Genetics Exam 2

Lyon hypothesis - proposed mechanism of X chromosome inactivation to explain cat fur

***CLASS NOTES Coat color is determined by an X-linked gene Heterozygous female cats (XX) can be calico or tortoiseshell, expressing patches of orange (recessive) and black (dominant) fur Coat color in cats determined by x linked- hetero XX females can be calico or tortoise What phenotype would you product for a clone of a calico or tortoise cat? Answer: either orange or black fur depending on x inactivation of donor cell **wont be same pattern -bummer Made first cat- copy cat Rainbow cat was cloned donor but copy cat was black fur only— so entirety of organism are from donor cell that had orange gene on inactivated copy of x (soo no expression of orange on x that copy cat got!!) some of other rainbow cells were active for orange that's why shows this pattern but bb didn't get that specific cells!! for cloning of calico- taking one cell to begin so entirety of clone is based on that one cell, why will just give you phenotype of whatever not silenced in that particular cell The default setting for most eukaryotic genes is "off" Different tissue types have distinct patterns of silencing to maintain cell/tissue identity and function Therefore, any process that disrupts establishment or maintenance of silencing can lead to disease or altered function Animals vary in their X-inactivation during cloning and development cats maintain X-inactivation of the donor cell mice show full independent mosaicism consistent with re-establishment of X-inactivation in a clone cow clones show partial mosaicism indicating preference for inactivation of the X that was inactivated in the donor Cloned mice show full independence mosaicism bc x enact is undone and then can be reactivated so baby shows the maniac too epigentetics— not as permanent as changing dan sequence can be changed and reactivated over time!! To explain pattern of black and orange fur on cats Due to ransom x-chromsome inactivation during embryonic development Orange patches of fur are due to the inactivation of the X chromosome that carries a black allele (so gene not being read) and vise versa for black — SO in general only heterozygous female cats can be calico!! atch pattern can not occur in male cats bc don't have 2 versions of allele to be silenced and not silenced in diff cells Lyon hypothisis- proposed mechanism of XCI So gets B from mom b from dad and then one of them becomes Barr body in each growing cell but diff ones may become Barr bodies in diff cells **this makes sense bc the methylation has to reoccur again At what stage of development does X chrome inactivation occur? 1. Initially both X chromosomes are active 2.At early stage of embryonic development one of the 2 x chromosomes is randomly inactivated in each somatic cell and becomes Barr body—during the inactivation the chromosomal DNA of the inactivated chromosome becomes highly compacted into a Barr body so most genes on that chrome cant be expressed 3. As embryo continues to grow and mature each embryonic cell divides and gives rise to millions somatic cells and the XCI is maintained in subsequent cell divisions 4.Bc primary event of XCI is random process that occurs at early stage, result is animal w some patched of white fur and others black *so if have 2 diff patches its bc embryonic cells of those 2 differed and then they each went on to continue to divide w the specific Barr body Mammals maintain one active X chromosome in their somatic cells Normal female chrom composition: XX Number of chromes: 2 Number of Barr bodies: 1 Normal maled Chrom composition: XY Number of X chromosomes: 1 Number of Barr bodies: 0 Turner syndrome (female) Chrom composition:XO Number of X chromosomes: 1 Number of Barr bodies: 0 Triple X syndrome (female) Chrom composition: XXX Number of X chromosomes: 3 Number of Barr bodies: 2— so still just presenting one Klinefelter syndrome Chrom composition:XXY Number of X chromosomes:2 Number of Barr bodies: 1 **key is that if number of chromosomes exceeds 2- the additional chromosomes are converted to Barr bodies depends on x inactivation center!! X chromosome inactivation in mammals depend on the X inactivation center and the Xist gene There is a short region on the X chromosome called the X inactivation center (xic) Counting of human X chromosomes is done by counting number of xics— a xic must be found on an inactive chromosome for inactivation to occur (so count xics and know how many inactive X chromes there are) Found that if a chrome is missing a xic then the inactivation does not occur— XX is a lethal condition Xic works how? Certain gene in Xic that is required for the compaction of the X chromosome into a Barr body — gene called Xist ( for X- inactive specific transcript) The Xist gene on the inactivated chromosome is active (bc has to help condense for whole chrome to be inactivated) whereas most other teams on the X chromosome are inactive Xist gene produced is an ran molecule that does not encode a protein but instead its role is to coat the chromosome and inactivate it!!— after it coats other proteins come in to help w compactions into Barr body X chrom inactivation occurs in 3 phases: intitation, spreading, maintenance The spreading begins by the Xic and spreads in both direction along x chromosome When cell divides, Barr body is replicated and remains compact in subsequent generations In humans around 1/4 of the genes escape the effects of the XCI and are expressed on the inactive chromosome

comparitive genomics

Comparative genomics- approach to understanding genomes We can use what is known about one system to infer what is likely in another Uses some genomes as model to understand other genomes For instance, if 2 genomes carry identical copies of the same gene, it is extremely likely that the gene has the same role in those 2 organisms If 2 genomes have highly similar versions of a gene, it is likely that the gene functions the same in those 2 organisms For both red bullets above— shows sequence alignments can show sequence similarity — this is what the BLAST tool does- shows sequence similarity It takes multiple sequences and tells us the similarity in the sequence but doesn't really tell us much about the structure If 2 genomes encode 2 proteins that take on the same structure in two diff organisms, the 2 proteins can have the same function in the 2 organisms SO lack of sequence homology does not mean 2 encoded proteins don't function the same way just means they don't have the exact same structure Degree of similarity can help us predict functions of genes in organisms BLAST SHOWS BLAST Is a simple tool that compares sequences and shows identity/similarity Likelihood that the observed similarity is due to chance Locations of similarity and dissimilarity BLASTp compares and aligns protein sequences BLASTn compares and signs nucleotide sequences **so diff degrees of sequencing based on if sequences in protein phase vs nucleotide phase bf protein made- can do in either port or nucl phase USing BLAST Puts in blast a long nucleotide sequence that is supposedly designated to correlate to a known gene Then she copied the same sequence into the next box and hit BLAST- quickly it realizes they are the same sequence — shows a diagram of the alignment, red is a very good alignment, pink not quite as good, green medium, last is black- some similarity but not very much You put in sequence and then it searched against the copy to find similarity Will tell you the number of how many nucleotides match — in this case it was every nucleotide a match is a called a hit! Then also has an expect column This gives a percentage that these sequences may be same just by chance!!- so for one she put in bc its a copy there is 0 expect that correlated by chance!! Program then lines up all sequences that match Then goes back and changes copy a bit to see what it would look like if some dissimilarity First deletes 6 nucleotides from copy and then later in sequence goes and adds some T's in and then somewhere in middle changes cagcag to gaggag— then sees if they can find eachother using the alignment tool Now system says bc not all nucleotide match its 99% identical and their are gaps in 12 nucleotides— the 6 you deleted and 6 you added — still o% chance that these sequences are arising due to chance The deletion looks like this- like a hole to show you that a change in the number of nucleotides (deletion) does not change the whole alignment-- creates the whole to keep the alignment in sequencing-- rest of genome could be same To show that a sequence differs bw 2 strands— will not show vertical lines connect the 2 strands at that point -- no vert, diff at that point Another way Can take sequence you are interested in and BLAST it against all known genotypes in the data base!— don't have to do parallized one at a time alignments, can just say is this gene anywhere else in the whole known collection of sequences So can either align to or more sequence or just send to all known in database Can also choose to compare to just all human genomes in data base or all mouse in database etc

X chromosome in activation -- type of imprinting, methylation!!

During this in embryonic development one of the 2 X chromosomes in each somatic cell of females is chosen for random inactivation during embyro development

forward genetics

Forward genetics allows the identification of mutants with phenotypes of interest and leads to the mechanistic understanding of biological processes. Start with a phenotype, then look for the sequence/gene that causes that phenotype.// The mutagenesis alters sequences, but at random. The selection is based on the phenotype and you start there to find the sequence/gene causing that phenotype. **so mutagenisis- even tho we are messing w genotypes, we don't know where specifically messing w genotype so after find weird phenobarbitals, go back and see what mutagenesis did forward- phenotype--->genotype we ask: why does this individual look different? THEN we identify or map the gene works if: phenotype is genetic (bc if caused by enviro may not be passed onto offspring if setting up experiment!) phenotype due to single genetic difference or "allele" for forward- need one gene affecting phenotype and not due to enviro collection of mutants are screened, then back cross- some mutations will go due to doom backcross while others stay- choose phenotype of one that stayed then self cross again to increase homozygity there etc until just have that one!!

ATP dependent chromatin remodeling

Is dynamic changes to structure of chromatin that occur during the life of a cell Could just be local alteration in positing of one or a few nucleosomes— or larger changes that affect whole chromatin structure over a long distance Carried out by: ATP dependent chromatin remodeling complexes — In eukaryotes:!! Changes in nucleosome position and histone composition are key features of gene regulation!!— alter chromatin so that not all proteins made bc don't need all proteins for all cells! (so regulation can be done using proteins that alter polymerase structure but also changes in chromatin structure that affect ability of transcription factors to gain access to and bind to target sequences in promotor region! If chromatin in Closed conformation Transcription may be difficult or impossible- so would close when don't want to encode proteins at that region Open conformation More easily accessible to transcription factors Histone composiiton and positioning of nucleosomes at or near promoters often play key role in euk gene regulation! A nucleosome is the basic unit of DNA packaging. It consists of a segment of DNA wound around a core ("octamer") of 8 histone proteins (two each of histones H2A, H2B, H3, and H4). ... Some biotinylated Histones and nucleosomes have been added to our continuously increasing product range.— both used in compacting DNA into cells!! Chromosome remodeling complexes alter the positions and compositions of nucleosomes So we know nucleosomes are part of DNa where bundled up in ball using histones — so these complexes come in and change where these bundles may be and compositions of them! There is an interconversion bc open and closed conformations Nucleosomes have been shows to have diff position in cells that normally express a particular gene compared to cell w gene inactive! Ex: in red blood cells that express B-globin genes, alteration in nucleosome positioning occurs in the promoter region from -500 to + 200 nucleotide— so nucleosome moved positioning of bundle and now either B globin is expressed or not Transcriptional factors: one of their role is changing from open to close by altering nucleosomes at diff sports ATP dependent Chromatin remodeling - one way to change chromatin structure ** a chromatin remodeling complex is a ATP dependent chromatin remodeling complex!! remodeling complex!! Energy derived from ATP hydrolysis is used to drive changes in the locations and or compositions of nucleosomes (either making DNA more or less likely to undergo transcription) ATP dependent chromatin remodeling complex- carries out reaction Recognizes nucleosomes and uses ATP to lather their configuration All have a catalytic ATPase subunit called DNA translocase (similar to what found in motor proteins)— moves along the DNA Names of complexes refer to effects of mutations in genes that encode them !!! Realize that these complexes to change chromatin structure also arise from mutations in dNA!!- so not direct mutation and then creation of diff protein— it is mutation, creates this complex to go silence diff parts of genome! How do chromatin remodelers change chromatin struxture? 3 effects are possible They can change the positions of the nucleosomes over a long stretch of DNA May evict histones from the DNA- creates gaps where nucleosomes are not found May change composition of nucleosomes by removing histones and replacing them w histone variants HISTONE VARIANTS Histone variants play specialized roles in chromatin structure and function Total number of histone encoding genes varies from species to species — humans have around 70 that encode standard histone proteins BUT some have accumulated mutations that change the amino acid sequence of histone proteins Altered histones called histone variants For eukaryotes histone variants have been identified for H1, H2A, H3, but NOT H4 In most cases the standard histones are incorporated into the nucleosomes while new DNA is synthesized during S phase of the cell cycle- then later some of the standard hsitoens are replaces by histone variants via chromatin remodeling complexes!!SO ITS THE COMPLEXES THAT CHANGE THEM— this how silencing— job of complex is to silence and this is one way of doing it— putting histone variant in Key role of histone variants: regulate structure of chromatin : so influences gene transcription!! Sometimes adding variation allows for gene activation whereas other times works as gene suppression!! The histone code also controls gene transcription Each of core histone proteins has globular domain and flexible charged amino terminus (amino-terminal tail) DNA wraps around the globular domain (helping to condense the DNA) and amino terminal tails protrude from the chromatin Have discovered that particular amino acids in the terminal tails of standard histones and histone variants are subject to several types of covalent modifications! (acetylation, methylation, phosphorolyation) — so amino acids of part f histone— their DNA sequences can be changed There are 50 diff modifying enzymes that have been identified in mammals that selectively modify amino terminal tails of histones!!— SO have ATP dependent chromatin remodeling complex that does the :change in nucleosome position,position, histone eviction, and replacement w histone varients— then also have modifying enzymes that are changing the tail part of histone complexes!! Ex: histone deacetylase removes acetyl groups which makes DNA less tightly bound to histones would now allow transcription in these areas Histone code hypothesis: Histone modifications occur in patterns that are recognized by proteins According to hypothesis - pattern of histone modification acts much like a language or code in specifying alterations in chromatin structure One pattern for ex could be phosphorylation of 5th and 8th position in H4, and acetylation in first position of H2A and then other pattern could be completely diff places in them -- all depending on what needs to be expressed Overall the code: plays important role in determining whether info w in genomes of euk species is accessed Many euk genes show a common pattern of nucleosome organization For Active genes, or genes that can be activated (so nucleosome not too tightly bound here)— promoter is found at nucleosome free region (NFR)

gene interaction transition

So far- have studies effect of single gene on outcome of trait—helps us understand various ways alleles can influence traits Mendel did this- looked at tall vs dwarf pea plants at single allele BUT more than one allele contributes to height so how did he do it? Answer is in genotypes of his chosen strains- although many genes affect the phenotype , he chose true breeding strains that differed w regard to only one of those genes ** so if both plants have all other genes same— only differing in one- then can isolate the effect of that gene on phenotype bc all other phenotypic aspects of other genes should be same Ex: if pea plants has 4 genes affecting heights , genotypes of 2 hypothetical strands would be Tall: KK LL MM NN Shortt: KK LL MM nn If cross these 2- will differ in genotype of only one gene- other 3 will be identical in all of them Researchers accept that essentially all traits are affected by the contributions of many genes - morph features like height, weight, growth rate etc all affected by expression of many genes in combo w enviro factors Epistasis: inheritance pattern where alleles of one gene mask the phenotypic effects of the alleles of a diff gene Complementation: phenomenon in which 2 diff parents that express the same or similar recessive phenotypes produce offspring w wild type phenotype Modifying genes: phenomenon in which allele of one gene modifies the phenotypic outcome of the alleles of a diff gene Gene Redundancy Pattern in which the loss of function in a single gene has no phenotypic effect, but the loss of function of 2 genes has an effect. Functionality of only one of the 2 genes is necessary for a normal phenotype: the genes are functionally redundant gene redundancy Phenomenon in which one gene can compensate for the loss of function of another gene. May be due to diff underlying causes Gene duplication: certain genes have been duplicated during evolution — so species may have 2 or more copies of similar genes (not identical bc random changes during evolution) — the genes are called paralogs When one gene misisng, parlor may be able to carry out the missing function May involve proteins that are involved in a common cellular function , when one protein missing due to mutated gene, other protein can be increased to compensate for missing protein and so we see no phenotypic defect Ex: Shull used a weed known as Shepards purse Trait he followed= shape of seed capsule Commonly triangular When capsule is smaller oval looking strain is homozygous for loss of function alleles in 2 diff genes (ttvv) Crossed true breeding plant w triangle capsules (TTVV) to plant having ovate capsules (ttvv)- F1 all had triangular (TtVv) BUT- when F1 self fert, F2: 15:1 ratio of triangle to ovate capsules Can be explained by gene redundancy Having at least 1 functional copy of either gene (T or V) is sufficient to produce the triangular phenotype! (SO EVEN IF HOMO RECESSIVE AT V BUT HAVE DOM T, THIS IS ENOUGH!) **so gene redundancy has opposite effect of epistasis- just need one dom and that is sufficient enough for expression of protein T and V are functional alleles of redundant genes — only one necessary for triangular shape Need function of both of them to be lost (ttvv) and capsule will become smaller

yeasts as model system

Tiny Super fast life cycle- 90 minutes to 4 weeks Small genomes, few introns 25% of yeast genes have human homologs **Haploids mate to form diploids and then those diploids can undergo meiosis and those products of meiosis stay together or can be separated to grow independently as haploids Yeast life cycle- A life cycle Single cell starts to replicate its genome and form a bud- bud grows and then buds off, so bud separates into 2 — this leaves 2 haploid yeasts as products— this can continue multiple times through multiple rounds of mitosis to grow into a yeast colony Second mating type: called alpha Can go throw same life cycle as one above- divide create bud and go through haploid vegetative life cycle A and alpha create 2 distinct buds in their haploid life cycles— then they can fuse together — mating (bc both haploid) to create diploid— their cells fuse and the nuclei fuse - so diploid has a/alpha Diploid can undergo mitosis and create diploid colony of yeast OR can be induced to form spores - undergo sporulation — involves diploid undergoing meiosis forming 4 spores each of which are haploid — then those spores can undergo the normal haploid life cycle like a and alpha think of meiosis regular where at end 4 haploids creates —but now those haploids created are haploid spores that can grow into independent colonies of yeast WHEN WANTING TO UNDERSTAND GENE INTERACTION WITH SPORES THIS IS HOW!! Consider a haploid yeast with 2 mutations, a and b (mutant alleles of the Land B genes) Mate the ab double mutant with wild type to form AaBb diploids **always get to diploid bc the interaction bw alleles will be seen when heterozygotes mate!! Induce meiosis in the diploid and observe a or A phenotypes , b or B phenotypes and frequency of ab, AB, aB, Ab phenotypes in the haploid products of meiosis Similar to a test cross where we are looking at the phenotype in the next generation but with out the need for future crosses - don't need to cross it to a homozygous animal bc its not an animal— its just a yeast can just induce meiosis in diploid and see what haploids look like w out having to cross to anything else! Because yeast are so small and easy to grow, it is possible to examine the products of multiple different meiosis events on a single agar plate — grow each meiotic product in a separate colony 4 products of first meiosis are 4 vertical white dots in a line and then of 2— ones next to it and so on— can then take this plate where all of the meiotic products are grown as their own colonies- they replicate and undergo mitosis— forming colonies Can then do what is called a replicaplating Where exact same arrangement of colonies is moved to a different plate which represents different conditions Can transfer them to a plate to test for A vs s phenotype— only ones showing A may appear now and same w B vs b on diff plate— then can look at ratios of Aa or Bb — the combos of alleles in phenotypes of haploid yeast— so don't need another cross just bring out their genotypes by putting them on plates in specific conditions!! Observable yeast phenotypes are often about colony growth Bc yeast are so small, their observable phenotypes are limited and usually are about colony growth — so we look at whether yeast can or cannot grow under specific conditions can/cannot grow under specific conditions Ability to make important molecules like amino acids— would be surveyed by growing them w out those amino acids and then seeing if they can make them — if can make it themselves can grow, if can't, cant grow Ability to grow at higher or lower temperatures Ability to grow on specific limited sugar sources Colony morphology or color can change based on build up of specific molecules that may be important in metabolic pathways So looking at pic above- would say a genotype can not grow at all whereas A can then tell based on test plate who got the a from heterozygote parent and who got A- or who got aa (no growth ) who got a A-- can find ratios based on growth based on how many dots of each we can tell ratios of A to a and B to b

Chromatin state and DNA methylation are examples of epigenetics

epi- because it is an effect "upon" the DNA sequence, silencing a gene that is perfectly functional as encoded -genetic because it is heritable over cell divisions once X-inactivation occurs in development of an XX individual, the same X will remain silenced in progeny cells once a sequence is methylated during gamete formation, it remains silenced in the somatic cells of the offspring Epigenetic effects are not as stable as DNA sequences can be un-done or established in response to some environmental conditions boundaries between heterochromatin and euchromatin are not perfect an can vary between cells

epigenetic

changing function by not actuallyy changing sequence Epigenetics: Heritable control of gene expression pattern, not just DNA sequence so its the heritable- bc can be passed on but has to be turned on again Silencing intact genes via heterochromatin methylation X-inactivation uses both Imprinting uses DNA methylation X-inactivation ensures that XX, XY, XO, XXX, XXY, XYY individuals express X-linked genes at the same levels X inactivation- 1st on pic- 1 X chromosomes is completely silenced in every cell- complete silencing is called inactivated and only the other copy is expressed C elegans- diff approach- XX individuals express X linked genes at half levels compared to expression level in XY- works too and both have same level of genes Drospohilia- per or higher transcription in the XY indivisual0 so twice as much df single X is same as regular 2 X's in humans: Xist RNA (labeled red) covers one of the two X chromosomes and recruits silencing factors (histone modifying enzymes, cytosine methyltransferases) Xist- gene that codes ran - pink is specific list ran which is localized around 1 copy of x chromosome and that mediates its silencing - list can help recruit silencing factors, include histone modifiers *xist can encode for proteins that recruit hsitonse factors to change histone shape X chosen randomly when X inactivation occurs- then occurs from there on Shown on left pic One of x chrome randomly chosen to be inactivated then when x cells keep dividing0 progeny cells keep maintaining inactivation — X cells maintained same choice so all those have to Random process that does happen at 4 cell stage !! Happens later and because of this individuals have very skewed values of which X's inactivated vs not sometime sin random choice there is this skew that leads to mostly expressing matters copy X on one side of skew and then most stressing patronal X on other side of skew— amnt of skew effects which phenotypes shown can either be from mom or dad one silenced- there is a skew Bc of this epigenetic effect every cell will only express one version of x linked genes!! (bc skew is so far towards!) So even if some cells are incapable of forming clots— if skewed towards wild type enough of the other types are capable of blood clotting that they will not express hemophilia trait After choice is made at early stage of development that is set X INACTIVATION MEANS THAT HETEROZYGOTES CAN SHOW RECESSIVE PHENOTYPES!! Images show diff tissues in a mous- red is one copy of x and green other copy of x being expressed Shows mosaicism in tissue- diff levels of prdominasim - variation from tissue to tissue - diff levels of mosaicism Opp of clumpiness— in cartilage tissue big chunks of green vs in cornea green sprinkled- just bc diff x inactivation patterns are diff in diff tissues Lab is looking at how this effects things like function in x linked genes

DNA methylation

one way to inhibit gene expression DNA methylation (inhibiting gene expression) One way to inhibit gene expression Cytosine bases in DNA can be methylated by methyltransferases Pic shows cytosine C base attached to a backbone — the DNA methyltransferases can specifically add a methyl group to the cytosine which alters its structure Methyltransferases target Cs when they occur in 5'CG3' sequences aka CpG (called CpG bc there is a phosphate group in between the C and the G)— the CG sequence will appear on both strands because G pairs with C (so if CG right by eachother then on opp strands it will be GC bc they pair w eachother!!) , so double stranded DNA can be unmethylated, hemi (half) methylated (one of C's is methylated) , or fully methylated (when both C's in the CG sequence are methylated) Methylation of cytosines alters the structure of the DNA double helix Methylation can prevent proteins such as transcription factors and activators from binding to CpG sequences — if they methyl is there and proteins cant bind, transcription can't occur Promoters and enhancers often have multiple (thousands) of CpG sequences nearby. These are called CpG islands— would make sense that the CpG sequences are by the promoters and enhancers bc they are going to dictate whether protein made or not— cells only want proteins specific to their function to be expressed so want some to be methykized and some to be demethylized Expression of the associated genes can be controlled by methylation (which silences expression) or demethylation (allowing expression to occur) CH3 is methyl- imagine it sticking off Housekeeping genes- Genes that encode proteins required in most cells of a multicellular organism — in these genes, the cytosine bases in the CpG islands are unmethylated (makes sense bc we need transcription of these genes)— so housekeeping genes therefore tend to be expressed in most cell types Tissue specific genes Genes that are highly regulated and may be expressed only in particular cells — expression of these may be silenced by the methylation of CpG islands SHOWS DNA methylation plays important rile in silencing of tissue specific genes to prevent them from being expressed in wrong tissue!! (so not always like thinking about it in terms of mutant!!) Methylation can affect transcription in 2 general ways 1) Alteration in the binding of regulatory transcription factors The methylation of CpG islands can prevent or enhance binding of regulatory transcription factors to promoter region Bc the methyl groups protrude into DNA- may prevent biding of activator protein to enhancer element (sequence on DNA) **See bc the CH3 sticks- the orange poly cant attach to DNA Binding or Methyl- CpG binding proteins 2)methylation can inhibit transcription through proteins known as methyl- CpG- binding proteins These proteins bind to methylated sequences and contain a methyl binding domain that specifically recognizes a methylated CG sequence Once bound to the DNA, methyl proteins recruit to the region other proteins that help to inhibit the transcription (for ex may recruit histone deacetylase— change histone so transc cant occur— makes ir more diff for nucleosomes to be removed from dNA so transcription can begin ) DNA methylation is Heritable The methylated DNA sequences are inherited during cell division How can methylation be inherited from cell to cell? 1. DNA in a particular cell may become methylated by de novo methylation - the methylation of DNA that was previously methylated 2. When a fully methylated segment of dNA replicated in preparation for cell division - newly made daughter strands contain unmethylated cytosines — now bc only one strand methylated— said to be semi methylated 3. This hemimethylated DNA is recognized by DNA methyltransferase which makes it fully methylated!— process called maintenance methylation (maintinang the full methylation) **maitanance methylation does not act on unmethylated DNA

suppression

when one mutation reverses the effect of another mutation, causing the double mutant to have a wild-type phenotype. **NOTE: in suppression double mutant has WT-- in epistasis double mutant would still cause mutant **if suppress your feelings, become doublyyy sad any time doing mating of one maybe that is ab and other AB-- don't think of filling up diploid in offspring -- look at haploid comobos-- so just diff ways of getting AB combos which we know is 4!!

RNA sequencing, reverse translation of protein sequences, homology searches for conservation in related species what each of these methods will help show

** whole point of this is that a single genome is not sufficient to confidently identify genes in a new eukaryotic genome-- so what you do is sequence the genome and then do another approach to compare- some types of genes identified by this approach and some missing RNA Sequencing RNA sequencing should show processesd transcripts sequences and in combo w genome sequence -- could know exon intron boundaries, if have full genome and also know where RNA sequencing is occur-- can see where boundaries are again, Reverse-translation of protein sequences will show what is encoded by exons. In combination with the genome sequence, you can see the boundaries. Homology searches would likely show higher homology in exons compared to introns.-- if looking at genes that are similar in another species-- our genes that are going to be similar w another species are our eons (bc imp part coding proteins-- doesn't matter what introns are-- those prolly through evolution of species) - will show you transcribed, expressed sequences-- so the exons and how they got together ** this still gives you more than just rna coding for protein (mrna)-- will give you trna, regulatory rna(HUGE RNA LETS U SEE REG RNA!- can see regulatory sequences) *if use multiple samples of rna sequences from diff tissues, can see alternative versions of spliced mrna (!! so some regions may be regulated (turned off in mrna of certain cells doing certain things-- can see that!) drawback: have to be careful about sample selection because may miss specifically regulated transcripts not expressed in this specific sample Isolate cellular proteins and reverse translate the amino acid sequences to find gene sequences **if can find protein (which this does) 100% sure that it is truly encoding a protein found in the cell- no doubtt drawbacks: the protein is cleaved when get it, only part in protein that you get is N terminus, C terminus is cleaved (sorry coops), processed proteins confound result, don't see all of protein, more than one way to encode amino acids (6 codons encode serine!), ONLY helps you find protein encoding genes, but a lot more genes effect phenotype! Identify locations in the genome where transcription machinery binds and look nearby for gene like sequences -thourough, all genes should be bound by transcription factors if will be expressed at some point!! and if not don't care about them anyway! Includes all types or rnas! not just protein encoding! BUT: just bc you find promotor (where poly binds) doesn't mean you know how far upstream the protein encoding sequence begins!- missing ones not expressed in particular samples- missing ones not expressed Identify sequences that are conserved in related organisms we have actual sequence of interest! comparison programs are pretty fast can also set program to include ones just identical or also ones that are pretty similar know these sequences important bc due to selection, utility -- maintained through evolution for a reason , if mouse needs it and we need it! prolly important!

exons, introns, translation IMPORTANT HW PICTURE

***from class- exons- encode proteins- first exon should start w start codon then after first exon, length of exon will be length of 3 nucleotides bc this is how ribosomes read mrna exons end w splice site donor -- will go to the acceptor which is start of next exon exons very short compared to introns in eukaryotic genomes introns then- intervening and not neck multiple of 3, bc who cares they will just be x'ed out anyway NOT part of processed mrna, not read by ribosome THEY DO THO contain sequences necessary for splicing- so have to have splice acceptor so splice donor on END of exon (bc will read exon then splice it) splice acceptor at END of intron- so that makes cut-- at 3' end of the intron, so when it gets sliced ***PIC We can get sequenced genome from various methods talked about - but then how do we go find the genes? 1 way- look for exons, open reading frames open reading frame- place where no stop codons for a while 3 rows- +1,2 and 3 are 3 reading frames and then -1 -2 -3 are 3 reading frames **bc we don't know where reading begins-- the triplets will change based on where you start reading !! the top 3 reading frames- the + direction are encoded in one direction (+ direction, right arrow- forward direction) and the other 3 are encoded in the reverse direction (why have the neg!!) BC remember there are 6 ways in which a reading frame can be seen-- we don't know if top or bottom strand is template strand so first 3 are if top strand is template strand- then they are starting at 3 diff genes, for first codon, then bottom 3 are if bottom strand is coding strand-- and same w its 3 options!! 2 exons can be in 2 diff frames as long as encoded on same mrna!!-- so think all just possible pieces going together so if creating couplets of 3 that would go together even tho started at diff reading frames, if in same direction, can go together but bottom exons are encoded by completely diff template- are not part of that same mrna -- so cant be spliced together!! **frameshift mutation moves mutation exon down a frame-- so goes from top row to row below it !! -1 framsefhit would move down 1 row, -2 frameshift would move down 2 take beginning of exon looking at and then move cursor down a row and if vert line bf vert line where originally was, then early stop codon this is true unreal dna- if take any sequence of dna and try to encode it, won't encode bc there are so many stop codons ORF- thinking exons, bc no stop codons in middle May or may not be included in all transcripts encoded by the gene (alternative splicing) May or may not be included bc of alternative splicing — sometimes when introns removed exons removed with them -- could mess up technicques like Rna identification "expressed" sequences found in mature processed mRNAs May or may not be included in all transcripts encoded by the gene (alternative splicing) May or may not be included bc of alternative splicing — sometimes when introns removed exons removed with them Include the sequences necessary to encode amino acid sequences -- so exons used for mrna exons have ot include promotor so tuna can bind to promoter! Activator proteins can bind at enhancers which favors transcription factor and RNA polymerase binding to the promoter Enhancers can be far away from promoters and or can be within coding region in in introns -enhancers, help bind to promoter A gene is double stranded DNA but the transcript is single stranded RNA Transcript synthesized from its 5' end to its 3' end Introns are removed during the synthesis of the mrna and spliced transcripts will have Exons only— so in that pic— huge gaps will go away bc spliced out **not every version of transcript will have every exon!— so could have DNA strand that we are looking at w 5 diff externs and in transcript maybe only 2 of those are included in mrna transcript ** each double stranded DNA could be read in any of 6 ways depending on- which strand is coding strand, where ribosome begins transcription **splice donors are at end of exons-- splice acceptors at beginning bc eaccpeting the fact their little region will be spliced

Illumina

ILLUMINA 4 steps: 1- library preparation dna sample broken down and ligatures glued to either side fragments then amplified on gel- like we know how 2- cluster generation fragments enter flow cell where adaptors are that complement adaptors we have ligated onto incoming fragments then each fragment undergoes bridge amplification cycle (creating clusters) 3- sequencing then throw in the nucleotides and they are fluorescent and as they attach down strand sticking up- will fluoresce their color as building strand!! then use a reference genome to align the diff fragments reference genome- manmade genome sequence that expresses a specific species set of genes program then goes and looks for differences bw reads and reference genomes 4- data analysis best for high volume of DNA (bc think how long it takes to create electrophoresis for each null for each strand)-- if have a lot to do-- this good 1- start off w pieces of DNA you want to sequence, — these sequences are small (SO DOWNISDE: to illumine is usually only sequence 100-200 basepairs) SO, can shine in high volume w low base pairs!! 100-200 bp per reaction, small pieces and get the pieces by sharing your dNA 1- get sequences and amplify (PCR) 2-have these adaptors (ligate adaptors— are known sequences - you know what sequence is on them — so don't know grey sequences (ones in bw them) but do know what pink and purple sequences are 3- physically covalently bond the pieces of DNA to glass in a sea of other adaptors which are also stuck to the glass— these are single strands of DNA bc the double strands you had were melted and covalently bound to glass — surrounded by sea of adaptors that compliment these strands— so what happen is is that DNA is not a rod, it is flexible piece of spaghetti so it flips around and wiggles around till it finds an adaptor it can base pair w — form these loops on DNA- all single stranded DNA loops over and base pair w the complimentary strand that is covalently bound to the surface (bc remember they are all single strand- so looking to pair) So in ex above the pink adaptor is attached to the surface but its other end is not, it is covalently bonded to another strand— so now that have loop can add polymerase and primers to create double strand— you use the ligate adaptors that have stuck to eachother as primer to create rest of complimentary strand NOW— last pic- have double stranded piece of DNA where one strand is covalently bound on one side and other on other -- one terminus is attached to glass other is free SO, when heat up again, now you have 2 strands of DNA that are covalently bound bc have copied single strand of DNA— essential performed PCR on a surface, but not have primers that are covalently bounded to glass ABOVE IS PCR PART- amplifying!! Then heat up- will separate again and go find other primers on surface, can repeat this again and again Create little islands that are result of single strand copied many times — each island single strand of DNA amplified **KEY- have start w low density of DNA strands that are fairly well separated (bc think only wanna start w so much on plate so that plate can be filled w amplification Each nucleotide has diff color bc flurophyll so could take picture and each little cluster would have diff color — so all added is just primer and 1 nuceleotide bc blockage THEN add chemical which cleaves off the dye (laser incisition, so taking off base that was just tagged and moving onto next!) , wash it away and now image black again , then add new polymerase and take another picture, every time you take picture , color of dots represents new nucleotide which was incorporated (so when washed first one took top nucleotide off strand that terminal dntp was bound to?— every time taking picture, taking pic of one of those reactions as colors change in each reaction, that can tell you which nucleotide was added So first pic certain dot may be red, then it is cleaved and that same space (next nucleotide down on strand) may show green which may mean T So you sequence by taking pics of slide and looking at how each dot changes color THEN have huge machine that can sequence many of the flow cells at once answer: 1 lane— huge improvement from Sanger sequencing which required thousands and thousands of wells — w single lane you can sequence entire genome!!bc you can get so many reads based on these tiny little dots SO this is really good at sequencing LARGE VOLUMES of dna SO, illumine is why on chart you see are drop in cost for sequencing DNA Sanger is still gold standard bc if just wanna sequence single gene or single portion of dna- illumina is overkill don't need all those dots If want whole genome- illumine way to go bc so many reads

pathway

Pathway is a set of events that connect together Biosynthetic pathway: a series of reactions that result in the formation of a biomolecule Metabolic pathway: a series of reactions that form or break down molecules Signaling pathway: a series of reactions and/ pr interactions that result in cells or organisms responding to their environment Any step in a generic pathway could be disrupted or any component of the pathway can also be diverted based on other mutations (so now arrow of diversion going a diff way than arrow of path) Important to remember that multiple genes contribute to the pathway and its associated phenotype and multiple mutations can affect the pathway and its associated phenotype

epistasis, complementation and suppression

epistasis- think arg pathway- 2 genes needed to be functional for pathway 2 work- if one is not working, pathway doesn't work-- is just need one double recessive and that dictates phenotype of other gene!!

SO, big thing above Is that sequence info alone and eyeballing regions is not enough to determine-- what else can we do-- how can we use sequence to find actual genes ?

identify RNAs and look for genomic sequences that match -will show u transcribed, expressed sequences -exact readout of which sequences are exons and how they go together (bc bf on comp couldn't even see exons) -going to give you data NOT just for mrnas-- NOT just for RNAs encoding proteins-- says RNAS in general , this can give you trans, regulator rna, ribosomal rna - if use multiple types of samples from multiple tissues, will show diff in diff genes expressed based on cell types - CAN SEE ALTERNATIVE SPLICING PATTERNS, ALTERNATIVE VERSIONS OF SPLICED TRANSCRIPTS!!-- CAN See what does this gene or region express in blood cells vs muscles diff cells have diff splicing patterns, think may splice something out in one cell if don't need for function CON- although can see gene diversity in cells, if gene is gene in some cells and is fully expressed but choose rna from cell where not expressed, will miss it COULD use blast and put the transcript on blast and go find where in genome it is!! could be rna sequence that matches w multiple places in genome and this could be confusing! isolate cellular proteins and then reverse translate amino acid sequence to find genes - if you can find protein, you are 100% sure it is expressed -- there is no actually this rna is used for something else, if know protein know its being used -pos sequence truly encodes protein that is found in sample - CON: if protein that is already cleaved in sample- then only n terminus is apparant and functional and stable -- missing c terminus (c terminus is regularly and often cleaved off-- can confound results if processed CON: more than one way to encode amino acid-- if you see a serine- there are 6 codons that do it- how do u know which one did it CON: only will help you find protein encoding genes, no reg, np tuna, no rib rna isolate places in genome where tractiptionary machinery binds and look for nearby gene like sequences VERY thourough, all genes should be found by transcription factors and rna polymerase at some point of they are going to be encoded CON: gene structure is diverse, we don't know how far away enhancer is from promoter- they are connected but could be far- just bc find promotor doesn't mean located immediately upstream from promotor - PRO- this also includes all diff types or rnas not just protein encoding ones like the protein one did BUT caveat, is if tracking specific conditions or expression, diff genes expressed in diff genes in diff samples at diff times in development so will only get rnas expressed in specific ex find sequences conserved in related organisms we have the sequence! comparison programs are pretty fast!- can sit parameters of how fast want it to be ! BC IN SOME SAMPLES SOME GENES COMPACT INTO CHROMATIN SO DONT SEE! maintained through evolution for a reason- due to selection or utility!! if important, if moss needs it we need it, prolly something functional like a gene! any gene type- need to include transcripts from diff cells at diff stages of devlopment so if looking at mrna sequence or protein sequence to understand functions of genes-look in all of these diff places bc gene may not be gen in one place but may be critical gene in another

complementation

so first thing you see is both parents recc- kid WT - this usually occurs bc ccBB CCbb-- 2 enzymes to create purple so lo Complementation: phenomenon in which 2 diff parents that express the same or similar recessive phenotypes produce offspring w wild type phenotype Refers to the production of offspring with a wild type phenotype from parents that both display the same or similar recessive phenotype ***LOOK BACK TO PEA PLANT EX— THIS CAN HAPPEN BC TO HALT PURPLE PRODUCTION JUST NEEDS TO BE HOMO RECESSIVE IN EITHER C OR P BUT COULD BE DOM IN OTHER ONE THEN WHEN MATED W SOMEONE WHO IS ALSO DOM— CAN CREATE PURPLE FLWOERS!! Typically occurs bc the recessive phenotype in the parents is due to homozygosity at 2 diff genes — one parent was CCpp and other ccPP

epistasis

when the genotype at one locus determines the phenotype regardless of the genotype at a second locus. Partial credit for partial answers "one gene controls another gene"

suppressors worksheet

in suppressors-- a double mutant - so homo recessive on both genes actually is creating the WT phenotype SO more ways to attain WT phenotype then just having dom allele- ones w dom allele will have WT, double recessive will have WT-- if just has first mutation- then will be mutated bc suppressor not yet found -- w suppressors always do possible gametes phenotype and genotype bc phenotype not always indicated by genotype bc of the double mutation (double recessive)-- double neg sign

how to know correct order of genes?

the lowest frequency recombinants will represent the gene order because a double crossover only changes the center one-- so know which is in center ** to figure out which is in middle-- first look at parental genotypes-- we know which are parental genotypes bc will be the 2 in highest frequency (bc w out recombination) THEN compare those highest frequency to the lowest frequency and see what changed in ex had Apt and and aPT highest APT and apt lowest **What changed was the A so it must be in the middle bc for a double crossover the allele in the middle is what is changing!!

23 and me and microarray technology, affymetrix

About half of the human genome consists of various repeating sequences. Only ~28% of the genome is transcribed to RNA Only 1.1% to 1.4% of the genome (~5% of the transcribed RNA) encodes protein. Only ~30,000 protein encoding genes (open reading frames or ORFs) identified. Predicted beforehand we would have 100,000 ORFs. Only a small fraction of human protein families are unique to vertebrates; most also found in other phyla Two randomly selected human genomes differ, on average, by only 1 nucleotide per 1250; that is, any 2 people are likely to be >99.9% identical. NEITHER NUMBER OF GENES NOR GENOME SIZE CORRELATE TO COMPLEXITY OF GENOME- also 2 genomes w really diff sizes can have same number of genes !! Wide range in genome size bw organisms but once get to multicellular organisms number of genes is relatively constant — diff in sizes of genomes has little to do w number of genes The large genome is not due to large number of genes when get to multicellular organisms but has to do w repeated sequences— so may have way bigger genome but w just a lot of repeat genes so actually actuall diff gene count may be same compared to smaller one! If barely represented in pop- mutation Polymorphism - allele that is NOT rare in the pop — more than one allele exists at the same locus in the pop—not rare To be considered polymorphism- just need 1% of pop to have it- seems small but of 7 bill enplane that means 70 million- single nucleotide polymorphism would just be diff in single nucleotide — lots of these in human pop SO when sequencing like 23 and me, focus on all the single nucleotide polymorphisms that are common in the population and which combo you have relative to others bc that is what is making you distinct SO— fi looking at these don't have to sequence whole genome bc if you have a reference genome, you know where all the single nucleotide polymorphism are so can just sequence that part of your genome and see which poly you have there!! Affymetrix snipshift- when just wanting to isolate loci where polymorphisms of interest are This 23 and me!!— uses this to genotype youRELATIVE to pop— so all a comparison of you vs pop Idea is that you have complimentary strands of DNA that base pair Idea is that you have complimentary strands of DNA that base pair You take probe array — this has the know sequences of DNA (where polys are) that we are wanting to look at Then can amplify strands of DNA that complement the strands on array YOu have tagged DNA (DNA coming from person and flow it over the probe and if they stick to the array— that means they are base pairing w the probe array at diff locations of interest- then take pics and those regions of the chip will be fluroeesecnt if the DNA bound bc DNA coming in has been tagged q fluorophyll If no pieces in genome that match probe array, that region would be dark! Sample dna fragments (purple get washed over DNA array) Some samples- if base pair match hybridize to DNA probe— if not mathc, wont stick The labeled strands coin in are carrying fluroresecnce on them so The squares are multiple repeats of same strand of DNA in cluster- usually about 25 bp long Each chip 6.5 mill features For humans usually a million or so snips of interest — these used to differentiate bw diff human populations Also certain snips we know of to look at for being susceptible to certain diseases When see chips w dots— those represent the little clusters we talked about earlier— if have millions of identical probes of same sequence— thats what allows for dot to be seen on chip! Millions of identical probes per feature and about 6500000 features!!- so that many diff snips and w in each snip is millions of identical copies!! Then as can see have put 4 probes on surface— all 4 proves compliment the reference sequence at the flanks - but differs at the polymorphism SO- if you had A here in reference sequence— your DNA should match the T row SOO you are the reference sequence and then have put in probe sequences of all same except diff at that poly location — put in each diff letter their in probe and then whichever your dna binds to u know it has complimentary IF have both (which will have 2 alleles at that location bc have allele from mom and dad, your DNA will match the T and C if have A and G The 2 A and G— why they are there?- act as controls bc what we are doing is having binding be done or not done only bc of single nucleotide so it shoullddd be that if you have diff nucleotide in a location, the whole strand wont bind BUT in some cases you get non specific binding where the DNA is only binding due to the common sequences around the poly— if thats the case you will get binding on all 4 — so if get binding on A row or G row- ones that don't correspond to either polymorphism— shows cant really trust binding bc prob a lot of unspecific binding going on!! If all 4 light up reddish— cant really trust data! If just one row lights up- means homozygous for that allele Can look at all these slips together on glass slip and see if you have 1 allele or 2 alleles— this what 23 and me does- can see whether homo or heater!

imprinting -- causes heterozygotes to be 2 distinct gametes!!!- due to methylation!

An imaginary gene (A) that is imprinted in male gametes The gene (we are calling A) is going to be silenced in male gametes How it works: Start with 2 heterozygous parents — both parents express the "A" gene and both Carry the mutant "a version" but it is not expressed — both express the A allele but both carry a version Then During gamete formation First methylation is removed - both versions of the gene are methylated in sperm development — bc this is male imprinted — paternal gametes (gametes that are getting copy of chromosome from dad, are going to have the gene imprinted) BUT during egg development those eggs develop without that methylation - both egg versions of the gene are not silenced THEN in the next generation, 4 combinations of gamete genotypes are possible (aa,aA,Aa,AA)— in blue is maternally inherited chrome and in green paternally inherited (bc each baby is getting one from mom and one from dad and each parent has 2 diff alleles to give) What you can tell: It matters which parent you got the various alleles from — ex: look at the heterozygotes that have same genotype (Aa) but diff phenotypes bc the A has been silenced if received it from dad!!— so not heterozygotes are 2 distinct gametes!! so no matter what copy you get from dad both his little a and big A are methyalted-- so if his A is contributing to heterozygote then that hetero will show mutant phenotype whereas if his little a is going into hetero- not functional so mom A will show phenotype! Note: silencing of alleles is not affected by the sex of the individual who inherited the genes, only the sex of the person who gave them the gene (in pic sex isn't even shown!! Not like more common in males or females like a sex linked pass on— just that it is passed on from one sex or the other of adult!!) ALSO NOTE: the 2 babies on the left (aa and aA) have different genotypes but the same phenotype bc that bigA in heterozygote is silenced!!— as long as you got that a from the maternal source, that is going to be one that is expressed 2 on right heterozygote Aa and AA— they have diff genotypes but same phenotypes EPigentic inheritance These are result of chromosomal modifications that occur during oogenesis, spermatogenesis or early stages of embryoogenesis —so occur when making egg and sperm — they are altered in the early stages of life like this and then these changes alter the expression of certain genes that can be changed over course of persons lifetime — they can permanently effect life of individual BUT are not permeant over course of many generations! This is bc—! They do not change the actual DNA sequence A gene could undergo an epigenetic change that inactivates it for the lifetime of an individual —- BUT when this individual makes gametes, the gene can become activated and remain operative during the lifetime of an offspring *no perm change to actual sequence In reg imprinting, birds identify marks on beaks to allow them to distinguish, during genomic imprinting— analogous situation where segment of DNA is marked and that mark is retained and recognize throughout the life of the organism inheriting the marked DNA Phenotypes caused by this follow mendelian inheritance because the marking process causes the offspring to distinguish between maternally and paternally inherited alleles (**depending on how each genes is marked, the offspring only express one of the 2 alleles— called monoalleic expression ) **for ex may be getting a from mom and A from dad but dads has a mark that silences it so inheritance of mom gene little a will show through **so now matter if dom or recc- silenced so doesn't show through what need to know for imprinted genes- can't use Punnett For imprinted genes: cant use pungent square for offspring phenotype — need 2 pieces of info 1: need to know if offspring expresses allele that is inherited from the mother or the father (bc if expressing it and know from father, then know it must not be silenced in father!!) 2: need to know which allele was inherited from the mother and which from the father Ex: if you know allele from father is whats being expressed if osspring inherits IGf2 from dad will be normal and if inherits Igf2- from dad will be dwarf bc mom input regardless is silenced The imprint is established during gametogenesis Imprinting can be divided into 3 stages 1: establishment of the imprint during gametogenesis 2: maintenance of the imprint during embryogenesis and in adult somatic cells —so as continue to grow cells all still have it 3: erasure and reestablishment of the imprint in germ cells imprinting control region (ICR)— located near imprinted gene— contains binding sites for one or more proteins that regulate transcription of the imprinted gene

Sanger Sequencing

heat DNA to get in single stranded form then have mixture containing DNA, polymerase deoxy NTPS (ATCG) and primer take equal amounts of mixture into 4 diff groups then each mixture has only one ddNTP placed in it SO the DNTPs are just based that can pair and are complimentary but the ddNTPs lack the 3' OH group so -- so some cut A's some cut C's etc so if have 4 separate solutions each w only one then the strand in one solution will be cut at all A' then strand in other at all G's So then when put into 4 separate wells-- bases will separate on cuts- each vertical well is a letter-- read from bottom to top upwards -- whichever you hit, read that letter corresponding to column then go up and read next etc and will build whole strand!! 3' at top of Gell

How transcriptional activators may facilitate transcription in eukaryotic gene (such as gene found in yeast)

1. Transcriptional activator binds to an enhancer in the NFR 2. Activator recruits chromatin-remodeling complexes and histone modifying enzymes to this region (complexes change nucleosomes enzymes change proteins of histone tails!) Chromatin remodelers may shift nucleosomes or temporarily evict nucleosomes from the promoter region. Histone modifying enzymes covalently modify histone proteins and may affect nucleosome contact w the DNA (if change DNA of nucleosome, changing parts of it so may not bind or function as well or may just function diff w new code) 3. Actions of chromatin remodeling complexes and histone modifying enzymes facilitate the binding of general transcription factors and rNA polymerase II to core promoter 4. For elongation to occur, histones are evicted, partially displaced, or destabilized so that RNA polymerase II can move along the DNA!! Evicted histones are transferred to histone chaperones and they then reassemble them and put them back on DNA behind moving RNA polymerase !

dosage compensation

Dosage compensation Refers to the phenomenon in which the level of expression of many genes on the sex chromosomes (X chromosome) is similar in both sexes, even though males and females have a diff complement of sex chromosomes —so females have XX and males have XY so would think in females there would be more expression of certain X linked genes bc have 2 of them— so this is case when one is silenced so only have one functional like the boys!! Saw Allele on X chromosome that made apricot eye produced a similar phenotype in females carrying 2 copies of the gene and in a male w just one BUT, females who had one copy of apricot allele and a deletion of apricot allele on other X chromosome has eyes paler in color SO, one copy of allele in female is not same as one copy of Allee in male rather 2 copies of allele in female produce phenotype similar to that produced by one copy in male 2 copy of allele in female= 2 copy of allele in male ey concept: difference in gene dosage (2 copies vs one) is being compensated at the level of gene expression Depending on the species, dosage compensation occurs via diff mechanisms Placental mammals Mechanism of compensation: one of the x chromosomes in the somatic cells of females is inactivated (either the paternal or the maternal X chromosome is inactivated randomly throughout the somatic cells of females— so either one they got from mom or one from dad gets inactivated so only inherits one active one) Marsupial mammals Mechanism of compensation: paternally derived X chromosome is inactivated in somatic cells of females (so always one coming from dad inactivates) Drosophila Mechanism of compensation:level of expression of genes on X chromosome in males is doubled — now equal to women Elegans Mechanism of compensation: the level of expression of genes on each X chromosome in hermaphrodites is decreased to 50% of the level occurring on the X chrome in males— now 2 w 50% function = one male X function In c elegans XX is hermaphrodite that produces both egg and sperm and animal carrying single X chrome is a male that produces only sperm Barr body — this is a highly condensed X chromosome (****OOOO SO IF HIGHLY CONDENSED STILL HAVE THAT SECOND CHROMOSOME BUT PROLLY SO CONDENSED THAT TRANSCROPTIN FACTORS CANT BIND TO INITIATE TRANSCRIPTION SO IT IS SILENCED!!)

imprinting

Pattern of methylation/silencing established during gamete development Occurs during one version of gamete development (egg or sperm, not both) Silencing established during gamete development for this and this is non random!! Specific loci w in chromosomes are affected in specific sexes Subset of genes or locations silenced in egg development and other locations that are targeted for silencing in sperm development Unlike in sex linked- in sex linked sex whas imp for genotype but here it is sex giving rise to offspring that is important!! At some point silencing happening in sex specific way- if this is egg development some silencing happening in particular way!! Will happen in same place regardless of patterned one or solid- location will be silenced in all copies at same place in all red and same for all green et Green circle- indicates bc this is not sperm development this is not silenced but if this gamete being formed was a sperm- it would be there if inherit 2 copies of silenced gene- not affecting pehnotype= only mothers will be affecting phenotype

suppressors

One way to find genes w functions related to known genes is to find "suppressors" "suppressor"- mutation that, in combo w another mutation, results in a wild type phenotype— Start w mutant you know about or are interested in, introduce a new mutant by mutagenesis or diversity in population over time and it converts that mutant into wild type phenotype So goes mutant—> (+new mutation — this new mutation is the suppressing mutation)—> wild type Suppressor possibility #1: Reversion Reversal of a mutation (G—T then later T—G)— mutagen hits the causal mutation (of original mutant) and reverses it —> wild type phenotype and return to original sequence ! --same gene scenario- would be if 0 recombinant!! Suppressor possibility #2: intragenic suppressor Compensating mutation within the same gene that fixes the defect caused by the first mutation In pic ex: intital mutation is insertion — can see it changes amino acid seq resulting after the insertion— also ends early bc now stop codon —trunkates the original protein and the short version may even be degraded — would probably be a loss of function mutation THEN second mutation is a deletion — could hit same gene but at diff location — the G from the stop codon from 1st mutagen gets deleted- then amino acids are now created after it — so now in new protein there is this region in the middle where there are differing amino acids but these couple changes may not be crucial in function of protein as whole So this ex of 2 mutations w in same gene that returns sequence to form where rest of protein should be able to be translated normally! **note for this way- wild type phenotype sequence does not match parental sequence note- not same as parental Suppressor possibility #3: compensating mutation in a different gene Second mutation is in the regulator of the original gene: Mutation #1 lowers the activity of a gene product Mutation #2 increases expression of that gene (can go and overexpress or increase the expression of that gene) SO still have mutant version of that protein but now have way more of it so a bunch of 40% active mutant can produce color more like OG green So WT—> 40% activity (mutant phenotype) Ex WT is green and then mutant has only 40% of activity so that is mutant phenotype which looks lighter green Another possibility: interacting protein Ex w 2 proteins that work together and together for a complex, if one of them is mutated, the entire complex is non functional but a second mutation in the other member of that complex will restore the association and restore the function!!— so if one mutated, need to mutate other and if both mutated can bind Protein that directly interacts with the protein encoded by the first gene Mutation 1: destroys a protein- protein binding site Mutation 2- changes the protein that binds the first so that it can bind the mutant version and creates a functional pair The suppressing mutation is mutation on second binding partner that restores the function of the protein complex Both genes remain mutated and both structures diff from original, the functional pair of them together leads to the wild type phenotype

BIG KEY TO SUPPRESSION REVIEW PROB

WE KNOW FROM HOW THE PROBLEM IS DESCRIBED THAT THE F2 GENERATION IS HETEROZYGOUS!! WE ALSO KNOW THAT HETEROZYGOUS CROSSES MAKE A RATIO OF 9:3:3:1 RATIO SO ALWAYS 16 POSSIBLE GAMETES 9 W G_H_ 3 JUST ONE DOM, 3 JUST OTHER DOM SO 3G_hh 3 ggH_ AND 1 REC BOTH gghh So, we know these ratios BUT now based on what they have given us for percentage of phenotypes-- we can determine if epistasis or suppression -if epistasis, even 1 double recc makes whole thing recc so ratio above would give 9 dom, 9 recessive-- the stats then would be 50-50 BUT it tells us phenotypically 10/16 WT and 6/16 mutant so -- has to be another combo that can give WT-- suppression and double recessive!! ALSO, we know it is a complex suppression bc if it was due to mutation in regulator- could be that the G_hh (over expression of gene already expressed) also shows WT phenotype which would mean 12 would show WT which also isn't true w the state

mutation in promoter that blocks transcription factors from binding

less of transcript mrna in mutant -- if factors can't bind- won't be shorter transcript will just be less transcript period bc at that promoter- not creating transcript so less promoters functioning less transcript if change intron- doesn't matter if intron can't be removed- longer transcript if mutation in splice acceptor- eons can't be attached so shorter transcript

how mutations would be discovered using complementation tests?

-- could get a bunch of diff mutants but hard to screen for whether the no growth in 2 diff colonies is due to mutation in same gene or not SO, to figure out how many genes are in a particular pathway 1- take all the mutant types 2- put them together 3- if still no growth - must be mutated at same gene bc the recessiveness of one is complementing recessiveness of other and have no dom (protein producing allele to come in and help) if growth!!- know that those 2 have 2 diff mutations that are complementing eachother bc where one may be recessive in c (could just have one c bc haploid worms)- the other may have a dom C there and be able to produce that protein in that particular pathway but not be able to produce another protein in pathway- but now other one can mask that **complmentations tests ALLOW: you to see how many genes responsible for traitr- in pathway -- one that can grow w any mutant has to be end of process bc doesn't matter If mutations bf arg-1 is required to convert _____some precursor molecule not explicitly tested in this experiment_____________ to ____ornithine—once it has been given ornithine or citrulline, it can make its own arginine________.-- bc has enzymes to cover the ornithine and citrulline functional -- if given the thing the mutated gene is supposed to make, can grow fuse the haploids of mutants discovered--- creat diploid self fusions will still all be unable to grow on arg plate- bc just increasing homozygity --- also these are all haploid so diploid would just get 2 of the already not functional one gene !\ ** so mutant 1 and 2 are said to complement eachother!! To figure out how many genes mutated: go through each cross but w eachother- if neg on growth, mark that those 2 are same gene-- then start to make groupings would say there are 3 complementation groups (however many genes mutated is however many groups) w complmentation- prolly is case all 3 genes leading to phenotype but on diff pathways

nanopore-- think pore , no polymerase

Each pore is a membrane and each membrane is a channel for bacteria which allows hydrophilic molecules to enter the cell — channel just large enough to allow single stranded DNA to pass through but not double (hole not big enough)— so bc whole not big enough, on top of pore is another protein which unwinds the doublstranded DNA Single stranded DNA is bias (wants to go through pore) bc you have a current going through it — like a circuit— havens chaired on other side— where DNA going towards - bc charge it goes through rapidly As the dNA goes through pore it changes resitance of the current as it goes through Single strand is acting as resistor, how well it resists the current depends upon which nucleotide going through the pore bc each nucleotide has diff resistance!! However much current passes through pore indicates which nucleotide passed through pore Diff nucleotide passes through channel each .02 seconds — if tried to go faster than .02 secs, would have difficulty resolving the current going through the channel - cant go through too fast or wont get a read - SO proteins in complex allowing it to go through help in slowing down the dNA just enough o that can get read So the plate has a bunch of diff pores each w own resistor, allows you to sequence REALL Really tiny- plug into your laptop and inject your sample in - and then can do sequencing on laptop!! Sequencing bf this had fundamental limit to how long sequence is they are sequencing, Sanger was short sequences and illumina long, this one—read length is variable, depends on sample — can produce really long reads if need be (so can do both!) The thing that is limiting is how quickly the pores clog up! -- so any length Q- nanopore always both!! ******* Illumina- 100 BP (so illumina can do high volume but not that many base pairs at once!! Sanger- 1000BP Nanopore- 10 100 thousand BP— so really really long For whole genome, 3 flow cells THis much cheaper than illuma!! Cheapest ! So progression— Sanger (have to sequence whole genome to do it— need whole facility to do it)—- to illumina (Can sequence only parts you are interested in (justnneed that one machine to do it , can sequence single flow cell — then to Oxford nanopore, load a few flow cells on this device and can sequence whole human genome!!— so now back to being able to do whole genome just way cheaper and faster! Improvements in feasibility, speed and cost ***went to few cells but not whole genome now back to whole genome!! Keep in mind: speed comes out of cost- so the Oxford nano pore is great but error rate is 1 in every 10 to 1 in every 100 (so fastest but most error— think bc may be missing the current going through so fast), illumina 1 in ever 100 to 1 in every 1000 error and Sanger 1 in every thousand to 1 in every 10000— SANGER MOST ACCURATE SLOWEST OXFORD NANOPRE LEAST ACCURATE FASTEST FUTURE: nanopore working on creating pore that doesn't need protein so that whole device wont require any biological components just the dNA going through!!

Sanger sequencing

First amplify DNA by PCR whereas in vitro means "within glass", usually in a cultured system. Sequencing relies on amplifying the source of DNA- why? Want to increase amount of DNA — ex if have small drop of blood at crime scene, not enough for sequencing so want to amplify— Amplify by PCR In vivo- in our cells they use a RNA primer but if doing this in vitro- in dish, primer will be DNA bc more stable dNTP's are nucleotides that will form the new complimentary strand Mg2+ helps neutralize the DNA bc it is super charged so that polymerase can do its job! dont use helices in vitro to unwind bc as building new DNA strand- helices will think needs to unwind the 2 bc its job is to unwind— so instead denature w temp Heat up to destroy enzymes — want the polymerase so use polymerase from hydrothermal events, that bacteria has, that can survive hot temps What do u need for DNA to replicate? DNA (template) DNA polymerase (enzyme that catalyzes DNA synthesis) primer - RNA in vivo, DNA for in vitro dNTPs Mg2+ (for polymerase activity) A way to melt the DNA to separate the strands Each cycle you double amount of strands you had n = cycle # DNA # = 2^n property of constant doubling is pretty effective!! n ~ 30 DNA # ~ 109 Usually goes through 30 cycles- if started w one piece— end w 10^9- massive increase Need 100,000 plates to sequence whole genome, a lot of plates!! This was typically used in Past to sequence genomes, pretty expensive 400$ a plate *10^5 40 mill dollars! So expensive Sanger produces anger bc so expensive!! -- bc think need all those rows for each letter of each strand!! The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. Putting it in a more sensible order, four separate reactions are needed in this process to test all four ddNTPs. Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. have 4 wells of electrophoresis -- each well corresponds to mixture that only got one dNTP so in electrophoresis bands will separate and parts will glow that have that corresponding letter- then put all rows next to eachother and can determine sequence Cost of sequencing got progressively cheaper as result of increase in technology Sanger is still gold stander (sometimes stick w ppl that make you most angry!!) and most common Other sequencing methods have now been developed, including single molecule methods

A gene interaction can exhibit epistasis and complementation -- purple flower example

Punnet discovered unexpected gene interaction when looking at crosses of sweet pea WT= purple flowers Crossed a true breeding purple flowered plant to a true breeding white flowered plant F1 - all purple (all heterozygotes) F2 (produced by self Fert of F1) had 3:1 ratio BUT suprise came when : crossed 2 diff varieties of white flowered plants P- 2 true breeding of 2 diff white variety ones F1- all purple!! F2- purple and white flowers in a 9: 7 ratio purple to white decided: 2 diff genes involved in relationship C (one purple color producing) allele is dominant to c (white) P (another purple color producing) allele is dom to p (white) BUT— cc masks the P allele and pp masks the C allele— gives the white color **so even tho may have big P or C— if have double recessive of other one, that P or C will be masked!! For Ex CCpp and CcPp and ccPp will all be white!! epistasis: when the alleles of one gene mask the phenotypic effects of the alleles of another gene at a diff locus It is described relative to particular phenotype- use WT as reference phenotype At Level of genotypes- cc epistatic to PP or Pp and pp epistatic to Cc or CC— this is ex of: Recessive epistasis Epistasis often occurs bc 2 or more diff proteins participate in a common function ex: 2 or more proteins may be part of enzymatic pathway leading to formation of single product Ex w peas Colorless precursor—- (enzyme C)—> colorless intermediate—-(enzyme P)—> Purple pigment Need both 2 get pigment! Colorless pecursor must be acted on by 2 diff enzymes to produce purple pigment Gene C encodes a functional protein called enzyme C- this converts colorless precursor into colorless intermediate— 2 copies of recessive (cc) result in lack of production of enzyme so lack of colorless intermediate Gene P encodes a functional enzyme P which converts the colorless intermediate into the purple pigment — like C, pp encodes non functional enzyme so colorless can be converted into purple **when one of the enzymes is missing, purple pigment cannot be made and flowers remain white— either doesn't make intermediate which halts process or cant go from intermediate to purple!! Complementation Refers to the production of offspring with a wild type phenotype from parents that both display the same or similar recessive phenotype Typically occurs bc the recessive phenotype in the parents is due to homozygosity at 2 diff genes — one parent was CCpp and other ccPP SO, in F1 gen, the C and P alleles which are WT compliment the c and p alleles (all hetro now)

Methylation of cytosines can coordinate with histone modification to silence genes further- the 2 working together!

Start w unmethylates sequence in euchromatin (so active not inhibited)— then CpG methyltransferase can turn off the sequence—> methylated sequence in euchromatin and thennnn methyl-c binding proteins that specifically bind to the methyl that is on the cytosines from the CpG methytransferase event can then recruit or bind to histone modifying enzymes that can increase the density of nucleosomes (changing it from euchromatin to heteochromatin!) So end resulted :!! Heterochromatin sequence that is now methylated **so became methylated and switched from not densely packed (euch) to densely packed (heterochrom) Methyaltion can be maintained through cell division cycles DNA methylation that is established during gamete formation will be maintained through subsequent cell divisions! Inheriting a methylated version of a gene from one parent leads to the silencing of that allele in the offspring Methylation inherited from parents CAN be un-done and restablished in different patterns during gametogenesis!!— so not like mutations which are passed down and undone— these can be undone!!

gene interactions epistasis

What are ratios of progeny phenotypes for the typical AaBbAaBb cross if unlinked genes that affect diff phenotypes? 9:3:3:1 typically think this when double hetero cross but epilepsy allows u to see how diff also diff depending on type of suppress This worked bc these traits had nothing to do with each other and they were completely independently derived0not linked— what happens when this note true? Gene intractions— when 2 genes involved in same pathway epic always think 2 genes involved in same pathway- dogs and expression of color, purpler plant If the encoded gene products (RNAs or proteins) influence one another or participate in common pathways, this is a "gene interaction" Interacting genes lead to progeny phenotypes and phenotypic ratios that deviate from the Mendelian expectations AND from predictions that can be explained by linked genes he F1 would be wild type but the F2 ratios would change to 9:7 because A_bb aabb would have the same phenotype — cc or ss- no circles and ccss definitely no circles— so just if has one recessive allele wont have color bc ss will stop to make square pp will stop to make circle so either way wont get to cricle- and then of course if both little- wont get circle BF B gene determines Black (dominant B) vs brown (recessive b) melanin Two important loci in Labrador retriever coat colors: B gene determines Black (dominant B) vs brown (recessive b) melanin E gene function needed for pigment to be deposited into the fur SO if have ee- the B status is irrelevant- can't deposit the fur so will be yellow lab B with E is black B e is yellow bb E is brown bbee is yellow— bc need that yellow to show color of B!! This is an example of recesisve epistasis bc it is the recessive ee that masks the B allele expression — can say that E is epistatic to B— bc the ee is one that wins- it is determining factor What would be result of test cross of BbEe to a bbee individual? BbEe- Black bbEe Bbee- yellow 1 black 2 yellow 1 brown Gametes bBEe bBee-yellow bbEe bbee Which of the following would be the simplest to breed? Yellow labs: yellow to yellow crosses will yield yellow puppies — you know ee *ee so all will be homozygous If breed chocolate w chocolate and black w black why not all same? Chocolate not great bc for brown we know it has to be bb but could be heterozygous at e location so Ee and Ee so could get ee and get yellow puppies Black labs— could be Bb and/or Ee— could end up w brown and yellow puppies too! This is when dominant version of alleles dictates the phenotype - presence of a dominant allele at one locus determines the phenotyoe controlled by another locus Ex: white squash plant That !2 is one of the 9:3:3:1 — 9 dominant at both 3 dominant at one 3 Dom at other allel 1 recessive of both Presence of that A allele that gives phenotype — still dealing w that 9:3:3:1 but phenotype diff bc based now on Dom dom eist if wat more than the 9-3 bc includes 3 dom at one and other

chromatin

chromatin- word that describes DNA in complex with proteins Core histones are proteins that assemble into an octamer Core histones have. Tail domain and a globular domain— there are 4 types of globular domains: H3, H4, H2A, H2B DNA then wraps around histone octamers to form nucleosomes — so FDNA octamar looks like ball and then DNA wrapped around it is strands wrapping all around it **each nucleosome contains 146 base pairs of DNA wrapped around that histone core A single DNA molecule (ex chromosome) forms multiple nucleosomes — this arrangement often called beads on a strand bc whole chrome has more than 146 nucleotides so has multiple chromatins for dna to wrap around histone complexes Nucleosome function Condense DNA so that it fits into the nucleus Control accessibility of DNA including ability of RNA polymerases to transcribe specific genes — allows polymerases to access certain sequences so that certain sequences can be transcribed Regulate chromatin structure nucleosome (whole complex- is regulating chromatin structure)-- changing bw hetero and euchrom 2 main categories of chromatin Heterochromatin Densely packed nucleosomes with little DNA that is not bound by protein Euchromatin: "open" conformation with fewer nucleosomes. Transcription machinery and other DNA binding proteins can access sequences Fewer nucleosomes scattered onto a DNA sequence DNA binding proteins and polymerase have easier access to the DNA when less tightly bound In euchromatic regions the RNA polymerase has access to the DNA and in the heterochromatin region it is so densely packed there is no access nucleosomes dictate hetero vs euchro bc of number of them there are in certain regions- more balls, more tightly packed Chromatin structure can be dynamic Sequences can be switched between euchromatin and heterochromatin via Moving nucleosomes from one sequence to another (sliding) Adding more or removing nucleosomes — to increase or decrease densiiy of nucleosomes Covalent modification of piston proteins (tails) within specific nucleosomes which can influence later protein binding events

Epistasis

from WS- fungi (including yeast) can synthesize all 20 amino acids which are important in protein synthesis -- they can grow on media that lacks the amino acid bc they can form amino acids from precursors -- diff enzymes in pathways involved in animo acid bitosynthesis-- think epistasis bc many genes (proteins) involved in attaining certain outcome (just like if had BBcc- the double cc didn't have enzyme to get to BB part of pathway, here if one enzyme not working for certain step towards amino acid- may not be able to create on own what they did: found a collection of yeast mutants that could not grow without arginine (so somewhere in their pathway they need arginine to move onto next step) narrowed to 3 distinct genes-- then looked at ability to grow w out arginine or biosynthetic precursors ** BIG THING: IF THE MOLECULE ADDED TO THE MEDIUM COMES AS A PRECURSOR TO THE GENE THAT IS MUTATED-- THE MUTATED STRAIN WILL NOT GROW -- THIS IS BC EVEN IF YOU GIVE MUTANT SOMETHING IT HAS ENZYME FOR TO CONVERT TO SOMETHING ELSE BUT IT IS MUTANT, IT WONT BE ABLE TO GROW-- IF THE MOELCULE ADDED COMES AFTER THE GENE (PROTEIN) MUTATED, THEN THERE WILL STILL BE GROWTH BC THE FUNCTION OF THAT SPECIFIC ENZYME HAS ALREADY PASSED AND NOW OTHER ENXYMES ARE NEEDED IN CONVERSION (LIKE ITS BEEN PLACED IN MEDIA 1 STEP IN FRONT OF MUTATED GENE!!) **IF MOLECULE SHOWS GROWTH ON ALL 3- IT IS END PT BC DONT NEED FUNCTIONING ENZYMES!! -- humans can synthesize 11/20 and must get other 9 from food sources or microbes in gut **in general epistasis is the interaction of genes that are not alleles, in particular the suppression of the effect of one such gene by another. interaction of genes that are not alleles- means 2 diff genes on diff loci influencing expression of eachother- many ways this could be done! suppression epistasis, type of epistasis often occurs bc 2 or more proteins participating in pathway--common function! when think epistasis ws-- it is pathway- why mutation on one gene affecting diff gene -- bc if can't get past 1 step of pathway- can't get pas other (arg!!) epistasis think 2 genes needed in pathway complementation, 2 genes involved but

how complementation goes w epistasis

in epistasis homo recessive of one of 2 alleles is dictating the recessive phenotype regardless if other gene has dom bc need 2 functional enzymes (think pathway) to create end product-- complementation comes into play bc it is in case where 2 phenotypically mutated individuals have WT offspring this would be like this ***think you 2 compliment each other (bc can create normal baby!) this would be like this wwPP WWpp (both would appear mutant but if mate---- WwPp-- WT!! bc no homo recessive SO in complementation the parents are showing epistasis (they are dom in one aspect but recc in other so appear recc) but then when mate and produce heterozygote-- this is complementation bc now hetero at each point and WT parent THEN from here is where need to establish whether suppression if f1 gen crosses -- if all heterozygotes write out the 9:3:3:1 ratios we usuallyyy know but then look at info and see whether someone that is homo recc may have to appear dom due to phenotype numbers bc more than 9 WT- know gghh has to be experiencing suppression (THEN THINK ABT 2) TYPES OF IT PRAC TEST

how to know linked vs unlinked and same vs diff gene!!

linked: if 2 genes are linked then the 2 parental gametes will be seen the most often in offspring (so if high percentage of WT phen offspring and both parents WT- then could be linked BUT also you will see some recombination due to crossover-- will not all be parents IF ALL WT or parent offspring then the mutation must be coming from the same gene and just be a reversion because even if linked, you see that little recombination if unlinked -- the %'s given to you will be relatively equal - bc assort independently so one not dominating the other -- this when do Aa Bb pick and choose 4 combos w all relatively same frequencies SO go through and do combinations of gametes -- BUT THEN HAVE to go after having done the genotypic combinations and see what the phenotypic ratios are -- get what they are describing to match up with how the segregation could have gone and from there can tell how many recombinant- if break into 4 (2 recombinant) and the genotypes of the breaking fit the described % phenotypes, then diff unlinked gene

mapping new mutation on same chromosome as mapping genes scenarios 1 and 2

so have these mapping genes we are interested -- now also incorporating a new mutation-- wanting to see what could occur if mutation lies bw to other genes or on outside p- 1 parent homo recessive -- so AB only (meaning recessive at A and B) other parent recessive at mutant m but dom at AB -- and both homo so that f1-- heterozygous! f1 hetero at each locus then when cross the f1 and wanting to know genotype and phenotypes of progeny -- this how you do we know most common will be parental so put each of those down bc those will occur at highest frequency, then put all that those can pair w-- including recombinants from possible diff crossover events -first match w eachother and self now mutation can also be in gene bw a and b (genes we have mapped) A only means only recessive at that locus -- this prolly good bc can really identify what is going on just when that one gene is mutated- not influenced by other mutations no double crossover in worms!! so if mutation is in middle will be on one parental +m+ so dommm on either side then other a+b-- so only way to get MAB would be DCO which not possible in worms! what need to do is take all recombinants- starting from parental and pair w all recombinants-- this gives you extensive ways of all pairings

synthetic phenotypes

synthetic phenotypes occur when the double mutant has a phenotype that neither single-mutant has Found by introducing new mutations into a single-mutant OR mating two mutants and analyzing double mutants sstt double mutant above has no circles (ss phenotype), no thinking (tt phenotype) and also no happiness = the synthetic phenotype Appear in double mutant diff than either single mutant We have talked about suppressors where in double mutant you don't get single mutant u get wild type But now the double mutant is showing completely new phenotype If this pathway functional- circle will be made — but what if circle was then precursor for another reaction - like happy face— we were not looking at this- we were just looking at end goal circle— missed whole bottom So now if have sstt— instead of just having no circle, also have no happy face! Synthetic phenotypes can show u other pathways that are independent but related to the happy face If faces pathway is totally active If studying somethin on top pathway- only going to be see those Called synthetic bc it has been created by both of those mutations at one Common one: common lethal phenotype- if both pathways mutant Can find these by doing suppressor screen- adding mutagen Or have 2 single mutants, cross them and see whether that other candidate gene is involved in the pathway

back crossing and test crossing explanations

the results of the self cross will be homozygous at many loci but not all-- what happens when you do these experiments is p0 are both true breeding so homo for everything-- then the Increases the likelihood of homozygosity at any site including the mutant ones. This is the ultimate inbreeding. !!!! this makes sense if want to look at mutant genes by crossing two of the same genotype increasing likelihood that double allele will show through self crossing increasing homozygosity at any site!! Want to have some way of purifying allele out- sow hat you do is back cross to wild type— bc want to isolate!! One back cross 1/2 will be wild type Have done one back cross- half wild type have mutations— if dom mutation heterozygous animal will produce m trait looking for — then pick m phenotype and back cross again— if keep back crossing you wikll jeep getting rid of other mutations- don't care about other mutations but keep selecting for ones w m mutation and back cross till just isllate m Then if you sequence it and do some tracking can find it BUT =- if recessive- harder Self fertilixe— choose m mutant and if has trait is homozygous Instead of just backcrossing to makes for RECESSIVE you have to do one round of self fertilization Back cross then self back cross then self- back cross to get rid of other mutations and seld to pick out homo recc that carry allele — after all end w one self cross to get final isolated mutant