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At least 1,369 records · Page 76Linked to original sources
PubMed1996

DNA sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata.

Ecteinascidin 743 is one of several related marine alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata. It is remarkably active and potent in a variety of in vitro and in vivo systems and has been selected for development as an anticancer agent. The present study investigates the interactions of ecteinascidin 743 with DNA. Ecteinascidin 743 retarded the electrophoretic migration of both supercoiled and relaxed simian virus 40 DNA even in the presence of sodium dodecyl sulfate and after ethanol precipitation, consistent with covalent DNA modifications. Similar results were obtained in a DNA oligonucleotide derived from ribosomal DNA. However, DNA denaturation reversed the DNA modifications. The homopolymeric oligonucleotide dG/dC was modified while neither the dI/dC nor the dA/dT oligonucleotides were, consistent with covalent attachment of ecteinascidin 743 to the exocyclic amino group at position 2 of guanine. Ecteinascidin 743 was then compared to another known DNA minor groove alkylating agent, anthramycin, which has also been shown to alkylate guanine N2. Footprinting analyses with DNase I and 1,10-phenanthroline-copper and exonuclease III digestions showed that ecteinascidin 743 covers three to five bases of DNA and exhibits a different sequence specificity than anthramycin in the Escherichia coli tyrosine tRNA promoter (tyrT DNA). The binding of ecteinascidin to DNA was abolished when guanines were substituted with inosines in this promoter. A band shift assay was designed to evaluate the influence of the bases flanking a centrally located guanine in an oligonucleotide containing inosines in place of guanines elsewhere. Ecteinascidin 743 and anthramycin showed similarities as well as differences in sequence selectivity. Ecteinascidin 743-guanine adducts appeared to require at least one flanking guanine and were strongest when the flanking guanine was 3' to the targeted guanine. These data indicate that ecteinascidin 743 is a novel DNA minor groove, guanine-specific alkylating agent.

Alkylation↗
PubMed2002

HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics.

Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.

Anti-Bacterial Agents↗
PubMed1982

Quantitative enzymatic hydrolysis of tRNAs: reversed-phase high-performance liquid chromatography of tRNA nucleosides.

A rapid quantitative method for enzymatic hydrolysis of microgram amounts of tRNA has been developed, specifically to take full advantage of our precise, accurate, and selective reversed-phase high-performance liquid chromatographic (HPLC) system for separation and measurement of the major and modified nucleosides in tRNA. After study of several enzyme systems, nuclease P1 and bacterial alkaline phosphatase were selected and the hydrolysis parameters were systematically studied. Optimized hydrolysis conditions give quantitative hydrolysis in 2 h and this short incubation time prevents loss of unstable nucleosides. The chromatographic system can tolerate relatively high levels of protein in the sample allowing high enzyme--substrate ratios and direct injection of hydrolysates. This enzymatic hydrolysis--HPLC method is the best described to date for quantitative determination of the nucleoside composition of tRNAs and has already provided important information for investigation of the role of modification in the function of RNAs.

Chromatography, High Pressure Liquid↗
PubMed2005

Complete translation of the hepatitis C virus genome in vitro: membranes play a critical role in the maturation of all virus proteins except for NS3.

We developed an in vitro translation extract from Krebs-2 cells that translates the entire open reading frame of the hepatitis C virus (HCV) strain H77 and properly processes the viral protein precursors when supplemented with canine microsomal membranes (CMMs). Translation of the C-terminal portion of the viral polyprotein in this system is documented by the synthesis of NS5B. Evidence for posttranslational modification of the viral proteins, the N-terminal glycosylation of E1 and the E2 precursor (E2-p7), and phosphorylation of NS5A is presented. With the exception of NS3, efficient generation of all virus-specific proteins is CMM dependent. A time course of the appearance of HCV products indicates that the viral polyprotein is cleaved cotranslationally. A competitive inhibitor of the NS3 protease inhibited accumulation of NS3, NS4B, NS5A, and NS5B, but not that of NS2 or structural proteins. CMMs also stabilized HCV mRNA during translation. Finally, the formyl-[35S]methionyl moiety of the initiator tRNA(Met) was incorporated exclusively into the core protein portion of the polyprotein, demonstrating that translation initiation in this system occurs with high fidelity.

Animals↗
PubMed1981

Tertiary structure of tRNAs in solution monitored by phosphodiester modification with ethylnitrosourea.

The alkylation by ethylnitrosourea of phosphodiester bonds in yeast tRNAPhe, tRNAVal and in Escherichia coli tRNAGlu, tRNAfMet, tRNAmMet and tRNAPhe was investigated under various conditions. In unfolded tRNAs the reactivities of phosphates in various positions toward the reagent were similar. In the folded tRNAs remarkable differences in reactivities of phosphates located in various positions of the molecules were observed. In yeast and E. coli tRNAPhe, reactivities of phosphates in positions 9, 10, 11, 19, 49, 58, 59 and 60 were found to be strongly decreased. Some decrease in reactivity was observed for phosphates 23 and 24. Spermine and ethidium bromide did not influence the pattern of phosphate alkylation in the T psi C arm of yeast tRNAPhe. Our solution results fit with the crystal structure of tRNAPhe with respect to the potential availability of the phosphates in this tRNA to solvent as shown by others. Judging from the pattern of phosphate reactivities, the structure of E. coli tRNAPhe is very similar to that of yeast tRNAPhe. Upon thermal denaturation of the yeast tRNAPhe, the reactivity of the low-reactive phosphates increased, demonstrating a cooperative melting curve. A comparison of the patterns of phosphate alkylation in several tRNAs, essentially in their T psi C arms, revealed a striking similarity, suggesting that the folding of these tRNAs is essentially similar.

Alkylation↗
PubMed1996

Site-specific ribose methylation of preribosomal RNA: a novel function for small nucleolar RNAs.

Eukaryotic cells contain many fibrillarin-associated small nucleolar RNAs (snoRNAs) that possess long complementarities to mature rRNAs. Characterization of 21 novel antisense snoRNAs from human cells followed by genetic depletion and reconstitution studies on yeast U24 snoRNA provides evidence that this class of snoRNAs is required for site-specific 2'-O-methylation of preribosomal RNA (pre-rRNA). Antisense sno-RNAs function through direct base-pairing interactions with pre-rRNA. The antisense element, together with the D or D' box of the snoRNA, provide the information necessary to select the target nucleotide for the methyltransfer reaction. The conclusion that sno-RNAs function in covalent modification of the sugar moieties of ribonucleotides demonstrates that eukaryotic small nuclear RNAs have a more versatile cellular function than earlier anticipated.

Base Sequence↗
PubMed2006

Yersiniabactin production by Pseudomonas syringae and Escherichia coli, and description of a second yersiniabactin locus evolutionary group.

The siderophore and virulence factor yersiniabactin is produced by Pseudomonas syringae. Yersiniabactin was originally detected by high-pressure liquid chromatography (HPLC); commonly used PCR tests proved ineffective. Yersiniabactin production in P. syringae correlated with the possession of irp1 located in a predicted yersiniabactin locus. Three similarly divergent yersiniabactin locus groups were determined: the Yersinia pestis group, the P. syringae group, and the Photorhabdus luminescens group; yersiniabactin locus organization is similar in P. syringae and P. luminescens. In P. syringae pv. tomato DC3000, the locus has a high GC content (63.4% compared with 58.4% for the chromosome and 60.1% and 60.7% for adjacent regions) but it lacks high-pathogenicity-island features, such as the insertion in a tRNA locus, the integrase, and insertion sequence elements. In P. syringae pv. tomato DC3000 and pv. phaseolicola 1448A, the locus lies between homologues of Psyr_2284 and Psyr_2285 of P. syringae pv. syringae B728a, which lacks the locus. Among tested pseudomonads, a PCR test specific to two yersiniabactin locus groups detected a locus in genospecies 3, 7, and 8 of P. syringae, and DNA hybridization within P. syringae also detected a locus in the pathovars phaseolicola and glycinea. The PCR and HPLC methods enabled analysis of nonpathogenic Escherichia coli. HPLC-proven yersiniabactin-producing E. coli lacked modifications found in irp1 and irp2 in the human pathogen CFT073, and it is not clear whether CFT073 produces yersiniabactin. The study provides clues about the evolution and dispersion of yersiniabactin genes. It describes methods to detect and study yersiniabactin producers, even where genes have evolved.

Amino Acid Sequence↗
PubMed2002

Unconventional rules of small nuclear RNA transcription and cap modification in trypanosomatids.

This review focuses on the spliced leader (SL) RNA and uridylic acid-rich small nuclear RNAs (U-snRNAs) involved in pre-mRNA processing in trypanosomatid protozoa, with particular emphasis on the mechanism of transcription and cap formation. The SL RNA plays a central role in mRNA biogenesis by providing the unique cap 4 structure to the 5' end of all mRNAs by trans-splicing. The trimethylguanosine capped U-snRNAs, on the other hand, represent an unusual example among eukaryotic snRNAs in that they are transcribed by RNA polymerase III. This implies the existence of a distinctive mechanism for capping enzyme selection by the transcriptional machinery. Furthermore, the transcription units of U-snRNA genes offer yet another example of the variety of choices that have been established during eukaryotic evolution, namely that an upstream tRNA gene or tRNA-like gene provides extragenic promoter elements for a downstream small RNA gene.

Animals↗
PubMed1987

Depurination of yeast 26S ribosomal RNA by recombinant ricin A chain.

The nature of the modification of yeast ribosomes by the recombinant form of the ricin A chain has been examined. Evidence is presented that the 26S rRNA molecule is depurinated at a specific site and that the activity is inhibited by antibody raised to ricin A chain. It thus appears that the recombinant form of this toxin retains the depurination activity of the native molecule. These results are consistent with the model that the site of depurination is in a highly conserved sequence forming a loop on the surface of the ribosome, a domain involved in elongation factor-dependent binding of aminoacyl-tRNA.

Purines↗
PubMed1999

Using novel phylogenetic methods to evaluate mammalian mtDNA, including amino acid-invariant sites-LogDet plus site stripping, to detect internal conflicts in the data, with special reference to the positions of hedgehog, armadillo, and elephant.

We look at the higher-order phylogeny of mammals, analyzing in detail the complete mtDNA sequences of more than 40 species. We test the support for several proposed superordinal relationships. To this end, we apply a number of recently programmed methods and approaches, plus better-established methods. New pairwise tests show highly significant evidence that amino acid frequencies are changing among nearly all the genomes studied when unvaried sites are ignored. LogDet amino acid distances, with modifications to take into account invariant sites, are combined with bootstrapping and the Neighbor Joining algorithm to account for these violations of standard models. To weight the more slowly evolving sites, we exclude the more rapidly evolving sites from the data by using "site stripping". This leads to changing optimal trees with nearly all methods. The bootstrap support for many hypotheses varies widely between methods, and few hypotheses can claim unanimous support from these data. Rather, we uncover good evidence that many of the earlier branching patterns in the placental subtree could be incorrect, including the placement of the root. The tRNA genes, for example, favor a split between the group hedgehog, rodents, and primates versus all other sequenced placentals. Such a grouping is not ruled out by the amino acid sequence data. A grouping of all rodents plus rabbit, the old Glires hypothesis, is also feasible with stripped amino acid data, and rodent monophyly is also common. The elephant sequence allows confident rejection of the older taxon Ferungulata (Simpson, 1945). In its place, the new taxa Scrotifera and Fereuungulata are defined. A new likelihood ratio test is used to detect differences between the optimal tree for tRNA versus that for amino acids. While not clearly significant as made, some results indicate the test is tending towards significance with more general models of evolution. Individual placement tests suggest alternative positions for hedgehog and elephant. Congruence arguments to support elephant and armadillo together are striking, suggesting a superordinal group composed of Xenarthra and African endemic mammals, which in turn may be near the root of the placental subtree. Thus, while casting doubt on some recent conclusions, the analyses are also unveiling some interesting new possibilities.

Animals↗
PubMed1997

Role of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in normal cell function and in cell pathology.

The glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be an archtypical protein of limited excitement. However, independent studies from a number of different laboratories reported a variety of diverse biological properties of the GAPDH protein. As a membrane protein, GAPDH functions in endocytosis; in the cytoplasm, it is involved in the translational control of gene expression; in the nucleus, it functions in nuclear tRNA export, in DNA replication, and in DNA repair. The intracellular localization of GAPDH may be dependent on the proliferative state of the cell. Recent studies identified a role for GAPDH in neuronal apoptosis. GAPDH gene expression was specifically increased during programmed neuronal cell death. Transfection of neuronal cells with antisense GAPDH sequences inhibited apoptosis. Lastly, GAPDH may be directly involved in the cellular phenotype of human neurodegenerative disorders, especially those characterized at the molecular level by the expansion of CAG repeats. In this review, the current status of ongoing GAPDH studies are described (with the exception of its unique oxidative modification by nitric oxide). Consideration of future directions are suggested.

Animals↗
PubMed2002

Methionine in and out of proteins: targets for drug design.

The increasing need for new antibiotics to overcome rapidly developing resistance mechanisms observed in clinical isolates of Gram-positive and Gram-negative eubacteria has placed critical emphasis on the search for new antibacterial enzyme targets and the structural and mechanistic investigation of such targets. Among these potential targets, the enzymes responsible for integrating the amino acid methionine into proteins, along with its subsequent post-translational modification and repair, have emerged as promising candidates for the development of novel antibiotics. As well, there is increasing evidence for the importance of several of these enzymes in the development of anti-cancer, anti-parasitic, and anti-atherosclerotic drugs. Within the last three years, the crystal structures of all of these enzymes have been determined, which offers an unprecedented source of structural information for inhibitor design. The development of combinatorial chemistry and high throughput screening procedures has quickly provided several potent, specific inhibitors for a number of these enzymes, particularly the peptide deformylase, methionine aminopeptidase, and methionyl-tRNA synthetase enzymes. This review critically analyzes the future potential for inhibition of enzymes in this pathway, allowing for a pragmatic view of the success of inhibitor developments and highlighting areas in which further investigations are warranted.

Amidohydrolases↗
PubMed1994

The Escherichia coli prr region encodes a functional type IC DNA restriction system closely integrated with an anticodon nuclease gene.

The prr locus was originally described as coding a ribonuclease that is activated after phage T4 infection to cut within the anticodon of a specific tRNA, inactivating protein synthesis and thus blocking phage development. Wild-type T4 phage has two genes coding the enzymes polynucleotide kinase and RNA ligase, whose only function seems to be to repair the damage done by the anticodon nuclease. As the only apparent function of the prr ribonuclease is to combat phage infection, it can be considered as an RNA-based restriction enzyme. In non-infected cells, the prr enzyme is kept inactive in a complex with three other proteins which were predicted on the basis of DNA homologies to be the subunits of a type IC DNA restriction and modification system. Unlike other type IC systems so far characterized, prr is chromosomally rather than plasmid coded. However, sequences upstream from prr also have homology with sequences from the plasmid R124 and the prophage P1. We have now investigated the prr system and shown that it is indeed a bona fide type IC system which we call EcoprrI, and which is active both in vivo and in vitro. The system is fully functional even in the absence of the anticodon nuclease and seems to be a typical type I enzyme. EcoprrI recognizes the sequence CCA(N7)RTGC. One peculiarity is that, with low efficiency, EcoprrI will recognize and methylate variants of its recognition sequence such as CCT(N7)ATGC, which is methylated in one strand of the DNA only.

Amino Acid Sequence↗
PubMed2004

Functional characterization of 2'-O-methylation and pseudouridylation guide RNAs.

Pseudouridines and 2'-O-methylated nucleotides are ubiquitous constituents of stable cellular RNAs. In eukaryotes, posttranscriptional synthesis of most pseudouridines and 2'-O-methylated nucleotides is directed by sequence-specific guide RNAs (gRNAs). In recent years, an enormous number of novel putative modification gRNAs have been identified in a broad variety of organisms by using bioinformatics and large-scale cDNA-sequencing approaches. Understanding of the function of the novel modification gRNAs as well as the functional importance of modified nucleotides requires techniques that support the site-specific detection of 2'-O-methylated nucleotides and pseudouridines. Here, we describe rapid, reverse transcription-based methods to map 2'-O-methylated nucleotides and pseudouridines in any cellular RNA.

DNA Primers↗
PubMed1997

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution.

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.

Base Sequence↗
PubMed1984

Characterisation and translation studies of messenger RNA from the human malaria parasite Plasmodium falciparum and construction of a cDNA library.

RNA was isolated from trophozoites, schizonts and mixed populations of Plasmodium falciparum. 5% of the total was poly(A+) message, of average length 1.2 kb (10-12 kb maximum) and a poly(A) content of 10%. The mRNA fractions could be translated in vitro by reticulocyte lysates supplemented either with exogenous or P. falciparum tRNA. The patterns from two independent isolates, one cloned (T9-94) and one uncloned (K1) were virtually identical. Major translation products from 16-230 kDa have been measured. The most abundant is lactate dehydrogenase (34.8 kDa). Trophozoite mRNA codes principally for proteins of less than or equal to 93 kDa, while schizont mRNA codes for additional proteins of higher molecular mass. There are marked similarities between the in vitro translation products and proteins synthesised in vivo in synchronous cultures. A number of schizont mRNA translation products (principally those of 230, 203, 185, 170, 115, 101 and 71 kDa) are specifically precipitated without post-translational modification by sera from humans exposed to malaria. A cDNA library has been constructed in phage lambda from total poly(A+) RNA and partially characterised. About 10% of the clones derive from abundant mRNA sequences. Putative actin clones have been isolated from this library and the parasite actin mRNA sized at approx. 2.8 kb.

Actins↗
PubMed1994

Proximal and distal effects of sequence context on ultraviolet mutational hotspots in a shuttle vector replicated in xeroderma cells.

Hotspots are a standard feature of mutational spectra induced by mutagens in a variety of marker genes. While it is generally believed that sequence context exerts an important influence on hotspot location, direct experimental evidence is quite limited. We have studied ultraviolet mutagenesis in a suppressor tRNA marker gene (supF) carried in a mammalian shuttle vector and replicated in Xeroderma pigmentosum cells in culture. We have now constructed a small family of functional variant suppressor tRNA marker gene which differ from one another by one or two nucleotide changes. UV mutational spectra were generated for each variant gene. We found that the change of a dipyrimidine from 5' TC to 5' CC eliminated a strong mutational hotspot. In addition a single base change in the supF gene was accompanied by the appearance of a new hotspot eight bases away. Finally, another single base change suppressed a major hotspot 48 bases away. Polymerase stop assays on the UV modified marker genes were used to measure the frequency and distribution of photoproducts. The differences in hotspot patterns could not be explained by differences in modification patterns. These results indicate that a change in sequence context can directly influence the probability of mutagenesis at specific sites.

Base Sequence↗
PubMed1996

Metabolism of pre-messenger RNA splicing cofactors: modification of U6 RNA is dependent on its interaction with U4 RNA.

The requirements for the formation of pseudouridine (psi) in U4 and U6 RNAs, cofactors in the splicing of pre-messenger RNA, were investigated in vitro using HeLa nuclear (NE) and cytoplasmic (S100) extracts. Maximal psi formation for both RNAs was extract order-dependent. Maximal psi formation in U4 RNA required incubation in S100 followed by the addition of NE, paralleling the in vivo maturation pathway of U4 RNA. In contrast, maximal formation of psi in U6 RNA required incubation in NE followed by the addition of S100 extract. Since U6 RNA does not exit the nucleus in vivo the contribution of S100 was investigated. In experiments where the extracts were treated with micrococcal nuclease to digest endogenous snRNAs, the efficient formation of psi in U6 RNA was dependent on the presence of U4 RNA, but not in U5 RNA or tRNA. When mutant U4 RNAs that inhibit or strengthen the interaction between U4 RNA, and U6 RNA were substituted for wild-type U4 RNA, the results confirmed the need for the interaction between these two RNAs for psi formation in U6 RNA. U6 RNA isolated from glycerol gradients after incubation in extracts had four times as much psi when associated with U4 RNA.

Base Sequence↗