review from last time - west virginia universitydray/219files/bio219_chapter_20_11-27-05.pdf ·...

Review from last time• Sanger dideoxy sequencing is the most common method

of DNA sequencing used these days– The method is analogous to LEGOs with no pegs on top– Be familiar with the details

• DNA engineering allows for detailed genetic manipulation including:– Determining when and where a gene is expressed– Creating novel DNA molecules– Expressing rare proteins

• Creating transgenic complex eukaryotes is a complicated process because the changed gene needs to be inserted into the germ line– Be familiar with the process

Genetics, Meiosis, and the Molecular Basis of Heredity

Theories on 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?

Theories on Inheritance

• Genetics = the science of heredity• This section will focus on the molecular

mechanisms of genetics

Genetics, Meiosis, and the Molecular Basis of Heredity

• Topics– Sexual reproduction (advantages,

disadvantages, meiosis)– Mendelian inheritance– Experimental genetics

Simple Inheritance

• Bacteria and some other organisms reproduce simply by making exact copies of themselves

• This is asexual reproduction• The basic mechanism for most unicellular

eukaryotes is mitosis

Mitosis

• Mitosis is part of the eukaryotic cell cycle, which consists of two growth phases (G1, G2), a synthesis phase (S), and an M phase during which cell division occurs

Mitosis

• During M-phase, a cell must complete a nuclear division and a cytoplasmicdivision and faithfully distribute replicated chromosomes to each daughter nucleus

Mitosis

• Although they overlap extensively, M-phase is typically divided into six phases

Mitosis• Although they overlap extensively, M-phase is

typically divided into six phases• Animal cells and plant cells differ with respect to

cytokinesis

• Movie

Mitosis

Sexual Reproduction• Sexual reproduction involves the mixing of genomes

from two individuals to produce offspring that are genetically distinct from either parent and from other offspring

• Disadvantages –– Half of your genes don’t make it to the next generation– very costly to produce specialized cells – interaction with other organisms is dangerous– mixing genes can produce unexpected results

• Advantages– One word – variation– The introduction of variation allows for better survival in a

changing environment and for the rapid spread or reduction of advantageous and deleterious genes – (see video on guppies)

Sexual Reproduction

• Sexual reproduction occurs in diploid organisms– Diploid organisms have two complete sets of

chromosomes, one from each parent– Diploid organisms therefore carry two copies

of most genes– Diploid organisms use haploid cells to

reproduce– Haploid cells contain only one copy of each

chromosome set

Sexual Reproduction• The basics of sexual reproduction

– The germ cells (gametes) are haploid– Gametes are generated through meiosis– There are typically two types in animals

• A large, immobile egg• Small, mobile sperm

– During sexual reproduction, the gametes fuse to produce a diploid zygote

Sexual Reproduction• Primordial germ cells

are produced by mitosis

• Ova and sperm by meiosis

• Spermatogenesis continues throughout male mammals life

• Increased mutation rate in males due to increased number of replication events

Review from last time• Mitosis (nuclear division) is the basic method of asexual

reproduction for eukaryotic cells• The process of mitosis is fluid but arranged into five phases,

prophase, prometaphase, metaphase, anaphase, and telophase• Cell division (cytokinesis) is the process by which the replicated

nuclei are distributed to daughter cells• Sexual reproduction has several major disadvantages and one

major advantage - variation• Sexual reproduction typically involves two haploid gametes fusing to

create a single diploid cell• The number of cell divisions in each process, oogenesis and

spermatogenesis, lead to more mutations in the male germ line

Meiosis• Meiosis is

the process by which gametes are produced

• Diploid cells reproduce through mitosis

• This NOT how gametes are made

Sexual Reproduction:Mitosis review

Sexual Reproduction:Meiosis (producing gametes)

• In mitosis, a diploid cell produces two diploiddaughter cells

• In meiosis, a diploid cell gives rise to four haploidcells


• Major differences between mitosis and meiosis– Mitosis – once cell division– Meiosis – two cell divisions– Mitosis – replicated chromosomes line up

‘single file’ during metaphase– Meiosis – replicated homologs line up in pairs

during metaphase I and ‘single file’ in metaphase II


• Variation is introduced to the offspring by combining the chromosomes of both parents into a single cell

• A second level of variation is introduced via recombination during meiosis (prophase I)

• Recombination is an exchange of material between homologous chromosomes via a process called ‘crossing over’

• Thus, the gametes you produce will be novel combinations of the chromosomes you received from your parents

Sexual Reproduction:Recombination

• The end result of meiosis is a pool of gametes in which the genetic information of the parent has been extensively rearranged

• Merely by combining different sets of paternal and maternal chromosomes, there are 223

(8,400,000) distinct gametes possible• By introducing recombination you increase that

number exponentially

Sexual Reproduction:Recombination

Sexual Reproduction:Creating variation

– A cell must keep track of 92 chromosomes (23 x 4) during meiosis and sometimes errors occur

– Nondisjunction – failure of chromosomes to separate properly

• Results in gametes with more or fewer than the standard number

Sexual Reproduction:Mistakes during meiosis

Sexual Reproduction:Nondisjunction

– Zygotes resulting from aneuploid(abnormal chromosome number) gametes typically don’t survive but sometimes do

– Down syndrome (trisomy 21)– Edward’s syndrome (trisomy 18)– Patau’s syndrome (trisomy 13)

Sexual Reproduction:Fertilization

• Fertilization is the union of two gametes to produce a diploid zygote

• ~200 of the 3 million sperm in a human male ejaculate reach the egg

• To ensure only one sperm fertilizes the egg, a chemical cascade ‘hardens’ the egg once one has fused with it

• Ca++ Wave During Sea Urchin Egg Fertilization– The sperm enters at about

the 2 o'clock position. Note the elevation of the fertilization membrane in the left panel and the calcium wave in the right panel.

Sexual Reproduction:Fertilization

• As a result of the processed described for sexual reproduction, the genomes of diploid organisms are a mixture of discrete segments of their parents’ genomes

• Some traits are inherited in a simple fashion through individual genes.

• Other traits are polygenic• Others are simple and/or polygenic and

influenced by the environment

Mendelian Inheritance

• Simple Mendelian inheritance– Attached earlobes– PTC (phenylthiocarbamide) tasting– ‘uncombable hair’

• Complex (polygenic) inheritance– Eye color– Height

• Studying inheritance in humans is difficult for ethical reasons but more easily done in other organisms


• Named for Gregor Mendel– 1822-1884– Studied discrete (+/-, white/black) traits in pea

plants


• Mendel began with true-breeding plants– True-breeding - when mated with themselves

or others of the same type, produce the same offspring

– Cross-pollinated these true breeding varieties– Crossed the offspring (F1’s) with each other

or back to the parents – Kept very detailed numerical records of the

offspring of each cross



• 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)

Review from last time

• Meiosis is the cellular process by which gametes are made

• Gametes are haploid cells which merge during fertilization to produce diploid cells

• Meiosis involves two nuclear and cytoplasmic divisions but only one chromosome replication – 1 diploid cell >>> 4 haploid cells

• Genotype vs. phenotype


• 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 (yellow is dominant vs. green is recessive)

• The F1’s pass on both of their alleles in a random manner

• Mendel’s Law of Segregation – the alleles for a 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 100% black trait offspring or ~50% black trait and ~50% white 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 (0.56 = 0.016)

• 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? B?B?

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

• Mendel’s Laws of Segregation and Independent Assortment are a result of the process of meiosis

• During meiosis, the chromosomes that carry alleles are distributed randomly among the resulting gametes – The law of segregation

• Traits (genes) residing on one chromosome are distributed independently of those on other chromosomes– The law of independent assortment


DNA Analysis: DNA Cloning

• Mendel’s Laws of Segregation and Independent Assortment are a result of the process of meiosis

• During meiosis, the chromosomes that carry alleles are distributed randomly among the resulting gametes – The law of segregation

• Traits (genes) residing on one chromosome are distributed independently of those on other chromosomes– The law of independent assortment

• What about genes that reside on the same chromosome? Do they also assort independently?


• Yes, generally genes on the same chromosome behave just like genes on different chromosomes – they assort independently

• How?• Remember recombination and

crossing-over?



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.

• By following the rates of recombination between genes on the same chromosome, we can determine where they are in relation to each other

• The results of these studies is called a linkage map

• Linkage maps are based on the frequency with which two genes are co-inherited. The closer they are to each other, the more often they are co-inherited.

Non-Mendelian Inheritance: Linkage Maps

DNA Analysis: Nucleic Acid Hybridization

• The genotype has an effect on the phenotype but not vice-versa

• Heterozygotes tell us whether an allele is dominant or recessive– Heterozygotes harbor two alternative alleles of a gene

• Why does the allele for round peas (dominant) mask the effect of the allele for wrinkled peas (recessive)?

Mendelian Inheritance: Genotypes and Phenotypes

RR rr

RrRrRrRrRr

• The gene in question encodes an enzyme that converts sugars into starch

• ‘R’ is the active allele, ‘r’ is an allele that doesn’t encode an active enzyme (a loss-of-function mutant)

• RR genotype > both alleles produce active enzyme > round pea phenotype

• rr genotype > neither allele produces active enzyme > wrinkled pea phenotype

• Rr genotype > the active allele produces enough enzyme to overcome the enzyme deficiency > round pea phenotype


• Gain-of-function mutants are usually dominant

• These types of mutations may cause a gene to produce hyperactive enzymes– Ex. One allele of the Ras gene in human is a gain-of-

function mutant that makes the enzyme active at inappropriate times. Cells grow out of control > cancer


• In chapter 9 we discussed how variation in the starting point for evolutionary change

• Most mutations are deleterious or neutral but some can increase an organism’s fitness


• Others can be deleterious in one environment but advantageous in another– Sickle cell disease– Advantageous in malaria prone

areas, deleterious elsewhere

• Once it became clear how traits were inherited it became possible to manipulate those traits in an effort to diagnose and treat human disease

Experimental Genetics

• The classical approach– Introduce mutations into an organism via

mutagenesis– Mutagens are factors (radiation, chemicals)

that can cause damage to DNA and chromosomes

– Once mutants are created, we then work backward from the phenotype to determine the genotype


• The classical approach– Not readily implemented in humans for

obvious reasons but still possible…– Using model organisms. Many genes are

shared between us and flies, mice and worms– Using cultured cells. The effected cells can

be removed, cultured and observed outside of the human body

– Using non-lethal traits that have arisen naturally (i.e. sickle cell)


• Genetic Screens– The more complex the genome, the harder it

is to locate the mutant phenotype of interest– Genetic screens make the process easier by

selecting for mutants of interest– Ex. Temperature sensitive mutants


Experimental Genetics• Temperature sensitive mutants contain alleles

that are inactive (often lethal) at certain temperatures– Usually the genes involve critical processes such as

RNA synthesis or cell cycle control– Isolating the mutant organisms is as simple as raising

the thermostat a degree or two

• Complementation tests– Complementation tests reveal whether two

mutations causing the same phenotype reside in the same gene

– This allows us to count the number of genes determining a particular phenotype


• Complementation tests


• Again, experiments like this on humans are frowned upon

• How do we investigate mutant human genotypes?

• One method involved haplotype blocks


• Haplotype blocks are groups of sequences along a chromosome that tend to be inherited as a unit as a result of linkage

• By tracking single nucleotide polymorphisms (SNPs) we can identify the linked mutations that underlie a disease


20_30_trace_inheritance.jpg

• Haplotype blocks can also be used to identify recent mutations and mutations that have been under positive selection

• The more recent the mutation, the larger the haplotype block since it has not been broken up through recombination

• Selectively advantageous mutations will spread more quickly through populations via large haplotype blocks


20_31_haplotype_blocks.jpg

• What about traits that ‘run in families’ but are not discrete?

• Traits that do not follow Mendel’s laws but do have an inherited component are called complex traits

• Reasons for complexity– They are polygenic– They have an environmental component– They are polygenic and have an environmental

component


• Polygenic traits may produce a continuum of traits instead of simple discrete states– Eye color – polygenic, numerous genes

contribute to the distribution of melanin in the iris and each gene has several alleles


• Environmental factors– Even if you have all the genes necessary to be a fine

athlete, if you don’t exercise and practice you won’t be

– Studies of identical twins adopted by different families have helped increase our understanding of the relative influence of genes and the environment


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