Notes (Genetics).docx

[Mendelian Genetics] - Law of Segregation = characteristics controlled by "factors" (monohybrid crosses), affected by non-Mendelian segregation (complex interactions) - Law of Independent Assortment = genes assort independently during meiosis (dihybrid crosses), affected by gene li...

[Mendelian Genetics] - Law of Segregation = characteristics controlled by "factors" (monohybrid crosses), affected by non-Mendelian segregation (complex interactions) - Law of Independent Assortment = genes assort independently during meiosis (dihybrid crosses), affected by gene linkages [Sex determination:] - Monoecious = hermaphrodites - Dioecious = M/F - Monoecious cycle: 1. Stamen (M) -\> diploid mother cell -- meiosis -\> haploid cells -- mitosis -\> pollen grain 2. Pistil (F) -\> diploid mother -- meiosis -\> 4 haploid nuclei -- 3 degenerate -\> one nucleus -- mitosis x3 -\> 8 haploid nuclei 3. Pollen stamen to stigma -\> 2 sperm nuclei w/ 3 embryo nuclei -\> 1x triploid (to form endosperm) + 1x diploid zygote formed (to form new plant) - Dioecious determination = XY/XX (mammal), ZW/ZZ (bird): 4. Klinefelter syndrome = 47, XXY (male, limited secondary sex. char.) 5. Turner syndrome = 45, X (female, limited SSC) 6. SRY encodes for TDF (testis determining factor) which causes M gonad formation 7. 46, XX (w/ SRY) = M; 46, XY (w/o SRY) = F - X chromosome has Xic which contains Xist = forms condensed DNA cannot be transcribed (Barr bodies which are n -- 1 where n = no. of x chromosomes) [Gene Interactions:] - Loss of function: 8. Amorphic -\> no protein produced at all/protein does not work (e.g. CFTR) 9. Hypomorphic -\> reduced protein function (e.g. C^ch^ chinchilla rabbit colour) - Gain of function: 10. Hypermorphic -\> increased protein function, **dom** (e.g. Hereditary pancreatitis) 11. Antimorphic -\> normal allele function disturbed, **dom** 12. Dom lethal -\> causes build-up of protein, occurs later in life thus rare 13. Neomorphic -\> alters function of protein (e.g. Legs on face of bug) - X-linkage = mostly males affected, no father to son, all daughters of affected fathers are carriers - Mitochondrial cytopathy is matrilineal - Epistasis (gene interactions): 14. Complementary gene action -\> both genes must be present to be expressed, *will be 9:7 of colour: albino* (e.g. plant colour) 15. Recessive epistasis -\> recessive locus affects other gene but not vice versa so phenotypes A-bb = aabb, *will be 9:3:4 of colour1: colour2: albino* (e.g. mouse colour where A-B- = agouti, aaB- = black, \--bb = albino) 16. Dom epistasis -\> dom locus affects other so phenotypes A-B- = aaB-, *will be 12:3:1 of albino: colour 1: colour 2* 17. Duplicate gene action -\> if two genes carry out same function so only aabb will have no function, *will be 15:1 of colour: albino* - Complementation = if crossing forms wild-type progeny, alleles of different genes exist, e.g. aa, BB + AA, bb = aA, Bb proves complementation [Cloning & Stuff] - RFLP = restriction fragment length polymorphism, restriction enzyme cannot recognise mutation so cuts into different length - Methylase = bacterial enzyme that methylates DNA so cannot be cut by restrictions - 100 -- 200 copies of recombinant plasmid in each bacteria - Multiple cloning sequence (MCS) placed in lacZ gene which usually produces blue bacteria but is disrupted by the recombinant plasmid - Cloning vectors: 18. Plasmids \< 20kb 19. Bacteriophage (based on viruses) = 25 - 30kb 20. Cosmids (modified plasmids) = 30 - 40kb 21. Bacterial artificial chromosomes = 100 -- 500kb 22. Yeast artificial chromosomes = 200kb -- 2Mb - Complementary DNA (cDNA) = DNA copy of mRNA produced using reverse transcriptase + w/o introns [DNA Sequencing & PCR] - DNA polymerases use dNTPs for DNA replication but is terminated by dideoxynucleotides, ddNTPs - Polymerase chain reaction: 23. Denaturation -\> 95˚C heat for 30s to separate DNA to single strands 24. Primer annealing -\> cooled to 58˚C for 30s to allow complementary primers to bind 25. Primer extension -\> heated to 72˚C for 1min to allow Taq polymerase function to synthesise DNA - But PCR can be affected by other sources however small and cannot use long DNA strands - dNTPs labelled with ^32^P or steroid digoxygenin - Southern blot: 26. DNA fragments undergo electrophoresis + denatured 27. DNA bound to nitrocellulose/nylon membrane 28. Labelled primers added which show up on the membrane - Northern blot = Southern blot for mRNA (no denaturing needed) - In situ hybridisations for developmental genetics - Transcriptome = total mRNA + all non-coding RNA [Linkage & Chromosome mapping] - Recombination Frequency (RF) = proportional to physical distance between two loci -- closer genes have less crossovers - RF = (no. of recombinants / total no. of progeny) x 100 - Dihybrid cross to find distance between two loci: 29. Fly Ww, Ee (normal wing, normal eye) crossed with ww, ee (weird wing, weird eye) 30. Produced 950 Ww, Ee; 950 ww, ee; 50 Ww, ee; 50 ww, Ee even though it should be 500; 500; 500; 500 therefore crossing over occurred 31. The recombinants Ww, ee and ww, Ee are not parental so input into equation (50 + 50 / 2000 x 100) = 5% 32. 5 out of 100 products of meiosis are recombinant, and the genes are 5 map units apart on the same chromosome - Trihybrid cross to find distance between three loci: 33. Cross Ww, Ee, Bb (normal wing, normal eye, normal colour) with ww, ee, bb (weird wing, weird eye, weird colour) to get - 775 E, B, W - 775 e, b, w - 40 E, b, w (single cross between E - W) - 40 e, B, W - 175 E, b, W (single cross between W - B) - 175 e, B, w - 10 E, B, w (double cross) - 10 e, b, W 34. The progeny with the least number (10 E, B, w + 10 e, b, W) has a double crossover event where the middle gene was swapped so order of chromosomes is E, W, B and e, w, b Picture 088 35. Single crosses can be found by seeing order as E, W, B so the one swapped is where the cross occurred (E, b, w + e, B, W have E swapped with e so crossover happened between E and W) 36. Distances (using equation) - between E and W (using both single and double cross so 40 + 40 + 10 + 10) to get 5% so 5 map units apart - between W and B (using single and double so 175 + 175 + 10 + 10) to get 18.5% so 18.5 map units apart - between E and B (using single EW, single WB and double so 175 + 175 + 40 + 40 +10 + 10) to get 23.5% so 23.5 map units [Bacterial Genetics] - Bacteria grown in nutrient broth (from yeast extract and casein) and minimal media (Na, K, Mg, Ca, Fe, NH~4~, Cl, PO~4~ buffer to pH 7, glucose) - E. coli can divide once every 20 min - Lag phase (acclimatisation of bacteria), log phase (rapid growth during doubling time), and stationary phase (less nutrients left) - Transformation (DNA uptake from environment): 37. DNA fragments from donor cells provided 38. Heat shock bacteria to make cell walls/membranes permeable to DNA 39. Bacteria takes donor DNA 40. DNA recombination occurs - Transduction (bacteriophage steals DNA and transfers it to someone else): 41. Phage adsorbs onto surface of host cell 42. Phage degrades host chromosome 43. Phage steals relevant genetic material + replicates 44. Fucks off and lyses host - Conjugation (transfer of plasmids between bacterial cells): 45. Donor cell forms hollow tube/conjugation pilus to another cell 46. Transfer of plasmids [Prokaryotic gene expression regulation:] - Constitutive gene expression = genes that produce proteins essential for survival undergo constant transcription (also called housekeeping genes) - Regulated gene expression = genes that are only needed depending on necessity and environment - Transcription control: 47. Negative = repressor binds to DNA sequence and prevents transcription 48. Positive = activator must bind to DNA to start transcription - Lac operon is inducible by lactose - Lac operon has: 49. Regulatory region with: - LacP promoter binding RNA polymerase - LacO operator binding lac repressor - CAP-cAMP region for CAP-cAMP binding 50. Coding genes: - LacZ for B-galactosidase - LacY for lactose permease and uptake - LacA for transacetylase 51. Lacl which codes for lac repressor (homotetramer with allosteric domain for allolactose binding) - No lactose environment: 52. Lacl expressed so repressor attaches to LacO 53. No allolactose to bind to repressor to prevent binding 54. No transcription occurs - Lactose in environment: 55. Allolactose produced which binds to repressor and stops it 56. RNA polymerase binds to LacO 57. Transcription occurs (though not enough) - No glucose in environment: 58. cAMP produced due to adenylate cyclase activity 59. cAMP binds to CAP forming CAP-cAMP 60. CAP-cAMP can bind to LacP to stimulate more transcription - Mutations and fuck-ups: 61. Lacl^-^ mutation -\> dodgy Lacl so repressor expressed cannot bind to operator B-galactosidase always present 62. LacO^c^ mutation -\> repressor cannot bind to LacO so B-galactosidase always present 63. Lacl^s^ mutation -\> super lac repressor always bound to LacO so no transcription at all - Arabinose operon has araC protein monomers that bind to aral and araO~2~ that forms DNA loop and so no transcription until arabinose breaks loop [Eukaryotic gene expression regulation] - RNA polymerase II = protein coding gene transcription - The order:  - Promoter region = specific DNA sequence that recruits RNA polymerase II + general transcription factors and directs them to Inr (initiator region) where transcription starts - Regulatory regions: 64. Proximal control elements that regulate start of transcription 65. CAAT box bound by CAAT-box Binding Protein (CBP) 66. GC box bound by SP1 67. CBP and SP1 initiate basal levels of transcription - General transcription factors: 68. Bind to promoters and known as TFIIA, TFIIB, etc 69. Separate DNA strands to allow transcription + form pre-initiation complex necessary for transcription 70. Exempli gratia = TATA Binding Protein (TBP), part of TFIID complex, binds to DNA minor groove and bends it to allow attachment of proteins 71. Different TFs have different DNA binding domains (e.g. Leucine zipper motif binds to major groove, and Helix-loop-helix motif) - Enhancer regions = in some tissues specific genes are expressed as dictated by the enhancer which recruits an activator protein that binds TFs via a mediator - Open promoters = promoter regions within nucleosome depleted regions accessible by TFs in any cell - Covered promoters = regulated genes packed into nucleosomes uncovered by activator proteins and chromatin remodelling by proteins - Histone acetylation = reducing +ve charge so histones relax to open config (added to specific lysines) - DNA methylation = forms 5-methylcytosine that silences transcription by closing chromatin structure - Somatic mutations = nonheritable, present in tissues - Germline = heritable, present in egg/sperm - BP sub mutation: 72. Silent 73. Missense = BP change resulting in AA change in protein - Conservative AA sub = change in AA which does not change function (neutral) - Nonconservative AA sub = change in AA that changes function 74. Nonsense = BP change that creates a stop codon creating an incomplete non-functional polypeptide - Regulatory mutations = promoter mutations (reduce/prevent transcription) - Origins of mutations: 75. Induced mutation = action of mutagen (environmental agent) which alters nucleotide sequence, known as mutagenesis - Deaminating agents - Alkylating agents - Hydroxylating agents - Oxidative agents - Intercalating agents (flat, planar molecules which add/remove nucleotides) - Radiation - Base analogues 76. Spontaneous mutation = without mutagens and caused by errors in DNA replication, providing background rate of mutation necessary for evolution - DNA bases can convert, forming tautomers, leading to dodgy base pairing: 77. Amino bases A & C -\> imino 78. Keto bases T & G -\> enol - DNA repair: 79. Photoreactivation = cleaves thymine dimer 80. Base excision = DNA glycosylase removes faulty pairing 81. Nucleotide excision = replacement of section of nucleotides with thymine dimer [Genomics] - ??? [Population genetics] - Frequency of alleles affected by: 82. Natural selection 83. Gene mutation 84. Migration 85. Genetic drift 86. Founder effects (small starting population leads to higher autosomal recessive disorders) 87. Genetic bottlenecks (population significantly decreased by natural disaster so more inbreeding) - Every human is a hetero carrier of 2-5 recessive alleles - Evolutionary clock = mutations occur at a steady rate over time so can be used to find time passed [Chromosomal mutations] - Chromosome structure: 88. p arm (short) + q arm (long) 89. Centromere = specialised DNA sequence joining two chromatids 90. Kinetochore = centromere associated protein allows for spindle fibre attachment 91. Telomere = specialised repeated DNA seq. protecting ends - Stains/dyes: 92. Heterochromatin = dark, repetitive DNA seq. 93. Euchromatin = light, protein encoding genes 94. Giemsa dye = stains chromosomes after trypsin activity 95. FISH = fluorescent in-situ hybridisation, add fluorescent marker to genes - Chromosome abnormalities: 96. Constitutive (global) vs acquired (one organ/tissue has it) 97. Homogenous (global) vs mosaic (only some have the mutation) 98. Numerical (total != 46) - Euploidy = normal 46 - Polyploidy \>= 3 sets of chromosomes - Triploidy = 23 x 3 = 69 - Tetraploidy = 23 x 4 = 92 - Aneuploidy = additional/missing individual chromosomes (most common, occurs during Anaphase I in Meiosis I) - Monosomy = 45 (Turner) - Trisomy = 47 (Down, Klinefelter) - Nullisomy = 44 99. Structural (missing/extra/translocated/inverted part of chromosome) - Balanced chromosomal rearrangements = no net gain/loss of chromatin - Translocation - Reciprocal = equal exchange of segments - Robertsonian = entire arm added to another - Inversion - Paracentric = w/o centromere - Pericentric = w/ centromere - Insertion - Ring formation of chromosome - Unbalanced = net loss/gain - Deletion - Duplication - Insertion 100. Uniparental disomy = two copies from one parent (instead of mix) [Cancer genetics] - Genetic disease arising from accumulations of mutations - Types: 101. Carcinomas = epithelial 102. Sarcomas = mesodermal (bone, tissue) 103. Adenocarcinomas = glandular tissue (breast) - Colon has 5 million cells die and replaced every minute, so cancer shows here: 104. Normal colonic crypts 105. Hyperplasia (small adenomatous crypt formation) 106. Dysplasia (polyp formation) 107. Metastasis to liver - Hallmarks of cancer cells: 108. Growth signal -- grow fast 109. Insensitive to anti-growth signals 110. Evade apoptosis 111. Limitless replication potential 112. Can trigger angiogenesis (formation of new blood vessels) 113. Metastasis - Proto-oncogenes (encode proteins for cell division) -- mutated -\> oncogenes (permanently active cell division proteins, **dom mutation**) - Tumour suppressor genes turned off (*recessive mutation*, both genes need to be off, e.g. p53) - Oncogenes: 114. Secreted growth factors (e.g. PDGF, IGF2) 115. Cell surface receptors 116. Signal transduction (e.g. Ras) 117. DNA binders (e.g. Myc) 118. Cell cycle regulators - Proto-oncogene activation: 119. Point-mutation (those in Ras are such) 120. Amplification = duplication of normal oncogenes, forming repeated banding patterns 121. Chromosomal translocation (forming novel fusion gene) 122. Chromosomal translocation (to a transcriptionally active region) [Genetics + Biology] - *C. elegans* detect diacetyl with AWA neuron - Odr-10 mutants cannot smell diacetyl + odr-7 needed to transcript odr-10 [Applications of genetics] - Insulin: 123. Preproinsulin -\> active insulin A diagram of a diagram of a bond Description automatically generated with medium confidence 124. Two separate plasmids with subunit A + B genes produce their respective units - Agriculture: 125. Selective breeding (directional) 126. Herbicide resistance: - Herbicide glyphosphate inhibits EPSP synthase in chloroplast (needed for AA synth) so make plants glyphosphate resistant - Use viral promoter from mosaic virus + EPSP synthase to inject into Ti plasmid in *R. radiobacter* which in plant leads to high levels of EPSP formation (enough to counter glyphosphate) 127. Insecticide resistance = cry toxin