INTEGRA PRINC BIOL 2
INTEGRA PRINC BIOL 2 BSC 2011
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This 46 page Class Notes was uploaded by Eugene Nicolas III on Friday September 18, 2015. The Class Notes belongs to BSC 2011 at University of Florida taught by Charles Baer in Fall. Since its upload, it has received 9 views. For similar materials see /class/206662/bsc-2011-university-of-florida in Biological Sciences at University of Florida.
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Date Created: 09/18/15
Microbial Genetics Ch 18 Genetics of Viruses Genetics of bacteria Recombination Viruses the Bottom Edge of Life Viruses are obligate parasites of cellular organisms Consist of a nucleic acid genome DNA or RNA enclosed in a protein coat Use the host39s cellular machinery transcriptional and translational to replicate themselves The viral genome encodes the information necessary to replicate the virus Viruses the Bottom Edge of Life Viruses are very useful genetic model organisms Very small genomes eg can sequence completely in a day Very fast generation time Undergo all the phenomena of interest to geneticists mutation recombination gene regulation Viruses Diversity of Genomes Genomic class i doublestranded dsDNA ii ssDNA iii dsRNA ssRNA IV genome can be mRNA V genome is template for mRNA Vl genome that is template for DNA synthesis quotretrovirusquot The dsDNA Virus Life Cycle Virus enters host cell method is variable involves host receptor molecule on cell surface Viral DNA replicated using the host39s DNA polymerase nucleotides etc DNA transcribed into mRNA using host39s RNA polymerase nucleotides mRNA translated using host39s ribosomes tRNAs amino acids capsid proteins GO to G 0 Ci r G QCv DCL n L U O u a quot100039 9 900 Doc00 ecu r j fip aw 39 0 Dquot pquot 2 1 3 quot Jquot The dsDNA Virus Life Cycle New DNA and capsid proteins assemble into new virus particles exit the cell in various ways 39 CO C Protein 0c be capsid 95 DNA CCU capsid proteins 000 0 0 con 0 D dry CV0 g JQ 900035 Iquot 000 a i 5 o vQOg o o 39v r 000 u 3 Ohm lb 5 u o 0 r3 a 00 90 b 060 Goo Goo Jame 6 a C r 9 0000 0300C K r 300 GOO q 39 Df 006 8 C The ssRNA tvpe V Virus gfe Cvcle K 1 Virus enters host cell 2 Capsid removed RNA released 3 complementary RNA made from genomic RNA by enzyme encoded in viral genome 4 new genomic RNA made from complementary strand 5 complementary strand is mRNA transcribed into viral proteins 6 Virus assembled exits cell by various means The Retrovirus Life Cycle 1 Virus enters host cell 2 Reversetranscriptase encoded in viral genome catalyzes synthesis of DNA complementary to the viral RNA cDNA 3 RTase catalyzes synthesis of 2nd strand 0 N complementary to the first 4 dsDNA incorporated into host genome quotprovirusquot provirus may remain unexpressed for a period of latency The Retrovirus Life Cycle 5 Proviral genes are transcribed by host39s transcriptional machinery into RNA RNA serves as mRNA for translation into viral proteins and as genomic RNA 6 New viruses are assembled containing genomic RNA and Reverse Transcriptase 7 Virus exits cell Phaqe Reproduction Lvtic and Lvsoqenic Recall Phage are viruses that attack bacterial hosts Lytic reproduction kills the host cell by Iysis Phage that reproduce ONLY by lytic reproduction are called virulent phage Lysogenic reproduction replicates phage genome along with host genome without killing host Phage that can reproduce by either lytic or lysogenic reproduction are called temperate phage Pha e Re roduction L ticC cle 1 Phage binds to surface receptor on host 2 Phage injects DNA into cell 3 Host39s DNA hydrolyzed degraded 1 LIA Pha e Re roduction L ticC cle 4 Host39s metabolic machinery used to make phage protein and DNA from free nucleotides Phage encodes lysozyme production cell lyses new phage released 5 1 LIA f g 3 1 gt quot E 4 7 ll Phage Regroduction Lysogenic Cycle 5 1 Phage attaches injects DNA Phage DNA circularizes Phage DNA integrates into bacterial chromosome by crossingover Phage Regroduction Lysogenic Cycle Integrated phage called quotprophagequot g may stay stably 1 integrated for many generations 4 4 Occasionally the I prophage is removed intact from the host genome lytic cycle ensues Genetics of Bacteria Bacteria have a single circular chromosome Many bacteria have plasmids Bacterial genomes contain between 700 to several thousand genes Genome is much smaller than eukaryotic genomes No introns Genetics of Bacteria Mutation Rate Mutation rate is 1 x 10397 per gene per generation in E coli 2 x 1010 bacteria in the average human gut 4300 genes Total of new mutations in the population is 1 x103972 x101 4300 9 million Genetics of HUMANS Mutation Rate an aside Assume mutation rate is 1 x 10397 per gene per generation in Humans 1 x 10396 is closer 4 x 109 humans on earth 25000 genes Total of new mutations in the population is 1 x103974 x10925000 10 million Genetics of Bacteria Recombination Bacteria reproduce by clonal fission asexually Yet recombination is extremely common in bacteria and in viruses Transformation recall Dr Griffiths39 Pneumococcus Uptake of naked DNA Transduction Phagemediated DNA transfer Conjugation quotMatingquot between bacteria Inference of Recombination Two strains of E coli one arg the other trp39 Plate on minimal medium Neither strain grows by itself but mixture grows Must be recombination not mutation blc mutation rate known mixture arg trp arg trp Transformation Bacterial cell takes up foreign DNA doublestranded DNA Integrated into the bacterial genome at a site of sequence homology similar sequences Many species of bacteria have receptor proteins on the cell surface that function in DNA uptake Transformation foreign dsDNA host DNA Transformation ssDNA aligns with homologous sequence one strand of host DNA displaced foreign sequence basepairs with complementarysequence Loose ends are cut and ligated to host DNA host DNA Transformation One daughter cell has donor strand one daughter cell has host strand N 9quot P T Phage infects bacteria enters lytic cycle DNA hydrolyzed phage DNA replicated Fragment of bacterial DNA incorporated into phage Phage infects new host 39 Phagemediated Generalized Transduction Pha emediated exchan e 4 Phage infects new host 5 Crossingover can occur between bacterial DNA fragment and homologous region of Crossover the chromosome J Recombinant genotype results Specialized Transduction Phaqemediated exchange Recall the lysogenic phase Prophage exits host genome before entry into the lytic cycle Occasionally the prophage takes some of the host genome with it Results as before recombination can occur Coniuqation and Plasmids Plasmids are essentially quotminichromosomesquot circular selfreplicating some can integrate into bacterial genome quotepisomesquot contain only a few usually nonessential genes Coniugation and Plasmids Conjugation is direct quoton purposequot transfer of DNA between bacterial cells One cell quotmalequot is donor one cell quotfemalequot is acceptor Sex pilus forms bridge between cells allo s cytoplasmic connection A Coniuqation and Plasmids In E coli ability to form sex pilus is encoded by a set of genes collectively called an quotFfactorquot which can exist as a plasmid F cells have F plasmids F39 cells do not F cells are male donors F39 cells are quotfemalequot acceptors The F plasmid may be integrated into the bacterial genome called Hfr male Coniuqation and Plasmids Integration of F plasmid into E coli genome to create an Hfr cell Similar to integration of prophage F plasmid Bacterial chromosome Coniuqation and Plasmids Replication and transfer of Hfr chromosome begins at a ecific sequence within the F factor black arrow Coniuqation and Plasmids Conjugation bridge usually breaks before entire genome transferred Recombination between homologous sequence as before For Tuesday Genetics of bacteria continued Transposable Elements Operons Announcement Instructor Evaluation Tuesdav Need volunteer to retrieve and deliver forms Change in Baer s office hours Monday 227 24 pm DNA Analys Southern Blotting mu 4 restriclinn Feltrlnllon onzyme fragmenls I U I quotI w o mammonmgmm preparation a Elacllophoresi g Elollmg Paper mm glad uquot rum mm away I unamch prom mm probe u in aululinn In mun N m n o Hybridiulian win radioaclivu probs e Aulurdiogrlphy Dideoxv Sanqer DNA sequencinq 1 Combine denatured DNA polymerase nucleotides and ONE primer 2 Divide reaction mix into four add ONE dideoxy nucleotide ddA ddC ddG ddT 3 Synthesis starts at the primer and continues until a ddNTP is incorporated 5 Singlestranded DNA with unknown sequence blue serves as a template 4 DNA polymeraaa dATP dETFquot and new 39I39 Hadiuacliwely labeled primer 139 I I I I r r I I I I 3 Prepare four reaction mixtures 5 r I W 3 tldA39l39P ddlcTP ddTlFP ddGTP l l I ll ll i Dideoxv Sanqer DNA sequencinq Synthesis starts at the primer and continues until a ddNTP is incorporated a T E A u T T E a A c A 1 Dideoxv Sanqer DNA sequencinq Separate DNA by polyacrylamide gel electrophoresis PAGE I l39I Q 1 n f I l I I 4 1 III 0 l 5 T 3 E 399 u 1 3 Longer 1 1 1 1 4 fragments 2 G and c 13 7 1 Head E deduce T 3 sequenae 1 sequence E C afnew T aquot A 43 strand G template c 35 39 quot g 1 2H G C fragments c G Physical Mapping Chromosome Walking Known gene DNA to b ed e mapp 1 Fonstruct two genomic gaming DNA libraries With different g 3 end of gene Probe 1 restriction enzymes Library 1 L39b 11Z I 2 395399 PTEe for one equot I my DNA of cloned fragments eg 39 o nown gene in GP b 1 one library 539 m e 3 i Probe 2 3 Probe the other library with the same probe to find fragments that 539 339 overlap the gene iprOM3 i 4 Design new probe at 3 Known gene end of second fragment r H Map Known sequences Genome Sequencing Mapbased method 1 Construct genetic linkage map using many markers eg microsatellites Data come from many pedigrees Relevant data are recombination frequencies mmnm quot WW III IIIIIquotFIlilTEI I Genome Sequencing Ma based method 2 Construct a Physical Map Cut genomic DNA into large fragments Clone fragments into YACs or BACs Determine order of fragments by chromosome walk Repeat with smaller fragments L Genome Sequencinq Mapbased method 3 DNA sequencing Clone 1 kb fragments into plasmid vectors Sequence each fragment Assemble quotcontigquot into complete sequence Genome Sequencinq Wholeqenome shotqun 1 Cut genomic DNA into small fragments 2 Clone fragments into plasmid vectors 3 Sequence each fragment 4 Assemble contig into finished sequence More difficult to deal with highly repetitive sequence Mnscriptomics DNA microarrays The sequence is just the beginning Phenotypic differences among organisms are ultimately the result of differences in gene expression protein functional RNA Differences in expression MAY be the result of sequence differences but often not eg environmental effects Mnscriptomics DNA microarrays Example Breast Cancer Different types of tumors have different prognoses eg In some cases very aggressive treatment is warranted in others it is not Is there a way to determine early in the disease what the longterm prognosis is