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At least 1,225 records · Page 68Linked to original sources
PubMed2005

Enhancement of the in vitro transcription by T7 RNA polymerase of short DNA templates containing oxidative thymine lesions.

5-(Hydroxymethyl)uracil, 5-formyluracil or 5-carboxyuracil can be formed in DNA upon exposure to ionizing radiations. We report the effect of the latter oxidative lesions on the in vitro transcription by T7 RNA polymerase using a single-stranded short model template. Kinetic experiments showed that the transcription yield was increased with the modified template. Due to the unforeseen presence of a hairpin structure within the transcribed RNA, we postulated that we are in the presence of a T7 RNA polymerase pausing signal and that the lesions within this critical DNA structure could interfere with normal regulation of RNA transcription.

Bacteriophage T7↗
PubMed2006

Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation.

The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373-13384], a new region between L amino acids 1450-1481 was identified which is critical for mRNA cap methylation.

Amino Acid Sequence↗
PubMed2003

Sequence dependence of substrate recognition and cleavage by yeast RNase III.

Yeast Rnt1p is a member of the double-stranded RNA (dsRNA) specific RNase III family of endoribonucleases involved in RNA processing and RNA interference (RNAi). Unlike other RNase III enzymes, which recognize a variety of RNA duplexes, Rnt1p cleaves specifically RNA stems capped with the conserved AGNN tetraloop. This unusual substrate specificity challenges the established dogma for substrate selection by RNase III and questions the dsRNA contribution to recognition by Rnt1p. Here we show that the dsRNA sequence adjacent to the tetraloop regulates Rnt1p cleavage by interfering with RNA binding. In context, sequences surrounding the cleavage site directly influence the cleavage efficiency. Introduction of sequences that stabilize the RNA helix enhanced binding while reducing the turnover rate indicating that, unlike the tetraloop, Rnt1p binding to the dsRNA helix may become rate-limiting. These results suggest that Rnt1p activity is strictly regulated by a combination of primary and tertiary structural elements allowing a substrate-specific binding and cleavage efficiency.

Base Sequence↗
PubMed1996

Chronic ethanol consumption increases the amount of mRNA for retinoic acid and triiodothyronine receptors in mouse brain.

It is known that alcohol induces disorders in the metabolism of retinoids and particularly in the biosynthetic pathways of retinoic acid (RA). Since RA has, along with other hormones and particularly triiodothyronine (T3), a physiological role in the adult brain, the effect of chronic exposure to alcohol on RA and T3 status was investigated. The amounts of RA receptor (RAR) and T3 receptor (TR) mRNAs were quantified and the activity of the 'tissue' transglutaminase (tTG; an RA-dependent enzyme) was assayed in the brain of mice following chronic ethanol consumption (CEC; 12% v/v for 6-10 months). Compared to controls, ethanol-treated mice exhibited increased amounts of RAR and TR mRNAs together with an increase in tTG activity. It is hypothesized that the enhanced cellular action of RA and T3 could play a role in the previously described brain damages induced by CEC.

Alcoholism↗
PubMed1996

High intrinsic rate of DNA loss in Drosophila.

Pseudogenes are common in mammals but virtually absent in Drosophila. All putative Drosophila pseudogenes show patterns of molecular evolution that are inconsistent with the lack of functional constraints. The absence of bona fide pseudogenes is not only puzzling, it also hampers attempts to estimate rates and patterns of neutral DNA change. The estimation problem is especially acute in the case of deletions and insertions, which are likely to have large effects when they occur in functional genes and are therefore subject to strong purifying selection. We propose a solution to this problem by taking advantage of the propensity of retrotransposable elements without long terminal repeats (non-LTR) to create non-functional, 'dead-on-arrival' copies of themselves as a common by-product of their transpositional cycle. Phylogenetic analysis of a non-LTR element, Helena, demonstrates that copies lose DNA at an unusually high rate, suggesting that lack of pseudogenes in Drosophila is the product of rampant deletion of DNA in unconstrained regions. This finding has important implications for the study of genome evolution in general and the 'C-value paradox' in particular.

Animals↗
PubMed2003

Retroelements containing introns in diverse invertebrate taxa.

We report that two structurally similar transposable elements containing reverse transcriptase (RT), Penelope in Drosophila virilis and Athena in bdelloid rotifers, have proliferated as copies containing introns. The ability of Penelope-like elements (PLEs) to retain introns, their separate phylogenetic placement and their peculiar structural features make them a novel class of eukaryotic retroelements.

Amino Acid Sequence↗
PubMed2002

Gamera, a family of LINE-like repetitive sequences widely distributed in medaka and related fishes.

A family of repetitive sequences, designated Gamera, has been identified in the genome of the Hainan medaka fish Oryzias curvinotus, a closely related species to the common medaka fish O. latipes. Sequencing and Southern blot analyses of this family revealed: (1) amino acid sequence similarity to reverse transcriptase domains of long interspersed nuclear elements (LINEs); (2) 5' truncation of dispersed copies; and (3) the disruption of another genetic element, indicating a past transposition event. These results suggest that Gamera belongs to the LINE superfamily. Gamera is widely distributed in the genus Oryzias, and the phylogenetic relationship might indicate its presence in the common ancestor of the genus.

Amino Acid Sequence↗
PubMed2002

Molecular cloning, expression and characterization of a novel class glutathione S-transferase from the fungus Cunninghamella elegans.

The structural gene for glutathione S-transferase (CeGST1-1) in the fungus Cunninghamella elegans was cloned by screening a cDNA library using a degenerate oligonucleotide probe based on the N-terminal sequence of the purified protein. Open reading frame analysis indicated that the cegst1 gene encodes a protein of 210 amino acid residues. The deduced amino acid sequence showed 25% sequence identity with the sequence of the Pi-class GST from Danio rerio (zebrafish). Similarity was also shown with the Alpha-class GST from Fasciola hepatica (liver fluke; 23% identity), the Mu class from Mus musculus (22%) and the Sigma class from Ommastrephes sloani (squid; 21%). Further screening of a cDNA library with the cegst1 gene probe revealed the presence of another GST isoenzyme (CeGST2-2) in this fungus, which shows 84% sequence identity with CeGST1-1 at the amino acid level. Reverse transcription PCR revealed that cegst2 was also expressed at the mRNA level in the fungus C. elegans. Both cegst genes were overexpressed in Escherichia coli using the expression vector pQE51, displaying specific activities with 1-chloro-2,4-dinitrobenzene of 2.04 and 0.75 micromol/min per mg of protein respectively. Both enzymes exhibited a similar substrate specificity and inhibition profile, indicating that CeGST1-1 and CeGST2-2 belong to the same GST class. Mutagenesis analysis revealed that Tyr(10) in the N-terminal region is essential for catalysis of CeGST1-1. We propose from these results that the CeGSTs are novel Gamma-class GSTs and designated as GSTG1-1 and GSTG2-2 respectively.

Amino Acid Sequence↗
PubMed1999

cDNA cloning, expression, and mutagenesis study of liver-type prostaglandin F synthase.

Prostaglandin (PG) F synthase catalyzes the reduction of PGD2 to 9alpha,11beta-PGF2 and that of PGH2 to PGF2alpha on the same molecule. PGF synthase has at least two isoforms, the lung-type enzyme (Km value of 120 microM for PGD2 (Watanabe, K., Yoshida, R., Shimizu, T., and Hayaishi, O. (1985) J. Biol. Chem. 260, 7035-7041) and the liver-type one (Km value of 10 microM for PGD2 (Chen, L. -Y., Watanabe, K., and Hayaishi, O. (1992) Arch. Biochem. Biophys. 296, 17-26)). The liver-type enzyme was presently found to consist of a 969-base pair open reading frame coding for a 323-amino acid polypeptide with a Mr of 36,742. Sequence analysis indicated that the bovine liver PGF synthase had 87, 79, 77, and 76% identity with the bovine lung PGF synthase and human liver dihydrodiol dehydrogenase (DD) isozymes DD1, DD2, and DD4, respectively. Moreover, the amino acid sequence of the liver-type PGF synthase was identical with that of bovine liver DD3. The liver-type PGF synthase was expressed in COS-7 cells, and its recombinant enzyme had almost the same properties as the native enzyme. Furthermore, to investigate the nature of catalysis and/or substrate binding of PGF synthase, we constructed and characterized various mutant enzymes as follows: R27E, R91Q, H170C, R223L, K225S, S301R, and N306Y. Although the reductase activities toward PGH2 and phenanthrenequinone (PQ) of almost all mutants were not inactivated, the Km values of R27E, R91Q, H170C, R223L, and N306Y for PGD2 were increased from 15 to 110, 145, 75, 180, and 100 microM, respectively, indicating that Arg27, Arg91, His170, Arg223, and Asn306 are essential to give a low Km value for PGD2 of the liver-type PGF synthase and that these amino acid residues serve in the binding of PGD2. Moreover, the R223L mutant among these seven mutants especially has a profound effect on kcat for PGD2 reduction. The Km values of R223L, K225S, and S301R for PQ were about 2-10-fold lower than the wild-type value, indicating that the amino acid residues at 223, 225 and 301 serve in the binding of PQ to the enzyme. On the other hand, the Km value of H170C for PGH2 was 8-fold lower than that of the wild type, indicating that the amino acid residue at 170 is related to the binding of PGH2 to the enzyme and that Cys170 confer high affinity for PGH2. Additionally, the 5-fold increase in kcat/Km value of the N306Y mutant for PGH2 compared with the wild-type value suggests that the amino acid at 306 plays an important role in catalytic efficiency for PGH2.

Amino Acid Sequence↗
PubMed2000

Coupling ribose selection to fidelity of DNA synthesis. The role of Tyr-115 of human immunodeficiency virus type 1 reverse transcriptase.

The catalytic efficiency of incorporation of deoxyribonucleotides by wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was around 100-fold higher than for dideoxyribonucleotides, in Mg(2+)-catalyzed reactions, and more than 10,000-fold higher than for nucleotides having a 2'-hydroxyl group in Mg(2+)- and Mn(2+)-catalyzed reactions. Mutant RTs with nonconservative substitutions affecting Tyr-115 (Y115V, Y115A, and Y115G) showed a dramatic reduction in their ability to discriminate against ribonucleotides in the presence of both cations. However, selectivity of deoxyribonucleotides versus ribonucleotides was not affected in mutants Y115W and F160W. The substitution of Tyr-115 with Val or Gly had no effect on discrimination against dideoxyribonucleotides, but these mutants were less efficient than the wild-type RT in discriminating against cordycepin 5'-triphosphate. We also show that Tyr-115 is involved in fidelity of DNA synthesis, but substitutions at this position have different effects depending on the metal cofactor used in the assays. In Mg(2+)-catalyzed reactions, removal of the side chain of Tyr-115 reduced misinsertion and mispair extension fidelity, while opposite effects were observed in Mn(2+)-catalyzed reactions. Our results indicate that the aromatic side chain of Tyr-115 plays a role as a "steric gate" preventing the incorporation of nucleotides with a 2'-hydroxyl group in a cation-independent manner, while its influence on fidelity could be modulated by Mg(2+) or Mn(2+).

Amino Acid Sequence↗
PubMed1991

A superfamily of Arabidopsis thaliana retrotransposons.

We describe a superfamily of Arabidopsis thaliana retrotransposable elements that consists of at least ten related families designated Ta1-Ta10. The Ta1 family has been described previously. Two genomic clones representing the Ta2 and Ta3 elements were isolated from an A. thaliana (race Landsberg erecta) lambda library using sequences derived from the reverse transcriptase region of Ta1 as hybridization probes. Nucleotide sequence analysis showed that the Ta1, Ta2 and Ta3 families share greater than 75% amino acid identity in pairwise comparisons of their reverse transcriptase and RNase H genes. In addition to Ta1, Ta2 and Ta3, we identified seven other related retrotransposon families in Landsberg erecta, Ta4-Ta10, using degenerate primers and the polymerase chain reaction to amplify a highly conserved region of retrotransposon-encoded reverse transcriptase. One to two copies of elements Ta2-Ta10 are present in the genomes of the A. thaliana races Landsberg erecta and Columbia indicating that the superfamily comprises at least 0.1% of the A. thaliana genome. The nucleotide sequences of the reverse transcriptase regions of the ten element families place them in the category of copia-like retrotransposons and phylogenetic analysis of the amino acid sequences suggests that horizontal transfer may have played a role in their evolution.

Amino Acid Sequence↗
PubMed1988

Primary structure and functional organization of Drosophila 1731 retrotransposon.

We have determined the nucleotide sequence of the Drosophila retrotransposon 1731. 1731 is 4648 bp long and is flanked by 336 bp terminal repeats (LTRs) previously described as being reminiscent of provirus LTRs. The 1731 genome consists of two long open reading frames (ORFs 1 and 2) which slightly overlap each other. The ORF 1 and 2 present similarities with retroviral gag and pol genes respectively as shown by computer analysis. The pol gene exhibits several enzymatic activities in the following order: protease, endonuclease and reverse transcriptase. It is possible that 1731 also encompasses a ribonuclease H activity located between the endonuclease and reverse transcriptase domains. Moreover, comparison of the 1731 pol gene with the pol region of copia shows similarities extending over the protease, endonuclease and reverse transcriptase domains. We show that codon usage in the two retrotransposons is different. Finally, no ORF able to encode an env gene is detected in 1731.

Animals↗
PubMed1999

The age and evolution of non-LTR retrotransposable elements.

A comprehensive phylogenetic analysis was conducted of non-long-terminal-repeat (non-LTR) retrotransposons based on an extended sequence alignment of their reverse transcriptase (RT) domain. The 440 amino acid positions used included a region proposed to be similar to the "thumb" of the right-handed RT structure found in retroviruses. All identified non-LTR elements could be grouped into 11 distinct clades. Using the rates of sequence change derived from studies of the vertical inheritance of R1 and R2 elements in arthropods as a comparison, we found no evidence for the horizontal transmission of non-LTR elements. Assuming vertical descent, the phylogeny suggested that non-LTR elements are as old as eukaryotes, with each of the 11 clades dating back to the Precambrian era. The analysis enabled us to propose a simple chronology for the acquisition of different enzymatic domains in the evolution of the non-LTR class of retrotransposons. The first non-LTR elements were sequence specific by virtue of a restriction-enzyme-like endonuclease located downstream of the RT domain. Evolving from this original group were elements (eight clades) that acquired an apurinic-apyrimidic endonuclease-like domain upstream of the RT domain. Finally, four of these clades have inherited an RNase H domain downstream of the RT domain. The phylogenies of the AP endonuclease and RNase H domains were also determined for this report and are consistent with the monophyletic acquisition of these domains. These studies represent the most comprehensive effort to date to trace the evolution of a major class of transposable elements.

Amino Acid Sequence↗
PubMed1993

Phylogenetic relationships of reverse transcriptase and RNase H sequences and aspects of genome structure in the gypsy group of retrotransposons.

The gypsy group of long-terminal-repeat retrotransposons contains elements having the same order of enzyme domains in the pol gene as do retroviruses. Elements in the gypsy group are now known from yeast, filamentous fungi, plants, insects, and echinoids. Reverse transcriptase and RNase H amino acid sequences from elements in the gypsy group--including the recently described SURL elements, TED, Cft1, and Ulysses,--were aligned and analyzed by using parsimony and bootstrapping methods, with plant caulimoviruses and/or retroviruses as outgroups. Clades supported at the 95% level after bootstrapping include (1) 17.6 with 297 and (2) all of the SURL elements together. Other likely relationships supported at lower bootstrap confidence intervals include (1) SURL elements with mag, (2) 17.6 and 297 with TED, and this collective group with 412 and gypsy, (3) Tf1 with Cft1, (4) IFG7 with Del, and (5) all of the retrotransposons in the gypsy group together, to the exclusion of Ty3. In contrast with an earlier analysis, our results place mag within the gypsy group rather than outside of a cluster that contains gypsy group retrotransposons and plant caulimoviruses. Several features of retrotransposon genomes provide further support for some of the aforementioned relationships. The union of SURL elements with mag is supported by the presence of two RNA binding sites in the nucleocapsid protein. Location of the tRNA primer binding site and the presence of a long open reading frame 3' to the pol gene support the 17.6-297-TED-412-gypsy cluster.

Amino Acid Sequence↗
PubMed2003

Molecular epidemiology of HIV-1 in Venezuela: high prevalence of HIV-1 subtype B and identification of a B/F recombinant infection.

The authors assessed HIV-1 variability in two distinct areas of Venezuela (the capital Caracas and Margarita Island) through the analysis of blood specimens and clinical and epidemiologic data of 72 persons. Proviral DNA was evaluated through heteroduplex mobility assay (HMA) based on the envelope region. Additionally, FOK I restriction enzyme digestion assay was performed in all subtype B ED31/33 amplified products to check the presence of the typical Brazilian subtype B GWGR variant. Sequencing and phylogenetic analysis for C2-V3 region of gp120 was performed in selected cases. The vast majority of samples were found to belong to subtype B, with a North American/European RFLP profile. An F subtype HIV-1 based on the region was identified for the first time in Venezuela. Genetic analyses of the protease and reverse transcriptase fragments of this sample depicted a recombinant B/F genetic profile. The discrimination capacity of HIV-1 subtypes using the primer set ED3/14-ED31/33 among the Caracas samples was found to be higher than for those from Margarita Island. The authors' results point to relevant differences between the samples of continental and Caribbean regions of Venezuela, requiring further evaluation of larger samples.

Adolescent↗
PubMed1997

Analysis of the sequence diversity of the P1, HC, P3, NIb and CP genomic regions of several yam mosaic potyvirus isolates: implications for the intraspecies molecular diversity of potyviruses.

Partial sequences from serologically characterized yam mosaic potyvirus (YMV) isolates were determined in conserved (helper-component proteinase, HC; nuclear inclusion b, NIb) and variable (first protein, P1; third protein, P3; and coat protein, CP) regions of the potyviral genome in order to investigate the intraspecies molecular diversity of YMV. Multiple sequence alignments and pairwise comparisons were used to quantify the sequence polymorphism in these regions. Two levels of diversity were observed among YMV isolates: above 90% nucleotide (nt) sequence identities were found between YMV isolates of the same group (intragroup) regardless of the region considered, whereas identities between isolates from different groups (intergroup) were lower and depended upon the protein chosen. For instance, the average intergroup nt sequence identity between YMV isolates was about 65% in the P1 protein and the N terminus of the CP while there was more than 80% nt identity in the HC, P3 and NIb proteins. Thus P3 appeared to be conserved between YMV isolates even though this region was variable between potyvirus species. Similar analysis of the intraspecies molecular diversity of other potyviruses (potato virus Y, zucchini yellow mosaic virus, plum pox virus, pea seed-borne mosaic virus) led to the same results: (i) two levels of intraspecies molecular diversity were found (intragroup and intergroup); (ii) intraspecies molecular diversity differed from interspecies molecular diversity in the P3, P1 and N-terminal regions.

Amino Acid Sequence↗
PubMed1987

Human endogenous retroviruslike genome with type C pol sequences and gag sequences related to human T-cell lymphotropic viruses.

We have cloned several prototypic members of the family of human endogenous retroviruslike elements having a histidine tRNA primer-binding site (RTVL-H) and have determined the nucleotide sequence of one of these clones (RTVL-H2). The RTVL-H2 sequence is 5,813 nucleotides long, with long terminal repeats of 450 nucleotides. Although this particular sequence contains no long open reading frames, computer searches have revealed several segments of amino acid homology with known retroviral gene products. In the gag region of RTVL-H2, there is a segment with significant homology to a region of the gag protein p30 of type C baboon endogenous virus. In the pol region of RTVL-H2, three segments similar to the Moloney leukemia virus (MLV) pol polyprotein were detected. These correspond to parts of the protease, reverse transcriptase, and endonuclease domains of the MLV pol gene. Interestingly, the last two pol domains are equidistant in RTVL-H2 and the type C murine retroviruslike DNA sequence (MuRRS), both having deletions of equal sizes relative to the MLV pol gene. One other segment similar to a retroviral gene product was identified in the RTVL-H2 gag region. This segment has 55 to 60% amino acid homology to a 50-amino-acid region of the gag nucleic acid-binding proteins encoded by human T-cell lymphotropic viruses types I and II and bovine leukemia virus. Thus, the RTVL-H2 genome harbors sequences related to evolutionarily distant retroviruses.

Amino Acid Sequence↗