MTC Block I

8/13/2019 MTC Block I

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Ch 1: Intro

Describe early pre-biotic environment and its eventual development into the biotic world

Atmosphere rich in CH4, ammonia, water, which in a primordial soup yielded carboxylic acids, nucleic acidbases, amino acids, and sugars

Started from RNA that both self-replicated and catalyzed protein synthesis, including the enzyme neededto transcribe RNADNA (reverse transcription), which became the predominant template for RNA

synthesis

Eubacteria became modern bacteria, but purple (aerobic) bacteria were swallowed by early eukaryotesand later became mitochondria

Apply biochemical and genetic models of disease to explain inborn errors of metabolism annotation

Studied Alkaptonuria and noted it to be recessively inherited (typical of siblings but not parents)o This disease has dark spots in urine and sun exposed places, as well as joints, cardiac valveso This is because of oxidized homogentisic acid

Missing homogentisic acid oxidase which converts to maleylacetoacetic acidDiscuss relevance of contributions by Osler and Garrod in history of medicine, genetics, and modern medical

curricula

Osler = art of medicine, physician-educator; Garrod = science of medicine, physician-scientist Garrod, through inborn diseases of metabolism, concluded that it is the biochemical diversity that we

should look for, whereas Osler relied on anatomy to verify his clinical judgement

o Concept of diathesis: that individuals may inherit predisposition to diseases

Ch 2: Gene Mapping, Genomics, Bioinformatics, Biodiversity

History of gene mapping and how it led to human genome project

For every Mendelian trait, there should be a locus within the chromosome where the entity of inheritance(gene) exists

This study was possible with linkage mapping and cytogenetic techniques, which allowed for gene maping With DNA cloning, it was possible to track specific chromosomal regions and gene maps were made Wave of new technologies created HGP, which finished early due to Celera

o Used DNA sequencing and took advantage of its reduction in costsPrinicples of bioinformatics and computational approaches to gene discovery and genome annotation

Provides identification and location of genes, establishment of domainso Requires decoding genome, identifying sequences defining beginning and end of genes and

between coding vs non-coding regions

Genome vs transcriptome vs proteome vs metabolome

Genome: total set of DNA molecules (for humans, this is 24 chromosomes and mitochondrialchromosome)

o We can compare our genome to other organisms for evolutionary studies Transcriptome: total set of RNA transcripts and their modifications within a cell type

o This helps determine how biological systems malfunction (like studying RNA expression duringcancer states to learn how we adapt)

Proteome: total set of protein products and their modifications within a cell typeo This helps us understand protein-protein interactions, structure of functional domains of proteins,

and protein expression in different states


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Metabolome: set of molecules that occur within a cell type, which provides description of metabolic stateof a cell

Nature of and application for various websites/databases

OMIM: useful and comprehensive, cumulative over time, extensive GeneTests & GeneReviews: helps find labs to test for genetic disorders; counseling guidelines HGMD: UK based, compiles all mutations reported in h umans UCSC Genome Browser: seen in class

Reasons why better genetic understanding of biodiversity may help medicine and public health in the future

Comparative genomic studies help identify genes uniquely human, which help us understandsusceptibilities in health in disease (example is dyslexia and behavioral traits)

Can use genetic approach to cure disease, such as bacterial antibiotic resistance Nutritional approach to genomics of our food Pharmaceuticals like hormone production by bacteria Metagenomics from our gut

Ch 3: Human Genome StructureBasic organization of human genome, define genome architecture

Haploid is 3,000 Mb, diploid is double thato Mitochondrial DNA is 16,500 base pairs

2% codes for proteins, the other 98%:o Is dispersed between introns (in between a gene, or intergenic) and in between genes (intragenic)o Is structural, making up telomeres or centromereso Codes for ribosomes or transfer RNAo 55% of the genome is repetitive non-coding DNA

See Satellite DNA below!!! Overall, 55% of DNA is repetitive, and most of this is non-coding

o 45% is present only once About 20,000 coding genes, most of which fall into the 45% of only present once 3-8% of non-coding DNA is highly conserved among many species, reason unclear

Explain basic structure of gene and identify its common elements

DNA reads 53, so the promoter will be at the 5 endo At the 3 end, there will be a poly-A tail

Has introns, which are typically larger than exons (avg 3000 bp for intron, 145 exon), average mRNA is2600 bp from 27,000 bp transcription unit

o There are untranslated regions on both ends of the gene 3 is avg 300 bp, 5 is avg 770 bp

Describe overlapping genes, nested genes, pseudogenes

Overlapping: can be in the same or opposite direction Nested: small genes nested in antisense strand of bigger gene of sense strand Pseudogenes: sequences that closely resemble known genes but are not functional due to lacking

transcriptional initiation elements (like promoters, etc). Denoted by psi.

o Considered by-products of evolutiono Found within gene clusters


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Modular construction of genes based on sequence motifs, how understanding sequence structure leads to

development of bioinformatics tools for analyzing human genome

Thematic nucleotide sequences are motifs, and provide specific functional/structural properties(conserved through evolution)

o For example, ATP-binding region motifs or transmembrane region sequence motifs (which codefor hydrophobic AAs like Phenylalanine, Leucine, or Isoleucine), or Zinc finger, leucine zipper, or

helix-loop-helix

Different motifs linked by connector sequences results in proteins with specific functional/structuraldomains

o By identifying all of a genes motifs, we should deduce the structure, function, and specificity ofthe proteins. This also opens up the possibility of adding function to a protein too!

Adjacent Cs and Gs (CpG-rich) are commonly found in promoter regions of genes that are constitutively(ALWAYS) expressed

o These are technically not coding sequences despite being key elementsExamples of homologous, paralogous, and orthologous genes, gene clusters, and gene families

Most genes are single copy per haploid, but some exist in multiple identical copies, like -globino Identical copies of the same gene are usually clustered in the same region (rarely on different

chromosome) MANY genes have SIMILAR (but not identical) genes: two or more similar genes are homologous or

homologs (and proteins may share similar functions). Two types of homologous genes:

o Paralogs: from the same species and have overlapping functions generally, but not identical Paralogous genes form a gene family(eg histone gene family, high degree of homology) Some gene families may not share homology in sequence, but in amino acid domains A gene superfamilyis paralagous genes that have weak homology but are generally

functionallyrelated (eg immunoglobulin genes)

If paralogous genes are in same chromosomal region, they are a gene clustero Orthologs: from different species (indicative of common ancestor), have identical functions

generally but this depends on how far the species is

There are 4 HOX gene clusters, present on 4 difference chromosomes. Gene family has a highly conservedsequence domain in all of its members. All genes in cluster are part of the same family, but homology

between paralogs at same position of each cluster is higher than that between these paralogs and other

members (so HoxA1 is more homologous with HoxB1 than HoxA1 is to HoxA2)

GC content determines gene-rich or gene-poor Gene desert is a gene with no other gene within 1 Mb (3% of genome)

o These contain big genes (0.5 Mb)transcription of these is slow and will not finish in time in acell that rapidly divides, so theyre usually only active in cells like neurons (non-dividing)

o These areas also house some genes that play critical roles in development and require regulationTypes of satellite and dispersed DNA sequences in human genome

Types of non-coding repetitive DNA (55% of genome):o Tandem repeats (satellite DNA) (10% of genome): these are contiguous with each other, many

repeating units, and can be direct or inverted

Alpha satellites: tandem array with repeat of 171 bp, has role in centromeric function byproviding binding sites for centromere-binding proteins


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Mini-satellites: Tandem arrays of 14-500 bp scattered throughout genome with totallength up to 20 kb. Near sub-telomeric regions. Can be further divided into GC or non-GC-

rich. Useful in fingerprinting and gene mapping

Microsatellites (3% of genome): arrays of 1-5 bp (2-4 common) length up to 300 bp,example of CA repeats

o Non-tandem (satellite) are dispersedrepeats are repeated at two or more locations (45% ofgenome!)

LINEs (long interspersed elements): 900bp avg (can be up to 7kb) units all over the place,with about 10^6 copies (21% of genome). Typically there are 2 ORFs, and one typicallycodes for reverse transcriptase???

SINEs (short interspersed elements): 90-500bp, common is the Alu repeat, ~300bp, isrecognition site for restriction enzyme AluI. Also 10^6 copies (13% of genome)

LTR Retroposons: repetitive sequences related to retroviruses although they lack genecoding for envelope protein. 5e5 copies (8% of genome)

DNA Transposons: Non-retrovirus but viral-like sequences (3e5 copies), 3% of genome Segmental Duplications: large size of repeat unit (>1kb) often reaches a few hundred kb

o May contain coding, non-coding, or repetitive DNA sequences (5% of genome)o Common in primates and has been occurring a long long time (highly conserved)o Can be on same chromosome or differento Concentrated in pericentromeric and subtelomeric regionso Can lead to duplication/deletion syndromes when recombination occurs between these

duplicated regions on same chromosome

Genetic polymorphism and explain classifications of DNA polymorphism

Presence of two or more alternative variants in phenotype of alleles, with frequency which cannot bemaintained by mutations alone (if something happens 1% or less of the time, it is a polymorphism)

o Polymorphism = change with no significant alteration in gene functiono Mutation = deleterious change in function of a coding sequence

Classificationo SNP: one base pair (estimated 1 SNP per 1000 bps or 3 million SNPs/haploid)

Can result in creation or destruction of a restriction enzyme recognition site and detectedby Southern analysis (and is known as a RFLP)

o Indel(insertion-deletion): presence or absence of a short segment of DNAo Mini-satellites/microsatellite polymorphisms: called short tandem repeat polymorphisms (STRPs)

Ex: (CA)no VNTRare many clustered mini-satellites (variable number of tandem repeats), have high variationo CNP or CNV (copy number polymorphism/variation) is variation in number of copies a

segmentally-duplicated chromosomal region has, range from 200bp to 2 Mb

How DNA polymorphisms are inherited and how they can be used as genetic markers in family studies

Most polymorphisms in mitochondrial DNA are mutations because mDNA is mostly coding Can be thought of as alleles, like Mother has (CA)7/(CA)7and Dad has (CA)7/(CA)10 Can be tracked through a family Linkage mapping is done this way, so for a phenotype, if the affected share the same allele at the

polymorphic marker locus, there is a high chance the phenotype allele is in that region

o It also allows us to see what parts of chromosomes came from a specific parent

Ch 4: DNA Cloning

Five general steps in DNA cloning


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(1) Restriction/Digestion: cutting DNA at precise locations that leaves specific joinable ends Done with restriction enzymes

(2) Vector: selecting small molecule of DNA capable of self-replication in a host(3) Ligation: joining the DNA fragment to be cloned, to, the vector

Done with DNA ligase(4) Transformation: transfer this joined DNA construct into the host organism(5) Selection: selecting for the host organism that has taken up the DNA construct Vector must contain:

o An origin (so replication takes place)o Restriction enzyme sites where DNA fragment can be added ino A gene critical to the survival of the host organism

Plasmid is a self-replicating structure in bacteriao Common one is pBR322, which is circular, 4361 bp, has a restriction enzyme site (EcoRI), ABx

resistance gene, and one origin of replication

How DNA fragment can be cloned into a plasmid vector

See above steps or the video in the syllabusHow genomic and cDNA libraries are constructed

Genomic library: total genomic DNA placed into a plasmid? cDNA library: mRNA isolated from a tissue, and a cDNA is created (using reverse transcriptase and DNA

pol), then the cDNA is cloned into vector molecules

o In this case, the cDNA will be cell-specific (brain vs skin is different)o Also remember there are no introns here

Ch 5: SSHuman Genome Evolution

Explain recombination and difference between non-homologous and homologous recombination

DNA sequences cleaved and rejoined in a way that new sequence combinations are formed, can behomologous or non-homologous

Homologous: recombination between DNA molecules that have sequence homology. Why?o DNA damage repair: double-stranded damage is repaired by transfer of a strand from the

homologous normal chromosome by forming Holliday structures, using various enzymes

o Meiotic recombination: double strand breaks are created, and chromosomal segments areexchanged between maternal and paternal homologs (aka crossing over)

Strand break created Ends digested, leaving single-stranded 3 ends Strand invasion when it finds a homolog 3 end ofinvading strand extended by DNA pol Ends ligated together and phosphodiester bonds cleaved and ends ligated

Non-homologous recombination doesnt require homologous sequences, andcan be either:

o Simple end-to-end rejoining of DNA fragments with broken ends by forming synapse wherecomplex with DNA-dependent protein kinase carries out reactions. Ligase then joins the ends

after processing by nucleases and this is associated with deleting a few bases. OR it can be:

o Site-specific recombination: mobile genetic elements (specialized sequences) are moved fromone location to another, which can add new information into a gene region or alter gene order

This is called a JUMPING GENE and the action of it inserting itself somewhere else non-homologously is called transposition, making the jumping gene segment called a

transposon (it uses enzymes called transposases)


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How properties of genetic elements and how transposable elements (transposons) are classified based on

structure, mechanism of transposition, and degree of autonomy

Autonomous: transposable elements that contain protein-coding sequences for key enzymes for carryingout the transposition

Non-autonomous: do not contain these and have to depend on other autonomous elements DNA transposon: remains a DNA molecule throughout the transposition Retroposon (retrotransposon): produces and RNA intermediate and is then reverse transcribed into cDNA

before re-attachment

o LTR transposon: retrovirus-like, using same mechanism as retroviruses to create DNA moleculesthat subsequently integrate into other chromosomal regions. The LTR stands for long terminal

repeats and are on either side of the central coding region (5 LTR is for promotor function, 3 is

for poly-AAA). They also have some target site repeats 5-10bp on either side

o Non-retroviral retrotransposons: dont have LTRs and are either LINEs or SINEs (non-tandemrepeats)

LINEs (long interspersed elements): 900bp avg (can be up to 7kb) units all over the place,with about 10^6 copies (21% of genome). Typically there are 2 ORFs, and one typically

codes for reverse transcriptase (autonomous)

SINEs (short interspersed elements): 90-500bp, common is the Alu repeat, ~300bp, isrecognition site for restriction enzyme AluI. Also 10^6 copies (13% of genome) (non-

autonomous)

How tandem gene duplication can occur through unequal homologous recombination; and its contribution to

genome evolution in terms of quantitative or qualitative alteration in gene content

Duplication: increasing sizeo Tandem gene duplication by unequal homologous recombination: probably how we got gene

clusters, -globin is a good example

If you have two similar sequences on either side of a gene (lets call them repeat 1 and 2),during meiosis, maybe repeat 1 on one copy will line up with repeat 2 on the other copy,

thinking they are perfectly homologous, and when they cross over, you end up with one

chromosome with two copies of the gene, and another with zero. Quality-wise, the gene

is not altered, but quanitity-wise it went from 1-2 copies

OR, if two almosthomologous genes are right next to each other, gene A might line upwith gene B, and then one daughter has A, B/A hybrid, and B, and the other has just A/B

hybrid. This changes quantity and maybe the quality, depending on the hybrids protein

expression

o The same thing can happen with sister chromatids, in which case the genes are from the sameparent, not a contribution from both as in the above

Two ways transposition-based sequence duplication can occur, and contribution of these mechanisms to

dispersed sequence duplication in the genome

Duplication (increasing size): Transposition-based sequence duplication (duplicative transposition)o This is where DNA is undergoing replication and a DNA transposon from a replicated part inserts

itself to a part that hasnt been replicated yet, resulting in one strand with 2 copies and one

strand with one copy

o OR, LINEs can create more copies of themselves (being transcribed, translated, and then usingreverse transcriptase), or by creating reverse transcriptase which helps non-autonomous SINEs

jump (like reverse transcriptase made by a LINE to help the SINE Alu work)

o The duplicated copies inserts itself into a new location, creating multiple copies of thosesequences. This can occur far from the original site, and typically occurs where there is active

gene transcription (because of more open chromatin activity). They can also insert themselves as


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an inverted repeat. You can also call it interchromosomal or intrachromosomal depending on

whether it inserts on the same or a different chromosome

How replication slippage can result in changed tandem repeat lengths, how this may have contributed to the

evolution of microsatellite polymorphism

Duplication (increasing size): Sequence Duplication Due to Replication Slippageo During replication in highly repetitive regions, a SS hairpin may occur if the DNA slips

backwards, and the new strand will have one extra copy of the repeated sequence. This is calledan expansion of the tandem repeat region

This can also occur on the template strand, (forward slippage) and will result in the newstrand missing a sequence. This is a contraction of the tandem repeat region

These mechanisms explain high degree of polymorphism of STR (short tandem repeats) inthe genome

Duplication of All or Part of a Chromosome by Cytogenic Events

Non-disjunction during meiosis results in chromosomal trisomy, which is a large scale gene duplication,although this is poorly viable, although partial trisomy can be thought of as a replication, and this may

have been a big part of the past, even though they dont seem to be advantageous now

Mechanism of genome evolution in terms of (1) duplication, (2) mutation, selection, and divergence, and the

effect of these on coding and non-coding sequences, and use this to explain why human genome seems so

organized and disorganized at the same time

Duplication, see above Mutation, Selection, & Divergence to increase genomic diversity

o Mutations lead to disorganization or instability, since they are subject to randomchance/probability. BUT, natural selection based on functional consequence of these mutations

get rid of the unstable ones and preserve the advantageous ones

Natural selection acts most strongly on coding sequences, the mutations that change oradd to normal protein function

o If duplicated genes each undergo independent mutations, the divergence rate will be slow withlow selective pressure, and will be fast with high selective pressure

o Mutations can result in inactivation of a duplicated gene copythis is how pseudogenes areformed!

o Repeated cycles of this is how paralogs are formedo Once reproductive identities are different (divergence to the point where mating is not possible),

that is speciation, or considered different species: after this point, the resulting species will share

orthologs, and the number of these are a measure of relatedness

Non-coding sequences: mutations here are not affected by natural selection, and these are typicallydisorganized (remember natural selection got rid of the unstable ones)

The five types of transposition-based events in human genome evolution and their significance in terms of

characteristics of modern human genome(1) With a transposition event, insertional mutagenesismay occur if re-integration occurs within a coding,

regulatory, or signal sequence of a gene, changing its ability to function

(2) Also consider that LINEs have weak poly-AAA signals, so a transcriptional read-throughcan occur, wheretranscription stops at the NEXT poly-AAA which might include another end of an exon for the next gene!

So the new site where the LINE is inserted will have a new exon region, and this is calledexon shuffling

(3) mRNA of a normal gene can be reverse transcribed by the reverse transcriptase of a LINE. The resultingTTTT tail on the cDNA will integrate into AT-rich regions of the genome. It will lack a promoter and

become a pseudo-gene (called aprocessedpseudogene because of its RNA splicing)


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(4) By increasing number of duplicative transposition (and copies of dispersed repeats), retrotranspositionincreases likelihood of recombination between different chromosomal regions

(5) Segmental Duplication (Ch. 3) suggests that there are preferred sites for re-integration of transposons,and these can be duplicated to create partial or full duplication

Ch 6: Structure of Chromosomes and the Nucleus

How double-stranded DNA molecules are organized into high-ordered structures such as an interphase

chromosome: from DNAinterphase chromosome

Histones help compact DNA by over 10k foldo These are 4 different histone molecules, and 2 each make a histone core, which DNA adds itself to

to make a nucleosome

Histones are rich in arginine and lysine (these are positively charged, and DNA backbone is negative, so ithelps the attachment)

146 bp of DNA wraps 1.75 times around the histone, and histones are connected by short linker DNAsegments, appearing like beads on a string

These are coiled into chromatin fibers, and nucleosomes are neatly packaged by stacking in a zig zagribbon, then twists into a double helix

200-100 kb loops of chromatin fibers appear to be attached to the chromosome scaffold although this isjust the SMC protein complex, which tethers chromatin fibers together

o These are called cohesins, and they act like a twisty-tie to loop around DNA Further condensing occurs from Interphaseprophasemetaphase, with metaphase being the most

condensed (visible under LM)

Chromosomal remodeling

In the compact interphase chromatin arrangement of zig zag ribbon and double helix, access to the DNA islimited, so the chromosome is inactive in replication, transcription, or repair, and it is said to be closed.

There are regions of the chromosome where little or no stacking of nucleosomes is seen, and this is an

open region. This allows DNA-binding proteins (other than histones) access to the DNA

o Control over the level of nucleosome packing is a major mechanism of regulating gene expressiono The transition from closed to open is chromosome remodeling

Large protein complexes (chromatin remodeling complex) carry out remodeling by covalently modifyinghistones (methylation), thus loosening the contact between histone and DNA

o These complexes may also move nucleosome position as another form of regulating expressionDifference between heterochromatin and euchromatin

Euchromatin: gene-rich, progressively condensed during cell division Heterochromatin: highly condensed throughout the cell cycle (10% of chromatin). Genes here are

typically not expressed due to inaccessibility. Position effectis how related gene expression of a particular

gene is to its position on a chromosome

oConstitutive heterochromatin: alwaysinactive and condensed, repetitive DNA, found incentromeres

Seen in non-centromeric regions of 1, 9, 16, Yo Facultative heterochromatin: condensed as needed

Example is the inactivated X chromosome Hetero can spread to Eu regions, inactivating the Eu genes, AND hetero regions can retreat, revealing Eu

regions, activating those Eu genes

o This allows prevention of invasion from foreign genes (viruses do this). We cannot preventincorporation, but if a newly incorporated gene is turned into heterochromatin, we can turn it

off, and this is position effect


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Heterochromatin genes can occasionally still be expressed if they possess one of two elements:o Locus control regions (LCRs): appear to be able to keep chromatin open for thousands of bps

away, and are a type of enhancer. They can also directly interact with a distant gene through

looping (with help from a SMC)

o Insulators: enhance gene expression by blocking influence of neighboring heterochromatin, likewhere the chromatin is attached to the scaffold?This is reserved for SERIOUSLY IMPT genes

Heterochromatin vs euchromatin regions are preserved in mitosisStructure of the nucleoplasm and how chromatins are localized within the nucleus; how these locations correlate

with transcription activity

Double membrane called nuclear envelope, the outer of which is continuous with rough endoplasmicreticulum

Nucleoplasm contains DNA (except mito DNA), nucleolus (involved in RNA processing), not membranebound

Nuclear matrix suggests high degree of organization as to each chromosomes chromosomal territorywithin the nucleus

o Genes being actively transcribed tend to be near periphery of that chromosomal territory (or evenjust outside of it), suggesting an out-looping

oIn between territories is interchromosomal domains which is where transcription, RNAprocessing, and transport take place

o Heterochromatin occupies regions next to the nuclear membrane, and euchromatin tend to bemore centrally located

Structure of the nuclear envelope, including nuclear pore complex

Inner membrane supported by fibrous nuclear lamina made up of lamin proteins (Hutchinson-GilfordProgeria is a mutation in the genes that create nuclear lamina lamins)

Space b/t membranes is perinuclear space, has nuclear pores where inner and outer membranes meetto form channels, and transcription factors can get into pores to have straight shot to regions to be

transcribed

Some lamins on intranuclear surface of inner membrane can bind to heterochromatin, which tethersindividual chromosomes and may explain the territories

Nuclear pore is formed by a nuclear protein complex (NPC) (made up of nucleoporins), filaments extendfrom the NPC and form terminal ring, which together make a nuclear basket. Filaments also extend into

cytoplasm

Ch 7: Chromosome Function & Cell Cycle Dynamics

Essential functional elements required to turn DNAchromosome (ex: yeast artificial chromosomes);

In yeast, DNA behaved like a chromosome when joined to two vector arms containing:o Telmeric sequences (TEL)o

Centromeric sequences (CEN4)o Autonomous replicating sequence (ARS1)

This was introduced into a yeast cell, making the YACStructure of centromere, telomere, and origin of replication

Centromere: essential for spindle formation, chromosome segregation, control of copy #during divisiono During metaphase, seen as highly condensed heterochromatin (-satellite)o On top of this is kinetochore, a complex protein structure that is the site of attachment for the

mitotic spindle

o In humans, centromeric sequences are tandem repeats of 171bp that are hundreds of kb


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o In yeast, the whole centromere is 110 bpo On either side of the centromere, it is bound by non-satellite heterochromatino Chromosome cannot tolerate two active centromereso Occasionally a neocentromere can form, which is when one arises in a region without any satellite

repeats

o Large # of proteins form kinetochore or mediate its functiono CLINICAL CORRELATES

Scleroderma, antibodies against centromeric proteins Dysfunction of centromeres can result in non-disjunction Abnormal recombination around centromeric regions can yield Robertsonian

translocations

Telomere: both ends capped by these, TTAGGG repeated thousands of timeso Highly conserved during evolutiono Serve as protection of the free endso At the end of the telomere, the 3 end overhangs and is single-stranded, which is protected

from degradation from a telomere-associated protein, which also brings chromosomes together

during meiosis

o Because RNA primers are used on the lagging strand during DNA replication, it always getschewed off, making the 3 end stick out a bit. Telomeres prevent progressive shortening of the

end of the strand

Telomeres shorten with age, but CA cells maintain long telomeres due to increasedtelomerase, which contains and RNA template that can extend the longer parent strand

and provide extra bases to synthesize more lagging strand

o CLINICAL CORRELATE: dyskeratosis congenital, inability to maintain telomere length Origin of replication: provide starting point for DNA replication, in yeast this is the ARS and is 11bp

sequence within a 50bp A-T rich region, although in humans this is not well-defined, as we have a lot of

origins of replication

o Euchromatin replicates in early S phase, heterochromatin is late Human artificial chromosomes (HACs) is possible, but complex and inefficient

Roles of cohesins and condensins in early mitosis

Cohesins are proteins that mediate binding of two sister chromatids along entire chromatid Since a metaphase chromosome is 50-fold shorter than interphase (recall, metaphase is most condensed,

which is why its visible!), there are proteins called condensinswhich use ATP to attach to different

regions of a chromosome and cause coiling and compaction

Both cohesins and condensins are dimeric complexes with a hinge region During prophase (before metaphase), cohesins are phosphorylated and dissociate from chromatins except

for centromere cohesins

Centrosome cycle and properties of microtubules, their classification in the mitotic spindle, and their action

during anaphase

Centrosome is the MTOC (microtubule-organizing center) Interphase: centrosome is near nucleus, consists of a pair of centrioles from which an array of

microtubules extend outwards (NEG end attached to centrosome, + end away from it)

Alpha and beta tubulin molecules make up microtubules, and their assembly begins at the centrioleo Then a nucleation event occurs, and they are then built (or removed) from the + endo They have a high turnover rate, based on GTP-dependent polymerization or depolymerization

Growth = rescue Shrinkage = catastrophe There is a dynamic equilibrium between these two states


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o CLINICAL CORRELATES: cancer drugs, including colchicine, vinblastine, and paclitaxel can interferewith microtubules (blocks disassembly, and because remodeling doesnt occur, cell is static and

undergoes apoptosis)

Centrosome cycle is its duplication, which occurs during S phaseo MTs shrink, the new ones form asters, and they migrate to opposite sides to form the bipolar

mitotic spindle

Mitotic spindle: three kinds of MTs(1) Astral MTs: extend towards cortex of cell, ensures spindle is correctly oriented(2) Kinetochore MTs: extend towards kinetochores of metaphase chromosomes, they seek out

kinetochores and attach, capturing them

(3) Polar MTs: extend toward opposite pole and overlap to form zone of interdigitationa. These push centrosomes apart and maintain structure of cell, also give the initial

force for separation during Anaphase

o Treadmilling: MTs are gaining tubulin at + end and losing at theendo Dynamic equilibrium (rescue vs catastrophe in balance)o NEG ends of kinetochore MTs PULL, while + ends of polar MTs PUSHo This tension creates stability called spindle-attachment checkpoint which stays there until

anaphase is ready to begin (scans for DNA errors, ensures enough energys around to proceed)

Sister chromatid separation (anaphase) occurs when protein called Anaphase Promoting Complex (APC) isactivated. This has many functions, including a cascade that cleaves centromeric cohesins that bind the

chromosomes together, and sister chromatids move away

o Early anaphase (A): kinetochore MTs shorten at both ends, giving initial forceo Later anaphase (B): polar MTs elongate, begin sliding towards their pole

At the same time, astral MTs shorten at NEG end, moving centrosomes further apartHow nuclear envelope is disassembled and re-assembled during mitosis and how cytokinesis is achieved

Nuclear envelope must go away in order for the microtubules to reach kinetochoreso This occurs quickly during prometaphaseo Triggered by phosphorylation of lamins by lamin kinase

Early telophase: envelope fragments re-assemble around de-condensing chromosomeso Thought that phosphates associated with chromatins remove phosphate group on lamins,

allowing them to re-polymerize and form the nuclear lamina

Late telophase: nuclear envelope is complete and cytokinesis beginso Possibly initiated by ER fragments and re-incorporation of NPC proteins (nuclear pore complex)o Contractile ring forms around cell, cleavage furrow appearso Ring tightens via actin-myosin skeletal muscle actiono ER and Golgi fragment and re-assemble, mitochondria remain intact, but all distribute among cells

Unique features of meiosis not seen in mitosis

Meiosis I: segregation of homologous chromosomes Meiosis II: segregation of sister chromatids Sister chromatids of a single chromosome are bound in prophase I, but homologous chromosomes are

also paired to form a bivalent chromosome or a tetrad with 4 sister chromatids

o Telomere clustering helps the homology pairing along with help of Synaptonemal complex (largeprotein complex)

At least one recombination between non-sister chromatids occurs, forming a chiasmao There are hot spots and cold spots for this, but it is unpredictableo In males, recombination occurs towards telomeres more, in females occurs near centromere

Two important consequences of meiotic crossing-over


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Holds homologous chromosomes together during metaphase I Contributes to genetic diversity

Ch 8: Basic Cytogenetics, Chromosomal Disorders & Aneuploidies I

Common staining techniques for cytogenetic studies

Banding patterns appear when digested with trypsin (remove chromosomal protein) and stained withGiemsa stain (called G-banding)

o Genes are located at GC-rich Giemsa-LIGHT bands, with small # of genes in Giemsa-dark (G) bandso Light-staining = euchromatin; dark-staining = heterochromatin

Q-banding is using quinacrine, and is similar to G-bands, but requires a UV microscope (rarely done) Prior to G-staining, chromosomes can be heat-denatured, and the banding pattern is the reverse, so this is

called R-banding

o This highlights the ENDS of chromosomes which are generally G-light Treating with alkali before G-staining results in staining constitutive regions near the centrosomes and is

called C-banding

Designate chromosomal regions with nomenclature

Short arm = p (petite) Long arm = q (comes after p) Acrocentric chromosomes have tiny p arms (13, 14, 15, 21, 22, Y)

o The short short arms contain ribosomal genes. These genes are redundant so they aredispensible (5*2 copies)

Metacentric have equal length arms, submetacentric have shorter p arms but not too short Locations described by #, arm, region #, band #, PERIOD, sub-band #

o 2q13.4 = chromosome 2, q arm, region 1, band 3, sub-band 4o ter = terminal end of that arm

+ is extra, - is missing Euploidy is the normal diploid 46 chromosomes (46,XX or 46,XY)

o Aneuploidy is anything else n-somy is n copies of a specific chromosome (trisomy, tetrasomy, pentasomy) n-ploidy is n copies of all of the chromosomes (triploidy = 69,XXX)

Impact of chromosomal abnormalities in pregnancy in terms of spontaneous miscarriage and morbidity among

liveborns

Many birth defects or miscarriages are related to chromosomal abnormalitiesNon-disjunction, how trisomies arise in terms of meiosis I and II non-disjunction

Two homologous chromosomes OR sister chromatids segregate to the same pole (meiosis I or IIrespectively)

o Meiosis I non-disjunction: results in 2 biparental aneuploid daughter cells, 2 empty daughterso Meiosis II: results in 2 normal daughter cells, 1 uniparental aneuploidy daughter, 1 empty

In actuality, it is more complex than this. Chiasmata stabilize bivalent chromosomes in prophase I, so thefewer chiasmata present, less chance of non-disjunction. If there are no chiasmata, each homologous

chromosome can behave as univalent and can drift randomly to the poles in anaphase I

OR, bivalent chromosomes might never form! This can be from:o Failure to form chiasmata, ORo Failure to pair homologous chromosomes

Non-disjunction increases with maternal age as crossing-over events decrease with age


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Non-disjunction in terms of failure of bivalent formation and premature separation of chromatids in meiosis I

Or a sister chromatids of one of two homologous chromosomes can separate prematurely (meiosis I), sogoing into meiosis II there is one more or less sister chromatid

Common clinical features of major autosomal aneuploidies

47,XX,+21 (trisomy 21, Down syndrome)o 21 is smallest chromosome, and the trisomy can be partial as wello There is a critical region on the chromosome that results in the phenotype of DSo Critical genes:

SOD1: superoxide dismutase, a step in converting free radical O to water. Extra enzymeperoxidizes lipids and lead to neural cell death (side note, ALS is heterozygous mutation)

MNB: minibrain, learning and memory problems when extra copy ETS2: increased risk of leukemia APP: amyloid precursor protein, contributes to Alz disease, heterozygous mutations of

this have familial early-onset Alz

o Clinical features: Upslanting eyes Flat nasal bridge Congenital heart disease 5thfinger clinodactyly Wide separation of 1stand 2ndtoes Mental retardation Simian crease (single transverse palmar crease) Risk for leukemia and dementia

47,XY,+13 (trisomy 13, Patau syndrome)o Clinical features:

Cutis aplasia (missing portion of skin/hair) Cleft lip & palate Micropthalmia (small eye) Hypotelorism (eyes close together) Holoprosencephaly (brain development) Polydactyly (postaxial) Cardiac & renal abnormalities Poor developmental High mortality rate (10% 1-year survival)

47,XY,+18 (trisomy 18, Edward syndrome)o Clinical features:

Intrauterine growth restriction Short palpebral fissures Micrognathia (small lower jaw) Short sternum Clenched hands Rocker-bottom feet 10% survival 1-year Poor developmental

Monosomies are uniformly first trimester miscarriages unless it is the X chromosome

Ch 10: Chromosomal Disorders IIX-inactivation, XY aneuploidies

Overview of sex chromosomes


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X: medium size, x-linked disorders (more common in males)o Females have second X chromosome inactivated, a form of dosage compensation

Y: not many genes, only a few homologous to X and a few unique ones like SRY and AZF (sexdetermination and sperm production)

o Two PAR homologous to PARs on Xo Mostly heterochromatino Transmission of Y is father-to-son only, and no recombination takes place

Lyon hypothesis

Lyon hypothesis: In any given cell, only one active X chromosome, the inactive one seen as a Barr bodyo Barr bodies are attached to nuclear envelope

Cells only know to express ONE X chromosome, so a 47,XXX female has two Barr bodiesCharacteristics and mechanism of x-chromosomal inactivation, # of active X chromosomes in patients with various

chromosomal disorders

Occurs at 1,0002,000 cell stage (late blastocyst)this is the 9thor 10thdivisiono The extra chromosome is important for development in the early stage

Paternal and maternal X have equal chance of being inactivated within each cell (so all females cells are amixture of paternal and maternal Xs being expressed)

o When normal cells undergo mitosis, the same X chromosome (maternal or paternal) is inactivatedo This random assortment is an example of mosaicism

The X chromosome is re-activated prior to meiosis in germ cells Unknown counting mechanism

o Triploidy69,XXX may have one OR two X chromosomes expressedo Tetraploidy92,XXXX have TWO active chromosomes

X-inactivation controlled by cis-acting elements (X-inactivation center geneXICon Xq13.2)o This gene inactivates the chromosome it resides on by producing an RNA that spreads from the

transcription site in a bidirectional manner, coating the whole chromosome

o Histone modification occurs, and it all becomes heterochromatin Two regions of X escape inactivation: pseudoautosomal regions (PARsPAR1 and PAR2)

o PAR1major PAR: telomere of Xp and Yp, 13 genes, recombination commono PAR2minor PAR: telomere of Xq and Yq, 4 genes, NOT often recombination

Clinical features of major sex chromosome aneuploidies

47,XXY (Kleinfelter Syndrome): no Leydig or Sertoli cells, LH & FSH high, one Barr bodyo Hypogonadism, small testes, infertileo Tall and lengthyo Gynecomastia (breasts)o Decreased body hair and muscle masso Sometime mild learning disabilities and behavioral problems

47,XYYo Tall and lengthyo Learning disabilities and behavioral problemso Criminal gene? Originally reported in prisoners

47,XXXtypically benigno Decreased fertilityo Menstrual instabilityo Seizure disordero Mild developmental problemso The more Xs you add, the more severe the phenotype is


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45,X (Turner Syndrome): other X important for early development, especially of germ cells. Could also beinactivation of PAR genes

o Treated early with growth hormone, but doesnt fix amenorrhea because its ovarian in origin, sono estrogen production, LH and FSH are high

o Short stature due to SHOX gene in PAR1: short! Heterozygous mutation of this gene (without Turner) causes Madelung deformity (radius)

called the Leri-Weill dyschondrosteosis, or dwarfism if homozygous (Langer mesomelic

dwarfism)o Clinical features

High frequency of fetal demise (9 or 10 miscarried for every 1 born) Lymphedema of hands and feet (cystic hydroma, can be Dxd on US) Aortic coarctation and bicuspid aortic valve Webbed neck VERY SHORT Amenorrhea Some behavioral problems and learning disabilities Nipples outside the midclavicular line

o Five windows of diagnosis1. Embryonic: cystic hydroma on US2. Infant: lymphatic obstruction, lose skin over previous cystic hydroma, puffy hands and

feet, and short limbs

3. Baby: systolic murmur, weak LE pulses, coarctation of aorta4. Toddler: short stature5. Preteen: no periods

Triploidy: 69,XXXo Lethal in neonatal period, growth deficiency, multiple malformations

Mixoploidypresence of two (or more) genetically different cell lineages in an individual. Two kinds:o Mosiacism: result of a mitotic non-disjunction event

Ex: 46,XX/45,X (Turner syndrome)a report would typically state % of each Phenotype is decreased severity

o Chimerism: individual with cells from two genetically different zygoteschimera Ex: 46,XX/46,XYa result of fusion from two zygotes All allograft transplant recipients are chimeras (could be found on a chromosome study of

a female who has had a bone marrow transplant from a male)

Ch 11: Chromosomal Disorders IIIDeletions, Duplications, Translocations

Nomenclature, background info

+ means theres something extra, - means something is missing (terminal deletion) del means deletion, r means ring chromosome, dup is a duplication, t is a translocation, der

means a derivative of a chromosome or derived chromosome

Centromere is proximal, telomere is distal or terminal Requires chromosomes breaking, which may result as a recombination event

Describe deletions & duplications, and their clinical consequences

Deletions: aka partial monosomyo Terminal deletion: remember, the telomere is still preserved!o 46,XX,5p- or 46,XX,del(5p) is Cri-du-chat syndrome

This does not indicate where the breakpoint is, but you know there is a terminal deletionon the p arm of chromosome 5, an example would be 46,XX,del(5)(p21)


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CLINICAL PICTURE: cat-like cry, microcephaly, growth delay, mental retardation,hypotonia

o Deletion can be in the middle: Ex. 46,XY,del(11)(q14.2q23.3) so he is monosomic for that regiono Ring chromosome: a bi-terminal deletion and a circular chromosome formstelomeres go away!

Ex: 46,XX,r(18)(p11.3q23) This is unstable during cell division

Duplication: ABCABBCo 46,XY,dup(5)(p13p15.3) shows the region that is duplicatedo Direct tandem is the same in a row (A B B C)o Inverted tandem duplication is backwards (A B B C)

Translocation: broken segment (not containing centromere) moves to a different chromosome. Typicallynot one-way, typically trades, and is reciprocal

Balanced vs unbalanced translocations

Balanced: no net gain or loss of chromosomal material from the genome, and no change in phenotype isexpected, however they can be carriers

o 46,XY,t(7;15) means there was a balanced translocation between material on 7 for 15 To show breakpoints: 46,XY,t(7;15)(p15;q15) showing breakpoints are 7p15 and 15q15

Unbalanced: could cause growth problem, developmental delay, retardation, or birth defects

o 46,XY,-7,+der(7)t(7;15)(p15;q15) indicates he is missing one normal 7 and has gained a 7 that hasa part of 15 on it which is derived from a 7;15 trade

Result is a partial trisomy of 15q and partial monosomy of 7po These can be inherited from a parent with a balanced translocation

Robertsonian translocation

Entire long arm of an acrocentric chromosome (13,14,15,21,22) attached to the long arm of anotherchromosome, with elimination of the short arms of both chromosomes

o 45,XY,-14,-21,rob(14q21q) is missing normal chromosome 14 and 21, but has gained one thatcontains both long arms

Since short arms of acrocentric chromosomes are disposable, its balancedo 46,XY,-14,rob(14q21q) is missing 14 and has gained one with 14+21 long arms. He has THREE

copies of 21q in his genome and will have Down syndrome phenotype

This is unbalancedReproductive consequences of balanced translocations (including Robertsonian)

Ch 13: SSDown Syndrome

Physical and psychosocial complications for patients with DS

PE:o Single palmar crease, 5thfinger clinodactylyo Separation of the first and second toeso Hypotonia (low muscle tone)o Upslanting palpebral fissures, flat nasal bridge, protruding tongue

Complications:o Developmental disabilityo Heart diseaseseptal defectso Duodenal atresia (GERD)o Hypothyroidism, cataracts, hearing loss, strabismus (cross-eyed)o Leukemia


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Geneticso Typically trisomy 21 (95%) or a mosaic of that, or a translocation (50% of these inherited)o Robertsonian carrier: 10% risk (16 from mother, 5 from father)o Non-disjunction event occurs about 1% of the time

Managemento PT/OTo Hearing, vision, thyroid checkso Educational intervention

Ch 14: Chromosomal Disorders IVStructural and Chromosomal Abnormalities II: Inversions and

other variations

Nomenclature for inversions

inv = inversion; i = isochromosome; h following an arm means extra heterochromatin on that armo Ex: 46,XX,inv(9)(p12q13)

Broken at two locations and it flips over, rejoining opposite terminal segmentso Generally result in no net loss or gain of genetic material, although carriers can produce

unbalanced gametes

Reproductive consequences of pericentric and paracentric inversions

Pericentric: breaks are on two different arms (break points indicated), and the inversion segment containsthe centromere

Paracentric: breaks on same arm of the chromosome, so no centromere involvemento Ex: 46,XY,inv(7)(q11q22)

Isochromosomes, complex chromosomal rearrangements, and chromosomal fragile sites

Isochromosome: an entire chromosome arm is duplicated, followed by displacement of the otherchromosome arm with this duplicated arm in the opposite direct. Essentially, the centromere splits

horizontally instead of vertically

o Ex: one of the X chromosomes is an isochromosome of the long arm 46,X,i(Xq)this patient has Turner syndrome (and is the genotype in 30% of TS)

Complex chromosomal rearrangement: more than 3 breakpoints = complex rearranagement Subtelomeric rearranagements: just proximal to telomeres are rich in genes (G-C), are hard to diagnose

using G-banding, and require FISH and/or microarray

Fragile sites: chromosomal region that appears almost brokenalthough this is just a small amount ofnon-staining chromatin

o On Xq27 can lead to Fragile X syndrome (mental retardation) Follows genetic anticipation, the idea that sxs become more apparent at an early age as it

is passed on

Chromosomal polymorphism

Chromosomal changes among normal individuals that are benign variationso Typically have extra heterochromatin (A-T gene-poor, dark-staining regions)o Designated 46,XX,9qh+

Ch 15: Molecular Diagnostics IChemical Principles

Define affinity, specificity, stringency, rate in DNA hybridization reaction

Affinityo When DS DNA is subject to heat or alkali, it will dissociate at a critical temperature or pH


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As temperature is lowered or pH neutralized, the strands will come back togethero High affinity = high critical deperatureo Determined by three factors

Strand Length: affinity proportional to hydrogen bonds Base composition: GC is stronger than A=T (hydrogen bonding), so if there are lots of

GCs it will take a higher temperature to dissociate than AT-rich region

Chemical environment: More H+ around, more bonding! Monovalent ions like Na+stabilize DNA duplex, whereas alkali, urea, formamide DE-stabilize it

Specificity & Stringencyo Affinity decreases with more mismatches, so the binding is less specifico Tmax = temperature at which 50% is bound, so GC-rich will have high Tmax, AT-rich lower Tmax

This is a measure of specificity, because the more mismatches, the lower Tmax will beo Reaction temperature defines stringencyof a hybridization reaction. More stringent conditions is

where only cDNA strands of high specificity will associate. LOW stringency will let anything

associate (ie bringing it back down to really cold instead of a moderately warm still)

pH of 9 is more stringent than 7.5 (more bonding at 7.5) Rate in DNA hybridization reaction:

o Affinity describes tendency for association and disassociation, but RATE of the reaction isdependent on the concentrations of the strands involved (more concentration, faster rate)

Highly redundant DNA fragments associated FAST (speed can be used to determine # ofcopies a gene in its genome)

Not linear repetitive DNA associates FAST and the rest slowly associatedExample of ASO Analysis (allele-specific oligonucleotide analysis): CF, p.F508del (3-bp deletion codon 508)

Add pts DNA onto solution of radioactively labeled probe that is perfectly complementary to normal CFTRgene, and another one complementary to del508 mutation

o Think of what a carrier would look like. It would be half lit up

Ch 16: Molecular Diagnostics IIDiagnosing Chromosomal Abnormalities using FISH, CGH-CMA

DNA hybridization assays & study of cytogenic abnormalities, predicting results of fluorescence in-situ

hybridization (FISH) studies based on probes used in patients with different abnormalities

FISH: chromosomes exposed to fluorescently-labeled DNA probe complementary to specific sequence.Hopefully they hybridize. Unbound probes washed off. Number of dots of light found in each genome

represent number of copies of the sequences in that genome.

o This can detect deletions as small as the probe (30bp smallest), although poor resolution due todetection of fluorescent dots

o Disadvantage: in order to use the correct probe, you must know what you are suspecting Ex: Williams syndrome, testing gene for elastin which is next to Williams

Side note: Williams syndrome

Round-ish face, upslanting, wide filtrum and mouthBasic principles and procedures of comparative genomics hybridization using chromosomal microarray analysis

(CGH-CMA): good for a wide range of diagnostic possibilities

Large number of probes on a big (tiny) grid, each probe being complementary to a defined chromosomalregion

o Up to 200,000 probes/chip! Label DNA from patient with RED, control DNA from normal individual with GREEN, and MIX equal

amounts together and hybridize on a chip, wash off unbound probes, and scan for amount of red and

green DNA in each probe area


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o Yellow = equal amount of red and green (equal gene dosage)o If patient has duplication syndrome, there will be more RED bound, and the scan will appear RED

Can take this data and enter it into the UCSC Genome Browser Advantage: exquisite resolution, versatile Disadvantage: only changes in copy # can be detected, but nature and context of change are not clearly

able to be known. Mosiacism and rearrangements like balanced translocations or inversions will not be

detected. MUST F/U with a FISH study

Ch 18: DNA Replication

DNA: anti-parallel, 10.4 bases/turn

How DNA replication is carried out using polymerases, nucleases, topoisomerases, helicases, and ligases

Origin of replication is typically A-T rich (weaker bonds). We have 10,000 of these among 46 chro Replication forks are the Y-shaped junctions where replication is occurring (two at each origin)

The two origins move in opposite directions, so DNA replication is bidirectional Rate of replication is 100 nt/sec (1000/sec in bacteria)possibly slower in mammals due to complexity DNA Polymerase is the enzyme that extends DNA, FROM the 3 hydroxyl group by attaching the 5

phosphate of the incoming nucleotide, thus DNA replication goes 53

o Because of this, the opposite strand in the fork goes in short 53 segments, moving backwardso The nucleotide added is a triphosphate, but only 1 phosphate is needed. Removing the two

provides energy

o DNA Pol is DNA-dependent, first isolated by Kornberg in 56o DNA Pol makes an error every 1 in 10^7 nucleotideso It has 35 exonuclease function which proof-reads

It scans one base pair back, and if there is an error, the nucleotide will NOT havehydrogen bonding to the base across from it, and this flips it down into the palm or

editing zone, fixes it, then goes back up

o What if there is no free hydroxyl group? How does DNA attach? Primaseis an enzyme that synthesizes RNA from a DNA template (DNA-dependent RNA

polymerase) This works because RNA has an extra hydroxyl group compared to DNA Primase makes errors but it doesnt matter because they are chewed up anyways

o What else is needed for the lagging strand? Ribonuclease: digests the RNA primer after its done Repair DNA Pol: to fill in the gap from the primer with new DNA Ligase: to join 5 and 3 ends of new DNA, requires energy

Other enzymes needed:o DNA Helicase: unwinds DNA strands, requires energyo SSBP(SS binding protein): binds to SS of helix, prevents DNA from re-annealing with opposite

strand of helix, blocking them from each other (preventing hairpins)

o Sliding clamp protein: attaches DNA polymerase to the template strando Topoisomerase: needed to restore replicated DNA to the supercoiled configuration

DNA POLYMERASE: at least 10 known, although many are for mitochondrial DNA. 3 are impt here (mostare complexes, not a single protein)

o DNA Polyermerase : this one synthesizes Okazaki fragments, and has a 53 RNA Pol activity aswell as 53 DNA Pol.

4 proteins: DNA Pol subunit, two primase subunits (RNA synthesis), structural subunit Synthesizes short stretches of 10nt RNA, then DNA Pol adds 30nt to existing primer NO exonuclease activity and cannot edit, but since the primer will be removed, no worries


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o DNA Polymerase delta: extends RNA/DNA primer on lagging-strand of replication fork, connectingOkazaki fragments (5-3 pol activity AND 3-5 exonuclease activity)

4 proteins: large catalytic subunit, 3 smaller ones that organize complex and enableinteraction with PCNA (proliferating cell nuclear antigen), which increases its processivity

by 50-fold!

PCNA is a trimer that enricles DNA helixlike a sliding clamp! This increases theinteraction b/t DNA Pol delta and the DNA

It needs help GETTING to the primer itself, so it uses replication factor C (RFC) RFC is a 5 protein complex that loads PCNA onto the template, using ATP

o DNA Polymerase epsilon: extends RNA/DNA primers and requires PCNA for activity soo similarto delta, found predominantly on the LEADING strand (not sure if the main one is delta or this)

What removes the primer? FEN1flap endonuclease 1Role of telomerase in completing DNA replication

Even if a primer is at the end of a DNA, it would get chewed off and not replicated, so how do we solvethis? TELOMERES! Telomerase adds repeat units to the 3 end of DNA, primase then adds a primer, and

Okazaki fragment is synthesized

o Telomerase decreases with age, so telomeres shorten agingo

Cancer models may be unregulated telomerase, so the cancer cells become immortal

Ch 19: Molecular Diagnostics IIIMutations and Overall Strategy in Molecular Diagnostics

Germ line vs somatic mutations

Germ-line: mutation present in eggs and sperm and can be transmitted to the next gen (vertical) Somatic: mutations in non-germ cells that are not transmitted, but are present in mitotic daughter cells

(horizontal)

Exogenous causes: ionizing radiation and chemical mutagenso Most mutations are endogenous and are from:

Failure to repair DNA replication error Defective chromosomal segregation Erroneous recombination Retrotransposition

Number of lifetime mutations in genome

10^17 cell divisions generate 10^14 cells in body Each division requires 6x10^9 nucleotides, so thats an incorporation of 6x10^26 nucleotides over a

lifetime. Since there is an error rate of 1 in 10^10, there will be a large number of mutations in a lifetime

Common types of mutations at DNA level, explain how small deletions, large deletions, duplications, and

inversions occur

g is genomic DNA; c for cDNA or coding sequence; m for mitochondrial DNA; r for RNA sequence;p for protein sequence

DNA uses capitals: A C G T; RNA uses lower case: a c g u; protein sequences use capital letters for AAs or Xfor stop codons

A range uses an underscore Two sequence variations in one allele are listed with SAME brackets, separated by + sign

o In different alleles it will be separate brackets, separated by + sign [g.76A>C + g.786_787insTG] on same allele [g.76A>C] + [g.786_787insTG] has two separate heterozygote mutations

+ after a # = position of nucleotide in an intron after splice donor site


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Minus sign = position of nt in an intron before the splice acceptor site IVS = intervening sequences or intron Single-Base substitutions

o Transition: pyrimidine (C,T) replaced by another one, or purine replaced by purine (A,G)o Transversion: pyrimidine replaced by purine, or vise versa

Since there are 4 ways a transversion can take place and 2 ways a transition can takeplace, one would think there would be 2:1 transversion to transition, but in actuality wesee the opposite. Why?

o Cytosineis most commonly mutated, since they tend to deaminate spontaneously, becoming auracil. However, this is quickly repaired normally, so there is a low mutation rate. EXCEPT.

Many Cs are next to Gs (CpG), which are common sites of methylation Here, the deaminated product is Thymine (not good repair, high mutation rate) This results in permanent TpG, and after the next round of replication, you get a TpG and

ApC with no methylation, where normally youd have 2 fully methylated daughter cells.

This may mess up gene expression.

Normally, after a round of DNA replication, methylated CpG become hemimethylated(one strand)

7:1 male to female ratio of C

T single base substitutions

This is because sperm is heavily methylated, where oocyte DNA is not Also, males have much higher # of cell divisions

Deletions: del of a few nts are common, many involved less than 5 nts, mostly in regions with directrepeats of 2bp or more (could be slippage mis-alignment)

o Remember, if not multiple of 3 it will cause a frame shift Small Insertions: small ones are less common than deletions. Sometimes they co-occur, this is an indel

o Remember, if not multiple of 3 it will cause a frame shift Trinucleotideexpansions is a type of insertion, but see Ch 24 on this Large deletions and duplications: larger deletions and duplications often occur as misalignment of

tandemly repeated sequences (mismatch), EXCEPT that crossing over within the misaligned region results

in unequal crossing b/t sister chromatids or homologous chromosomes (line up at the wrong part to cross

over, so one has a deletion and the other has a duplicationyikes!)

Large insertions: transposable elementsare a common one, like an Alu insertion. However, these arentvery mutagenic, so they are not as significant (theyre kinda dormant)

Inversions: this can occur by recombination between homologous sequences with opposite orientationson the same chromosome, which is like the inversion on Factor VIII gene that causes hemophilia A (big

gene on X chromosome that can curl up on itself)

Apply simple rules for designation of common sequence variations: substitutions, deletions, insertions, more than

one mutation in same allele or individual

Substitution = >; Ex: g.76A>C = substitution of A replaced by C at 76thnucleotideo The number should be the last nucleotide of the preceding exon or first of the next exono Ex: c.15+1G>C = G was replaced by C, one nt into the intron from the end of the 15thnt of the

coding sequence (this is a short exon I guess).

Since we dont know which exon is involved, we can say IVS1+1G>C, which means thesubstitution is in the first intron, one nt in from the splice donor site.

Another example: IVS1-2A>G


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Deletion/Insertion: ins or del after nt following by nts deleted. Ex: g.786_787insTG

Relationship between paternal age and rate of new mutations in Mendelian, and between maternal age and new

non-disjunction

Males are susceptible to single-base mutations like CT substitutions as described above due to highmethylation in sperm. They are also more susceptible due to increased # of cell replications

o These mutations related to paternal age are typically dominant or X-linked recessive Maternal associated w/ non-disjunction because eggs are formed early in life and held at meiosis I until

puberty

o The # of chiasmata recombinations between homologous chromosomes decreases, which holdchromosomes together the decrease makes them susceptible to randomly drifting to the same

pole during meiosis (unk why # of chiasmata decreases)

Some people argue that chiasmata dont decrease over time, but eggs with less chiasmataare ovulated last (which suggests selection for normal gametes)

Explain parameters used to determine whether DNA sequence alteration or structural rearrangement represents

a deleterious mutation

Population study data: taking into account pattern of inheritance, do a large # of affected people havethis alteration? Obviously very strong!!!

Family study data: is there a pattern of inheritance with this trait? Molecular data: coding region, regulatory region, or near splice site is more likely to cause significant

change than middle of an intron, Isoleucineleucine is not a big deal, but something else might be

Functional data:cell biological, histological, or biochemical studies may reveal functional derangement ofprocesses

Phylogenetic data: DNA alterations occurs in position that is highly conserved throughout evolution(more likely to be deleterious)

Experimental organism data: creation of model organisms according to the altered DNA sequence info, tosee if it results in a mutant organism with a phenotype

If insufficient evidence to classify a mutation, it could be a VUS (variant of unknown significance) whichcould favor deleterious or become deleterious when additional info is discovered. In the opposite

vain, it could favor polymorphism or become benign polymorphism

o Note the PS consequences of finding a VUSGeneral approach to choosing a molecular diagnostic technique

Four common reasons for using molecular diagnostic testing:o To define causative mutation in an individual diagnosed with a disorder based on clinical or lab

informationuseful when the gene that causes the disorder is well defined

Ex: sickle cell (single mutation in single gene accounts for all pts with the disease) Ex: CF (common mutation accounts for majority, but remaining have diverse # of

mutationsso use a graded approach)

Mutations in a # of genes cause same disorder, so do Tiered Reflex Testing (timeconsuming) or Simultaneous Paneled Testing (testing all genes at the same timecost

consuming)


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Ch 20: Mutation Detection Using Simple Non-Sequencing Techniques

Basic principles, advantages, limitations of: electrophoresis, PCR, restriction enzyme analysis, Southern analysis

Electrophoresiso Advantage: simpleo Disadvantage: primary information is only size and # of fragments, so secondary information is

deducted

o Disadvantage: agarose gel has limitations to the range of DNA fragment sizes it can handle PCR:

o Advantage: easy to obtain large quantities of a DNA fragmento Limitation: must know sequence of the fragment of interest so you can design a primero Limitation: polymerases used in PCR can only extend DNA a finite amounto Disadvantage: so sensitive that it can produce erroneous results due to minor contamination of

the template DNA

o Disadvantage: if primers are not specific enough, unintended gene regions can be amplifiedwithout realizing

o Disadvantage: polymerase used have low but definite error rateso Common use: can determine an individual genotype at a short tandem repeat polymorphic

marker locus Restriction enzymes and DNA ligase

o Advantage: inexpensive reagents that recognize specific sequenceso Disadvantage: # available are limited, preference for palindromic sequences

Southern Analysiso Advantage: specific DNA fragments of interest in a complex mixture can be examinedo Disadvantage: only DNA sequences complementary to the probe can be examined, availability of

probes specific for a sequence is a challenge, although it is not sensitive to quantitative changes,

and it is labor intensive

Apply electrophoresis, PCR, restriction enzyme analysis, Southern analysis in determining genotype at loci with

microsatellite repeat polymorphism or restriction fragment polymorphism

General approach to choosing a molecular diagnostic technique

Single mutation: use PCR Kilobase: use Southern analysis Multimegabse to whole chromosome: cytogenic techniques, like chromosomal microarray

Apply basic techniques above to detect known small and large mutations

Single nucleotide substitutions, small deletions and insertions, trinucleotide expansionso Small mutations are detected by PCR and electrophoresis (for something like a tri-nt expansion,

just electrophorese the product for a single-nt substitution, you will need a restriction enzyme

that cuts at the suspected substitution, like in sickle cell

Large deletion and duplication, gross rearrangement, inversiono If its too big for PCR, Southern analysis can be used. If there is a deletion, and a restriction

enzyme cuts it in half then is blotted, the resulting piece will be shorter than expected due to the

deletion. If you see no result, the part where the RE typically cuts was probably deleted

Basic principles and applications of pulsed-field gel electrophoresis and real-time PCR

PFGE: Pulsed voltage from different directions helps large DNA fragments separate, this process calledreputation

RTPCR: quantity of product monitored during reaction. Why is this useful?


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o Product increases slowly until threshold is reachedo You can know exactly how much mRNA is in a certain tissue or cell typeo Also if there is a 3 mismatch in a primer region, the product will be low, and you will be able to

detect this

Ch 21: Molecular Diagnostics IVDNA Sequencing

Principles of Sanger sequencing, its improvement by the use of fluorescent tags, and capillary sequencing

Pure, isolated DNA molecule required (need cloning/PCR of discrete regions, this is the template) Divide into 4 aliquots, all with DNA Pol, primer, bases (one of which is labeled to be detected by

autoradiography)

o One with dideoxyATP (ddATP), another add ddCTP, another add ddTTP, another add ddGTP You want some probability of the reaction continuing, so you need a relatively smaller

concentration of ddNTP, and every lab uses a different ratio cause its tricky

o Dideoxy compound only has hydrogens instead of a hydroxyl in the 3, so elongation stopso After the reaction, restriction enzyme cleaves off newly made DNA and are separated by

polyacrylamide gel electrophoresis (length, conformation and charge of the molecule, good for

small molecules)

Since large molecules move slower, you read it bottom to top The smallest one is the 5 end (since DNA synthesized 53) Gels are hard to pour!

Improvements with fluorescent tags, easier to interpret Capillary sequencing: they used differentially fluorescent tagged dideoxynucleotides, so with a detector

and laser, you could read the sequence as it traveled

o You mix all the stuff together and electrophorese on a long skinny thing (the capillary)o This decreases human error of reading the results

Iterative pyrosequencing, NextGen sequencing, and single-molecule sequencing

Iterative pyrosequencing: each time a nucleotide is incorporated into a replicating DNA, a pyrophosphateis released (remember it only uses 1 of the 3 phosphates)

o Adding each dNTP at a time and see if a pyrophosphate is released! Detect pyrophosphate release using sulurylase which converts it to ATP. Luciferase then

reacts with ATP and emits light (brighter if 2 nucleotides in a row)

NextGen sequencing (massively parallel): no more electrophoresis!o Fragment DNA into 300-500 bp long, add primer to the endso DNA emulsified in water-oil with microbeads to bind to DNA (one strand and one bead each)o PCR amplifies these (1 million molecules of DNA to one microbead)o Then place each bead in individual well and do the same pyrosequencing thing with one base at a

time

Single-molecule sequencing: tries not to amplify itExome sequencing

This focuses on exons (exome = genome of only exons)

Ch 22: DNA Repair & Related Disorders

Importance of DNA repair, clinical consequence of genetic defect in DNA repair pathways

Exogenous causes: ionizing radiation (UV light), chemical mutagenso Most mutations are endogenous and are from:


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Failure to repair DNA replication error, free radicals, defective chromosomal segregation,erroneous recombination, retrotransposition

Mutations in genes that code for DNA repair enzymes often results in altered functiono Growth deficiencyo Premature agingo Photosensitivityo Immunodeficiency/hematologicalo Cancer predisposition

Base excision repair

Nts can become oxidized, methylated, or deaminated (C deaminated to U, or CpG methylated isdeaminated to T)

In BASE EXISION REPAIR (BER), the bad base distorts DNA, and this is detected by glycosylase, removesdamaged base, leaving an apurinic or apyrimidinic nt

o Next, an apurinic/apyrimidinic or AP endonuclease cleaves at that site, removing the sugar. Thegap is filled in and strand is ligated by DNA Pol and ligase

Two types:o Short-patch (for single-base damage)major pathwayo

Long patch (for 2-10 nts)also involved but not as major Genetic defect: hyper-IgM syndrome (not in this course, but only problem known with BER)

Nucleotide excision repair, clinical manifestations of XP

UV light can induce pyrimidine dimer (T=T, T=C, C=T, C=C) These cause bulky distortion in the double helix which is detected

o 25nt containing the dimer is removedo PCNA and ligase involved in repairing the gap

How are distortions detected? Two ways.o Global genome pathway: both transcriptionally active and inactiveo Transcriptional-coupled pathway: only transcriptionally active gene regions

Disorder when NER is messed up: xeroderma pigmentosum (XP)o Profound sensitivity to lightredness, blistering, dryness, high risk of skin CA, eye involvement,

hearing loss, cognitive impairment

o Many different types of XP, all autosomal recessiveMismatch repair, clinical manifestations of Lynch syndrome

Backwards/forwards slippage during tandem microsatellite repeats Also, point mutation or small insertion/deletion not repaired by BER will result in mismatch between two

complementary strands, and distortion will result in two SS mismatch loops formed

MMR consists of a # of proteinso MSH6 recognizes single nt mismatcheso MSH3 recognizes small insertions or deletions

These then mark out site for nuclease that cuts bad nt DNA pol and ligase repair Unk how the system knows which strand is defective This protects against expansion of genome in somatic cells

Clinical disorderso Lynch syndrome (HNPCC)

Tumor cells from these patients demonstrate microsatellite instability (MSI)this meanshigh frequency of variability in length of alleles at microsatellite loci as a consequence of

the deficiency


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Parts of telomerase complex ALSO part of small nucleolar ribonucleoproteins (snoRNPs)these are usedin ribosomal RNA processing

Telomerase deficiency leads to tell cycle arrest and death of progenitor cells like in bone marrow (thiscould be augmented by impaired snoRNP function)

Telomeric shortening causes chromosomal/genome instability because of fusion bridges when thechromosomal ends become exposed (repeat DNA ends join), then they break later on (breakage-fusion-

bridge cycle), which is a chromosomal rearrangement and is instable, increases malignancy risk

NOT premature aging (weird?!), AND female carriers of the x-linked form shows skewed inactivation

Ch 23: Molecular Diagnostics VMore Mutations; Genotype, Phenotype, & Genetic Counseling

Biochemical consequences of common mutations at RNA and protein levels (transcription, translation, splicing,

protein stability/folding)

Transcriptional mutations:o 150 identified mutations of promoter regions of genes, many of which are of TATA box and CCATT

motif (which are not present in all promoters)this decreases ability of transcription factors to

bind (can be totally silent or slightly reduced level).

o Also, if a mutation increases distance b/t promoter and transcription start site, this can alsosilence transcription

o Occasionally, mutation of promoter region can increase transcription (ex: persistent expression offetal Hb in adults from promoter mutation of G(gamma) and A(gamma) genes

o Enhancer mutations (remote from gene) can effect transcription. Ex: Beta-globin enhanced by LCR60kb away

Translational mutationso Missense: single base substitution causing a codon change (common 1stor 2ndbase of codon), ex:

sickle cell mutation in beta-globin gene

Nomenclature: p.N39K means normal protein N at residue 39 has been substituted for a K When at third base of codon, typically does not result in AA substitution a silent

mutation (or synonymous)

When a non-synonymous substitution is present, it can be conservative (similar inchemical property, eg p.I306L isoleucine replaced by leucine); or it can be non-conservative (different in chemical property, eg p.Y15N)

o Nonsense: single base substitution causing premature STOP codon to replace a AA. Ex:Hemoglobin McKees Rock

This can lead to unstable mRNAif at least 50 bases upstream of last splice junction, isdegraded by nonsense-mediated decay

If a gene lacks introns, the polypeptide will just be truncated Exon skipping - ??? rare event when truncated polypeptide is mitigated by alternative

splicing that eliminated premature stop codon

Nomenclature: AA replacing is X, p.W26Xo Frameshift: shift reading frame due to change in DNA sequencethis could cause change in

location of stop codon (proximal or distal), which can lead to nonsense-mediated decay or

unstable/altered protein

o Codon deletions or insertions: deletion/insertion in DNA of a multiple of 3 Ex: del508Fp.F508del means 3 bases got deleted and an F in the 508thposition was del

Splicing Mutationso Nt substitution (or insertion or deletion) at splice donor/acceptor sitesabnormal RNA

transcript splicing. Ex: Alu repeat found in neurofibromatosis type I deletes all of exon 6

o Another type may involve creating new splice site or cryptic splice site (new sequence that lookslike a splice site)


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Other RNA Instability Mutationso Mutations in poly-AAA site (AAUAAA) instable mRNA

Mutations of 3-untranslated region can have the same effecto Mutations in 5 UTRpoor translational initiation

Trinucleotide expansion: next lecture Mismatch repair gene mutations: cells with these mutations have mutation rates 100-1000 fold over

normal. Mutations beget more mutationsinc CA risk

Loss of function/gain of function mutations

Loss of function: mutation results in loss of gene function (the degree to which its lost depends oninheritance pattern)

o For many enzymes, having 50% is sufficient, so most enzyme deficiencies are recessiveo Haploinsufficiencyis when heterozygous loss of function mutation is phenotypically abnormal

(these are dominant inheritance)

o If a mutation reduces normal function AND makes something new to mess it up, that is dominantnegative mutation, which follows dominant pattern

o Allele that produces no product is null or amorphic (generally most gene deletions)o Allele that produces reduced amount is hypomorphico

Alleles that antagonize normal products are antimorphic Gain of function: positively abnormal due to quantity or quality (aka activating mutations)

o Often seen in CA state, due to transposition of a strong promoter upstream of a coding sequencethat normally produces gene product enhancing cell growth (double promoter!)

o This can also be seen when there is a mutation of a transducer gene that are receptorsresponding to signals to increase or decrease expression, which could be constantly repressed or

activated

o Allele that produces increased amount is hypermorphico Quantity gain of function mutations typically near promoter regiono Quality gain of function mutations typically missense mutations

Penetrance vs expressivity

Penetrance: probability that a disease phenotype will appear when genotype is presento Incomplete penetrance is when this is less than 100%

Expressivity: range of possible phenotypes given a genotype (variability) The variability of phenotype and probability it will appear given a genotype is dependent on the clinical

criteria used to define the phenotype

Co-dominance (ex of ABO)

You could describe this as incomplete dominance failure of the dominant allele to completely over-ride the recessive allele

o This is the case where homozygous is more severe than hetero (eg. Homozygous forachondroplasia is lethal)

ABO is partially co-dominanto A and B are co-dominant in relation to each other, but are both dominant in relation to O

A blood = AA or AO B blood = BB or BO AB blood = AB O blood = OO

How new mutations in stages of germ cell development should be considered in some pedigrees

Skewed X-chromosomal inactivation and clinical consequence


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Causes of non-random X-chromosomal inactivation

Sex effects and embryonic lethality

Consider above factors in genetic counseling

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