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PubMed1984

The small nuclear RNAs of Drosophila.

We have investigated the sequences of the major small nuclear RNAs of Drosophila cultured cells, with the objective of elucidating phylogenetically conserved primary and secondary structures by comparison of the data with previously determined sequences of these RNAs in vertebrate species. Our results reveal striking degrees of conservation between each Drosophila RNA and its vertebrate cognate, and also demonstrate blocks of homology among the Drosophila small nuclear RNAs, as previously described for vertebrates. The most conserved features include the 5' terminal region of U1 RNA, though to function in pre-mRNA splicing, most of the regions of U4 RNA recently implicated in 3' processing of pre-mRNA, and the major snRNP protein binding site ("domain A") that is also shared by vertebrate U1, U2, U4 and U5 RNAs. Several other conserved features have been revealed, suggesting additional regions of functional significance in these RNAs and also providing further insights into the evolutionary history of the small nuclear RNAs.

Animals↗
PubMed2006

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland.

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni(2+) chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.

Amino Acid Sequence↗
PubMed2005

Cloning and characterization of Xenopus beta2-microglobulin.

cDNAs for Xenopus beta2-microglobulin (beta2m), the obligatory light chain of most vertebrate Major Histocompatibility Complex (MHC) class I molecules, were isolated and ESTs were identified. Alignment of the deduced amino acid sequence to other species' beta2m showed that the overall structure is evolutionarily conserved, and phylogenetic analysis showed that the Xenopus beta2m sequence is intermediate between fish and bird/mammal beta2m. The Xenopus beta2m mRNA is expressed ubiquitously with highest expression in intestine, spleen, and thymus, correlating well with classical class Ia expression. beta2m mRNA and protein were also detected in Xenopus thymic tumor and kidney cell lines. Segregation analysis on a tetraploid Xenopus laevis family revealed two independently segregating, non-MHC-linked loci. As expected, only one locus was found in the diploid Xenopus tropicalis, strongly suggesting that the two beta2m loci in the tetraploid species were generated by genome-wide duplication, and did not undergo diploidization unlike many other MHC genes.

Amino Acid Sequence↗
PubMed2003

Identification, molecular characterization and expression of the gene encoding the epidermal growth factor receptor orthologue from the fox-tapeworm Echinococcus multilocularis.

Receptor tyrosine kinases (RTKs) are crucially involved in the development of metazoan organisms and possible mediators of cell-cell communication that occurs between eukaryotic parasites and hosts. We have now cloned and characterized the complete complementary DNA (cDNA) molecule encoding a novel receptor tyrosine kinase, EmER, of the human parasite Echinococcus multilocularis. EmER shared significant amino acid sequence homologies with members of the epidermal growth factor (EGF) receptor family of different phylogenetic origin, exhibited a domain structure which is typical for this group of membrane receptors and contained all catalytically important residues at the corresponding positions. Highest homologies were detected between EmER and a previously identified receptor kinase, SER, from the parasitic trematode Schistosoma mansoni. The EmER encoding gene, emer, spans a chromosomal region of 39 kb and is composed of 23 exons. Structural comparisons indicated that emer and the EGF receptor encoding genes from mammals derive from a common ancestor. DNA/DNA hybridization experiments demonstrated that emer is present as a single copy locus in the parasite. Transcriptional analyses on in vitro cultivated parasite larvae revealed that emer is expressed in both the metacestode and the protoscolex stage, although about 10-fold higher transcription levels were detectable for the protoscolex. Northern blot experiments further indicated that emer is expressed as a single 5.2-kb transcript in parasitic larvae during an infection of the intermediate host. These results suggest an involvement of EmER in echinococcal differentiation processes towards the protoscolex stage during natural infections and provide, for the first time, structural information on an epidermal growth factor receptor-like kinase from a cestode.

Amino Acid Sequence↗
PubMed2006

Molecular cloning and characterisation of a proteinase inhibitor, alpha 2-macroglobulin (alpha2-M) from the haemocytes of tiger shrimp Penaeus monodon.

An alpha 2-macroglobulin (alpha2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA consists of 4876 bp with an open reading frame (ORF) of 4494 bp, a 52 bp 5'-untranslated region, and a 327 bp 3'-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence. The predicted molecular mass of the mature protein (1480 amino acids) is 167.7 kDa with an estimated pI of 5.30. The P. monodon alpha2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate alpha2-Ms. Sequence comparison showed that alpha2-M deduced amino acid sequence of P. monodon has an overall similarity of 85, 52 and 49% to that of kuruma shrimp Marsupenaeus japonicus, American horseshoe crab Limulus polyphemus and mud crab Scylla serrata, respectively. Alignment of the deduced amino acid sequence to other species alpha2-M showed that the overall structure is evolutionarily conserved and phylogenetic analysis revealed that P. monodon alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of P. monodon increased significantly in 12, 24 and 48 h post-peptidoglycan (PG) injection, but returned to the original values in 72 h post-PG injection.

Amino Acid Sequence↗
PubMed2005

Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance.

A new species, Enterobacter ludwigii, is presented on the basis of the characteristics of 16 strains, which were isolated from clinical specimens. These bacteria form a distinct genetic cluster in phylogenetic analyses of the population structure of the Enterobacter cloacae complex. As determined by DNA-DNA cross-hybridization experiments in microplates, this genetic cluster can be delineated from the other species of the E. cloacae complex with deltaTm values equal to or above 5 degrees C with Enterobacter hormaechei being the closest relative. The bacteria are gram-negative, fermentative, motile rods with the general characteristics of the genus Enterobacter and the E. cloacae complex in particular. E. ludwigii can be differentiated from the other Enterobacter species by its growth on myo-inositol and 3-0-methyl-D-glucopyranose. The type strain is EN-119 (= DSM 16688T = CIP 108491T).

Bacterial Typing Techniques↗
PubMed2003

Genome sequence and organization of a nucleopolyhedrovirus isolated from the smaller tea tortrix, Adoxophyes honmai.

Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) has a distinctive pathology in A. honmai larvae, killing the host more slowly than other NPVs. To further understand the pathology of AdhoNPV, its genome was completely sequenced and compared with those of other baculoviruses. The AdhoNPV genome is 113,220 bp, with a G + C content of 35.6%. It contains 125 putative open reading frames (ORFs), of which 8 are unique to AdhoNPV, and 4 homologous regions. The other 117 ORFs display similarity to previously characterized baculovirus genes involved in early and late gene expression, DNA replication, and structural and auxiliary functions. The phylogenetic position of AdhoNPV, in relation to 15 other baculoviruses whose genomes have been completely sequenced, was assessed by three different analyses: gene sequence, gene order, and gene content. Although gene content analysis failed to support the group II NPVs, phylogenetic trees based on gene sequence and gene order showed AdhoNPV to be closely related to the group II NPVs.

Amino Acid Sequence↗
PubMed2002

Molecular cloning and characterization of a new linalool synthase.

Mentha citrata Ehrh. (bergamot mint; Lamiaceae) produces an essential oil containing only the acyclic monoterpenol (-)-3R-linalool and its acetate ester. A cloning strategy based upon the assumption that the responsible monoterpene synthase would resemble, in sequence, monoterpene cyclases from this plant family yielded a cDNA encoding the (--)-3R-linalool synthase. The nucleotide sequence of this monoterpene synthase is similar to those of several monoterpene cyclases from the mint (Lamiaceae) family (62-72% identity), but differs substantially from that of 3S-linalool synthase from Clarkia (41% identity; this composite gene appears to be of recent origin) and from that of 3R-linalool synthase from Artemisia (52% identity; the functional role of this gene is uncertain). Heterologous expression in Escherichia coli of a truncated version of the cDNA (in which the plastidial transit peptide was deleted) allowed purification and characterization of the enzyme, which was shown to possess most properties similar to other known monoterpene cyclases, but with a K(m) value for the natural substrate, geranyl diphosphate, of 56 microM with k(cat) of 0.83 s(-1). These kinetic constants for this 3R-linalool synthase are higher than those of any defined monoterpene cyclase, but the kinetic efficiency does not approach that reported for the 3S-linalool synthase from Clarkia. Although linalyl diphosphate is an enzyme-bound intermediate of monoterpene cyclase reactions, this tertiary allylic isomer of the geranyl substrate is not an efficient precursor of linalool with the M. citrata synthase. Modeling of the active site of this linalool synthase from Mentha and comparison to the modeled active sites of phylogenetically related monoterpene cyclases revealed structural differences in the binding of the diphosphate moiety which initiates the ionization step of the electrophilic reaction sequence and in the access of water to the active site to permit stereoselective quenching of the initially formed carbocationic intermediate to produce 3R-linalool.

Binding Sites↗
PubMedDate not supplied

Recombinant Plasmodium falciparum NADP-dependent isocitrate dehydrogenase is active and harbours a unique 26 amino acid tail.

During infection, Plasmodium spp. require reducing equivalents such as NADPH to support the function of specific enzymes in overcoming oxidative stress. The catalysis of isocitrate by the NADP-dependent isocitrate dehydrogenase of Plasmodium falciparum (pfICDH) generates NADPH and is thus crucial for the parasite's survival and pathogenecity. In this study, pfICDH was cloned from a clinical isolate of P. falciparum. This was facilitated by designing primers based on the P. falciparum genome sequence resource PlasmoDB. DNA sequence of the cloned gene revealed an ORF that encodes a protein of 468 aa. Furthermore, after expression in Esherichia coli BL21, enzyme assays of cell-free extracts confirmed the overexpression and function of pfICDH. Further, pfICDH purified by affinity chromatography retained its enzyme activity. Substitution of NADP for NAD, or the use of EDTA, in enzyme assays abolished pfICDH activity. ATP and chloroquine, as well as cupric and argentic ions, inhibited pfICDH activity. Phylogenetic analysis revealed high primary structure homology (45-97%) among genes coding for eukaryal NADP-dependent ICDH, and the occurrence of three subfamilies of ICDH genes. Interestingly, there were significant sequence dissimilarities between pfICDH and its mammalian or bacterial homologs, particularly at the N- and C-termini. Confirming the functionality of the cloned pfICDH, and asserting its distance from the human homolog by molecular definitions, are important prerequisites for promoting this gene as a drug target screen.

Amino Acid Sequence↗
PubMed2000

Adenylylsulfate reductases from archaea and bacteria are 1:1 alphabeta-heterodimeric iron-sulfur flavoenzymes--high similarity of molecular properties emphasizes their central role in sulfur metabolism.

Highly active adenylylsulfate (APS) reductase was isolated under N(2)/H(2) from sulfate-reducing and sulfide-oxidizing bacteria and archaea. It was a 1:1 alphabeta-heterodimer of molecular mass approximately 95 kDa, and two subunits (alpha approximately 75, beta approximately 20 kDa). The specific activity was 11-14 micromol (min mg)(-1); cofactor analysis revealed 0.96+/-0.05 FAD, 7.5+/-0.1 Fe and 7.9+/-0.25 S(2-). The photochemically reduced enzyme had a multiline EPR spectrum resulting from two interacting [4Fe-4S] centers. The properties of the different APS reductases were remarkably similar, although the enzyme is involved in different metabolic pathways and was isolated from phylogenetically far separated organisms. A structural model is proposed, with FAD bound to the alpha-subunit, and two [4Fe-4S] centers located in close proximity on the beta-subunit.

Adenosine Monophosphate↗
PubMed1998

African origin of GB virus C/hepatitis G virus.

Ninety-four GB virus C/hepatitis G virus (GBV-C/ HGV) RNA-positive serum samples were obtained from all over the world. We found that all 15 GBV-C/HGV isolates from the Pygmies and the Bantu in the Central African region had a 12-amino acid indel (i.e. insertion or deletion) in the non-structural protein (NS) 5A region. Phylogenetic analyses of the NS5A region, using GBV-A as an outgroup, showed that these 15 isolates had diverged from the common ancestor much earlier than the remaining isolates, indicating an African origin of GBV-C/HGV.

Africa↗
PubMed2002

Investigating patterns may reveal processes: evolutionary ecology of ectoparasitic monogeneans.

We reviewed several published and ongoing studies concerning monogenean communities. Patterns of species richness, host specificity, community structure and host--parasite coevolutionary interaction were carefully analysed, and hypotheses of evolutionary processes are proposed. The structuring of monogenean communities seems to be related to both ecological and historical constraints. The database supports an absence of intra- and interspecific competition in monogeneans. Species richness seems to be more due to host characteristics than to parasite interactions. Monogeneans seem to specialise on large hosts, leading to greater species richness on those hosts. The morphometric evolution of attachment and copulatory organs support the hypothesis of a reproductive segregation among conspecifics parasitising the same host(s). It also suggests the existence of concurrent adaptive and non-adaptive processes. The general absence of a coevolutionary pattern between host and parasites also suggests the constraints of history without dismissing the influences of ecological factors in the structuring of the communities. More generally, we strengthen the need to study the structure of communities in a phylogenetic context.

Animals↗
PubMed2005

The genetics of ATP-binding cassette transporters.

The ATP-binding cassette (ABC) superfamily consists of membrane proteins that transport a wide variety of substrates across membranes. Mutations in ABC transporters cause or contribute to a number of different Mendelian disorders, including adrenoleukodystrophy, cystic fibrosis, retinal degeneration, cholesterol, and bile transport defects. In addition, the genes are involved in an increasing number of complex disorders. The proteins play essential roles in the protection of organisms from toxic metabolites and compounds in the diet and are involved in the transport of compounds across the intestine, blood-brain barrier, and the placenta. There are 48 ABC genes in the human genome divided into seven subfamilies based in gene structure, amino acid alignment, and phylogenetic analysis. These seven subfamilies are found in all other sequenced eukaryotic genomes and are of ancient origin. Further characterization of all ABC genes from humans and model organisms will lead to additional insights into normal physiology and human disease.

ATP-Binding Cassette Transporters↗
PubMed1975

Psychobiology of partnership behaviour.

Animal experiments demonstrate that it is not only the quality of transmitted and received social signals that is important, but also their frequency and the timing of the information transmitted. In order for progress to be made in the investigation of human social behaviour and its disorders, methods must be developed which allow the transmission of verbal and non-verbal information to be measured. Experiments carried out with healthy adults and healthy and disturbed children to investigate human eye contact and distance behaviour are reported, along with experiments on the influence of gaze and body posture on spoken communication. Finally, a report on the use of behaviour therapy for an autistic child is outlined in order to explore the psychobiological correlations between social behaviour and language, which concur with extensive experiments on brain stimulation. It is suggested that there is a cerebral representation for species-specific social behaviour and a vocalization system embedded in these brain structures which is a phylogenetically-patterned prerequisite for the development of human language.

Animals↗
PubMed2002

Identification and quantification of feathers, down, and hair of avian and mammalian origin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We describe a fast method for the identification of the origin of native and chemically processed feathers, down, and hair and for distinguishing closely related species using enzyme digestion and MALDI-TOF mass spectrometry. Additionally we present two methods for the quantification of different identified bird and mammalian samples, respectively, in binary species mixtures. Without any prior cleaning or isolation of single proteins, enzymatical digests of feathers, down, and hair are performed. Fragments generated are analyzed by MALDI-TOF mass spectrometry, and peak groups of different selectivity are established for every animal species. For the identification of individual animal species, only unique species-specific peaks are accepted while for species classification of tinted or bleached hair, the group of semispecific peaks (SEMPs) is used. Samples from native animal species show a higher coincidence of digest peak masses of SEMPs than far-related species, indicating a phylogenetical relationship of the investigated structure proteins. Quantification of mixed binary avian samples is performed by identification of preweighed single feathers and down, followed by calculation of the gravimetric mass fractions. The composition of mixed binary mammalian samples is calculated from correlation of the quotient of the relative peak intensities or areas of these species and the quotient of corresponding gravimetric mass fractions. High accuracy is achieved by both quantification methods. The fast methods are well suited for industrial quality control for example.

Animals↗
PubMed1999

Tribosphenic mammal from the North American Early Cretaceous.

The main groups of living mammals, marsupials and eutherians, are presumed to have diverged in the Early Cretaceous, but their early history and biogeography are poorly understood. Dental remains have suggested that the eutherians may have originated in Asia, spreading to North America in the Late Cretaceous, where an endemic radiation of marsupials was already well underway. Here I describe a new tribosphenic mammal (a mammal with lower molar heels that are three-cusped and basined) from the Early Cretaceous of North America, based on an unusually complete specimen. The new taxon bears characteristics (molarized last premolar, reduction to three molars) otherwise known only for Eutheria among the tribosphenic mammals. Morphometric analysis and character comparisons show, however, that its molar structure is primitive (and thus phylogenetically uninformative), emphasizing the need for caution in interpretation of isolated teeth. The new mammal is approximately contemporaneous with the oldest known Eutheria from Asia. If it is a eutherian, as is indicated by the available evidence, then this group was far more widely distributed in the Early Cretaceous than previously appreciated. An early presence of Eutheria in North America offers a potential source for the continent's Late Cretaceous radiations, which have, in part, proven difficult to relate to contemporary taxa in Asia.

Journal Article↗
PubMed1998

Molecular cloning and nucleotide sequence of a cytochrome P450 cDNA from a pyrethroid-resistant mosquito, Culex quinquefasciatus say.

A novel cDNA clone encoding a cytochrome P450 was screened from a gut cDNA library of permethrin-resistant Culex quinquefasciatus larvae. Nucleotide sequence of the clone designated CYP6E1 was determined. This is the first full-length sequence of a mosquito P450 cDNA. Comparison of the deduced amino acid sequence of CYP6E1 to those of other known cytochrome P450s indicates that this clone belongs to CYP6 family. In addition, a phylogenetic analysis of CYP6E1 primary structure showed that it is related to CYP6A and CYP6C subfamilies. Polymerase chain reaction (PCR) using primers designed on the basis of the nucleotide sequence of the cDNA showed that mRNA encoding CYP6E1 is present in permethrin-susceptible larvae as well as -resistant larvae, suggesting the ubiquitous distribution of CYP6E1.

Amino Acid Sequence↗
PubMed1999

Amylase and 16S rRNA genes from a hyperthermophilic archaebacterium.

A hyperthermophilic and amylolytic prokaryote, designated Rt3, was isolated from a thermal spring near Rotorua, New Zealand. The 16S rRNA gene of Rt3 was cloned and sequenced with the aim of determining its phylogenetic affiliations. The phylogenetic analysis of this sequence, which included a selection of archaebacterial and eubacterial 16S rRNA sequences, indicates that Rt3 most likely belongs to the archaebacterial order Thermococcales. An amylase gene (amyA) from Rt3, encoding a highly thermostable amylase activity, was cloned and its DNA sequence determined. Transcriptional signals typical of archaebacteria were evident in this sequence. The sequence is homologous to a broad range of enzymes from the AMY superfamily and contains a typical N-terminal signal peptide. Phylogenetic analysis and comparison of structural features with other AMY superfamily enzymes reveals that, firstly, the closest homologues of the Rt3 amylase are members of the Bacillus and Plant alpha-amylase groups; and secondly, that the Rt3 amylase is closely related to only one other currently known archaebacterial enzyme, i.e. an (AMY superfamily) alpha-amylase from Natronococcus.

Amino Acid Sequence↗