Welcome to Part 2 of Bio 219 Lecturer – David Ray

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 – ext 31454 – Lectures and other resources are available online at Go to ‘Courses’ link

Chapter 10: The Nature of the Gene and the Genome

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?

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 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 (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)

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 10_05_gel.electrophor.jpg

Named for Gregor Mendel Studied discrete (+/-, white/black) traits in pea plants

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

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)

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)

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

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

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?

BB 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

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

Using the information from the previous slides we can deduce most individual’s genotypes Bb BB bb B? Bb bb Bb Bb Bb Bb Bb Bb

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?

A dihybrid cross

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

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 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 (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)

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

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

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?

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

Chromosomes Are chromosomes important for inheritance?
Whatever the genetic material is, it must behave in a manner consistent with Mendelian principles Ascaris egg & sperm nuclei had 2 chromosomes each before fusion Somatic cells had 4 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

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 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) Hypothesized that homologous pairs correlated with Mendel's inheritable pairs of factors

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 a a Aa

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

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

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 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

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

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

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

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

Review of nucleic acid structure: Sugar-phosphate backbone Nitrogenous base rungs Directional – 5’ to 3’

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

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’ 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.

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
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?

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

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’

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

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

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

Review from last time The structure of DNA was resolved by Watson and Crick and was aided by the observation of Chargaff’s Rule DNA is a directional double helix The genome is the complete DNA complement of an organism Information on genome content and complexity can be obtained by DNA denaturation/renaturation experiments A Cot curve can provide details about the size and complexity of a genome Eukaryotic genomes typically have three Cot fractions, highly repetitive, middle repetitive and single copy The highly repetitive portions are made up primarily of micro and minisatellites Satellite DNA is important as a functional component and also as a genetic marker The middle repetitive portion can be coding or non-coding Genomes are dynamic and can undergo genome duplication

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)

Genome Stability Gene duplication – the globin cluster in primates
Each gene is built of 3 exons (coding sequences) & 2 introns (noncoding intervening sequences)

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 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 in the human genome Transposase gene XXX DR DR DR DR ITR ITR ITR ITR 1 – 3 kb <1kb Autonomous transposon hAT, mariner, Tc1, piggyBac, etc. Nonautonomous transposon MERs (100+ types), Arthur1, FordPrefect

Class II elements – cut and paste mobilization

Class I elements in the human genome LTR vs non-LTR retrotransposons LTRs, such as HERVs are relatively quiet in the human genome but do occasionally retrotranspose LTR Retrotransposon gag pol env TSD TSD LTR LTR kb LINE ORF1 ORF2 (A)n TSD TSD 5’UTR 3’UTR 6 – 8 kb SINE (A)n A B TSD TSD 0.1 – 0.5 kb

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

Generating Genetic Variation:
Exon shuffling via SINE mobilization exon 1 SINE exon 2 intron SINE exon 2 DNA copy of transcript SINE transcription can extend past the normal stop signal Reverse transcription creates DNA copies of both the SINE and exon 2 Reinsertion occurs elsewhere in the genome

Genome Analysis How many human genes?
80,000 Antequera F & Bird A, “Number of CpG islands and genes in human and mouse”, PNAS 90, (1993). 120,000 Liang F et al., “Gene Index analysis of the human genome estimates approximately 120,000 genes”, Nat. Gen., 25, (2000) 35,000 Ewing B & Green P, “Analysis of expressed sequence tags indicates 35,000 human genes”, Nat. Gen. 25, (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, (2000). 41,000-45,000 Das M et al., “Assessment of the Total Number of Human Transcription Units”, Genomics 77, (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 (~ %) 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 protein-coding 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 Most of the intergenic & intronic DNA of genome does not contribute to the reproductive fitness of an individual Not subject to any significant degree of natural selection Thus, intergenic & intronic sequences tend to change rapidly as organisms evolve; are not conserved Portions of the genome that code for protein sequences & regulatory sequences that control gene expression are subject to natural selection; tend to be conserved among related species. … …

Genome Analysis Comparative genomics
Finding the conserved regions tells us much about what makes organisms similar What tells us what makes us (or other organisms) different from one another?

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

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)

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 years; around the time modern humans may have emerged

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