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L Simpson

Publications and source records attributed to L Simpson.

At least 73 records · Page 4Linked to original sources
PubMed1997

The genomic organization of guide RNA genes in kinetoplastid protozoa: several conundrums and their solutions.

The guide RNA (gRNA) paradigm states that the uridine (U) insertion/deletion type of RNA editing is mediated by short 3' uridylylated gRNAs that are complementary to specific blocks of mature edited sequence. These gRNAs contain the edited sequence information in the form of guiding purine residues that can base pair with the inserted U's and do not base pair with encoded U's that are to be deleted. The minicircle gRNA genes in trypanosomatids are localized at specific sites within the variable region, with the number and the precise localization of genes also being species-specific. The total number of minicircle sequence classes and thereby minicircle-encoded gRNAs varies greatly between species and even between different strains of the same species, with the greatest number being in the trypanosome species. Several conundrums which appeared to raise problems for the gRNA paradigm arose during comparative analysis of minicircle gRNA gene organization. The solution of these conundrums has led to a better understanding of the function and evolution of this RNA modification phenomenon.

Animals↗
PubMed1997

The mitochondrion in dividing Leishmania tarentolae cells is symmetric and circular and becomes a single asymmetric tubule in non-dividing cells due to division of the kinetoplast portion.

Kinetoplastid protozoa have a single mitochondrion that extends throughout the cell. The disk-shaped portion of the mitochondrion adjacent to the basal body of the flagellum contains the kinetoplast DNA nucleoid body which consists of thousands of catenated minicircles and a smaller number of catenated maxicircles. The maxicircles contain structural genes and cryptogenes, rRNA genes, and a few guide RNA genes The minicircles contain the majority of the guide RNA genes. The long slender non-dividing stationary phase Leishmania tarentolae cells in culture have an asymmetric mitochondrion that consists of a single tubule extending from one edge of the kinetoplast portion. This presents a problem for cell division, in that one daughter cell will receive significantly less mitochondrial membranes than the other cell. We show in this paper that the solution to this problem is that dividing cells, which are normally shorter and rounder than stationary phase cells, possess a symmetric circular mitochondrion that has mitochondrial tubules extending from both edges of the kinetoplast which are joined in the posterior region of the cell. This implies that growth of the mitochondrion occurs after cell division, either from elongation of the longitudinal tubule towards the anterior of the cell, or from elongation of the kinetoplast portion of the mitochondrion towards the posterior region and fusion of the tubules.

Animals↗
PubMed1997

Gene gun-based nucleic acid immunization alone or in combination with recombinant vaccinia vectors suppresses virus burden in rhesus macaques challenged with a heterologous SIV.

Gene gun-based DNA immunization alone or in combination with recombinant vaccinia vectors was evaluated for the ability to elicit protective immune responses in rhesus macaques challenged with a pathogenic, heterologous simian immunodeficiency virus (SIV). Six monkeys primed with seven consecutive doses of DNA encoding SIVmac239 gp120 and gp160 (DNA + DNA) were divided into two groups. Three of these animals received another DNA booster immunization and the remaining three received a booster immunization containing a homologous, live recombinant vaccinia virus expressing SIVmac251 gp160 (DNA + VAC). In addition, a group of 15 animals primed with recombinant vaccinia vectors were divided into two groups. One group of six monkeys received another immunization of vaccinia (VAC + VAC) and the other nine animals received a DNA (mac239) booster immunization (VAC + DNA). Geometric mean end-point IgG titres in the DNA + VAC and VAC + DNA groups were substantially higher than the responses seen in the VAC + VAC and DNA + DNA groups, demonstrating a synergistic relationship between DNA-based vaccines and recombinant vaccinia virus-based vaccines. All vaccinates and five naive controls were challenged 19 weeks after the final booster immunization with 10 animal infectious doses of SIVDelta/B670. The vaccines did not prevent infection. However, all vaccine groups showed significant virus load reductions from seven to 56 days post challenge when compared to controls. Although the DNA + DNA group developed the lowest prechallenge antibody responses, the most significant reduction (200-fold) in virus load was associated with this group. In addition, a significant delay in CD4+ T cell loss relative to controls was observed in the DNA + DNA group. These results demonstrate that a gene gun-based DNA vaccine provided some attenuation of infection and CD4+ T cell loss after a heterologous challenge.

Animals↗
PubMed1997

Mitochondrial glutamate dehydrogenase from Leishmania tarentolae is a guide RNA-binding protein.

To identify specific proteins interacting with guide RNAs (gRNAs) in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [alpha-32P]UTP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyltransferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by coimmunoprecipitation and competitive gel shift analyses. Complex formation was strongly inhibited by competition with poly(U) or by deletion or substitution of the gRNA 3' oligo(U) tail. Also, addition of a 3' oligo(U) tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.

Amino Acid Sequence↗
PubMed1996

Guide RNA-directed uridine insertion RNA editing in vitro.

Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa. The Us are proposed to be derived either from UTP by two successive cleavage-ligations or transesterifications, or from the 3' end of the gRNA by the same mechanisms. We have demonstrated gRNA-dependent U insertions into a specific editing site of a pre-edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA-mRNA anchor duplex was necessary for activity. UTP and alpha-beta bond hydrolysis of ATP were required, and the activity was inhibited above 50-100 mM KCl. A gRNA-independent insertion of up to approximately 13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA-independent U insertion activity and had no effect on the gRNA-dependent activity. Blocking the 3' OH of the gRNA had little effect on the gRNA-dependent U insertion activity. The data are consistent with a cleavage-ligation model in which the Us are derived directly from UTP.

Animals↗
PubMed1996

Detection and identification of human pathogenic Leishmania and Trypanosoma species by hybridization of PCR-amplified mini-exon repeats.

A single pair of PCR primers within a conserved region of the mini-exon repeat was used to amplify the repeats from 10 species of pathogenic Leishmania belonging to four major clinical groups and also from three species of Trypanosoma. Oligonucleotide hybridization probes for the detection and identification of the PCR-amplified repeats were constructed from alignments of mini-exon intron and intergenic sequences. The probes generated from mini-exon intergenic regions of the L. (V.) braziliensis, L. (L.) donovani, and L. (L.) mexicana species hybridized specifically to their cognate groups without discriminating between the species within the groups. The probes for L. (L.) major and L. (L.) aethiopica were species-specific, while the L. (L.) tropica probe also hybridized with the L. (L.) aethiopica mini-exon repeat. The mini-exon intron-derived probes for T. cruzi, T. rangeli, and T. brucei were species-specific. This method involving the detection of specific PCR-amplified products produced using a single primer set represents a novel sensitive and specific assay for multiple trypanosomatid species and groups.

Animals↗
PubMed1996

Phylogeny of trypanosomes as inferred from the small and large subunit rRNAs: implications for the evolution of parasitism in the trypanosomatid protozoa.

Sequences of the small rRNA genes and partial sequences of the large rRNA genes were obtained by PCR amplification from a variety of vertebrate trypanosomes. The trypanosome species and hosts included Trypanosoma avium from a bird, T. rotatorium from an amphibian, T. boissoni from an elasmobranch, T. triglae from a marine teleost and T. carassii from a freshwater teleost. Phylogenetic relationships among these species and other representatives of the family Trypanosomatidae were inferred using maximum likelihood, maximum parsimony and evolutionary parsimony. The trypanosomatid tree was rooted using rRNA sequences from two species from the suborder Bodonina. All methods showed that the mammalian parasite, Trypanosoma brucei, constitutes the earliest divergent branch. The remaining trypanosomes formed a monophyletic group. Within this group, the bird trypanosome was grouped with T. cruzi, while the elasmobranch trypanosome and the two fish trypanosome species formed a group with an affinity to T. rotatorium. Our results provide no evidence for co-evolution of trypanosomatids and their hosts, either vertebrate or invertebrate. This suggests that evolution of trypanosomatids was accompanied by secondary acquisitions of hosts and habitats.

Animals↗
PubMed1996

Uridine insertion into preedited mRNA by a mitochondrial extract from Leishmania tarentolae: stereochemical evidence for the enzyme cascade model.

An RNA editing-like internal uridine (U) incorporation activity (G. C. Frech, N. Bakalara, L Simpson, and A. M. Simpson, EMBO J. 14:178-187, 1995) and a 3'-terminal U addition activity (N. Bakalara, A. M. Simpson, and L. Simpson, J. Biol. Chem. 264:18679-18686, 1989) have been previously described by using a mitochondrial extract from Leishmania tarentolae. Chiral phosphorothioates were used to investigate the stereoconfiguration requirements and the stereochemical course of these nucleotidyl transfer reactions. The extract utilizes (SP)-alpha-S-UTP for both 3' and internal U incorporation into substrate RNA. The internal as well as the 3' incorporation of (SP)-alpha-S-UTP proceeds via inversion of the stereoconfiguration. Furthermore, internal U incorporation does not occur at sites containing thiophosphodiesters of the RP configuration. Our results are compatible with an enzyme cascade model for this in vitro U insertion activity involving sequential endonuclease and uridylyl transferase directly from UTP and RNA ligase steps and are incompatible with models involving the transfer of U residues from the 3' ends of guide RNAs.

Animals↗
PubMed1996

RNA editing.

RNA editing is a term describing a variety of novel mechanisms for the modification of nucleotide sequences of RNA transcripts in different organisms. These editing events include (a) the U-insertion and -deletion type of editing found in the mitochondrion of kinetoplastid protozoa, (b) the C-insertion editing found in the mitochondrion of Physarum, (c) the C-to-U substitution editing of the mammalian apoB mRNA, (d) a similar C-to-U substitution editing of mRNAs in higher plant mitochondria and chloroplasts and in tRNAs of marsupials and rats, (e) a diverse nucleotide substitution editing of tRNAs in Acanthomoeba mitochondria, and (f) the A-to-I type of editing found in the mammalian glutamate receptor subunits. These diverse phenomena involve several different enzymatic mechanisms. In several cases, duplex RNAs with internal or external guide sequences help determine the site specificity of editing. The A-to-I editing observed in RNAs encoding non-NMDA glutamate receptor subunits may be due to the actions of a double-stranded RNA-specific adenosine deaminase that is widespread in higher organisms. Although the function of many RNA editing events is unclear, the biological importance of RNA editing in other systems may prove as significant as the nucleotide modifications regulating the cation selectivity and electrophysiological profiles elaborated by non-NMDA glutamate receptors in the mammalian brain.

Animals↗
PubMed1996

Sequence-dependent in vivo importation of tRNAs into the mitochondrion of Leishmania tarentolae.

Sequence determinants for the importation of tRNAs into the mitochondrion of Leishmania tarentolae in vivo were investigated. tRNA(Ile)(UAU) is exclusively localized within the mitochondrion and tRNA(Gln)(CUG) exclusively in the cytosol (Lye LF, Chen DHT, Suyama Y, 1993, Mol Biochem Parasitol 58:233-246; Shi X, Chen DHT, Suyama Y, 1994, Mol Biochem Parasitol 65:23-37). L. tarentolae cells were transfected with plasmids encoding either tRNA(Ile) or tRNA (Gln) that were tagged with altered sequences in the D loop, permitting discrimination from the endogenous tRNAs. Primer extension analysis was used to show that the plasmid-encoded genes were expressed and that the tagged tRNAs showed a similar intracellular localization as the endogenous tRNAs. Exchange or deletion of the 5'-flanking genomic sequences had no effect on the expression or mitochondrial localization of the tagged tRNA(Ile) or on the expression or cytosolic localization of the tagged tRNA(Gln), suggesting that the signals for importation are localized within the tRNA itself. Swapping the D loop+stem from the exclusively cytosolic tRNA(Gln) with that from the tRNA(Ile) produced a partial mitochondrial localization of the plasmid-expressed mutated tRNA(Gln). However, D loop exchange did not eliminate the mitochondrial localization of the plasmid-expressed mutated tRNA(Ile), suggesting that tertiary structure or additional sequence elements may be involved in the importation signal.

Animals↗
PubMed1996

Guide RNAs and guide RNA genes in the cryptobiid kinetoplastid protozoan, Trypanoplasma borreli.

Trypanoplasma borreli belongs to the bodonid/cryptobiid group of kinetoplastid protozoa, which represents a sister group to the trypanosomatids. RNA transcripts from several mitochondrial genes in this organism undergo the trypanosomatid type of uridine addition/deletion RNA editing. A guide RNA (gRNA) cDNA library was constructed and five gRNAs were identified, one for editing the ribosomal protein S12 mRNA, three for editing the cytochrome oxidase subunit I mRNA, and one for editing the cytochrome b mRNA. All of the gRNAs contained nonencoded oligo[U] sequences at the 3' end, as is common with gRNAs in trypanosomatids, but also contained nonencoded oligo[U] sequences at the 5' end. The mechanism for addition of the 5' nonencoded oligo[U] sequence and the function of this sequence are unknown. The T. borreli gRNAs were shorter (25-35 nt, excluding the 5' oligo[U]) than gRNAs in trypanosomatids (45-50 nt), indicating a smaller size of editing blocks in this organism. Genomic sequences for two gRNAs were cloned and sequenced. These two gRNA-encoding sequences were shown to originate from the 180-kb Component I molecules, which represent a possible homologue of minicircle DNA in trypanosomatids, and not from the 80-kb Component II molecules, which contain the structural genes and cryptogenes.

Animals↗
PubMed1996

Atomic force microscopy of DNA, nucleoproteins and cellular complexes: the use of functionalized substrates.

Progress towards rapid and simple characterization of biomolecular samples by scanning probe microscopy is impeded mainly by limitations of the current approach to sample preparation. We are working on approaches based on chemical functionalization of mica. Treatment of mica with aminopropyltriethoxy silane (APTES) makes the surface positively charged (AP-mica) and able to hold DNA in place for imaging, even in water. We have shown that AP-mica is an appropriate substrate for numerous nucleoprotein complexes as well. The AFM images of the complex of DNA with RecA protein are stable and indicate a structural periodicity for this filament. AP-mica holds strongly such large DNA complexes as kinetoplast DNA (kDNA) and is an appropriate substrate for their imaging with AFM. We have further develop this approach for making hydrophobic substrates. Silylation of mica surface with hexamethyldisilazane (Me-mica) allowed us to get AFM images of chlorosomes, an antenna complex isolated from green photosynthetic bacteria. Me-mica may be converted into a positively charged substrate after treatment with water solutions of tetraethylammonium bromide or cetyltrimethylammonium bromide. These activated surfaces show high activity towards binding the DNA molecules.

Aluminum Silicates↗
PubMed1995

In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae.

A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre-edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. Inhibition by micrococcal nuclease suggests a requirement for some type of endogenous RNA. The limitation of internal U-incorporation to the pre-edited region in the CYb mRNA and the inhibition by deletion or substitution of both mRNA anchor sequences for CYb gRNA-I and -II could be consistent either with a gRNA-mediated process or a secondary structure-mediated process. A low level of incorporation of [alpha-32P]CTP occurs at the same sites as UTP. Internal U-incorporation activity is selectively inhibited by heterologous RNAs, suggesting an involvement of low affinity RNA-binding proteins which can be competed by the added RNA.

Animals↗
PubMed1995

Erythrocyte insulin and insulin-like growth factor-I receptor tyrosine kinase activity in hypertension in pregnancy.

We have shown that preeclampsia is associated with insulin resistance. In the present study, we examined young normal, preeclamptic (PE), and gestational hypertensive (GH) nulliparous African-American women at term to investigate cellular determinants of this resistance and insulin and insulin-like growth factor-I (IGF-I) binding to partially purified erythrocyte receptors and receptor tyrosine kinase activity (TKA). Blood pressure was significantly elevated in PE and GH subjects as compared with controls. Insulin binding was similar in number and affinity in the three groups (femtomoles per microgram). IGF-I binding was increased in PE subjects as compared with either normals or GH subjects (0.2 +/- 0.02, 0.15 +/- 0.01, and 0.14 +/- 0.02 fmol/microgram protein). Insulin receptor TKA was increased in PE subjects as compared with normals when assessed either per microgram protein or per femtomole insulin binding (P < .01). In contrast, IGF-I-potentiated TKA was elevated in PE subjects only when assessed per microgram protein (P < .03). Thus, the increased number of IGF-I receptors in erythrocytes of PE subjects yields a net increase in receptor tyrosine kinase. Also, there is an augmentation of insulin receptor TKA in PE subjects. Together, these two alterations may be a compensatory mechanism for the insulin resistance associated with hypertensive diseases of pregnancy.

Adult↗
PubMed1995

Characterization of two nuclear-encoded protein components of mitochondrial ribonucleoprotein complexes from Leishmania tarentolae.

Two mitochondrial proteins with molecular masses of 18 and 51 kDa were isolated from Leishmania tarentolae, and N-terminal amino-acid sequences were obtained. The cDNAs and genes encoding these proteins were cloned using RT-PCR. The proteins were identified as components of the previously characterized mitochondrial ribonucleoprotein complexes, T-Ia and T-VI, by comigration in native gels. The p18 and p51 genes contain 17 and 9-amino-acid N-terminal sequences, which are not present in the mature proteins and may represent cleavable mitochondrial targeting sequences. There are two identical p18 genes separated by 1.7 kb in tandem array and both are transcribed. The p18 amino-acid sequence is not similar to any sequence in the database. Antiserum to p18 expressed in Escherichia coli reacts with the entire tubular mitochondrion. The p51 gene is single copy, and the amino-acid sequence is similar to mitochondrial aldehyde dehydrogenases from other organisms. The N-terminal amino-acid sequences of 71 and 62-kDa mitochondrial proteins which co-migrated in native gels with several other T-complexes were also obtained. The p71 sequence proved to be similar to hsp70 sequences from other organisms. The p62 sequence was identical to an hsp60 sequence from Trypanosoma brucei.

Amino Acid Sequence↗