Qualifications and Experience
Michael E. Brown, Ph.D.
PUBLICATIONS WITH ABSTRACTS
|1987f||Brown, M. B., Isolation and Cloning of fX174 n' Recognition Site: Characterization of Mutant Forms. Ph.D. Dissertation, Loma Linda University.
The n' recognition site within the F-G intercistronic region of the fX174 DNA genome was isolated as a 183 bp fragment by successive digestions with the blunt cutting restriction enzymes AluI and NruI. The three largest fragments from the AluI digest (1007 bp, 853 bp, 662 bp) were selectively precipitated from the 22 smaller fragments with 7% (w/v) polyethyleneglycol (PEG). All three fragments were digested with NruI creating four fragments. A second 7% (w/v) PEG precipitation removed the three largest fragments (1007 bp, 670 bp, 662 bp) from this digestion leaving the desired 183 bp product in the supernatant.
The 183 bp fX174 DNA fragment containing the n' site was ligated into the HinCII restriction site of M13mp7, transfected into E. coli JM103 cells and plated on media containing IPTG and X-Gal. Colorless plaques were selected to test for the insertion of the 183 bp fX174 fragment. One plaque, designated J11, was isolated and shown by restriction analysis to contain the 183 bp fX174 segment. The orientation of the cloned fragment was determined by HinfI digestion and shown to be in the non-functional (-) orientation. In order to obtain a phage with the n' sequence in the (+) orientation, the cloned sequence in J11 was subcloned into M13. DNA from isolates containing the 183 bp fragment were subsequently sequenced in the region of the insert using the enzymatic dideoxy-terminator method.
One subclone, M13mp7M24, contained the original 183 bp sequence of fX174 DNA in the functional (+) orientation. Eighteen other subclones contained the 183 bp sequence but nucleotide analysis indicated that substitutions had occurred near the 3' end of the cloned segment.
Thus, all clones containing the fX174 sequence in the (+) orientation had a nucleotide substitution in the insert sequence or a change in the cloning vector. Both events may have occurred in some clones. These results suggested that the two orientations of the sequence have significantly different biological consequences.
These clones can be used as a DNA substrate for the study of the n' recognition process.
Brown, M. B.; Bauer, C. A.; Eaton, D. O. W.; Zuccarelli, A. J.: Biological activity of an origin of DNA replication cloned in bacteriophage M13. Clinical Research, Vol. 31, No. 1, 1983 pp. 35A. Was selected for presentation at the annual meetings of the American Federation for Clinical Research (Western student medical research forum) in Monterey California.
An origin of complementary strand DNA synthesis, isolated as a 201 base-pair fragment from the bacterial virus fX174, has been cloned in an M13mp7 virus vector. The fX174 fragment contains the recognition site for the Escherichia coli replication protein n'. This protein initiates a cascade of biochemical events involving the bacterial enzyme dnaG ("primase") resulting in the synthesis of the fX174 complementary strand. Insertion of the fX174 fragment into the M13 genome was initially screened by interruption of the function of the b-galactosidase gene in the vector.
M13 clones obtained from the screening were subjected to restriction enzyme analysis, first, to determine if the insert size was the same as the original fX174 segment and, second, to discover the orientation of the site.
Ordinarily, M13 DNA synthesis is initiated at a specific location by the action of E. coli RNA polymerase. The antibiotic rifampicin specifically blocks its activity. The fX174 insert provides M13 with an alternative DNA origin mediated by dnaG which is unaffected by rifampicin.
We have devised a rapid biological assay using gel electrophoresis to detect rifampicin-resistant DNA synthesis of the MB complementary strand. Appearance of such synthesis in the presence of rifampicin in some of our clones is confirmation of the successful transfer of the n' site. We intend to probe the function of this DNA origin by site-directed mutagenesis.
Brown, M. B.; Zuccarelli, A. J.: Specific interactions of the Escherichia coli n' protein with a DNA fragment isolated from bacteriophage fX174. Clinical Research, Vol. 30, No. 1, 1982 pp. 41A.
Interactions between regulatory proteins and specific DNA sequences underlie many importnat cellular processes. The recognition site of the Escherichia coli DNA replication protein n' has been isolated from the double-stranded replicative form (RF) of the bacterial virus fX174. A 201 base-pair fragment was excised from the RF by successive exposure to two different restriction endonucleases, HinfI and MboII.
One milligram of RF digested to completion with HinfI was resolved into 18 bands by electrophoresis on 5% polyacrylamide gels. The fifth band, containing 427, 417, and 413 base-pair fragments was cut from the gel, the DNA eluted, concentrated, and then digested with the second enzyme MboII. After electrophoresis the third band in the gel contained a 201 base-pair fragment which spanned the n' recognition site. The fragment was retreived from the gel, heat denatured, and the complementary strands were resolved on SDS-polyacrylamide gels. The viral (or "+" strand) was selected and purified.
To accomplish this isolation we devised techniques to visualize DNA bands in gels under conditions which minimize UV photodamage. Gels stained with ethidium bromide were photographed on a 302 nm transilluminator with Tri-X film at an exposure index of 1600. Exposure to the UV light was less than one second. Enlargements of the photograph were used as templates for cutting out bands.
We have studied the conditions under which n' protein will interact with its DNA recognition site in vitro. Complexes between the protein and radioactively labeled single-stranded fX174 DNA were detected by passing the mixture through a nitrocellulose filter. Under the conditions of the assay the n' binds to the filter while free DNA passes through unless it has interacted with the protein.