Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The LaBelle-1b strain of Neurospora intermedia contains a 4.1-kb closed-circular mitochondrial plasmid DNA, which encodes a single long open reading frame of 1,151 amino acids reported to have sequence similarity to reverse transcriptases. Here, we show that the LaBelle strain contains a novel DNA polymerase activity that is highly specific for the endogenous LaBelle plasmid DNA in nucleoprotein particles and can be distinguished from the mitochondrial DNA polymerase by several characteristics. Photolabeling experiments indicate that the LaBelle-specific DNA polymerase activity is associated with a polypeptide of 120 kDa, which is in good agreement with the size predicted for the protein encoded by the LaBelle plasmid open reading frame (132 kDa). This 120-kDa polypeptide is found only in the LaBelle strain that contains the mitochondrial plasmid, and it cosegregates with mitochondria in sexual crosses, suggesting that it is encoded by the plasmid. The LaBelle-specific DNA polymerase efficiently uses the artificial DNA substrates, poly(dA)-oligo(dT) and poly(dC)-oligo(dG), but despite its reported sequence similarity to reverse transcriptases, it has very low activity with analogous RNA substrates, poly(rA)-oligo(dT), poly(rC)-oligo(dG), or poly(rCm)-oligo(dG). Considered together with the previous sequence comparisons, our results suggest that the LaBelle plasmid encodes a novel DNA polymerase, which was derived from a protein that was at one time a reverse transcriptase but lost its ability to use RNA templates. This DNA polymerase now presumably functions in replication of the plasmid. Our results constitute the first biochemical evidence for a DNA polymerase activity associated with a mitochondrial plasmid. Further, they may provide insight into the evolution of DNA polymerases from reverse transcriptases, as presumably occurred in the course of evolution following the transition from the so-called RNA world to the present DNA world.
Mol Cell Biol 1991 Mar
PMID:The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase. 170 12

Topoisomerase cDNA and various fragments thereof generated by the DNA polymerase chain reaction were cloned into plasmid expression vectors (pET series) and the expressed polypeptides were probed with scleroderma sera from seven different patients immunoreactive with topoisomerase I. All sera reacted selectively with a region between amino acid residues 405 and 484 of human topoisomerase I. This conclusion is based on loss of reactivity when this region was omitted from larger pieces. Other portions of topoisomerase I were not reactive with these autoantibodies. At least two different epitopes appear to be recognized within this region by different sera based on differences in immunoreactivity of the 405-484 region when expressed as C-terminal, N-terminal or internally within a peptide.
Mol Immunol
PMID:An antigenic region of topoisomerase I in DNA polymerase chain reaction-generated fragments recognized by autoantibodies of scleroderma patients. 171 70

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
J Mol Biol 1991 Aug 05
PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5

The APN1 gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apn1 protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism. We show here that yeast strains lacking Apn1 (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl- and ethylmethane sulfonate) agents that damage DNA. These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apn1 mutant yeast cells. Hydrogen peroxide-treated APN1+ but not apn1 mutant cells regenerate high-molecular-weight DNA efficiently after the treatment. The DNA strand breaks that accumulate in the Apn1-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apn1. An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apn1 mutant cells. The rate of spontaneous mutation in apn1 mutant S. cerevisiae was 6- to 12-fold higher than that measured for wild-type yeast cells. This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apn1 is substantial and that such lesions can be mutagenic when left unrepaired.
Mol Cell Biol 1991 Sep
PMID:Cellular role of yeast Apn1 apurinic endonuclease/3'-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation. 171 20

Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on primary sequence. Under the appropriate conditions, all base pair (bp) substitutions, frame-shifts, and deletions less than about 10 bp can be resolved from the wild type sequence using DGGE. Polymerase chain reaction (PCR) permits facile amplification of a given region of the genome. We have combined PCR and DGGE to: (i) Localize mutations in the X-linked human androgen receptor gene. PCR/DGGE was used to screen the individual exons in the 2757-bp coding region of the gene in afflicted individuals as well as in potential carriers. Inheritance of a mutant allele has been demonstrated in several cases; (ii) Analyze thousands of thioguanine-resistant mutants simultaneously. The in vitro mutational spectra of MNNG, ICR-191, and cisplatin at the human HPRT locus have been examined by this method. The compounds all have mutational hotspots in a GGGGGG sequence in exon 3; however, the particular mutations induced by the agents were different; (iii) Examine the fidelity of several DNA polymerases used in PCR. The fidelity of Thermus aquaticus DNA polymerase (Taq) is 1-2 x 10(-4) misincorporations/bp/replication. Problems with Taq polymerase arise in the analysis of complex mutant populations by DGGE because the Taq-induced errors reduce the sensitivity of the system. To circumvent this, it had been necessary to use Sequenase, a modified T7 DNA polymerase with a higher fidelity. However, Sequenase is not thermostable and must be added every PCR cycle. A thermostable DNA polymerase from Thermococcus litoralis (Vent) is now available, and we have examined the fidelity of Vent, Taq, and Sequenase polymerase in PCR using DGGE. The fidelity of Vent, Taq, and Sequenase polymerase was 2.4 x 10(-5), 8.9 x 10(-5), and 4.4 x 10(-5) errors/bp, respectively. Vent polymerase had the highest fidelity of the three enzymes tested.
Environ Mol Mutagen 1991
PMID:Analysis of mutations using PCR and denaturing gradient gel electrophoresis. 174 86

DNA content and DNA polymerase activity were measured on rabbit blastocysts removed from the uterus at 24-hr intervals over the period of days 4-7 postcoitum (pc). Median DNA content increased 53 times over the 72-hr period, from 25.3 ng on day 4 to 1,360 ng on day 7. Median DNA polymerase activity (fmole of radiolabeled nucleotide incorporated in 30 min at 37 degrees C) increased 393-fold from day 4 to day 7: 32.8 to 12,900. These embryos also increased in surface area and volume by 334-fold and 6,078-fold, respectively. Litters containing individuals with high DNA content also tended to have similar individuals with high DNA polymerase activity. Therefore, DNA polymerase activity may be a useful measure of the potential for the next cell division. A large amount of variation existed between blastocysts in all parameters measured. An analysis of variance, conducted to partition variation between litters and within litters, determined that within-litter variation was actually greater than that between litters, resulting in intraclass correlation coefficients less than 0.5. There was also a positive regression of DNA content and DNA polymerase activity on surface area in 6- and 7-day-old blastocysts after eliminating variation attributable to litters. The developmental pattern of DNA polymerase activity in the rabbit may be quantitatively different from that described in the mouse. The pattern in mammals is very different from that described in several nonmammalian species.
Mol Reprod Dev 1991 Dec
PMID:Growth and DNA replication in rabbit blastocysts. 175 Oct 36

The use of the polymerase chain reaction was proposed for intron excision from genomic genes with known nucleotide sequences. Three exons (5, 6 and 7) of genomic interleukin 1 beta gene were amplified by means of thermostable DNA polymerase TthI from Thermus thermophilus on the base of cloned in M13 phage human genomic interleukin 1 beta gene. Synthetic oligonucleotides complementary to sequences flanking exons were used as primers. The fragments obtained by exon DNA amplification were joined in the correct order due to reciprocal complementation of end sequences, that was foreseen during synthesis of oligonucleotide primers followed by amplification of the enlarged fragments. As a result the structural interleukin-1 beta gene consisting of three exons was assembled. DNA sequences carrying the ATG initiation codon and XbaI recognition site at the 5'-end, and PstI recognition site at the 3'-end (essential for insertion into the expression vector) were formed by the additional end sequences of primers. The nucleotide sequence analysis of the obtained structural gene revealed its complete identity with natural interleukin 1 beta human gene. We created the expression vector pPR114 with phage lambda promoter PR thermo-inducible in case of the cIts857 repressor presence in cells. It was used for expression of the present gene. The interleukin 1 beta synthesized in E. coli had biological activity.
Mol Biol (Mosk)
PMID:[Elimination of introns from the interleukin 1 beta genome by DNA amplification and expression of interleukin 1 beta in Escherichia coli]. 175 56

We report the isolation and sequencing of genomic DNA clones that encode the 1094-amino acid catalytic subunit of DNA polymerase delta from the human malaria parasite Plasmodium falciparum. Protein sequence comparison to other DNA polymerases revealed the presence of six highly conserved regions found in alpha-like DNA polymerases from different prokaryotic, viral, and eukaryotic sources. Five additional regions of amino acid sequence similarity that are only conserved in delta and delta-like DNA polymerases, so far, were present in P. falciparum DNA polymerase delta. P. falciparum DNA polymerase delta was highly similar to both Saccharomyces cerevisiae DNA polymerase delta (DNA polymerase III; CDC2) and Epstein-Barr virus DNA polymerase at the amino acid sequence, and the predicted protein secondary structure levels. The gene that encodes DNA polymerase delta resides as a single copy on chromosome 10, and is expressed as a 4.5-kb mRNA during the trophozoite and schizont stages when parasite chromosomal DNA synthesis is active.
Mol Biochem Parasitol 1991 Dec
PMID:The primary structure of Plasmodium falciparum DNA polymerase delta is similar to drug sensitive delta-like viral DNA polymerases. 177 72

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [HPMPA] belongs to a class of nucleoside analogues originally described as having potent activity against a broad spectrum of DNA viruses. We examined the effects of this class of drugs on the growth of cultured Plasmodium falciparum. Strong inhibition was observed by HPMPA (ID50 = 47 nM) at concentrations more than 1000-fold less than the cytotoxic dose for human cells. 3-deaza-HPMPA was even more strongly inhibitory (ID50 = 8 nM), whereas several other acyclic nucleosides were not effective. In mice infected with Plasmodium berghei, increase of parasitaemia can be blocked for 4-6 days by a single injection of HPMPA. Repeated drug administration blocks parasite growth for prolonged periods at doses that are clinically feasible. We also determined the inhibition of several purified Plasmodium DNA polymerases by diphosphorylated HPMPA (HPMPApp). DNA polymerase alpha-like enzymes of P. falciparum and P. berghei are inhibited with an IC50 = 40 microM and a gamma-like DNA polymerase from P. falciparum is even 40-fold more sensitive to the drug. The inhibition by HPMPApp is competitive with dATP, strongly suggesting that Plasmodium DNA polymerases are targets for this class of nucleotide analogue.
Mol Biochem Parasitol 1991 Jul
PMID:Inhibition of the growth of Plasmodium falciparum and Plasmodium berghei by the DNA polymerase inhibitor HPMPA. 185 84

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
Plant Mol Biol 1991 Jun
PMID:Pea chloroplast DNA primase: characterization and role in initiation of replication. 186 57


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>