Rhipicephalus (Boophilus) microplus: Clotting time in tick-infested skin varies according to local inflammation and gene expression patterns in tick salivary glands

Ticks deposit saliva at the site of their attachment to a host in order to inhibit haemostasis, inflammation and innate and adaptive immune responses. The anti-haemostatic properties of tick saliva have been described by many studies, but few show that tick infestations or its anti-haemostatic components exert systemic effects in vivo. In the present study, we extended these observations and show that, compared with normal skin, bovine hosts that are genetically susceptible to tick infestations present an increase in the clotting time of blood collected from the immediate vicinity of haemorrhagic feeding pools in skin infested with different developmental stages of Rhipicepahlus microplus; conversely, we determined that clotting time of tick-infested skin from genetically resistant bovines was shorter than that of normal skin. Coagulation and inflammation have many components in common and we determined that in resistant bovines, eosinophils and basophils, which are known to contain tissue factor, are recruited in greater numbers to the inflammatory site of tick bites than in susceptible hosts. Finally, we correlated the observed differences in clotting times with the expression profiles of transcripts for putative anti-haemostatic proteins in different developmental stages of R. microplus fed on genetically susceptible and resistant hosts: we determined that transcripts coding for proteins similar to these molecules are overrepresented in salivary glands from nymphs and males fed on susceptible bovines. Our data indicate that ticks are able to modulate their host`s local haemostatic reactions. In the resistant phenotype, larger amounts of inflammatory cells are recruited and expression of anti-coagulant molecules is decreased tick salivary glands, features that can hamper the tick`s blood meal. (C) 2010 Elsevier Inc. All rights reserved.

Analysis of the Nicotiana tabacum Stigma/Style Transcriptome Reveals Gene Expression Differences between Wet and Dry Stigma Species

The success of plant reproduction depends on pollen-pistil interactions occurring at the stigma/style. These interactions vary depending on the stigma type: wet or dry. Tobacco (Nicotiana tabacum) represents a model of wet stigma, and its stigmas/styles express genes to accomplish the appropriate functions. For a large-scale study of gene expression during tobacco pistil development and preparation for pollination, we generated 11,216 high-quality expressed sequence tags (ESTs) from stigmas/styles and created the TOBEST database. These ESTs were assembled in 6,177 clusters, from which 52.1% are pistil transcripts/genes of unknown function. The 21 clusters with the highest number of ESTs (putative higher expression levels) correspond to genes associated with defense mechanisms or pollen-pistil interactions. The database analysis unraveled tobacco sequences homologous to the Arabidopsis (Arabidopsis thaliana) genes involved in specifying pistil identity or determining normal pistil morphology and function. Additionally, 782 independent clusters were examined by macroarray, revealing 46 stigma/style preferentially expressed genes. Real-time reverse transcription-polymerase chain reaction experiments validated the pistil-preferential expression for nine out of 10 genes tested. A search for these 46 genes in the Arabidopsis pistil data sets demonstrated that only 11 sequences, with putative equivalent molecular functions, are expressed in this dry stigma species. The reverse search for the Arabidopsis pistil genes in the TOBEST exposed a partial overlap between these dry and wet stigma transcriptomes. The TOBEST represents the most extensive survey of gene expression in the stigmas/styles of wet stigma plants, and our results indicate that wet and dry stigmas/styles express common as well as distinct genes in preparation for the pollination process.

Non-coding transcript in T cells (NTT): Antisense transcript activates PKR and NF-kappa B in human lymphocytes

T cell activation is a complex process involving many steps and the role played by the non-protein-coding RNAs (ncRNAs) in this phenomenon is still unclear. The non-coding T cells transcript (NTT) is differentially expressed during human T cells activation, but its function is unknown. Here, we detected a 426 m NTT transcript by RT-PCR using RNA of human lymphocytes activated with a synthetic peptide of HIV-1. After cloning, the sense and antisense 426 nt NTT transcripts were obtained by in vitro transcription and were sequenced. We found that both transcripts are highly structured and are able to activate PKR. A striking observation was that the antisense 426 nt NTT transcript is significantly more effective in activating PKR than the corresponding sense transcript. The transcription factor NF-kappa B is activated by PKR through phosphorylation and subsequent degradation of its inhibitor I-kappa B beta. We also found that the antisense 426 nt NTT transcript induces more efficiently the degradation Of I-kappa B beta than the sense transcript. Thus, this study suggests that the role played by NTT in the activation of lymphocytes can be mediated by PKR through NF-kappa B activation. However, the physiological significance of the activity of the antisense 426 nt NTT transcript remains unknown. (c) 2007 Elsevier Inc. All rights reserved.

Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil

We have studied the variability of glutamate dehydrogenase (gdh) and small subunit ribosomal (SSU) rRNA coding genes of Giardia species in fecal samples isolated from wild and exotic animals in Brazil, and compared with homologous sequences of isolates from human and domestic animals characterized in previous studies. Cysts of Giardia duodenalis were obtained from feces of naturally infected monkeys (Alouatta fusca) (n = 20), chinchillas (Chinchilla lanigera) (n = 3), ostriches (Struthio camelus) (n = 2) and jaguar (Panthera onca) (n = 1). Assemblage AI was assigned to the unique isolate of jaguar. All the samples from monkeys, chinchillas, and ostriches were assigned to Assemblage B. There was little evolutionary divergence between the referred isolates and isolates described elsewhere. The Assemblage B isolates identified in this study were closely related to Assemblage BIV isolated from humans. The molecular identification of Assemblages A and B of G. duodenalis isolates from exotic and wild animals demonstrates that such hosts may be a potential reservoir for zoonotic transmission of G. duodenalis. (C) 2011 Elsevier B.V. All rights reserved.

Numerous gene rearrangements in the mitochondrial genome of the wallaby louse, Heterodoxus macropus (Phthiraptera)

The complete arrangement of genes in the mitochondrial (mt) genome is known for 12 species of insects, and part of the gene arrangement in the mt genome is known for over 300 other species of insects. The arrangement of genes in the mt genome is very conserved in insects studied, since all of the protein-coding and rRNA genes and most of the tRNA genes are arranged in the same way. We sequenced the entire mt genome of the wallaby louse, Heterodoxus macropus, which is 14,670 bp long and has the 37 genes typical of animals and some noncoding regions. The largest noncoding region is 73 bp long (93% A+T), and the second largest is 47 bp long (92% AST). Both of these noncoding regions seem to be able to form stem-loop structures. The arrangement of genes in the mt genome of this louse is unlike that of any other animal studied. All tRNA genes have moved and/or inverted relative to the ancestral gene arrangement of insects, which is present in the fruit fly Drosophila yakuba. At least nine protein-coding genes (atp6, atp8, cox2, cob, nad1-nad3, nad5, and nad6) have moved; moreover, four of these genes (atp6, atp8, nad1, and nad3) have inverted. The large number of gene rearrangements in the mt genome of H. macropus is unprecedented for an arthropod.

Optical encoding of microbeads for gene screening: alternatives to microarrays

Rapid access to genetic information is central to the revolution presently occurring in the pharmaceutical industry, particularly In relation to novel drug target identification and drug development. Genetic variation, gene expression, gene function and gene structure are just some of the important research areas requiring efficient methods of DNA screening. Here, we highlight state-of-the-art techniques and devices for gene screening that promise cheaper and higher-throughput yields than currently achieved with DNA microarrays. We include an overview of existing and proposed bead-based strategies designed to dramatically increase the number of probes that can be interrogated in one assay. We focus, in particular, on the issue of encoding and/or decoding (bar-coding) large bead-based libraries for HTS.

The evolution of controlled multitasked gene networks: The role of introns and other noncoding RNAs in the development of complex organisms

Eukaryotic phenotypic diversity arises from multitasking of a core proteome of limited size. Multitasking is routine in computers, as well as in other sophisticated information systems, and requires multiple inputs and outputs to control and integrate network activity. Higher eukaryotes have a mosaic gene structure with a dual output, mRNA (protein-coding) sequences and introns, which are released from the pre-mRNA by posttranscriptional processing. Introns have been enormously successful as a class of sequences and comprise up to 95% of the primary transcripts of protein-coding genes in mammals. In addition, many other transcripts (perhaps more than half) do not encode proteins at all, but appear both to be developmentally regulated and to have genetic function. We suggest that these RNAs (eRNAs) have evolved to function as endogenous network control molecules which enable direct gene-gene communication and multitasking of eukaryotic genomes. Analysis of a range of complex genetic phenomena in which RNA is involved or implicated, including co-suppression, transgene silencing, RNA interference, imprinting, methylation, and transvection, suggests that a higher-order regulatory system based on RNA signals operates in the higher eukaryotes and involves chromatin remodeling as well as other RNA-DNA, RNA-RNA, and RNA-protein interactions. The evolution of densely connected gene networks would be expected to result in a relatively stable core proteome due to the multiple reuse of components, implying,that cellular differentiation and phenotypic variation in the higher eukaryotes results primarily from variation in the control architecture. Thus, network integration and multitasking using trans-acting RNA molecules produced in parallel with protein-coding sequences may underpin both the evolution of developmentally sophisticated multicellular organisms and the rapid expansion of phenotypic complexity into uncontested environments such as those initiated in the Cambrian radiation and those seen after major extinction events.

Mitochondrial gene content, arrangement and composition compared in African and Asian schistosomes

Complete sequences were obtained for the coding portions of the mitochondrial (mt) genomes of Schistosoma mansoni (NMRI strain, Puerto Rico; 14415 bp), S. japonicum (Anhui strain, China; 14085 bp) and S. mekongi (Khong Island, Laos; 14072 bp). Each comprises 36 genes: 12 protein-encoding genes (cox1-3, nad1-6, nad4L, atp6 and cob); two ribosomal RNAs, rrnL (large subunit rRNA or 16S) and rrnS (small subunit rRNA or 12S); as well as 22 transfer RNA (tRNA) genes. The atp8 gene is absent. A large segment (9.6 kb) of the coding region (comprising 14 tRNAs, eight complete and two incomplete protein-encoding genes) for S. malayensis (Baling, Malaysian Peninsula) was also obtained. Each genome also possesses a long non-coding region that is divided into two parts (a small and a large non-coding region, the latter not fully sequenced in any species) by one or more tRNAs. The protein-encoding genes are similar in size, composition and codon usage in all species except for cox1 in S. mansoni (609 aa) and cox2 in S. mekongi (219 an), both of which are longer than homologues in other species. An unexpected finding in all the Schistosoma species was the presence of a leucine zipper motif in the nad4L gene. The gene order in S. mansoni is strikingly different from that seen in the S. japonicum group and other flatworms. There is a high level of identity (87-94% at both the nucleotide and amino acid levels) for all protein-encoding genes of S. mekongi and S. malayensis. The identity between genes of these two species and those of S. japonicum is less (56-83% for amino acids and 73-79 for nucleotides). The identity between the genes of S. mansoni and the Asian schistosomes is far less (33-66% for amino acids and 54-68% for nucleotides), an observation consistent with the known phylogenetic distance between S. mansoni and the other species. (C) 2001 Elsevier Science B.V. All rights reserved.

Non-coding RNAs: the architects of eukaryotic complexity

Around 98% of all transcriptional output in humans is noncoding RNA. RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, imprinting, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signaling. I suggest that the central dogma is incomplete, and that intronic and other non-coding RNAs have evolved to comprise a second tier of gene expression in eukaryotes, which enables the integration and networking of complex suites of gene activity. Although proteins are the fundamental effectors of cellular function, the basis of eukaryotic complexity and phenotypic variation may lie primarily in a control architecture composed of a highly parallel system of trans-acting RNAs that relay state information required for the coordination and modulation of gene expression, via chromatin remodeling, RNA-DNA, RNA-RNA and RNA-protein interactions. This system has interesting and perhaps informative analogies with small world networks and dataflow computing.

Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Characterization of a novel gene, STAG1/PMEPA1, upregulated in renal cell carcinoma and other solid tumors

Using differential display-polymerase chain reaction, we identified a novel gene sequence, designated solid tumor-associated gene 1 (STAG1), that is upregulated in renal cell carcinoma (RCC). The full-length cDNA (4839 bp) encompassed the recently reported androgen-regulated prostatic cDNA PMEPA1 and so we refer to this gene as STAG1/PMEPA1, Two STAG1/PMEPA1 mRNA transcripts of approximately 2.7 an 5 kb, with identical coding regions but variant 3' untranslated regions, were predominantly expressed in normal prostate tissue and at lower levels in the ovary. The expression of this gene was upregulated in 87% of RCC samples and also was upregulated in stomach and rectal adenocarcinomas. In contrast, STAG1/PMEPA1 expression was barely detectable in leukemia and lymphoma samples, Analysis of expressed sequence tag databases showed that STAG1/PMEPA1 also was expressed in pancreatic, endometrial, and prostatic adenocarcinomas. The STAG1/PMEPA1 cDNA encodes a 287-amino-acid protein containing a putative transmembrane domain and motifs that suggest that it may bind src homology 3- and tryptophan tryptophan domain-containing proteins. This protein shows 67% identity to the protein encoded by the chromosome 18 open reading frame 1 gene. Translation of STAG1/PMEPA1 mRNA in vitro showed two products of 36 and 39 kDa, respectively, suggesting that translation may initiate at more than one site. Comparison to genomic clones showed that STAG1/PMEPA1 was located on chromosome 20q13 between microsatellite markers D20S183 and D20S173 and spanned four exons and three introns. The upregulation of this gene in several solid tumors indicated that it may play an important role in tumorigenesis. (C) 2001 Wiley-Liss, Inc.

Complete DNA sequence and gene organization of the mitochondrial genome of the liverfluke, Fasciola hepatica L. (Platyhelminthes; Trematoda)

The complete nucleotide sequence of the mitochondrial (mt) DNA molecule of the liverfluke, Fasciola hepatica (phylum Platyhelminthes, class Trematoda, family Fasciolidae), was determined, It comprises 14462 bp, contains 12 protein-encoding, 2 ribosomal and 22 transfer RNA genes, and is the second complete flatworm (and the first trematode) mitochondrial sequence to be described in detail. All of the genes are transcribed from the same strand. Of the genes typically found in mitochondrial genomes of eumetazoans, only atp8 is absent. The nad4L and nad4 genes overlap by 40 nt. Most intergenic sequences are very short. Two larger non-coding regions are present. The longer one (817 nt) is located between trnG and cox3 and consists of 8 identical tandem repeats of 85 nt, rich in G and C, followed by 1 imperfect repeat. The shorter non-coding region (187 nt) exhibits no special features and is separated from the longer region by trnG. The gene arrangement resembles that of some other trematodes including the eastern Asian Schistosoma species (and cyclophyllidean cestode species) but it is strikingly different from that of the African schistosomes, represented by Schistosoma mansoni. The genetic code is as inferred previously for flatworms. Transfer RNA genes range in length from 58 to 70 nt, their products producing characteristic 'clover leaf' structures, except for tRNA(S-VON) and tRNA(S-AGN) lacking the DHU arm.

The murine chaperonin 10 gene family contains an intronless, putative gene for early pregnancy factor, Cpn10-rs1

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. Human platelet-derived EPF shares amino acid sequence identity with chaperonin 10 (Cpn10), a mitochondrial matrix protein which functions as a molecular chaperone. The striking differences in cellular localization and function of the two proteins suggest differential regulation of production reflecting either alternative transcription of the same gene or transcription from different genes. In mammals and more distantly related genera, there is a large gene family with homology to CPN 10 cDNA, which includes intronless copies of the coding sequence. To determine whether this could represent the gene for EPF, we have screened a mouse genomic library and sequenced representative Cpn10 family members, looking for a functional gene distinct from that of Cpn 10, which could encode EPF. Eight distinct genes were identified. Cpn10 contains introns, while other members are intronless. Six of these appear to be pseudogenes, and the remaining member, Cpn10-rs1, would encode a full-length protein. The 309-bp open reading frame (ORF) is identical to that of mouse Cpn10 cDNA with the exception of three single-base changes, two resulting in amino acid changes. Only one further single nucleotide difference between the Cpn10-rs1 and Cpn10 cDNAs is observed, located in the 3' UTR. Single nucleotide primer extension was applied to discriminate between Cpn10-rs1 and Cpn10 expression. Cpn10, which is ubiquitous, was detected in all tissue samples tested, whereas Cpn10-rs1 was expressed selectively. The pattern was completely coincident with known patterns of EPF activity, strongly suggesting that Cpn10-rs1 does encode EPF. The complete ORF of Cpn10-rs1 was expressed in E. coli. The purified recombinant protein was found to be equipotent with native human platelet-derived EPF in the bioassay for EPF, the rosette inhibition test.

Cloning and functional analysis of the Sry-related HMG box gene, Sox18

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii);ln intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor-2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue. (C) 2001 Elsevier Science B.V. All rights reserved.

Characterization of the mouse hnRNP A2/B1/B0 gene and identification of processed pseudogenes

The mouse hnRNP A2/B1/B0 gene has been cloned using a PCR-based strategy and sequenced. Analysis of this sequence showed that the gene organization closely follows that of the human orthologue with 12 exons and 11 introns. The hnRNP A2/B1/B0 gene gives rise to four splice variants through alternative splicing of exons 2 and 9. RT-PCR assays indicated that all splice variants were expressed in mouse brain, skin, and stomach tissues of varying ages, although their ratios to one another varied with age and tissue type. We also identified a small subset of all polyadenylated splice variants that included intron 11, which shows 94% sequence identity between human and mouse. Several processed pseudogenes were identified in the mouse genome. A search of the mouse genome databases located five pseudogenes, four of. which are presumed to be non-functional because of the presence of premature stop codons, large deletions or rearrangements within the coding region. The fifth, which possesses putative promoter elements and has a coding sequence identical to that of the hnRNP A2 mRNA, variant, may be functional. (C) 2002 Elsevier Science B.V. All rights reserved.