Genetics 2581b

1. Genetics 2581b Ian Grant rgrant5@uwo.ca

2. Chromosomal Mutations • Change the Chromosomes themselves • Inversions, Deletions, Insertions, translocations etc. • Change the number of Chromosomes in a Cell or an animal • Note: Large proportion of Chromosomal modifications results in the prenatal death of an organism. Surviving individuals well most likely have various defects.

3. Changing the number of Chromosomes • Autopolyploidy (within a species) and Allopolyploidy (hybridization of 2 species • Common in plants • Results in larger plants due to increased cell size (necessary for holding more DNA) • Rare in animals • Due to problems that occur during meiosis when chromosomes need to pair.

4. 2n 2n 2n Generating Polyploid +colchicine + 0n -colchicine 1n 4n 1n

5. Effect of being a polyploid • Autopolyploids are usually infertile • When a cell has more than 2 sets of chromosomes, many chromosomes will try to hybridize during meiosis. During chromosome segregation, unequal division can occur, creating gametes with various numbers of chromosomes. • Allopolyploids can be fertile • When two diploid species contribute their genomes in species hybridization, a tetraploid will be created. Because each chromosome can pair with a like chromosome, segregation can occur equally, creating fertile gametes. A B

6. Chromosome doubling 4n - Fertile Allotetraploid 4n = 36 gametes n = 9 Hybrid Species Diploid hybrid 2n =18 gametes n = 9 SPECIES A 2n = 18 STERILE Species B 2n = 18 Modified from Dr. A Day lecture

7. X colchicine 2n 3n=11 6n=22 n Y Question 1 • Two species X (tetraploid 4n=16) and Y (diploid 2n=6) are crossed; the hybrid is infertile. How would you make this hybrid plant fertile, and what would be the chromosome content of the fertile hybrid plant?

8. Aneupoidy • When you are missing a chromosome or have an extra chromosome. (2n-1, 2n+1) • This has dire effects, usually a lethal phenotype • This is due to the imbalances in Gene Expression

10. Aneuploids can be caused by nondisjunction events in both meiosis I or meiosis II

11. Chromosomal Rearangements • Translocations • Inversions • Deletions • Duplications Gustav J. V. Nossa lNature 421, 440-444(23 January 2003

12. Translocations • Can happen after chromosomal breakage • Results in changes in gene expression or if breakpoint is in a gene, gene function

13. Inversions • If homozygous, can function normally • If heterozygous, can have problems during meiosis, and therefore altered fertility

14. Heterozygous inversions • Cause chromosomal loops that effect the gametes function • Pericentric inversions give you two good and two bad gametes • Paracentric inversions give you two good and bad gametes as well

16. Deletions • Usually are lethal. Same effect as aneuploidy. • Cause looping structures during meiosis • EFFECT GENE BALANCE

17. Less severe problems than Deletions Can disturb gene balance Can happen in tandem (at the process of crossing over in meiosis) Can have insertion events (Piece of DNA inserts into the chromosome doubling the genetic material present) e.g start with 4 copies each end with 5 copies in one; 3 in the other Duplications

18. Genetic techniques • Genetic Technologies • Genomic Approaches • Gene Mutation • Genotyping

19. Genetic techniques • Genetic Technologies • Genomic Approaches • Gene Mutation • Genotyping

20. Isolating DNA • Grind up tissue • Add a detergent (Sodium Dodecyl Sulfate) to break up membranes • Add proteinase K to chew up all unwanted protein • Add phenol:chloroform to separate DNA from excess protein • Add salt and alcohol to precipitate DNA

21. Restriction Endonuclease Digestion • RE are enzymes that recognize palindromic DNA sequences • They cut the sequence in a particular way leaving 5’ or 3’ overhangs 5'GAATTC 3'CTTAAG 5’ G AATTC 3' 3’ CTTAA G 5' EcoRI 5'GGTACC 3'CCATGG 5’ GGTAC C 3’ 3’ C CATGG 5’ KpnI

22. Calculating the average size of fragments digested with RE • What if the proportion of the nucelotides in the sequence isn’t 1:1:1:1? • Eg. What is the average size of a DNA fragment cut with the RE EcoRI (GAATTC) if the proportion of nucleotides is G(1/3), C(1/3), A(1/6), T(1/6)? • Ans. • G A A T T C • 1/3 x 1/6 x 1/6 x 1/6 x 1/6 x 1/3 • 1/11644 • Therfore the average fragment length should be 11644bp

23. Gel Electrophoresis • Separates genetic material based on SIZE • Send Genetic material through a polymer (agarose or polyacrylamide) using an electric current • Because DNA is negatively charged it will move from the -(anode) to +(cathode). • Visualize the DNA in the gel with Ethidium Bromide and UV light.

24. DNA cut with RE *Note the very specific pattern DNA that has been randomly Broken (sheared)

25. Blotting

26. Blotting } • Northern=RNA • Southern=DNA • Western=protein Detect using radiolabeled probes } Detect using radiolabeled antibodies

27. Where do you get DNA from? • Genomic (PCR) • cDNA (using RNA as a template)

28. Recombinant Engineering • Goal is to express your gene of interest in a model system (mamalian cells, bacterial cells, whole organisms) • Start with a source of DNA (cDNA or Genomic) • Put that DNA into a suitable vector • Transfect (eukaryotic), or Transform (prokaryotic) your vector with your DNA into a host system

29. Cloning

30. Transformation • Insert your cloned DNA into bacteria • Bacteria will amplify your DNA many times

31. Cloning using Bacteriophage vectors • Insert your favorite DNA sequence into the dispensable region of Phage DNA • Then use the phage to infect cells you want to express your DNA • Phages will kill whatever cell you infect leaving a plaque where an infection occurred.

32. Selectable markers • Let you know which cells contain your plasmid • Each plasmid contains a antibiotic resistance gene LB plate with apicillin and tetracyclin

33. What makes a good vector? • Host specific promoter • Origin of replication (for independent replication) • Selectable marker • Unique cloning sites • Small size ORI

34. Is your gene working? • Transfect or transform your cells • If it is expressing your protein should be present • You can lyse your cells, on a membrane, and use antibodies to detect your protein

35. Polymerase Chain Reaction • Amplifies your piece of DNA exponentially • Remember DNA is amplified in a 5’-3’ manner ONLY • For PCR you need, DNA, dNTP’s, Polymerase and Primers 5’ 3’ 3’ 5’

36. Start by denaturing your DNA with high temp (94 degrees) • Anneal your primers at a lower temp (50 degrees) • Extend your DNA at a medium temp (72 degrees)

37. Question 3 • If you expect only one DNA amplicon after a PCR reaction, but you detect multiple, what could be wrong? • Ans. • Anealing temp too low • Contamination in your reaction (more than one DNA source)

38. DNA sequencing • Very similar to PCR • Need DNA, dNTP’s, Primer, Polymerase • Add small number of dideoxy nucleic acids (ddNTPs) OH H dNTP ddNTP

39. Start with 4 different reactions containing either :ddTTP, ddATP, ddGTP or ddCTP Eg. Reaction with ddTTP

40. What is your sequence? Nascent sequence ddTTP ddATP Template sequence ddGTP ddCTP 3’ 5’ T A G T A A T G C C G C G G C G C T A A T G C 5’ 3’

41. Genetic techniques • Genetic Technologies • Genomic Approaches • Gene Mutation • Genotyping

42. Mapping the Genome

43. Molecular markers • Restriction fragment length polymorphisms • Simple Sequence Repeats • (microsatelites, minisatelites) • Single Nucleotide plolymorphisms

44. probe probe RFLP • Principle is that different alleles may contain different RE cut sites RE RE RE RE RE A B BB AB AA

45. SSR • Microsatelites • CT CT CT CT CT CT CT CT CT CT • Minisatelites • Eg. Variable number tandem repeats (vntr) • CTAGCTTAGAGAG CTAGCTTAGAGAG • Detect differences between people by using PCR to amplify a region that you know contains these repeats

46. So what? • The goal of finding these markers is to use them to map the genome. • Can tell which ones are linked to each other by looking at inheritance patterns on a pedigree This is a method of genotyping Or simply telling individuals apart based on variation in genes

47. Physical mapping • Ordering DNA based on overlaping regions

48. Genetic techniques • Genetic Technologies • Genomic Approaches • Gene Mutation • Genotyping

49. Where do mutations come from? • Exogenous mutations (UV light, chemicals) • Endogenous (part of the cellular process, mistakes in DNA replication)

50. Point Mutations • Insertions (Frame Shift) • Substitutions • Transversions (Pyrimidine for a Purine or Purine for a Pyrimidine) • Transitions (Pyrimidine for Pyrimidine or Purine for a Purine) • Deletions (Frame shift)