18 - polyoma - notes.docx

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Department
Molecular Genetics and Microbiology
Course
MGY378H1
Professor
Alan Cochrane
Semester
Winter

Description
MGY378 – MARCH 2, 2012 POLYOMAVIRUSES - Nonenveloped, icosahedral capsid, - Circular dsDNA – complexed with histones – chromatin structure formed - Two examples widely studied o Polyomavirus – infects mice o SV40 – infects monkey  Used to understand replication  A DNA tumour virus – but not tumour virus in permissive host – like adenovirus Genome is in complex with histones – nucleosome structure – Organization - Into early half and late half – separated by region called regulatory region where have origin of DNA replication and promoters and enhancers - Does not encode its own - Early genes called T antigens – for tumour o Two – small T and large T o Also have a protein in between in size – called middle T – determined that also very small T antigen, called tiny T – produced by same initial transcript o Alternative splicing to make the final message - Transcription goes in two opposite directions – divergent transcription – uses two different strands of dNA otherwise couldn’t go in opposite directions - Small T middle T is between and Large T goes around – tiny T is TTAg – gaps between are splicing T antigens - Tiny T – unclear function o Only in polyomavirus – not SV40 - Small T – n-terminal part of large T with some slight sequence difference – C-terminus known to bind to cellular protein called phosphatise PP2A - Can contribute to tumourgenesis – follow cell transformation – interaction contributes to that - Protein also has role in infection but unclear - Middle T – o Contains small T in addition to some amino acids o Results in phosphorylation of middle T – sets off signalling cascade – important for cell transformation by polyoma virus - Large T - nuclear protein , sequence specific DNA binding protein – binds to sequences within regulatory region and has many functions – FUNCTIONS OF LARGE T - Stimulates resting cells to enter cell cycle – need S phase – like adenovirus o Carry proteins that do this o Different mechanisms  Family of pRb proteins – Rb released from E2F – activation of genes involved in cell proliferation –  Also binds P53 – one protein doing both functions  Decreases expression of cell cycle inhibitors – and T is interfering with that  P53 involved in triggering apoptosis – turning on specific genes and T antigen tends to also inhibit P mediated apoptosis  Third protein interaction is with histone acetyl transferase –  Bind to cellular hsp70 – involved in assisting the folding of other proteins – chaperone – N terminal region of T antigen – contributes to cell transformation - Large T antigen – also involved in regulating early mRNA production – early infection proceeds, level of T antigen builds up – turn off early gene expression – involves binding to site 1 in regulatory region and that interferes with recruitment of RNA polymerase and transcription factors to promoter – so shut down - Required for viral DNA Replication – origin binding protein – recognizes origin sequence, especially site 2 – must bind in order to being replication – melt origin DNA to separate strands, and act as DNA helicase at replication fork – interact with DNA replication proteins in particular DNA polymerase alpha complex – recruit it – NEED CELLULAR polymerase, which are not encoded by virus DNA REPLICATION - Initiation from single origin of replication and then get replication bidirectionally around cicle – theta structure replication - Reinitiation from same origin many many times – so amplification of viral genome – - Origin divided into core and auxiliary sequences – core are eSSENTIAL for replication and auxiliary sequences are if replication works better - Core sequences consist of T antigen binding site 2 and on either s
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