Bms2042 Usg Part 1

Autosomal Inheritance

Autosome • Non-sex chromosome • Same in both sexes • 22 pairs of autosomes in humans (the other will be the sexchromosome X or Y)

Fields of genetics • Transmission genetics – how traits are passed • Molecular genetics – how hereditary material controls expression of genes (and thus traits) • Population genetics – genetic variation

Mendelian inheritance • Experiments on monohybrid crosses (where one characteristic differs) revealed the law of segregation • Reciprocal crosses disproved the notion of one parent contributing more to the offspring • Used pure lines – Parental generation = P – First filial generation = F1 – Intercrossed F1s => 2nd filial generation (F2)

Mendel’s hypothesis • Hereditary factor (gene) is necessary for displaying particular traits • Each ‘parent’ has a pair of this type of gene • Alleles = variants of the gene • Each gamete only contains one member of each gene pair – Mendel’s first law • These gametes fuse RaNdOmLy at fertilization to form the ZYGOTE • Zygote = first cell that develops into a progeny individual • Genes do not act in a vacuum; they depend on the environment for their effects

Some terms y0 • Haplosufficiency – one ‘dose’ of the wildtype allele is enough to allow full expression • Homozygote – plant with a pair of identical alleles – Homozygote dominant (Y/Y)

• Heterozygote – plant in which the alleles of the pair differ – Heterozygote for one gene is sometimes referred to as ‘monohybrid’ – Heterozygous (Y/y) – Heterozygous recessive (y/y) Note: Y/Y, Y/y, y/y are all GENOTYPES

Autosomal dominant inheritance • Males and females equally affected • Affected people are heterozygous for the abnormal allele • Transmission by both sexes to both sexes – I.e. Huntington disease (neurodegenerative disease)

Autosomal recessive inheritance • • • •

Rare Skips in generation Males and females equally affected Occur in individuals who are homozygous for a particular gene mutation • Common for parents to be related (consanguineous) – Cystic fibrosis – Albinism – Sickle cell anaemia

Monohybrid – 3:1

Dihybrid – 9:3:3:1

Allowed Mendel to infer that gene pairs on different chromosome pairs assort independently during gamete formation

Dihybrid – 9:3:3:1

Allowed Mendel to infer that gene pairs on nd different chromosome Mendel’s 2 Law pairs assort independently during gamete formation

Example 1 •

Albinism is an autosomal recessive disorder. Two heterozygous parents are expecting a baby. A/a . A/a – Probability that the baby is affected? 0.25 – Probability that the baby is a homozygote? 0.50 – Probability that this baby is affected, and they then have a second baby who is unaffected? 0.19 – If they have 5 children, what is the probability that the children will have the following phenotypes in the order stated; 1. unaffected, 2. albino, 3. albino, 4. unaffected, 5. unaffected? 0.026

Example 2 • A male of genotype Aa:Bb and a female of genotype Aa:bb decide to have children (the two traits are autosomal) What is the probability of obtaining a child of the genotype aa:bb? Probability is ¼ for aa Probability is ½ for bb Overall probability is ¼ x ½ = 1/8

Example 3 • Plants of the genotype Aa:bb ; Cc:Dd and Aa:Bb; Cc:dd are crossed. What is the probability of obtaining a progeny plant of the genotype aa; bb; cc; dd? Probability of aa = ¼ Probability of bb = ½ Probability of cc = ¼ Probability of dd = ½ Product of these values gives us: 1/64

Sex-linked Inheritance

Sex-linked Inheritance • Regularly shows different phenotypic ratios in the two sexes of progeny, as well as different ratios in reciprocal crosses • X-linked traits do not give same results with reciprocal crosses • w = recessive mutant allele; w+ = dominant wild-type allele • Males are HEMIZYGOUS for X-lines genes • I.e. they only have one allele of a gene rather than the usual two – just 1 ‘X

X-linked recessive inheritance • Males usually affected – Only need ONE copy of the mutant allele in order to exhibit mutant phenotype; they can only be HEMIzygous – Females would require BOTH parents to bear the allele in order to inherit it i.e. (X^A . X^a) x (X^a . Y)

• Transmitted through unaffected females • Females affected through affected fathers • No male to male transmission – Father’s X chromosome isn’t inherited

• • • •

Hemophilia (failure of blood to clot) Colour Blindness Duchenne Muscular Dystrophy Fragile-X syndrome (x-linked mental retardation)

X-linked dominant inheritance • Rare • Both genders affected • Females less severely affected than males (due to X inactivation) • Affected heterozygous females married to unaffected males pass the condition to half their sons and daughters • Affected males transmit to all daughters but not sons – Because sons don’t receive the X chromosome from the father

• Hypophosphatemia (vit-D resistance rickets) • Rett Syndrome (neurodevelopmental disorder)

Y-linked inheritance • Very rare – VLittle genetic information on Y chromosomes

• Only males affected

Molecular basis of dominance/recessiveness of alleles

• Dominance is a result of interactions between genes at the SAME locus • Dominance does not affect the way in which genes are inherited; it only influences the way genes are expressed • Dominance/recessiveness depends on the FUNCTION OF THE GENE and the EFFECT that the mutation has on this function

• Recessive mutant alleles – Most new mutations cause LOSS OF FUNCTION of the gene – Complete loss of function = NULL mutation • I.e. ALBINISM (protein simply doesn’t function)

• Dominant mutant allele – Rare – Haploinsufficiency = loss of function mutation. 1 wild-type dose (50%) is NOT enough to achieve normal levels of function  • I.e. Tailless mice

• Dominant-negative mutations – Loss of function mutation – Protein made but non-functional – Mutant allele inhibits function of normal protein in heterozygotes • ‘Spoiler’ polypeptide distorts or interferes with the function of the wild-type polypeptide

Gain function mutations - New function of the gene product - Protein always active - Increased levels of expression - Inappropriate expression

Extensions to Mendelian Inheritance

Extensions to Mendel for single genes Recall – Complete dominance is the interaction between alleles on a single gene in a heterozygote whereby a single copy of a dominant allele hides the recessive allele. 50% of the gene product is enough to produce wild-type phenotype. Transmission of many traits doesn’t produce Mendelian ratios however, Mendelian

Variations on Dominance • Arise from different types of interaction between alleles • Incomplete dominance

• Co-dominance

Variations on Dominance • Arise from different types of interaction between alleles • Incomplete dominance

• Co-dominance

Incomplete Dominance • The occurrence of an intermediate phenotype observed (between the corresponding homozygotes) • Phenotypic ratio = genotypic ratio i.e. 1:2:1

Each wild-type allele produces a set dose of its protein product. The number of doses of the wildtype allele determines the concentration of a chemical made by the protein, i.e. PIGMENT.

Genes – code for enzymes Alleles – determines total level of the enzyme in the cell/organism

Each wild-type allele produces a set dose of its protein product. The number of doses of the wildtype allele determines the concentration of a chemical made by the protein, i.e. PIGMENT.

Genes – code for enzymes Alleles – determines total level of the enzyme in the cell/organism

Each wild-type allele produces a set dose of its protein product. The number of doses of the wildtype allele determines the concentration of a chemical made by the protein, i.e. PIGMENT.

Genes – code for enzymes Alleles – determines total level of the enzyme in the cell/organism

Incomplete dominance can be observed on the microscopic level with peas ->

Codominance • Expression of both alleles of a heterozygote, i.e. blood type AB, where I^A and I^B alleles are codominant because both transferases are active and both antigens are present – Blood type is determined by the types of polysaccharide antigen present on the RBC surface – In blood, all individuals have the O (aka. Substance H) surface antigen on the RBC Universal surface Donor

Univers al Recipie nt

Codominance • Expression of both alleles of a heterozygote, i.e. blood type AB, where I^A and I^B alleles are codominant because both transferases are active and both antigens are present – Blood type is determined by the types of polysaccharide antigen present on the RBC surface – In blood, all individuals have the O (aka. Substance H) surface antigen on the RBC Universal surface Donor

Protein made by that immune system that is capable of binding to a stimulating molecule (antigen)

Univers al Recipie nt

nullmutation

Agglutination is undesirable as it BLOCKS blood vessels and the recipient of the transfusion may go into shock and may die. We see, A can’t accept blood from B.

So…? More than 2 alleles may be present within a group of individuals, although each diploid individual still only has two alleles at that locus

Bear in mind… Variations in dominance do not negate Mendel’s law of segregation. They reflect the differences in the way GENE PRODUCTS control the production of PHENOTYPES and the effect of the mutant allele on protein function

Lethal alleles • Usually recessive – Generally cause death in homozygotes – Can be dominant, where heterozygotes don’t survive

• Capable of causing the death of an organism – Mutation of an essential gene – Manx cat (a homozygote, tailless cat)

• May produce ratios that deviate from Mendelian ratios • Pleiotropic – A gene that affects multiple traits

Penetrance • Penetrance – percentage of individuals with a given allele who exhibit the phenotype associated with that allele • Incomplete penetrance – phenotype expected from a particular genotype is not expressed • Phenotype may not be expressed due to – Environmental factors i.e. a person genetically predisposed to lung cancer may not get it if he/she doesn’t smoke; temperature; chemicals

Expressivity • Expressivity – degree to which a trait is expressed • Quantifies the modification of gene expression by varying environment and genetic background

Sex-influenced and sexlimited traits • Sex-influenced trains – gender governs inheritance pattern. Allele is dominant in one sex and recessive in another – I.e. Males have a greater propensity to baldness if this autosomal trait is passed onto them as they only require a single allele

• Sex-limited traits are limited to one

And then…

Extensions to Mendel – Gene Interation • Most traits are genetically heterogeneous – controlled by multiple genes (and often the environment too) – Thalassemia – Deafness

Complementation test • Can determine whether two mutations with the same phenotype affect the same gene or different gene – Determines whether or not mutations occur at the same locus

• Applied only to recessive mutations • Two homozygous mutant lines are crossed to produce a heterozygous mutant line • When organisms homozygous for mutations that show the same phenotype and are in: – Different genes -> Wild-type observed, mutations complement one another – Same gene -> mutant, fail to complement

Two genes controlling a single trait give rise to novel phenotypes in the F2

• When two genes affect the same phenotypic trait, we get a novel phenotype (a double homozygous mutant)

Complementary gene action • Dominant allele from both genes required to produce the trait • 9:7 ratio in the F2 generation is observed when a homozygous mutation in either or both of two different genes results in the same mutant phenotype. • i.e. In sweet peas, genotypes that are C- for the C gene and P- for the P gene have purple flowers

Two individuals heterozygous for two loci are crossed

• Let’s say, pigement (compound C) is produced ONLY after compound A has been converted to compound B by enzyme I and after compound B is converted to compound C by enzyme II…

At least one dominant allele A at the first locus

Enzyme I

At least one dominant allele B at the second locus At least one dominant allele A at the first locus

Enzyme I

Enzyme II

Compound C

At least one dominant allele B at the second locus At least one dominant allele A at the first locus

Enzyme I

Enzyme II

Compound C

So, if there are two recessive alleles at the first locus. i.e. aaB_ = no pigementation A_bb = no pigmentation Aabb = no pigmentation

Epistasis • Masking of the expression of one gene by another gene at a different locus (unlike ‘dominance’ where masking occurs at the same locus)

– Masking gene = epistatic gene • Can be dominant or recessive • Can mask when recessive homozygous

– Masked gene = hypostatic gene

• Normally occurs because genes act in the same pathway for producing a phenotype • F2 ratio is 9:3:4 [3pist4sis (9 letters)]

“Golden Labrador retrievers are homozygous for the recessive e allele. Because this ee genotype masks the effects of the B coat color gene, golden retrievers may be of any genotype—BB, Bb, or bb—at this other gene. In Edogs, a B- genotype produces black and a bb genotype produces brown”

Individual can still pass on IA or IB allele

Omg, no substance H! – needed for addition of A / B sugars at the surface of the RBC

Suppression • A suppressor is a mutant allele of a gene that reverses the effect of a mutation of another gene and restores the corresponding wild-type phenotype • Only 2 phenotypes segregate (unlike 3 as in epistasis) • Implies that gene products normally interact

Non-Mendelian Inheritance • Extranuclear inheritance • Maternal effect (nuclear genes) • Imprinting – Expression of a gene is influenced by the sex of the parent who transmits the gene to the offspring.

Extranuclear inheritance • Inheritance of genetic material not located within the nucleus (aka. Cytoplasmic inheritance) • Main organelles – mitochondria and chloroplasts – Uniparental inheritance – Each mitochondria contains approx. 15,000 nucleotides that encode for 37 genes – Nuclear DNA contains some 3 billion nucleotides encoding for 35,000 genes

• Contain circular chromosomes, resemble smaller versions of bacterial chromosomes • •

Rely largely on nuclear genes for their function Replicate autonomously

• Encode products which function within the organelles • Multiple chromosome per organelle

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Extranuclear inheritance continued… • Mitochondria is inherited from the mother in most cases • Variegation in plants is caused by mixture of chloroplasts (heteroplasmic vs homoplasmic) – White leaves caused by mutations in chloroplast genes that control the production and deposition of chlorophyll – White-leaved plants cannot live

• During somatic cell division, organelles separated more or less evenly into daughter cells • Paternal (and biparental) inheritance is much rarer as the egg is the larger gamete and is able to provide far more cytoplasm to

Mutations in mitochondrial DNA • Often in genes that code for the components of the electron-transport chain, which generates most of the ATP in aerobic cellular respiration • Disease only inherited maternally • Can cause disease that are chronic degenerative disorders affecting the brain, kidneys, heart and other high energy requiring tissue – Myopathy – Leber’s Hereditary Optic Neuropathy • Affects cells in the optic nerve, leading to loss of vision • Point mutation in the gene encoding NADH

Maternal effect • PHENOTYPE of the offspring determined by GENOTYPE of mother • Genes inherited from BOTH parents (unlike in cytoplasmic inheritance) • Phenotype is not affected by the genotypes of the father, and of the offspring themselves – Expression is too late in embryogenesis

Maternal effect

Snail-shell coiling

Sinistral coiling is recessive Direction of the coiling is determined by the nuclear genotype of the mother and NOT the snail itself or by extranuclear inheritance Obviously no Medelian inheritance patterns here…

Maternal effect

Snail-shell coiling – closer look

The direction of coiling is affected by the way in which the cytoplasm divides soon after fertilization, which in turn is predetermined by a substance produced by the mother and passed to the offspring in the cytoplasm of the egg

Defective control of cell cycle -> cancer

Errors in mitosis -> cancer

Accidents in cell division -> aneuploidy

Cell division and chromosome theory

Errors in meiosis -> developmental defects and mental retardation

Change in number of chromosom es that can lead to chromosom al abnormality

Chromosomes • Singular piece of DNA constituted by genes, regulatory elements and other nucleotide sequences • Linear structures in eukaryotes – Highly folded and condensed – Packed around histone proteins – Consist of a centromere, a pair of telomeres and origins of replications

• Most eukaryotes are DIPLOID (contain two similar sets of chromosomes, one is maternal and the other paternal), each chromosomes is a member of a pair called HOMOLOGUES. They each carry copies of the same genes but are NOT identical to each other • Our gametes are HAPLOID

Also participate in limiting cell division and play important roles in aging and cancer

Cell division • Mitosis: Same number and types of chromosomes as the original mother cell (genetically identical) i.e. diploid cells • Meiosis: Half the number of chromosomes as the mother cell, one member of each chromosome pair. I.e. haploid cells • Division occurs via CELL CYCLE, alternates between – Interphase • DNA replication that produced an identical pair of sister CHROMATIDS which are to end up in daughter cells • Sister chromatids become visible at the beginning of mitosis

– Mitosis (the phase of actual division) • Resulting daughter cells have the same genomic

More annoying terms… • Dyad = replicate (two) sister chromatids • Bivalent = pair of synapsed dyads • Tetrad = 4 chromaTIDS that make up the bivalent

Major events of cell cycle

Major events of cell cycle Doubling of the genome

Halving of the genome

Major events of cell cycle Synthesis (of DNA) 10 – 12 hours

Nuclear division -Anaphas e -Telophas Cytoplasmice division

Mitosis 30 mins Chromosome segregation Cell division

Phases of the cell cycle •

Gap phases G1 and G2 allow time for the cell to grow and double their mass of proteins and organelles

•

They also provide time for the cell to monitor the internal and external environment to ensure that conditions are suitable and preparations and complete before the cell commits itself to the S phase or mitosis

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Cell growth does not occur during mitosis

Cell-cycle control system • •

• •

Operates much like a timer or oscillator that triggers the events of the cell cycle in a set sequence Control system is independent of the events in controls but does utilize sensors to detect the completion of processes and will delay progression of the cycle if for example DNA damage occurs Highly adaptable and can be modified to suit specific cell types or to respond to specific intracellular or extracellular signals Triggers cell-cycle progression at 3 major regulatory transition (checkpoints). - 1st checkpoint @ Start(restriction point), late in G1 - 2nd checkpoint @ G2/M where the control system triggers early mitotic events that lead to chromosome alignment on the specific spindle in the metaphase - 3rd checkpoint @ metaphase-to-anaphase transition where the control system stimulates sister-chromatid separation -> completion of mitosis and cytokinesis

Cell-cycle control system

Cell-cycle control system Feedback signals are received at checkpoints Checkpoints allow the cellcycle control system to receive information about the environment If damage is irreversible, apoptosis will be triggered

Checkpoints • Signals that arrest the cycle usually occur at G1 • During infection of T-cells with HIV-1, signal is arrested at G2 – this allows for greater virus production!

Interphase • DNA is being synthesized • RNA and proteins are being produced • G1 (gap 1) – material required for survival and growth is made • S – chromosomes replicated to form sister chromatids • G2 (gap 2) – synthesis of proteins needed for divison – i.e. microtubules, centrosomes etc…

Mitosis • •

Separation of sister chromatids to provide a complete set of genetic information for each of the resulting cells Involves 6 stages – Prophase – Prometaphase – Metaphase – Anaphase – Telophase – Cytokinesis

Mitosis • •

Separation of sister chromatids to provide a complete set of genetic information for each of the resulting cells Involves 6 stages – Prophase – Prometaphase – Metaphase – Anaphase – Telophase – Cytokinesis

Mitosis • •

Separation of sister chromatids to provide a complete set of genetic information for each of the resulting cells Involves 6 stages – Prophase – Prometaphase – Metaphase – Anaphase – Telophase – Cytokinesis

Mitosis • •

Separation of sister chromatids to provide a complete set of genetic information for each of the resulting cells Involves 6 stages – Prophase – Prometaphase – Metaphase – Anaphase – Telophase – Cytokinesis

Mitosis - Prophase • Chromosomes condense and become visible • Each chromosomes possesses two chromatids – Each pair of chromatids is the product of the duplication of one chromosome in the S period of the interphase – Chromatids are held together at the centromere by cohesin

• The mitotic spindle (a bipolar bundle of microtubules that move the chromosomes in mitosis) forms – Nuclear envelope disintegrates

Before we move to Prometaphase…

Centrosome •

Regulator of the cell-cycle progression

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Centrosomes surround the centrioles which organize the mitotic spindles and are involved in the completion of cytokinesis

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They form the two poles of the mitotic spindle which serve to pull two sets of sister chromatids to opposite ends of the cell during the anaphase

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Eukaryote cells must duplicate its centrosome to provide one for each of its two daughter cells

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Must replicate only once per cell cycle to ensure that the cell enters mitosis with only 2 copies. Incorrect number of centrosomes could lead to defects in spindle assembly and thus errors in chromosome segregation

Mitosis - Prometaphase • Spindle microtubules which have thus far been outside the nucleus, enter the nucleus • The ends of certain microtubules make contact with the chromosome and anchor to the kinetochore of one of the sister chromatids • A microtubule from the opposite centrosome then attaches to the other sister chromatid and so each chromosome is anchored to both of the centrosomes • The microtubules lengthen and shorten,

Mitosis - Metaphase • Both sister chromatids attached to kinetochore microtubules and chromosomes line up on the metaphase plate • Basically, this metaphase plate is an imaginary plane that’s equidistant from the two poles of the mitotic spindle where all the kinetochores lie on • Chromosomes reach their maximum contraction on this plate

Anaphase • Centrosomes divide • Each sister chromosome moves to opposite poles of the spindle and regarded as a separate chromosome in its own right • Chromosome movement results in part from progressive shortening of the kinetochores attached to the centromeres • This movement is mediated by KINESINS (motor proteins) which and results in chromosomes being pulled

Telophase • Chromosomes at poles begin to decondense • Spindle disappears • Nuclear envelope forms around each compact group of chromosomes -> 2 nuclei • Chromosomes decondense until they’re no longer visible as discrete entities

Cytokinesis ‘Cytoplasm division’

• Fiber ring composed of actin around the center of the cell contracts pinches the cell into two daughter cells • Important organelles (i.e. ribosomes, mitochondria) are divided between daughter cells

Summary

Meiosis • Introduces GENETIC VARIATION – If we all reproduced mitotically, we’d essentially be clones of one another and would not evolve – Cells produced by meiosis are genetically different from one another and the parental cell

• Longer process than mitosis – spans for several days or even weeks • Only one member of each pair of chromosome is present in the premeiotic cell • Consists of two successive nuclear divisions (meiosis I and meiosis II) that lead to four HAPLOID cells – Meiosis I: the homologous chromosomes disjoin = reductive division – Meiosis II: the sister chromatids separate (similar to mitosis BUT there is no chromosome replication)

• Occurs during GAMETE formation in GERM CELLS • Diploid chromosome number re-established at fertilization (23 in gamete, 46 in zygote)

More on meiosis… • Independent assortment of maternal and paternal chromosomes, and cross-over of genetic material between homologous chromosomes – Mendel’s law of independent assortment • Begins after cells have progressed through G1, S and G2 phases of the cell cycle, thus chromosomes have been replicated • The cells enter meiosis containing pairs of sister chromatids • Major differences occur during prophase I (prophase of meiosis I): – Homologous chromosomes pair – Exchange between non-sister chromatids

Prophase I • •

Leptotene ‘thin thread’ – chromosomes begin to condense (become visible) Zygotene ‘paired threads’ – pairing (or synapsis) of the homologous chromosomes – Recall! Each pair of synapsed homologous chromosome is referred to as a ‘bivalent’ or ‘tetrad’

•

Pachytene ‘thick thread’ – condensation of the chromosome continues – Crossing over occurs – physical exchange of chromosome segments (genetic info) – Site of crossing-over is called CHIASMA which is formed by a breakage and rejoining by non-sister chromatids

• •

Diplotene ‘double thread’ and diakinesis ‘moving apart’ – homologous chromosomes move apart, except at segments connected by chiasmata (cross-over points) At the end of diakinesis, the formation of a spindle is initiated, and the nuclear envelope breaks down

Exchange of genes between non-sister chromatids generates even more diversity

Metaphase I • Homologous pairs of chromosomes align along the metaphase plate • Arrangement of chromosomes within this double row is RANDOM – 50-50 chance for daughter cells to get either the mother’s or father’s homologue for each chromosome

• A microtubule from one pole attaches to one chromosome of a homologous pair

Anaphase I • Homologous chromosomes separate from each other and migrate to opposing poles • Centromeres and sister chromatids still intact with each other • Alignment of chromosomes in metaphase I ensures each daughter cell receives a RANDOM assortment of maternal and paternal chromosomes

Telophase I • The chromosomes arrive at the spindle poles • Spindle breaks down and the cytoplasm divides (this overlaps with the short INTERKINESIS stage) • Chromosomes enter phase II of mitotic division after only a limited uncoiling

Meiosis II • Similar to mitosis, except half the number of chromosomes are invovled – In mitosis, we have 46 chromosomes, containing 92 sister chromatids joined into 46 pairs – In meiosis II, we have 46 sister chromatids joined as 23 pairs

Stages of Meiosis I Simplified Version

• Prophase I - Chromosomes condense, homologous pairs of chromosomes synapse, crossing over takes place, nuclear envelope breaks down, and mitotic spindle forms • Metaphase I - Homologous pairs of chromosomes line up on the metaphase plate • Anaphase I - The two chromosomes (each with two chromatids) of each homologous pair separate and move toward opposite poles • Telophase I - Chromosomes arrive at the spindle poles • Cytokinesis The cytoplasm divides to produce two cells, each having half the original number of chromosomes • Interkinesis In some cells the spindle breaks down and chromosomes relax

Stages of Meiosis II Simplified Version

• Prophase II - Chromosomes condense, the spindle forms, and the nuclear envelope disintegrates • Metaphase II - Individual chromosomes line up on the metaphase plate • Anaphase II - Sister chromatids separate and migrate as individual chromosomes toward the spindle poles • Telophase II - Chromosomes arrive at the spindle poles; the spindle breaks down and a nuclear envelope re-forms • Cytokinesis The cytoplasm divides

Genetic variation • Crossing-over • Random distribution of chromosomes

Crossing-over

Random distribution of chromosomes

Chromosome theory Chromosome theory of inheritance states that genes are located on chromosomes. The two alleles of a genotype segregate during ANAPHASE I of meiosis, when homologous chromosomes separate. The alleles may also segregate during anaphase II of meiosis if crossing over has taken place Btw, in drosophila, sex is determined by number of X chromosomes. 1 = male, 2

Gene mutation and Gene Function

Yay, even more definitions… • Forward mutation = Change from WT -> mutant • Reverse mutation = Change back to WT • Spontaneous mutation – Natural changes to DNA structure – Very rare – Arise from altered base structures and from wobble base pairing – Small insertions and deletions may occur through strand slippage in replication and through unequal crossing over – Depurination / deamination -> alter pairing properties of bases

• Mutagen = substance that increases rate of mutation; causes point mutation

Luria and Delbruck experiment • TonR = resistance mutants • The hypothesis: – If an ADAPTIVE RESPONSE let to tonR – we’d expect resistance only when the cells are exposed to the adverse agent – If RANDOM MUTATION let to tonR – we’d expect that the mutation could occur any time in the growth of the culture

• Results: Large variation in number of T1 resistance colonies between the 20 individual cultures • Conclusion: Heritable variants are produced by mutations occurring spontaneously at random – Specific mutations are not responses to the

Function of a gene • Genes encode RNAs (and not proteins) • Chemical synthesis in cells is by pathways of sequential steps catalyzed by enzymes • The genes encoding the enzyme of a specific pathway constitute a functionally interacting subset of the genome

Types of mutation – levels of mutation The following types are stably inherited: • Changes to the genome – changes in chromosome number. i.e. Down syndrome, trisomy etc… • Changes to a chromosome – structural changes in chromosomes • Single gene mutations – relatively small changes within a particular gene

Types of mutation – What can a single gene mutation do?

• Mutation within the coding sequence

– May or may not affect AA sequence – Effect on protein function depends on the role of the altered amino acid(s) in the protein

• Mutations in non-coding regions – Promoter region – mutation may result in an increase or a decrease of gene transcription – Splice recognition sites – pre-mRNA may not be spliced correctly – similar result to a frameshift mutation – Alteration in ability of mRNA to be translated

•

Types of mutation – Mutation can occur in germ-line or somatic cells Mutations are the primary tools of genetic

analysis • Most mutations seen are GERM-LINE (cells that produce gametes) mutations which can be inherited • Somatic mutations = mutation in the somatic cells; not passed from parent to offspring, but will be passed from cell to cell during cell division – Cells with somatic mutations that that stimulate cell division can increase in number and spread • Basis for all cancers!

• Only dominant mutations will affect the phenotype of cells within a somatic mutant clone • Genetic mosaic = individual who has a

How does genotype affect phenotype? • Effect ranges from trivial -> lethal • Depends on function of gene in the cell • Depends on effect of the mutation on the gene product • Deleterious - i.e. disease genes • Beneficial – enhances survival of organism and is favored by evolution • Conditional – mutations affect phenotype only under defined conditions

How does genotype affect phenotype? Loss of gene function vs. gain of gene function

• Loss of gene function mutations

– Alteration of the gene product most often leads to loss of wild-type function – Null mutation = total loss of function – HYPOMORPHIC mutation = partial loss of function – I.e. cystic fibrosis, albinism – Normally recessive as 50% of gene product is often enough – Can be dominant • Haploinsufficiency (50% not enough) • Dominant-negative – mutant protein is nonfunction and interferes with function of normal protein in heterozygotes

How does genotype affect phenotype? Loss of gene function vs. gain of gene function

• Gain of gene function mutations – Relatively uncommon

• Mutant protein has a new function • Mutant protein is always active • Mutant gene is expressed at increased levels or in different areas

– Generally dominant

Using mutations to understand gene function • Forward genetics – Phenotype first – DNA sequence later

• Reverse genetics – DNA sequence first – phenotype later

Population and Quantitative Genetics

Population genetics • Is concerned with the distribution patterns of alleles and the factors that alter or maintain their frequencies • Application of genetic principles to entire populations of organisms • The gene pool of a population can be described by the frequencies of genotypes and alleles in the population

Genotype frequencies depend on allele frequencies

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Genotype frequencies depend on allele frequencies Can be calculated from a random sample of genotypes - AA = 60, Aa = 30, aa = 10 - P = freq (A) = (twice no. homozygotes + no. of heterozygotes) / (total no. of alleles sampled) = [(2x60) + 30] / (2x100) = 0.75

- I.e. allele frequency of A = 0.6 and allele frequency of a is 0.4

Hardy-Weinberg principle – these frequencies result from random mating for a gene with two alleles

Principles of Hardy-Weinberg Equilibrium are founded on

6 key assumptions • The population is sufficiently large that the frequencies of alleles do not change from generation to generation because of chance • Allelic frequencies of the population are not affected by natural selection, migration and mutation(no gene flow) • Mutation does not occur • Mating is random; there are no subpopulations that differ in allele frequency • All the genotypes are equal in viability and fertility; selection does not operate • Allele frequencies are the same in males and females

Hardy-Weinberg Equilibrium

2 predictions • The allelic frequencies of a population do not change • The genotypic frequencies stabilize after one generation [in the proportions p2 (the frequency of AA),

2pq (the frequency of Aa), and q2 (the frequency of aa), where p equals the frequency of allele A and q equals the frequency of allele a]

• So… when these assumptions are met, reproduction alone does not alter allelic or genotypic frequencies and the allelic frequencies determine the frequencies of genotypes • A population cannot evolve if it meets the Hardy-Weinberg assumptions since evolution consists of change in allelic frequencies of a population

i.e. when q = 0.01, aa = 1/10,000

Calculating carrier frequencies for rare recessive gene defects

• I.e. Cystic fibrosis affects 1 in 2,500 live births = 0.0004 • Q^2 = 0.0004, Q = 0.02 • Since p + q = 1, p = 0.98 • Almost 4% of the population! = 0.0392 (almos

1 in a million disease, we’d have q^2 = 1 x 10^-6, q = 0.001, p = 0.999 2 x 0.001 x 0.999 = 0.001998, about 1 in

How to check if a gene in a population is in HardyWeinberg equilibrium? • Use the known allele frequencies to predict the genotype frequencies for a population in the H-W equilibrium • Compare these to the known genotype frequencies • Use chi-squared test to see if excepted frequencies = observed frequencies

Genetic drift (sampling error)

• The random, undirected changes in allele frequency that occur by chance in all populations, but particularly in small ones • Amount of change in allelic frequency due to genetic drift is inversely related to the effective population size • Effective population size decreases when there are unequal numbers of males and females

Bottlenecks • Temporary Founder effect that can have long-lasting effects – Founder effect: establishment of a population by a small number of individuals • I.e. Cheetahs, elephant seals

Therefore, allele has reached frequency of 1

Loss of an allele = reduction in genetic diversity

Risks: - Higher incidence of rare genetic disease - All individuals may be susceptible to an infectious disease - Individuals may have reduced fitness because of ‘inbreeding

Assortative mating • Bias towards choosing a similar mate – positive assortative mating – Increases the homozygosity of the population

• Bias towards choosing a dissimilar mate – negative assortative mating – I.e. short people mating with tall people

Inbreeding • Non-random mating between related individuals – Alters the frequency of the genotypes but not the frequency of the alleles

• Decreases proportion of heterozygotes – Boosts the probability that deleterious and lethal recessive alleles will combine to produce homozygotes with a harmful trait

• When inbreeding occurs, the genotypic frequency will be: – F(A,A) = p^2 + Fpq – F (A,a) = 2pq – 2Fpq – F (a,a) = q^2 + Fpq

Mutation in population genetics • Mutations create VaRiAtioN – Can influence the rate at which one genetic variant increases at the expense of another

• Mutation rate = µ = probability of mutation to a different allele per gene per generation; mutation rates are around 10^-5 to 10^-8 • Mutation is extremely slow at changing allele frequencies, and so cannot account for rapid genetic changes – Very few mutations are favorable for the organism and contribute to evolution – If mutation rates were very high, a species would suffer excessive genetic damage due to preponderance of harmful mutations

Migration (gene flow) • Influx of genes from other populations causing change in allelic frequency; causes gene pools from two populations to become more similar • Prevents genetic divergence between populations • Increases genetic variation within populations • I.e. I^B allele of ABO blood groups is highest in frequency in Eastern

Natural Selection • Changes allelic frequencies – Direction and magnitude depends on the intensity of the selection, dominance relations among the genotypes and the allelic frequencies – Environmental factors can also influence selection pressure

• Selection works on GENOTYPES (not alleles) • Alleles that give more fit genotypes, will be represented in higher frequencies in the following generation – Fitness (relative reproductive success of a genotype) measured from 0 (none) to 1 (maximum) – Fitness of 1 = selection of 0 – Fitness of 0 = selection of 1 – This selection coefficient (s) measures the relative intensity of selection against the genotype – I.e. People with achrondroplasia produce only about 74% as many children as those without it, so fitness of people without achrondroplasia averages 0.74, selection

Frequency of single-gene disorders Mutation – selection balance • Mutation and natural selection act as opposing forces on detrimental alleles – Mutations tend to increase frequency – Natural selection tends to decrease frequency – Equilibrium formed

Heterozygote (overdominance) advantage • Heterozygote has higher fitness than homozyogetes… • Explains why some gene disorders have much higher incidence • In some cases, natural selection works to maintain a deleterious phenotype – I.e. Cystic fibrosis, incidence much higher in Europeans due to heterozygote advantage

Quantitative genetics • A character which is continuous over a range – I.e. height, weight, color, metabolic rate etc...

• Many genes contribute to one trait = polygenic inheritance, i.e. height • Trait can be affected by environment, i.e.

Heritability of a quantitative trait • Heritability = proportion of phenotypic variance that is due to genetic variance • A trait is heritable if some of the variation can be accounted for by the genetics of the system • Narrow heritability = h^2 = measure of how heritable a trait is, using family data = (additive genetic variance ÷ phenotypic variance)

• Familial = trait shared by a family, even if they do not share the same genotype • Heritable = trait shared by people with the same genotype

Regression analysis

Regression analysis

Identifying the genes that affect a qualitative trait • Educated guess • Quantitative trait loci (QTL) mapping using DNA markers – QTL = genes that control polygenic characteristics

Autosomal Chromosomal Disorders Changes in chromosome number

Human cytogenetics • Cytogenetics = study of the genetic implications of chromosome structure and behavior – Can be performed on blood, amniotic fluid, placental tissue, bone marrow aspirates etc…

Karyotype • Karyotype = complete set of chromosmes • In a standard karyotype, chromosomes are arranged according to: – Position of the centromere – Size

Karyotype nomenclature • • • •

Normal male – 46, XY Normal female – 46, XX Female trisomy 21 – 47, XX + 21 P = short arm of the chromosome – 13p = short arm of chromosome 13

• Q = long arm of the chromosome

Chromosomal abnormalities • Responsible for a large proportion of spontaneous miscarriage and childhood disabilities • Types – Chromosome number i.e. aneuploidy, polyploidy – Mixed cells i.e. mosaicism, chemerism – Chromsome structure i.e. translocations, deletions – [Diagnostic tools i.e. FISH, chromosome paint] – Abnormalities of autosomes

Changes in chromosome number

Polyploidy • The presence of more than two genomic sets of chromosomes – – – –

Normal karyotype (46 chromosomes) Triploidy (69 chrmosomes) Tetraploidy (92 chromsomes) Pentaploidy (115 chromosomes)

• Caused by failure of meiotic division during formation of ovum or sperm or by fertilization of an ovum by two sperm • Phenotype – very severe – Polyploid individuals almost always die midpregnancy – Polyploidy is a common cause of spontaneous miscarriage – Polyploid individuals never survive beyond birth

Changes in chromosome number

Aneuploidy • Loss or gain of one or more entire chromosome (not sets of chromosomes!) – Monosomy – loss of one chromosme – Trisomy – gain of one chromosome – Tetrasomy – gain of two chromosomes

• Usually caused by non-disjuction • More than one aneuploid mutation may occur in the same individual • Phenotype – relatively mild to severe – Depends on which chromosome

Learn to draw this!

Failure of separation of a pair of homologous chromosomes

Failure of a pair of sister chromatids to separate

Monosomy One chromosome missing

• Missing a copy of one of the chromosomes • Monosomy of an autosome – severe – Usually do not survive to term (miscarriage)

• Monosomy of sex chromosomes milder • Monosomy uncovered deleterious recessive alleles • Some genes are haploinsufficiency • Small deleterious effects of incorrect

Trisomy One extra chromosome

• Trisomy of an autosome – moderate to severe – Three trisomies can survive to term • Patau syndrom (trisomy 13) • Edwards syndrome (trisomy 18) • Down syndrome (trisomy 21)

– Survival perhaps due to fewer genes on affected chromosomes • I.e. chromosome 21 is thought to contain less than 300 genes of a total 30,000-35,000 for the entire genome

• Trisomy of sex chromosomes – mild

Patau Syndrome and Edwards Syndrome • Incidence of 1 in 5000 • Increased incidence with maternal age • Phenotype – severe – Very poor prognosis (most infants die in a few days) – Long term survivors suffer severe learning difficulties and cardiac abnormalities

• Cleft palate, harelip, polydactyly • Growth failure, skull elongation

Down Syndrome – Trisomy 21 •

Caused by nondisjunction – 1 gamete will contain two copies of chromosome 21 and the other will carry none – If this gamete with two copies participates in fertilization, then we’ve got ourselves a zygote with trisomy 21

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1 in 700 80% spontaneously lost in pregnancy Increased incidence with maternal age – For some unknown reason, nondisjunction of chromosome 21 is more likely to occur in oogenesis than in spermatogenesis

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Most common and least detrimental of the autosomal trisomies Phenotype – moderate – Low IQ – Usual life expectancy (if no cardiac defect) – Most adults develop Alzheimer’s disease • APP gene affected

Clinical features of Down syndrome Can also be caused by: - Translocation - Movement of a chromosome segment to a nonhomologous chromosome or region of the same chromosome - Mosaicism - regions of tissue with different chromosome constitutions Upward sloping palpebral fissures, small ears

Atrial and ventral septal defect – results in early death in 15-20% of

Small middle phalanx of 5th finger

Clinical features of Down syndrome Can also be caused by: - Translocation - Movement of a chromosome segment to a nonhomologous chromosome or region of the same chromosome - Mosaicism - regions of tissue with different chromosome constitutions

Diagnostic tools for chromosomal disorders Fluorescent in situ hybridization (FISH)cytogenetics • Diagnostic tool combining

(karyotype) with molecular genetics • Allows us to determine the chromosomal location of a particular gene • We need to now the sequence of a gene to make a probe [1] Make a single-stranded DNA probe – designed so it binds only to the target sequence – labeled with a fluorophore [2] Hybridize the DNA probe to chromosomes in a metaphase spread [3] The area of the chromosome with the target sequence will fluoresce under a fluorescent microscope Allows us to detect the presence or absence of

A fluorophore is a chromosome-specific DNA probe that contains fluorescent dyes. It anneals with its complementary target sequence wherever it is located on a metaphase spread

Different types of FISH • Centromere probes and telomere probes – Useful for identification of the whole chromosome – Ideal for rapid diagnosis of aneuploidy syndromes

• Whole chomosomes probes (chromosome paint) – Ideal to identify translocations – Induces different color images of each chromosome pair

• Locus-specific probes to show just one

Different types of FISH

Multiplex-FISH (M-FISH) uses multiple colors - Only 5 different colour fluorophores are required to label all the chromosomes differently -A CCD camera can generate a composite image of each chromosome in a pseudocolor visualized by appropriate software - Ideal for detecting subtle chromosome rearrangements i.e.

Physical chromosomal mapping • Genetic maps reveal the relative positions of genes on a chromosome on the basis of frequencies of crossing over, but they don’t provide information that can allow us to place groups of linked genes on a particular chromosome • Due to these limitations, physical mapping, that don’t rely on rates of crossing over was developed • Physical mapping of a gene is an essential first step in its identification and cloning • Identification will enable an understanding of the developmental basis of the disease with the prospect of the possibility of therapeutic interventions • All that needs to be known is the DNA sequence

Types of physical chromosomal mapping

• Somatic cell hybridization

– Involves the fusion of cell from two different species(a process facilitated by the Sendai virus which alters plasma membranes ) – Requires gene product to be identifiable in cell culture and different in the two species – After fusion, the cell possesses two nuclei and is called a heterokaryon – The two nuclei eventually fuse, generating a hybrid cell that contains chromosomes from both cell lines – The hybrid cell eventually begins to lose chromosomes as they divide and chromosomes from one of the species are lost preferentially • I.e. in the human-mouse somatic-cell hybrid, the human chromosomes tend to be lost • The presence of “extra” human chromosomes in the mouse genome makes it possible to assign human genes to specific chromosomes

Types of physical chromosomal mapping • Radiation hybrids – Human cell cultures are irradiated with lethal levels of X-rays, which causes many chromosome breaks – This cell is fused to a rodent cell – The fragments of broken chromosomes join up with the mouse chromosomes, stay as mini chromosomes, or are lost – Panel of hybrids is produced, each carrying a small proportion of the human genome – Genes are ‘mapped’ to each hybrid cell by PCR identification

Autosomal Chromosome Disorders (structural)

Structural abnormalities in chromosomes

• Caused by breakage followed by reunion in a different configuration • OR crossing over between repetitive (duplicated) regions • Only survive meiosis if abnormal chromosome still has one centromere and two telemores • Can be balanced or unbalanced • Balanced – No gain or loss of genetic material – Chromosome complement is complete – Generally harmless unless breakpoint disrupts via a vital gene – Carriers at risk of producing offspring with an unbalanced complement

• Unbalanced – Clinically serious

Four basic types of rearrangement

Translocation • Transfer of genetic material from between two chromosomes or even the same chromosome • Can affect a phenotype – Create new linkage relations that affect gene expression, as in… genes translocated to new locations may come under the control of different regulatory sequences or other genes that affect their expression – Chromosomal breaks that bring about translocations may take place within a gene and disrupt its function

Types of translocation •

Reciprocal – Breakage of at least two chromosomes with exchange of material – Chromosome number usually remains unchanged – Indentified by high resolution banding studies or FISH – Usually does not cause clinical signs

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Non-reciprocal – Genetic material moves from one chromosome to another without any reciprocal exchange

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Robertsonian – Specific type of reciprocal translocation – Breakpoints are located at / close to centromeres of two acrocentric chromosomes – Generates a metacentric chromosome and another chromosome with two short arms – This small chromosome fails to segregate, leading to an overall reduction in chromosome number – Functionally balanced translocation, with an incidence of 1 in 1000 – 6 possible games: 1 normal, 1 balanced, 4 unbalanced

Robertsonian translocation Can also happen in chromosome 13, 15, 21 and 22

Pachytene quadrivalent in meiosis • At meiosis, incorrect segregation can generate significant chromosome imbalance – Early pregnancy loss – Infant with multiple abnormalities

• Chromosomes with translocations cannot pair properly to form normal bivalents • Cluster to form a pachytene quadrivalent

Formation of a pachytene quadrivalent

Formation of a pachytene quadrivalent

Robertsonian translocation is one cause of Down syndrome

• Robertsonian translocation can predispose to birth of babies with Down syndrome – 3 copies of long arm of chromosome 21 – 2 copies of normal chromosome 21 + 14/21 translocation

• 2/3 cases – translocation is de novo – has occurred only in the gamete that gave rise to the child • 1/3 of the cases – parent is the carrier of the translocation – Parents have high risk of having further affected children or spontaneous abortion due to other chromosomal abnormalities

Chromosome deletions – large or small • Loss of part of a chromosome resulting in monosomy for that • • • • •

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segment Phenotypic consequences depend on which genes are located in the deleted region If the deletion includes the centromere, the chromosome will not segregate in meiosis or mitosis and will usually be lost Generally deletions larger than 2% of total haploid genome will be lethal Defects due to haploinsufficiency of genes In individuals heterozygous for a deletion, the normal chromosome loops out during prophase I of meiosis. Deletions do not undergo reverse mutation. They cause recessive genes on the undeleted chromosome to be expressed and cause imbalances in the gene product Large chromosomal deletions – Karyotyping – Wolf-Hirschhorn and Cri du chat syndromes

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Microdeletions – Detectable by FISH – Cause of Prader-Willi and Angelman syndromes – Even more sensitive technique – CGH (comparative genomic

Wolf-Hirschhorn and Cri du chat • Very rare • Visible deletions of tips of chromosomes 4 and 5 respectively • Mortality rate of 34% as infants, usually due to heart defect, pneumonia, infection or seizure • Cause severe growth, statomotoric and mental retardation • ‘Cry of the cat’ • 10% mortality rate as infant, due to heart defects • Causes severe and variable mental retardation • Poor concentration between extent of deletion and phenotype

Microdeletion syndromes • Detectable with high-resolution karyotyping or FISH • May result in loss of only a few genes at adjacent loci-contiguous gene syndromes • Usually presents with characteristics facial features, aortic stenosis (narrowing of the aorta), growth retardation and impaired mental development • I.e. WAGR (Wilm’s tumour, Aniridia – absence of colored part of iris, Genitourinary abnormalities and Retardation of growth and development), DiGeorge syndrome

Chromosome insertions • Segment of one chromosome becomes inserted into another chromosome • Balanced – Inserted material has moved from elsewhere in another chromosome (deletion-insertion rearrangment) – Breakpoints can disrupt important genes – Carriers have 50% risk of producing unbalanced gametes (random chromosome segregation at meiosis will result in 50% of the gametes inheriting either the deletion or the insertion but not both)

• Unbalanced – Likely to be clinically severe

Chromosome inversions with or without the centromere • • • • • • • •

A segment of a chromosome in which the order of the genes is the reverse of the normal order No gain/loss of genetic material Chromosome must break in two places for inversion to take place Paracentric (para means ‘next to’) inversion = inversions that do not include the centromere Pericentric (peri means ‘around) inversion = inversions that include the centromere In heterozygotes for a chromosome inversion, the chromosomes form loops in prophase I of meiosis When crossing over takes place within the inverted region, nonviable gametes are usually produced, resulting in a depression in observed recombination frequencies. Balanced – Breakpoints can disrupt important genes, one part can move to a new location and destroying the function of that gene – phenotypic effects arise! – Many genes are regulated in a position-dependent manner; if their positions are altered by an inversion, they may be expressed at inappropriate times or in inappropriate tissues – Carriers of inversions have a variable risk of producing unbalanced gametes

In both cases, individual has one inverted chromosome and one normal chromosome

Paracentric inversions: crossovers in inversion can cause inviable gametes

• Crossover in the inverted segments results in recombinant chromosomes that are either acentric or dicentric • Acentric (chromosome fragment, no centromere) – Cannot attach a spindle – randomly segregate at meiosis, but cannot segregate at mitosis – Usually early pregnancy loss

• Dicentric (two centromeres) – Forms a dicentric bridge during meiosis, which breaks as the two centromeres are pulled further apart – Usually early pregnancy loss

Pericentric inversions: • • •

•

crossovers in inversion can cause inviable gametes Crossover within inversion loop results in recombinant chromosomes with complementary duplications / deletions No acentric fragments or dicentric bridges produced Recombinant chromosomes have too many copies of some genes and no copies of others; so gametes that receive recombinant chromosomes cannot produce viable progeny The larger the inversion, the more likely crossovers will occurs, leading to gametes with duplication/deletion

Ring chromosomes – rare and severe • Break occurs on each arm of a chromosome leaving two sticky ends that reunite as a ring • Often involves the loss of the chromosome ends (two deletions) • Very unstable during mitosis • Survivors have severe mental retardation

Iso-chromosomes: lose one arm and duplicate the other – usually the X • Loss of one arm of a chromosome and duplication of the other – Unbalanced duplication / deletion • Accounts for 15% of cases of Turner’s syndrome

Mixoploidy – Mosaicism A mix of two genotypes • Mosacism – presence of two or more cell types differing in genetic composition but derived from a single zygote • Usually caused by non-disjunction in an early embryonic mitotic division • Accounts for 1-2% of all cases of Down

The earlier the occurrence of disjunction, the more severe the effects

Mixoploidy – Chimerism A mix of two embryos • Presence of two different cell lines derived from fusion of two zygotes • Dispermic chimeras – Double fertilization – If zyogotes are different sexes, a true hermaphrodite forms! (XX / XY karyotype)

• Blood chimeras – Exchance of blood cells between nonidentical twins via placenta – This can happen because immune system hasn’t fully developed at that stage

Stuff I’ve missed… • In reciprocal translocation – 2:2 segregation = normal, balanced, unbalanced, unbalanced – 3:1 segregation = unbalanced, tertiary trisomy

Sex chromosome disorders

Sex chromosomes •

Presence of SRY gene on the Y chromosome causes human embryo to develop as a male – SRY gene encodes a protein that binds to DNA and causes a sharp bend in the molecule. This alteration of DNA structure affects the expression of other genes that encode testis formation – There have been rare cases of XX males with the SRY gene attached to one of the X chromosomes! (also XY females who lack the SRY gene in their Y chromosome)

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X and Y share small homologous pseudoautosomal regions required for chromosome pairing which contains some genes Genes in pseudoautosomal regions are not subject to X inactivation

X chromosome inactivation

Dosage compensation • X chromosomes have approx. 1000 genes • Dosage imbalance is corrected by dosage compensation – Otherwise, females would be producing twice as much gene product and protein concentration (which plays a critical role in development) would be affected

• Achieved by random inactivation of one of two X chromosomes in each cell early in female development • Inactive state is propagated to all progeny cells • Inactivated chromosome is called a Barr body – Darkly stained, highly condensed structure

• Most genes on the inactivated chromosomes are silenced

If a female is heterozygous for a recessive allele X-linked disorder, she will be mosaic for the disorder (may reduce severity) and can make the disorder harder to diagnose Cells that have inactivated the normal allele may have a selective advantage, and not be 50:50 i.e. Duchenne Muscular Dystrophy

Female cell w/ Barr body

Male cell w/o Barr body

The Tortoise Shell Cat

Once an X chromosome becomes inactive in a cell, it remains inactivated and is inactive in all somatic cells that descend from the cell. Thus, neighboring cells tend to have the same X chromosome inactivated, producing a patchy pattern (mosaic) for the expression of an X linked characteristic. The particular X chance that remains activefame is afor correcting a I regret not attending this lecture because I missed the rare of getting ephemeral lecturer  matter of chance Lecturer says “when you see this type of cat, you can impress people at parties by saying, if a cat looks like this , it MUST be a female”

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Some regions on the X chromosome are not inactivated Approx. 200 X chromosome genes remain active – It’s probably because of these that patients with say Turner syndrome differ from normal females even despite the dosage compensation… or something might be happening in the short period of development where all X chromosomes are active

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XIST (X inactive-specific transcript) gene required for inactivation – Mechanism not fully understood, but entails the addition of methyl groups (-CH3) to the DNA – Only one copy is expressed, and it continues to be expressed during inactivation – XIST does not encode a protein; it produced an RNA molecule that binds to the inactivated X chromosome – this prevents the attachment of other proteins that participate in transcription

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Genes in the pseudoautosomal region (also found on Y) Genes for which gene dosage is not an issue (may be genes which feminize)

Klinefelter Syndrome (47,XXY – male) • Can also be XXXY, XXXXY, XXYY – More Xs -> greater degree of mental retardation

• Arises by non-disjunction, thus the additional X chromosome is equally likely to be maternally or paternally derived • Phenotype – Individuals are slightly taller than average – Sterile – Slightly lower IQ

• Testosterone treatment can improve 2nd degree sexual characteristics

Turner Syndrome (45,X or 45,XO – female) • • • • • • •

Generally arises from meiotic non-disjunction High fetal mortality rate Infertile (unless they have the mosaic 46XX/45X – mitotic non-disjunction) Slightly lower IQ Estrogen treatment can improve 2nd degree sexual characteristics and prevent osteoporosis IVF treatment with donor cell may allow patient to conceive as they still have a normal womb Phenotype – – – – –

Immature secondary sex characteristics Shorter than normal Broad chest Folds of skin on the neck Coarction of the aorta

XXX females (47,XXX) and XYY males (47,XYY) • Caused by extra chromosome from nondisjunction • Phenotype – Occasional behavioral problems – Mild reduction in IQ

• Normal fertility as additional chromosomes does not pair during meiosis, is subsequently lost and therefore the condition is not passed on

XXX and dosage compensation • In triploidy (69,XXX), cells randomly contain 1 or 2 Barr bodies • Autosome: X ratio is abnormal and variable – lethal • In trisomy (47,XXX), cells contain 2 Barr bodies • Autosome: X ratio is normal – phenotype is very mild

Trinucleotide Repeat Disease / Disorder • Changes to a gene i.e. missense mutations are stably inherited • Structural alterations to the chromosome are sometimes stably inherited • Trinucleotide repeat expansions are NOT stably inherited – Several genes are known to contain regions of trinucleotide repeats. The number of repeats varies from person to person in the general population, but within the normal range, these repeats are stably repeated. When the number of repeats is increased beyond the normal range, this region becomes unstable with a tendency to increase in size when transmitted to offspring – In some conditions, there’s a clear distinction between normal and pathological alleles. In others, the expanded alleles may act either as premutations (no phenotypic effects) or as full pathological mutations – Premutations can -> Mutations as they can increase in

Fragile X syndrome A trinucleotide repeat disorder

• Caused by a mutation in a single gene on the X chromosome • Phenotype – High forehead, large jaw, learning difficulties

• Female carriers sometimes show some facial features • A fragile X contains a ‘fragile site’ at Xq27-Xq28 that tends to break in cultured cells that are starved for DNA precursors, such as nucleotides • Mutation causes trinucleotide expansion of CGG nucleotide repeat in

Linkage, Recombination and Genetic Mapping

Crossing over produces recombinant chromosomes

• Linkage can be altered during gamete formation by crossing over • New combination of alleles generated – genetic recombination • Same allele combination as parents (of the F1) = parental gametes • New combination of alleles = recombinant (or non-parental) gametes – Involves a physical exchange between homologous chromosomes

• If two genes lie close together on the same chromosome, they do not assort independently, therefore independent assortment ratios will not be observed

Recall

Crossing over takes place in meiosis and is responsible for recombination – it breaks up associations of genes imposed by linkage

Linkage between genes causes them to be inherited together and reduces recombination; crossing-over breaks up the association of such genes. In a testcross for two linked genes, each crossover produces two recombinant gametes and two non-recombinant gametes. The frequency of recombinant gametes is half the frequency of the crossing over, and the maximum frequency of recombinant gametes is 50%

Crossing over produces half non-recombinant games and half recombinant gametes

With linked genes and some crossing over, we observe inheritance patterns that deviate from Mendel’s 2nd law

• Was Mendel wrong? Nah – Recombination is the sorting of alleles into new combinations. – InTERchromosomal recombination, produced by independent assortment, is the sorting of alleles on different chromosomes into new combinations – InTRAchromosomal recombination, produced by crossing over, is the sorting of alleles on the same chromosome into

Physical basis of recombination Creighton and McClintock’s study on corn Studied the inheritance of two traits in corn determined by genes on chromosome 9: at one locus, a dominant allele (C) produced colored kernels, recessive alleles (c) produced colorless kernels Wx – starchy kernels ; wx – waxy kernels

Homozygous for colorless and heterozygous for waxy

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Gene mapping with Recombination Frequencies

Physical distance between genes on a chromosome are related to the rates of recombination

– The further apart the genes are, the more likely they are to crossover

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Genetic maps = chromosome maps calculated by using recombination frequencies – Distances on genetics maps are measured in centimorgans (cM) or map units (m.u. 1 cM = 1 m.u.)

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Genetic distances measured with recombination rates are approximately additive – i.e. if the distance from gene A to gene B is 5 m.u. and the distance from gene B to gene C is 10 m.u., the distance from gene A to gene C is 15 m.u. (= recombination frequency of 15%) or

Double crossovers make long distances inaccurate

• A double crossover arises when two separate crossover events take place between the same two loci – Single crossover just switches the alleles on the homologous chromosome, producing combinations of alleles that were not present on the original parental chromosomes

• The further apart the two loci are, the fewer the recombinants observed compared with the number expected – Genes very far apart on the same chromosome act as though they’re on separate chromosomes, hence appear to segregate independently

• The second crossover between the same two genes reverses the effects of the first, restoring the original parental combination of alleles – This is why recombination frequencies will be underestimated – Makes it harder to distinguish it between progeny

Why generate genetic maps • Allows us to determine whether human mutations affect different genes or not • We can identify genes using their map position – i.e. Cystic fibrosis gene

• It can enhance our ability to predict inheritance patters • Generating a high res. Genetic map is the first step in sequencing a genome

Gene mapping in humans • • • • • •

Humans have 1-2 recombination events per pair of chromosomes in meiosis I, total approx. 40 1 cM approx. = 1000kb Relationship between map units and physical distance is not entirely linear Recombination events are rare close to centromeres ; occurs more often in female meiosis Genetic mapping in human genes hampered by the inability to perform desired crosses and the small number of progeny in most human families Geneticists are restricted to analyses of pedigrees – Have to combine data from many families and calculate the odds of linkage (LOD score – calculation of the most likely degree degree of linkage)

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Make use of molecular markers, mostly consisting of RFLPs (restriction fragment length polymorphisms) – Cosegregation of two or more markers is studied and map distances are based on the rates of recombination between the

Genetic linkage and mapping

Trihybrid crosses • A more efficient mapping technique than dihybrid crosses – Numerous dihybrid crosses must be carried out to establish the order of genes and because double crossovers are missed

• For each locus, two types of progeny will be produced: progeny that are heterozygous, displaying the dominant trait, and progeny that are homozygous, displaying the recessive trait – With two classes of progeny possible for each of the three loci, there will be 2^3 = 8 classes of phenotypes possible

• In test-cross progeny, phenotypes reflect genotypes of gametes of F1 parent (two most numerous phenotypes will be that of the parents’)

3 types of crossover can take place among three linked loci

NonRecombinant Recombinant Recombinant NonRecombinant

Determining gene order • Method 1 – Consider two genes at a time and determine the map distance between them – Divide number of recombinants from all crossovers (smaller numbers) by the total number of progeny tested – i.e. 10% recombinant frequency corresponds to map distance of 10 m.u. (cM) – Map distance between the two outer loci may be less than the sum of the two internal regions, this is due to double crossovers – Double the map distance for double crossovers (usually smallest numbers) to resolve this

Determining gene order •

Method 2 – First determine which progeny are the non-recombinants, they will be the two most numerous classes of progeny – Identify the double crossover progeny, almost always the leastnumerous phenotypes (because the probability of a double crossover is always less than the probability of a single crossover) – The locus with differing gene in the double crossover progeny is the locus in the middle – i.e. our double crossover recombinants have the following: (st+ e+ ss) and (st e ss+) Three possible gene orders and the types of progeny produces by the double crossover are:

Determining gene order •

Method 2 – First determine which progeny are the non-recombinants, they will be the two most numerous classes of progeny – Identify the double crossover progeny, almost always the leastnumerous phenotypes (because the probability of a double crossover is always less than the probability of a single crossover) – The locus with differing gene in the double crossover progeny is the locus in the middle – i.e. our double crossover recombinants have the following: (st+ e+ ss) and (st e ss+) Three possible gene orders and the types of progeny produces by the double crossover are:

Probability of double crossover = (st-ss recombination frequency) x (ss-e recombination frequency) Here’s our gene order!

Interference messes with double recombination • Interference: degree to which one crossover interferes with additional crossovers in the same region – 1 minus coefficient of coincidence – i.e. a value of 0.4 tells us that 40% of the double crossover progeny expected will not be observed because of interference. Value of 1 would indicate that no double crossover progeny are observed – A value of say -0.3 means that more doublecrossover progeny appear than expected, which happens when a crossover increases the probability of another crossover occurring nearby (coefficient of coincidence > 1) – negative interference is rare

• Coefficient of coincidence: ratio of observed double crossovers to expected double crossovers

Q and A 2 from Griffiths

Just draw a Punnett square and you’ll see…

Chromosomal inversions affect recombination and fertility • Heterozygote carriers of inversions have reduced fertility due to effect of crossing over within the inversions – Homozygotes are fertile

• Crossing over within a pericentric (exchange over centromere) inversion – duplications and deletions • Crossing over within a paracentric inversion – dicentric and acentric chromosomes • For genes within the inversions, no recombinants will ever be seen in the progeny • There is no looping in meiosis, crossing over occurs normally and all gametes carry the inverted chromosme

Molecular Mapping

Molecular markers can be used for linkage mapping • Facilitates examination of variations in DNA itself (even if it is not a gene) • Genetic mapping of the loci of genes using classical mapping requires alleles that give phenotypic differences

Single Nucleotide Polymorphisms (SNPs)

• Simples variant of a DNA sequence is a one base pair difference (SNP) – Polymorphic DNA marker

• Sometimes it will create a difference in ability of a restriction enzyme to cut the surrounding sequence – Even if only one base is different, the sequence is not cut

• Good for mapping because an SNP occurs about once every 100bp in a human genome – Can generate high resolution genetic maps

• When a SNP is physically close to a disease-causing locus, it will tend to be inherited along with the disease-causing allele. Thus the SNP marks the location of a genetic locus that causes the disease • A SNP can also be useful for determining family relationships—most SNPs are unique within a population, having arisen only once by mutation. Thus the presence of the same SNP in two persons often indicates that they have a common ancestor

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Restriction Fragment Length Polymorphisms (RFLPs) RFLPs are polymorphisms in the patterns of

fragments produced when DNA molecules are cut with the same restriction enzyme Provide a large number of genetic markers that can be used in mapping If DNA from two persons is cut with the same restriction enzyme and different patterns of fragments are produced, these persons must posses differences in their DNA sequence RFLPs are detected restriction enzymes (RE) digestion followed by Southern Blot OR by polymerase chain reaction (PCR) amplification followed by RE digestion and agarose gel electrophoresis If the disease and the RFLP are inherited together, they must be physically linked

Recall

(a) Restriction fragment length polymorphisms can be used to detect linkage

In this pedigree, the father and half of the children are affected (red circles and squares) with Huntington disease (autosomal dominant disease). The father is heterozygous (Hh) and will pass the chromosome with the Huntington gene to approximately half of his offspring. The father is also heterozygous for RFLP alleles A and C; each child receives one of these two alleles from the father. The mother is homozygous for RFLP allele B, so all children receive the B allele from her (a) In this case, there is no correspondence between the inheritance of the RFLP allele and inheritance of the disease—children with the disease are just as likely to carry the A allele as they are the C allele. Thus the disease gene and RFLP alleles segregate independently and are not closely linked (b) In this case, there is a close correspondence between the inheritance of the RFLP alleles and the presence of the disease—every child who inherits the C allele from

Simple Sequence Length Polymorphisms (SSLPs) • A variable number of copies of a short sequences occurring in tandem repeats • Used commonly in DNA fingerprinting to detect genetic differences among people • Also called Mini- and Micro- satellite markers – Mini-satellite markers are based on variation in the number of tandem repeats of a repeating unit from 15 – 100 nucleotides long – Micro-satellite markers based on even simpler sequence, usually dinucleotide repeats (VNTR)

• Detected by gel electrophoresis followed by Southern hybridization, using the repeat as a probe OR PCR, using sequences on each side of the repeat as primers followed by gel

Human pedigree showing segregation of VNTR alleles. Six alleles (1–6) are present in the pedigree, but any one person can have only one allele (if homozygous) or two alleles (if heterozygous)

Why are SSLPs good for mapping? • Satellites scattered throughout the genomes, occurs once every 10,000bp in humans (so roughly 3 million SNPs in the human genome) • Can often identify both alleles of an individual • Ideal for DNA profiling • Downside is that there are not as many SSLPs as SNPs

How DNA markers are used in human gene mapping • Can test of DNA markers are linked to each other • Can generate a genetic map – the human one has 100s of 1000s of DNA markers

• Can use these markers to map human disease genes – using multiple pedigrees to gain sufficient information • I.e. Huntingtons Disease – Mapped using molecular markers, found to be closely linked to a marker on 4p. 10 years later the gene was cloned

How are DNA markers made / discovered? • Detection is by trial and error • Process: – Isolate genomic DNA from many members of a population and run on a Southern blot – Probe with many different random probes (i.e. from a genomic library); by chance, some will detect a polymorphism – OR, try many different random PCR primers; by chance, some will detect a SNP or satellite polymorphism

Using molecular mapping to clone human genes • If a gene is known only by its mutant phenotype, the next stage is to identify the gene: – In chromosome walking, a gene is first mapped in relation to a previously cloned gene. A probe made from one end of the cloned gene is used to find an overlapping clone, which is then used to find another overlapping clone. In this way, it is possible to walk down the chromosome to the gene of interest – The candidate is the gene of interest if it is • Never separated from the disease phenotype by crossing over • Expressed in tissues affected

Applications of genetic mapping • High res. Genetic map of DNA markers is the essential starting point for a genome sequencing project • A gene is defined by its map position which can be used to determine whether a disorder is caused by the one gene, or by different genes in different pedigrees

DNA profiling

DNA fingerprinting • Involves profiling the individual’s genetic information from DNA test results of genetic markers, placed in highly variable regions of the genome, in order to locate characteristics unique to the individual • Most commonly used in forensics and criminal investigation – PCR can be used to amplify the DNA if there’s too little for testing – PCR is extremely sensitive and will amplify intact DNA; degraded DNA will not amplify

• Identical twins are monozygotic and fraternal twins are dizygotic • Possible because: – DNA sequence is stable and sequence remains the same – All progeny have the same DNA (unless mutations

DNA fingerprinting

Basically… each DNA sample is cut with one or more restriction enzymes, and the resulting DNA fragments are separated by gel electrophoresis. The fragments in the gel are denatured and transferred to nitrocellulose paper by Southern blotting. One or more radioactive probes is then hybridized to the nitrocellulose and detected by autoradiography. In a crime scene, the patterns of bands produced by DNA from the sample is then compared with patterns produced by DNA from the suspects

Genetic diversity in humans • 3 billion base pairs in the human genome – Individuals are 99.9% identical at the DNA sequence level

• Differences occur in both coding (some which can be observed at the phenotypic level) and non-coding regions (require specialized techniques to detect them)

DNA Polymorphisms

• Difference in DNA sequence at the same locus – Variation occurs too frequently to be labeled as a mutation – >1% of the population – When studied using the Southern blot, we may see restriction fragments complementary to a probe that differ in size among individuals. These differences arise from differences in the location of restriction sites along the DNA

• Single nucleotide polymorphisms (SNPs) • Mini- and Micro-satellites • Insertions and deletions

DNA Polymorphisms • •

Simple sequence length polymorphisms (SSLPs) Microsatellite (CA repeats) – 2-10 bp repeats – Simple sequence repeats – Difference in number of microsatellites at any one site between individuals in highly polymorphic and these have shown to be inherited in a Mendelian codominant manner

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Minisatellite – 10 – 100 bp repeats – Clustered repeats of specific DNA sequences – Variable Number of Tandem Repeats (VNTR) • This same repeat of around 10bp is usually in many places in the genome • Highly polymorphic compared to RFLPs, which is due to the presence of variable number of tandem repeats over a short DNA sequence that has been inherited in a Mendelian fasion

• •

Many different alleles and lots of polymorphisms Co-dominant

Problems with DNA fingerprinting (multi locus) using minisatellites

• Southern blot requires large amounts of DNA • DNA must be intact • Cannot tell which pairs of bands represent alleles

Potential sources of error • False inclusion – Relatives likely to share alleles – Some alleles more frequent in specific populations

• False exclusion – Contamination or mixed source of DNA – Technical problem such as ‘allele dropout’

Multilocus Single locus Cannot determine which allele comes from Partially degraded DNA can be used which locus

Polymorphisms in mitochondrial DNA and Y chromosome DNA

• Y chromosome DNA

– Passes to male children from father only – No recombination – Polymorphisms can be followed through many generations

• Mitochondrial DNA – Passed to children from mother only – No recombination – Polymorphisms can be followed through many generations

Developmental Genetics

Content in this lecture was straightforward, the notes are mostly copied off lecture notes

Dysmorphology

Study of congenital birth defects that alter the shape of one or more body parts

• Malformation, primary structural defect of an organ or part of an organ. Their presence suggests that the early development of a particular tissue or organ has been arrested or misdirected • Intrinsic genetic abnormality • Common examples of malformations include congenital heart disease, cleft lip and neural tube defects (i.e. anencephaly) • Most malformations involving only a single organ show multi-factorial inheritance, implying an interaction of many genes with environmental factors

Dysmorphology

Study of congenital birth defects that alter the shape of one or more body parts

• Deformation, defect which results from an abnormal mechanical force which distorts an otherwise normal structure, i.e. dislocation of the hip which can be caused by lack of amniotic fluid or intra-uterine crowding due to twinning or a structurally abnormal uterus • Usually occur late in pregnancy • Generally resolved soon after birth

Dysmorphology

Study of congenital birth defects that alter the shape of one or more body parts

• Disruptions, abnormal structure of an organ or tissue as a result of external factors (such as ischemia – restriction of blood supply, infection and trauma) disturbing the normal developmental process • Not genetic, although occasionally, genetic factors can predispose to disruptive events – I.e. small proportion of amniotic bands are caused by an underlying, genetically determined defect in collagen which weakens the amnion, making it more liable to tear or rupture spontaneously

Major causes of malformations • 25% autosomal trisomies • 20% single gene mutation – i.e. achondroplasia

• 50% multifactorial – I.e. cleft lip, congenital heart defects

• 5% environmental teratogens – Drugs, infections, chemicals, radiation

Plieotropy • Single underlying cause results in abnormalities in more than one system of the body • A syndrome – multiple abnormalities in parallel – Branchial arch defects and renal defects

• A sequence – only one organ system is affected, with secondary pleiotropic effects – I.e. Robin sequence, where the mutant collagen causes primary defect in jaw

Developmental Genetics • Study of the cellular processes – Division – Migration – Differentiation – Apoptosis

Mosaic development • Later in development, some cells have already developed distinct fates – the embryo only appears to be homogenous • Loss of part an embryo after this point could lead to failure of development • Conjoined twins – if cleavage occurs after progression from regulative mosaic development, two fetuses share body structures / organs

Fate, specification and determination • Differentiation – process where cells undergo a series of discrete steps (acquiring distinct functions / attributes) until they reach a final fate – Stepwise acquisition of a stable cellular phenotype of gene expression

• Specification – cell acquires specific characteristics, but it can still be influenced by environmental cues such as signaling molecules, neighboring cells, positional information • Determination – cell has irreversibly committed to acquire final traits / attributes – i.e. nerve cells making synaptic proteins, RBCs making hemoglobin

Homeobox (HOX) gene system Plays a crucial role in early morphogenesis

• These genes were shown to be transcription factors that determine segment identity and developmental fate along an axis • In drosophila, mutations in these genes have resulted in major structural abnormalities, such as development of a leg instead of an antenna • 38 HOX genes in mammals; most mutations are lethal – I.e. mutation in HOXD13 will result in synpolydactylyl, resulting in an extra digit

• Order of the HOX gene parallels: – Position in the embryo in which that gene is expressed – The time in development when it is expressed

Antennapedia, left is the normal fruit fly, right is the antennapedia mutant

Pattern – positional cues •

Cell-to-cell communication conveys positional information – Can be direct or via short/long-range signals – In a concentration dependent manner, Hedgehog proteins play a major role in the development of the ventral neural tube with loss-of-function mutations resulting in a serious and often lethal malformations

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Signaling cells produce ligands which bind to the cell surface receptors leading to transmission of a signal in the receiving cell – i.e. fibroblast growth factors (receptors) FGF(R) – 23 known human FGFs – mutation causes diseases such as achondroplasia

Arrows indicate the location of mutations that lead to achondroplasia

Signaling Centers • Diffusible ligands / signals produced at signaling centers act as sources of positional information • Cells can detect how far from the signal source they are and which direction the signal source lies

Immunogenetics

Content in this lecture was straightforward, the notes are just copied of lecture notes

The immune system • Protection mechanisms: – Physical barriers • skin, mucous membranes, respiratory cilia

– Immediate general response – innate immune response • Inflammation, phagocytes, cytokines

– Specific acquired defense that identifies selfantigens from foreign antigens

• Antigens – substances that elicit immune reaction – proteins or parts of proteins often on the surface of bacteria, viruses, pollens etc… • Reacts against a foreign substance, destroying it • Remembers a foreign substance and responds more strongly to a later exposure

• • • •

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Acquired immune response

Third line of defense Slow to respond as it must be stimulated Macrophages – large lymphocytes that engulf and digest foreign substances B-cells – produce antibody proteins and are activated by T-cells in the humoral immune response – Antibodies are proteins that circulate in the blood and other body fluids, binding to specific antigens and marking them for destruction by phagocytic cells T-cells – After a pathogen such as a virus has infected a host cell, some viral antigens appear on the cell surface. Proteins, called T-cell receptors, on the surfaces of T cells bind to these antigens and mark the infected cell for destruction. T-cell receptors must simultaneously bind a foreign antigen and a self-antigen called a major histocompatibility complex (MHC) antigen on the cell surface. Not all T cells attack cells having foreign antigens; some help regulate immune responses, providing communication among different components of the immune system – Killer T-cells – do not make antibodies, but destroy antigen carriers by direct cell-to-cell contact

Antibodies IgM IgD IgE IgG IgA

• Immunoglobulins (Ig), consisting of four polypeptide chains – two identical heavy chains liked by disulfide bonds + two identical light chains – Each chain has a constant region and a variable region (where there are variations in amino acid sequence) – The variable regions of both heavy and light chains make up the antigen-binding region and specify the type of antigen that the antibody can bind

Variable regions of Antibodies • Amino acids in the variable regions of different amino acids differ from each other – Variation is restricted to a few subregions called the hypervariable regions – These regions form the antigen binding cavity – Different antibodies are characterized by their antigen-binding characteristics

Generation of antibody diversity

Immune system capable of making 10^15 antibody molecules, yet human genome only contains 3 x 10^9 base pairs, how’s this possible?

• Antibody GENES are composed of segments. There are a number of copies of each type of segment, each differing slightly from the others. In the maturation of a lymphocyte, the segments are joined to create an immunoglobulin gene. The particular copy of each segment used is random and, because there are multiple copies of each type, there are many possible combinations of the segments. A limited number of segments can therefore encode a huge diversity of antibodies.

Generation of antibody diversity

Immune system capable of making 10^15 antibody molecules, yet human genome only contains 3 x 10^9 base pairs, how’s this possible?

• Somatic recombination – Permanent change at DNA level – B and T cells use alternate RNA splicing and somatic recombination to generate diversity – Diversity generated by changing the pieces of these genes – Heavy chains have 4 sections, V, D, J and C regions – Kappa and lambda light chains have 3 sections V, J and C

Somatic recombination producing variation in the heavy chain

Immature B-cells Clonal theory • Each B cell makes one type of antibody • Contains a heavy chain and either a kappa or lambda chain • C region of the heavy region determine antibody class • Variable regions of antibody determine antigen specificity… • Each cell has different arrangement of V-(D)-J

Preprogrammed B-cells • If antigen is present: – It binds to the antibody and is taken up by cells – Antigen is processed and peptide displayed on cell surface (with MHC protein) – Altered MHC is recognized by T cells • The MHC genes encode proteins that provide identity to the cells of each individual organism. To bring about an immune response, a T-cell receptor must simultaneously bind both a histocompatibility (self) antigen and a specific foreign antigen

– T cells stimulates B cell to differentiate causing clonal expansion of the antibody secreting cell

Antibody diversity by class switching • Class switching generates the different classes of antibody, all with the same variable domains as the original antibody generated by V(D)J recombination, but with distinct constant domains in their heavy chains • Naïve mature B-cells produce both IgM and IgD with identical antigen binding regions

Classes of antibody Defined by heavy chain C region

• IgA – Milk, saliva, urine – protects against pathogens at point of entry into cell

• IgD - on B cells in blood – stimulates B cells to make other types of antibodies

• IgE - in secretion with IgA and in mast cells – cause mast cells to secrete allergy mediators

• IgG - blood plasma (can pass to fetus) – especially involved in secondary immune response

Antibody diversity by somatic hypermutation • V-(D)-J recombination (+ alternate splicing) creates initial antibody diversity allowing recognition of various antigens • Base substitutions occur at high rate in V-(D)-J DNA during differentiation of activated B cells to plasma cells • Especially high mutation rate in hypervariable regions • Process leads to variant antibodies with an enhanced ability to recognize and bind a specific foreign antigen

T cell response • Involved in cell-mediated responses • Important for combating viral infections • Allows recognition of self from nonself • T cell receptors – Structurally similar to immunoglobulins – Like the genes that encode antibodies, the genes for the T-cellreceptor chains consist of segments that undergo somatic recombination, generating an enormous diversity of antigenbinding sites – Don’t undergo somatic hypermuation

Major Histocompatibility Complex (MHC) • Important for recognizing self from non-self • Required for presenting antigen on B cells • Required for stimulating T cells • Important antigens in transplantation – Rejection of donor tissue due to non-self antigens