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 • It was clear for millennia that offspring resembled their parents, but how this came about was unclear. • Do males and females harbor homunculi? • Do the components of sperm and egg mix like paint? • What role do gametes and chromosomes play?

4. The Gene • A review of Gregor Mendel’s work • Goal was to mate or cross pea plants having different inheritable characteristics & to determine the pattern by which these 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 (into that organism’s gametes , see point 2). • 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)

5. 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

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

7. Mendelian Inheritance • A classic experiment • What did it tell Mendel? • That pod color was inherited as a discrete trait, inheritance was not ‘blended’ for this trait • That one trait was ‘dominant’ over the other • yellow + green ≠ yellow-green • yellow + green = yellow

8. Mendelian Inheritance • By continuing the experiment, more can be learned • The trait that was ‘lost’ in the first generation (F1) was regained by the second (F2) • yellow + yellow = yellow and green • The cause of the trait was not destroyed, but was harbored unseen in the parent • There was a definite mathematical pattern to the occurrence of the traits (3:1)

9. Mendelian Inheritance • Mendel concluded: • Heredity was caused by discrete ‘factors’ (genes) • These ‘factors’ remain separate instead of blending • The ‘factors’ came in different ‘flavors’ (alleles) • Each offspring must inherit one gene from each parent (2 total) • The phenotype (appearance) of the plants was determined by the genotype (actual combination of alleles)

10. Mendelian Inheritance • The true-breeders only had one type of allele (homozygous) • Each parent passes on one of the alleles they have to the offspring • 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 in a random manner • Mendel’s Law of Segregation – two alleles for each trait separate randomly during gamete formation and reunite at fertilization

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 • 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?

13. 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

14. 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

15. 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

16. 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?

17. Mendelian Inheritance • A dihybrid cross

19. 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 during gamete formation

20. The Gene • A review of Gregor Mendel’s work • Goal was to mate or cross pea plants having different inheritable characteristics & to determine the pattern by which these 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 (into that organism’s gametes , see point 2). • 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)

21. 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

22. Review from last time • Office hours are MWTh, not MTW • Mendel crossed pea plants with easily discernible traits to develop four ideas • Genes are the carriers of inheritable traits • Genes can come in different versions – alleles • Law of Segregation – the alleles separate when gametes are formed • Law of Independent assortment – the alleles of one gene segregate without regard to the alleles of other genes • All of these ideas are important to our understanding of chromosomes and genome structure

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 of: • 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? • Uhhh, yeah. • Theodore Boveri (German biologist) - studied sea urchin eggs fertilized by 2 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 depends upon a particular combination of chromosomes & that each chromosome possesses different qualities • There is a qualitative difference among chromosomes

25. Chromosomes • Are chromosomes important for inheritance? • Whatever the genetic material is, it must behave in a manner consistent with Mendelian principles • Ascaris egg&spermnuclei had 2chromosomeseach before fusion • Somatic cells had4 chromosomes • 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? • meiotic division must include a reduction division during which chromosome number was reduced by half before gamete formation • If no reduction division, union of two gametes would double chromosome number in cells of progeny • Double chromosome number with every succeeding generation

27. Chromosomes • Are chromosomes the carriers of genetic information • Whatever the genetic material is, it must behave in a manner consistent with Mendelian principles • Walter Sutton (1903) –pointed directly to chromosomes as the carriers of genetic factors • Studiedgrasshopperspermformation and observed: • 23 chromosomes(11homologouschromosomepairs&extraaccessory(sexchromosome)) • 2 differentkinds of cell division in spermatogonia • mitosis (spermatogonia make more spermatogonia) • meiosis (spermatogonia make cells that differentiate into sperm) • Hypothesized that homologous pairs correlated with Mendel's inheritable pairs of factors

28. 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

29. 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? • Crossing over and Recombination to the rescue! Human chromosome 2

30. Chromosomes • What about Mendel’s Law of Independent Assortment? • 1909 –homologous chromosomes wrap around each other during meiosis • breakage & exchange of pieces of chromosomes

33. 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.

34. Chromosomes • Chromosome mapping via linkage maps • 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

35. 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

36. Review from last time • Based on Mendel’s work, people now had a conceptual framework on which to base ideas on the physical nature of inheritance • One of the potential locations for genes was on chromosomes • During meiosis, chromosome behave much like the hypothesized genes appear to behave • Chromosomal abnormalities have severe effects on organismal development and survivability • The law of independent assortment at first appeared to be a problem for chromosomal inheritance • Recombination and crossing over allow for independent assortment to occur in most cases • Tracking linked genes allowed for the first genome ‘maps’ • Despite the fact that proteins look like better candidates for the genetic material, DNA actually is • DNA is a polymer made up of deoxyribose (sugar), phosphate, and a nitrogenous base

37. 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

38. Chemical Nature of the Gene • Review of nucleic acid structure: • 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

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

40. Chemical Nature of the Gene • Review of nucleic acid structure: • Is DNA the genetic material? • What must the genetic material do? • Store genetic information • Be replicable and inheritable • Be able to express the genetic message • DNA fits the bill for two of these

41. 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

42. 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.

43. 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

44. 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

45. Genome Structure • Complexity in bacterial and viral genomes • MS-2 virus – 4000 bp genome • T4 virus – 180,000 bp genome • E. coli – 4,500,000 bp genome A Cot curve uses the Concentration and time necessary for a genome to renature to characterize a genome Simple genomes have simple Cot curves Why do the smaller genomes renature more quickly?

46. Genome Structure • Complexity in eukaryotic genomes • Eukaryotic Cot curves are more complex because the genomes consist of different fractions

47. Genome Structure • Complexity in eukaryotic genomes • Highly repetitive DNA – Satellite DNAs - ~1-10% of eukaryotic genomes • Identical or nearly identical, tandemly arrayed sequences • Minisatellites – 10 – 100 bp repeats • 5’- ATCAAATCTGGATCAAATCTGGATCAAATCTGG-3’ • Microsatellites – 1 – 10 bp repeats • 5’-ATCATCATCATCATCATCATC-3’

48. Genome Structure • Complexity in eukaryotic genomes • Highly repetitive DNA – the importance of satellite DNA • Centromeric DNA – the sections of chromosomes essential for proper cell division are mostly microsatellite DNA • DNA fingerprinting utilizes polymorphic micro- and minisatellite DNA – CODIS loci

49. Genome Structure • Complexity in eukaryotic genomes • Repeat expansion and human pathogenicity • A CAG expansion in the huntingtin gene is associated with severity of Huntington’s disease • CAG expansion produces long runs of glutamates in proteins • Polyglutamate chains tend to aggregate. • Inverse relationship between CAG repeat size and severity of disease. • Normal range = (CAG)6 – (CAG)39 • Disease range = (CAG)35 – (CAG)121

50. Genome Structure • Complexity in eukaryotic genomes • Moderately repetitive DNA – 10-80% of eukaryotic genomes • Coding repeats – Ribosomal RNA genes • rRNA is necessary in large amounts • Genes are arrayed tandemly • Noncoding repeats – Interspersed aka mobile aka transposable elements • ~1/2 of your genome • More on these later