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L Simpson

Publications and source records attributed to L Simpson.

At least 145 records · Page 8Linked to original sources
PubMed1989

Structure, genomic organization and transcription of the bifunctional dihydrofolate reductase-thymidylate synthase gene from Crithidia fasciculata.

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene from the monogenetic kinetoplastid protozoan, Crithidia fasciculata, was isolated and characterized. The gene is located on a single chromosome of approximately one megabase, and shows significant sequence similarity to other eukaryotic and prokaryotic DHFR and TS genes. There is a single low-abundance polyadenylated DHFR-TS transcript of approximately 3100 nt. One major miniexon splice site was identified by primer extension analysis. The 5' flanking region of the gene is divergently transcribed and shows strong similarities to a consensus DHFR promoter as well as to other eukaryotic 'housekeeping' gene promoter regions. A sequence downstream of the DHFR promoter consensus region is complementary to the 3' end of the C. fasciculata miniexon-derived RNA. This suggests a means by which the two separately transcribed RNAs may be juxtaposed for trans-splicing. In the 3' flanking region of the DHFR-TS gene, there is a sequence that is present in all of the chromosomes from this species and also from Leishmania tarentolae.

Amino Acid Sequence↗
PubMed1989

Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: use in diagnosis of Chagas' disease.

Amplification of DNA sequences from the kinetoplast minicircle DNA was employed as a method for the detection and classification of small numbers of Trypanosoma cruzi cells. Two overlapping fragments from the conserved 120 bp minirepeat regions of the minicircle DNA and one fragment covering the adjacent variable regions were amplified. The minimal amount of minicircle DNA required to detect a product by hybridization with an oligonucleotide probe was 0.015 fg, which represents approximately 10 molecules or 0.1% of the minicircle DNA component of a single cell. The amplification worked equally well with kDNA from several strains of T. cruzi and did not occur with kDNA from several other kinetoplastids. kDNA recovered from less than 10 trypanosomes in whole blood could be used as a template for amplification; the presence of a several billion fold excess of human DNA had no effect on the amplification process. Schizodeme analysis by hybridization with specific oligonucleotides or by direct restriction enzyme digestion could be performed on the amplified fragments representing the minicircle conserved region or variable regions. This method should prove useful as a rapid, specific and sensitive assay for Chagas' disease in chronic patients as well as for epidemiological studies of infected animals and insects.

Animals↗
PubMed1989

Characterization of a protein fraction containing cytochromes b and c1 from mitochondria of Leishmania tarentolae.

A soluble red band fraction was obtained from Leishmania tarentolae cells by sucrose gradient sedimentation of a Triton X-100 lysate. Spectral analysis indicated that cytochrome b was present in the red band: the reduced minus oxidized difference spectra revealed absorption maxima at 562,527, and 431 nm at room temperature and 562, 530, and 422 nm at 77K. In addition, a 28-kDa protein was identified in this fraction which retained heme-associated peroxidase activity even after denaturation on SDS-polyacrylamide gels. The amino acid composition of this protein showed a strong similarity to cytochrome c1 of both bovine and yeast.

Amino Acids↗
PubMed1989

Genomic organisation of nuclear tRNAGly and tRNALeu genes in Trypanosoma brucei.

We have isolated a 0.3-kb HaeIII restriction fragment from Trypanosoma brucei which contains two tRNA genes. Secondary structure models predict that the two genes identified encode tRNA molecules which specify glycine (anticodon UCC) and leucine (anticodon CAG). The two genes are separated by 86 nucleotides, transcribed in the same direction and contain features of conventional RNA polymerase III transcription units. Southern blot analysis indicates the presence of multicopy tRNa gene families in T. brucei.

Animals↗
PubMed1989

Internal frameshifts within the mitochondrial genes for cytochrome oxidase subunit II and maxicircle unidentified reading frame 3 of Leishmania tarentolae are corrected by RNA editing: evidence for translation of the edited cytochrome oxidase subunit II mRNA.

The Leishmania tarentolae cytochrome oxidase (EC 1.9.3.1) subunit II (COII) and maxicircle unidentified reading frame 3 (MURF3) mRNAs are edited internally by the addition of four and five uridine residues, respectively, which eliminate -1 and +1 reading frameshifts in the gene sequences. The editing events in COII are conserved in three kinetoplastid species, and those in MURF3 are conserved in two species. A primer extension assay showed that the ratio of edited to unedited RNA differed for each gene: 89% of the COII and 36% of the MURF3 transcripts are edited. Preliminary evidence was obtained for translation of the edited COII transcript into protein: antibodies generated against a synthetic peptide with the predicted carboxyl-terminal amino acid sequence reacted with a polypeptide of the correct molecular weight in immunoblots of a mitochondrial lysate.

Amino Acid Sequence↗
PubMed1989

Similar-sequence and similar-structure in retarded and non-retarded children's development.

Three questions important in relation to the developmental-difference controversy were addressed. The first, to what extent do retarded and non-retarded children show a similar sequence in their development of concepts, was answered using scalogram analyses which supported a common order of acquisition of conservation concepts when intermediate as well as conservation and non-conservation categories were used. There was a high degree of similarity in sequence within as well as between attributes. The second question, how far does the similar-structure hypothesis hold, was answered in the terms set by the developmental theorists. Multiple regression analyses and comparisons of mean mental ages for each of the conservation subgroups supported the similar-structure hypothesis. Non-retarded and retarded children with no known organic condition revealed similar structures. The third question concerned findings of volume conservation in retarded children and how these might be interpreted. It is argued that recognition of conservation in displacement volume tasks need not imply a generalised stage of formal operations nor be interpreted in terms of ceiling in development. Overall the findings have implications for ordering and providing appropriate levels of experiences for retarded children without assuming fixed limits in their development.

Adolescent↗
PubMed1988

Editing of kinetoplastid mitochondrial mRNAs by uridine addition and deletion generates conserved amino acid sequences and AUG initiation codons.

RNA editing is a novel type of mRNA processing in the kinetoplastid mitochondrion that involves the co- or posttranscriptional addition of uridine residues within transcripts. The result is an mRNA nucleotide sequence which differs from that of the corresponding gene. We have found that RNA editing may also occur through the deletion of genome-encoded uridines from the RNA sequence. Uridine additions and deletions in the 5' ends of the COIII, MURF2, and MURF3 transcripts create new N-terminal amino acid sequences that are conserved between species, and new AUG initiation codons in several cases. These results suggest that the creation of new amino acid sequences and methionine initiation codons may be important functions of RNA editing in the kinetoplastid mitochondrion.

Amino Acid Sequence↗
PubMed1988

Comparison of several lizard Leishmania species and strains in terms of kinetoplast minicircle and maxicircle DNA sequences, nuclear chromosomes, and membrane lipids.

Eight strains of a lizard Leishmania species, L. tarentolae, were compared with four other saurian species [L. hoogstrali, L. adleri, L. agamae and Leishmania sp. LizS], with L. major from man and with Trypanosoma platydactyli, a putative lizard trypanosome, in terms of kinetoplast DNA minicircle and maxicircle sequences and in terms of nuclear chromosome patterns on orthogonal gel electrophoresis. The L. tarentolae strains fell into two major groups, one (group A) consisting of the L. tarentolae strains, UC, Krassner and Trager, derived from an Algerian gecko isolate and the other (group B) consisting of five L. tarentolae LEM strains isolated from geckos in southern France. T. platydactyli TPCL2, which was postulated by Wallbanks et al. to represent the lizard form of a French L. tarentolae strain, was closely related to the UC strain and not to the LEM strains, in all respects analyzed. Leishmania sp. LizS from a Mongolian gecko and L. hoogstrali from a Sudanese gecko showed some sequence similarities to the L. tarentolae strains, but the leishmanias said to be L. adleri from a Kenyan lacertid and L. agamae from an Israeli agamid showed no minicircle sequence similarities with lizard Leishmania and in fact were probably the same species. The maxicircle divergent region was larger in the group B strains than in the group A strains, but there were sequences in common with both groups, and not with L. hoogstrali and L. major. Four strains of L. tarentolae, the four other supposed saurian Leishmania species, three mammalian leishmanias, T. platydactyli and four other trypanosomes, T. cyclops (Malaysian macaque), T. conorrhini (Hawaiian reduviid bug), T. cruzi (man) and T. lewisi (feral rat) were analyzed for their contents of sterols and phosphoglyceride fatty acyl groups. T. platydactyli TPCL2 contained a sterol (5-dehydroepisterol), a phosphatidylcholine fatty acyl group (alpha-linolenic acid) and a phosphatidylethanolamine fatty acyl group (dihydrosterculic acid) characteristic of members of the genus Leishmania and not the genus Trypanosoma. The proportions of those lipids in the free sterol and phosphoglyceride fractions of T. platydactyli TPCL2 most closely resembled those seen in the Leishmania strains from Algerian, French, Mongolian and Sudanese geckos.

Animals↗
PubMed1988

Peculiar sequence organization of kinetoplast DNA minicircles from Trypanosoma cruzi.

The sequences of two minicircles from the kinetoplast DNA of the CL strain and one of the Y strain of Trypanosoma cruzi are reported. These 1.4 kb molecules have a peculiar sequence organization, the most distinctive feature being the occurrence of a 120 bp sequence repeated four times, located at 0, 90, 180 and 270 degrees along each circle. We have termed these conserved regions in this species 'minirepeats'. Minirepeats have a 3-fold higher concentration of cytosine residues in comparison with the variable regions and contain the universal 12-mer motif GGGGTTGGTGTA present in all sequenced minicircles and which was shown to be involved in DNA replication. A consensus sequence of T. cruzi minirepeats was determined using the 20 minirepeats present in five known T. cruzi minicircle sequences. This consensus sequence contains regions which have been remarkably well preserved in strains which show great biological diversity. In addition a low level of intraminicircle sequence similarity was also observed within the variable region, but this similarity did not extend between strains. The abundance of conserved minirepeat sequences containing invariant restriction sites in T. cruzi cells may prove valuable for the development of new direct diagnostic methods for Chagas' disease based on DNA probe technology.

Animals↗
PubMed1988

Evolution of parasitism: kinetoplastid protozoan history reconstructed from mitochondrial rRNA gene sequences.

A phylogenetic tree for the evolution of five representative species from four genera of kinetoplastid protozoa was constructed from comparison of the mitochondrial 9S and 12S rRNA gene sequences and application of both parsimony and evolutionary parsimony algorithms. In the rooted version of the tree, the monogenetic species Crithidia fasciculata is the most deeply rooted, followed by another monogenetic species, Leptomonas sp. The three digenetic species Trypanosoma cruzi, Trypanosoma brucei, and Leishmania tarentolae branch from the Leptomonas line. The substitution rates for the T. brucei and T. cruzi sequences were 3-4 times greater than that of the L. tarentolae sequences. This phylogenetic tree is consistent with our cladistic analysis of the biological evidence including life cycles for these five species. A tentative time scale can be assigned to the nodes of this tree by assuming that the common ancestor of the digenetic parasites predated the separation of South America and Africa and postdated the first fossil appearance of its host (inferred by parsimony analysis). This time scale predicts that the deepest node occurred at 264 +/- 51 million years ago, at a time commensurate with the fossil origins of the Hemiptera insect host. This implies that the ancestral kinetoplastid and its insect host appeared at approximately the same time. The molecular data suggest that these eukaryotic parasites have an evolutionary history that extends back to the origin of their insect host.

Animals↗
PubMed1988

Creation of AUG initiation codons by addition of uridines within cytochrome b transcripts of kinetoplastids.

The cytochrome b gene of Trypanosoma brucei has an ATG codon near its 5' end but the cytochrome b genes of the related kinetoplastids Leishmania tarentolae and Crithidia fasciculata lack an ATG. Recent results have shown that 34 uridines that are not encoded in the genome are added within the 5' end of T. brucei cytochrome b transcripts during or after transcription. These additions create an AUG in the transcript that is 20 amino acids upstream of the AUG predicted from the genomic sequence. We report here that the cytochrome b transcripts of L. tarentolae and C. fasciculata also contain added uridines within their 5' ends. The additions occur in similar numbers and positions and an in frame AUG is created at a similar site in all three species. These data strongly suggest that the created AUG functions as the initiation codon for cytochrome b in these species. Since some other kinetoplastid mitochondrial genes also lack conventional initiation codons, creation of initiation codons may be an important function of uridine addition.

Amino Acid Sequence↗
PubMed1987

T-independent and T-dependent steps in the murine B cell response to antiimmunoglobulin.

Sepharose-anti-Ig and purified populations of small, high-density B cells have been used to study the formation and function of B lymphoblasts. Sepharose-anti-Ig converts small, Ia-poor B cells with a high-buoyant density to large, Ia-rich, B blasts with a low-buoyant density. We find that this response proceeds efficiently in the absence of IL-4 (BSF-1) as well as most T cells, macrophages, and dendritic cells. Further development of the blasts requires an additional stimulus, such as LPS or the conditioned medium of stimulated EL-4 thymoma cells. Within 6 h, blasts begin to enter S phase and within 24 h most divide. At later times (48-72 h) most of the blasts are actively secreting IgM. Recombinant IL-1, -2, -3, and -4 have little or no effect on the B blasts, and a neutralizing mAb to IL-4 does not block the response to EL-4 Sn. We conclude that Sepharose-anti-Ig induces B cell blastogenesis in a T-independent fashion and that these blasts represent a highly enriched population of cells that respond to distinct, T cell-derived lymphokines.

Animals↗
PubMed1987

Comparison of the maxicircle (mitochondrial) genomes of Leishmania tarentolae and Trypanosoma brucei at the level of nucleotide sequence.

The entire 16.7-kilobase (kb) transcribed region of the Leishmania tarentolae maxicircle was compared to the entire 15-kb transcribed region of the Trypanosoma brucei maxicircle at the nucleotide sequence level by dot matrix analysis and by alignments of individual genes. The L. tarentolae NADH dehydrogenase subunit 1 (ND1) gene was identified in a newly obtained 2.9-kb sequence. All but two regions which flank the cytochrome b gene are highly conserved in both species. One 3.1-kb region in L. tarentolae that contains the cytochrome oxidase subunit III (COIII) gene and several open reading frames corresponds to a 2-kb sequence in T. brucei with limited sequence homology that lacks the COIII gene. Another 0.6-kb region that comprises an unidentified open reading frame (open reading frame 12) in L. tarentolae is substituted by a nonhomologous 0.4-kb open reading frame in T. brucei. A short intergenic region between the ND1 gene and the maxicircle unidentified reading frame 1 gene shows limited sequence homology, and the regions between the ND4 and ND5 genes and between the COI and ND4 genes are not conserved. All of the intergenic regions share G + C richness and a similar pattern of G versus C strand bias. 1.8 kb of the L. tarentolae divergent region (DV) and around 3 kb of the T. brucei DV were also obtained. The T. brucei DV sequences were not homologous to the L. tarentolae DV sequence but were organized in a similar fashion with tandem repeats of varying complexity.

Animals↗
PubMed1987

The monogenetic kinetoplastid protozoan, Crithidia fasciculata, contains a transcriptionally active, multicopy mini-exon sequence.

A repeated sequence from the Crithidia fasciculata nuclear genome has been isolated which is homologous to the mini-exon genes of other kinetoplastid protozoa. Sequence analysis of the 417 bp monomeric unit confirmed the presence of a 35 nt sequence within the repeat that is 77% homologous with the Trypanosoma brucei 35-mer mini-exon or spliced leader sequence. The repeat is present at approximately 250 copies per cell and is organized into one, or a few, large head to tail tandem clusters predominantly on a single chromosome. The mini-exon repeat unit hybridizes to a major 84 nt and a minor 87 nt poly (A)- steady state transcript, the first 35 nts of which comprise the mini-exon sequence found at the 5' end of mRNAs in several other kinetoplastid species. The 3'-termini of the transcripts map to positions on the DNA sense strand directly preceeding a stretch of 8 thymidine residues. Crithidia represents the most primitive kinetoplastid species which apparently possesses a discontinuous type of mRNA processing, implying that this represents a conserved feature in possibly all genera of kinetoplastid protozoa.

Animals↗