MGY200 Final Exam Study Sheet Page

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University of Toronto St. George
Molecular Genetics and Microbiology
Deborah Cowen

MGY200 Final Exam Study Sheet P a g e | 1 Genetic Interactions Synthetic Lethality: when combination of mutations in two or more genes is lethal although the mutants alone are healthy o Synthetic sickness is when the double mutant can survive but its growth is impaired Importance of genetic interactions o Genotype phenotype problems o Most diseases are multigenic Synthetic Genetic Array (SGA) analysis o Recall yeast cells have a and haploids that fuse to create diploid One diploid usually makes 2 a and 2 haploids o Query gene and every other possible gene each with their own reporters need to end up in the same double mutant Yeast genetic interaction map o Genetic interactions are rare Usually occur among functionally related genes o SGA screens identify complexes/pathways Similar patterns of genetic interaction identify pathways or complexes o These interactions can predict functions for previously unknown genes Generally the predicted double mutant fitness level is the product of the two mutants fitness levels alone Negative genetic interactions occur in case of synthetic lethality/sickness (double mutant worse off than usual mutants) o Occur between pathways/complexes MGY200 Final Exam Study Sheet P a g e | 2 Positive genetic interactions occur when the fitness of double mutant is more than that of the mutants alone; usually due to suppression o Usually indicates co-equal genes in the same pathway (a positive control and a negative control) Integrating genetic and chemical interactions o Drugs mimic loss of function in target gene Microbial Replication Chromosomes: DNA organized structure Chromatin: Protein-DNA complex Sister Chromatid pair: Replicated X-like structure baring two identical chromatids Cell Cycle: o G 1 Gap 1 phase cell grows; checkpoint is that DNA can be synthesized o S phase is when DNA replication occurs o G 2 Gap 2 phase cell grows; checkpoint is that everything is ready to inter mitosis o M stage is for mitosis; checkpoint during metaphase to see that cell is ready to divide Prophase: Chromatin condenses and centrioles move to poles Metaphase: Kinetochores created by protein attacking to centromeres while chromosomes align at metaphase plate Anaphase: Paired chromosomes separate at kinetochores Telophase: Chromosomes arrive at opposite poles and cytokinesis occurs Faithful Genetic Inheritance o Cohesin causes cohesion of chromosomes Starts in 1 , established and maintained in S a2d G then removed in Metaphase-anaphase Required for sister chromatid cohesion (discovered through 1 spot-2 spot assay of single marked chromosomal locus) Either embrace model of handcuff model o Condensin causes condensing of chromosomes Also needed for sister chromatid cohesion (two spots for inactive condensin) Not required in prophse but required in metaphase MGY200 Final Exam Study Sheet P a g e | 3 Cohesion at multiple arm loci requires condesin (different throughout chromosome) Double mutant significantly worse o Cohesin and Condensin bind independently at URA3 o Cohesion is locus specific; also lots of mechanisms around it Replication and mitotic segregation of Epstein Barr o Recall EBNA1 DNA replication- origin binding Mitotic segregation of EBV episomes Episomes complexed with histones to form nucleosomes o OriP (Origin of Plasmid Replication) DS- four EBNA1 binding sites Initiates DNA replication FR (Family of repeats)- 20 EBNA1 binding sites Segregation element Transcriptional enhancer o EBNA1 recruits origin recognition complex (ORC) to DS Is made of two dimmers that assumble on 2 adjacent sites in DS separated by 3bp Tethers viral episomes to host cellular chromosome Binds EBP2 (EBNA1 Binding Protein 2) Human protein that binds all over mitotic chromosomes like EBNA1 EBP2 binds DNA with or without EBNA1 Plasmid partition in bacteria o Plasmids symbiotic and rely on bacterial cell for maintenance o Plasmid P1 par genes encode partition proteins (ParA and ParB) and centromere-like DNA site (parS) ParA (ATPase) positions plasmids in bacterial cells ParB DNA binding protein binds to pars o ParA-GFP binds to non specific DNA on bacterial chromosome Regulation of Gene expression Recall Central Dogma MGY200 Final Exam Study Sheet P a g e | 4 Promoter is DNA sequence where transcription factors (TF) and polymerase bind Alterations in chromatin structure can influence transcription o Remodeling nucleosomes (by Chromatin Remodelling Complex [CRC]) o Histone removal (CRC) o Histone replacement (CRC) o Histone modification patterns We can predict promoters by genome sequence But motif matches often weak prediction of TF binding in vivo o TF binding may not cause regulatory activity Protein-binding microarrays (Hughes Lab) o Have all 10-bp binding sites on microarray and add protein of interest o Each protein binds several binding sites o Secondary Motifs: Sequence logos show how well a motif is conserved Rsc3 for targeting Nucleosome Exclusion at Promotors o Rsc3 binding sequence CGCG usually found ~100 bp upstream of start site o Nucleosome occupancy presents Thermodynamic problem DNA bending Decoding problem Regulates global access of DNA polymerase (Heterochromatin vs euchromatin) Regulates local TF access, heterochromatin silencing, chromatin remodelers and histone modifications o Cross-linked proteins with DNA and formaldehyde o Added nuclease to isolate the nucleosomes o Marked DNA on nucleosomes over total DNA shows what is being enriched to the nucleosomes o Promoters are not enriched to nucleosomes (Abf1 and Reb1 binding sites) o Mutations of TF leads to increase in promoters at nucleosomes o Rsc3 binds like Abf1 and Reb1 which is why CGCG is the sequence used Captializing Protein Structure Prion is protein that exists in two stable forms, one is aggregating prone and infectious o Prion form can appear due to genetic predisposition or uptake from environment Levels of Protein Structure o Primary is amino acid order Predicted from DNA sequence or Edman Degradation or mass spectrometry o Secondary is local folding of residues Alpha-helix (i bonds to i+4) Beta-sheet (Carbonyl oxygen and amides form H-bonds) Beta-turns (involves 4 residues, oxygen bonds to amide) o Teriary is global folding of protein o Quaternary is higher-order assembly of proteins (tetramer, filament)
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