124 Class Note for BIOL 222 at PSU
124 Class Note for BIOL 222 at PSU
Popular in Course
Popular in Department
verified elite notetaker
One Day of Notes
verified elite notetaker
verified elite notetaker
One Day of Notes
verified elite notetaker
verified elite notetaker
verified elite notetaker
This 26 page Class Notes was uploaded by an elite notetaker on Friday February 6, 2015. The Class Notes belongs to a course at Pennsylvania State University taught by a professor in Fall. Since its upload, it has received 19 views.
Reviews for 124 Class Note for BIOL 222 at PSU
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 02/06/15
Chapter 119Gene Isolation and Manipulation 1024051 Problems 12 13 21 25 26 31a b c Recombinant DNA Technology Recombinant DNA technology allows us to isolate specific genes from any genome so that we can study their function Recombinant DNA molecules can be made from any organism by inserting DNA fragments into a cloning vector Figure 11 3 Vector Plasmid or Virus Contains an origin of replication for gene amplification Contains an antibiotic resistance gene or some other gene for selection Restriction Enzymes Make sequence specific cuts in DNA by cleaving phosphodiester bonds of each strand of the DNA duplex quotdigestionquot e g EcoRI cohesive quotstickyquot ends xl 5 GAATTC3 G AATTC 3 CTTAAG5 CTTAA G I eg Smal blunt ends xl 5 CCCGGG3 CCC GGG 3 GGGCCC5 GGG CCC 1 Characteristics of Restriction Sites A 180 axis of symmetry B Usually a 4 1256 or 6 nt sequence 14096 Gene Cloning Figure 1 1 5 1 Digest chromosomal and vector DNA with an enzyme 2 Mix together Sticky ends m hybridize base pair due to complementarity Can also use blunt ends 3 Seal the phosphodiester bonds quotnicksquot with DNA Li gase 4 Transform E coli and select for drug resistance or by complementation compensation of a mutant defect Figure 11 6 5 Amplify and purify recombinant DNA THEN 1 Determine the DNA sequence and identify the Open Reading Frame ORF 2 Engineer mutations to study gene function and gene regulation 3 Overexpress gene and purify the protein to study function Animation Vectors A Plasmids Figure 11 7 Small circular origin of replication antibiotic resistance gene Can clone several kb B Expression Vectors Specialized plasmids that contain transcription and translation signals to allow overproduction of the protein encoded by the gene Express eukaryotic genes in bacteria eg human insulin C Shuttle Vectors Contain origins of replication for two organisms eg E coli and SV40 Monkey virus Clone in E coli purify DNA transfect mammalian cell line D Bacterial Artificial Chromosomes BACs Based on the F plasmid 150 kb can be cloned E Yeast Artificial Chromosomes Y ACs Contain an origin of replication telomeres and a centromere 1000 kb can be cloned cDNA complementary DNA DNA generated from mRNA and reverse transcriptase thus no introns 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 cloning by complementation or screening Cloning by complementation 1 Isolate a mutant strain giving the desired phenotype 2 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 that is complementary to the gene you want to clone Can be the homologous gene from a related organism eg clone a human gene using the cloned mouse gene as a probe Electrophoresis Used to fractionate DNA RNA or proteins based on their size eg Digest DNA and run on a gel Figure 11 13 Southern Blot Probing for a DNA fragment using a DNA probe Figure 11 14 Northern Blot Probing for an RNA fragment using a DNA probe Western Blot Probe for a protein using antibodies Restriction Mapping Restriction sites in a DNA fragment can be used to subclone fragments within the fragment 1 M WP 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 1 1 16 Sample Problem A linear 13 kb fragment of DNA is digested with various restriction enzymes The results of single and double digests are shown below Enszes 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 Chapter 119Gene Isolation and Manipulation 1028051 DNA Seguencing Used to determine the nt sequence of any gene Can resolve DNA fragments differing by 1 nt Gilbert and Sanger shared the Nobel Prize A Dideoxy Sequencing Dideoxy nts lacking a 3 OH group can t be extended by DNA pol once incorporated Random incorporation because of a mixture of dNTPs and one ddNTP ddATP ddCTP ddGTP ddTTP Figures 11 17 and 11 18 B Automated Sequencing Uses uorescent dyes Figure 11 19 Polymerase Chain Reaction 1ECRMullis Nobel Prize Used to amplify specific regions of DNA Uses a thermostable DNA polymerase eg Taq Can amplify DNA from a single cell Figure 11 21 Animation SiteDirected Mutagenesis Directing point mutations insertions or deletions into cloned DNA fragments by PCR Gene Inactivation Suicide vector 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 1 Clone the regulatory region eg promoter adjacent to a lm gm a 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 Human Genetic Disorders Recessive disorders cause over 500 genetic diseases Would like to determine if individual carries mutant genes Restriction Fragment Length Polymorphism BFLPz Sickle cell anemia affects 025 of US African Americans GAG HbA9GTG Hbs mutation eliminates an Mstll restriction site Change detected by Southern blotting Change in banding pattern diagnostic for sickle allele Glu Val change alters Hb structure Figure 11 24 DNA Fingerprinting Used in forensic medicine Variable Number Tandem Repeats VNTRs 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 39gtE H Pattern on autoradiograph is highly individualistic DNA samples can be amplified by PCR using trace amounts of blood semen hair Eukaryotic Transgenic Technology E coli946 million bp Human93 billion bp Plants Some even larger Specialized techniques were developed to handle large genomes e g YACs Transgenic Technology Methods used to transfect eukaryotic cells Transgenic Organism Organism that develops from the transfected cell Transgenic Plants Ti Tumor Inducing Plasmid from Agrobacterium tumefaciens Causes crown gall plant tumors Figure 11 28 Bacteria infects plant and transfers part of plasmid called T DNA Ttransfer into plant genome Clone gene in middle of T DNA so that the gene is inserted into plants with T DNA eg Fire y luciferase gene glow in the dark plants Transgenic Animals Applying similar techniques to study the function of animal genes Can be used for gene therapy in humans Human Gene Therapy Correct genetic defects by transferring W genes into the germ line gametes or other actively dividing tissue e g stem cells SCID Severe Combined Immunodeficiency Disease quotBoy in the Bubble Diseasequot No functional immune system Has been cured in 9 of 11 individuals in a clinical trial with gene therapy France One individual developed leukemia due to the point of insertion in the genome Chapter 12Genomics 103105 Problems none Genomics The study of entire genomes gt 250 completed bacterial genomes Theoretically possible to complete a bacterial genome in a single day using automated technology Figure 12 3 Human Drosophia yeast C elegans etc also sequenced 1 Sequence the entire genome A Shotgun sequencing Figure 12 2 Sequence random clones and then assemble the sequence into a complete chromosome by looking for overlaps within the sequenced clones Requires more sequencing 10 genome equivalents but no need to order clones B Sequence ordered clones Less sequencing and no need to assemble the genome but it takes time to order the clones 2 Bioinformatics Annotate the genome Figure 12 22 A Identify all of the Open Reading Frames ORFs encoded in the genome 1 Computationally remove introns in higher eukaryotes 2 Compare to full length cDNA sequences 3 Predict regulatory regions quotdocking sites for regulatorsquot comparative genomics 4 BLAST Basic Local Alignment Search Tool search Search public databases for similar DNA andor protein sequences A hit suggests that the gene is real comparative genomics is a powerful approach Figures 12 25 and 12 26 5 Codon bias Human Genome 25 000 genes There are about 3 alternative splicing pathways per gene Thus the proteome is thought to be about 3 times the size of the genome ie 75000 proteins 3 Functional Genomics Using genomic approaches to study all of the gene products simultaneously A Transcriptome analysis Analyze the expression patterns of all genes simultaneously under various growth conditions times during development when a regulatory protein is mutated etc Microarray studies ie gene chips Figure 12 27 Animation B Proteome analysis Using 2 D gels and mass spectroscopy to identify changes in protein levels under various growth conditions times during development when a regulatory protein is mutated etc Complementary to transcriptome analysis but also identified translationally controlled genes C Interactome analysis Identification of the complete set of physical interactions between proteins and DNA proteins and RNA and between proteins Yeast 2 hybrid assay Figure 12 29 D Phenome analysis Identification of all phenotypes associated with the inactivation of each gene Chapter 139Transposable Elements 1112051 Problems 2 3 6 7 Transposable Genetic Elements quotJumping Genesquot Genetic elements than can move or quottransposequot from one position to another Barbara McClintock won the Nobel Prize 1983 for her pioneering work 1940s on Maize AcDs elements Transposable elements are present in essentially all organisms Bacteria 1 Insertion Sequences IS 2 Transposons Tn When IS elements insert in the middle of a gene it inactivates that gene eg IS1 1S2 IS3 etc tnp lt gt lRgtlnverted Repeats IR IR tnp encodes transposase the enzyme responsible for transposition IS elements are sites where crossing over X over can occur F fertility factor integration into the chromosome to generate Hfr strains occurs Via X overs between IS elements in the F plasmid and chromosomal IS elements Figure 13 8 Transposons Composite elements that contain tnp and additional genes typically a drug resistance gene A Composite Transposon Figure 13 9a Composed of 2 IS elements and a drug resistance gene between them eg Tn10 contains 2 IS10 elements and a tetracycline resistance gene Transposase comes from one of the IS elements and the IS elements also function as the IRs B Simple Transposon Figure 13 9b Composed of a transposase gene and typically a drug resistance gene IRs similer to those for IS elements Mechanism of Transposition Transposition involves cleavage of the DNA target followed by insertion of the transposable element Subsequent filling in of the resulting single stranded gaps generates target site duplications Characteristic of transposable elements Transposase is responsible for target site selection and DNA cleavage A Replicative One copy of the transposable element remains in the original site and a second copy inserts into a new site Requires DNA replication B Conservative nonreplicative No DNA replication The transposable element is excised and moved to a new site Disrupted genes at the original site revert to wild type Figure 13 1 1 Multiple Antibiotic Resistance Transposons can jump from a naturally occurring plasmid to a chromosome or from plasmid to plasmid The plasmids can move from bacteria to bacteria via transformation andor conjugation Can lead to multiple antibiotic resistant strains of bacteria Huge problem in a clinical setting Figure 13 10 Eukaryotic Transposable Elements Can inactivate a gene cause chromosome breaks and transpose to new locations replicative or non replicative A Class I Retro transposons RNA elements Must be transcribed into RNA then reverse transcribed to DNA then inserted Class I elements can39t excise and restore function to an interrupted gene e g yeast Ty Drosophila copia human Alu elements These elements are similar to retroviruses both have gag and pol genes gag involved in RNA maturation pol reverse transcriptase The absence of the env envelope gene prevents packaging of the genome into a vial particle Instead the DNA is inserted into a new place in the genome Figure 13 15 Retroviruses ssRNA animal viruses e g HIV 1 Eject RNA genome 2 Reverse transcriptase synthesizes dsDNA from the ssRNA A First DNA strand synthesized using RNA as template B Second DNA strand synthesized using the first DNA strand as template while simultaneously degrading the RNA strand 3 Integration into the mammalian genome B Class II DNA transposons Similar to bacterial transposable elements encode transposase eg Maize Ac and Drosophila P elements Maize Ds elements are defective Ac elements no functional transposase Requires transposase from Ac to transpose Figure 13 19 Excision from a gene can lead to reversion to wild type Figures 13 5 and 13 6 Autonomous Elements Fully functional can transpose by themselves eg Ac elements of Maize Nonautonomous elements Requires an autonomous element to supply transposase for transposition eg Ds elements of Maize Humans Transposable elements can be VERY abundant in genomes 50 of the human genome is derived from transposable elements gt1 million Alu sequences alone There is 20 times more human DNA corresponding to transposable elements than protein coding DNA LINEs autonomous and SINEs nonautonomous are retro transposons Alu is a SINE The majority of transposable elements are inserted in introns or between genes Natural selection reduces exon insertions being fixed in the population Chapter 14Gene Mutation 1114051 Problems 2 17 21 24 Gene Mutation A mutation in a specific gene resulting in a new allele Wild type WT compared to a mutant or variant Mutant An individual or strain carrying a mutation Muta en Agents that increase the rate of mutations 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 Results in the loss of activity Gain of Function Mutation Results in a new or altered activity Transition Substitution of a pyrimidine for the other pyrimidine OR a purine for the other purine Transversion Substitution of a pyrimidine for a purine and vice versa Point Mutations AE Single nucleotide changes A Silent QSynonymous Substitution The mutation changes one codon for an amino acid into another codon for the same amino acid Missense Mutation The codon for one amino acid is replaced by a codon for another amino acid Nonsense Mutation The codon for an amino acid AA is replaced by a stop codon Deletion Removal of one nucleotide Insertion Addition of one nucleotide D and E will lead cause frameshifts if in a protein coding region Table 14 2 1 A C INDUCED MUT ATIONS Produced when a cell or organism is exposed to a mutagen eg chemicals or UV light Base Replacement Molecules that are similar in structure to bases base analogs but have different pairing properties can replace the normal base in the DNA during replication Base Alteration Figure 14 9 Mutagens that alter the structure of a base lead to mispairing eg alkylating agents Base Damage Figure 14 12 Damage so severe that pairing can39t take place Will lead to a replication block lethal if not repaired eg UV induced cyclobutane pyrimidine dimmers OR 6 4 photoproduct II SPONTANEOUS LESIONS Occur in all cells without a mutagen A D Depurination When the N glycosidic bond between the base and the sugar is broken Put in part of Figure 14 14 The resulting apurinic site AP site can t specify a complementary base during replication Deamination Loss of an amino group from the base Deamination of dC yields dU dU pairs with dA Figure 14 18 DNA repair required Errors in DNA Replication 1 KetoEnol Shift Normally A C G and T are in the keto form Figure 14 5 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 These tautomers pair with the wrong base Figure 14 6 The DNA pol III editing function removes mismatches when the rare forms change back unless polymerization has already moved past the mismatch DNA Repair required 2 Replication Slippage Results in deletions or insertions that can cause frameshifts Figure 14 21 Animation Triplet repeat expansion diseases in humans is thought to occur Via replication slippage e g Fragile X syndrome Huntingtons disease myotonic dystrophy OXidative Damage Byproducts of aerobic metabolism produce compounds that cause oxidative DNA damage eg superoxide radicals and hydrogen peroxide H202 Chapter 14DNA Repair 11705 1 Several enzymatic systems exist to repair various types of DNA damage In humans several disorders are caused by defects in DNA repair systems that can lead to cancer Classes of Repair Pathways Systems 1 Prevention of Errors Before they Happen Some enzymes neutralize damaging compounds e g 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 A Cyclobutane pyrimidine dimers are repaired by photolyase Requires visible light for the enzyme to work Figure 14 26 B Removal of alkyl groups added to bases Alkyltransferases responsible for direct reversal e g Methyltransferase of E coli III ExcisionR epair Pathways A Nucleotide Excision Repair gGeneral Excision Repair Removal of damaged bases along with several neighboring bases and then repairing the gap by DNA synthesis eg cyclobutane pyrimidine dimers large base aducts Figure 14 28 E cali An excinuclease excision nucleasecuts 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 B AP Endonuclease Repair Pathway AP endonuclease removes AP site by breaking a phosphodiester bonds at the AP site Then the general excision repair pathway takes over Figure 14 27 C 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 e g Uracil DNA Glycosylase The resulting AP site is cleaved by AP endonuclease Then the general excision repair pathway takes over Figure 14 27 IV Postreplication Repair A Mismatch Repair Figure 14 30 DNA editing by DNA pol 111 did not occur 1 Recognition of the mismatch 2 Determine which mismatched base is incorrect Crucial step 3 Excise the incorrect base 4 General excision repair takes over Adenine methylase methylates A residues following replication in the sequence 5 GATC 3 Since 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 Chapter 15 Changes in Chromosome Number 1119051 Problems 2 3 9 15 16 23 32 36a b Haploid Cells n9Cells with one chromosome set eg gametes Diploid Cells 2n9Cells with two chromosome set e g somatic cells Monoploid Number n9Number 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 1n Diploid 2n Triploid 3n etc Monoploid organisms male bees wasps ants Males develop parthogenetically from unfertilized eggs These organisms produce gametes via mitosis Plant Engineering Generate a monoploid plant from a diploid Figure 15 1 1 Then generate a drug resistant diploid from the monoploid plant 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 e g cotton wheat 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 Figure 15 6 Polyploidy in animals Leeches brine shrimp atworms Common in amphibians and reptiles Salmon and trout originated through polyploidy Oysters 3n9No spawning palatable all year Most human triploids die in utero If born none survive Aneuploidy An individual whose chromosome number differs from WT by part of a chromosome set Usually one chromosome Caused by nondisjunction during meiosis Figure 15 13 If an n l gamete is involved in fertilization the resulting zygote will be monosomic for that particular chromosome Zn 1 If an n1 gamete is involved in fertilization the resulting zygote will be trisomic for that particular chromosome 2n1 Animation Monosomics Zn 1 1 Deleterious Missing chromosome disturbs homeostasis The individual is hemizygous for that chromosome Deleterious because recessive alleles are expressed phenotypically Chapter 15Chr0m0s0me Mutation 1111051 Human Aneuploidy 10 of all human conceptions have a major chromosome abnormality Most are spontaneously aborted A Monosomic 1 Turner Syndrome 15000 females 44 autosomes with 1X chromosome Sterile normal intelligence ALL MONOSOMICS FOR AUTOSOMES DIE IN UTERO B Trisomic 1 Klinefelter Syndrome 11000 males XXY Lanky builds retarded sterile Mean Man Syndrome 11000 males XYY Fertile Aggressive behavior XXX Phenotypically normal female Fertile Patau Syndrome Trisomy 13 Severe physical and mental abnormalities 3 month survival Edward Syndrome Trisomy 18 Severe physical and mental abnormalities survive a few weeks 6 Down Syndrome 1 5 1000 births Trisomy 21 Most common human aneuploid This more common than the translocation form No family history Older mothers at greater risk Figure 15 18 Mental retardation Males infertile Females may be fertile producing normal and trisomic children Acute Myeloid Leukemia 4758 patients were aneuploid for either chromosome 8 9 or 21 Chromosomal Rearrangements A Deletions9Loss of a chromosomal region 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 Figure 15 28 Pseudodominance I h I I h I A H I A H Deletion Will uncover recessive alleles thus the recessive phenotype is expressed Small deletions can be mapped due to pseudodominance Humans Usually caused by a neW germinal mutation in one parent e g cri du chat syndrome Tip of chromosome 5 deleted Figure 15 30 B Duplications Gain of a chromosomal region Can be adjacent to each other or the second copy may be in a novel location in the genome 3 copiescell in a diploid Usually difficult to detect phenotypically A loop structure may be detected during meiosis l Homologous Chromosomes Tandem Duplication A B C B C D Reverse Duplication A B C C B D Human homozygous duplications have never been detected probably lethal Chapter 15Chr0m0s0me Mutation 1114051 C Inversions Chromosomal region rotated 180 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 15 21b Paracentric Inversion Centromere outside of the inversion eg gC D E Fgt C E D F X overs between a paracentric inversion and a WT chromosome result in a dicentric and an acentric chromosome Figure 15 22 Acentric fragment is lost Dicentric breaks randomly in bridge Animation Pericentric Inversion lnversion spans the centromere eg ABCDE gtACBDE X overs between a pericentric inversion and a WT chromosome result in products with a deletion and a duplication of different parts of the chromosome Figure 15 23 Animation Inviable deletion products and inhibition of pairing in the inverted region reduces the number of X over recombinants among the progeny of inversion heterozygotes Diagnostic features of inversions 1 Decreased recombinant frequency 2 Inversion loops 3 Inverted arrangements of chromosomal landmarks eg Centromere position 3 Normal 41 ratio Inversion 1 1 ratio D Translocations Exchange of parts of non homologous chromosomes 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 generated simultaneously most common Figure 17 23 Animation 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 Human Translocations Always in heterozygous state Cri du chat syndromeMissing the tip of chromosome 5 due to translocation Down syndrome Two normal chromosomes 21s and an additional region of 21 due to translocation Results in high occurrence in family tree Philadelphia Chromosome Part of chromosome 22 is translocated to chromosome 9 Often found in individuals with chronic myeloid leukemia 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 Figure 15 27
Are you sure you want to buy this material for
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'