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dc.contributor.advisorEdward G. Fey
dc.contributor.advisorJeanne B. Lawrence
dc.contributor.authorSparks, Cynthia A.
dc.date2022-08-11T08:08:41.000
dc.date.accessioned2022-08-23T16:04:20Z
dc.date.available2022-08-23T16:04:20Z
dc.date.issued1995-09-01
dc.date.submitted2006-06-28
dc.identifier.doi10.13028/a5ym-tg75
dc.identifier.urihttp://hdl.handle.net/20.500.14038/31668
dc.description.abstractThe overall objective of this study was to identify novel proteins of the nuclear matrix in order to contribute to a better understanding of nuclear structure and organization. To accomplish this, a monoclonal antibody specific for the nuclear matrix was used to screen a human λgt11 expression library. Several cDNAs were isolated, cloned, sequenced, and shown to represent NuMA, the nuclear mitotic spindle apparatus protein. Further characterization of the gene and RNA was undertaken in an effort to obtain information about NuMA. The NuMA gene was present at a single site on human chromosome 11q13. Northern and PCR analysis of NuMA mRNA showed a major 7.2 kb transcript and minor forms of 8.0 and 3.0 kb. The minor forms were shown to be alternatively spliced although their functional significance is not yet understood. Immunofluorescence microscopy demonstrated that NuMA oscillates between the nucleus and the microtubule spindle apparatus during the mitotic cell cycle. NuMA appeared as a 200-275 kDa protein detectable in all mammalian cells except human neutrophils. To determine whether NuMA's changes in intracellular distribution correlated with post-translational modifications, the protein's phosphorylation state was examined through the cell cycle using highly synchronized cells. NuMA was a phosphoprotein in interphase and underwent additional phosphorylation events in mitosis. The mitotic phosphorylation events occurred with similar timing to lamin B (G2/M transition) and were concomitant with NuMA's release from the nucleus and its association with the mitotic spindle. However, the mitotic phosphorylation occurred in the absence of spindle formation. Dephosphorylation of NuMA did not correlate with reassociation with the nuclear matrix but occurred in two distinct steps after nuclear reformation. Based on the timing of these events, phosphorylation may playa role in nuclear processes. In conclusion, the work in this dissertation identified NuMA, a nuclear matrix protein and showed that it is phosphorylated during the cell cycle and may be important for nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G1/S.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectNuclear Matrix-Associated Proteins
dc.subjectPhosphoproteins
dc.subjectNuclear Matrix
dc.subjectMitotic Spindle Apparatus
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectCell Biology
dc.subjectCells
dc.subjectGenetic Phenomena
dc.subjectNucleic Acids, Nucleotides, and Nucleosides
dc.titleCloning and Cell Cycle Analysis of NuMA, a Phosphoprotein That Oscillates Between the Nucleus and the Mitotic Spindle
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1035&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/35
dc.legacy.embargo2017-04-24T00:00:00-07:00
dc.identifier.contextkey176655
refterms.dateFOA2022-08-27T04:44:56Z
html.description.abstract<p>The overall objective of this study was to identify novel proteins of the nuclear matrix in order to contribute to a better understanding of nuclear structure and organization. To accomplish this, a monoclonal antibody specific for the nuclear matrix was used to screen a human λgt11 expression library. Several cDNAs were isolated, cloned, sequenced, and shown to represent NuMA, the nuclear mitotic spindle apparatus protein. Further characterization of the gene and RNA was undertaken in an effort to obtain information about NuMA. The NuMA gene was present at a single site on human chromosome 11q13. Northern and PCR analysis of NuMA mRNA showed a major 7.2 kb transcript and minor forms of 8.0 and 3.0 kb. The minor forms were shown to be alternatively spliced although their functional significance is not yet understood. Immunofluorescence microscopy demonstrated that NuMA oscillates between the nucleus and the microtubule spindle apparatus during the mitotic cell cycle. NuMA appeared as a 200-275 kDa protein detectable in all mammalian cells except human neutrophils. To determine whether NuMA's changes in intracellular distribution correlated with post-translational modifications, the protein's phosphorylation state was examined through the cell cycle using highly synchronized cells. NuMA was a phosphoprotein in interphase and underwent additional phosphorylation events in mitosis. The mitotic phosphorylation events occurred with similar timing to lamin B (G<sub>2</sub>/M transition) and were concomitant with NuMA's release from the nucleus and its association with the mitotic spindle. However, the mitotic phosphorylation occurred in the absence of spindle formation. Dephosphorylation of NuMA did not correlate with reassociation with the nuclear matrix but occurred in two distinct steps after nuclear reformation. Based on the timing of these events, phosphorylation may playa role in nuclear processes. In conclusion, the work in this dissertation identified NuMA, a nuclear matrix protein and showed that it is phosphorylated during the cell cycle and may be important for nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G<sub>1</sub>/S.</p>
dc.identifier.submissionpathgsbs_diss/35
dc.contributor.departmentCell Biology
dc.description.thesisprogramCell Biology


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