No description available.

No description available.

No description available.

T5 DNA polymerase, a highly processive single polypeptide enzyme, and PRD1 DNA polymerase, a protein-primed DNA polymerase, have been analyzed for their primary structural features. The amino acid sequence of T5 DNA polymerase reveals a high degree of homology with DNA polymerase I (Pol I) of Escherichia coli and retains many of the amino acid residues which have been implicated in the 3'-5' exonuclease and DNA polymerase activities of that enzyme. Alignment with sequences of polymerase I and T7 DNA polymerase (family A polymerases) was used to identify regions possibly involved in the high processivity of this enzyme. Further amino acid sequence comparisons of T5 DNA polymerase with a large group of DNA polymerases (family B) previously shown to exhibit little similarity to Pol I, indicate certain sequence segments are shared among distantly related DNA polymerases. These shared regions have been implicated in the 3'-5' exonuclease function of Pol I which suggests that the proofreading domains of all these enzymes may be related. Mutations in these segments in T5 DNA polymerase (family A) and PRD1 DNA polymerase (family B) greatly decrease the exonuclease activity of these enzymes but leave the polymerase activities intact. Additionally, an exonuclease deficient T5 DNA polymerase is used in DNA sequencing reactions and yields consistent results with low background contamination on autoradiographs of polyacrylamide/urea gels. PRD1 mutants defective in 3 regions which are highly conserved among family B DNA polymerases, are deficient in DNA polymerase activity but retain exonuclease activity.

CBP1 is a yeast nuclear gene encoding a mitochondrial protein that stabilizes the 5' end of cytochrome b (cob) pre-mRNA. Cytochrome b is the only mitochondrially synthesized component of the respiratory chain Complex III. Since the nuclearly-encoded subunits of this complex are regulated at the transcriptional level by catabolite repression, we hypothesized that CBP1 might be similarly regulated. To test the idea that transcriptional regulation of CBP1 could coordinate an increase in cytochrome b mRNA stability with an increase in nuclearly-encoded Complex III subunit production, we characterized the change in abundance of CBP1 mRNA during derepression on a non-fermentable carbon source. Poly A⁺ RNA from derepressed yeast was examined by Northern analyses with cRNA probes from CBP1. Both 2.2 kb and 1.3 kb transcripts were detected. The 1.3 kb mRNA lacks approximately 900 base-pairs of the 3'-end of the 2.2 kb mRNA, which encodes the carboxyl-terminal 250 amino acid residues of the CBP1 coding sequence. Northern analyses of RNA isolated from deletion/insertion mutants of CBP1 and from strains which overexpress CBP1 mRNA demonstrated that both mRNAs are transcribed from the CBP1 gene. Furthermore, we have demonstrated that the levels of the two CBP1 mRNAs are reciprocally regulated by the carbon source in the growth medium. Having proposed that regulation of 3' end formation dictates the amount of each CBP1 transcript we now show that a 146 bp fragment from the middle of CBP1 is sufficient to direct carbon source-regulated production of two transcripts when inserted into the yeast URA3 gene. This fragment contains seven polyadenylation sites for the wild-type 1.2 kb mRNA, as mapped by sequence analysis of CBPl cDNA clones. Deletion mutations upstream of the polyadenylation sites abolished formation of the 1.2 kb transcript, whereas deletion of three of the sites only led to a reduction in abundance of the 1.2 kb mRNA. Though none of the experiments showed whether the 1.2 kb mRNA is formed solely by 3' processing, or if processing at this site is coupled to premature transcription termination, our results do indicate that regulation of the abundance of both CBP1 transcripts is contro11ed by elements in a short segment of the gene that directs 3' end formation of the 1.2 kb transcript, a unique case in yeast.

A 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.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces an altered program of gene expression, followed by DNA synthesis and cell division, in quiescent murine fibroblasts. The phosphorylation of DNA topoisomerase I, a nuclear enzyme involved in transcription and replication, was characterized in quiescent 3T3-L1 cells treated with TPA. An anti-DNA topoisomerase I antibody from sera of patients with the autoimmune disease diffuse scleroderma was used to isolate phosphorylated DNA topoisomerase I. Phosphorylation was stimulated slightly within 10 min and 6-fold by 2 h of TPA treatment; TPA at 100 ng/ml maximally enhanced phosphorylation. DNA topoisomerase I was modified primarily on serine, with a minor phosphothreonine component. The half-life of incorporated phosphate on DNA topoisomerase I was approximately 40 min in both TPA-treated and control cells, suggesting that stimulation of a kinase was the mechanism for increased phosphorylation. In addition, the phosphorylation of DNA topoisomerase I was enhanced in rat H-35 hepatoma cells treated with insulin as well as Swiss/3T3 fibroblasts treated with epidermal growth factor. DNA topoisomerase I phosphorylation in vitro by protein kinase C, the cellular receptor for TPA, was also characterized. DNA topoisomerase I from HeLa cells was purified to apparent homogeneity and was phosphorylated in a Ca²⁺ and phospholipid-dependent fashion by type III protein kinase C, purified 860-fold from mouse brain; the reaction was stimulated by TPA. Approximately 1.3 moles of phosphate were incorporated per mole of substrate, predominantly on a tryptic peptide that comigrated with the major in vivo phosphopeptide, although other sites were phosphorylated to a lesser extent. Serine was the primary amino acid modified, however phosphothreonine was also detected. The incorporation of phosphate into DNA topoisomerase I was linear in the range of time and protein kinase C examined. The apparent K(m) and V(max) for the reaction were 0.4 μM and 0.7 μmol phosphate per min per mg, respectively. Thus, phosphorylation, possibly mediated by protein kinase C, was postulated to be a physiologically significant means of regulating DNA topoisomerase I during mammalian cell proliferation.

The rat ventral prostate (RVP) has served as a model system for investigating steroid hormonal control of development and differentiation. This male specific organ is dependent on androgens for both its development and the maintenance of its normal secretory function. Androgen effects on the prostate are mediated through the androgen receptor (AR). In order to gain a better understanding of the molecular nature of androgen regulation in the RVP I have cloned the AR cDNA, mapped structural domains responsible for AR function, compared the level of transcription induction by the glucocorticoid receptor (GR) to that of the AR on several hormone response element (HRE) containing promoters, compared the strength and specificity of DNA binding by AR and GR, and established a panel of RVP epithelial cell lines that will be used to investigate androgen regulation in prostate cells. I have found that the AR is a member of the steroid receptor family of ligand dependent transcription regulatory proteins containing a structural organization similar the GR. AR binds to glucocorticoid response element (GRE) consensus DNA sequences with approximately a two-fold lower affinity than the GR and increases transcription from prostate and non-prostate expressed genes. AR does not require cell specific factors for its function; however, the level of transcription enhancement by AR on different genes varies compared to GR suggesting that gene specific and/or receptor specific proteins affect AR activity. Using the SV40 large T-antigen I have immortalized a panel of RVP epithelial cell lines. All of these cell lines maintain a morphology typical of RVP epithelial cells, express a nuclear localized T-antigen, tartrate inhibitable acid phosphatase, and the GR. The cell lines express a low level of the AR which can be complemented by introduction of the AR cDNA. I conclude that these cell lines most closely resemble RVP basal epithelial cells which may have been preferentially immortalized through transfecting primary cultures of epithelial cells. These cell lines will be used to further investigate the role of AR specific factors which allow androgen responses to prevail in the RVP.

In the yeast Saccharomyces cerevisiae the production of respiratory competent mitochondria requires the activity of several hundred proteins, the vast majority of which are encoded by nuclear genes and imported into mitochondria. A subset of these nuclear gene products are required for the expression of individual mitochondrial genes. Expression of the mitochondrial gene cob, encoding cytochrome b, requires the activity of at least seven nuclear gene products, including CBP1, CBS1 and CBS2. CBP1 is required for the stability of cob transcripts, while CBS1 and CBS2 are required for translation of cob mRNA. Previous analyses of mitochondrial rearrangements that suppress cbp1, cbs1, or cbs2 mutations have led to the hypothesis that CBP1, CBS1 and CBS2 interact with the 5' untranslated leader (UTR) of cob transcripts. To further define cob sequence important for stability or translation of cob transcripts, the expression of mutant cob genes, in which portions of the cob coding sequence or 5' UTR have been deleted, has been analyzed in vivo. Quantitation of steady-state levels of cob transcripts in wild-type CBP1 and mutant cbp1 strains carrying deletion mitochondrial genomes leads to the conclusion that a 63 nucleotide sequence is sufficient for the CBP1-dependent stability of cob transcripts. This sequence encompasses the site of cleavage that produces the mature 5' end of cob mRNA from precursor transcripts. Determination of the 5' ends of mature cob mRNAs in the deletion strains indicated that this sequence is also sufficient for correct positioning of the cleavage. The data suggest that the cleavage is CBP1-dependent and occurs at a specific distance 5' of a recognition site located within the defined 63 nucleotides. In addition, the data suggest that the stability of the mature cob mRNA produced by the cleavage is dependent on sequence at its 5' end. Lastly, an analysis of the structure of a high molecular weight cob transcript present in a respiratory incompetent deletion strain leads to the hypothesis that 5' UTR sequence between -200 and -4 is required for interaction with factors, such as CBS1 or CBS2, required for translation of cob transcripts.

A small lipid-containing bacteriophage PRD1 encodes a DNA polymerase that utilizes a protein primer for the initiation of DNA replication. The purification of PRD1 DNA polymerase has been hampered by the insolubility of the overexpressed enzyme in E. coli cells. A simple and rapid procedure for purification of the overexpressed PRD1 DNA polymerase has been developed. This method is based on guanidine hydrochloride denaturation and renaturation of the insoluble PRD1 DNA polymerase overexpressed in E. coli containing recombinant plasmid pEJG. The purified DNA polymerase was extensively characterized and found to be indistinguishable from the normal soluble PRD1 DNA polymerase as judged by enzymatic properties. These properties include: protein-primed initiation of PRD1 DNA replication, strand-displacement DNA synthesis, DNA polymerase processivity, 3' to 5' exonuclease activity and filling-in repair type DNA synthesis. Furthermore, the kinetic parameters determined for dNTPs and primer-terminus were of the same order of magnitude. The availability of a simple purification procedure for PRD1 DNA polymerase should permit detailed structure-function analysis of this enzyme. All known family B DNA polymerases contain a conserved region of amino acids, KX₆₋₇YG, which appears to be correspond to the "finger" alpha helix O of the Klenow fragment of E. coli DNA polymerase I, a family A DNA polymerase. A site-directed mutagenesis study has been applied to access the functional role of the invariant amino acid lysine-340 of PRD1 DNA polymerase. Mutant DNA polymerases were overexpressed and purified to near homogeneity. The results showed that the modification of the lysine-340 of PRD1 DNA polymerase abolishes the polymerase activity without affecting the 3' to 5' exonuclease activity. Site-directed mutagenesis studies revealed that residues important for the 3' to 5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in PRD1 DNA polymerase as well. Although PRD1 DNA polymerase has a smaller 3' to 5' exonuclease domain, active sites appear to be very similar to those of the Klenow fragment. Moreover, the metal binding ligands were also found to be important for the strand-displacement activity, a unique feature of PRD1 DNA polymerase.