Welcome to Part 2 of Bio 219

Welcome to Part 2 of Bio 219Lecturer – David Ray

Contact info:Office hours – 1:00-2:00 pm MWTh

Office location – LSB 5102Office phone – 293-5102 ext 31454E-mail – [email protected]

Lectures and other resources are available online athttp://www.as.wvu.edu/~dray.

Go to ‘Courses’ link

mailto:[email protected]

http://www.as.wvu.edu/~dray

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)

• 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

• Named for Gregor Mendel– 1822-1884– 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

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)

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)

• 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

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?


• 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

BBbb

Bbbb

Bb Bb

Bb

BbBbBbBb

Bb Bbbb 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?

bbBB

Bb Bb BbBbBbBb

Mendelian Inheritance• Using the information from the previous slides we can

deduce most individual’s genotypes

Bb

BBbb

BbBbBbBbBb

Bb

bb

bb

bb

bbbbbb

bb

bb

B?

Bb

BbB?B? 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

• 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

ChromosomesA a

AA aa

AA aa

A A a a

Aa

• 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!

Chromosomes

Human chromosome 2

• What about Mendel’s Law of Independent Assortment?– 1909 –homologous chromosomes

wrap around each other during meiosis

– breakage & exchange of pieces of chromosomes

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-inheritedthan 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 notstrictly follow the Law of Independent Assortment.

• 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

Chromosomes

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?

1

1

1

2

2

2

3

3

3

A.

B.

C.

1 2 3D.

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

• 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:– 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 – 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

Genome Structure

Clicker Question• Which of the following 12 bp double helices will denature most

quickly?

5’-AATCTAGGTAC-3’3’-TTAGATCCATG-5’

5’-GGTCTAGGTAC-3’3’-CCAGATCCATG-5’

A.

D.C.

B.

5’-AATTTAGATAT-3’3’-TTAAATCTATA-5’

They are all DNA, they will all denature at the same rate.

• 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

Genome Structure

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?

• 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 micro- and minisatellite DNA – CODIS loci

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

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 Structure

• 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

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

• 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)

Genome Stability

– 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

– 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

Transposable Elements and the Human Genome

• Class II elements in the human genome


Transposase gene1 – 3 kbITR DR DRITR

XXX<1kbITR DR DRITR

Autonomous transposonhAT, mariner, Tc1, piggyBac, etc.

Nonautonomous transposonMERs (100+ types), Arthur1, FordPrefect

• Class II elements – cut and paste mobilization

• http://www.public.iastate.edu/~jzhang/Transposition.html

• 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


gag pol env1 - 11 kbLTR LTRTSD TSD

TSDORF1 ORF2 (A)n

6 – 8 kbTSD 3’UTR5’UTR

(A)nA B0.1 – 0.5 kb

TSDTSD

LTR Retrotransposon

LINE

SINE

Pol III transcription

Reverse transcription and insertion

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:Normal SINE mobilization

Promoter

disrupts reading frame

disrupts splicing

no disruption

Mobile Element Insertions and Mutation

alters gene expression

ALU INSERTIONS AND DISEASE

LOCUS DISTRIBUTION SUBFAMILY DISEASE REFERENCEBRCA2 de novo Y Breast cancer Miki et al, 1996Mlvi-2 de novo (somatic?) Ya5 Associated with

leukemiaEconomou-Pachnis andTsichlis, 1985

NF1 de novo Ya5 Neurofibromatosis Wallace et al, 1991APC Familial Yb8 Hereditary desmoid

diseaseHalling et al, 1997

PROGINS about 50% Ya5 Linked with ovariancarcinoma

Rowe et al, 1995

Btk Familial Y X-linkedagammaglobulinaemia

Lester et al, 1997

IL2RG Familial Ya5 XSCID Lester et al, 1997Cholinesterase one Japanese family Yb8 Cholinesterase

deficiencyMuratani et al, 1991

CaR familial Ya4 Hypocalciurichypercalcemia and

neonatal severehyperparathyroidism

Janicic et al, 1995

C1 inhibitor de novo Y Complement deficiency Stoppa Lyonnet et al, 1990ACE about 50% Ya5 Linked with protection

from heart diseaseCambien et al, 1992

Factor IX a grandparent Ya5 Hemophilia Vidaud et al, 19932 x FGFR2 De novo Ya5 Apert’s Syndrome Oldridge et al, 1997GK ? Sx Glycerol kinase

deficiencyMcCabe et al, (personalcomm.)

Generating Genetic Variation:Exon shuffling via SINE mobilization

exon 1 SINE

intron

exon 2

SINE transcription can extend past the normal stop signalReverse transcription creates DNA copies of both the SINE and exon 2

DNA copy of transcript

Reinsertion occurs elsewhere in the genome

SINE exon 2

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 numberProvided 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 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?

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

Genome Analysis

Documents

Welcome to Part 2 of Bio 219