Mitochondrial DNA content and oxidation in bipolar disorder and its role across brain regions.

The underlying pathology of bipolar disorder remains unknown, though evidence is accumulating to support a role of mitochondrial dysfunction. In this study, we aim to investigate electron transport chain complex I subunit NDUFS7 protein expression; mtDNA content; common deletion; and oxidation in the Broadmann area 24 (BA24), cerebellum, hippocampus, and prefrontal cortex from patients with bipolar disorder, schizophrenia, and non-psychiatric controls. Here, we demonstrate no changes in NDUFS7 in BA24, cerebellum or hippocampus, increases in mtDNA content in hippocampus of patients with bipolar disorder, and decreases in mtDNA oxidation in patients with bipolar disorder and schizophrenia, respectively. Paired analysis between BA24 and cerebellum reveal increases within NDUFS7 levels and mtDNA content in cerebellum of patients with bipolar disorder or schizophrenia. We found a positive correlation between NDUFS7 and mtDNA content (ND4 and ND5) when combining brain regions. Our study supports the involvement of mitochondrial dysfunction in bipolar disorder and schizophrenia.

In vitro antimicrobial activities of novel anilinouracils which selectively inhibit DNA polymerase III of gram-positive bacteria.

The 6-anilinouracils are novel dGTP analogs that selectively inhibit the replication-specific DNA polymerase III of gram-positive eubacteria. Two specific derivatives, IMAU (6-[3'-iodo-4'-methylanilino]uracil) and EMAU (6-[3'-ethyl-4'-methylanilino]uracil), were substituted with either a hydroxybutyl (HB) or a methoxybutyl (MB) group at their N3 positions to produce four agents: HB-EMAU, MB-EMAU, HB-IMAU, and MB-IMAU. These four new agents inhibited Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium. Time-kill assays and broth dilution testing confirmed bactericidal activity. These anilinouracil derivatives represent a novel class of antimicrobials with promising activities against gram-positive bacteria that are resistant to currently available agents, validating replication-specific DNA polymerase III as a new target for antimicrobial development.

Inhibitors of DNA polymerase III as novel antimicrobial agents against gram-positive eubacteria.

6-Anilinouracils are selective inhibitors of DNA polymerase III, the enzyme required for the replication of chromosomal DNA in gram-positive bacteria (N. C. Brown, L. W. Dudycz, and G. E. Wright, Drugs Exp. Clin. Res. 12:555-564, 1986). A new class of 6-anilinouracils based on N-3 alkyl substitution of the uracil ring was synthesized and analyzed for activity as inhibitors of the gram-positive bacterial DNA polymerase III and the growth of gram-positive bacterial pathogens. Favorable in vitro properties of N-3-alkyl derivatives prompted the synthesis of derivatives in which the R group at N-3 was replaced with more-hydrophilic methoxyalkyl and hydroxyalkyl groups. These hydroxyalkyl and methoxyalkyl derivatives displayed K(i) values in the range from 0.4 to 2.8 microM against relevant gram-positive bacterial DNA polymerase IIIs and antimicrobial activity with MICs in the range from 0.5 to 15 microg/ml against a broad spectrum of gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Two of these hydrophilic derivatives displayed protective activity in a simple mouse model of lethal staphylococcal infection.

6-Anilinouracil-based inhibitors of Bacillus subtilis DNA polymerase III: antipolymerase and antimicrobial structure-activity relationships based on substitution at uracil N3.

6-Anilinouracils (6-AUs) are dGTP analogues which selectively inhibit the DNA polymerase III of Bacillus subtilis and other Gram-positive bacteria. To enhance the potential of the 6-AUs as antimicrobial agents, a structure-activity relationship was developed involving substitutions of the uracil N3 position in two 6-AU platforms: 6-(3,4-trimethyleneanilino)uracil (TMAU) and 6-(3-ethyl-4-methylanilino)uracil (EMAU). Series of N3-alkyl derivatives of both 6-AUs were synthesized and tested for their ability to inhibit purified B. subtilis DNA polymerase III and the growth of B. subtilis in culture. Alkyl groups ranging in size from ethyl to hexyl enhanced the capacity of both platforms to bind to the polymerase, and with the exception of hexyl, they also significantly enhanced their antimicrobial potency. N3 substitution of the EMAU platform with more hydrophilic hydroxyalkyl and methoxyalkyl groups marginally enhanced anti-polymerase III activity but enhanced antibacterial potency severalfold. In sum, the results of these studies indicate that the ring N3 of 6-anilinouracils can tolerate substituents of considerable size and structural variety and, thus, can be manipulated to significantly enhance the antibacterial potency of this novel class of polymerase III-specific inhibitors.

DNA polymerase III of Gram-positive eubacteria is a zinc metalloprotein conserving an essential finger-like domain.

DNA polymerase III (pol III) of Gram-positive eubacteria is a catalytically bifunctional DNA polymerase:3'-5' exonuclease [Low, R. L., Rashbaum, S. A., and Cozzarelli, N. R. (1976) J. Biol.Chem. 251, 1311-1325]. The pol III protein conserves, between its exonuclease and dNTP binding sites, a 35-residue segment of primary structure with the potential to form a zinc finger-like structure [Berg, J. M. (1990) Ann. Rev. Biochem. 19, 405-421]. This paper describes results of experiments which probe the capacity of this segment to bind zinc and the role of this segment in enzyme function. The results of metal and mutational analysis of a model pol III derived from Bacillus subtilis indicate that (i) the Gram-positive pol III is a metalloprotein containing tightly bound zinc in a stoichiometry of 1, (ii) the zinc atom is bound within the 35-residue segment, likely in one of two probable finger-like structures, and (iii) the integrity of the zinc-bound structure is specifically critical to the formation and/or function of the enzyme's polymerase site.

Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones.

A flow cytometry-based, ultrasensitive fluorescence detection technique is used to size individual DNA fragments up to 167 kb in length. Application of this technology to the sizing of P1 artificial chromosomes (PACs) in both linear and supercoiled forms is described. It is demonstrated that this method is well suited to characterizing PAC/BAC clones and will be very useful for the analysis of large insert libraries. Fluorescence bursts are recorded as individual, dye stained DNA fragments pass through a low power, focused, continuous laser beam. The magnitudes of the fluorescence bursts are linearly proportional to the lengths of the DNA fragments. The histograms of the burst sizes are generated in <3 min with <1 pg of DNA. Results on linear fragments are consistent with those obtained by pulsed-field gel electrophoresis. In comparison with pulsed-field gel electrophoresis, sizing of large DNA fragments by this approach is more accurate, much faster, requires much less DNA, and is independent of the DNA conformation.

Measuring outcomes in nursing centers: otitis media as a sample case.

While it is generally recognized that NPs offer affordable, quality health care, few studies have measured outcomes of clients who seek primary care services from NPs. This pilot study describes the outcomes of children with otitis media who received care from NPs employed in an academic nursing center. Outcome measurements included issues related to timing, level of analysis, and attribution. Parents of 27 children participated in a telephone survey consisting of seven questions relating to the care their children received from NPs and their recovery path. Although every respondent reported having a positive visit at the nursing center, concerns for NPs surfaced during the process of measuring outcomes. This study emphasizes the need for measuring outcomes in nursing clinics and demonstrates one way to measures client outcomes, revealing both general health care and specific nursing practice implications.

The 3'-5' exonuclease site of DNA polymerase III from gram-positive bacteria: definition of a novel motif structure.

The primary structure of the 3'-5' exonuclease (Exo) site of the Gram+ bacterial DNA polymerase III (Pol III) was examined by site-directed mutagenesis of Bacillus subtilis Pol III (BsPol III). It was found to differ significantly from the conventional three-motif substructure established for the Exo site of DNA polymerase I of Escherichia coli (EcPol I) and the majority of other DNA polymerase-exonucleases. Motifs I and II were conventionally organized and anchored functionally by the predicted carboxylate residues. However, the conventional downstream motif, motif III, was replaced by motif III epsilon, a novel 55-amino-acid (aa) segment incorporating three essential aa (His565, Asp533 and Asp570) which are strictly conserved in three Gram+ Pol III and in the Ec Exo epsilon (epsilon). Despite its unique substructure, the Gram+ Pol III-specific Exo site was conventionally independent of Pol, the site of 2'-deoxyribonucleoside 5-triphosphate (dNTP) binding and polymerization. The entire Exo site, including motif III epsilon, could be deleted without profoundly affecting the enzyme's capacity to polymerize dNTPs. Conversely, Pol and all other sequences downstream of the Exo site could be deleted with little apparent effect on Exo activity. Whether the three essential aa within the unique motif III epsilon substructure participate in the conventional two-metal-ion mechanism elucidated for the model Exo site of EcPol I, remains to be established.

Characterization and overexpression of the gene encoding Staphylococcus aureus DNA polymerase III.

The polC gene specifying DNA polymerase III (PolIII) of Staphylococcus aureus (Sa), was cloned with a novel strategy and found to contain a 4305-bp open reading frame (ORF) encoding a polypeptide of approx. 162 kDa. The 1435-codon ORF was engineered into an Escherichia coli (Ec) expression plasmid under the control of the lac promoter and its repressor. Derepression of Ec transformants carrying the recombinant (re-) vector generated high-level synthesis of active re-Sa PolIII. The re-PolIII was purified to > 98% homogeneity and was shown by N-terminal amino acid sequence analysis to be the bona fide product of the Sa polC ORF. The physical and catalytic properties of re-Sa PolIII and its responsiveness to inhibitors of the HPUra type were generally similar to those of Bacillus subtilis (Bs) PolIII. Comparative analysis of the primary structures of Sa PolIII, Bs PolIII and Mycoplasma pulmonis PolIII indicated strong conservation of essential catalytic domains and a novel zinc-finger motif. Comparison of the primary structures of Ec PolIII and these three Gram+ enzymes revealed a region of novel homology and reinforced the likelihood of a specific evolutionary relationship between PolIII of Gram+ and Gram- eubacteria. The polC gene mapped between omega 1074 [Tn551] and recA/ngr on the Sa NCTC 8325 genome.

DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the enzyme and its structural gene, polC.

Mycoplasmas have originated from Gram-positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram-positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)-positive enzymes to a single exo-negative species. The objective of this work was to rigorously investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram-positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III-specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram-positive bacterial gene encoding Pol III. M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram-positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram-positive Pol III-selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra-resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram-positive bacteria than previously thought.

Construction and characterization of partial digest DNA libraries made from flow-sorted human chromosome 16.

In this report, we present the techniques used for the construction of chromosome-specific partial digest libraries from flow-sorted chromosomes and the characterization of two such libraries from human chromosome 16. These libraries were constructed to provide materials for use in the development of a high-resolution physical map of human chromosome 16, and as part of a distributive effort on the National Laboratory Gene Library Project. Libraries with 20-fold coverage were made in Charon-40 (LA16NL03) and in sCos-1 (LA16NC02) after chromosome 16 was sorted from a mouse-human monochromosomal hybrid cell line containing a single homologue of human chromosome 16. Both libraries are approximately 90% enriched for human chromosome 16, have low nonrecombinant backgrounds, and are highly representative for human chromosome-16 sequences. The cosmid library in particular has provided a valuable resource for the isolation of coding sequences, and in the ongoing development of a physical map of human chromosome 16.

Properties of the nuclear P1 protein, a mammalian homologue of the yeast Mcm3 replication protein.

Polyclonal antibodies were raised against a multiprotein 'holoenzyme' form of calf thymus DNA polymerase alpha-primase and used to probe a human cDNA-protein expression library constructed in the lambda gt11 vector. The probe identified a series of cDNA clones derived from a 3.2 kb mRNA which encodes a novel 105 kDa polypeptide, the P1 protein. In intact cells, the P1 protein was specifically associated with the nucleus, and in cell extracts, it was associated with complex forms of DNA polymerase alpha-primase. The synthesis of human P1-specific mRNA was stimulated upon addition of fresh serum to growth-arrested cells, and RNA blot analyses with the human P1-cDNA probe indicated that P1 is encoded by a strictly conserved mammalian gene. The amino acid sequence deduced from a 240-codon open reading frame resident in the largest human P1-cDNA (0.84 kb) displayed greater than 96% identity with that deduced from the equivalent segment of a 795-codon open reading frame of a larger mouse P1-cDNA (2.8 kb). Throughout its length, the primary structure of mammalian P1 displayed strong homology with that of Mcm3, a 125 kDa yeast protein thought to be involved in the initiation of DNA replication (Gibson et al. 1990. Mol. Cell. Biol. 10: 5707-5720). The P1-Mcm3 homology, the strong conservation of P1 among mammals, its nuclear localization, and its association with the replication-specific DNA polymerase alpha strongly suggest an important role of the P1 protein in the replication of mammalian DNA.

Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III.

Structural gene mutants were cloned and exploited to identify the major catalytic domains of Bacillus subtilis DNA polymerase III (BsPolIII), a 162.4-kDa [1437 amino acids (aa)] polymerase: 3'-5' exonuclease (Exo) required for replicative DNA synthesis. Analysis of the sequence, mutagenicity, and catalytic behavior of natural and site-directed point mutants of BsPolIII unequivocally located the domain involved in exonuclease catalysis within a 155-aa residue segment displaying homology with the Exo domain of Escherichia coli DNA polymerase I. Sequence analysis of four structural gene mutations which specifically alter then enzyme's reactivity to the inhibitory dGTP analog, 6-(p-hydroxyphenylhydrazino)uracil, and the inhibitory arabinonucleotide, araCTP, defined a domain (Pol) involved in dNTP binding. The Pol domain was in the C-terminal fourth of the enzyme within a 98-aa segment spanning aa 1175-1273. The primary structure of the domain was unique, displaying no obvious conservation in any other DNA polymerase, including the distantly related PolIIIs of the Gram- organisms, E. coli and Salmonella typhimurium.

Overproduction and purification of Bacillus subtilis DNA polymerase III.

The objectives of this work were to engineer the cloned polC gene encoding Bacillus subtilis DNA polymerase III for controlled overexpression in Escherichia coli and to devise a facile purification scheme permitting the large-scale production of pure recombinant polymerase. The translational signals of polC were restructured by expression cassette PCR (MacFerrin et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1937-1941), and the modified gene was inserted into the expression plasmid, pKC30 (Rosenberg et al., 1983, in "Methods in Enzymology," Vol. 101, pp. 123-138, Academic Press, San Diego), under the strict control of the coliphage lambda pL promoter and its repressor, cI. When the system was derepressed at 32 degrees C, soluble DNA polymerase III accumulated at levels approximating 2% of total cellular protein. The recombinant protein was purified to greater than 99% purity by utilizing a tandem combination of Cibacron blue agarose, phenyl-Sepharose, and MonoQ FPLC chromatography. The properties of the purified recombinant protein were indistinguishable from those of native B. subtilis DNA polymerase III.

The molecular mechanism of inhibition of alpha-type DNA polymerases by N2-(butylphenyl)dGTP and 2-(butylanilino)dATP: variation in susceptibility to polymerization.

Calf thymus DNA polymerase alpha (pol alpha) and bacteriophage T4 DNA polymerase (pol T4) were exploited as model enzymes to investigate the molecular mechanism of inhibitory action of N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butyl-anilino)dATP (BuAdATP) on the BuPdNTP-susceptible alpha polymerase family. Kinetic analysis of inhibition of pol alpha with mixtures of complementary and noncomplementary template:primers indicated that both nucleotides induced the formation of a polymerase: inhibitor:primer-template complex. Primer extension experiments using the guanine form as the model analog indicated that pol alpha cannot utilize these nucleotides to extend primer termini. In contrast, pol T4 polymerized BuPdGTP, indicating that resistance to polymerization is not a common feature of the inhibitor mechanism among the broad membership of the alpha polymerase family.

Bacillus subtilis DNA polymerase III: complete sequence, overexpression, and characterization of the polC gene.

Genomic DNA encompassing polC, the structural gene specifying Bacillus subtilis DNA polymerase III (PolIII), was sequenced and found to contain a 4311-bp open reading frame (ORF) encoding a 162.4-kDa polypeptide of 1437 amino acids (aa). The ORF was engineered into an Escherichia coli expression plasmid under the control of the coliphage lambda repressor. Derepression of E. coli transformants carrying the recombinant vector resulted in the high-level synthesis of a recombinant DNA polymerase indistinguishable from native PolIII. N-terminal aa sequence analysis of the recombinant polymerase unequivocally identified the 4311-bp ORF as that of polC. Comparative aa sequence analysis indicated significant homology of the B. subtilis enzyme with the catalytic alpha subunit of the E. coli PolIII and, with the exception of an exonuclease domain, little homology with other DNA polymerases. The respective sequences of the mutant polC alleles, dnaF and ts-6, were identified, and the expression of specifically truncated forms of polC was exploited to assess the dependence of polymerase activity on the structure of the enzyme's C terminus.

Use of sex-linked minisatellite fragments to investigate genetic differentiation and migration of North American populations of the peregrine falcon (Falco peregrinus).

The M13 repeat detects different levels of genetic variation in falcons. First, this minisatellite probe reveals typically highly variant restriction fragments that show no apparent unequal distribution between the sexes. Secondly, the M13 repeat detects sets of fragments that are only present in DNAs from female falcons. The level of polymorphism displayed by the sex-linked fragments is greatly reduced relative to most autosomal minisatellites. In addition, the size of these fragments (in kilobase pairs) is species-specific among Mauritius kestrels (Falco punctatus) and peregrines (Falco peregrinus). Variation observed at one o of the sex-linked fragments in peregrines has proven to be useful in distinguishing a subset of the tundrius subspecies of this endangered raptor. This correlation has enabled a genetic test to be used to examine the representation of tundrius peregrines during mass migration.