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At least 991 records · Page 55Linked to original sources
PubMed1994

Three mitochondrial unassigned open reading frames of Podospora anserina represent remnants of a viral-type RNA polymerase gene.

The mitochondrial DNA of Podospora anserina is complex, consisting of a characteristic set of genes with a large number of introns and a substantial amount of sequence of unknown function and origin. In addition, as indicated by various types of reorganization, this genome is highly flexible. Here we report the identification of three unassigned mitochondrial open reading frames (ORF P', ORF Q', ORF 11) as remnants of a rearranged viral-type RNA polymerase gene. These ORFs are not transcribed and may be derived from the integration of a linear plasmid of the type recently identified in a mutant of P. anserina.

Amino Acid Sequence↗
PubMed1999

Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules.

Single molecules of DNA or RNA can be detected as they are driven through an alpha-hemolysin channel by an applied electric field. During translocation, nucleotides within the polynucleotide must pass through the channel pore in sequential, single-file order because the limiting diameter of the pore can accommodate only one strand of DNA or RNA at a time. Here we demonstrate that this nanopore behaves as a detector that can rapidly discriminate between pyrimidine and purine segments along an RNA molecule. Nanopore detection and characterization of single molecules represent a new method for directly reading information encoded in linear polymers, and are critical first steps toward direct sequencing of individual DNA and RNA molecules.

Bacterial Toxins↗
PubMed1991

A C-nucleotide base pair: methylpseudouridine-directed incorporation of formycin triphosphate into RNA catalyzed by T7 RNA polymerase.

With templates containing 2'-deoxy-1-methylpseudouridine (dm psi), T7 RNA polymerase catalyzes the incorporation of either adenosine triphosphate (ATP) or formycin triphosphate (FTP) into a growing chain of RNA with the same efficiency as with templates containing thymidine (dT). In each case, the overall rate of synthesis of full-length products containing formycin is about one-tenth of the rate of synthesis of analogous products containing adenosine. Analysis of the products of abortive initiation shows that incorporation of FMP into the growing oligonucleotide by T7 RNA polymerase is more likely to lead to premature termination of transcription than is incorporation of AMP. Nevertheless, the results demonstrate that T7 RNA polymerase tolerates the formation of a C-nucleotide transcription complex in which the nucleoside bases on both the template and the incoming nucleotide are joined to the ribose by a carbon-carbon bond. This result increases the prospects for further expanding the genetic alphabet via incorporation of new base pairs with novel hydrogen-bonding schemes (Piccirilli et al., 1990).

Adenosine Triphosphate↗
PubMed1991

A transposition of the reverse transcriptase gene reveals unexpected structural homology to E. coli DNA polymerase I.

The rational design of antiviral agents targeting the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) would greatly benefit from a more intimate knowledge of the structure of RT. Until now, the degree of sequence similarity between RT and E. coli DNA polymerase I (Pol I) has been thought to be confined to several small regions, suggesting little basis for homology molecular modeling. However, we have found that a region in the C terminal of the RT polymerase domain is homologous to a central region of Pol I that lies between the universal polymerase motifs A and C (specifically, helices N-O-P of the Pol I crystal structure); a single transposition closely aligns the RT and Pol I genes, revealing a similar domain structure with 20% residue identity, as well as the possible structural correlates of several RNA-dependent polymerase motifs. The RT from Myxococcus xanthus (a bacterium believed to have diverged from other species 2 billion years ago), if similarly transposed, shows homology to both HIV-1 and E. coli, suggesting the possibility of a very ancient divergence between the RT and Pol I polymerase genes. A second even more significant match to this E. coli region was found in the retroviral ribonuclease H (RNase H) domain, and corresponds precisely to a region that has been aligned by previous investigators with the E. coli RNase H, suggesting that Pol I helices O and P are homologous to helices A and D of the RNase H crystal structure, respectively. These results are consistent with a modular theory of molecular evolution.

Amino Acid Sequence↗
PubMed1989

The intron of a plastid gene from a green alga contains an open reading frame for a reverse transcriptase-like enzyme.

Plastid (pt) and mitochondrial (mt) DNA were isolated from the unicellular green alga KS3/2, which is presumed to be a species of the genera Ankistrodesmus or Monoraphidium. The DNA species are characterized by their different densities (pt, 1.685 g/ml; mt, 1.695 g/ml), individual restriction patterns, and their respective sizes of 130 and 47 kb. Using an intronic sequence from fungal mitochondria as a hybridization probe, intron sequences from mtDNA and from ptDNA were identified. DNA sequencing of the cloned ptDNA intron revealed that the plastid gene for subunit IV of the cytochrome b6/f petD complex is interrupted by a group II intron of unusual length (3533 bp). This was confirmed by transcript analysis. The intron encodes an open reading frame (ORF) which shows significant homology with reverse transcriptase genes from various genetic elements. This discovery is unique for plastomes and indicates that introns, retrotransposons, insertion elements and retroviruses may have a common evolutionary origin.

Amino Acid Sequence↗
PubMed2006

Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples.

Long RT-PCR (LRP) amplification of RNA templates is sometimes difficult compared to long PCR of DNA templates. Among RNA templates, hepatitis C virus (HCV) represents an excellent example to challenge the potential of LRP technology due to its extensive secondary structures and its difficulty to be readily cultured in vitro. The only source for viral genome amplification is clinical samples in which HCV is usually present at low titers. We have created a comprehensive optimization protocol that allows robust amplification of a 9.1 kb fragment of HCV, followed by efficient cloning into a novel vector. Detailed analyses indicate the lack of potential LRP-mediated recombination and the preservation of viral diversity. Thus, our LRP protocol could be applied for the amplification of other difficult RNA templates and may facilitate RNA virus research such as linked viral mutations and reverse genetics.

Amino Acid Sequence↗
PubMed2003

The detection of recombinant, tuber necrosing isolates of Potato virus Y (PVY(NTN)) using a three-primer PCR based in the coat protein gene.

A simple and reliable procedure for reverse transcription-polymerase chain reaction (RT-PCR) detection and strain differentiation of Potato virus Y (PVY) was developed. Three primers were designed within the coat protein (CP) and nuclear inclusion protein b (NIb) region, exploiting a single base polymorphism identified as being present in all the recombinant PVY(NTN) isolates published. Samples infected with PVY produce a single band of 569 bp, while isolates belonging to PVY(NTN) strain give an additional band of 334 bp. The technique was tested on a collection of well-characterised isolates of PVY from a range of strains and was found to detect all of the isolates reported as belonging to the PVY(NTN) strain. All of the isolates detected possess a recombination event within the coat protein. Further sequence analysis revealed that all the recombinant PVY(NTN) isolates reported thus far would be detected using this assay, whilst isolates thought to be PVY(NTN) that do not possess the coat protein recombination event would not be detected.

Base Sequence↗
PubMed1997

Expression of amphiregulin in the sheep mammary gland.

The reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify, from sheep mammary gland total RNA, a 280 bp sequence of amphiregulin cDNA. Cloned and sequenced, it corresponded to the 78 amino acids of the major secreted form of amphiregulin, showing 81, 70 and 69% identity with human, rat and mouse amphiregulin, respectively. Expression of amphiregulin was detected by RT-PCR in the mammary gland at several developmental stages (fetal, lamb, early and late pregnant and lactating ewes) and in isolated myoepithelial cells. By Western blotting with an antiserum to human amphiregulin, two molecular weight forms, 27 and 51 kDa were detected in sheep mammary gland microsomal preparations, in a mammary gland extract after heparin affinity chromatography and in a medium conditioned by mammary epithelial cells. By immunocytochemistry, amphiregulin was detected in the cytoplasm and nuclei of luminal epithelial cells, myoepithelial cells and in intralobular stroma. An autocrine/paracrine role in sheep mammary growth is indicated.

Amino Acid Sequence↗
PubMed2003

DNA interactions of monofunctional organometallic ruthenium(II) antitumor complexes in cell-free media.

Modifications of natural DNA in a cell-free medium by antitumor monodentate Ru(II) arene compounds of the general formula [(eta(6)-arene)Ru(en)Cl](+) (arene = biphenyl, dihydroanthracene, tetrahydroanthracene, p-cymene, or benzene; en = ethylenediamine) were studied by atomic absorption, melting behavior, transcription mapping, circular and linear dichroism, plasmid unwinding, competitive ethidium displacement, and differential pulse polarography. The results indicate that these complexes bind preferentially to guanine residues in double-helical DNA. The data are consistent with DNA binding of the complexes containing biphenyl, dihydroanthracene, or tetrahydroanthracene ligands that involves combined coordination to G N7 and noncovalent, hydrophobic interactions between the arene ligand and DNA, which may include arene intercalation and minor groove binding. In contrast, the single hydrocarbon rings in the p-cymene and benzene ruthenium complexes cannot interact with double-helical DNA by intercalation. Interestingly, the adducts of the complex containing p-cymene ligand, which has methyl and isopropyl substituents, distort the conformation and thermally destabilize double-helical DNA distinctly more than the adducts of the three multiring ruthenium arene compounds. It has been suggested that the different character of conformational alterations induced in DNA, and the resulting thermal destabilization, may affect differently further "downstream" effects of damaged DNA and consequently may result in different biological effects of this new class of metal-based antitumor compounds. The results point to a unique profile of DNA binding for Ru(II) arene compounds, suggesting that a search for new anticancer compounds based on this class of complexes may also lead to an altered profile of biological activity in comparison with that of metal-based antitumor drugs already used in the clinic or currently on clinical trials.

Animals↗
PubMed1998

sigA is an essential gene in Mycobacterium smegmatis.

sigA encodes a sigma factor of the sigma70 family, sigmaA, that is found in all mycobacterial species. As sigmaA shows high similarity to the primary sigma factor in Streptomyces coelicolor, it was postulated that sigmaA has the same role in mycobacteria. However, a point mutation in sigA, resulting in the replacement of arginine 522 by histidine, was found responsible for the attenuated virulence of the Mycobacterium bovis strain ATCC 35721. This raised the possibility that sigmaA was an alternative sigma factor specifically required for virulence gene expression. In this work, we show that sigA can not be disrupted in Mycobacterium smegmatis unless an extra copy of the gene is provided at another chromosomal site, which demonstrates that sigA is essential. To characterize the pattern of sigA expression during exponential and stationary phase in M. smegmatis, we measured the beta-galactosidase activity in a strain carrying a sigA-lacZ transcriptional fusion and monitored sigmaA levels using Western blotting. Our results indicate that sigA is expressed throughout the growth of the culture. The essential character of sigA and its pattern of expression corroborate the hypothesis that sigA codes for the primary sigma factor in M. smegmatis and, most likely, in all mycobacteria.

Base Sequence↗
PubMed1995

Catalytically critical nucleotide in domain 5 of a group II intron.

Domain 5 (D5) is a small hairpin structure within group II introns. A bimolecular assay system depends on binding by D5 to an intron substrate for self-splicing activity. In this study, mutations in D5 identify two among six nearly invariant nucleotides as being critical for 5' splice junction hydrolysis but unimportant for binding. A mutation at another site in D5 blocks binding. Thus, mutations can distinguish two D5 functions: substrate binding and catalysis. The secondary structure of D5 may resemble helix I formed by the U2 and U6 small nuclear RNAs in the eukaryotic spliceosome. Our results support a revision of the previously proposed correspondence between D5 and helix I on the basis of the critical trinucleotide 5'-AGC-3' present in both. We suggest that this trinucleotide plays a similar role in promoting the chemical reactions for both splicing systems.

Base Composition↗
PubMed2004

Synonymous genetic polymorphisms within Brazilian human immunodeficiency virus Type 1 subtypes may influence mutational routes to drug resistance.

BACKGROUND: Most published data on antiretroviral-drug resistance is generated from in vitro or in vivo studies of subtype B virus. However, this subtype is associated with <10% of HIV infections worldwide, and it is essential to explore subtype-specific determinants of drug resistance. One potential cause of the differences between subtypes is the synonymous codon usage at key resistance positions. METHODS: We investigated the nucleotide sequences at drug resistance-related sites, for all major Brazilian subtypes (B, C, and F1) of human immunodeficiency virus type 1 (HIV-1) group M. RESULTS: We identified a change at positions 151 and 210 of the reverse-transcriptase region in subtype F1, such that the emergence of these key nucleoside/nucleotide analogue resistance mutations required an extra nucleotide change in subtype F1, compared with subtypes B and C. The clinical significance of position 210 was confirmed within a large Brazilian database, in which we identified a lower prevalence of the L210W mutation in subtype F1 virus, compared with subtype B virus, in patients matched for thymidine-analogue experience. An inverse relationship between the L210W and K70R mutations was also observed. CONCLUSIONS: The findings of the present study illustrate an important mechanism by which a subtype may determine genetic routes to resistance, with implications for treatment strategies for populations infected with HIV-1 subtype F.

Base Sequence↗
PubMed1993

Reversion of a polymerase-defective integrated HIV-1 genome.

The 8E5 clonal cell line, derived from HIV-1-infected CEM cells, carries a single, reverse transcriptase (RT)-defective copy of an integrated HIV genome. The absence of RT production is a consequence of a frame shift in the pol gene, due to the addition of a single base at position 3241. We report here that 8E5 cells produce an infectious virus that can be serially passaged on CD4+ lymphoid cells. This virus (8E5R) is RT positive, but displays a slow replication profile, together with a reduced cytopathic effect. The nucleotide sequence of a segment of the pol region produced by PCR amplification of DNA from 8E5R-infected cells shows that the single nucleotide insertion characteristic of the 8E5 genome had been corrected. The same reversion event was also found to occur in most single-cell clones derived from the 8E5 cell line. Because this cell line is used in many laboratories, notably as a standard for PCR quantitation, and is generally considered as unable to produce infectious virus, our findings should prompt investigators to use particular care in the handling of these cells.

Base Sequence↗
PubMed1989

The mitochondrial plasmid from Neurospora intermedia strain Labelle-1b contains a long open reading frame with blocks of amino acids characteristic of reverse transcriptases and related proteins.

We have determined the DNA sequence of the 4070 base pair mitochondrial plasmid from the Labelle-1b strain of Neurospora intermedia. Analysis of the sequence revealed that the plasmid contains a long open reading frame (ORF) that could encode a protein of up to 1151 amino acids. Codon usage in the long ORF shows no clear relationship to Neurospora mitochondrial genes, nuclear genes, nor to the ORF of a different Neurospora mitochondrial plasmid. The long ORF contains regions of similarity to yeast mitochondrial RNA polymerase as well as blocks of amino acids that are characteristic of reverse transcriptases and the ORFs of certain group II mtDNA introns (Michel and Lang, (1985) Nature 316,641). The plasmid gives rise to specific transcripts, some of which may be unit length, and which carry the information for expression of the long ORF. The genetic organization and content of the plasmid suggest that it is related to mobile genetic elements.

Amino Acid Sequence↗
PubMed1994

The 'helix clamp' in HIV-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases.

Amino acid sequences homologous to 259KLVGKL (X)16KLLR284 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) are conserved in several nucleotide polymerizing enzymes. This amino acid motif has been identified in the crystal structure model as an element of the enzyme's nucleic acid binding apparatus. It is part of the helix-turn-helix structure, alpha H-turn-alpha I, within the 'thumb' region of HIV-1 RT. The motif grasps the complexed nucleic acid at one side. Molecular modeling studies on HIV-1 RT in complex with a nucleic acid fragment suggest that the motif has binding function in the p66 subunit as well as in the p51 subunit, acting as a kind of 'helix clamp'. Given its wide distribution within the nucleic acid polymerases, the helix clamp motif is assumed to be a structure of general significance for nucleic acid binding.

Algorithms↗
PubMed1995

Luteinizing hormone/chorionic gonadotropin bioactivity in the common marmoset (Callithrix jacchus) is due to a chorionic gonadotropin molecule with a structure intermediate between human chorionic gonadotropin and human luteinizing hormone.

Chorionic gonadotropin (CG), a pregnancy-specific heterodimeric hormone found in primates, is responsible for CL rescue with pregnancy maintenance. Of the primates, the human and baboon gene sequences are the only structures so far determined. In order to study the structure and function of CG in other primates, we have isolated and sequenced the coding regions for the two subunits of marmoset CG (mCG) by the reverse transcription/polymerase chain reaction method. Study of multiple clones confirmed a high degree of homology with the human sequences (88% and 80% for the alpha and beta nucleotide sequences, respectively). Marmoset CG alpha has an extra four amino acids compared to hCG alpha, whereas the mCG beta sequence has a 3-bp deletion that maintains the reading frame and C-terminal amino acid sequence. Most of the differences between hCG beta and mCG beta peptides occur in the C-terminal region, which includes the loss of two of the O-linked glycosylation consensus sequences and the presence of an N-linked glycosylation consensus sequence. When mCG alpha and beta were co-expressed in CHO cells, assembly of biologically active hormone was confirmed by induced steroid secretion by MA10 cells. Partially purified mCG beta was used to raise anti-mCG antibodies. To date, an antibody has been obtained that is capable of detecting recombinant mCG beta, recombinant mCG dimer, and mCG dimer secreted by cultured marmoset trophoblast. Marmoset CG alpha and beta were also detectable at the transcriptional level in cultured trophoblast by in situ hybridization. This suggests that the LH/CG bioactivity reported from marmoset placentae and embryos is due to a molecule with structural features common to hLH (glycosylation pattern) and hCG (CG beta C-terminal structure).

Amino Acid Sequence↗