Identification and cloning of amplified DNA sequences by a modified-in-gel renaturation technique: Isolation of an amplified DNA sequence from a human sarcoma.
AuthorCoccia, Marco Anthony.
AdvisorTrent, Jeffery M.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractA modified in gel renaturation technique has been developed which detects DNA sequence amplifications of ≥10-fold without prior identification of the target gene. In order to determine the sensitivity of detection and applicability of modified in gel renaturation, primary human tumor cultures and drug resistant human cell lines with identified cellular gene amplifications were assayed. The modified in gel renaturation technique was then used to screen 71 human tumor cell lines and direct tumor biopsies for amplified sequences. Particular emphasis was placed on screening cell lines derived from human melanoma and direct tumor samples from adult sarcomas. Four of 55 cell lines tested had amplification ≥10-fold of previously known cellular oncogenes. None of the 25 melanoma short term cultures tested positive by this assay. One of 16 adult sarcoma samples (a malignant fibrous histiocytoma (MFH)) tested positive for DNA sequence amplification >10-fold. The amplified DNA present in this MFH, (termed ST-23987), was not identified by Southern blot analysis with a panel of oncogene probes previously reported as amplified in other human malignancies. In order to further characterize the amplified DNA in ST-23987, DNA was subjected to in gel renaturation cloning procedures followed by PCR modified cloning. Of 55 clones isolated and analyzed, four cloned inserts (termed pMAC895, pMAC15, pMAC20, and pMAC30) were amplified between 10-to-15-fold in ST-23987DNA. Several other adult sarcomas were probed with these four clones and three other instances of amplification were identified by Southern blot analysis with probes generated from pMAC15 and pMAC30 (found in tumors ST-24609, 870141 and ST-23493). A bacteriophage lambda genomic library was constructed from DNA isolated from a ST-23493. Four clones representing ∼34 kb of the amplified domain (designated 493.1, 493.2, 493.4, and 493.5) were isolated using clones pMAC15 and pMAC30 as probes. Plasmid subclones generated from the EcoRI fragments of the amplified bacteriophage lambda clones (termed p10.5, p6.0, p5.2, p5.0, p4.7, p2.8, and p0.5) were used as probes against Northern blots of sarcoma mRNA in an attempt to identify transcribed sequences within the cloned regions of the amplification unit. The hypothesized target gene carried within the amplification unit will likely be important to the progression, diagnosis and treatment of adult sarcoma, irrespective of the target gene identity.
Degree ProgramMicrobiology and Immunology