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This 14 page Class Notes was uploaded by Lisa Montanez on Friday April 22, 2016. The Class Notes belongs to 300 at Edinboro University of Pennsylvania taught by Dr. William J. Mackay in Winter 2016. Since its upload, it has received 42 views. For similar materials see Genetics in Biology at Edinboro University of Pennsylvania.
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Date Created: 04/22/16
GENETICS NOTES CHAPTERS 20-24 RECOMBINANT DNA TECHNOLOGY -enables individual fragments of DNA from any genome to be inserted into vector DNA molecules (e.g. plasmids, viruses) and these fragments can be introduced into bacteria (e.g. transformation) and then amplified DONOR ORGANISM- organism under study that donates DNA -ex: fruit fly Drosophila melanogaster VECTOR DNA MOLECULES- plasmids/viruses that can accept foreign DNA sequences; capable of replicating TRANSFORMATION- introduction of vector DNA (plasmid DNA) into a bacterial cell TRANSFECTION- introduction of a virus into a host cell -ex: introducing lambda bacteriophage (virus that infects bacteria) into bacterial cells RECOMBINANT DNA(CHIMERIC DNA; after Greek monster Chimera)- vector molecule + DNA insert RECOMBINANT (DNA) CLONE- a large population of identical DNA inserts STEPS IN PRODUCING RECOMBINANT DNA MOLECULES: 1) Isolating DNA- both donor and vector DNA -donor DNA isolated by “genomic prep” procedures -vector DNA: Plasmid also isolated using a variety of procedures “mini”, “midi”, “maxi” preps Viruses can also be purified using “phage preps” 2) Cutting DNA: -EXONUCLEASES- cleave nucleotides one at a time from the end of a polynucleotide chain; they may be specific for either 5’ or 3’ end of DNA or RNA -ENDONUCLEASES- cleave bonds within a nucleic acid chain -RESTRICTION ENZYMES- endonucleases that recognize specific short sequences of (usually) unmethylated DNA and cleave the double-stranded molecule -produced by bacteria as a defense mechanism against phages (part of the restriction/modification system) -enzymes cut DNA into fragments of a size suitable for cloning -enzymes can make “staggered” cuts which generate single-stranded “sticky” ends conducive to the formation of recombinant DNA -most restriction enzymes either recognize a specific 4 bp “four cutters” or a specific 6bp “six cutters” DNA sequence 4 -“4 cutters” will cut once, on average, every 4 bases = 256 bases 6 -“6 cutters” will cut once every 4 bases = 4096 bases 2 EXAMPLES OF RESTRICTION ENZYMES: -named after the organism from which it was identified or purified from 1)ECO R1 - from bacterium Escherichia coli 2)HIND III - from bacterium Haemophilus influenzae -both are 6 cutters -ECO R1 = recognizes a 6 bp palindrome sequence -both DNA strands have the same nucleotide sequence but in antiparallel orientation 5’ - GAATTC - 3’ => 5’ - G AATTC - 3’ 3’ - CTTAAG - 5’ => 3’ - CTTAA G - 5’ -STAGGERED CUT- leaves a pair of identical single-stranded “sticky ends” -ends can H bond to a complementary sequence 3) Joining DNA: -donor DNA (foreign DNA) + vector DNA = digested with restriction enzyme/mixed => recombinant DNA -join fragments (via H bonding of complementary sequences)(annealing); catalyzed by enzyme DNA ligase -seals nicks that have available 3’-OH and 5”-PO4termini -uses ATP as an energy source 4) Amplifying recombinant DNA: -recombinant plasmid DNA is introduced into host cells by transformation -vector will replicate (donor DNA will also replicate) -will have multiple copies of vector in cell -cell will also replicate (cell division) -from 1 to 10 cells/ml CLONING A SPECIFIC GENE: -choosing a cloning vector: -must be small (for manipulation/convenience) -must be able to replicate -must contain convenient restriction sites -must be able to identify/recover recombinant molecules CLONING VECTORS: I. Plasmids: -autonomous, self-replicating, extrachromosomal circular DNA -distinct from bacterial chromosomes -1 plasmid that was identified was the F plasmid -EPISOME- can exist in 2 forms a) Self-replicating b) Integration into chromosome -sex plasmid involved in bacterial conjugation- the union of 2 bacterial cells, during which chromosomal material is transferred from the donor(Hfr) to the recipient(F-) cell EXAMPLES OF PLASMIDS: 1) PBR 322- contains 2 drug resistance genes a) TETR = tetracycline resistance -TETRACYCLINE- binds to a protein on the 30S subunit of the ribosome and inhibits ribosomal translocation b) AMPR = ampicillin resistance -AMPICILLIN- inhibits enzyme that are involved in the synthesis of the cell wall -BLA- codes for an enzyme that is secreted to cell wall and breaks down amp R -donor DNA can be inserted into tet gene = insertional inactivation -select for tet /amp colonies 2) pUC plasmids: a) plasmids have a mutation in a replication gene (rop gene) which leads to increased copy number b) Has a polycloning site (polylinker; multiple cloning site) -a DNA sequence that was genetically constructed in vitro and contains many sites which are recognized by restriction endonucleases b) polylinker inserted (insertion as in-frame with ß-gal coding sequence) in the lac Z gene (ß-galactosidase) -vector contains DNA sequence encoding the 1 146 amino acids of ß-galactosidase, thus, pUC vectors express the NH t2rminal of ß-galactosidase c) RECIPIENT CELLS (e.g. DH5 alpha) express the COOH terminal fragment e) active ß-galactosidase protein is a HOMOTETRAMER -multimeric enzyme with each polypeptide consisting of 1173 amino acids in length f) the 2 fragments will then join together in trans to form a functional gene product (partial proteins coded by 2 fragments unite to form a functional ß-gal protein) = called ALPHA-COMPLEMENTATION g) thus, one can detect lacZ expression (ßgal activity) 1) vector + cells (e.g. DH5 alpha)(lac-) => ß-gal activity (lac+) -ß-gal activity in the presence of a colorless substrate called X-GAL (5-bromo-4- chlero-3-indoyl-B-D-galactoside) will produce a colored (chromogenic-produces a colored pigment)(blue) product 2) Vector + insert + cells => no ß-gal activity = white colonies -COLONY- a visible clone of cells h) Insertion of a fragment of foreign DNA into the polycloning site of pUC results in production of an amino-terminal fragment that is not capable of alpha- complementation i) pUC plasmids usually accept foreign DNA up to 10 kb I. Bacteriophages: -viruses that infect bacteria A) LAMBDA-convenient cloning vector 1) Accepts foreign DNA usually 10-15 kb 2) central portion of phage genome not required for replication packaging -can be removed using restriction enzyme 3) lambda can be amplified to get large amounts of foreign DNA CLONING IN LAMBDA: 1) nonessential region of genome discarded (leaving left/right arms) 2) foreign DNA digested into 10-15 kb fragments 3) foreign DNA ligated into arms (ends of arms connected to form a long linear molecule consisting of multiple phage genomes (concatenated DNA) -CONCATEMER-series of unit genomes repeated in tandem 4) DNA then placed into a viral head (CAPSID = composed of viral proteins) in vitro packaging B) Cosmids: -hybrid vectors of lambda phages + plasmids -can replicate in a cell like a plasmid or be packaged like a phage -Cosmids can accept foreign DNA up to 45 kb (reason that most of the lambda genome has been deleted) -however, cosmids still contain the signal sequences that promote phage headstuffing (cos sites) -SHUTTLE VECTORS- is a plasmid constructed to have origins for DNA replication for 2 hosts (E.coli/S.cerevisiae) so that it can be used to carry foreign sequences in either prokaryotes or eukaryotes C) Single-stranded phages (M13) -useful in DNA sequencing -EXPRESSION VECTORS- where cloned genes can be transcribed and translated into a protein -OPEN READING FRAME (ORF)- a DNA sequence that can be converted to an amino acid sequence (via RNA) -can synthesize a foreign protein in a bacterial cell (remember, bacteria cannot process introns = thus, clone cannot contain introns) II.YAC’s -yeast artificial chromosomes -foreign DNA + yeast centromere sequences + yeast telomere sequences -can be assembled into artificial chromosomes (up to 1000 kb) -YAC’s use yeast cells as hosts III. BAC’s - bacterial artificial chromosomes -based on F factor (sex factor) -accept up to 300 kb (usually 100 kb) -one advantage of BAC’s over YAC’s is that one uses bacterial cells rather than yeast cells CLONING/CONSTRUCTING A DNA LIBRARY: -source of foreign DNA LIBRARY- is a set of cloned fragments together representing the entire genome “SHOTGUN” CLONING- experimenter clones (isolates) a large sample of DNA fragments hoping that one of the clones will contain a “hit” the desired gene DIFFERENT TYPES OF LIBRARIES: -based on: -type of vector -source of DNA -different cloning vectors carry different amounts of DNA -choice of vector depends on size of genome a) Small genomes = plasmids/phages b) Middle genomes = cosmids c) Largest genomes = YAC’s/BAC’s 1) GENOMIC LIBRARY- contains entire genome (introns, exons, etc.) 2) CDNA LIBRARY (COMPLEMENTARY DNA)- is synthetic DNA made from mRNA (catalyzed by an enzyme called reverse transcriptase- originally isolated from retrovirus (tumor virus) mRNA –reverse transcriptase ssDNA –DNA pol dsDNA -thus, for example, if a scientist knows that a particular gene is highly expressed (i.e. there is lots of protein) in a particular tissue of an organism, then one can construct a cDNA library using the mRNA from that tissue USING PROBES TO ISOLATE SPECIFIC CLONES: 32 3 -PROBE- a nucleic acid molecule that can be detected by radioactive ( P/ H) methods or chemiluminescent methods -probes depend on the natural tendency of one single-strand of nucleic acid to find and base-pair (hybridize) to a complementary base sequence via H bonding WHERE DOES PROBE COME FROM? 1) You can synthesize it 2) You can get it from a friend “clone by phone” CLONING STEPS: 1) transfer colonies/plaques - containing foreign DNA of interest form the petri dishes to membrane filters composed of nitrocellulose or nylon = colony lifts/plaque lifts -DNA must be denatured on filters often by chemical treatment (e.g. NaOH) 2) Bath membrane with solution containing probe -Probe must be denatured first- heat it 3) Remove probe/ develop filters to detect presence of complementary DNA sequences SOUTHERN BLOTTING (DNA-DNA HYBRIDIZATION)- describes the procedure for transferring denatured DNA from an agarose gel to a nitrocellulose filter where it can be hybridized with a complementary nucleic acid -restriction digests of genomic DNA produces a wide array of DNA fragments differing in size -will look like a smear on an agarose gel -a probe can detect a specific DNA restriction fragment 1) separate digested genomic DNA using agarose gel electrophoresis (gel fractionation)(DNA fractionation) 2) lay absorbent membrane (nitrocellulose/nylon)on top of the gel -DNA transferred/”blotted” from gel to membranes by capillary action (DNA stays in the same position) 3) expose membrane to a probe of interest (radioactive probe) 4) remove probe and lay a photographic film over membrane -expose and develop film -will see a specific band on this film AUTORADIOGRAPHY- process that detects radioactively labeled molecules by their effect in creating an image on photographic film -SOUTHERN BLOTTING = DNA-DNA hybridization -NORTHERN BLOTTING = DNA-RNA hybridization -WESTERN BLOTTING = protein-AB interactions FINDING SPECIFIC CLONES BY FUNCTIONAL COMPLEMENTATION: -cloned genes can be detected through their ability to confer a missing function on a transformation recipient -ECTOPIC EXPRESSION- describes the expression of a gene in a tissue in which it is not usually expressed -ECTOPIC INSERTION- insertion of recombinant DNA (vector + insert) into a recipient’s genome at a location that is different from its original site -POSITIONAL CLONING- any method that uses information about a gene’s location or position on a chromosome in order to isolate that gene -CHROMOSOME WALKING- describes the sequential isolation of clones carrying overlapping sequences of DNA allowing large regions of the chromosome to be spanned. Walking is often performed in order to reach a particular locus of interest CLONING A GENE BY TAGGING- can isolate a specific gene by inducing a mutation in that gene (insertional mutagen = transposable element) 1)P-element - clone genes in Drosophila 2)Ty1 element - clone genes in yeast CANCER CELLULAR REPRODUCTION: -according to cell theory, new cells originate from other living cells -process = cell division = cellular reproduction Mother (parental) cell daughter (offspring) cell REVIEW CELL CYCLE: Control of cell cycle (2 control events in cell cycle): 1) Initiation of DNA replication which occurs at the transition between G1/S 2) Initiation of mitosis which occurs at the transition between G2/M -research on factors controlling the cell cycle began in yeast with the discovery of a gene called cdc2 -budding yeast Saccharomyces cerevisiae -fission yeast Schizosaccharomyces pombe -revealed a large # of genes whose products function to maintain the proper cell cycle -were identified ( > 80 genes identified) as conditional mutations (ts = temperature sensitive) called cdc mutations (cell cycle division) -will grow normally at low temps (permissive temp - 23’C) -will stop growing at higher temp (restrictive temp 36’C) -a particular cdc mutant will stop growing at a specific time in the cell cycle (all cells will exhibit a similar morphological phenotype) -in contrast to a typical ts mutation, cell will stop at varying points in the cell cycle (numerous phenotypes) -RESTRICTION (“START”) POINT- during the G1 phase of the cell cycle, the point at which the cell is committed to divide FACTORS INFLUENCING RESTRICTION POINT: 1) CELL SIZE- ratio of cytoplasmic volume to genomic size 2) REGULATORY PROTEINS- function as a molecular clock to carry out the cell cycle TYPES OF REGULATORY PROTEINS: A) PROTEIN KINASES -an enzyme that regulates the activity of another protein, or target molecule, by adding a PO 4erived from ATP -CYCLIN-DEPENDENT KINASES (CDK’S)- a protein that is active only when attached to a particular cyclin B) CYCLINS- proteins whose concentration fluctuates over time (amount of protein varies in a cycle = periodic fluctuations) -entry into M phase (mitotic phase) triggered by the activation of a protein kinase called maturation-promoting factor (MPF) MPF CONTAINS 2 SUBUNITS: 1) catalytic subunit ATP ADP + PO (s4r/thr) 2) regulatory subunit = cyclin -First yeast cdc mutation = cdc2 (S. pombe)/cdc28 (S. cerevisiae) -when mutated, cell stops growing at: 1) Either just prior to DNA replication or 2) Just prior to mitosis -product of cdc2 is catalytic unit of MPF -cdc2 regulated at end of G1 and at end of G2(determined by temperature-shift experiment) -both cell cycle stages represent points in the cell cycle when a cell becomes committed to beginning crucial events a) DNA REPLICATION b) NUCLEAR DIVISION -passage through these points requires the transient activation of Cdk’s by specific cyclins -passage through START (G1/S restriction point) requires the activation of cdc2 by a G1 cyclin (cig2) -CDC2 CIG2 DIMER -passage through the second point of commitment just before the end of G2 requires activation of cdc2 by a different group of cyclins (mitotic cyclins)(cdc13) -CDC2 CDC13 DIMER Progression through the cell cycle requires the phosphorylation and dephopsphorylation of certain critical amino acids -Prior to mitosis(G2/M checkpoint)- Cdk subunit must be phosphorylated at threonine-161 = catalyzed by CAK (Cdk-activating kinase) -there is also phosphorylation at threonine 14/tyrosine15 = catalyzed by wee1 and mik1 -this inhibits CAK activity -active wee1 or mik1 produces an inactive cdc2 cdc13 enzyme complex -tyrosine15/threonine 14 PO4’s can be removed by a phosphatase cdc25 -thus, the cdc25 phosphatase and wee1/mik1 kinases act as competitors to one another, one stimulating cdc2 and the other inhibiting its activity IN SUMMARY, PROTEIN KINASES/CONTROL OF CELL DIVISION: 1) The cyclin is synthesized throughout the cycle and accumulates during interphase 2) Cyclin attaches to Cdk (cdc2) and the protein complex is activated at the end of interphase 3) The active complex, MPF (maturation promoting factor), coordinates mitosis by phosphorylating various proteins which include protein kinases phosphatases 4) One of the proteins activated by MPF is a cyclin degrading enzyme that destroys MPF activity 5) The Cdk component of MPF is recycled, its kinase activity restored by association with a new cyclin that accumulates during interphase CANCER: INTRODUCTION: -Cancer is the leading cause of death for women in the U.S.- due to the consistent decline in heart disease mortality in the last 40 years -Cancer will be the leading cause of death in the U.S. by the year 2000. -Most common cancers are caused by tobacco and alcohol CANCER- a malignant tumor resulting from a progressive series of genetic and cellular events which occur in a single clone of cells due to alterations in a limited number of specific genes -oncogenes -tumor suppresser genes -tumor susceptibility genes -Cancer, or THE PROCESS OF CARCINOGENESIS, can be divided into 2 or more stages depending on the type of cancer - INITIATION-is a clonal expansion of cells that gain a selective advantage by genetic changes in the cells (mutations) pre-cancerous state - PROMOTION (PROGRESSION) - when there is an accumulation of genetic alterations in these pre-cancerous cells -these alterations are caused by initial genetic changes and can also occur by exposure of the cells to tumor promoting compounds such as phorbol esters -these alterations cause the phenotype of these cells to change into more malignant ones -Measurement of age-dependent cancers suggest that at least 2 or more than 7 independent events are necessary for the development of malignant tumors There are two types of tumors: 1) BENIGN (PRIMARY OR PREMALIGNANT) tumors are growths which remain confined to its normal location -ex: warts 2) MALIGNANT (CANCEROUS) tumors are growths which can invade non tumorous tissues via the circulatory or lymphatic systems -movement of cancerous tumors to non-tumor tissues is called METASTASIS -More than 100 different kinds of human cancers are recognized and historically classified according to their cellular origin. MOST CANCERS ARE DIVIDED INTO 3 MAJOR GROUPS 1) Carcinomas 2) Sarcomas 3) leukemias/lymphomas CARCINOMAS: -comprise approximately 90% of human cancers -arise from epithelial cells SARCOMAS, LEUKEMIAS, LYMPHOMAS: -sarcomas are cancers of connective tissue or muscle tissue; develop from mesodermal cells and/or circulatory cells of the blood and lymph system -leukemias are cancers derived from hemopoietic cells (blood bone marrow cells) -lymphomas are cancers derived from lymph cells -Tumors are further classified to their tissue of origin -Next slide is a multistep pathway which leads to colon cancer THIS PATHWAY INCLUDES 5 GENES: -p53, DCC, APC, ras k -in this pathway there must be at least 7 independent genetic events (2 each for p53, DDC, and APC and one for ras k) for a normal cell to change to a metastatic tumor -It has been known for several years that changes in DNA and not changes in other subcellular components such as lipids and proteins are responsible for the formation of cancerous tumors -Next slide illustrates an experiment where DNA was extracted from human cancer cells and this DNA was introduced into non tumor mouse cells -These TRANSGENIC mouse cells changed into tumor cells -It was further shown that the DNA in the human cancer cells contained an activated oncogene which was not present in the non-tumor mouse cells. However, the transgenic tumor mouse cells contained the activated human oncogene
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