Lectures 3/9-3/16 Bios 312
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This 11 page Class Notes was uploaded by Cara Cahalan on Wednesday April 6, 2016. The Class Notes belongs to Bios 312 at University of Nebraska Lincoln taught by Karrie Weber in Spring 2016. Since its upload, it has received 33 views. For similar materials see Microbiology in Biology at University of Nebraska Lincoln.
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Date Created: 04/06/16
3/911: Microbial Genomics: Gene Transfer Lecture: A base pair substitution occurs resulting in a change in the coding region from GAG (glutamic acid) to GGG (glycine) resulting in a nonfunctional (defective) protein. This is an example of a ________. o silent mutation o nonsense mutation o missense mutation o reversion o gene duplication Horizontal gene transfer: transfer of genetic information between organisms, may cross domains o Transformation DNA is incorporated into a recipient cell and brings genetic change Competent cell bind more DNA than noncompetent cell Natural and artificial (bacteria chemically treated to induce competence, induced electrically) o Transduction o Conjugation pilus produced by donor cell and attaches to recipient cell Pilus contracts recipient and retracts and subunits disassembled Cell pair stabilized (TraI: nicking enzyme, nicks one strand of F plasmid) Transfer of one strand, synthesis of complementary strand begins in recipient cell Vertical gene transfer: inheritance from parental organisms Transformation is the _______________ to a recipient cell. o transfer of genes from a donor cell by a virus o transfer of genes in free DNA o transfer of genes on a plasmid from a donor cell o all of the above o none of the above Key concepts: Significance of conjugation: o Plasmids can spread rapidly through populations o Additional genomic information (conferring physiological traits) acquired through conjugation via the plasmid or the donor’s chromosome (this includes genes for antibiotic resistance) Significance of genetic exchange: o Additional genomic information acquired through conjugation, transduction, and transformation. o Manipulated as genetic tools. 3/9 Lecture continued: Fplasmid can integrate into the chromosome o Cells possessing a nonintegrated F F . Integrated F plasmid Hfr (high frequency of recombination) o Presence of the F plasmid results in alterations in cell properties Ability to synthesize F pilus, mobilization of DNA for transfer to another cell Alteration of surface receptors so that cell can no longer act as a recipient in conjugation o Recipient cell does not become Hfr, only some is integrated F plasmid is transferred by the donor Transduction: transfer of any gene or random genes from one bacterium to another o Virulent or temperature virus involved o Host DNA incorporated virus during assembly (low rate), genetic marker can be included o Specialized: transfers small region of bacterial chromosome Cell lysis and release phage carrying host DNA capable of transduction Temperature virus integrated into host chromosome, excision takes portion of host Virushost DNA packaged Phage conversion: phenotypic change of bacterial cell through lysogenization by normal temperate phage o Immunity to infec15on by same type of bacteriophage Phage e infection change in cell surface (alters binding site) Genetic exchange: once DNA is transferred, DNA can be o Degraded by restriction enzymes, selfreplicate, recombine into host chromosome Homologous recombination o Endonuclease nick one strand, strand displaced from other by enzymes with helicase activity which separate two strands o Binding of SSB protein to prevent DNA from annealing (binding to another DNA strand) o RecA binds to SS region. Complex facilitates annealing with complementary sequence in second DNA duplex which displaced recipient strand (strand invatriosn) o New genotype the two sequences have to be related by genetically different Resolvases cut and rejoin strands, scissors, cut vertically and horizontally Cross strand exchange: o Holliday junction visualized by rotating onehalf of molecule o Resolvases cleave junction in two ways (one strand remains original) Which of the following occur during transformation, transduction, and conjugation? i. Unidirectional transfer of genes ii. Incomplete gene transfer iii. Homologous recombination iv. Rolling circle replication a. i, ii b. iii, iv c. i, ii, iii d. all features are common Quiz 3 Questions: 1. Which of the following terms describes a mutant that has a nutritional requirement for growth? a. Heterotroph b. Auxotroph c. Organotroph d. Autotroph 2. E. coli is exposed to radiation which causes double strand breaks resulting in a pause in DNA replication. What repair mechanism would be upregulated? a. Errorfree light repair system b. Errorfree excision repair system c. Errorfree methyl mismatch repair d. Error prone SOS system e. Error prone reverse transcriptase 3. An open reading frame encodes for ________. a. A gene b. A polypeptide c. mRNA d. protons 4. The F (fertility) plasmid contains a set of genes that encode for the ____ proteins that are essential in conjugative transfer of DNA. a. Pili b. SOS repair c. Transduction d. Transformation e. Transfection 5. How do bacteriophage influence bacterial evolution? a. Bacteriophage causes cleavage and rearrangement of bacterial genomes, thus accelerating bacterial evolution b. Bacteriophage lyse mutated bacterial cells, preenting them from replicating and passing on genetic information c. Bacteriophage decrease the size of bacterial populations and thus decreasing genetic diversity and slowing down bacterial evolution. d. Bacteriophage transfer genetic information between bacterial cells through transduction, thus increasing the genetic diversity of bacterial populations. e. Bacteriophage transfer genetic information between bacterial cells through transduction, thus increasing the genetic diversity of bacterial populations. 6. Consider a mutation in which the change is from UAC to UAU. Both codons specify the amino acid tyrosine. Which type of point mutation is this? a. Missense b. Nonsense c. Silent d. Frameshift 7. The Ames test is commonly used to test whether a particular chemical is mutagenic. When the Ames test is conducted, two different groups of plates of culture medium in which histidine is omitted are inoculated. One group of plates is inoculated with bacteria that are exposed to the chemical of interest, while the other plates are inoculated with bacteria that are not exposed to the chemical. Why is the latter group needed if you are just interested in the rate of mutation caused by the chemical? a. It is important to compare the rate of mutations with the chemical to the normal background mutation rate. Mutations occur spontaneously even without the presence of a mutagen. b. The group without the chemical can be exposed to mammalian liver extract to see the effects of liver enzymes on the bacteria as an additional treatment c. The second group is used as a control to make sure that the replica plating technique is done correctly. d. Two groups are not needed. It is only necessary to count the number of colonies that form on the plate that has the chemical of interest. 8. Aquifex aeolicus is a ___________________. a. Archaeum b. Eukaryote c. Bacterium d. Virus 9. A point mutation refers to _______________________. a. Change in a single base pair b. Insertion of single base pair c. Deletion of a single base pair d. All of the above 10. Horizontal gene transfer refers to the transfer of genes from one organism to another, as occurs during conjugation between bacteria. How does this differ from vertical gene transfer? a. In vertical gene transfer, genes are transferred from one organism to another with or without a vector. b. Conjugation can involve horizontal or vertical gene transfer, depending on the physical orientation of the bacteria involved. c. In horizontal gene transfer, genes are transferred from one mature cell to another mature cell. In vertical gene transfer, genes are transferred from the mother cell to the daughter cell (offspring). d. In horizontal gene transfer, genes are transferred between two mature cells of the same species. In vertical gene transfer, genes are transferred between two mature cells of different species. 3/9: Horizontal Gene Transfer (Synthetic Biology) Readings: Ch. 11.111.10, 11.17 I: Methods of Manipulating DNA 11.1 Restriction Enzymes and Nucleic Acid Separation Restriction enzymes recognize base sequences, cut phosphodiester bond (2x stranded breaks) o Type I and III: bind and cut some distance away o Type II: cleave DNA within recognition sequences, most useful Modification enzymes protects cell from own restriction enzymes o Each restriction enzyme has matching modification enzyme that share recognition sequence o Modify recognition sequences so restriction enzymes can no longer cut the DNA Gel electrophoresis separates charged molecules by migration in an electrical field o Smaller molecules migrate faster than larger 11.2 Nucleic Acid Hybridization Hybridization ssDNA form hybrid dsDNA by base pairing Probes ss nucleic acids with known identities, can be radioactive or colored 11.3 Polymerase Chain Reaction Polymerase chain reaction (PCR) copy segments to amplify DNA, yield large amounts of genes, requires DNA pol and primer, in thermocycler. Repeat to make many copies. Steps: o Denaturation heat to break H bonds between DNA strands (9496 C) o o o Annealing 5065 Co o Extension 7580 C (optimal temperature of Taq polymerase) Enzymes used in PCR: o Taq polymerase stable at 95 C, unaffected by denaturation o Pfu polymerase stable at 100, also proofreads Reverse transcription PCR DNA from mRNA template, detect if a gene is expressed or produce an intronfree eukaryotic gene 11.4 Essentials of Molecular Cloning Molecular cloning DNA replicated to isolate the desired gene and move it around o Vector manipulable genetic elements, plasmids and viruses Foreign gene expressed in cloning host, encoded protein screened for, so host cannot produce protein Replica plating duplicate master plate and colonies lysed to release antigen of interest o Antibody added and binds antigen, unbound antigen washed away o Radioactive colonies appear as spots on Xray film after developed o Limitation specific antibody must be in experimental animal against antigen in question used 11.5 Molecular Methods of Mutagenesis Site directed mutagenesis mutation at precise sites, allows for change to any base pair in a specific gene, can manipulate protein characteristics. Process: o Cloning target gene inserted vector denatures resulting in ssDNA oligonucleotide binds with target gene daughter cell either has wild type or mutant gene DNA synthesized 1 nucleotide fastened to insoluble support, nucleotides added and cleaved at desired length Cassette mutagenesis DNA cassettes mutate DNA, can replace sections of DNA of interest o Gene disruption cassette inserted into middle of gene, disrupting coding sequence Knockout mutations: Gain antibiotic resistance as well as lose function of gene 11.6 Gene Fusions and Reporter Genes Reporter gene encodes protein that is easy to detect Gene fusions coding sequence fuse to different regulatory region o Operon fusing retains translational start site and signals fused to transcriptional signals of another gene o Protein fusion proteins fused, share transcriptional/translational start and stop signals Yield a single hybrid polypeptide II: Gene Cloning 11.7 Plasmids as Cloning Vectors Vectors modified to abolish conjugative transfer preventing unwanted movement of vector Ideal cloning vector: small, high copy number, easy to isolate, known base sequence and inserted easily by transformation Vector and foreign DNA are cut linearized DNA inserted into open cut ligated o Without cloned DNA blue o With cloned DNA do not form betagal and are white 11.8 Hosts for Cloning Vectors Disadvantages of E. coli as host o Found in human intestine, lack system to correctly modify eukaryotic proteins, outer membrane hinders protein secretion (gram ) B. subtilis gram positive, overcome problem Eukaryotic host: o Advantage already possess systems required for protein synthesis o Disadvantage expensive and difficult to produce largescale 11.9 Shuttle and Expression Vectors Shuttle vectors moving cloned genes between organisms of different species Expression vectors optimize expression of cloned genes in given host, control expression in cell o Regulatory sequences allowing manipulation of gene expression o Promoter that functions efficiently in host to control transcription of cloned gene Avoid damaging cell expression vector grown without expression of foreign gene large population obtained genetic switch Codon usage pattern different in gene from host inefficient translation 11.10 Other Cloning Vectors Phage lambda: 1/3 genome is not essential so large fragments can be inserted Cosmids contain cos site yielding cohesive ends when cut, required for packing DNA o Advantage clone large fragments, fewer clones are needed to cover whole genome Artificial chromosomes: o Carry large segments of DNA, size of initial library stays manageable Bacterial artificial chromosomes (BAC) Yeast AC (YAC) replicate in yeast with normal chromosomes, sites for very large fragments of DNA to be inserted. 11.17 Synthetic Biology Synthetic biology genetic engineering to create novel biological systems out of available parts Lecture: Manipulation tools in genetic engineering: Synthesizing DNA o Oligonucleotides synthesis of 100 bases can be made Multiple oligonucleotides can be ligated together o PCR: amplify target DNA a million fold from a small amount of template DNA Manipulation o Restriction enzymes protect against hostile foreign DNA in prokaryotes, rare in eukaryotes, essential for in vitro DNA manipulation Recognize inverted repeats (palindromes), 48 bp long Molecular cloning isolation/incorporation of a piece of DNA into a vector for replication/manipulated o Three main steps of gene cloning Isolation and fragmentation of source DNA Source DNA can be genomic DNA, RNA, or PCR amplified fragments Genomic DNA must be restriction digested Inserting DNA fragment into cloning vector Most vectors from plasmids or viruses, DNA inserted in vitro Works with sticky or blunt ends Introduction of cloned DNA into host organism Transformation to get recombinant DNA into host Some cells contain desired cloned gene, others have cloned genes Gene library: mixture of cells containing a variety of genes Shotgun cloning: gene libraries made by cloning random genome fragments o Recombinant vector hybrid DNA molecular from restriction enzymes o Plasmids as a cloning vector: small, easy to isolate DNA, independent origin of replication, multiple copy number, presence of selectable markers (i.e. pUC19) o Vector transfer carried out by chemical transformation or electroporation o Antibiotic resistance genes as “selective markers” to identify bacterial cells with the plasmid. Blue colonies no vector with foreign DNA White colonies have foreign DNA inserted Insertional activation: lacZ inactivated no Xgal blue color does not develop Bacteriophage Lambda as cloning vector. Viral cloning vector advantages over plasmids: o Hold larger amounts of DNA and packaged more efficiently into phage particles, infect host o Charon phages: modified lambda phages, unwanted restriction enzyme sites have been removed by mutation Lambda cloning: o Isolating vector DNA from phage particles and cutting into fragments Maximum size of inserted DNA is ~ 20 Kbp o Connect two lambda fragments to fragments of foreign DNA using DNA ligase o Packaging DNA: add cell extracts with head and tail proteins, spontaneous phage formation o Infecting E. coli cells, isolate phage clones by picking plaques on a host strain o Checking the recombinant phage for the presence of foreign DNA using nucleotide acid hybridization, DNA sequencing, or observation of genotypic properties Cosmid Vectors use specific lambda genes and are packaged into lambda virions. o Containing cos site from the lambda genome required for packaging DNA into lambda virions o Advantage of cosmids: used to clone large fragments of DNA Selection of recombinants is less of a problem with lambda replacement vectors than with plasmids: o Efficiency of transfer of recombinant DNA into the cell by Lambda is very high o Lambda fragments that have not received new DNA are too small to be incorporated into phage particles every viable phage will contain cloned DNA Molecular cloning hosts: o E. coli common host due to wealth of knowledge, potentially pathogenic, gram negative o B. subtilis easily transformed, nonpathogenic, naturally secrete proteins, genetically unstable, genetics less developed than E.coli o S. cervaisiae well developed genetics, nonpathogenic, can process mRNA and proteins Question: A gene responsible for Ampicillin resistance is used on a plasmid as a selective marker. After transforming cells of E. coli with the plasmid, you cultivate the cells on culture medium containing Ampicillin. What does the growth of E. coli on the culture medium indicate? o The foreign DNA was inserted into the plasmid. o The plasmid was successfully transformed into E. coli. o a and b 3/16: Origin of Life and Microbial Evolution Readings: 12.112.7 12.2 Photosynthesis and the Oxidation of Earth UV radiation converts O to2 (o3one), forms ozone shield and protects earth from sun’s radiation 12.3 Endosymbiotic Origin of Eukaryotes Endosymbiotic hypothesis: mitochondria arose from the stable incorporation of respiring bacteria into other cells, chloroplasts with prospective cyanobacteria o Bacteria engulfed by preeukaryotic cells, symbiotic relationship neither capable of living independently. Phototrophic eukaryote similar evolution manner with cyanobacteria Eukaryotic cell formation hypotheses: o From nucleusbearing cell line, arose of cells that split from Archaea o Hydrogen hypothesis: eukaryotes from association between H consumin2 Archaea host II: Living Fossils 12.5 Molecular Phylogeny Sequence alignment adds gaps to sequence to establish positional homology Homoplasy organisms share a trait that was not inherited from a common ancestor III: Microbial Evolution 12.7 Evolution of Microbial Genomes Core genome genes shared by all members of a species Pan genome core genome + genes not shared by all members, through horizontal gene transfer o Keeps genomes dynamic, deletions are favored to keep genome size small (nonessential or functional genes deleted) Lecture: Life on earth began with microorganisms o 4.6 bya: origin of earth o 4 bya: shaped the geology and chemistry of earth Bacteria/Archaea o 3.86 bya: first evidence of microbial life in rocks Early earth: anoxic, energygenerating metabolism exclusively anaerobic and likely chemotrophic o Obtained C from CO and H2 rom H generat2d by H S reacting2with itself or UV light Origin of life in the Hadean: veritable hell o Atmosphere: CH , N ,4wat2r vapor, CO , SO /S2 1 2ol L HCl, small amounts of H and Ar 2 N 2 ed as N O2by lightning Oxygen produced in the upper atmosphere but likely consumed by the reduced gases o Oceans: earth cooled and began to condense to form oceans Acidic rain from HCl in the atmosphere, ocean pH was 56, retained transition metals and P in solution Evidence of early microbial life look in oldest rocks (Stelly Pool Formation, Australia) o Identify cellular structures that resemble microorganisms, some S metabolizing cells 3.4 bya o Barberton Greenstone Belt, South Africa: Oldest microbial fossils: 3.4453.334 Ga. From volcanic sediments, not BIF Hypothesis of common ancestry: all living things on earth arose from one ancestor o Progenote common ancestor from which life arose (LUCA 4.2) How and when did life on early earth begin? 2 hypotheses: o De novo origin(favored hypothesis) life began as organic soup or surface metabolism Prebiotic chemistry of early earth set stage for selfreplicating systems (1 RNA based) RNA binds small molecules (ATP, nucleotides), catalyzed its own synthesis o Panspermia how microbial life arose Life arrives on extraterrestrial body Spore survives harsh conditions (space travel) De novo origin of life o Surface origin hypothesis 1st membraneenclosed, selfreplicating cells from primordial soup rich in organic and inorganic compounds in ponds on Earth’s surface Against: dramatic temperature change/ mixing meteor impacts, dust clouds, and storms o Subsurface origin hypothesis Life originated at hydrothermal springs on ocean flood, more stable conditions Steady and abundant supply of energy (e.g., H and H S) 2 2 Miller/Urey experiment components of early atmosphere, lightning could have formed amino acids/other C sources that are required for life to begin o 1 week: 1015% of C was in the form of organic compounds, 2% amino acids First genetic material and enzymes both may have been RNA o Popular hypothesis: first genes short strands of RNA, selfreplicated Formation of membrane vesicles o Buildup of lipids o Synthesis of phospholipid vesicles, enclosed biochemical/replication machinery in cell Earth metabolism: need energy to start life o Wächtershäuser early metabolism predated genetics, energy produced that can be used for other processes once a primitive metabolic cycle was established more complex compounds o Key idea: chemistry occurred on mineral surfaces near deep submarine vents where Crich molecules could accumulate on iron crystals and acquire new C from material o First cells lipid bubbles on mineral surface Pyrite theory life started as metabolic process that could occur on iron pyrite surfaces Provides positive charges for bonding of phosphates Polymerization of lipids formed semipermeable membranes, proton gradients could be generated, providing energy for synthetic reactions involving organic compounds generated either inside or outside of these membranes o O may not have been involved in generation of BIF, anaerobic metabolisms maybe Early Photosynthetic microbial life o Stromatolites fossilized microbial mats of filamentous prokaryotes and trapped sediment Anoxygenic phototrophic filamentous bacteria formed ancient stromatolites Oxygenic phototrophic cyanobacteria dominate modern stromatolites o ~2.7 billion years ago, cyanobacteria developed a photosystem using H O n2t H S, g2nerating O 2 o Great oxidation event: 2.4 billion years ago, O 2oncentrations raised to 1 part per million O 2could not accumulate until it reacted with abundant reduced materials in the oceans o Banded iron formations: laminated sedimentary rocks; prominent feature in geological record Metabolic diversification: early microbial metabolism were anaerobic o Development of oxic atmosphere new metabolic pathway yielding more energy Formation of eukaryotic cells hypotheses: o Began as nucleusbearing lineage later acquired mitochondria and chloroplasts by endosymbiosis o From intracellular association between O c2nsuming bacterium (the symbiont), mitochondria and an Archaean host o Support: eukaryotes have similar lipids and energy metabolism to bacteria and have transcription/translation machinery similar to archaea Life on Earth originated __________________. o On another plant and was transported to Earth on an extraterrestrial body o In ponds or ice on the surface of the Earth o In hydrothermal springs or vents at the bottom of the ocean o all of the above have been proposed to described the origin of life on Earth Microbial Evolution Mutations o Changes in nucleotide sequence, occurs because of replication errors/radiation, adaptive mutation improve fitness of organism increasing survival o o At 31,500 generations1 line evolved ability to use citrate (medium buffer) as glucose alternative Selection/Genetic drift survival of the fittest/natural selection Key Concepts: Early life forms: selfreplicating RNA’s using anaerobic chemolithotrophic metabolism (H an2 or S may have served as an electron donor) or possibly chemoorganotrophs. Evolution of oxygenic photosynthesis gave rise to the O in Earth’s atmosphere. 2 Bacteria, Archaea, and Eukaryote evolved from LUCA, eukaryotes chimera of Bacteria and Archaea. Mutations can result in metabolic changes that may result in one species to behave like two.
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