced translocation gametes; Adjacent seg. 1 - Trisomy 14 and Monosomy 14 gametes; Adjacent seg. 2 - Unbalanced translocation (DS) and Monosomy 21.

105

What are fragile sites?

Regions of chromosomes that do not stain.

106

How are flagile sites associated with diseases?

Some coincide with cancer breaks. Some associated with inherited diseases e.g.: X-linked mental retardation.

107

What causes X-linked mental retardation?

Expression of folate sensitive fragile sites distal of Xq (FMR1 gene).

108

Describe the differences in wt and mutant FMR1.

FMR1 is made up of CGG repeats. Normal alleles contain 6-52 repeats. Premutation is 50-230. Full mutants have 100s/1000s of CGGs causing abnormal chromatin structure.

109

Why does the number of repeats correlate with mutation severity?

Increase in length increases risk of germline mutations to form a full mutant.

110

Describe the main differences in karyotype between higher primates.

Human chr.2 seems to come from a fused pair of chromosomes which explains why human karyotype has 48chrs and not 48. Chr.5 differs strongly only in chimpanzees (pericentric inversion shown by banding pattern). Chr.6 of chimps and gorillas shows duplications at telomeres (Reciprocal inversion is also possible).

111

Describe how chromosome rearrangement may cause cancer.

Proto-oncogenes may become oncogenes - activated by mutation/overexpression/amplification causing transformation of cell to tumour.

112

What are proto-oncogenes?

Genes involved in cell proliferation.

113

Give 2 examples of cancer caused by chromosomal rearrangements.

Burkitt's Lymphoma; Chronic Myelocytic Leukemia.

114

What causes Burkitt's Lymphoma?

Reciprocal Translocation in somatic cells - 8q:14q. 8q contains MYC gene - made up of 3 exons. Exon 1 is non-coding. Translocation of exons 2 and 3 only. Causes upregulation of MYC hence overexpression of Myelocytomatosis protein.

115

What is the function of Myelocytomatosis?

Regulates expression of 15% of all genes via association to enhancers and recruitment of HATs.

116

What causes Chronic Myelocytic Leukemia?

Reciprocal Translocation 9q:22q causing a Philadelphia chromosome. Causes fusion of ABL (Abelson Murine Leukemua) gene to the BCR (Breakpoint Cluster Region) on 22q. Novel transcript forms - p210 fusion protein - a constantly active tyrosine kinase.

117

Give an example of cancer caused by loss of Tumour Supressor genes?

Retinoblastoma - loss of retinoblastoma protein (TS).

118

Describe the phenotype of a Retinoblastoma.

Sporadic unilateral tumour if de novo. Bilateral if familial (40%). Inherited as AR but phenotype AD (haplosufficient allele).

119

Describe the expression of the 3 possible genotypes of retinoblastoma gene?

wt||wt - Normal somatic cell; -||wt - Rare in normal persons/ all somatic cells in familial offspring; -||- - Founder cell of tumour.

120

How is loss ot TS detected?

Loss of heterozygosity confirmed by M-FISH - markers near TS loci - No markers in tumour-potent cells.

121

What mechanisms may give rise to loss of TS?

Non-disjunction; Non-ds and duplication; Mitotic recombination; Deletion; Gene mutation; Epigenetic modification.

122

What is CGH?

Comparative Genome Hybridisation.

123

What is CGH used for?

Detection of unbalanced rearrangements in tumour DNA.

124

Describe how CGH is performed.

Tumour DNA labeled with green fluorophore; Normal DNA labeled with red fluorophore; Each sample hybridised to metaphase chromosomes; Image analysed and quantified based on fluorescence ratio.

125

What are the limitations of CGH?

Limited resolution on metaphase chromosomes - 5-10Mb regions detcted.

126

Describe legacy Array-CGH.

Co-hybridisation of samples onto an array of BACs. BACs chosen because of large inserts.

127

Describe new method of Array-CGH.

Hybridisation onto high density arrays of oligonucleotides.

128

What are the advantages of Array-CGH?

Higher resolution (smaller gains/losses detected).

129

Describe the process of Northern Blotting.

mRNA extracted from cells. Electrophoresis used to separate by size. Gel blotted onto nitrocellulose membrane. Probe with labeled marker introduced to membrane. Autoradiography - X-ray used to visualise film.

130

What is northern blotting used for?

Observation of gene expression patterns at different stages of cellular development.

131

Describe the process of Southern Blotting.

Restriction nucleases cut DNA into small chunks. Electrophoresis. Blotted onto nitrocellulose sheet. Hybridisation of labeled probe. Film washed and viewed on X-ray using autoradiography.

132

Describe the process of Western Blotting.

Proteins extracted. Electrophoresis by 3D structure or polypeptide length if denatured. Blotted onto nitrocellulose. Antibodies used to stain.

133

Describe the process of Eastern Blotting.

Uses antibodies designed to detect post-translational modifications (ER/Golgi).

134

Gvie examples of post-translational modifications detected by Eastern blotting.

Lipids; Phosphomoieties; Glycoconjugates; Carbohydrate epitopes.