Genetics - Appunti esame

Mendel

• Blending theory (inheritance theory of the time): the offspring is a mixture of the parental traits, you will never go back to the parental lines
• Proposed the concept of gene/elements (inheritance of characters) through scientific technique: using a model system (peas: large offspring, easy, clear phenotypes, cheap, short generation time), he studied 7 independent (≠ chromosomes) phenotypes, 1 at a time, starting from pure lines (always give the = offspring), and counted the observed phenotypes and their proportions (quantitative approach, large data → statistic)
• Exp: purple (remove anthers → contains pollen, male) x white flower (take the pollen) → F1 is all purple
• Exp: round x wrinkled → F1 all round → round is dominant (repeat for all 7 characters). Studied this character bc 1 depended on both parents 2 it’s easier to study the seeds than the flowers
• Exp: round x wrinkled → F1 round → F2 3 (round) : 1 (wrinkled) → self pollinate → F3 1 (pure round) : 2 (mixture round and wrinkled), came from ¾ round : 1 (wrinkled, came from ¼ wrinkled in F2)
• Explanation: existence of hereditary determinants (later called genes); gene pairs: alternative phenotypes of a character (round/wrinkled) are determined by ≠ forms of 1 type of gene (alleles), so in pea plants a gene is presented twice in each cell (gene pair)
• Gamete exp: RR gives R gametes, rr gives r. R x r → Rr which is 50/50 R or r gametes → 1 RR : 2 Rr : 1 rr
• Explanation: members of gene pairs segregate into gametes (which carry 1 of 2 members) and the union of gametes is random
• From all this he determined 3 laws:
  • Segregation: in production of gametes the 2 members of each gene segregate so the offspring only gets 1 of 2 members from a parent (this is why he’s the father of genetics)
  • Independence: alleles of genes get sorted into gametes independently of one another
  • Dominance: one factor (allele) dominates the other

Hereditary material is DNA

• Now: genetic material is 50/50 DNA and protein → chromatin
• 1869: Miescher found a new weak acidic chemical (DNA) present in huge quantities in nuclei of leucocytes
• Nuclei became studied: observed that in fertilisation nuclei of sperm and egg cells fuse
• Chromosome discovery: their number is specific for species, constant in cells and they are equally separated during divisions → all this supported the idea that they were at the basis of heredity
• 1920: evidence that chromosomes contain DNA
• 2 theories:
  • It’s all protein (establishments): there were known functions and they are variable in structure
  • It’s all DNA: it’s very stable, its amount is = in all cells except for gametes which have half → they had to prove it’s all DNA and that it can’t be protein
• 1928: Exp Griffith with bacteria in colonies and mice
  • Streptococcus pneumoniae: divide by binary scission (colony comes from 1 cell); have 2 strains S-III (smooth bc contains a polysaccharide which protects it from immune system of mice, virulent, lethal in mice) and R-II (not virulent)
  • Took S and injected in mice → dead. Took R and injected in mice → alive. Took S and killed them with heat, then injected in mice → alive. Took dead S and alive R and injected in mice → dead (samples contained living S). Result: the character for the polysaccharide was passed from S to R and made them S
• 1944: Exp Avery, Mac Leod and McCarty with R colonies and heated S strains exam:
  • 1st, 2nd, 3rd tubes with heated S: added RNAses in 1, proteases in 2, DNAses in 3. They used these tubes on the R colonies → the first 2 became S, the last one (R + dead S + DNAses) stayed R.
  • Result: if you degrade DNA there is no way R can become S, but if you degrade RNA or proteins they can → DNA is the hereditary material
• 1952: Exp Hershey, Chase with T2 phages (1st type contains S³⁵ to label proteins, 2nd P³² to label DNA):
  • T2 with S³⁵ is put on colonies of E.coli → injects its DNA → the solution is put into a blender to shake off all phages from bacteria (they become ghosts) → E. coli is left to grow → radioactivity is found in ghosts
  • T2 with P³² same procedure → radioactivity found in E.coli and even in progeny of the phage
  • Result: T2 injects DNA not protein, and it’s all info needed for progeny → DNA is the hereditary material
• DNA theory wins → new question: what structure allows the ability to encode info and to duplicate it precisely? Now we know it’s: pentose (ribose or deoxy) + PO4 + nitrogen base (purines: 2 aromatic rings, pair with pyrimidines: 1 ring
• Franklin, Wilkins did an X ray diffraction analysis on a sample of DNA: it’s long, skinny, has 2 parallel similar parts, it’s helical
• Chargaff analysed DNA from different organisms and established rules about quantities: n purines = n pyrimidines; T = A; C = G; A + T not always = G + C.
• 1953: Watson and Crick suggest DNA structure
  • There is a sugar-phosphate backbone (with phosphodiester bonds) and hydrogen pairing of bases (A-T 2, G-C 3). There is a major (used by proteins) and minor groove. Helix is antiparallel, DNA is oriented 5’-3’, each cell has 2m of DNA, and only 10-15% of it gives protein
  • 3 forms of DNA: A form (compact, similar to double strand RNA), B form (most frequent in living cells, not compact, 10 bps per turn), and Z form which is very rare
  • Replication: the 2 strands are separated and each becomes a template for new daughter cell

DNA replication

Discovery of semi-conservative process

• It could be semi conservative (each strand is a template: ½ helix is parental), conservative (the original strand gives a new identical one: 100% parental) or dispersed (mosaic of old and new pieces)
• 1958: Exp Meselson, Stahl with E.coli and N¹⁵ (heavy isotope of normal N¹⁴)
  • Grew E.coli in N¹⁵ medium for many generations (so that their DNA contained only N¹⁵ in nitrogen bases), washed them and put on N¹⁴ medium, taking 3 samples (t=0, after 1 division, after 2).
  • Samples were mixed with CsCl (creates density gradient) and were put in centrifuge. At t=0, all the DNA is N¹⁵ (heavy band), after 1 gen one strand is N¹⁵ and one N¹⁴ (intermediate band), after 2 gens they got 1 light (N¹⁴) and 1 intermediate band (N¹⁴ + N¹⁵) → never will get a heavy band
  • If it were conservative: t=0 heavy band, after 1 gen one heavy and one light band. Result: it’s semi conservative
• Replication process: a DNAse opens the helix forming a fork, which is kept open by proteins that destabilize the helix and unwound by topoisomerase (1 strand break). RNA polymerase puts a primer on 3’ (template strand 3’-5’), so that DNA polymerase can start 5’-3’: on 3’ it puts a nucleotide 3 phosphate (α,β,γ) forming a phosphodiesterase bond with a release of the pyrophosphate (β,γ), so the DNA is labelled with P³² on α phosphate. The strand that has 3’ on the opening of the fork is lagging, it needs many primers and its Okazaki fragments have to be united after. The primers are degraded by a nuclease, the DNA polymerase elongates them and ligase unites the pieces with phosphodiesterase bond with ATP use.
• Denaturation: DNA has Tm (melting temperature), it then has to cool slowly and at 20° it renaturates. It can be hybridized with a probe (small DNA piece) which will bind to its complementary, so you can analyse it.
• PCR: primers + DNA polymerase + 5kpbs DNA → 36 cycles, amplification of the DNA

Chromosomes

• A chromosome is 1 DNA molecule, 46 of them in humans make up 2m of DNA. Giemsa staining to see the karyotype in mitotic metaphase: chromosome banding with G-light (usually contain active genes, chromatin is less compact, but bands are very generic so it’s not universal) and G-dark stains (more compact).
• Packaging: DNA is winded in nucleosomes (creating a pearl chain) of 145 bps + connection sequence = 200bps (H2A, H2B, H3, H4 histones, 2 each, charged + bc are 20% lysine and arginine) held together by H1. This makes up the chromatin, divided in eu and hetero (constitutive, in centromeric and telomeric regions, or facultative, where it can switch, epigenetic regulation). To separate histones from DNA → put a high salt solution to eliminate the +/- attraction. The pearl chain rolls over H1 forming a solenoid, which is then looped, condensed and winded in a compact chromosome (present only in cell division).
• Histone modifications: acetylation and methylation make chromatin accessible
• Bacteria DNA is located in the nucleoid and has a packaging in loops by basic similar proteins (not histones)
• Chromosomes are: metacentric (centromere in the middle), sub metacentric (between middle and end), acrocentric (close to end) and telocentric (at end)

Diploid life cycle

• Normally they have 2n chromosomes with gametogenesis (formation of gametes through meiosis). The life cycle is G1 (only here cell is 2n, there is normal function and checkpoint), S (DNA replication and quality checkpoint), G2 (structure proteins for M are produced, G2-M checkpoint) and M. In the interphase there are no chromosomes, just chromatin.
• In prophase the cell is 4n, the centrioles split and move to the poles, the chromosomes get packed. In metaphase they are aligned thanks to the spindle and in telophase the cells are separated by cytokinesis.
• Meiosis is a reduction division (product: gametes) of meiocytes found in gonads (testes and ovaries), with the difference in orientation of chromosomes (not chromatids are pulled away but homologous chromosomes). From spermatogenesis 4 spermatozoa, from oogonia 1 oocyte and 3 polar bodies. Meiocytes are formed from spermatogonia and oogonia (formed in embryo in high amount from primordial germ cells).
• In plants: production of germ cells is constant until plants flower (sensed by light and temperature)

Haploid life cycle

Fungi and algae

• Yeast has mating type A and α and goes in a temporary diploid state by fusion to form a diploid meiocyte.
• Neurospora crassa: has mating type A and a (can’t mate A-A) → mitosis → cross fertilization → fusion into a 2n → synchronous divisions to get 4 cells → 1 mitosis → 8 sexual spores (n) ordered in perithecia with asci

Plants have 2 alternating life cycles/generations

• Sporophytic (2n) and gametophytic (n). Zygote (2n) → seed with embryo → plant (sporophyte) → flower (ovule and anthers) with meiosis → meiospores (n) → mitosis → male and female gametophyte, pollen, embryo sack → fertilization (egg + pollen) → zygote (2n)
• Maize: tassel (male) and ear shoot (female). From ear a megaspore mother cell is obtained (2n) → meiosis → microspores (n) → only 1 functioning microspore mother cell (2n), 4 pollen grains in the male
• Sporogenesis (formation of spores) in specific cells of sporophyte undergo meiosis → haploid spores → cell proliferation/gametogenesis → multicellular gametophyte → gametes (sperm + egg cell)
• Angiosperms (flowering plants) are heterosporous, they have megasporocytes (2n) and microsporocytes (2n) which undergo meiosis and form megaspores (→ gametophyte → egg cell) and microspores (→ pollen)
• Primitive plants: sporophyte forms spores through meiosis and releases them in the soil → gametophyte (smaller). In evolution, the gametophyte got smaller till it could stay inside the flower for protection
• Male gametophyte (pollen) develops in the anther → 2 cell divisions → 2 sperm cells in 1 vegetative cell
• Female gametophyte (embryo sac) develops in the ovule → 3 cell divisions and 1 fusion → 3 antipodal cells, 1 central cell (2n), 2 synergid cells and 1 egg cell. Double fertilization: 1 sperm fertilises the egg and 1 central cell → endosperm (3n)

Test cross

• Mendel used testers (homozygous individuals for the character he studied) to see the genotype of a plant: RR x rr = Rr; Rr x rr = 1:1; Rr x Rr = 3:1 2(−)
• If ratio is not clear, use chi-square test (reject hp below 5%): .The value is then converted into p-value through a table with degrees of freedom (n phenotypic classes – 1)
• Mendel worked not only with monohybrid crosses (Aa x Aa) but with dihybrid (AaBb x AaBb) also (seed shape x seed colour): ratio 9:3:3:1
• Punnet square: write all gametes (M/F) combinations in a square to predict the outcome

Interactions between alleles of 1 gene

• Incomplete dominance: two alleles of one gene are both dominant, F1 is the result of their mixture (intermediate phenotype). Self-crossing F1, there’s Mendel’s 1:2:1
• Codominance: both alleles are expressed (F1 is heterozygous, so not always the heterozygous has to have recessive and dominant alleles)
• 1900: Landsteiner - Human blood groups are defined by modifications on red blood cell polysaccharides (antigens) with a transferase. Anti A bodies recognise A antigen and bind to it (same with B). In O, transferase is not functional. Possible genotypes: AA/Ai → A is dominant, BB/Bi → B is dominant; AB → codominant; ii → O
• Process (eg A group): adding anti A antibodies, an agglutination of red cells form; adding anti B antibodies, nothing happens. AB blood group doesn’t produce antibodies or will die of agglutination.
• Production of antibodies: A → anti B; B → anti A; AB → NO; O → anti A and anti B. Donation: A to A and AB; B to B and AB; O to everyone; AB only to AB.
• O is most common and recent (suffers less from malaria); A is oldest and mutated into B
• Multiple alleles of 1 gene: there is a scale of dominance, behaving in Mendelian way

Chromosomal basis of heredity

• Human mating follows Mendel in autosomal inheritance but there is also sex linkage. No experiments here, but family trees are used. Proband/propositus comes to geneticist, gets a pedigree analysis. People outside the family are considered sane homozygous.
• 4 patterns of inheritance: autosomal recessive (generation jumps, frequent after inbreeding) and dominant (no jumps, frequent), X linked recessive and dominant.

Discovery that genes are on chromosomes

• Cytological evidence:
  • Henkings studied firebugs → meiotic nuclei 11 pairs of chromosomes + 1 unpaired element (X body)
  • Wilson studied Proteanor → F 6 pairs of chromosomes, M 5 + unpaired element (X chromosomes)
  • Stevens studied beetles → common chromosomes in M and F (X) except for 1 in M (Y chromosome)
• 1902: Sutton, Boveri propose chromosome theory bc: chromosomes behave similar to genes (independent assortment in divisions) + all are needed for an embryo development
• 1910: Exp Morgan, fly lab with male Drosophila with white eyes
  • Females are larger than males, normally red eyes → easy to study
  • White male x wt female = F1 all red eyes → white is recessive. → self-cross F1 (expect a 3:1) → F2 females all red eyes, males 1:1 ratio → ≠ ratio for M and F = sex linkage → the gene is on a sex chromosome. Y has few genes, so it’s X. Result: genes are on chromosomes.
  • Explanation: F (w+ w+) gives a w+ to F progeny, M (wY) gives w → females heterozygous (w+ w) red, males w+ Y . In next gen, F can give w+ or w, while M only w+ or Y → F all red, M 1:1
  • Reciprocal cross (white F, wt M) → F1 M all white, F all red → M gives only w, F w+ or w → F2 both sexes are 1:1 → to know genotype of mother, use a male tester
• Exp Bridges with red eyed M x white eyed F
  • Expected all red eyed F and white eyed M but found F with white eyes and M with red (exceptional progeny) → red eyed males are sterile bc F progeny can only be white if gotten both ww from the mother. M progeny got their X from the father (wt) and not mother (otherwise would be white).
  • Explanation:
    • Non-disjunctive meiosis I → in telophase I homologous chromosomes are not separated → 2 cells, 1 with an extra chromosome, 1 empty. In telophase II there will be 2 cells with 1 extra and 2 empty
    • Non-disjunctive meiosis II → in telophase II chromatids are not separated, 50% gametes are ok, 1 cell is empty and 1 with an extra chromosome
    • Y in Drosophila doesn’t define maleness, but only male fertility, autosomes define maleness
    • F white progeny were XXY, M red progeny were X
• How to analyse sex linked patterns: genotype of the mother? → look at ratio of male progeny (1:1, hetero). Dominant allele? → look at female progeny. Male phenotype = male genotype
• Birds also have heteromorphic sex chromosomes but system is inverted: ZW female, ZZ male
• X chromosome contains PAR1 and PAR2 regions, homologous to the ones on the Y (to pair in meiosis → ½ gametes get X, ½ Y). Y contains also SRY (sex determination region on Y) to determine male sex in humans.

Sex determination

• Results of non-disjunctive meiosis in humans
  • Turner syndrome (XO) → monosomy that doesn’t