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 acid bases, amino acids, and sugars Started from RNA that both self-replicated and catalyzed protein synthesis, including the enzyme needed to transcribe RNA DNA (reverse transcription), which became the predominant template for RNA synthesis Eubacteria became modern bacteria, but purple (aerobic) bacteria were swallowed by early eukaryotes and 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 valves o This is because of oxidized homogentisic acid Missing homogentisic acid oxidase which converts to maleylacetoacetic acid Discuss 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 costs Prinicples of bioinformatics and computational approaches to gene discovery and genome annotation Provides identification and location of genes, establishment of domains o 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 mitochondrial chromosome) 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 during cancer states to learn how we adapt) Proteome: total set of protein products and their modifications within a cell type o This helps us understand protein-protein interactions, structure of functional domains of proteins, and protein expression in different states

Metabolome: set of molecules that occur within a cell type, which provides description of metabolic state of 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 understand susceptibilities 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 Structure

Basic organization of human genome, define genome architecture Haploid is 3,000 Mb, diploid is double that o 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 centromeres o Codes for ribosomes or transfer RNA o 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 5 3, so the promoter will be at the 5 end o 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 is 2600 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 evolution o Found within gene clusters

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 code for 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/structural domains o By identifying all of a genes motifs, we should deduce the structure, function, and specificity of the 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 elements Examples 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 -globin o 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 superfamily is paralagous genes that have weak homology but are generally functionally related (eg immunoglobulin genes) If paralogous genes are in same chromosomal region, they are a gene cluster o 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 conserved sequence 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 a cell 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 regulation Types 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 by providing binding sites for centromere-binding proteins

Mini-satellites: Tandem arrays of 14-500 bp scattered throughout genome with total length up to 20 kb. Near sub-telomeric regions. Can be further divided into GC or non-GCrich. 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 dispersed repeats are repeated at two or more locations (45% of genome!) 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??? SINEs (short interspersed elements): 90-500bp, common is the Alu repeat, ~300bp, is recognition site for restriction enzyme AluI. Also 10^6 copies (13% of genome) LTR Retroposons: repetitive sequences related to retroviruses although they lack gene coding 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 different o Concentrated in pericentromeric and subtelomeric regions o 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 be maintained 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 function o Mutation = deleterious change in function of a coding sequence Classification o 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 detected by Southern analysis (and is known as a RFLP) o Indel (insertion-deletion): presence or absence of a short segment of DNA o Mini-satellites/microsatellite polymorphisms: called short tandem repeat polymorphisms (STRPs) Ex: (CA)n o VNTR are many clustered mini-satellites (variable number of tandem repeats), have high variation o 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)7 and 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

(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 in o A gene critical to the survival of the host organism Plasmid is a self-replicating structure in bacteria o 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 syllabus How 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: SS Human 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 be homologous 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 are exchanged 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 of invading strand extended by DNA pol Ends ligated together and phosphodiester bonds cleaved and ends ligated Non-homologous recombination doesnt require homologous sequences, and can be either: o Simple end-to-end rejoining of DNA fragments with broken ends by forming synapse where complex 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 from one 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 nonhomologously is called transposition, making the jumping gene segment called a transposon (it uses enzymes called transposases)

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 carrying out 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 molecules that 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-tandem repeats) 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, is recognition site for restriction enzyme AluI. Also 10^6 copies (13% of genome) (nonautonomous) 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 size o 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 almost homologous genes are right next to each other, gene A might line up with 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 same parent, 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 using reverse 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 those sequences. 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

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 Slippage o 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 called an expansion of the tandem repeat region This can also occur on the template strand, (forward slippage) and will result in the new strand missing a sequence. This is a contraction of the tandem repeat region These mechanisms explain high degree of polymorphism of STR (short tandem repeats) in the 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 random chance/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 or add to normal protein function o If duplicated genes each undergo independent mutations, the divergence rate will be slow with low selective pressure, and will be fast with high selective pressure o Mutations can result in inactivation of a duplicated gene copy this is how pseudogenes are formed! o Repeated cycles of this is how paralogs are formed o 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 typically disorganized (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 mutagenesis may 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-through can occur, where transcription 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 called exon shuffling (3) mRNA of a normal gene can be reverse transcribed by the reverse transcriptase of a LINE. The resulting TTTT tail on the cDNA will integrate into AT-rich regions of the genome. It will lack a promoter and become a pseudo-gene (called a processed pseudogene because of its RNA splicing)

(4) By increasing number of duplicative transposition (and copies of dispersed repeats), retrotransposition increases 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 DNA interphase chromosome Histones help compact DNA by over 10k fold o 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 it helps the attachment) 146 bp of DNA wraps 1.75 times around the histone, and histones are connected by short linker DNA segments, appearing like beads on a string These are coiled into chromatin fibers, and nucleosomes are neatly packaged by stacking in a zig zag ribbon, then twists into a double helix 200-100 kb loops of chromatin fibers appear to be attached to the chromosome scaffold although this is just 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 Interphase prophase metaphase, 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 is limited, 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 expression o The transition from closed to open is chromosome remodeling Large protein complexes (chromatin remodeling complex) carry out remodeling by covalently modifying histones (methylation), thus loosening the contact between histone and DNA o These complexes may also move nucleosome position as another form of regulating expression Difference 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 effect is how related gene expression of a particular gene is to its position on a chromosome o Constitutive heterochromatin: always inactive and condensed, repetitive DNA, found in centromeres Seen in non-centromeric regions of 1, 9, 16, Y o 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 prevent incorporation, but if a newly incorporated gene is turned into heterochromatin, we can turn it off, and this is position effect

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, like where the chromatin is attached to the scaffold? This is reserved for SERIOUSLY IMPT genes Heterochromatin vs euchromatin regions are preserved in mitosis

Structure 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 endoplasmic reticulum Nucleoplasm contains DNA (except mito DNA), nucleolus (involved in RNA processing), not membrane bound Nuclear matrix suggests high degree of organization as to each chromosomes chromosomal territory within the nucleus o Genes being actively transcribed tend to be near periphery of that chromosomal territory (or even just outside of it), suggesting an out-looping o In between territories is interchromosomal domains which is where transcription, RNA processing, and transport take place o Heterochromatin occupies regions next to the nuclear membrane, and euchromatin tend to be more centrally located Structure of the nuclear envelope, including nuclear pore complex Inner membrane supported by fibrous nuclear lamina made up of lamin proteins (Hutchinson-Gilford Progeria 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 meet to 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 tethers individual chromosomes and may explain the territories Nuclear pore is formed by a nuclear protein complex (NPC) (made up of nucleoporins), filaments extend from 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 DNA chromosome (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 YAC Structure of centromere, telomere, and origin of replication Centromere: essential for spindle formation, chromosome segregation, control of copy # during division o 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

In yeast, the whole centromere is 110 bp On either side of the centromere, it is bound by non-satellite heterochromatin Chromosome cannot tolerate two active centromeres 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 function o 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 times o Highly conserved during evolution o Serve as protection of the free ends o 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 gets chewed 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 increased telomerase, 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

o o o o

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 condensins which 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 centriole o Then a nucleation event occurs, and they are then built (or removed) from the + end o 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

CLINICAL CORRELATES: cancer drugs, including colchicine, vinblastine, and paclitaxel can interfere with microtubules (blocks disassembly, and because remodeling doesnt occur, cell is static and undergoes apoptosis) Centrosome cycle is its duplication, which occurs during S phase o 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 interdigitation a. 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 the end o Dynamic equilibrium (rescue vs catastrophe in balance) o NEG ends of kinetochore MTs PULL, while + ends of polar MTs PUSH o 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) is activated. 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 force o Later anaphase (B): polar MTs elongate, begin sliding towards their pole At the same time, astral MTs shorten at NEG end, moving centrosomes further apart

How 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 kinetochores o This occurs quickly during prometaphase o Triggered by phosphorylation of lamins by lamin kinase Early telophase: envelope fragments re-assemble around de-condensing chromosomes o 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 begins o Possibly initiated by ER fragments and re-incorporation of NPC proteins (nuclear pore complex) o Contractile ring forms around cell, cleavage furrow appears o Ring tightens via actin-myosin skeletal muscle action o 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 (large protein complex) At least one recombination between non-sister chromatids occurs, forming a chiasma o There are hot spots and cold spots for this, but it is unpredictable o In males, recombination occurs towards telomeres more, in females occurs near centromere Two important consequences of meiotic crossing-over

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 with Giemsa stain (called G-banding) o Genes are located at GC-rich Giemsa-LIGHT bands, with small # of genes in Giemsa-dark (G) bands o 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 are dispensible (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 4 o 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 abnormalities Non-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 II respectively) o Meiosis I non-disjunction: results in 2 biparental aneuploid daughter cells, 2 empty daughters o 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 the fewer 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, OR o Failure to pair homologous chromosomes Non-disjunction increases with maternal age as crossing-over events decrease with age

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), so going 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 well o There is a critical region on the chromosome that results in the phenotype of DS o Critical genes: SOD1: superoxide dismutase, a step in converting free radical O to water. Extra enzyme peroxidizes 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 5th finger clinodactyly Wide separation of 1st and 2nd toes 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 II X-inactivation, XY aneuploidies

Overview of sex chromosomes

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 (sex determination and sperm production) o Two PAR homologous to PARs on X o Mostly heterochromatin o 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 body o Barr bodies are attached to nuclear envelope Cells only know to express ONE X chromosome, so a 47,XXX female has two Barr bodies Characteristics 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 9th or 10th division o 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 a mixture of paternal and maternal Xs being expressed) o When normal cells undergo mitosis, the same X chromosome (maternal or paternal) is inactivated o This random assortment is an example of mosaicism The X chromosome is re-activated prior to meiosis in germ cells Unknown counting mechanism o Triploidy 69,XXX may have one OR two X chromosomes expressed o Tetraploidy 92,XXXX have TWO active chromosomes X-inactivation controlled by cis-acting elements (X-inactivation center gene XIC on 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 (PARs PAR1 and PAR2) o PAR1 major PAR: telomere of Xp and Yp, 13 genes, recombination common o PAR2 minor 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 body o Hypogonadism, small testes, infertile o Tall and lengthy o Gynecomastia (breasts) o Decreased body hair and muscle mass o Sometime mild learning disabilities and behavioral problems 47,XYY o Tall and lengthy o Learning disabilities and behavioral problems o Criminal gene? Originally reported in prisoners 47,XXX typically benign o Decreased fertility o Menstrual instability o Seizure disorder o Mild developmental problems o The more Xs you add, the more severe the phenotype is

45,X (Turner Syndrome): other X important for early development, especially of germ cells. Could also be inactivation of PAR genes o Treated early with growth hormone, but doesnt fix amenorrhea because its ovarian in origin, so no 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 diagnosis 1. Embryonic: cystic hydroma on US 2. 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 aorta 4. Toddler: short stature 5. Preteen: no periods Triploidy: 69,XXX o Lethal in neonatal period, growth deficiency, multiple malformations Mixoploidy presence 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 zygotes chimera Ex: 46,XX/46,XY a 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 III Deletions, 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 monosomy o 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 deletion on the p arm of chromosome 5, an example would be 46,XX,del(5)(p21)

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 region o Ring chromosome: a bi-terminal deletion and a circular chromosome forms telomeres go away! Ex: 46,XX,r(18)(p11.3q23) This is unstable during cell division Duplication: ABC ABBC o 46,XY,dup(5)(p13p15.3) shows the region that is duplicated o 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. Typically not 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 is expected, 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 has a part of 15 on it which is derived from a 7;15 trade Result is a partial trisomy of 15q and partial monosomy of 7p o 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 another chromosome, 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 that contains both long arms Since short arms of acrocentric chromosomes are disposable, its balanced o 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 unbalanced Reproductive consequences of balanced translocations (including Robertsonian)

Ch 13: SS Down Syndrome

Physical and psychosocial complications for patients with DS PE: o Single palmar crease, 5th finger clinodactyly o Separation of the first and second toes o Hypotonia (low muscle tone) o Upslanting palpebral fissures, flat nasal bridge, protruding tongue Complications: o Developmental disability o Heart disease septal defects o Duodenal atresia (GERD) o Hypothyroidism, cataracts, hearing loss, strabismus (cross-eyed) o Leukemia

Genetics o 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 Management o PT/OT o Hearing, vision, thyroid checks o Educational intervention

Ch 14: Chromosomal Disorders IV Structural and Chromosomal Abnormalities II: Inversions and other variations
Nomenclature for inversions inv = inversion; i = isochromosome; h following an arm means extra heterochromatin on that arm o Ex: 46,XX,inv(9)(p12q13) Broken at two locations and it flips over, rejoining opposite terminal segments o 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 contains the centromere Paracentric: breaks on same arm of the chromosome, so no centromere involvement o 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 other chromosome 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 broken although this is just a small amount of non-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 variations o Typically have extra heterochromatin (A-T gene-poor, dark-staining regions) o Designated 46,XX,9qh+

Ch 15: Molecular Diagnostics I Chemical Principles

Define affinity, specificity, stringency, rate in DNA hybridization reaction Affinity o When DS DNA is subject to heat or alkali, it will dissociate at a critical temperature or pH

As temperature is lowered or pH neutralized, the strands will come back together High affinity = high critical deperature 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 & Stringency o Affinity decreases with more mismatches, so the binding is less specific o 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 be o Reaction temperature defines stringency of 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 is dependent on the concentrations of the strands involved (more concentration, faster rate) Highly redundant DNA fragments associated FAST (speed can be used to determine # of copies a gene in its genome) Not linear repetitive DNA associates FAST and the rest slowly associated o o

Example 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 CFTR gene, 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 II Diagnosing 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 to detection 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 mouth Basic 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 chromosomal region 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

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 chr o 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 backwards o 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 56 o DNA Pol makes an error every 1 in 10^7 nucleotides o It has 35 exonuclease function which proof-reads It scans one base pair back, and if there is an error, the nucleotide will NOT have hydrogen 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? Primase is 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 energy o 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 strand o 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 (most are complexes, not a single protein) o DNA Polyermerase : this one synthesizes Okazaki fragments, and has a 53 RNA Pol activity as well 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

DNA Polymerase delta: extends RNA/DNA primer on lagging-strand of replication fork, connecting Okazaki fragments (5-3 pol activity AND 3-5 exonuclease activity) 4 proteins: large catalytic subunit, 3 smaller ones that organize complex and enable interaction with PCNA (proliferating cell nuclear antigen), which increases its processivity by 50-fold! PCNA is a trimer that enricles DNA helix like a sliding clamp! This increases the interaction 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 similar to delta, found predominantly on the LEADING strand (not sure if the main one is delta or this) What removes the primer? FEN1 flap endonuclease 1

Role 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 solve this? 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 aging o Cancer models may be unregulated telomerase, so the cancer cells become immortal

Ch 19: Molecular Diagnostics III Mutations 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 mutagens o 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 X for 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

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 take place, one would think there would be 2:1 transversion to transition, but in actuality we see the opposite. Why? o Cytosine is most commonly mutated, since they tend to deaminate spontaneously, becoming a uracil. 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 CT 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 direct repeats 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 Trinucleotide expansions 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 duplication yikes!) Large insertions: transposable elements are a common one, like an Alu insertion. However, these arent very mutagenic, so they are not as significant (theyre kinda dormant) Inversions: this can occur by recombination between homologous sequences with opposite orientations on 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 76th nucleotide o The number should be the last nucleotide of the preceding exon or first of the next exon o Ex: c.15+1G>C = G was replaced by C, one nt into the intron from the end of the 15th nt 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 the substitution is in the first intron, one nt in from the splice donor site. Another example: IVS1-2A>G

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 high methylation 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 hold chromosomes 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 chiasmata are 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 have this 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 of processes 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, to see 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) which could 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 VUS General 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 information useful 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 mutations so use a graded approach) Mutations in a # of genes cause same disorder, so do Tiered Reflex Testing (time consuming) or Simultaneous Paneled Testing (testing all genes at the same time cost consuming)

Ch 20: Mutation Detection Using Simple Non-Sequencing Techniques

Basic principles, advantages, limitations of: electrophoresis, PCR, restriction enzyme analysis, Southern analysis Electrophoresis o Advantage: simple o 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 fragment o Limitation: must know sequence of the fragment of interest so you can design a primer o Limitation: polymerases used in PCR can only extend DNA a finite amount o 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 amplified without realizing o Disadvantage: polymerase used have low but definite error rates o 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 sequences o Disadvantage: # available are limited, preference for palindromic sequences Southern Analysis o Advantage: specific DNA fragments of interest in a complex mixture can be examined o 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 expansions o 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, inversion o 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 called reputation RTPCR: quantity of product monitored during reaction. Why is this useful?

o o o

Product increases slowly until threshold is reached You can know exactly how much mRNA is in a certain tissue or cell type 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 IV DNA 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 stops o 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 pyrophosphate is 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 ends o 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 it Exome 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 mutagens o Most mutations are endogenous and are from:

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 function o Growth deficiency o Premature aging o Photosensitivity o Immunodeficiency/hematological o Cancer predisposition

Base excision repair Nts can become oxidized, methylated, or deaminated (C deaminated to U, or CpG methylated is deaminated to T) In BASE EXISION REPAIR (BER), the bad base distorts DNA, and this is detected by glycosylase, removes damaged base, leaving an apurinic or apyrimidinic nt o Next, an apurinic/apyrimidinic or AP endonuclease cleaves at that site, removing the sugar. The gap is filled in and strand is ligated by DNA Pol and ligase Two types: o Short-patch (for single-base damage) major pathway o 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 removed o PCNA and ligase involved in repairing the gap How are distortions detected? Two ways. o Global genome pathway: both transcriptionally active and inactive o Transcriptional-coupled pathway: only transcriptionally active gene regions Disorder when NER is messed up: xeroderma pigmentosum (XP) o Profound sensitivity to light redness, blistering, dryness, high risk of skin CA, eye involvement, hearing loss, cognitive impairment o Many different types of XP, all autosomal recessive Mismatch 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 proteins o MSH6 recognizes single nt mismatches o 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 disorders o Lynch syndrome (HNPCC) Tumor cells from these patients demonstrate microsatellite instability (MSI) this means high frequency of variability in length of alleles at microsatellite loci as a consequence of the deficiency

Strand break repair by homologous recombination, clinical manifestation of Bloom, Warner, and Fanconi anemia Single-strand breakage: repaired by BER (since SS breakage is part of BER) o One difference is the role of PARP (poly-ADP-ribose-polymerase), which detects SS break, binds to DNA, and begins synthesis of a poly-ADP-ribose chain as a signal to attract other repair enzymes Maybe PARP inhibitors could be used for cancer therapy Double-strand breakage: need DSBR this is harmful to DNA, and even if repair there can be consequences. Two types: o Non-homologous end joining (NHEJ): if broken ends are uneven, the protruding ones are removed, and then even ends are ligated with ligase this does not req homologous pairing, but it typically results in removing bases. This is kind of okay because of the % of non-coding DNA, but it could be serious when it goes occur in an exon this is acceptable because DSB is not ok and cell will die Deficient gene XRCC4 shows severe combined immunodeficiency o Homologous recombination repair (HRR): this knows that when there is DSB, theres a normal copy of the chromosome nearby. A segment of the normal chromosome transfers over to replace damaged segment via homologous recombination (Holliday junction), using a large # of enzymes, factors, helicases One risk is going from heterozygous homologous Genetic defects involving homologous recombination repair (HRR) o Bloom syndrome (BS autosomal recessive): BLM gene codes for a protein like DNA helicase and helps unwind the DNA. It also prevents crossing-over in mitosis (and because of this, a defect shows mitotic hyper-recombination, shown on SCE test, sister chromatin exchange when exposed to chemicals that cause DSB, suggesting chromosome instability). BLM prevents excessive illegitimate crossing-over. Also has some role in meiosis since patients have decreased fertility. Growth restriction, photosensitivity (face), immunodeficiency, pulmonary disease, DM, learning disability, infertility/early menopause, high CA rate o Werner syndrome (WS autosomal recessive): WRN gene encodes a DNA helicase. Decreased HRR. WRN protein interacts with telomeric binding factors, so they have short telomeres Premature aging, cataracts, retinal degeneration, scleroderma-like skin, ulcers, hair loss, atherosclerosis, DM, hypogonadism, osteoporosis, increased risk of soft tissue sarcomas, bird-like face o Fanconi anemia (FA autosomal recessive): FA-A through FA-N so many subtypes, caused by 13 genes. The FA proteins are complex, and together minimize or repair DSB. Its thought that mutation makes the cell favor alternative repair pathways (like NHEJ) Bone marrow failure (pancytopenia), radial anomaly (missing thumb), short stature, pigmentation abnormality, high malignancy rate (lymphoma) Familial Breast, Ovarian, Prostate, Pancreatic Cancer Syndrome o BRCA1 or BRCA2 mutation: proteins made by these genes provide scaffold for cell-signaling of proteins part of DSBR if not successful in repairing DSB, signals apoptosis

How telomerase deficiency causes genome instability, clinical manifestations of dyskeratosis congenita Dyskeratosis congenital (DC AD, AR, & X-linked forms of this): pigmentation of skin (reticular), dystrophic nails, oral leukoplakia (white patchy lesions), bone marrow failure. 6 genes linked to this o Cells have short telomeres and are premature replicative senescence (old?) o Spontaneous chromosome breaks Telomerase has two core components o TERC (telomerase RNA component) o TERT (telomerase reverse transcriptase) Telomerase requires dyskerin (coded by x-chromosome gene)

Parts of telomerase complex ALSO part of small nucleolar ribonucleoproteins (snoRNPs) these are used in ribosomal RNA processing Telomerase deficiency leads to tell cycle arrest and death of progenitor cells like in bone marrow (this could be augmented by impaired snoRNP function) Telomeric shortening causes chromosomal/genome instability because of fusion bridges when the chromosomal ends become exposed (repeat DNA ends join), then they break later on (breakage-fusionbridge 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 V More 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 also silence transcription o Occasionally, mutation of promoter region can increase transcription (ex: persistent expression of fetal 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 LCR 60kb away Translational mutations o Missense: single base substitution causing a codon change (common 1st or 2nd base 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 in chemical property, eg p.I306L isoleucine replaced by leucine); or it can be nonconservative (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 mRNA if at least 50 bases upstream of last splice junction, is degraded 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.W26X o Frameshift: shift reading frame due to change in DNA sequence this 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: del508F p.F508del means 3 bases got deleted and an F in the 508th position was del Splicing Mutations o Nt substitution (or insertion or deletion) at splice donor/acceptor sites abnormal 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 looks like a splice site)

Other RNA Instability Mutations o Mutations in poly-AAA site (AAUAAA) instable mRNA Mutations of 3-untranslated region can have the same effect o Mutations in 5 UTR poor 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 mutations inc 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 on inheritance pattern) o For many enzymes, having 50% is sufficient, so most enzyme deficiencies are recessive o Haploinsufficiency is 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 dominant negative 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 hypomorphic o 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 sequence that 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 receptors responding to signals to increase or decrease expression, which could be constantly repressed or activated o Allele that produces increased amount is hypermorphic o Quantity gain of function mutations typically near promoter region o Quality gain of function mutations typically missense mutations Penetrance vs expressivity Penetrance: probability that a disease phenotype will appear when genotype is present o 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 override the recessive allele o This is the case where homozygous is more severe than hetero (eg. Homozygous for achondroplasia is lethal) ABO is partially co-dominant o 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

Causes of non-random X-chromosomal inactivation Sex effects and embryonic lethality Consider above factors in genetic counseling