| Demonstration of osteonectin mRNA in megakaryocytes: the use of the polymerase chain reaction. | |
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MedLine Citation:
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PMID: 1878589 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Platelets have been shown to release osteonectin on thrombin stimulation. The origin of platelet osteonectin was unclear as it may have been synthesized by megakaryocytes or it may have been endocytosed from plasma as other platelet alpha-granule constituents are. Platelet osteonectin has a larger apparent molecular size than the bone species, although the molecular basis for this difference has not been elucidated. These two issues have been addressed here by (1) examining the potential for osteonectin biosynthesis in human megakaryocytes by demonstrating the presence of osteonectin mRNA in purified megakaryocytes, and (2) comparing the coding portion of osteonectin transcript in megakaryocytes to the size of its bone counterpart. Because of the limitations of cell population purity and in obtaining sufficient numbers of megakaryocyte cells for Northern analysis, we have used the polymerase chain reaction (PCR) to detect the presence of human osteonectin mRNA in megakaryocyte and megakaryocyte-depleted bone marrow cells. Isolation of RNA, cDNA synthesis, and PCR were performed on human osteosarcoma SaOS-2 cells, enriched megakaryocytes, and megakaryocyte-depleted cells. Restriction enzyme analysis of PCR DNA products confirmed the identity of the products as those encoding osteonectin for all three cell populations studied. In addition, the sizes of DNA indicate that osteonectin genomic DNA, nuclear RNA, or altered transcript were not amplified, and that the transcript from megakaryocytes is the same size as that from bone cells. These data suggest that the difference in protein size between platelet and bone osteonectin is due to posttranslational modification. To overcome the possibility that megakaryocyte signal originated from contaminating cells (less than 5% by cell count), all three cell populations were diluted to less than one cell per tube and PCR amplification was performed. Limiting dilution analyses demonstrated the presence of osteonectin mRNA in single megakaryocytes as well as in single cells from the cell population depleted of megakaryocytes, suggesting the capacity for osteonectin biosynthesis in all cells studied. The procedure we describe in this report can be used to examine specific characteristics of mRNA molecules in heterogeneous cell populations and in situations where only small quantities of cells can be obtained. |
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Authors:
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X C Villarreal; B W Grant; G L Long |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Blood Volume: 78 ISSN: 0006-4971 ISO Abbreviation: Blood Publication Date: 1991 Sep |
Date Detail:
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Created Date: 1991-09-27 Completed Date: 1991-09-27 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 7603509 Medline TA: Blood Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 1216-22 Citation Subset: AIM; IM |
Affiliation:
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Department of Biochemistry, University of Vermont, Burlington 05405. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Blotting, Southern Humans Megakaryocytes / chemistry* Nucleic Acid Amplification Techniques Osteonectin / genetics*, isolation & purification Osteosarcoma / chemistry, pathology Poisson Distribution Polymerase Chain Reaction RNA, Messenger / analysis*, isolation & purification Tumor Cells, Cultured / chemistry |
| Grant Support | |
ID/Acronym/Agency:
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HL 01607/HL/NHLBI NIH HHS; HL 34282/HL/NHLBI NIH HHS; HL 38899/HL/NHLBI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Osteonectin; 0/RNA, Messenger |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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