Computational identification of four spliceosomal snRNAs from the deep-branching eukaryote Giardia intestinalis

RNAs processing other RNAs is very general in eukaryotes, but is not clear to what extent it is ancestral to eukaryotes. Here we focus on pre-mRNA splicing, one of the most important RNA-processing mechanisms in eukaryotes. In most eukaryotes splicing is predominantly catalysed by the major spliceosome complex, which consists of five uridine-rich small nuclear RNAs (U-snRNAs) and over 200 proteins in humans. Three major spliceosomal introns have been found experimentally in Giardia; one Giardia U-snRNA (U5) and a number of spliceosomal proteins have also been identified. However, because of the low sequence similarity between the Giardia ncRNAs and those of other eukaryotes, the other U-snRNAs of Giardia had not been found. Using two computational methods, candidates for Giardia U1, U2, U4 and U6 snRNAs were identified in this study and shown by RT-PCR to be expressed. We found that identifying a U2 candidate helped identify U6 and U4 based on interactions between them. Secondary structural modelling of the Giardia U-snRNA candidates revealed typical features of eukaryotic U-snRNAs. We demonstrate a successful approach to combine computational and experimental methods to identify expected ncRNAs in a highly divergent protist genome. Our findings reinforce the conclusion that spliceosomal small-nuclear RNAs existed in the last common ancestor of eukaryotes.     

Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia

Giardia is a eukaryotic protozoal parasite with unusual characteristics, such as the absence of a morphologically evident Golgi apparatus. Although both constitutive and regulated pathways for protein secretion are evident in Giardia, little is known about the mechanisms involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle-associated membrane protein and syntaxin families play essential roles in these processes. In this work we identified and characterized genes for 17 SNAREs in Giardia to define the minimal set of subcellular organelles present during growth and encystation, in particular the presence or not of a Golgi apparatus. Expression and localization of all Giardia SNAREs demonstrate their presence in distinct subcellular compartments, which may represent the extent of the endomembrane system in eukaryotes. Remarkably, Giardia SNAREs, homologous to Golgi SNAREs from other organisms, do not allow the detection of a typical Golgi apparatus in either proliferating or differentiating trophozoites. However, some features of the Golgi, such as the packaging and sorting function, seem to be performed by the endoplasmic reticulum and/or the nuclear envelope. Moreover, depletion of individual genes demonstrated that several SNAREs are essential for viability, whereas others are dispensable. Thus, Giardia requires a smaller number of SNAREs compared with other eukaryotes to accomplish all of the vesicle trafficking events that are critical for the growth and differentiation of this important human pathogen.     

Chromatin and histones from Giardia lamblia: a new puzzle in primitive eukaryotes

The three deepest eukaryote lineages in small subunit ribosomal RNA phylogenies are the amitochondriate Microsporidia, Metamonada, and Parabasalia. They are followed by either the Euglenozoa (e.g., Euglena and Trypanosoma) or the Percolozoa as the first mitochondria-containing eukaryotes. Considering the great divergence of histone proteins in protozoa we have extended our studies of histones from Trypanosomes (Trypanosoma cruzi, Crithidia fasciculata and Leishmania mexicana) to the Metamonada Giardia lamblia, since Giardia is thought to be one of the most primitive eukaryotes. In the present work, the structure of G. lamblia chromatin and the histone content of the soluble chromatin were investigated and compared with that of higher eukaryotes, represented by calf thymus. The chromatin is present as nucleosome filaments which resemble the calf thymus array in that they show a more regular arrangement than those described for Trypanosoma. SDS-polyacrylamide gel electrophoresis and protein characterization revealed that the four core histones described in Giardia are in the same range of divergence with the histones from other lower eukaryotes. In addition, G. lamblia presented an H1 histone with electrophoretic mobility resembling the H1 of higher eukaryotes, in spite of the fact that H1 has a different molecular mass in calf thymus. Giardia also presents a basic protein which was identified as an HU-like DNA-binding protein usually present in eubacteria, indicating a chimaeric composition for the DNA-binding protein set in this species. Finally, the phylogenetic analysis of selected core histone protein sequences place Giardia divergence before Trypanosoma, despite the fact that Trypanosoma branch shows an acceleration in the evolutionary rate pointing to an unusual evolutionary behavior in this lineage.     

Core histone genes of <Emphasis Type="Italic">Giardia intestinalis</Emphasis>: genomic organization,promoter structure,and expression

Background  

Giardia intestinalis is a protist found in freshwaters worldwide, and is the most common cause of parasitic diarrhea in humans. The phylogenetic position of this parasite is still much debated. Histones are small, highly conserved proteins that associate tightly with DNA to form chromatin within the nucleus. There are two classes of core histone genes in higher eukaryotes: DNA replication-independent histones and DNA replication-dependent ones.     

Giardia lamblia -- a model organism for eukaryotic cell differentiation

Giardia lamblia is a binucleated, flagellated protozoan parasite that inhabits the upper small intestine of its vertebrate hosts. The entire life cycle, which can be completed in vitro, is simple with cycling between a vegetative trophozoite and a highly resistant cystic form. The parasite is one of the earliest diverging eukaryotes known and more than 95% of the genome is sequenced. This makes Giardia an excellent model system for studies of basic eukaryotic processes like cell differentiation. In this review we will discuss recent data concerning Giardia differentiation with a focus on DNA replication and cytokinesis.     

Expression of tubulin polyglycylation in Giardia lamblia

By means of immunofluorescence, immunoelectron microscopy and immunoblotting, we show that polyglycylation, a posttranslational modification of tubulin widely spread among eukaryotes, is present in the diplomonad, Giardia lamblia, a putative ancestral cell possessing a highly developed microtubular cytoskeleton. This modification was recently discovered in the ciliated protist, Paramecium, and was not found in the Euglenozoa, a lineage considered as ancient. We used two monoclonal antibodies (mAbs), TAP 952 and AXO 49, specifically recognizing mono- and polyglycylated tubulin isoforms, to detect this modification in Giardia extracts and to localize it in the different classes of microtubules within the cell. The alpha- and beta-tubulin subunits were recognized by the two mAbs, indicating that both tubulin subunits are glycylated, in agreement with lately reported mass spectrometry results. Noticeably, Giardia tubulin was much more reactive with AXO 49 than with TAP 952. In situ, AXO 49 intensely labeled the microtubules present in the four pairs of flagella and the median body, and lightly decorated the microtubules from the adhesive disc. In contrast, TAP 952 intensely labeled only the microtubules of the median body. The results indicate a differential expression of glycylated isoforms within various microtubular structures of Giardia lamblia. They also suggest that the complete set of enzymes required for polyglycylation is expressed in very divergent eukaryotes.     

从基因组分析贾第虫的核糖体合成系统

为探讨贾第虫细胞核内核糖体合成系统,及与典型的真核生物有何差异,首先,确定在典型真核生物中参与核糖体合成的129条共有的保守蛋白,然后用这些蛋白搜索贾第虫基因组以调查它们在贾第虫中的直系同源蛋白的情况,以对贾第虫的核糖体合成系统作一了解。结果表明：贾第虫具有89条这些蛋白的直系同源蛋白,包括参与rRNA甲基化和假尿嘧啶化的蛋白复合体成员,以及存在于90S、40S和60S复合体中的蛋白。贾第虫的核糖体合成系统与典型的真核生物相似,但还有40条蛋白在贾第虫基因组中找不到同源蛋白。这意味着贾第虫的核糖体合成系统较典型的真核生物简单。贾第虫虽然没有核仁结构,但其核糖体亚基合成的途径和机制可能与真核细胞相似,参与的成分不同于无核仁结构的原核生物,可能相对简单。     

生物素标记贾第虫全基因组DNA探针的制备及其特异性敏感性测定

用生物素标记的贾第虫全基因组ＤＮＡ探针,在斑点杂交试验中显示高度的敏感性和特异性。用它可检出１０ｎｇ贾第虫ＤＡ,１０＾３个贾第虫滋养体或包囊,且不与阴道毛滴虫、溶组织内阿米巴、弓形虫和ＢＡＢＬ／ｃ小鼠肝细胞ＤＮＡ,以及贾第虫患者粪便上清液发生交叉反应。本探针可用于贾第虫病病原体检测和虫株鉴定研究。     

DNA “fingerprints” and paternity ascertainment in chimpanzees (Pan troglodytes)

Highly variable regions of DNA are found in a wide diversity of organisms and are typically composed of alleles consisting of a variable number of tandem repeats (VNTRs) of a short core sequence. DNA fingerprinting probes are VNTR probes that simultaneously detect a large number of similar VNTRs in the target DNA. The highly polymorphic pattern observed in a DNA fingerprint allows resolution of questions concerning individual identification. M13 phage was used to fingerprint captive chimpanzees for paternity ascertainment. Although the probability of band sharing among captive chimps appears to be higher than among some other reported captive and feral animal populations, the probe is highly useful and can be expected to become more widely used in the genetic management of captive populations.     

Pyruvate-phosphate dikinase of oxymonads and parabasalia and the evolution of pyrophosphate-dependent glycolysis in anaerobic eukaryotes

In pyrophosphate-dependent glycolysis, the ATP/ADP-dependent enzymes phosphofructokinase (PFK) and pyruvate kinase are replaced by the pyrophosphate-dependent PFK and pyruvate phosphate dikinase (PPDK), respectively. This variant of glycolysis is widespread among bacteria, but it also occurs in a few parasitic anaerobic eukaryotes such as Giardia and Entamoeba spp. We sequenced two genes for PPDK from the amitochondriate oxymonad Streblomastix strix and found evidence for PPDK in Trichomonas vaginalis and other parabasalia, where this enzyme was thought to be absent. The Streblomastix and Giardia genes may be related to one another, but those of Entamoeba and perhaps Trichomonas are distinct and more closely related to bacterial homologues. These findings suggest that pyrophosphate-dependent glycolysis is more widespread in eukaryotes than previously thought, enzymes from the pathway coexists with ATP-dependent more often than previously thought and may be spread by lateral transfer of genes for pyrophosphate-dependent enzymes from bacteria.     

A novel molecular fingerprint probe based on the endogenous avian retroviral element (EAV) of chickens

We have developed a novel molecular probe that is useful for DNA fingerprint analysis in chickens. The probe is based on the middle-repetitive, chicken endogenous retroviral (EAV) element. It consists of 1503 bp of the 3′ portion of the EAV element, extending from the downstream end of the envelope gene to the beginning of the downstream long terminal repeat (LTR). Unlike other probes that are currently in use for fingerprint analysis with chicken DNA, the EAV-based probe works well at normal levels of stringency, and with standard hybridization buffers. Digestion of chicken genomic DNA with a variety of restriction enzymes routinely yields up to 30 resolvable bands per bird in the 500 bp to 20kbp range. In order to test the efficacy of the EAV-based fingerprint probe, we have used it to estimate the degree of inbreeding in the inbred WG strain of White Leghorns. We find that the estimates derived with the EAV probe are very similar to those reported previously for the WG strain. These results suggest that molecular probes based on endogenous retroviruses and other middle-repetitive DNA elements should be useful for fingerprint analysis in chickens, and in vertebrates in general.     

Evolution of the Isd11-IscS complex reveals a single alpha-proteobacterial endosymbiosis for all eukaryotes

Giardia and Trichomonas are eukaryotes without standard mitochondria but contain mitochondrial-type alpha-proteobacterium-derived iron-sulfur cluster (ISC) assembly proteins, located to mitosomes in Giardia and hydrogenosomes in Trichomonas. Although these data suggest a single common endosymbiotic ancestry for mitochondria, mitosomes, and hydrogenosomes, separate origins are still being proposed. Here, we present a bioinformatic analysis of Isd11, a recently described essential component of the mitochondrial ISC assembly pathway. Isd11 is unique to eukaryotes but functions closely with the alpha-proteobacterium-derived cysteine desulfurase IscS. We demonstrate the presence of homologues of Isd11 in all 5 eukaryotic supergroups sampled, including hydrogenosomal and mitosomal lineages. The eukaryotic invention of Isd11 as a functional partner to IscS directly implies a single shared alpha-proteobacterial endosymbiotic ancestry for all eukaryotes. This pinpoints the alpha-proteobacterial endosymbiosis to before the last common ancestor of all eukaryotes without ambiguity.     

Giardia sp.: comparison of electrophoretic karyotypes

Species in the genus Giardia have been named on the basis of host specificity, cell dimensions, and median body morphology. Despite these criteria, the species taxonomy of Giardia is still in question. To investigate Giardia taxonomy on a molecular level, Giardia chromosomal DNA was analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE) and transverse alternating field electrophoresis (TAFE). Chromosomal DNA of G. duodenalis isolates (human, muskrat, sheep, dog, beaver), G. muris (mouse), and G. ardeae (great blue heron) were subjected to OFAGE and TAFE analyses. Comparable DNA patterns were obtained by both electrophoretic methods, but OFAGE required 8 days while TAFE required only 3 days. DNA patterns among all G. duodenalis isolates, although quite similar to each other, were distinctly different from those of G. muris and G. ardeae; G. muris and G. ardeae DNA patterns were distinctly different from each other. A G. duodenalis (Portland 1) total DNA probe hybridized to the DNA of all G. duodenalis isolates on Southern blots, but not detectably to G. muris and G. ardeae DNA. Similarly, G. muris and G. ardeae total DNA probes only hybridized detectably to their respective DNA. One probe that appears to hybridize to the DNA of all G. duodenalis and to G. ardeae DNA rather than G. muris DNA has been developed. Another probe that hybridizes only to G. muris and G. ardeae DNA has been developed. These data suggest that the differentiation of Giardia isolated from host and environmental samples may eventually be accomplished by DNA probes. Additionally, these techniques perhaps combined with other criteria may lead to the establishment of a sound taxonomic scheme for this genus.     

Evaluation of ��-Tubulin as an Antigenic and Molecular Probe to Detect Giardia lamblia

The α/β-tubulin heterodimer is the basic subunit of microtubules in eukaryotes. Polyclonal antibodies specific to recombinant α-tubulin of Giardia lamblia were made, and found effective as a probe to specifically detect G. lamblia by immunofluorescence assays. Nucleotide sequences of α-tubulin genes were compared between G. lamblia WB and GS strains, prototypes of assemblage A and assemblage B, respectively. A set of primers was designed and used to amplify a portion of the α-tubulin gene from G. lamblia. PCR-RFLP analysis of this α-tubulin PCR product successfully differentiated G. lamblia into 2 distinct groups, assemblages A and B. The results indicate that α-tubulin can be used as a molecular probe to detect G. lamblia.     

DNA fingerprinting analysis of vegetative compatibility groups in Aspergillus caelatus

Forty-three isolates of Aspergillus caelatus, whose vegetative compatibility groups (VCGs) have been identified, were assessed by DNA fingerprinting using a repetitive sequence DNA probe (pAF28) cloned from A. flavus. Thirteen distinct DNA fingerprint groups or genotypes were identified among the 43 isolates. Twenty-four isolates belonging to VCG 1 produced identical DNA fingerprints and included isolates from the United States and Japan. Four other DNA fingerprint groups had multiple isolates sharing identical fingerprints corresponding to VCGs 2, 3, 12 and 13. Eight of the 13 fingerprint groups corresponding to VCGs 4-11 were represented by a single isolate with a unique fingerprint pattern. These results provide further confirmation that the pAF28 probe can distinguish VCGs of species within Aspergillus section Flavi based on DNA fingerprint patterns and that the probe can be used to estimate the number of VCGs in a sample population. Most of the A. caelatus isolates produced fewer restriction fragments and weakly hybridized with the repetitive DNA probe pAF28 compared to hybridization patterns obtained with A. flavus, suggesting less homology of the probe to A. caelatus genomic DNA.     

WhatEntamoeba histolytica andGiardia lamblia tell us about the evolution of eukaryotic diversity

Entamoeba histolytica andGiardia lamblia are microaerophilic protists, which have long been considered models of ancient pre-mitochondriate eukaryotes. As transitional eukaryotes, amoebae and giardia appeared to lack organelles of higher eukaryotes and to depend upon energy metabolism appropriate for anaerobic conditions early in the history of the planet. However, our studies have shown that amoebae and giardia contain splicoeosomal introns, ras-family signal-transduction proteins, ATP-binding casettes (ABC)-family drug transporters, Golgi, and a mitochondrion-derived organelle (amoebae only). These results suggest that most of the organelles of higher eukaryotes were present in the common ancestor of all eukaryotes, and so dispute the notion of transitional eukaryotic forms. In addition, phylogenetic studies suggest many of the genes encoding the fermentation enzymes of amoebae and giardia derive from prokaryotes by lateral gene transfer (LGT). While LGT has recently been shown to be an important determinant of prokaryotic evolution, this is the first time that LGT has been shown to be an important determinant of eukaryotic evolution. Further, amoebae contain cyst wall-associated lectins, which resemble, but are distinct from lectins in the walls of insects (convergent evolution). Giardia have a novel microtubule-associated structure which tethers together pairs of nuclei during cell division. It appears then that amoebae and giardia tell us less about the origins of eukaryotes and more about the origins of eukaryotic diversity.     

Fibrillarin, A Conserved Pre-ribosomal RNA Processing Protein of Giardia

ABSTRACT The flagellated protozoan Giardia has been shown by 16S rRNA sequence analysis to be one of the most primitive of the eukaryotes. A gene encoding the protein fibrillarin, a pre-rRNA processing protein implicated in rRNA methylation and ribosome assembly, has been isolated. A genomic DN'A fragment 1,240 base pairs long containing an open reading frame of 981 base pairs (327 amino acids) was sequenced. The deduced protein sequence of 35.3 kDa is similar to other known fibrillarin sequences. The Giardia sequence includes the amino terminal glycine/arginine rich domain characteristic of eukaryotic fibrillarins but is unique in having a large number of acidic residues in this domain. Phylogenetic analysis of the available fibrillarin sequences is consistent with the assignment of Giardia to a position close to the most primitive of the eukaryotes. A monoclonal antibody to yeast fibrillarin crossreacts with a 36 kDa polypeptide from Giardia on western blots and diffusely stains both nuclei of the organism by immunofluorescence microscopy. This result is consistent with the absence of well defined nucleoli in this organism. The evolutionary conservation of fibrillarin suggests an important function for this protein in ribosome biosynthesis, and this function appears to be maintained from the archaebacteria, which lack a nucleus, to Giardia , which contains a nucleus but lacks a prominent nucleolus, to higher mammals, which have both nucleus and nucleolus.