Biomedical subjects

David A Campbell

Publications and source records attributed to David A Campbell.

At least 19 recordsLinked to original sources

PubMed2006

Further evidence of association of OPRD1 & HTR1D polymorphisms with susceptibility to anorexia nervosa.

BACKGROUND: A recent study reported strong evidence for the involvement of a region on human chromosome 1 and genetic susceptibility to anorexia nervosa (AN). A more detailed analysis of this region has suggested 2 genes that may account for this susceptibility. These data suggest that polymorphisms in both the serotonin 1D (HTR1D) and opioid delta 1 (OPRD1) receptor genes show a significant association with restricting AN (RAN). METHODS: In the current study, we have conducted an independent association study on 226 females meeting DSM-IV criteria for AN and 678 matched volunteers. RESULTS: We genotyped 4 SNPs in HTR1D and 6 SNPs in OPRD1. 3 SNPs were found to be associated with both RAN and binge-purge AN (BPAN) within the gene for OPRD1. We also found evidence of association between 2 polymorphisms within HTR1D and RAN. CONCLUSIONS: These data support the hypothesis that polymorphisms within this region form a component of the genetic basis to susceptibility to RAN. However, further work is required to understand the processes that may be mediated by these genes.

Adult↗

PubMed2006

A non-universal transcription factor? The Leishmania tarentolae TATA box-binding protein LtTBP associates with a subset of promoters.

In kinetoplastids a 39-nucleotide spliced leader RNA is trans-spliced to the 5' end of nuclear mRNAs before they can be translated, thus the spliced leader is central to gene expression in kinetoplastid biology. The spliced leader RNA genes in Leishmania tarentolae contain promoters with important sites at approximately -60 and -30. A complex forms specifically on the -60 element as shown by electrophoretic mobility shift. The -60 shift complex has an estimated mass of 159 kDa. An L. tarentolae homologue of TATA-binding protein, LtTBP, co-fractionates with the -60 shift complex. Inclusion of anti-LtTBP antiserum in the shift assay disrupts the shift, indicating that LtTBP is a component of the complex that interacts with the TATA-less -60 element of the spliced leader RNA gene promoter. Both LtTBP and LtSNAP50 are found near the spliced leader RNA gene promoter and the promoters important for tRNAAla and/or U2 snRNA gene transcription, as demonstrated by chromatin immunoprecipitation. The LtTBP appears to interact with a subset of promoters in kinetoplastids with an affinity for short transcription units.

Animals↗

PubMed2006

Trypanosoma congolense: paraoxonase 1 prolongs survival of infected mice.

In vitro studies have suggested that a fraction of human high density lipoprotein (HDL), termed trypanosome lysis factor (TLF), can protect against trypanosome infection. We examined the involvement of two proteins located in the TLF fraction, apolipoprotein A-II (apoA-II) and paraoxonase 1 (PON1), against trypanosome infection. To test whether PON1 is involved in trypanosome resistance, we infected human PON1 transgenic mice, PON1 knockout mice, and wild-type mice with Trypanosoma congolense. When challenged with the same dosage of trypanosomes, mice overexpressing PON1 lived significantly longer than wild-type mice, and mice deficient in PON1 lived significantly shorter. In contrast, mice overexpressing another HDL associated protein, apoA-II, had the same survival as wild-type mice. Together, these data suggest that PON1 provides protection against trypanosome infection. In vitro studies using T. brucei brucei indicated that HDL particles containing PON1 and those depleted of PON1 did not differ in their lysis ability, suggesting that protection by PON1 is indirect. Our data are consistent with an in vivo role of HDL protection against trypanosome infection.

Animals↗

PubMed2006

Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region.

BACKGROUND: The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. RESULTS: We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. CONCLUSION: The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.

Amino Acid Sequence↗

PubMed2006

Complete cap 4 formation is not required for viability in Trypanosoma brucei.

In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5' ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m(7)G cap, ribose 2'-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2'-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A(2) and is not required for subsequent steps; TbMT511 methylates C(3), without which U(4) methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m(7)G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A(2), C(3), and U(4) methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.

Amino Acid Sequence↗

PubMed2005

Trypanosoma cruzi 5S rRNA arrays define five groups and indicate the geographic origins of an ancestor of the heterozygous hybrids.

Isolates of the etiological agent of Chagas disease, Trypanosoma cruzi, have been subdivided into six subgroups referred to as discrete typing units. The subgroups are related through two distinct hybridisation events: representatives of homozygous discrete typing units I and IIb fused to form discrete typing units IIa and IIc, whose homozygous genotypes have features of both ancestral types; a second fusion between strains of homozygous discrete typing units IIb and IIc created the heterozygous hybrid strains discrete typing units IId and IIe. The intergenic region of the tandemly repeated 5S rRNA array displays four variant sequence classes, allowing the discrimination of five discrete typing units. The genome project reference strain, CL Brener, is a hybrid discrete typing unit IIe strain that contains both discrete typing unit IIb and IIc classes of 5S rRNA repeats in distinct arrays present on different chromosomes. The CL Brener discrete typing unit IIb-type array contains approximately 193 repeated units, of which about one-third contain a 129 bp sequence that replaces a majority of the 5S rRNA sequence. The 129 bp 'invader' sequence was detected within the arrays of all hybrid discrete typing unit IId and IIe strains and in a subset of discrete typing unit IIb strains. This array invader replaces the internal promoter elements conserved in 5S rRNA. The discrete typing unit IIb Esmeraldo strain contains approximately 135 repeats and shows a region of homology to the array invader in the 5' flank of the array, but no evidence of the invading sequence element within the array. A survey of additional discrete typing unit IIb strains revealed a split within the subgroup, in which some strains contained invaded arrays and others were homogeneous for the 5S rRNA. The putative discrete typing unit IIb ancestor of the hybrid discrete typing units IId and IIe more closely resembles the extant Bolivian/Chilean IIb isolates than the Brazilian IIb isolates based on the correlation with the array invader.

Animals↗

PubMed2005

The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease.

Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.

Animals↗

PubMed2005

Two hybridization events define the population structure of Trypanosoma cruzi.

Genetic variation in Trypanosoma cruzi is likely a key determinant in transmission and pathogenesis of Chagas disease. We have examined nine loci as markers for the extant T. cruzi strains. Four distinct alleles were found for each locus, corresponding to the sequence classes present in the homozygous discrete typing units (DTUs) I, IIa, IIb, and IIc. The alleles in DTUs IIa and IIc showed a spectrum of polymorphism ranging from DTU I-like to DTU IIb-like, in addition to DTU-specific sequence variation. DTUs IId and IIe were indistinguishable, showing DTU homozygosity at one locus and heterozygosity with DTU IIb and IIc allelic sequences at eight loci. Recombination between the DTU IIb and IIc alleles is evidenced from mosaic polymorphisms. These data imply that two discrete hybridization events resulted in the formation of the current DTUs. We propose a model in which a fusion between ancestral DTU I and IIb strains gave rise to a heterozygous hybrid that homogenized its genome to become the homozygous progenitor of DTUs IIa and IIc. The second hybridization between DTU IIb and IIc strains that generated DTUs IId and IIe resulted in extensive heterozygosity with subsequent recombination of parental genotypes.

Animals↗

PubMed2005

Intragenomic spliced leader RNA array analysis of kinetoplastids reveals unexpected transcribed region diversity in Trypanosoma cruzi.

The spliced leader RNA gene (SL RNA) repeat is present in large multicopy arrays and has been used as a marker for the diversity of kinetoplastid protozoans. Intra-array variation could affect conclusions made using a randomly isolated repeat as a marker. We examined the Leishmania major (Friedlin) and Trypanosoma cruzi (CL Brener) genome projects for SL RNA repeat sequences in order to assess their homogeneity and the possible effects of sequence variation on taxonomic interpretation. Of the dozens of distinct sequence classes examined, no single copy would bias clustering analyses with regard to other closely related species or isolates. Six dimorphic sites within the T. cruzi transcribed region were found to be linked and are predicted to yield a heterogeneous SL RNA population. The variation that exists among the repeats paints a picture of the broad mechanisms of array maintenance and evolution where site-specific mutations in a single repeat may be spread throughout the array and recombined with existing repeats to create new sequence classes, all occurring under selective pressure to maintain or increase the fitness of the cell line in which these events occur.

Animals↗

PubMed2005

The Trypanosoma brucei La protein is a candidate poly(U) shield that impacts spliced leader RNA maturation and tRNA intron removal.

By virtue of its preferential binding to poly(U) tails on small RNA precursors and nuclear localisation motif, the La protein has been implicated for a role in the stabilisation and nuclear retention of processing intermediates for a variety of small RNAs in eukaryotic cells. As the universal substrate for trans-splicing, the spliced leader RNA is transcribed as a precursor with just such a tail. La protein was targeted for selective knockdown by inducible RNA interference in Trypanosoma brucei. Of three RNA interference strategies employed, a p2T7-177 vector was the most effective in reducing both the La mRNA as well as the protein itself from induced cells. In the relative absence of La protein T. brucei cells were not viable, in contrast to La gene knockouts in yeast. A variety of potential small RNA substrates were examined under induction, including spliced leader RNA, spliced leader associated RNA, the U1, U2, U4, and U6 small nuclear RNAs, 5S ribosomal RNA, U3 small nucleolar RNA, and tRNATyr. None of these molecules showed significant variance in size or abundance in their mature forms, although a discrete subset of intermediates appear for spliced leader RNA and tRNATyr intron splicing under La depletion conditions. 5'-end methylation in the spliced leader RNA and U1 small nuclear RNA was unaffected. The immediate cause of lethality in T. brucei was not apparent, but may represent a cumulative effect of multiple defects including processing of spliced leader RNA, tRNATyr and other unidentified RNA substrates. This study indicates that La protein binding is not essential for maturation of the spliced leader RNA, but does not rule out the presence of an alternative processing pathway that could compensate for the absence of normally-associated La protein.

Animals↗

PubMed2004

HygR and PurR plasmid vectors for episomal transfection of Trypanosoma cruzi.

This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.

Animals↗

PubMed2004

The 3' termini of small RNAs in Trypanosoma brucei.

Post-transcriptional 3' end formation is an essential step in the maturation of most small RNAs. Knowledge of the precise 3' ends of mature RNAs is essential for defining 3'-processing activities. We have mapped the mature 3' ends of Spliced Leader RNA, the U1, U2, U4, U5, and U6 small nuclear RNAs, the U3 small nucleolar RNA, and the 5S and 5.8S ribosomal RNAs by ligation-mediated PCR in Trypanosoma brucei. With the exception of U5, two classes of 3' ends were observed: flush with the base of a stem-loop structure, and 1-2 nt extended from a stem-loop. Multiple mechanisms and structural features are likely to influence 3' end maturation of RNAs.

Animals↗

PubMed2004

SmD1 is required for spliced leader RNA biogenesis.

The Sm-binding site of the kinetoplastid spliced leader RNA has been implicated in accurate spliced leader RNA maturation and trans-splicing competence. In Trypanosoma brucei, RNA interference-mediated knockdown of SmD1 caused defects in spliced leader RNA maturation, displaying aberrant 3'-end formation, partial formation of cap 4, and overaccumulation in the cytoplasm; U28 pseudouridylation was unaffected.

Alternative Splicing↗

PubMed2004

3'-End polishing of the kinetoplastid spliced leader RNA is performed by SNIP, a 3'-->5' exonuclease with a Motley assortment of small RNA substrates.

In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3' extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 3'-->5' exonuclease required for SL RNA 3'-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3' processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 3'-->5' proofreading epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3' extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one- to two- nucleotide 3' extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3'-end formation is a multistep process in which SNIP provides the ultimate 3'-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.

Animals↗

PubMed2004

Molecular pathology in oncology--the AstraZeneca perspective.

Growth of the oncology portfolio remains of strategic importance to AstraZeneca, and the adoption of new technologies to allow us to enhance this portfolio is central to this strategy. With the move away from classical hormonal and cytotoxic therapies to the development of more targeted approaches for the treatment of cancer, an understanding of the molecular pathology of the disease state is becoming vital. Our understanding of the pathogenesis of cancer has increased dramatically over the last few decades and with the publication of the human genome and the resultant explosion in the field of genetics and genomics, AstraZeneca is turning its attention to using these new technologies to enhance the oncology R&D platform. In particular, the fields of pharmacogenetics and pharmacogenomics in relation to oncology have received much attention and this has been mirrored externally both within the pharmaceutical/biotechnology and academic sectors. Future products from the AstraZeneca oncology portfolio will increasingly rely on the use of genetics and genomics for patient identification and stratification, whilst these technologies will also provide a source of novel biomarkers and diagnostics that may allow us to streamline the R&D process and help us to better understand the biological basis of the diseases we are aiming to treat. The AstraZeneca perspective is, however, pragmatic enough to appreciate the practical challenges involved in applying pharmacogenetics and genomics not only for early drug development, but also in the organization of the healthcare infrastructure to undertake timely and complex laboratory investigations. Finally, validation of this approach will require carefully controlled clinical studies.

Drug Industry↗

PubMed2004

The determinants of Chagas disease: connecting parasite and host genetics.

As a consequence of infection by Trypanosoma cruzi, 30% of victims may develop chronic Chagas disease, which presents a spectrum of pathology including cardiomyopathy, megacolon and megaesophagus. The outcome of infection in a particular individual is the result of a set of complex interactions among the host genetic background, environmental and social factors, and the genetic composition of the parasite, all of which can be complicated by mixed infections and re-infections. Initially we consider what is known about the genetic structure and biological properties of the protozoan. Currently, six distinct subgroups have been characterized by different combinations of four distinct genotypic classes. The recent demonstration of genetic exchange via non-meiotic cell fusion illustrates a mechanism by which maintained heterogeneous polyploidy may have been generated in these parasites. Subsequently, we consider factors in humans and in experimental mouse-infection and tissue culture models that have contributed to our understanding of the host's susceptibility or resistance to disease. Identification of the direct players in host-pathogen interactions at the establishment and chronic phases of the disease is perhaps the best hope of a clinical handle for treatment. At some point in the future, these disparate areas of study will have to come together. It is to be hoped that this scientific fusion will result in better prognosis and treatment of Chagas disease.

Animals↗

PubMed2003

The Leishmania tarentolae spliced leader contains determinants for association with polysomes.

In kinetoplastids, every nuclear-derived mRNA contains an identical 39-nucleotide (nt) spliced leader at its 5'-terminus. The spliced leader is derived from substrate spliced leader RNA and joined to pre-mRNA by trans-splicing, thus providing mature mRNAs with an m7G cap and additional methylations referred to as cap 4. It was shown previously that mutations spanning nucleotides 10-39 of the spliced leader did not affect substrate spliced leader RNA transcription or trans-splicing in Leishmania tarentolae (Saito, R. M., Elgort, M. G., and Campbell, D. A. (1994) EMBO J. 13, 5460-5469). In this study we examined these sequences for a possible role in translation by assaying the association of mRNAs, which possess mutated spliced leaders, with polysomes. For the nt 28-39 mutated spliced leaders, both the substrate spliced leader RNA and the spliced leader demonstrated a wild-type methylation pattern; spliced nt 28-39 mRNA was found in polysomes. Thus, the nt 28-39 region conserved primary sequence is not a determinant of polysome association. An undermethylated cap 4 structure was present on substrate and mRNA spliced leaders in nt 20-29 mutated exons; nt 20-29 mRNA was not present in polysomes. A differential pattern of cap 4 methylation was seen between the nt 10-19 substrate spliced leader RNA and the nt 10-19 spliced leaders found in the poly(A)+ population of RNA; the nt 10-19 mRNA was not seen in polysomes. Undermethylated spliced leaders did not associate efficiently with polysomes, suggesting a requirement for the cap 4 and/or primary sequence of the spliced leader in translation. This is the first report demonstrating that the spliced leader contains critical structural or sequence determinants for association with polysomes and, hence, translation.

5' Untranslated Regions↗

PubMed2003

Transcription in kinetoplastid protozoa: why be normal?

Transcription in the kinetoplastid protozoa shows substantial variation from the paradigms of eukaryotic gene expression, including polycistronic transcription, a paucity of RNA polymerase (RNAP) II promoters, no qualitative regulated transcription initiation for most protein-coding genes, transcription of some protein-coding genes by RNAP I, an exclusive subnuclear location for VSG transcription, the dependence of small nuclear RNA gene transcription on an upstream tRNA gene, and the synthesis of mitochondrial tRNAs in the nucleus. Here, we present a broad overview of what is known about transcription in the kinetoplastids and what has yet to be determined.

Animals↗