15 Class Note for BIOL 222 at PSU
15 Class Note for BIOL 222 at PSU
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Date Created: 02/06/15
Recombinant DNA Technology Recombinant DNA technology allows us to isolate specific genes from the genome so that we can study their function Recombinant DNA molecules can be made from any organism by inserting DNA fragments into a gene cloning vector Figure 12 1 Vector plasmid or Virus Contains an origin of replication for gene amplification Usually contains an antibiotic resistance gene for selection Restriction Enzymes Make sequence specific cuts in DNA by cleaving each strand of the duplex Digestion e g EcoRI cohesive quotstickyquot ends xl 5 GAATTC3 G AATTC 3 CTTAAG5 CTTAA G I eg Hindll blunt ends xl 5 GTPyPuAC3 GTPy PuAC 3 CAPuPyTG5 CAPu PyTG 1 Characteristics of Restriction Sites A 180 axis of symmetry B Usually a 4 1256 or 6 nt sequence 14096 Gene Cloning Figure 12 4 1 Digest chromosomal and vector DNA with an enzyme 2 Mix together Sticky ends anneal due to base complementarity Can also use blunt ends 3 Seal the quotnicksquot with DNA Ligase 4 Transform E coli and select for drug resistance or by complementation compensation of a mutant defect Figure 12 5 5 Amplify and purify recombinant DNA Vectors A Plasmids small circular origin of replication antibiotic resistance gene Can clone several kb Figure 12 6 B Expression Vectors contain transcription and translation signals to allow overproduction of the protein encoded by the gene Express eukaryotic genes in bacteria C Shuttle Vectors contains origins for two organisms eg E coli and SV40 virus Clone in E coli purify DNA transfect mammalian cell line D Yeast Arti cial Chromosomes Y ACs9contain an ARS telomeres and a centromere 1000 kb can be cloned DNA Library Random chromosomal or cDNA fragments cloned into one of the above vectors A random population of clones should contain every gene Isolate a specific gene by selection or screening Cloning by complementation 1 2 Isolate a mutant strain giving the desired phenotype Transform the mutant strain with a DNA library and directly select for the positive clone by its ability to complement the mutant defect DNA Probe A radioactive DNA fragment complementary to the gene you want to clone Can be from a related organism Electrophoresis Used to fractionate DNA RNA or proteins based on their size eg Digest DNA and run on a gel Southern Blot Probing for a DNA fragment using a DNA probe Figures 12 18 12 20 Northern Blot Probing for an RNA fragment using a DNA or RNA probe Figure 12 20 Western Blot Probe for a protein using antibodies Figure 12 20 DNA Sequencing Used to determine the nt sequence of any gene Can resolve DNA fragments differing by 1 nt Gilbert and Sanger shared the Nobel prize Dideoxy Seguencing Dideoxy nts lacking a 3 OH group can t be extended by DNA pol once incorporated Random incorporation ddATP ddCTP ddGTP ddTTP Figure 12 23 Automated Seguencing Uses fluorescent dyes Figure 12 24 Polymerase Chain Reaction PCR9Mullis Nobel Prize Used to amplify specific regions of DNA Uses a thermostable DNA pol Taq Vent Pfu Figure 12 26 Can amplify DNA from a single cell Animation 1202 Restriction Mapping Restriction sites in a DNA fragment can be used to subclone fragments within the fragment 1 9WP Digest DNA with one of several enzymes Run digested DNA on an agarose gel to separate fragments Stain DNA with ethidium bromide EtBr which intercalates between bases View under UV light EtBr fluoresces Single double or partial digests Figure 12 27 A linear 13 kb fragment of DNA is digested with various restriction enzymes The results of single and double digests are shown below Here is a sample problem to work on your own Enzvmes Fragment Sizes kb BamHI 3 and 10 EcoRI 6 and 7 HindIII 1 and 12 BamHI and EcoRI 3 4 and 6 BamHI and HindIII 1 3 and 9 What fragment sizes are expected if the 13 kb fragment is digested with EcoRI and HindIII A 13and9 B 34and6 C 15and7 D 14and8 E 44and5 SiteDirected Mutagenesis Directing point mutations insertions or deletions into cloned DNA fragments Figure 13 1 Eukaryotic Gene Expression in Bacteria Use specialized vectors to specifically overexpress biologically important human proteins in bacteria usually E coli eg Insulin Eukaryotic Transgenic Technology E coli942 million bp Human93 billion bp Plants Some even larger Specialized techniques were developed to handle large genomes Transgenic Technology Methods used to transfect eukaryotic cells Transgenic Organism Organism that develops from the transfected cell Gene Inactivation Suicide vector Figure 13 12 1 Clone selectable marker in the middle of a gene 2 Linearize with restriction enzyme 3 Transform organism 4 Double X over results in replacement of WT gene with disrupted gene 5 Study the effect of the mutation Studying Gene Regulation Figure 13 13 1 Clone regulatory 5 region adjacent to a reporter gene gene whose protein is easy to assay Expression of reporter gene depends on cloned regulatory elements 2 Study regulation 3 Repeat with deletions or point mutations in regulatory region Transgenic Plants Ti Plasmid from Agrobacterium tumefaciens Causes crown gall plant tumors Figure 13 14 Bacteria infects plant and injects part of plasmid called T DNA Ttumor Clone gene in middle of T DNA so that the gene is inserted into plants with T DNA eg Firefly luciferase gene glow in the dark plants Chapter 13 cover Transgenic Animals Applying similar techniques to study the function of animal genes Can be used for gene therapy in humans Gene Therapy Correct genetic defects by transferring WT genes into the germ line gametes of animals or other actively dividing tissue e g stem cells SCID Severe Combined Immunodeficiency Disease has been cured in 10 individuals with gene therapy but one developed leukemia due to the point of insertion in the genome Human Genetic Disorders Recessive disorders cause over 500 genetic diseases Would like to determine if individual carries mutant genes Restriction Fragment Length Polymorphism BFLPI Sickle cell anemia affects 025 of US African Americans GAG GTG mutation eliminates an MstII site Figure 13 29 Change detected by Southern blotting Change in banding pattern diagnostic for sickle allele DNA Fingerprinting Used in forensic medicine Variable Number Tandem Repeats VNTRs Figure 14 4b Humans91 5 kb sequences consisting of repeats 15 100 nt long Digest DNA with restriction enzyme that does not cut within VNTRs Run DNA on gel Southern blot with VNTR probe Pattern on autoradiograph is highly individualistic Figure 14 3 PWNH DNA samples can be amplified by PCR using trace amounts of blood semen hair Wild type WT compared to a mutant or variant Mutant An individual or strain carrying a mutation Mutation Change from one hereditary state to another Used to genetically dissect biological functions and to study the process of mutation Gene Mutation A mutation in a specific gene resulting in a new allele Forward Mutation Any change from the WT allele Reverse Mutation Change to the WT allele true reversion Second Site Suppressor A change in the same gene or a second gene resulting in a complete or partial phenotypic reversion to W second site reversion Loss of Function Mutation A Null mutation No activity B Leaky mutation Some residual activity Gain of Function Mutation Results in a new activity Silent Substitution The mutation changes one codon for an AA into another codon for the same AA Missense Mutation The codon for one AA is replaced by a codon for another AA Nonsense Mutation The codon for an AA is replaced by a stop codon Somatic Mutation A mutation in any tissue other than the germinal tissue Clone Population of identical cells derived from one mutant progenitor asexual ie Mitosisnot transmitted to progeny Often visualized as a sector Figures 15 3 to 15 5 Germinal Mutation9A mutation in tissue that forms gametes An individual with the new germinal mutation will not show the phenotype but the mutation can be transmitted to progeny Figure 15 8 Conditional MutationThe allele only expresses the mutant phenotype under certain environmental conditions e g 9 ts temperature sensitive Auxotrophic Mutation9The individual must be supplied with certain nutrients amino acids nucleotides vitamins Commonly used when studying microorganisms WT is prototrophic nutritionally self sufficient Resistance Mutation Confers the ability to grow in the presence of an inhibitor eg antibiotic or pathogen Point MutationSingle base pair change in DNA Deletion Removal of one or more bases of DNA Insertion Addition of one or more bases of DNA Mutation Rate of mutations OR of mutations cell division gamete Human Genetics Germinal mutations are detected by the sudden appearance of the abnormal phenotype in a pedigree with no previous record of abnormality Dominant mutations are easy to detect Recessive mutations can go unnoticed for several generations X linked recessive mutations are easier to detect than autosomal Queen Victoria Hemophilia Different genes have different mutation rates See Tables 15 2 amp 15 3 Mutant Hunts Experiments designed to isolate mutants that affect a specific biological function Selective System9A technique designed to separate rare mutant individuals from WT Need a selectable phenotype Mutagens Used to increase mutation rates e g chemicals radiation Diploid oLganism germinall aabb X aabb xl a a bb aw b b mutant 1 mutant 2 Diploid ogganism somaticL Look for sectoring in a heterozygote Single cell haploid organisms Advantages 1 Grow as single cells in liquid culture or as colonies 2 Easy to examine millions of individuals 3 Isolated single cells generate a clonal population colony of genetically identical cells 4 Mutants are easily identified dominant or recessive Detection of Reverse Mutations gauxotrophz 1 Grow mutant culture in minimal medium supplement 2 Plate cells on minimal medium without supplement 3 Survivors are prototrophs Penicillin Enrichment Auxotrophic selection in bacteria Penicillin kills actively growing cells 1 Grow cells in rich medium 2 Transfer to minimal medium 3 Add penicillin prototrophs die auxotrophs survive 4 Plate cells on minimal medium supplement Resistance Mutations 1 Grow cells in liquid culture 2 Plate cells on selective medium drug or virus Fluctuation Test Used to determine if resistant mutations were due to random mutations or changes in bacterial physiology 1 Liquid culture of E coli 2 Mix individual cultures with bacteriophage T1 3 Plate cell phage mixture 4 WT cells killed by phage T1 5 Resistant mutants form colonies Fluctuation Test Predictions A Random mutation Rare mutations could occur early or late in the culture Predict a large variation in the number of resistant colonies B Physiological change Time for physiological adaptation would be relatively constant Predict small variation in number of colonies Answer Random Mutation Figure 15 21 Table 15 4 Somatic Cell GeneticsApplying mutagenic and selective techniques to animal and plant cell cultures Often only identify dominant mutations because the organism is diploid Mutation and Cancer Cancer is a genetic disease caused by mutations in proto oncogenes dominant or tumor suppressor genes recessive Protooncogenes and Tumor Suppressor Genes Normally carry out functions related to regulation of cell division A mutation in a proto oncogene can lead to uncontrolled cell division mutant clone resulting in a tumor cancer Cancer can spread by metastasis Genetic Predjsposition A mutant gene causes an increase in mutation frequency of other genes leading to cancer Spontaneous Mutations Occur in all cells without a mutagen Errors in DNA Replication Normally A C G and T are in the keto form Figure 16 1 Errors occur during DNA replication when rare imino forms of A and C or rare enol forms of T and G are incorporated by DNA polymerase Fair with the wrong base Figure 16 2a b The DNA pol III editing function removes mismatches when the rare forms change back unless polymerization has already moved past the mismatch Mutations will result if editing does not occur unless the change is repaired by another mechanism called mismatch repair Transition purine for the other purine or pyrimidine for the other pyrimidine Transversion Pyrimidine substituted for a purine and Vice versa Frameshift Caused by deletion or insertion of one to a few nts Can occur Via slipped mispairing during DNA replication eg Fragile X syndrome myotonic dystrophy etc Figure 16 4 Animation 1602 Spontaneous Lesions Mutations can occur due to DNA damage Depurination When the N glycosidic bond between the base and the sugar is broken The resulting apurinic site AP site can t specify a complementary base during replication 10 000cell generation in mammals DNA repair required Deamination Loss of an amino group from the base Deamination of dC yields dU Figure 16 8a dU pairs with dA GC gtAT transition DNA repair OXidative Damage Byproducts of aerobic metabolism produces compounds that cause oxidative DNA damage Induced Mutations Produced when a cell or organism is exposed to a mutagenic agent mutagen Replace a base in DNA9 Molecules which are similar in structure to bases base analogs but have different pairing properties can replace the normal base in the DNA during replication Speci c Mispairing Some chemicals alter the structure of a base resulting in mispairing during replication Intercalating Agents Chemicals that are planar can mimic bases and slip in intercalate between bases in the double helix Results in frameshifts Loss of Pairing due to Chemical Alteration Chemical structure is altered so it can39t pair with any base Results in a replication block Lethal unless the block is bypassed UV light can cause several types of DNA damage eg UV light can generate cyclobutane pyrmidine dimers which distorts the structure of DNA such that base pairing is not possible Most carcinogens result in chemical alteration of DNA Cancer can be caused by mutations in genes whose protein products regulate cell division If this check point is abolished it will lead to uncontrolled cell division ie cancer 13539 About 50 of all cancerous tissues contain mutations in the p53 gene p53 arrests cell division when DNA mismatches are recognized ie involved in cell cycle control DNA Repair Several enzymatic systems exist to repair various types of DNA damage In humans several disorders are caused by defects in DNA repair systems which can lead to cancer Classes of Repair Pathways Systems 1 Prevention of Errors Before they Happen Some enzymes neutralize damaging compounds eg Detoxification of molecules that cause oxidative damage Superoxide dismutase converts oxygen radicals to hydrogen peroxide Then catalase converts hydrogen peroxide to water 11 Direct Reversal of DNA Damage Cyclobutane pyrimidine dimers UV light9Repaired by photolyase Requires visible light for the enzyme to work Figure 16 25 Removal of alkyl groups added to bases Alkyltransferases responsible for direct reversal eg Methyltransferase of E coli III ExcisionR epair Pathways General Excision Repair Removal of altered bases along with several neighboring bases and then repairing the gap by DNA synthesis Figure 16 27 E cali An endonuclease cuts on both sides of the damaged base removing ssDNA containing the damaged bases Gap filled in by DNA pol 1 DNA ligase seals the nick Animation 1601 Specific Excision Repair AP Endonuclease Repair Pathway AP endonuclease removes AP site by breaking a phosphodiester bonds at the AP site Figure 16 30 Then the general excision repair pathway takes over DNA Glycosylase Repair Pathway DNA glycosylases recognize certain damaged bases and cleave the N glycosidic bond between the base and the sugar leaving an AP site Figure 16 29 eg Uracil DNA Glycosylase The resulting AP site is cleaved by AP endonuclease Then the general excision repair pathway takes over IV Postreplication Repair Figure 16 32 Mismatch Repair DNA editing by DNA pol 111 did not occur 1 Recognition of the mismatch 2 Determine which mismatched base is incorrect 3 Excise the incorrect base 4 General excision repair takes over Adenine methylase methylates A residues following replication in the sequence 5 GATC 339 But it takes a few minutes for the newly synthesized strand to be methylated The old methylated strand is distinguished from the newly synthesized unmethylated strand An enzyme introduces a cleavage in the backbone of the unmethylated strand near the mismatch and adjacent to the nearest GATC sequence ssDNA gap is filled in by DNA pol 1 Li gase seals the nick Repair Defects and Human Disorders Often leads to an increased incidence of cancer Usually autosomal recessive See table 16 3 Xeroderma Pigmentosum Caused by a mutant excision repair enzyme Most people die by the age of 30 from skin cancer Bloom syndrome Caused by a DNA ligase deficiency Greatly increased cancer rate usually die by 30 Hereditary Nonpolyopsis Colorectal Cancer HNPCC9 Caused by the loss of the mismatch repair system Chromosomal Rearrangements Deletions9Loss of a region caused by a chromosomal break Duplication Reciprocal change of a deletion Inversion9Chromosomal region rotated 180 Translocation Exchange of parts of non homologous chromosomes Deletions Usually fatal if homozygous Often fatal if heterozygous Some small deletions are Viable as a heterozygote Deletions can never revert to WT Visualized as a deletion loop during meiosis Homologous Chromosomes Pseudodominance9Deletion Will uncover recessive alleles on the other chromosome thus the recessive phenotype is expressed lhl Ill u A H I A H Small deletions can be mapped due to pseudodominance Useful in correlating linkage and cytological maps Figure 17 4 Humans Usually caused by a neW germinal mutation in one parent e g cri du chat syndrome Tip of chromosome 5 deleted Duplications Can be adjacent to each other or the second copy may be in a novel location on the same or a different chromosome 3 copiescell in a diploid Duplication heterozygotes can result in unusual pairing structures during meiosis Figure 17 9 Duplications can arise from breaking adding new DNA then rejoining OR by unegual crossing over Figure 17 11 Usually difficult to detect phenotypically A loop structure may be detected during meiosis l Homologous Chromosomes Tandem Duplication ABCBCD Reverse Duplication ABCCBD The hemoglobin Hb gene family provides evidence for duplications generated by unequal crossing over Figure 17 13 Human homozygous duplications have never been detected probably lethal Inversions 2 of humans carry inversions No net change of genetic material Viable without phenotypic abnormalities unless breakage occurs in an essential gene Then they are lethal if homozygous Paired homologs form an inversion loop during meiosis if heterozygous Figure 17 15ab Paracentric Inversion Centromere outside of the inversion eg ABCDE gtABDCE X overs between a paracentric inversion and a WT chromosome results in a dicentric and an acentric chromosome Acentric fragment is lost Dicentric breaks randomly in bridge Figure 17 16 Animation 1701B Pericentric Inversion lnversion spans the centromere eg ABCDE gtACBDE X overs between a pericentric inversion and a W chromosome result in products with a deletion and a duplication of different parts of the chromosome Figure 17 17 Inviable deletion products and inhibition of pairing in the inverted region reduces the number of recombinants among the progeny of inversion heterozygotes Diagnostic features of inversions 1 Decreased recombinant frequency 2 Inversion loops 3 Partial sterility 4 Inverted arrangements of chromosomal landmarks eg Centromere position 3 Normal 41 ratio Inversion 1 1 ratio Translocations When two nonhomologous chromosomes mutate by exchanging parts Viable unless the breakpoint is in an essential gene Reciprocal Translocation A region from one chromosome is exchanged with a region from another nonhomologous chromosome so that two translocation products are simultaneously generated most common Figure 17 23 Diagnostic features of translocations 1 Establishes new linkage groups ie gene is now on a different chromosome 2 May alter the size of chromosomes and centromere position 3 Causes semisterility950 of plant gametes and mammalian zygotes are inViable Animation 1702C Humans Always in heterozygous state Cri du chat syndrome if the child is missing the tip of chromosome 5 due to translocation Down syndrome if two normal chromosomes 21s and additional segment of 21 due to translocation Results in high occurrence in family tree Position Effect Variegation When a gene is translocated to a region near heterochromatin of another chromosome In some cells the heterochromatin will engulf the gene shutting off expression leading to mutant phenotype Burkitt s Lymphoma in humans is usually caused by the translocation of an oncogene on the tip of chromosome 8 next to an antibody gene enhancer on chromosome 14 Monoploid Number Number of chromosomes in the basic set of an organism Euploid Organisms with multiples of the monoploid number Polyploid Euploid with more than two sets of chromosomes Monoploid 1x Diploid 2x Triploid 3x etc n haploid number9The number of chromosomes in gametes n X for most organisms Monoploid organisms male bees wasps ants Males develop parthogenetically from unfertilized eggs These organisms are sterile because meiosis is not possible no pairing Plant Engineering Generate a monoploid plant from a diploid Then generate a drug resistant diploid from the monoploid plant Figure 18 3 Colchicine inhibits mitotic spindle formation After mutant selection and growth use colchicine for one cell division Autopolyploids Multiple chromosome sets from within one species Allopolyploids 9Multiple chromosome sets from closely related species Triploids 135119 Problems during meiotic segregation sterile 4x Tetraploid X 2x Diploid xl 3x Triploid eg Seedless watermelons and bananas Autotetraploids Arise spontaneously from accidental doubling 2X to 4X or use colchicine Advantages larger plant and fruit Polyploidy in animals Leeches brine shrimp Common in amphibians and reptiles Salmon and trout originated through polyploidy Oysters 3x9No spawning palatable all year Most human triploids are spontaneously aborted If born none survive Aneuploidy Number of one or more chromosomes change during formation of an individual Caused by nondisjunction during meiosis Figure 18 16 If an n 1 gamete is involved in fertilization the resulting zygote will be monosomic for that particular chromosome 2n 1 If n1 gamete the zygote will be trisomic 2n1 A Zn 2 individual is nullisomic A 2n11 individual is a double trisomic Monosomics Zn 1 1 Deleterious Missing chromosome disturbs overall balance of chromosomes ie disturbs homeostasis Hemizygous for that chromosome Deleterious because recessive alleles are expressed phenotypically Humans About 10 of all human conceptions have a major chromosome abnormality Most are spontaneously aborted Turner Syndrome944 autosomes with 1X chromosome Sterile normal intelligence 15000 females ALL MONOSOMICS FOR AUTOSOMES ABORT Klinefelter Syndrome XXY Lanky builds retarded sterile 1 1000 males Mean Man Syndrome9XYY A gressive behavior Fertile 11000 males Patau Syndrome Trisomy 13 Severe physical and mental abnormalities 130 day survival Edward Syndrome Trisomy 18 Severe physical and mental abnormalities survive a few weeks Down Syndrome Trisomy 21 15 1000 births Most common human aneuploid More common than the translocation form No family history Older mothers at greater risk Figure 18 22 Mental retardation Males infertile Females may be fertile producing normal and trisomic children Somatic Aneuploids Aneuploid cells that arise spontaneously in somatic tissue genetic mosaics eg Sexual mosaics XOXYY Caused by an XY zygote in which the Y chromosome fails to disjoin at an early mitotic division The phenotypic sex depends on where the male and female sectors end up in the body If nondisjunction occurs later in development a three way mosaic arises XYXOXYY XOXY Probably due to the loss of a chromosome in the male zygote XXXY Probably due to a double fertilization fused twins Acute Myeloid Leukemia94758 patients were aneuploid for either chromosome 8 9 or 21 Philadelphia Chromosome Part of chromosome 22 is translocated to chromosome 9 Often found in individuals with chronic myeloid leukemia
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