Welcome to Part 2 of Bio 219

1. Welcome to Part 2 of Bio 219 Lecturer – David Ray Contact info: Office hours – 1:00-2:00 pm MWTh Office location – LSB 5102 Office phone – 293-5102 ext 31454 E-mail – david.ray@mail.wvu.edu Lectures and other resources are available online at http://www.as.wvu.edu/~dray. Go to ‘Courses’ link

2. Chapter 10:The Nature of the Gene and the Genome

3. Inheritance • Observation: Offspring resemble their parents • Question: How does this come about? • Innumerable potential explanations can be proposed: • Homunculi? • Components of sperm and egg mix like paint? • Are gametes and chromosomes involved?

4. The Gene • A review of Gregor Mendel’s work • Goal: to determine the pattern by which inheritable characteristics were transmitted to the offspring • Four major conclusions

5. Mendelian Inheritance • Named for Gregor Mendel • 1822-1884 • Studied discrete (+/-, white/black) traits in pea plants

6. Mendelian Inheritance • A classic experiment • What did it tell Mendel? • What conclusions can be drawn? • Pod color was inherited as a discrete trait, inheritance was not ‘blended’ for this trait • Organism characteristics may be carried as discrete ‘factors’ (now known as ‘genes’)

7. Mendelian Inheritance • By continuing the experiment, more can be observed • The trait that was ‘lost’ in the first generation (F1) was regained by the second (F2), but in smaller numbers • yellow + yellow = yellow and green • The ‘factors’ come in different versions (alleles) • ‘Factors’ can mask one another – dominant/recessive – but they are not destroyed • Further support for the discrete gene hypothesis

8. Mendelian Inheritance • By continuing the experiment, more can be observed • There was a definite mathematical pattern to the occurrence of the traits (3:1) in F2 • Comparison with mathematics suggests that each offspring inherits one allele from each parent (2 total) • The phenotype (appearance) of the plants was determined by the genotype (actual combination of alleles)

9. Mendelian ‘Model’ of Inheritance • The true-breeders had two copies of one type of allele (homozygous) • Each parent passes on one of the alleles to the offspring randomly • The first generation will all be heterozygous (have two different alleles) • One of the alleles is able to block the other (is dominant vs. being recessive) • The F1’s pass on both of their alleles randomly • Simple math provides the expected ratios of phenotypes and genotypes

10. The Gene • A review of Gregor Mendel’s work • Goal: to determine the pattern by which inheritable characteristics were transmitted to the offspring • Four major conclusions • 1. Characteristics were governed by distinct units of inheritance (genes) • Each organism has 2 copies of gene that controls development for each trait, one from each parent • Thetwogenes may be identicalto one anotheror nonidentical (may havealternateforms or alleles) • One of the two alleles can be dominant over the other and mask recessive alleles when they are together in same organism • 2. Gametes (reproductive cells) from each plant have only 1 copy of the gene for each trait; plants arise from union of male & female gametes • 3. Law of Segregation - an organism's alleles separate from one another during gamete formation and are carried in that organism’s gametes.

11. Mendelian Inheritance • Mendel’s results held true for other plants (corn, beans) • They can also be generalized to any sexually reproducing organism including humans

12. Mendelian Inheritance • Simple Mendelian inheritance • Attached earlobes • PTC (phenylthiocarbamide) tasting • ‘uncombable hair’ • Complex (multigenic) inheritance • Eye color • Height • Studying inheritance in humans is difficult for ethical reasons but more easily done in other organisms

13. Mendelian Inheritance • Humans don’t typically have families large enough to see mendelian ratios • Inheritance can be tracked through the use of pedigrees • Are the traits in white and black dominant or recessive?

14. Mendelian Inheritance • If the trait indicated in black is dominant we would expect the cross between 2 and 3 to produce either ~50% black trait and ~50% white trait offspring or 100% black trait offspring • That ain’t the case BB bb Bb Bb Bb Bb Bb Bb Bb bb Bb bb bb bb Bb Bb

15. Mendelian Inheritance • If the trait indicated in black is recessive we would expect the cross between 2 and 3 to produce all white trait offspring • Although it is possible for individual 3 to have a Bb genotype, it is unlikely • What is the genotype of #2’s sister? bb BB Bb Bb Bb Bb Bb Bb

16. B? B? B? Bb Bb bb Bb Bb bb Bb Bb bb bb bb bb bb bb Mendelian Inheritance • Using the information from the previous slides we can deduce most individual’s genotypes Bb BB bb Bb Bb Bb Bb Bb Bb

17. Mendelian Inheritance • The examples above are referred to as monohybrid crosses since they deal with only one trait at a time • Mendel also followed dihybrid crosses in which two traits are followed at once • Would the traits segregate as a single unit or independently?

18. Mendelian Inheritance • A dihybrid cross

20. Mendelian Inheritance • A dihybrid cross produced all possible phenotypes and genotypes • Thus, all of the alleles behaved independently of one another • Mendel’s Law of Independent Assortment – Each pair of alleles segregates independently from other pairs during gamete formation

21. The Gene • A review of Gregor Mendel’s work • Goal: to determine the pattern by which inheritable characteristics were transmitted to the offspring • Four major conclusions • 1. Characteristics were governed by distinct units of inheritance (genes) • Each organism has 2 copies of gene that controls development for each trait, one from each parent • Thetwogenes may be identicalto one anotheror nonidentical (may havealternateforms or alleles) • One of the two alleles can be dominant over the other and mask recessive alleles when they are together in same organism • 2. Gametes (reproductive cells) from each plant have only 1 copy of the gene for each trait; plants arise from union of male & female gametes • 3. Law of Segregation - an organism's alleles separate from one another during gamete formation and are carried in that organism’s gametes. • 4. Law of Independent Assortment - segregation of allelic pair for one trait has no effect on segregation of alleles for another trait. (i.e. a particular gamete can get paternal gene for one trait & maternal gene for another)

22. Clicker Question • Like most elves, everyone in Galadriel’s family has pointed ears (P), which is the dominant trait for ear shape in Lothlorien. Her family brags that they are a “purebred” line. She married an elf with round ears (p), which is a recessive trait. Of their 50 children (elves live a long time), three have round ears. • What are the genotypes of Galadriel and her husband? • ♀ = Galadriel; ♂ = husband • A. ♀ PP; ♂PP • B. ♀ pp; ♂ pp • C. ♀ PP; ♂ Pp • D. ♀ Pp; ♂ pp

23. Chromosomes • Mendel made no effort to describe what carried the genes, how they were transmitted, or where they resided in an organism • 1880s – Chromosomes are discovered because : • 1. Improvements in microscopy led to… • 2. observing newly discernible cell structures.. • 3. and the realization that all the genetic information needed to build & maintain a complex plant or animal had to fit within the boundaries of a single cell • Walther Flemming observed: • 1. During cell division, nuclear material became organized into visible threads called chromosomes (colored bodies) • 2. Chromosomes appeared as doubled structures, split to single structures & doubled at next division • Were chromosomes important for inheritance?

24. Chromosomes • Are chromosomes important for inheritance? • Hypothesis: If chromosomes are important for reproduction and inheritance, altering the number of chromosomes delivered to offspring should screw up the process. • Theodore Boveri (German biologist) - studied sea urchin eggs fertilized by two sperm (polyspermy) instead of the normal one single sperm • 1. Disruptive cell divisions & early death of embryo • 2. Second sperm donates extra chromosome set, causing abnormal cell divisions • 3. Daughter cells receive variable numbers of chromosomes • Conclusion - normal development (reproduction/inheritance) depends upon a particular combination of chromosomes & that each chromosome possesses different qualities

25. Chromosomes • Are chromosomes important for inheritance? • Do chromosomes carry the genes? • Whatever the genetic material is, it must behave in a manner consistent with Mendelian principles • Hypothesis: If chromosomes carry the genes necessary for inheritance, they should mimic the theoretical behavior of genes • Two copies per organism, Discrete units, Segregate independently into gametes

26. Chromosomes • Are chromosomes important for inheritance? • Hypothesis: If chromosomes carry the genes necessary for inheritance, they should mimic the theoretical behavior of genes • Two copies per organism, Discrete units, Segregate independently into gametes • Experimental observations: • Egg&spermnuclei had twochromosomeseach before fusion; Somatic cells had4 chromosomes • Walter Sutton (1903) – Studied grasshopper sperm formation and observed: • 23 chromosomes (11 homologous chromosome pairs & extra accessory (sex chromosome)) • 2 different kinds of cell division in spermatogonia • mitosis (spermatogonia make more spermatogonia) • meiosis (spermatogonia make cells that differentiate into sperm)

27. Chromosomes • Are chromosomes important for inheritance? • Haploid vs. Diploid • Haploid – having a single complement of chromosomes in a cell • Diploid – having a double set of chromosomes in a cell • Humans gametes? Human somatic cells? • 23 chromosomes, 46 chromosomes

28. Chromosomes • Are chromosomes important for inheritance? • Hypothesis: There must be some mechanism to divide up the chromosomes in the formation of gametes • Experimental observations: • Meiotic division (only observed in the formation of gametes) includes a reduction division during which chromosome number was reduced by half • Two different kinds of cell division in spermatogonia • mitosis (spermatogonia make more spermatogonia) • meiosis (spermatogonia make cells that differentiate into sperm) • If no reduction division, union of two gametes would double chromosome number in cells of progeny • Double chromosome number with every succeeding generation

30. Chromosomes • In meiosis, members of each pair associate with one another then separate during the first division • This explained Mendel's proposals that : • hereditary factors exist in pairs that remain together through organism's life until they separate with the production of gametes • gametes only contain 1 allele of each gene • thenumberof gametes containing 1 allele was equal to the number containing the other allele • 2 gametes that united at fertilization would produce an individual with 2 alleles for each trait (reconstitution of allelic pairs) • Law of segregation Aa AA aa AA aa A A a a Aa

31. Chromosomes • What about Mendel’s Law of Independent Assortment? • Having traits all lined up on a chromosome suggests that they would assort together, not independently…. • as a linkage group • Experiments in Drosophila showed that most genes on a chromosome did assort independently… how? • Is there some mechanism to allow neighboring genes to assort independenty? Human chromosome 2

32. Chromosomes • What about Mendel’s Law of Independent Assortment? • Hypothesis: If neighboring genes on a chromosome can assort independently, there must be some observable mechanism to separate them Human chromosome 2

33. Chromosomes • What about Mendel’s Law of Independent Assortment? • Hypothesis: If neighboring genes on a chromosome can assort independently, there must be some observable mechanism to separate them • Experimental observations: • 1909 – homologous chromosomes wrap around each other during meiosis • During this process there is breakage & exchange of pieces of chromosomes • Crossing-over and recombination

36. Chromosomes Typically, several cross-over events will occur between well-separated genes on the same chromosome. Therefore, genes E and F or D and F are no more likely to be co-inherited than genes on different chromosomes. Genes that are very close together (A and B), on the other hand, are less likely to have cross-over events occur between them. Thus, they will often be co-inherited (linked) and do not strictly follow the Law of Independent Assortment.

37. Chromosomes • Hypothesis: If the frequency of independent assortment is related to physical distance on the chromosome, we can predict how close two genes are by measuring frequency of recombination. • Since the likelihood of alleles being inherited together is influenced by their proximity… • Genetic maps were possible by determining the frequency of recombination between traits

38. Clicker Question • Three genes (1, 2, and 3) are present on a chromosome. The recombination frequencies between them are: • 1-2 = 11% • 1-3 = 2% • 2-3 = 13% • Which diagram best approximates the relative locations of the genes on the chromosome? A. 1 2 3 B. 2 1 3 C. 1 2 3 D. 1 2 3

39. Chemical Nature of the Gene • What is the genetic material? • Observations: • Chromosomes are likely the carriers • Chromosomes consist primarily of three components • Protein, RNA and DNA • Are any of these the genetic material?

40. Chemical Nature of the Gene • Which one (DNA, RNA or protein) is the actual genetic material? • Let’s narrow it down by hypothesis and experimentation • Early experiments had shown that pneumonia causing bacteria that are normally nonvirulent (R; rough) can be ‘transformed’ into the virulent (S; smooth) type by some ‘transforming factor’ – the likely genetic material Rough Smooth

41. Chemical Nature of the Gene • What was the ‘transforming’ or ‘genetic material’? • Hershey and Chase (1952) – ‘blender experiment’ • Observations: • Phage viruses consist of only two chemical components – DNA and protein • When a virus infects a cell, the cell makes many new virus particles • Thus, genes must enter the cell and direct it to make new virus particles • Which one enters the cell and actually becomes a part of the new viruses?

42. Chemical Nature of the Gene • What was the ‘transforming’ or ‘genetic material’? • Avery et al. 1944 set up a multi-level hypothesis • Extracted and separated DNA, RNA, and protein from smooth (S; virulent) bacteria • Three hypotheses: • If protein is the genetic material, combining S-derived protein with R bacteria will transform the R bacterial into the S strain • If DNA is the genetic material, combining S-derived DNA with R bacteria will transform the R bacterial into the S strain • If RNA is the genetic material, combining S-derived RNA with R bacteria will transform the R bacterial into the S strain • Experimental observation: • Only DNA was able to transform the strains

43. Chemical Nature of the Gene • Label the phosphates in DNA radioactively (32P) – no phosphate in the protein • Label the sulfur in the protein (35S) – no sulfur in the DNA • Hypothesis: If the DNA enters the cell, we should find 32P in the infected cells but not 35S (and vice versa) • Observation: 32P in the infected cells • Animation online

44. Chemical Nature of the Gene • Review of nucleic acid structure: • Phosphate • Sugar • Ribose or deoxyribose • Nitrogenous base • Purines • Adenine and Guanine • Pyrimidines • Cytosine andThymine/Uracil

45. Chemical Nature of the Gene • Review of nucleic acid structure: • Observation: Chargaff’s rules • [A] = [T], [G] = [C] • [A] + [T] ≠ [G] + [C] • Suggested base pairing to Watson and Crick, who later went on to describe the overall structure of DNA in vivo

46. Chemical Nature of the Gene • Review of nucleic acid structure: • Sugar-phosphate backbone • Nitrogenous base rungs • Directional – 5’ to 3’

47. Genome Structure • Genome – the complete genetic complement of an organism; the unique content of genetic information • Early experiments to determine the structure of the genome took advantage of the ability of DNA to be denatured • Denaturation – separation of the double helix by the addition of heat or chemicals • How to monitor this separation? • DNA absorbs light at ~260nm • ss DNA absorbs more light, dsDNA less light

48. Clicker Question • Which of the following 12 bp double helices will denature most quickly? A. 5’-AATCTAGGTAC-3’ 3’-TTAGATCCATG-5’ B. 5’-GGTCTAGGTAC-3’ 3’-CCAGATCCATG-5’ C. 5’-AATTTAGATAT-3’ 3’-TTAAATCTATA-5’ D. They are all DNA, they will all denature at the same rate.

49. Genome Structure • DNA renaturation (reannealing) – the reassociation of single strands into a stable double helix • Seems unlikely give the size of some genomes but it does happen. • What does renaturation analysis allow? • Investigations into the complexity of the genome • Nucleic acid hybridization – mixing DNA from different organisms • Most modern biotechnology – PCR, northern blots, southern blots, DNA sequencing, DNA cloning, mutagenesis, genetic engineering

50. Genome Structure • Genome complexity - the variety & number of DNA sequence copies in the genome • Renaturation kinetics – what determines renaturation rate? • Ionic strength of the solution • Temperature • DNA concentration • Incubation length • Size of the molecules