Molecular phylogeny of parasitic Platyhelminthes based on sequences of partial 28S rDNA D1 and mitochondrial cytochrome c oxidase subunit I

The phylogenic relationships existing among 14 parasitic Platyhelminthes in the Republic of Korea were investigated via the use of the partial 28S ribosomal DNA (rDNA) D1 region and the partial mitochondrial cytochrome c oxidase subunit 1 (mCOI) DNA sequences. The nucleotide sequences were analyzed by length, G + C %, nucleotide differences and gaps in order to determine the analyzed phylogenic relationships. The phylogenic patterns of the 28S rDNA D1 and mCOI regions were closely related within the same class and order as analyzed by the PAUP 4.0 program, with the exception of a few species. These findings indicate that the 28S rDNA gene sequence is more highly conserved than are the mCOI gene sequences. The 28S rDNA gene may prove useful in studies of the systematics and population genetic structures of parasitic Platyhelminthes.     

Expression of myelin protein genes in the developing brain

The major myelin proteins fall into two classes, the basic proteins and the proteolipid proteins. In mice, five forms of the myelin basic protein (MBP) have been identified with apparent molecular masses of 21.5 kD, 18.5 kD, 17 kD and 14 kD. The 17 kD MBP variant consists of two molecular forms with similar molecular masses but different amino acid sequences. Cell-free translation studies and analyses of MBP cDNAs have shown that each of the MBP variants is encoded by a separate mRNA of approximately 2 000 bp. The five mouse MBP mRNAs appear to be derived by alternative splicing of exons 2, 5, and 6 of the MBP gene. cDNAs encoding four forms of MBP have been isolated from a human fetal spinal cord library. The mRNAs corresponding to these cDNAs are probably derived by alternative splicing of exons 2 and 5 of the human MBP gene. Proteolipid protein (PLP) cDNAs have been isolated from several species and used to establish that the size of the major PLP mRNA is approximately 3 kb. Multiple size classes of the PLP mRNAs exist in mice and rats whereas the 3 kb mRNA is the predominant form in the developing human spinal cord. In normal mice, maximal expression of the PLP gene lags behind that of the MBP gene by several days. Studies on dysmyelinating mutants have determined some of the molecular defects with respect to these two classes of myelin proteins. For example, there is a deletion of a portion of the MBP gene in the shiverer mutant. In the quaking mutant, the expression of both classes of myelin proteins is significantly reduced prior to 3 weeks. However, after 3 weeks, MBP expression approaches normal levels but the newly synthesized protein fails to be incorporated into myelin. In the jimpy mutant, although the expression of both classes of proteins is reduced, PLP expression is most severely affected.     

Analysis of 18S rRNA gene sequences suggests significant molecular differences between Macrodasyida and Chaetonotida (Gastrotricha)

Partial 18S rRNA gene sequences of four macrodasyid and one chaetonotid gastrotrichs were obtained and compared with the available sequences of other gastrotrich species and representatives of various metazoan phyla. Contrary to the earlier molecular data, the gastrotrich sequences did not comprise a monophyletic group but formed two distinct clades, corresponding to the Macrodasyida and Chaetonotida, with the basal position occupied by the sequences of Tetranchyroderma sp. and Xenotrichula sp., respectively. Depending on the taxon sampling and methods of analysis, the two clades were separated by various combinations of clades Rotifera, Gnathostomulida, and Platyhelminthes, and never formed a clade with Nematoda. Thus, monophyly of the Gastrotricha is not confirmed by analysis of the presently available molecular data.     

Expressed sequence tags from feline uterine library.

Partial nucleotide sequences of 634 cDNAs randomly isolated from a feline uterine cDNA library (Stratagene) were determined by single pass sequencing. Homology search of the sequences to the non-redundant nucleotide databases revealed that 83% of the cDNAs matched registered feline or non-feline genes. Based on the gene identifications, these genes were predicted to be related with immunological, biochemical and regulatory functions in cats. Interestingly, the rest 17% of the cDNAs did not show homology to gene or EST sequence present in the nucleotide and protein databases, suggesting that these cDNAs include novel genes expressed only in the Felidae. This large scale sequencing of uterine cDNA will provide a useful molecular source for research not only towards health and disease conditions in cats but also in different fields of science where genetic information from cats will be of interest.     

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.     

Mitochondrial genome data support the basal position of Acoelomorpha and the polyphyly of the Platyhelminthes

We determined 9.7, 5.2, and 6.8 kb, respectively, of the mitochondrial genomes of the acoel Paratomella rubra, the nemertodermatid Nemertoderma westbladi, and the free-living rhabditophoran platyhelminth Microstomum lineare. The identified gene arrangements are unique among metazoans, including each other, sharing no more than one or two single gene boundaries with a few distantly related taxa. Phylogenetic analysis of the amino acid sequences inferred from the sequenced genes confirms that the acoelomorph flatworms (acoels+nemertodermatids) do not belong to the Platyhelminthes, but are, instead, the most basal extant bilaterian group. Therefore, the Platyhelminthes, as traditionally constituted, is a polyphyletic phylum.     

Differentially expressed genes in response to amoxicillin in Helicobacter pylori analyzed by RNA arbitrarily primed PCR

Because the molecular mechanism of amoxicillin resistance in Helicobacter pylori seems to be partially explained by several mutational changes in the pbp1A gene, the aim of the present study was to evaluate the gene expression pattern in response to amoxicillin in the Amx(R) Hardenberg strain using RNA arbitrarily primed PCR (RAP-PCR). In the experiments, c. 100 differentially expressed RAP-PCR products were identified using five arbitrary primers. The cDNAs that presented the highest levels of induction or repression were cloned and sequenced, and the sequences were compared with those present in databases using the blast search algorithm. The differential expression of the isolated cDNAs was confirmed by real-time PCR. The preliminary results showed that amoxicillin alters the expression of five cDNAs involved in biosynthesis, two involved with pathogenesis, four related to cell envelope formation, two involved in cellular processes, three related with transport and binding proteins, one involved with protein degradation, one involved with energy metabolism and seven hypothetical proteins. Further analysis of these cDNAs will allow a better comprehension of both the molecular mechanism(s) of amoxicillin resistance and the adaptative mechanism(s) used by H. pylori in the presence of this antibiotic.     

Probing recalcitrant problems in polyclad evolution and systematics with novel mitochondrial genome resources

For their apparent morphological simplicity, the Platyhelminthes or “flatworms” are a diverse clade found in a broad range of habitats. Their body plans have however made them difficult to robustly classify. Molecular evidence is only beginning to uncover the true evolutionary history of this clade. Here we present nine novel mitochondrial genomes from the still undersampled orders Polycladida and Rhabdocoela, assembled from short Illumina reads. In particular we present for the first time in the literature the mitochondrial sequence of a Rhabdocoel, Bothromesostoma personatum (Typhloplanidae, Mesostominae). The novel mitochondrial genomes examined generally contained the 36 genes expected in the Platyhelminthes, with all possessing 12 of the 13 protein-coding genes normally found in metazoan mitochondrial genomes (ATP8 being absent from all Platyhelminth mtDNA sequenced to date), along with two ribosomal RNA genes. The majority presented possess 22 transfer RNA genes, and a single tRNA gene was absent from two of the nine assembled genomes. By comparison of mitochondrial gene order and phylogenetic analysis of the protein coding and ribosomal RNA genes contained within these sequences with those of previously sequenced species we are able to gain a firm molecular phylogeny for the inter-relationships within this clade.Our phylogenetic reconstructions, using both nucleotide and amino acid sequences under several models and both Bayesian and Maximum Likelihood methods, strongly support the monophyly of Polycladida, and the monophyly of Acotylea and Cotylea within that clade. They also allow us to speculate on the early emergence of Macrostomida, the monophyly of a “Turbellarian-like” clade, the placement of Rhabditophora, and that of Platyhelminthes relative to the Lophotrochozoa (=Spiralia). The data presented here therefore represent a significant advance in our understanding of platyhelminth phylogeny, and will form the basis of a range of future research in the still-disputed classifications within this taxon.     

Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes.

Two cDNAs encoding variants (alpha 1 and alpha 2) of the strychnine binding subunit of the inhibitory glycine receptor (GlyR) were isolated from a human fetal brain cDNA library. The predicted amino acid sequences exhibit approximately 99% and approximately 76% identity to the previously characterized rat 48 kd polypeptide. Heterologous expression of the human alpha 1 and alpha 2 subunits in Xenopus oocytes resulted in the formation of glycine-gated strychnine-sensitive chloride channels, indicating that both polypeptides can form functional GlyRs. Using a panel of rodent-human hybrid cell lines, the gene encoding alpha 2 was mapped to the short arm (Xp21.2-p22.1) of the human X chromosome. In contrast, the alpha 1 subunit gene is autosomally located. These data indicate molecular heterogeneity of the human GlyR at the level of alpha subunit genes.     

cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis

Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro anti-haemocytic properties, and disrupts host encapsulation responses. By sequencing 500 cDNAs randomly isolated from a venom gland library, we have identified 60 clones that encode proteins containing potential secretory signal sequences. To identify cDNAs encoding particular venom proteins, N-terminal amino acid sequences were determined for large (>30 kDa) venom proteins that had been separated using a combination of gel filtration and SDS-PAGE. We describe five of these cDNAs, which encoded residues that matched with the N-terminal sequences of previously undescribed venom proteins. cDNAs vpr1 and vpr3 encoded related proteins of approximately 32 kDa that were found in widely different fractions of gel filtration-separated venom. Neither vpr1 nor vpr3 were closely related to any other protein in the GenBank database, suggesting that they are highly specialised venom components. vpr2 encoded a 57-kDa polypeptide that was similar to a Drosophila protein, of unknown function, which lacks a signal sequence. A fourth clone, tre1, encoded a 61-kDa protein with extensive sequence similarity to trehalases. The 76-kDa sequence encoded by lac1 contained three regions which were very similar to histidine-rich copper-binding motifs, and could be aligned with the laccase from the fungus Coprinus cinereus. This study represents a significant step towards a holistic view of the molecular composition of a parasitoid wasp venom.     

Disentangling taxonomy within the Rhabditis (Pellioditis) marina (Nematoda, Rhabditidae) species complex using molecular and morhological tools

Correct taxonomy is a prerequisite for biological research, but currently it is undergoing a serious crisis, resulting in the neglect of many highly diverse groups of organisms. In nematodes, species delimitation remains problematic due to their high morphological plasticity. Evolutionary approaches using DNA sequences can potentially overcome the problems caused by morphology, but they are also affected by theoretical flaws. A holistic approach with a combination of morphological and molecular methods can therefore produce a straightforward delimitation of species. The present study investigates the taxonomic status of some highly divergent mitochondrial haplotypes in the Rhabditis ( Pellioditis ) marina species complex by using a combination of molecular and morphological tools. We used three molecular markers (COI, ITS, D2D3) and performed phylogenetic analyses. Subsequently, morphometric data from nearly all lineages were analysed with multivariate techniques. We included R. ( P. ) mediterranea and R. (R.) nidrosiensis to infer species status of the observed lineages. The results showed that highly divergent genotypic clusters were accompanied by morphological differences, and we created a graphical polytomous key for future identifications. This study indisputably demonstrates that R. ( P. ) marina and R. ( P. ) mediterranea belong to a huge species complex and that biodiversity in free-living marine nematodes may be seriously underestimated.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society , 2008, 152 , 1–15.     

Processing of rabbit hemorrhagic disease virus polyprotein.

Expression of rabbit hemorrhagic disease virus (RHDV) cDNAs in vitro with rabbit reticulocyte lysates and in Escherichia coli have been used to study the proteolytic processing of RHDV polyprotein encoded by ORF1. An epitope tag was used for monitoring the gene products by a specific antibody. We have identified four gene products with molecular masses of 80, 43, 73, and 60 kDa, from the amino to the carboxy terminus of the polyprotein. The amino-terminal sequences of the 43- and 73-kDa products were determined and indicated that RHDV 3C proteinase cleaved Glu-Gly peptide bonds.