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Title: A viral DNA-protein complex in SV40 infected cells
Author: White, Martin
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1973
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SV40 deoxyribonucleic acid (DNA) can be extracted with Triton X-I00 from productively infected cells in the form of a DNA-protein complex which is distinct from the virus and which has a sedimentation coefficient of 44s. Analysis of the proteins present in the complex is complier by the presence in the extracts of large amounts of a ribonucleoprotein component with a sedimentation coefficient close to that of the complex. However, a nuclear extraction procedure coupled with the use of actinomycin D abolished the synthesis the ribonucleoprotein component and, as a consequence of this, radio-chemical purification of 3H leucine labelled complex could be achieved. Analysis of 3H-leucine labelled complex on sodium dodecylsulphate-polyacrylamide gels revealed that the complex contained all of the virus capsid proteins (I, II, III, IV, V, VI) but in greatly altered relative proportions from those observed in mature virus Particles. The complex appears to particularly rich in the small basic proteins (IV,V,VI ) SV40 'empty' particles possess all of the structural proteins observed to be present in SV40 virus but do not contain viral DNA and are deficient in proteins IV, V, and VI. The above deficiencies would be corrected if the complex were to combine with an 'empty' particle to yield mature virus experiments with 3H thymidine provided further data which was Pulse chase consistent with the possibility that the complex is a precursor of mature virus. Short term labelling experiments with 3H-thymidine demonstrate that SV40 DNA molecules in the course of replication were also present as DNA-protein complexes (90S) SV40 DNA synthesised in the presence of neomycin was found to be present in a DNA-protein complex which had a greatly reduced sedimentation coefficient (25S) and also to have very few superhelical turns. If the puromycin is subsequently removed, the SV40 DNA is observed to recover both its normal protein complement (44S ) and its normal superhelix density. Since this recovery process can take place in the presence or arabinocytosinyl furanoside (a potent inhibitor of SV40 DNA replication), it therefore appears that introduction of superhelical turns into SV40 DNA can occur by a hicking reclosing process which is independent of DNA replication and that in association with this process, binding of proteins takes place. Puromycin inhibits the appearance of 3H thymidine into mature virus particles, despite the presence of labelled 'puromycin-DNA' in the cells. Experiments were performed which demonstrate, that virus particle assembly occurs under conditions of puromycin treatment, but that 'puromycin-DNA was not encapsulated. It probable that binding of protein to SV40 DNA is required to produce a condensed structure for encapsulation and that steric prevent packaging of the 'puromycin complex'.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available