RESUMO
Population and species persistence in a rapidly warming world will be determined by an organism's ability to acclimate to warmer conditions, especially across generations. There is potential for transgenerational acclimation but the importance of ontogenetic timing in the transmission of environmentally induced parental effects remains mostly unknown. We aimed to disentangle the effects of two critical ontogenetic stages (juvenile development and reproduction) to the new-generation acclimation potential, by exposing the spiny chromis damselfish Acanthochromis polyacanthus to simulated ocean warming across two generations. By using hepatic transcriptomics, we discovered that the post-hatching developmental environment of the offspring themselves had little effect on their acclimation potential at 2.5 months of life. Instead, the developmental experience of parents increased regulatory RNA production and protein synthesis, which could improve the offspring's response to warming. Conversely, parental reproduction and offspring embryogenesis in warmer water elicited stress response mechanisms in the offspring, with suppression of translation and mitochondrial respiration. Mismatches between parental developmental and reproductive temperatures deeply affected offspring gene expression profiles, and detrimental effects were evident when warming occurred both during parents' development and reproduction. This study reveals that the previous generation's developmental temperature contributes substantially to thermal acclimation potential during early life; however, exposure at reproduction as well as prolonged heat stress will likely have adverse effects on the species' persistence.
Assuntos
Aclimatação , Recifes de Corais , Animais , Reprodução , Aquecimento Global , Perciformes/fisiologia , Transcriptoma , Oceanos e Mares , Peixes/fisiologia , TemperaturaRESUMO
The environment experienced by one generation has the potential to affect the subsequent one through non-genetic inheritance of parental effects. Since both mothers and fathers can influence their offspring, questions arise regarding how the maternal, paternal and offspring experiences integrate into the resulting phenotype. We aimed to disentangle the maternal and paternal contributions to transgenerational thermal acclimation in a reef fish, Acanthochromis polyacanthus, by exposing two generations to elevated temperature (+1.5°C) in a fully factorial design and analysing the F2 hepatic gene expression. Paternal and maternal effects showed not only common but also parent-specific components, with the father having the largest influence in shaping the offspring's transcriptomic profile. Fathers contributed to transcriptional transgenerational response to warming through transfer of epigenetically controlled stress-response mechanisms while mothers influenced increased gene expression associated with lipid metabolism regulation. However, the key to acclimation potential was matching thermal experiences of the parents. When both parents were exposed to the same condition, offspring showed increased expression of genes related to structural RNA production and transcriptional regulation, whereas environmental mismatch in parents resulted in maladaptive parental condition transfer, revealed by translation suppression and endoplasmic reticulum stress. Interestingly, the offspring's own environmental experience had the smallest influence on their hepatic transcription profiles. Taken together, our results show the complex nature of the interplay among paternal, maternal and offspring cue integration, and reveal that acclimation potential to ocean warming might depend not only on maternal and paternal contributions but importantly on congruent parental thermal experiences.
RESUMO
The genomic sequences of 20 Leishmania infantum isolates collected in northeastern Brazil were compared with each other and with the available genomic sequences of 29 L. infantum/donovani isolates from Nepal and Turkey. The Brazilian isolates were obtained in the early 1990s or since 2009 from patients with visceral or non-ulcerating cutaneous leishmaniasis, asymptomatic humans, or dogs with visceral leishmaniasis. Two isolates were from the blood and bone marrow of the same visceral leishmaniasis patient. All 20 genomic sequences display 99.95% identity with each other and slightly less identity with a reference L. infantum genome from a Spanish isolate. Despite the high identity, analysis of individual differences among the 32 million base pair genomes showed sufficient variation to allow the isolates to be clustered based on the primary sequence. A major source of variation detected was in chromosome somy, with only four of the 36 chromosomes being predominantly disomic in all 49 isolates examined. In contrast, chromosome 31 was predominantly tetrasomic/pentasomic, consistent with its regions of synteny on two different disomic chromosomes of Trypanosoma brucei. In the Brazilian isolates, evidence for recombination was detected in 27 of the 36 chromosomes. Clustering analyses suggested two populations, in which two of the five older isolates from the 1990s clustered with a majority of recent isolates. Overall the analyses do not suggest individual sequence variants account for differences in clinical outcome or adaptation to different hosts. For the first known time, DNA of isolates from asymptomatic subjects were sequenced. Of interest, these displayed lower diversity than isolates from symptomatic subjects, an observation that deserves further investigation with additional isolates from asymptomatic subjects.
Assuntos
Doenças do Cão/parasitologia , Leishmania infantum/genética , Leishmaniose Visceral/veterinária , Animais , DNA de Protozoário/genética , Doenças do Cão/epidemiologia , Cães , Variação Genética , Genoma de Protozoário , Humanos , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
African trypanosomes evade the immune response of their mammalian hosts by sequentially expressing genes for different variant surface glycoproteins (VSGs) from telomere-linked VSG expression sites. In the Trypanosoma brucei clone whose genome is being sequenced (GUTat 10.1), we show that the expressed VSG (VSG 10.1) is duplicated from a silent donor VSG located at another telomere-linked site. We have determined two 130 kb sequences representing the VSG 10.1 donor and expression sites. The telomere-linked donor VSG 10.1 resembles metacyclic VSG expression sites, and is preceded by a cluster of 35 or more tandem housekeeping genes, all of which are transcribed away from the telomere. The 45 kb telomere-linked VSG 10.1 expression site contains a promoter followed by seven expression site-associated genes (ESAGs), three pseudo ESAGs, two pseudo VSGs and VSG 10.1. The 80 kb preceding the expression site has few, if any, functional ORFs, but contains 50 bp repeats, INGI retrotransposon-like elements, and novel 4-12 kb repeats found near other telomeres. This analysis provides the first look over a 130 kb range of a telomere-linked donor VSG and its corresponding telomere-linked VSG expression site and forms the basis for studies on antigenic variation in the context of a completely sequenced genome.
Assuntos
Regulação da Expressão Gênica , Genes Duplicados/genética , Genes de Protozoários/genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular , DNA Complementar/genética , Ordem dos Genes/genética , Ligação Genética/genética , Dados de Sequência Molecular , Família Multigênica/genética , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Pseudogenes/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retroelementos/genética , Análise de Sequência de DNA , Sequências de Repetição em Tandem/genética , Telômero/genética , Trypanosoma brucei brucei/imunologiaRESUMO
In yeast and mammalian cells, the cell cycle-dependent histone genes are typically expressed at a 15- to 35-fold higher level during S phase than during other phases of the cell cycle due to increases in both their transcription rates (three- to 17-fold) and the stabilities of their mRNAs (three to fivefold). In the protozoan trypanosomatids, most life cycle stage-specific genes are not regulated by changes in transcription rates, but are controlled entirely by post-transcriptional events. In contrast, little is known about cell cycle-dependent regulation of trypanosomatid genes. To examine cell cycle-associated expression of histone genes in a trypanosomatid, Trypanosoma cruzi epimastigotes were synchronized with hydroxyurea. The steady state levels of histone mRNAs in the G1, S and G2 phases of the cell cycle were found to vary only two- to fourfold, peaking in S phase. Nuclear run on assays showed that the histone genes are transcribed by RNA polymerase II and that their transcription rates do not increase in S phase relative to G1 and G2. Thus, during S phase of T. cruzi the increase in histone mRNA stability is about the same as in mammals and yeast, but no corresponding increase in the transcription rates of the histone genes occurs.
Assuntos
Ciclo Celular/fisiologia , Genes de Protozoários , Histonas/genética , Histonas/metabolismo , Processamento Pós-Transcricional do RNA , Trypanosoma cruzi/genética , Animais , Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Hidroxiureia/farmacologia , RNA Mensageiro/metabolismo , Trypanosoma cruzi/citologia , Trypanosoma cruzi/metabolismoRESUMO
We identified in a Trypanosoma brucei brucei strain (AnTat 1) an expression site for a metacyclic variant surface glycoprotein (MVSG) gene (MVSG) that was previously characterized in a T. b. rhodesiense strain (WRATat 1.1). The 3.4 kb sequences of the two expression sites are 99.6% identical, with no differences in the sequence of the 1.5 kb MVSG. Two other MVSGs in the WRATat 1.1 genome are not present in the AnTat 1 genome. In addition, five other T. b. brucei and T. b. rhodesiense strains, isolated in the same geographic region as the two former strains, do not contain any of these three MVSGs. Two of these five strains, however, appear to possess a very similar MVSG expression site, but with different MVSGs in it. Thus, the presence of the same MVSG in the same expression site in two different isolates is unusual and may be the result of genetic exchange in the field between T. b. brucei and T. b. rhodesiense isolates. Analysis of other African trypanosome strains for the presence of the three WRATat 1.1 MVSG expression sites demonstrated that the expression sites' promoter sequences are much more likely to be present than are specific MVSGs, suggesting that loss of MVSGs is the result of replacement by other VSGs. The promoter region of the MVSG expression site active in the WRATat 1.1 MVAT7 variant was found to be highly conserved among T. b. brucei, T. b. rhodesiense and T. b. gambiense group 2 isolates, whereas it does not occur in the T. b. gambiense group 1 isolates tested. A phylogenetic analysis of this promoter region sequence shows that the T. b. gambiense group 2 isolates form a monophyletic clade well separated from the T. b. brucei/T. b. rhodesiense isolates. Thus, whilst the T. b. brucei, T. b. rhodesiense and T. b. gambiense group 2 isolates are closely related but heterogenous, molecular tools may be developed to distinguish T. b. gambiense group 2 isolates from the others.
Assuntos
Genoma de Protozoário , Trypanosoma/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , África , Animais , Variação Genética/genética , Humanos , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas , Trypanosoma/classificação , Trypanosoma/imunologia , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismoRESUMO
GP63 is a 63-kDa glycoprotein that is abundantly expressed on the surface of all Leishmania species and is involved in several steps of promastigote infection of host cells. Leishmania chagasi has at least 18 haploid msp (major surface protease) genes encoding GP63 that are divided into three classes, mspS, mspL or mspC, according to their unique 3' UTR sequences and differential expression. All three msp classes are constitutively transcribed during virulent promastigote growth in vitro, although mspL mRNA is most abundant during logarithmic phase and mspS mRNA predominates in stationary phase. Thus, the steady state levels of the mspL and mspS mRNAs are post-transcriptionally regulated. Using Actinomycin D to arrest transcription, we found that in virulent promastigotes the half-life (t(1/2)) of mspL mRNA is coordinately modulated with growth phase, decreasing from a mean of 84 min during early logarithmic growth to a mean of 17 min at a stage intermediate between logarithmic and stationary phase. However, in attenuated promastigotes, the t(1/2) of mspL RNA remains the same throughout parasite growth. In contrast to mspL RNA, the t(1/2) of mspS and mspC RNA is constant throughout all growth phases of both virulent and attenuated promastigote growth. The presence of the translation inhibitor cycloheximide increases the t(1/2) of mspL RNA 4-6-fold in both virulent and attenuated promastigotes at all growth phases. These results indicate that the t(1/2) of mspL RNA is maintained by at least two distinct mechanisms - one activated during growth to stationary phase and the other dependent on a labile negative regulatory protein factor(s).
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Leishmania/patogenicidade , Metaloendopeptidases/genética , Estabilidade de RNA , RNA Mensageiro/genética , Animais , Cricetinae , Cicloeximida/farmacologia , Humanos , Leishmania/genética , Leishmania/crescimento & desenvolvimento , Leishmania/metabolismo , Leishmaniose Visceral/parasitologia , Metaloendopeptidases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Transcrição Gênica , VirulênciaRESUMO
The discovery of double-stranded RNA interference (dsRNAi) in Trypanosoma brucei provides a convenient method to generate knockout phenotypes in this protozoan parasite [Ngo H, Tschudi C, Gull K, Ullu E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 1998;95:14687-14692]. The presence of double-stranded RNA (dsRNA) dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous RNA to be degraded. To simplify the generation of knockout phenotypes in T. brucei via dsRNAi, we used two promoters arranged as an inverted repeat on a plasmid. This promoter arrangement generates transcripts of both strands of DNA inserted between the promoters, which then form dsRNA. We have used plasmids encoding either two T. brucei ribosomal RNA promoters or two bacteriophage T7 promoters to interfere with expression of alpha-tubulin (TUB), green fluorescent protein (GFP), paraflagellar rod protein A (PFRA), flagellum-adhesion glycoprotein 1 (FLA1), and histone 2B (H2B) in T. brucei. We show here that FLA1 is required for flagellar attachment in T. brucei and that H2B is required for parasite growth. Thus, the two-promoter approach efficiently generates dsRNAi in T. brucei and can be used to produce both specific and random knockout phenotypes in T. brucei. This approach should be useful in generating knockout phenotypes in other kinetoplastid parasites including Trypanosoma cruzi and Leishmania.
Assuntos
Inativação Gênica , Genes de Protozoários , Regiões Promotoras Genéticas , RNA de Cadeia Dupla/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Animais , Bacteriófago T7/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde , Histonas/genética , Histonas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fenótipo , Plasmídeos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoAssuntos
Regulação da Expressão Gênica no Desenvolvimento , Genes de Protozoários , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Northern Blotting , DNA Complementar , Dados de Sequência Molecular , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
The trypanosome cytoskeleton consists almost entirely of microtubule-based structures. Although alpha- and beta-tubulin from Trypanosoma brucei have been well characterized, much less is known about other cytoskeleton-associated proteins in trypanosomes. Using biochemical fractionation, we demonstrate here that T lymphocyte-triggering factor (TLTF) from T. brucei is a component of the detergent-resistant and Ca(2+)-resistant fraction of the parasite cytoskeleton. This fraction contains the flagellar apparatus and a subset of cytoskeletal protein complexes that together function in cell motility, cytokinesis, and organelle inheritance. We also show that TLTF-related genes are present in several highly divergent eukaryotic organisms. Although the function of the corresponding proteins is not known, the mammalian TLTF-like gene (GAS11; growth arrest-specific gene 11) is up-regulated in growth-arrested cells and is a candidate tumor suppressor (Whitmore, S. A., Settasatian, C., Crawford, J., Lower, K. M., McCallum, B., Seshadri, R., Cornelisse, C. J., Moerland, E. W., Cleton-Jansen, A. M., Tipping, A. J., Mathew, C. G., Savnio, M., Savoia, A., Verlander, P., Auerbach, A. D., Van Berkel, C., Pronk, J. C., Doggett, N. A., and Callen, D. F. (1998) Genomics 52, 325-331), suggestive of a role in coordinating cytoskeleton activities. Consistent with this possibility, we show that the human GAS11 protein contains a 144-amino acid domain that co-localizes with microtubules when fused to the green fluorescent protein and expressed in mammalian cells. These findings suggest that TLTF represents a newly defined protein family, whose members contribute to cytoskeleton function in species as diverse as protozoa and mammals.
Assuntos
Citoesqueleto/metabolismo , Flagelos/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/fisiologia , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Células COS , Cálcio/metabolismo , Proteínas do Citoesqueleto , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Microtúbulos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Regulação para CimaRESUMO
The genome of Trypanosoma cruzi contains tandem arrays of alternating genes encoding amastin and tuzin. Amastin is a surface glycoprotein abundantly expressed on the intracellular mammalian amastigote form of the protozoan parasite, and tuzin is a G-like protein. We demonstrated previously that the amastin-tuzin gene cluster is polycistronically transcribed to an equal extent in all parasite life cycle stages. The steady state level of amastin mRNA, however, is 68-fold more abundant in amastigotes than in epimastigotes. Here we show that the half-life of amastin mRNA is 7 times longer in amastigotes than in epimastigotes. Linker replacement experiments demonstrate that the middle one-third of the 630-nucleotide 3'-untranslated region (UTR) is responsible for the amastin mRNA up-regulation. This positive effect is dependent on the distance of the 3'-UTR segment from the stop codon and the polyadenylation site as well as on its orientation. A protein or protein complex more abundant in amastigotes than in epimastigotes binds to this minimally defined 3'-UTR segment and may be involved in its regulatory function.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Glicoproteínas de Membrana/genética , Proteínas de Protozoários/genética , RNA Mensageiro/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Sequência de Bases , Northern Blotting , Dactinomicina/farmacologia , Meia-Vida , Dados de Sequência Molecular , Inibidores da Síntese de Proteínas/farmacologia , Termodinâmica , Trypanosoma cruzi/genética , Regulação para CimaRESUMO
The haploid nuclear genome of the African trypanosome, Trypanosoma brucei, is about 35 Mb and varies in size among different trypanosome isolates by as much as 25%. The nuclear DNA of this diploid organism is distributed among three size classes of chromosomes: the megabase chromosomes of which there are at least 11 pairs ranging from 1 Mb to more than 6 Mb (numbered I-XI from smallest to largest); several intermediate chromosomes of 200-900 kb and uncertain ploidy; and about 100 linear minichromosomes of 50-150 kb. Size differences of as much as four-fold can occur, both between the two homologues of a megabase chromosome pair in a specific trypanosome isolate and among chromosome pairs in different isolates. The genomic DNA sequences determined to date indicated that about 50% of the genome is coding sequence. The chromosomal telomeres possess TTAGGG repeats and many, if not all, of the telomeres of the megabase and intermediate chromosomes are linked to expression sites for genes encoding variant surface glycoproteins (VSGs). The minichromosomes serve as repositories for VSG genes since some but not all of their telomeres are linked to unexpressed VSG genes. A gene discovery program, based on sequencing the ends of cloned genomic DNA fragments, has generated more than 20 Mb of discontinuous single-pass genomic sequence data during the past year, and the complete sequences of chromosomes I and II (about 1 Mb each) in T. brucei GUTat 10.1 are currently being determined. It is anticipated that the entire genomic sequence of this organism will be known in a few years. Analysis of a test microarray of 400 cDNAs and small random genomic DNA fragments probed with RNAs from two developmental stages of T. brucei demonstrates that the microarray technology can be used to identify batteries of genes differentially expressed during the various life cycle stages of this parasite.
Assuntos
Genoma de Protozoário , Trypanosoma brucei brucei/genética , Animais , Variação Antigênica , Etiquetas de Sequências Expressas , CariotipagemRESUMO
Cellular immune responses are required for protective immunity against Leishmania chagasi. Immunization strategies using live intracellular bacteria (e.g., bacille-Calmette Guerin strain of Mycobacterium bovis) expressing recombinant antigens can induce cellular immune responses to these antigens. Previous studies demonstrated that the L. chagasi antigen LCR1 stimulates IFN-gamma production from T cells of infected BALB/c mice, and immunization with recombinant LCR1 partially protects against L. chagasi infection. To determine whether live bacteria could enhance the immunization potential of LCR1, we engineered BCG expressing LCR1 (BCG-LCR1). Subcutaneous immunization with BCG-LCR1, but not with BCG containing plasmid only (BCG-pMV261), elicited better protective immunity against L. chagasi infection than LCR1 protein alone. BCG-LCR1 administered intraperitoneally did not protect. Splenocytes from mice immunized s.c. with either BCG-LCR1 or BCG-pMV261 and then infected with L. chagasi promastigotes had increased antigen-induced IFN-gamma and reduced IL-10 production compared to splenocytes of control mice. We propose that BCG-LCR1 promotes a Th1-type protective immune response, and it may be a useful component of a Leishmania vaccine.
Assuntos
Antígenos de Protozoários/imunologia , Vacina BCG , Leishmania infantum/imunologia , Leishmaniose Visceral/prevenção & controle , Vacinas Protozoárias , Vacinas Sintéticas , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Western Blotting , Células Cultivadas , Citocinas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologiaRESUMO
A search was conducted for transcriptional promoters in Trypanosoma congolense. A promoter test plasmid was constructed utilising the luciferase coding region flanked by the intergenic regions of a T. congolense gene encoding GARP, the glutamic acid and alanine rich protein on the surface of procyclic organisms. Using this plasmid, sequences located upstream of an 18S rRNA gene were tested in transient transfection assays for their ability to promote luciferase expression. A rRNA promoter fragment of 377 bp was identified that increases luciferase activity by as much as 35,000-fold above background levels. The rRNA transcription initiation site is located 961 bp upstream of the 18S rRNA gene and immediately downstream of 6 bp imperfect repeats. The plasmid was also used to examine sequences upstream of a GARP gene cluster in two different T. congolense strains for promoter activity. In contrast to the findings of another group, we were unable to detect promoter activity upstream of these GARP genes in either strain. We conclude that the GARP gene promoter, if it exists, has less than 0.03% (1/3000) of the activity of the rRNA promoter in this luciferase-based assay.
Assuntos
Genes de Protozoários , Glicoproteínas de Membrana/genética , Regiões Promotoras Genéticas , Proteínas de Protozoários/genética , RNA Ribossômico/genética , Trypanosoma congolense/genética , Animais , Sequência de Bases , Genes Reporter , Biblioteca Genômica , Dados de Sequência Molecular , Família Multigênica , Alinhamento de SequênciaRESUMO
An expression vector was constructed to express foreign genes in Trypanosoma congolense. The foreign gene and a neomycin phosphotransferase (NPT) gene are flanked by glutamate and alanine rich protein (GARP) gene processing signals and their expression is driven by a ribosomal RNA gene promoter. The plasmid is not maintained as an episome in T. congolense, but the NPT gene permits selection of cells in which the plasmid has integrated into the genome. We used this plasmid to express luciferase, green fluorescent protein and a surface protein of Trypanosoma brucei, glycine-proline-glutamate glutamate threonine procyclic acidic repetitive protein (GPEET PARP). The plasmid-derived GPEET PARP is expressed on the surface of procyclic T. congolense and comigrates on a polyacrylamide gel with native GPEET PARP from T. brucei procyclic cells. We also attempted to use the plasmid to overexpress a previously identified T. congolense cysteine protease. The plasmid-derived cysteine protease mRNA species occurs in the transfected cells, but we were unable to detect increased levels of protein or protease activity.
Assuntos
Vetores Genéticos , Proteínas Recombinantes/biossíntese , Trypanosoma congolense/genética , Sequência de Aminoácidos , Animais , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Gentamicinas/farmacologia , Canamicina Quinase/genética , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Oligopeptídeos/biossíntese , Oligopeptídeos/genética , Plasmídeos , Biossíntese de Proteínas , Proteínas/genética , Proteínas de Protozoários/genética , Recombinação Genética , Homologia de Sequência de AminoácidosRESUMO
Teixeira, S. M. R., Kirchhoff, L. V., and Donelson, J. E. 1999. Trypanosoma cruzi: Suppression of tuzin gene expression by its 5'-UTR and spliced leader addition site. Experimental Parasitology 93, 143-151. The genome of the protozoan parasite Trypanosoma cruzi contains a tandemly repeated array of two alternating genes, one encoding amastin and the other encoding tuzin. Amastin is an abundant amastigote surface protein, whereas tuzin is thought to be a rare protein whose location and function are unknown. The 137-nucleotide 5' untranslated region (5'-UTR) of the tuzin mRNA has a 22-codon open translation reading frame containing 3 methionine codons followed by a stop codon that overlaps the methionine start codon of the tuzin coding region. A fragment containing the tuzin 5'-UTR and upstream intergenic region was placed in front of a luciferase reporter gene in a plasmid for transient transfection assays of luciferase activity. By mutating the three upstream ATGs in the tuzin 5'-UTR and replacing the tuzin spliced leader (SL) acceptor site with that of the amastin gene, we found that the 22-codon reading frame and the tuzin SL acceptor site combine to substantially reduce expression of the luciferase gene. These results indicate that expression of the multicopy tuzin gene is posttranscriptionally suppressed by both inefficient RNA processing and poor translation initiation, resulting in a low level of tuzin.
Assuntos
Regiões 5' não Traduzidas/fisiologia , Regulação da Expressão Gênica , Proteínas de Protozoários/genética , RNA Líder para Processamento/fisiologia , Trypanosoma cruzi/genética , Animais , Western Blotting , Doença de Chagas/imunologia , Sequência Consenso , Genes Reporter , Soros Imunes/imunologia , Luciferases/genética , Luciferases/metabolismo , Glicoproteínas de Membrana/genética , Família Multigênica , Fases de Leitura Aberta/genética , Plasmídeos , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , RNA Mensageiro/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transfecção , Trypanosoma cruzi/metabolismoRESUMO
The flagellar pocket of African trypanosomes is a critical sorting station for protein and membrane trafficking, and is considered to be an Achilles' heel of this deadly pathogen. Although several proteins, including receptors for host-derived growth factors, are targeted specifically to the flagellar pocket, the signals responsible for this restricted subcellular localization are entirely unknown. Using T lymphocyte triggering factor-green fluorescent protein (TLTF(1)-GFP) fusion proteins, we demonstrate that an internal 144 amino acid domain of TLTF from Trypanosoma brucei is sufficient for directing GFP to the cytoplasmic side of the anterior flagellar pocket. Immuno-gold electron microscopy reveals that the TLTF-GFP fusion protein is located in an electron dense structure that immediately abuts the anterior flagellar pocket membrane. The amino acid sequence of the TLTF targeting domain does not resemble previously characterized protein trafficking signals, and random mutagenesis reveals that flagellar pocket targeting is conferred by a structural motif, rather than a short, contiguous array of amino acids. The aberrant sorting of two mutant proteins into the flagellum, and the targeting of a related human protein to the plus end of the trypanosome's cytoskeletal microtubules, lead us to suggest that flagellar pocket targeting involves interactions with the trypanosome cytoskeleton. The finding that TLTF-GFP is restricted to the anterior, cytoplasmic face of the flagellar pocket membrane, suggests that there is structural heterogeneity in the membrane of this organelle.
Assuntos
Flagelos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Citoesqueleto/metabolismo , Primers do DNA/genética , Flagelos/genética , Flagelos/ultraestrutura , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestruturaRESUMO
African trypanosomes evade the immune response of their mammalian hosts by switching the expression of their variant surface glycoprotein genes (vsg). The bloodstream trypanosome clone MVAT4 of Trypanosoma brucei rhodesiense expresses a metacyclic vsg as a monocistronic RNA from a promoter located 2 kilobases (kb) upstream of its start codon. Determination of 23 kb of sequence at the metacyclic variant antigen type 4 (MVAT) vsg expression site (ES) revealed an ES-associated gene (esag) 1 preceded by an ingi retroposon and an inverted region containing an unrelated vsg, short stretches of 70-bp repeats and a pseudo esag 3. Nuclear run-on experiments indicate that the 18-kb region upstream of the MVAT4 vsg promoter is transcriptionally silent. However, multiple members of different esag families are expressed from elsewhere in the genome. The MVAT4 vsg promoter is highly repressed in the procyclic stage, in contrast to the known polycistronic vsg ESs which undergo abortive transcription. Activation of the MVAT4 vsg ES occurs in situ without nucleotide sequence changes, although this monocistronic ES undergoes a pattern of base J modifications similar to that reported for the polycistronic ESs. The relative simplicity of the MVAT4 vsg ES and the uncoupled expression of the vsg and esags provide a unique opportunity for investigating the molecular mechanisms responsible for antigenic variation in African trypanosomes.
Assuntos
Genes de Protozoários , Proteínas de Protozoários , Transcrição Gênica , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Animais , Variação Antigênica , Sangue/parasitologia , Diferenciação Celular , Sequência Conservada , Dosagem de Genes , Regulação da Expressão Gênica , Ligação Genética , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Retroelementos , Telômero/genética , Trypanosoma brucei brucei/citologiaRESUMO
Trypanosoma brucei undergoes antigenic variation by periodically switching the expression of its variant surface glycoprotein (VSG) genes (vsg) among an estimated 20-40 telomere-linked expression sites (ES), only one of which is fully active at a given time. We found that in bloodstream trypanosomes one ES is transcribed at a high level and other ESs are expressed at low levels, resulting in organisms containing one abundant VSG mRNA and several rare VSG RNAs. Some of the rare VSG mRNAs come from monocistronic ESs in which the promoters are situated about 2 kilobases upstream of the vsg, in contrast to the polycistronic ESs in which the promoters are located 45-60 kilobases upstream of the vsg. The monocistronic ES containing the MVAT4 vsg does not include the ES-associated genes (esag) that occur between the promoter and the vsg in polycistronic ESs. However, bloodstream MVAT4 trypanosomes contain the mRNAs for many different ESAGs 6 and 7 (transferrin receptors), suggesting that polycistronic ESs are partially active in this clone. To explain these findings, we propose a model in which both mono- and polycistronic ESs are controlled by a similar mechanism throughout the parasite's life cycle. Certain VSGs are preferentially expressed in metacyclic versus bloodstream stages as a result of differences in ESAG expression and the proximity of the promoters to the vsg and telomere.