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 Chapter 10: The Nature of the Gene and the Genome 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? 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 Mendelian Inheritance • Named for Gregor Mendel – 1822-1884 – Studied discrete (+/-, white/black) traits in pea plants 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’) 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 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) 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 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 • The two genes may be identical to one another or nonidentical (may have alternate forms 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. Mendelian Inheritance • Mendel’s results held true for other plants (corn, beans) • They can also be generalized to any sexually reproducing organism including humans 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 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? Mendelian Inheritance BB bb Bb Bb Bb Bb Bb Bb bb Bb bb bb bb Bb • If the trait indicated in black is dominant we would expect the cross Bb between 2 and 3 to produce either ~50% black trait and ~50% white trait offspring or 100% black trait offspring Bb • That ain’t the case Mendelian Inheritance bb BB Bb Bb Bb Bb Bb Bb • 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? Mendelian Inheritance • Using the information from the previous slides we can deduce most individual’s genotypes Bb BB B? bb Bb B? B? Bb Bb bb Bb Bb Bb Bb Bb Bb Bb bb Bb bb bb bb bb Bb bb bb 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? Mendelian Inheritance • A dihybrid cross 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 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 • The two genes may be identical to one another or nonidentical (may have alternate forms 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) 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 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? 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 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 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 & sperm nuclei had two chromosomes each before fusion; Somatic cells had 4 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) 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 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 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 – the number of 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 A a AA aa AA aa A A Aa a a 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 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 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 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. 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 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. C. D. 1 2 2 1 2 3 1 3 3 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? 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 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? 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 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 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 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 Chemical Nature of the Gene • Review of nucleic acid structure: – Sugar-phosphate backbone – Nitrogenous base rungs – Directional – 5’ to 3’ 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 Clicker Question • Which of the following 12 bp double helices will denature most quickly? A. 5’-AATCTAGGTAC-3’ 3’-TTAGATCCATG-5’ C. 5’-AATTTAGATAT-3’ 3’-TTAAATCTATA-5’ B. 5’-GGTCTAGGTAC-3’ 3’-CCAGATCCATG-5’ D. They are all DNA, they will all denature at the same rate. 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 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 Genome Structure • Complexity in bacterial and viral genomes A Cot curve uses the Concentration and time necessary for a genome to renature to characterize a genome Simple genomes have simple Cot curves • MS-2 virus – 4000 bp genome • T4 virus – 180,000 bp genome • E. coli – 4,500,000 bp genome Why do the smaller genomes renature more quickly? Genome Structure • Complexity in eukaryotic genomes • Eukaryotic Cot curves are more complex because the genomes consist of different fractions 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’ 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 microand minisatellite DNA – CODIS loci Genome Structure • Complexity in eukaryotic genomes • Repeat expansion and human pathogenicity • • • • • • 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 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 Genome Stability • Eukaryotic genomes are very dynamic over long and short periods of time • Whole genome duplication aka polyploidization • offspring are produced that have twice the number of chromosomes in each cell as their diploid parents • May occur in either of two ways: • • Two related species mate to form a hybrid organism that contains the combined chromosomes from both parents (occurs most often in plants) Single-celled embryo undergoes chromosome duplication but duplicates are not separated into separate cells, but are retained in single cell that develops into viable embryo (most often in animals) Genome Stability • Whole genome duplication aka polyploidization • Polyploidization provides HUGE evolutionary potential • "extra" genetic information can: - be lost by deletion - be rendered inactive by deleterious mutations - evolve into new genes that possess new functions Genome Stability • Gene duplication - duplication of a small portion of a single chromosome • Much more common than whole genome duplication • Thought to occur most often via unequal crossover • • Misalignment of chromosomes during meiosis Genetic exchange causes one chromosome to acquire an extra DNA segment (duplication) & the other to lose a DNA segment (deletion) Genome Stability • Gene duplication – the globin cluster in primates • Hemoglobin consists of 4 globin polypeptides • (2 pairs: 1 pair always in ά-family, 1 in β-family) • combinations differ with developmental stage (embryonic, fetal, adult) Transposable Elements and the Genome – Transposable elements are sequences that are interspersed throughout all eukaryotic genomes examined. – They play a role in the structure, function, and evolution of the genome Transposable Elements and the Genome – Imagine a sequence that can copy itself and then insert that copy somewhere else in the genome – What would expect to find when you line some of them up? Transposable Elements and the Human Genome – Types of transposable elements • Class I – Retrotransposons – LINEs, SINEs, SVA, LTR, ERV – Defined as having an RNA intermediate • Class II – DNA transposons – Mariner, hAT, piggyBac – Defined as having a DNA intermediate • Class II elements – cut and paste mobilization • http://www.public.iastate.edu/~jzhang/Transposition.html Generating Genetic Variation: Normal SINE mobilization Reverse transcription and insertion Pol III transcription 1. Usually a single ‘master’ copy 2. Pol III transcription to an RNA intermediate 3. Target primed reverse transcription (TPRT) – enzymatic machinery provided by LINEs Mobile Element Insertions and Mutation Promoter alters gene expression disrupts reading frame disrupts splicing no disruption ALU INSERTIONS AND DISEASE LOCUS BRCA2 Mlvi-2 DISTRIBUTION de novo de novo (somatic?) SUBFAMILY Y Ya5 de novo Familial Ya5 Yb8 about 50% Ya5 Familial Y Familial one Japanese family Ya5 Yb8 familial Ya4 C1 inhibitor ACE de novo about 50% Y Ya5 Factor IX 2 x FGFR2 GK a grandparent De novo ? Ya5 Ya5 NF1 APC PROGINS Btk IL2RG Cholinesterase CaR Sx DISEASE Breast cancer Associated with leukemia Neurofibromatosis Hereditary desmoid disease Linked with ovarian carcinoma X-linked agammaglobulinaemia XSCID Cholinesterase deficiency Hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism Complement deficiency Linked with protection from heart disease Hemophilia Apert’s Syndrome Glycerol kinase deficiency REFERENCE Miki et al, 1996 Economou-Pachnis and Tsichlis, 1985 Wallace et al, 1991 Halling et al, 1997 Rowe et al, 1995 Lester et al, 1997 Lester et al, 1997 Muratani et al, 1991 Janicic et al, 1995 Stoppa Lyonnet et al, 1990 Cambien et al, 1992 Vidaud et al, 1993 Oldridge et al, 1997 McCabe et al, (personal comm.) Genome Analysis How many human genes? 80,000 Antequera F & Bird A, “Number of CpG islands and genes in human and mouse”, PNAS 90, 11995-11999 (1993). 120,000 Liang F et al., “Gene Index analysis of the human genome estimates approximately 120,000 genes”, Nat. Gen., 25, 239-240 (2000) 35,000 Ewing B & Green P, “Analysis of expressed sequence tags indicates 35,000 human genes”, Nat. Gen. 25, 232-234 (2000) 28,000-34,000 Roest Crollius, H. et al., “Estimate of human gene number Provided by genome-wide analysis using Tetraodon nigroviridis DNA Sequence”, Nat. Gen. 25, 235-238 (2000). 41,000-45,000 Das M et al., “Assessment of the Total Number of Human Transcription Units”, Genomics 77, 71-78 (2001) Genome Analysis • Sequencing a eukaryotic genome has become relatively easy • Figuring out what it all means is the hard part • Human genome - ~25-30,000 genes (latest estimate) • Nematode worm - ~25,000 genes • Mustard plant - ~25,000 genes • Puffer fish - ~25,000 genes • What explains the differences in complexity and function among different genomes? • Comparative genomics suggests: • Alternative splicing (more later) • Differential regulation (more later) Genome Analysis • What explains the differences in complexity and function among different genomes? • The protein-coding portion of the human genome represents a remarkably small percentage of total DNA (~1.1-1.6%) • • The great majority of the genome consists of DNA that resides between the genes & thus represents intergenic DNA Each of the 25-30,000 or more proteincoding genes consists largely of noncoding portions (intronic DNA) • How do we figure out what is a gene and what isn’t? Genome Analysis • How do we determine what is important in a genome? • Comparative genomics • Conserved vs. nonconserved • What are the “important” parts of a genome? • Is most of the intergenic/intronic DNA subject to natural selection? • Are the intergenic/intronic portions conserved or nonconserved • Protein coding and genetic control sequences tend to be ____. … … Genome Analysis • Comparative genomics • The chimpanzee genome sequence was completed in 2005 • Much of what makes us human is likely to be determined through finding differences between our genome and that of the chimp Genome Analysis • Comparative genomics • FOXP2 a regulatory gene common to many vertebrates • 2 amino acid differences are human specific (found only in humans, not chimps or any other studied organism) Genome Analysis • Comparative genomics • FOXP2 a regulatory gene common to many vertebrates • Persons with mutations in FOXP2 gene suffer from a severe speech & language disorder • They are unable to perform the fine muscular movements of lips & tongue that are required to engage in vocal communication • Changes in FOXP2 that distinguish it from the chimp version were fixed in human genome in the past 120,000 - 200,000 years; around the time modern humans may have emerged Review of human genome complexity at: http://www.dnalc.org/ddnalc/resources/chr11a.html