genetics 2581b

Genetics 2581bIan [email protected]

Chromosomal MutationsChange the Chromosomes themselves Inversions, Deletions, Insertions, translocations etc.Change the number of Chromosomes in a Cell or an animal

Note: Large proportion of Chromosomal modifications results in the prenatal death of an organism. Surviving individuals well most likely have various defects.

Changing the number of ChromosomesAutopolyploidy (within a species) and Allopolyploidy (hybridization of 2 species

Common in plants Results in larger plants due to increased cell size (necessary for holding more DNA)Rare in animalsDue to problems that occur during meiosis when chromosomes need to pair.

Generating Polyploid0n1n1n+4n+colchicine-colchicine

Effect of being a polyploidAutopolyploids are usually infertileWhen a cell has more than 2 sets of chromosomes, many chromosomes will try to hybridize during meiosis. During chromosome segregation, unequal division can occur, creating gametes with various numbers of chromosomes. Allopolyploids can be fertileWhen two diploid species contribute their genomes in species hybridization, a tetraploid will be created. Because each chromosome can pair with a like chromosome, segregation can occur equally, creating fertile gametes.AB

SPECIES A 2n = 18Species B 2n = 18STERILEModified from Dr. A Day lecture

Question 1Two species X (tetraploid 4n=16) and Y (diploid 2n=6) are crossed; the hybrid is infertile. How would you make this hybrid plant fertile, and what would be the chromosome content of the fertile hybrid plant?

AneupoidyWhen you are missing a chromosome or have an extra chromosome. (2n-1, 2n+1)This has dire effects, usually a lethal phenotypeThis is due to the imbalances in Gene Expression

Aneuploids can becaused by nondisjunctionevents in both meiosis Ior meiosis II

Chromosomal RearangementsTranslocationsInversions DeletionsDuplicationsGustav J. V. Nossa lNature 421, 440-444(23 January 2003

TranslocationsCan happen after chromosomal breakageResults in changes in gene expression or if breakpoint is in a gene, gene function

InversionsIf homozygous, can function normallyIf heterozygous, can have problems during meiosis, and therefore altered fertility

Heterozygous inversionsCause chromosomal loops that effect the gametes functionPericentric inversions give you two good and two bad gametesParacentric inversions give you two good and bad gametes as well

DeletionsUsually are lethal. Same effect as aneuploidy.Cause looping structures during meiosisEFFECT GENE BALANCE

DuplicationsLess severe problems than DeletionsCan disturb gene balanceCan happen in tandem (at the process of crossing over in meiosis)Can have insertion events (Piece of DNA inserts into the chromosome doubling the genetic material present)

Genetic techniquesGenetic TechnologiesGenomic ApproachesGene MutationGenotyping

Isolating DNAGrind up tissueAdd a detergent (Sodium Dodecyl Sulfate) to break up membranesAdd proteinase K to chew up all unwanted proteinAdd phenol:chloroform to separate DNA from excess proteinAdd salt and alcohol to precipitate DNA

Restriction Endonuclease DigestionRE are enzymes that recognize palindromic DNA sequences They cut the sequence in a particular way leaving 5 or 3 overhangs5'GAATTC3'CTTAAG5 G AATTC 3'3 CTTAA G 5'EcoRI5'GGTACC3'CCATGG5 GGTAC C 33 C CATGG 5KpnI

Calculating the average size of fragments digested with REWhat if the proportion of the nucelotides in the sequence isnt 1:1:1:1?Eg. What is the average size of a DNA fragment cut with the RE EcoRI (GAATTC) if the proportion of nucleotides is G(1/3), C(1/3), A(1/6), T(1/6)?Ans.G A A T T C1/3 x 1/6 x 1/6 x 1/6 x 1/6 x 1/31/11644Therfore the average fragment length should be 11644bp

Gel ElectrophoresisSeparates genetic material based on SIZESend Genetic material through a polymer (agarose or polyacrylamide) using an electric currentBecause DNA is negatively charged it will move from the -(anode) to +(cathode).Visualize the DNA in the gel with Ethidium Bromide and UV light.

DNA cut with RE*Note the very specific patternDNA that has been randomlyBroken (sheared)

Blotting

BlottingNorthern=RNASouthern=DNA

Western=proteinDetect using radiolabeled probes}}Detect using radiolabeled antibodies

Where do you get DNA from?Genomic (PCR)cDNA (using RNA as a template)

Recombinant EngineeringGoal is to express your gene of interest in a model system (mamalian cells, bacterial cells, whole organisms)Start with a source of DNA (cDNA or Genomic)Put that DNA into a suitable vectorTransfect (eukaryotic), or Transform (prokaryotic) your vector with your DNA into a host system

Cloning

TransformationInsert your cloned DNA into bacteriaBacteria will amplify your DNA many times

Cloning using Bacteriophage vectorsInsert your favorite DNA sequence into the dispensable region of Phage DNAThen use the phage to infect cells you want to express your DNAPhages will kill whatever cell you infect leaving a plaque where an infection occurred.

Selectable markers Let you know which cells contain your plasmidEach plasmid contains a antibiotic resistance geneLB plate with apicillin and tetracyclin

What makes a good vector?Host specific promoterOrigin of replication (for independent replication)Selectable markerUnique cloning sitesSmall sizeORI

Is your gene working?Transfect or transform your cellsIf it is expressing your protein should be presentYou can lyse your cells, on a membrane, and use antibodies to detect your protein

Polymerase Chain ReactionAmplifies your piece of DNA exponentiallyRemember DNA is amplified in a 5-3 manner ONLYFor PCR you need, DNA, dNTPs, Polymerase and Primers5533

Start by denaturing your DNA with high temp (94 degrees)Anneal your primers at a lower temp (50 degrees)Extend your DNA at a medium temp (72 degrees)

Question 3If you expect only one DNA amplicon after a PCR reaction, but you detect multiple, what could be wrong?Ans.Anealing temp too lowContamination in your reaction (more than one DNA source)

DNA sequencingVery similar to PCR Need DNA, dNTPs, Primer, PolymeraseAdd small number of dideoxy nucleic acids (ddNTPs)ddNTPOHHdNTP

Start with 4 different reactions containing either :ddTTP, ddATP, ddGTP or ddCTPEg. Reaction with ddTTP

ddTTPddATPddGTPddCTPWhat is your sequence?35GATGGCCGATGT53CTTAAACCCGGTemplate sequenceNascent sequence

Mapping the Genome

Molecular markersRestriction fragment length polymorphismsSimple Sequence Repeats(microsatelites, minisatelites)Single Nucleotide plolymorphisms

RFLPPrinciple is that different alleles may contain different RE cut sitesREREREREREABAABBAB

SSRMicrosatelitesCT CT CT CT CT CT CT CT CT CTMinisatelitesEg. Variable number tandem repeats (vntr)CTAGCTTAGAGAG CTAGCTTAGAGAGDetect differences between people by using PCR to amplify a region that you know contains these repeats

So what?The goal of finding these markers is to use them to map the genome.Can tell which ones are linked to each other by looking at inheritance patterns on a pedigreeThis is a method of genotypingOr simply telling individuals apart based on variation in genes

Physical mappingOrdering DNA based on overlaping regions

Where do mutations come from?Exogenous mutations (UV light, chemicals)Endogenous (part of the cellular process, mistakes in DNA replication)

Point MutationsInsertions (Frame Shift)SubstitutionsTransversions (Pyrimidine for a Purine or Purine for a Pyrimidine)Transitions (Pyrimidine for Pyrimidine or Purine for a Purine)Deletions (Frame shift)

What does a mutation do?Insertions or Deletions will generally change the function of the gene Substitutions can change the function of the gene product depending on the particular substitutionOccasionally a gene mutation can cause a gain of function.This means the gene product has an alternate functionOr it means the gene product has an enhanced function

Deletions and DuplicationsHappen in a process called slippageIn a repetitive stretch of DNA, the nascent strand slips, youll get a duplication. If the template strand slips, youll get a deletion

Spontaneous mutationsDepurinationDeaminationCytosine to Uracil5-methylcytosine to thymineTransition (purine for a purine)Transversion (pyrimidine for a purine)

DepurinationLoss of a purineCauses DNA repair systems (SOS) to replace the missing base with a new one

G:CO:C DNA Replication SOSO:A DNAreplicationT:AThe result of losinga guanine is the Transversion toThymineG to T transversiondepurination

DeaminationC:GdeaminationU:GDNA replicationU:AC:GDNA replicationU:AT:AResults in a C to T transition

Exogenous MutationsInsult on DNA from another sourceUV, X-rays, toxinsPyrimidine DimersAdducted Guanine

Pyrimidine DimersWhen two Cytosines or Thymines are physically linked together upon UV irradiation of DNASimilar mutation process to depurinationC:GC:GUV lightCC}SOSO:AO:ADNA replicationT:AT:ACC dimer to TT

SEX DETERMINATION

STUDY HARD!!ANY Questions?

More QuestionsWhich of the following could explain the origin of humans that are XYY trisomics?A. Non-disjunction in dad at meiosis IB. Non-disjunction in dad at meiosis IIC. Non-disjunction in Mom at meiosis ID. Non-disjunction in Mom at Meiosis II

What is the most likely to be sterile?

A. a allotetraploidB. an autotriploidC. a diploid

If you partially digest a circular piece of DNA with a RE that cuts 3 places on the DNA, how many different sized fragments could you end up with?

A. 5B. 6C. 7D. 8

GlossaryPloidyNumber of sets of COMPLETE CHROMOSOMESEg. 1X Haploid (monoploid), 2X diploid, 3X triploid, 4X tetraploid, 6X hexaploidPolyploid Multiple sets of chromosomesEg. Triploid, TetraploidsAneuploidIncomplete sets of chromosomesAutosomal Trisomies, sex chromosomes abnormalities (normal=XY abnormal=XXY)Monosomy (2n-1), trisomy (2n+1), Tetrasomy (2n+2)

AutopolyploidyChange that happened within the speciesAllopolyploidyChange that happened upon mating of 2 different speciesColchicineDrug, inhibits spindle formationNon-disjunctionWhen 2 chromosomes separate inappropriately during meiosis I or II (two of the same chromosome in 1 gamete)

MosaicOrganism with patches of cells that have different chromosomal arrangements (non-disjunction event in mitosis)Chromosomal RearrangementsChanges in a chromosomes physical structureInversionA section of DNA becomes reversedPericentric (Involves the Centromere)Peracentric (outside of the Centromere) (ABCDACBD)

TranslocationReciprocal (exchange of fragments between two non-homologous chromosomes) (Ch1. ABCD ABCH)(Ch2. EFGH EFGD)DeletionRemoval of a large fragment of DNA from a chromosome (not just a bp deletion)(ABCDACD)DuplicationMultiplication of a region of DNA on a chromosome(ABCDABBCD)

SDS (sodium dodecyl sulphate)Soap that will solubilize biological membranesProteinase KChews up protein in DNA extractionsPhenol:chloroformProvides organic solvent to separate DNA from Protein in DNA extraction ComplementaryBasepair alignment (AT, GC)AntiparallelOrientation of Double Stranded DNA based on 5 3 phosphate organization on nucleic acides5 33 5

PalindromeSame forwards as reverse (RACE CAR or in terms of DNA GAATTCCTTAAGRestriction Endonuclease (Restricion enzyme)Protein that finds specific DNA sequences and cuts specific DNA in a predictable manner (GAATTCG/AATTC)(CTTAAGCTTAA/G)OverhangsThe non paired DNA left over after a RE cut5 overhangs (non-paired DNA on 5 strand after RE cut)3 overhangs(non-paired DNA on 3 strand after RE cut)Both refered to as sticky ends

Blunt EndNo 3 or 5 overhang after RE cutComplete RE digestionevery RE site is cut by the RE enzyme. There are no intermediate fragments leftPartial RE digestionRE has cut some RE sites but others have not yet been cleaved. Presence of int. fragments

PolymerLarge molecule comprised of many repeating unitsEg. Agarose, PolyacrylamideEthidium bromideMolecule that intercalates (inserts and binds to) DNA.Fluoresces upon UV light excitationSouthern BlotUsing probes to detect DNA on a nitrocellulose membrane

Northern BlotUsing probes to detect RNA on a nitrocellulose membraneWestern BlotUsing Antibodies to detect Protein on a nitrocellulose membranecDNA (complementary DNA)DNA synthesized from RNAVectorBacteriophageProteinacious infectious particle that can insert DNA into host genomePlasmidCircular piece of DNA that can be cut, and have other DNA inserted into it.

Origin of ReplicationSignal that tells bacteria to reproduce a DNA sequenceSelectable MarkerDNA sequence that when expressed, will provide a cell with resistance to a drugAmpicillin, Tetracyclin, KanamycinDrugs used for expression vector selection

MappingDetermining the structural, regional, and sequence information of ChromosomesIdiogram: Idealized DiagramRepresentation of Chromosomal structureCytogenetic MappingStaining chromosomes and looking at banding patternsGenetic High Resolution MappingMolecular markers with higher resolution than genesSingle Sequence Repeats (SSR), Micorsatelites, Minisatelites, Restriction enzyme sitesMultiallelicMany different forms of gene (more than one allele)

FISH (Fluroescent in situ hybridization)Fluorescent probe used to locate a specific sequence on a chromosome Variable Number Tandem Repeat (VNTR)Repeated sequence of DNA Single Nucleotide polymorphisim (SNP snip)Variablity at a nucleotide depending on alleleRAPD Random amplification of Polymorphic DNAGenetic technique used to identify

Point MutationsChanges in DNA sequence at a single nucleotide levelSingle nucleotide substitutionRepalcement of one nucleotide with anotherSingle nucleotide deletionSelf explanatorySingle nucleotide insertionIntegration of a nucleotide into a DNA sequence

Base SubstitutionsTransitions (Changing a Purine for a Purine or Pyrimidine for a Pyrimidine)Transversions (Changing a Purine for a PyrimidineSpontaneous mutation Mutation resulting from biological processes without insult from exogenous affectors (10-5-10-8/ bp)Induced MutationMutation resulting from a toxic agent (UV light, chemicals) from the environment (dose dependent mutation rate)

Loss of function mutationMutation causes the reduced (hypomorph) fuction of a geneMutation causes the complete loss of function (null/amorphic)Gain of function mutationEnhanced function (hypermorph)Different function (neomorph)Non-synonomous mutationPoint mutation resulting in the change in amio-acid constitution of a protein. Amino acid substitutionConservative similar amino acidNonconservative different amino acid

genetics 2581b

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