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A new DNA hybridization technique, based on chromatographic migration of DNA on a nitrocellulose strip passing through an immobilized probe area, is described. The new paper chromatography hybridization assay (PACHA) is faster and simpler to use than the conventional dot hybridization assay. In this assay, an aliquot of biotinylated, PCR-amplified target DNA is applied to one end of a nitrocellulose strip. The DNA migrates to the opposite end of the strip by capillary forces and hybridizes to a specific DNA probe immobilized in a reaction zone (RZ), located in the middle of the strip. Unhybridized DNA migrates away from the RZ. The biotinylated hybrid is visualized by a color reaction employing a streptavidin-alkaline phosphatase (SA-AP) conjugate and a specific chromogenic substrate. The new PACHA technique allows for detection of as little as 1-5 pg of specific human papilloma virus 16 (HPV16) DNA in 25 min of hybridization. In this system, the hybridization efficiency is controlled by the flow velocity of the hybridization solution (HS) and by the volume of the amplified labeled DNA migrating across the immobilized probe. Glycerol (30%) or polyvinyl pyrrolidone (PVP) (1%) reduces the flow rate by a factor of 2.5-3 and increases the sensitivity of the assay by a factor of 5.2 for glycerol and 2.6 for PVP. This novel method ensures efficient hybridization to multiple probes and appears to be superior to currently available solid-phase hybridization techniques.

Human papillomavirus (HPV) was detected by using two sets of deoxyribonucleotide primers for differentiating between 'low-risk' types (HPV11 and HPV6) and 'high-risk' (hri) types (HPV16, HPV18 and HPV33). A new application of the Chemiprobe method for labeling DNA was used to detect products of the polymerase chain reaction (PCR) from 36 cervical biopsies. This method, first demonstrated by Uchimura et al. (submitted), is based on the sulfonation of a polycytidylic acid tail of 5-20 monomers attached to the 5' end of either one or both of the PCR primers. This procedure can increase the sensitivity of detection of PCR products more than 100-fold with respect to ethidium bromide (EtdBr) staining. Various methods were used to detect hri HPV DNA in the 36 clinical samples. The number of positive results obtained was as follows, two by Southern-blot hybridization; five by PCR amplification followed by electrophoresis and detection of products by EtdBr staining; six by PCR amplification using one or two sulfonated C-tailed primers followed by electroblotting and immunoenzymatic visualization; and five by hybridization of sulfonated genomic viral recombinant with a PCR product immobilized on a membrane. The yield of the PCR product was significantly greater when one of the primers was C-tailed than when both or neither of the primers were C-tailed. PCR employing sulfonated C-tailed oligo primers is very specific and sensitive, and the entire procedure can be employed as a nonradioactive substitute for radioactive dot-blot or Southern-blot hybridization procedures, routinely used for detection of HPV in clinical samples.

The activation of plasminogen plays a crucial role in a variety of extracellular proteolytic events such as, fibrinolysis, cell migration, ovulation, involution of the mammary gland and the activation of other protease classes and growth factors. In this paper we describe the isolation of the full-length cDNAs of ovine urokinase plasminogen activator (u-PA) and its receptor (u-PAR) using a polymerase chain reaction based strategy. The ovine u-PA cDNA comprised of 2350 bp and it is characterized by a coding region of 1302 bp, and 5′- and 3′-UTR regions of 129 and 919 bp, respectively. The deduced amino acid sequence consists of 433 amino acids. The ovine u-PAR cDNA is comprised of 1247 bp and it is characterized by a coding region of 957 bp and 5′- and 3′-UTR regions of 44 and 246 bp respectively. The deduced amino acid sequence consists of 318 amino acids. Three-dimensional models of the putative protein products of both cDNAs showed that the proteins bear a high similarity with their human counterparts. Real-time PCR revealed high levels of u-PA expression in the adipose tissue, followed by that in mammary gland and kidney. Lower levels of expression were detected in the adrenal glands, heart, ovaries, spleen, liver and cerebellum. A similar pattern was observed in u-PAR expression with noticeably lower levels of expression in heart, liver and cerebellum. To the best of our knowledge, this is the first paper reporting expression of u-PA and u-PAR in the adipose tissue. These data strengthen the suggestion that adipose tissue functions as an endocrine organ besides an energy storage organ. Furthermore, u-PA and u-PAR mRNA levels were 7 and 8.5 fold higher respectively in involuting mammary tissue obtained from non-lactating ewes compared to that detected in mammary tissue obtained from lactating ewes. These data are consistent with the notion that upregulation of u-PA and u-PAR expression may play a key role in the process of involution of the mammary gland.

The satellite DNA of ants Messor bouvieri, M. barbarus and M. structor, studied in a previous paper, is organized as tandemly repeated 79-bp monomers in the three species showing high sequence similarity. In the present paper, a mariner-like element (Mboumar) and a new MITE (miniature inverted-repeat transposable element) called IRE-130, inserted into satellite DNA from M. bouvieri, are analyzed. The study of Mboumar element, of its transcription and the putative transposase that it would encode, suggests that it could be an active element. Mboumar elements inserted into IRE-130 elements have also been detected. It is the first time, to our knowledge, that a MITE has been described in Hymenoptera and it is also the first time that a mariner-like element inserted into a MITE has been detected. A mariner-like element, inserted into satellite DNA from M. structor and in M. barbarus, also has been found. The results seem to indicate that transposition events have participated in the satellite DNA mobilization and evolution.

Analysis of the huge volume of data generated by large scale sequencing projects requires the construction of new, sophisticated computer systems. These systems should be able to manage the biological data as well as the results of their analysis. They should also help the user to choose the most appropriate methods, and to string them together in order to solve a global analysis task. In this paper we present the prototype of a software system providing an environment for the analysis of large-scale sequence data. As a first step toward this end, this environment has been put to the test within the Bacillus subtilis genome sequencing project. This system integrates both the descriptive knowledge of the entities involved (genes, regulatory signals and the like) and the methodological knowledge comprising an extensible set of analytical methods. A knowledge representation based on two existing object-oriented models is used to implement this integrated system. In addition, the present prototype provides a suitable user interface both for displaying simultaneously the results generated by several methods and for interacting with the objects. We present in this paper the analysis of a B. subtilis genome fragment, present in data libraries but not annotated. Annotation of the genes present in the fragment allowed us to combine the results of several methods used for predicting coding sequences, and to characterize it as comprising a cryptic phage, the skin element. Comparison between the annotation of the skin element and a standard region of the chromosome indicated that local features of the nucleotide sequence could discriminate between phage and non-phage DNA sequence.

This paper describes systematic sequence studies of repetitive DNA in and around translocation breakpoints on chromosomes 9 and 22, which are involved in the formation of the Philadelphia chromosome in acute leukemias. In addition to Alu repeats described in previous studies, the breakpoint regions appear to contain many other repetitive elements, including a member of a new repetitive family (MER34) reported in this paper. Identification of these repeats broadens current studies on the possible involvement of repetitive DNA in this intensely studied chromosomal translocation.

The allergenic Tha p 2 protein has been extracted recently from the urticating setae of the pine processionary moth Thaumetopoea pityocampa. In the present paper, we test for the occurrence of this protein in other Thaumetopoeinae, with a particular focus on members of the genus Thaumetopoea, as well as unrelated moth species, to better understand the physicochemical properties of the protein, the nature of encoding genes and their evolutionary history. Tha p 2 is encoded by the intronless gene Tha p 2 that is restricted to the processionary moths (Thaumetopoeinae, Notodontidae, Lepidoptera). Most of the species present two isoforms of Tha p 2 that can be interpreted as the result of heterozygosity in the single gene. The only exception is represented by Thaumetopoea wilkinsoni, in which 20 different isoforms occur in a single specimen, leading to the conclusion that, at least in this species, multiple copies of Tha p 2 exist. Serine, glycine, cysteine and leucine are abundant in Tha p 2, a protein well conserved among processionary moths. The predicted secondary structures of Tha p 2 indicate the presence of 3 α-helices and six β-barrels. Finally, the evolution of the gene and the protein was characterized by a combination of positive and negative selection, with the latter being more evident.

Background The naked mole rat (NMR, Heterocephalus glaber) is a long-lived rodent model with multiple extraordinary traits. They live underground and have been known to live for up to thirty years, much longer than similar-sized mice. Moreover, congenital cancer or experimentally induced cancer genesis could not been observed in this rodent so far. Such unique biochemical and physiological characteristics lead them to become a popular model for cancer research. Results In this paper, a genome-wide comparative analysis was conducted based on the genomes of NMR and several other mammals. First, all the annotated proteins of NMR were searched against 11 selected mammalian genomes to verify their occurrence in these organisms. Among them, 66 NMR genes were not detected in other 11 mammals, almost all of which present alkalinity isoelectric points. In contrast, a total of 89 genes that are present in all of the 11 organisms could not be found in NMR genome. Among them, 3 genes are known to be related to cancer development. Finally, we identified NMR-specific single amino acid change (SAAC) events for the proteins that are present in both NMR and other mammals. KEGG pathway database was also used to investigate the metabolic processes in which these SAAC proteins may be involved. These genes were significantly enriched in two known cancer pathways, “Pathways in cancer” and “Pancreatic cancer”. In the “Pancreatic cancer” pathway, 3 out of 6 paths leading to DNA duplication appeared to be affected by direct connection to the SAAC genes in NMR. In addition, a significant number of other SAAC genes enriched in several cancer-related pathways have been known to be associated with a variety of cancers, implying that many of them may be also related to tumor genesis in mammals. Conclusions Overall, our results not only can be used to find possible genes involved in physiological mechanism of NMR but also provide new clues for the anti-cancer mechanism of NMR.

CHARGE syndrome is an autosomal-dominant disorder involved in multiple organs. Loss-of-function mutations in CHD7, a member of the chromodomain helicase DNA-binding (CHD) protein family, are known to cause the CHARGE syndrome. The purposes of this paper were to affirm the diagnosis and to identify the molecular basis of one atypical CHARGE syndrome patient from China, where only one CHARGE case was reported before. We employed the Verloes criteria to make a preliminary clinical diagnosis, and performed mutation screening of CHD7 via Ion Torrent semiconductor sequencing. The patient was preliminary diagnosed as atypical CHARGE syndrome according to Verloes criteria with a novel heterozygous small deletion of CHD7 (CHD7: c.3462_3471delTCGCTTCCCT). As the second reported case of CHARGE syndrome in China, it was caused by one novel heterozygous mutation of the CHD7 gene. Our findings further reveal the relationship between CHD7 and CHARGE syndrome and provide a potential clinical diagnosis for CHARGE syndrome.

Neuromedin B (NMB) is a highly conserved bombesin-related neuropeptide found in mammals. Neuromedin B (NMB) executes its effect by binding to the cell surface receptor, neuromedin B receptor (NMBR). In this study, we cloned the rabbit NMB and NMBR genes. The similarity and phylogenetic analyses of NMB and NMBR gene sequences were performed. The expression of NMB and NMBR mRNA in the rabbit was investigated using real-time RT-PCR. Our bioinformatic analysis demonstrated that the cloned rabbit NMB precursor cDNA encoded Gly-His-Phe-Met-NH2 amino acids at the C-terminus, and that its receptor possessed typical transmembrane features. The NMB mRNA was highly expressed in the CNS, while the NMBR mRNA was widely expressed in many tissues, with the highest expression in the gastrointestinal tract. The studies on the NMB distribution and function are limited by the lack of a specific antibody to this neuropeptide. In this paper, polyclonal NMB antibody was generated in mice. Western blotting analysis revealed that the prepared antibody could specifically recognize the recombinant and the endogenous NMB proteins. Immunohistochemistry analysis indicated that the NMB protein was localized in the cytoplasm of the pituitary cells. The existence of NMB protein in the hypothalamic-pituitary-gonadal axis suggests that NMB might function in rabbit reproduction.

This paper describes the complete genomic sequences of two virulent Newcastle disease virus (NDV) isolates, Shaanxi06 (prevalent genotype VIId) and Shaanxi10 (novel sub-genotype VIi), from sick crested ibises. The genomes of both isolates were 15,192 nt long and consisted of six genes in the order of 3′-NP-P-M-F-HN-L-5′. The genomes of the two isolates were highly similar to other reference NDV strains. However, some unique features were found in the HN protein of Shaanxi06 and the F gene end of Shaanxi10. Shaanxi06 and Shaanxi10 shared the same virulent motif 112 −R-R-Q-K-R-F− 117 at the F protein cleavage site, which coincided with previous pathogenicity test results. Phylogenetic analysis revealed that both isolates were clustered within class II NDV, with Shaanxi06 in genotype VII and Shaanxi10 in genotype VI. Both isolates shared high homology with the prevalent genotype NDV strains that circulate in fowls and waterfowls. This study is the first to provide genomic information about a novel sub-genotype VIi NDV strain and another genotype VIId virus, which will be useful for subsequent investigations.

Cotton is an important fiber plant, and it's attractive to elucidate the molecular mechanism of anther development due to the close relationship between the anther fertility and boll-setting, and also fiber yield. In the present paper, 47.2 million paired-end reads with average length of 82.87 bp from the anthers of TM-1 (Gossypium hirsutum L.), a genetic standard line, were generated through transcriptome sequencing, and 210,965 unigenes of more than 100 bp were obtained. BLAST, KEGG, COG, and GO analyses showed that the genes were enriched in the processes of transcription, translation, and post-translation as well as hormone signal transduction, the transcription factor families, and cell wall-related genes mainly participating in cell expansion and carbohydrate metabolism. Further analysis identified 11,153 potential SSRs. A suit of 5122 primer pair sequences were designed, and 82 of 300 randomly selected primer pairs produced reproducible amplicons that were polymorphic among 22 cotton accessions from G. hirsutum, Gossypium barbadense and Gossypium arboreum. The UPGMA clustering analysis further confirmed high quality and effectiveness of these novel SSR markers. The present study provided insights into the transcriptome profile of the cotton and established a public information platform for functional genomics and molecular breeding.

CD2BP2 (CD2 cytoplasmic tail binding protein 2), one of several proteins interacting with the cytoplasmic tail of CD2, plays a crucial role in CD2-triggered T cell activation and nuclear splicing. The studies on CD2BP2 have tended to be confined to a few mammals, and little information is available to date regarding fish CD2BP2. In this paper, a CD2BP2 gene (On-CD2BP2) was cloned from Nile tilapia, Oreochromis niloticus. Sequence analysis showed that the full length of On-CD2BP2 cDNA was 1429 bp, containing a 5'untranslated region (UTR) of 111 bp, a 3'-UTR of 193 bp and an open reading frame of 1125 bp which is encoding 374 amino acids. Two important structural features, a GYF domain and a consensus motif GPFXXXXMXXWXXXGYF were detected in the deduced amino acid sequence of On-CD2BP2, and the deduced genomic structure of On-CD2BP2 was similar to the known CD2BP2. The mRNA expression of On-CD2BP2 in various tissues of Nile tilapia was analyzed by fluorescent quantitative real-time PCR. In healthy Nile tilapia, the On-CD2BP2 transcripts were mainly detected in the head kidney and spleen. While vaccinated with inactivated Streptococcus agalactiae, the On-CD2BP2 mRNA expression was significantly up-regulated in the head kidney, spleen and brain 48 h post immunization. Moreover, there was a clear time-dependent expression pattern of On-CD2BP2 after immunization and the expression reached the highest level at 24h in the brain and 48 h in the head kidney and spleen. This is the first report of proving the presence of a CD2BP2 ortholog in fish, and investigating its tissue distribution and expression profile in response to bacterial stimulus. These findings indicated that On-CD2BP2 may play an important role in the immune response to bacteria in Nile tilapia.

Postembryonic development of insects is highly dependent on ecdysteroid hormones ecdysone (E) and 20-hydroxyecdysone (20E). A cytochrome P450 monooxygenase CYP306A1, the product of the Halloween gene phantom (phm), is involved in the ecdysteroidogenesis in representative insects in Diptera, Lepidoptera and Orthoptera. In the present paper, Sfphm was cloned from a hemipteran insect species, the white-backed planthopper Sogatella furcifera. SfPHM has five insect conserved P450 motifs, i.e., Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal and spatial expression patterns of Sfphm were evaluated by q-PCR. Sfphm showed three expression peaks in late second-, third- and fourth-instar stages. In contrast, the expression levels were lower and formed three troughs in the newly-molted second-, third- and fourth-instar nymphs. The relative 20E levels exhibited similar temporal patterns to Sfphm expression levels. On day 3 of the fourth-instar nymphs, Sfphm clearly had a high transcript level in the thorax where PGs were located. Dietary introduction of double-stranded RNA of Sfphm into the second instars successfully knocked down the target gene, and greatly reduced 20E level and ecdysone receptor (EcR) expression level. Moreover, knockdown of Sfphm caused lethality and slowed down ecdysis during nymphal stages. Furthermore, ingestion of 20-hydroxyecdysone did not alter Sfphm expression level, but almost completely rescued SfEcR expression level, and relieved the negative effects on nymphal survival and ecdysis in Sfphm-dsRNA-exposed planthoppers. Thus, our results suggest that Sfphm plays a critical role in ecdysteroidogenesis in S. furcifera.

Congenital disorders of glycosylation (CDG) are a growing group of inherited metabolic disorders where enzymatic defects in the formation or processing of glycolipids and/or glycoproteins lead to variety of different diseases. The deficiency of GDP-Man:GlcNAc2-PP-dolichol mannosyltransferase, encoded by the human ortholog of ALG1 from yeast, is known as ALG1-CDG (CDG-Ik). The phenotypical, molecular and biochemical analysis of a severely affected ALG1-CDG patient is the focus of this paper. The patient's main symptoms were feeding problems and diarrhea, profound hypoproteinemia with massive ascites, muscular hypertonia, seizures refractory to treatment, recurrent episodes of apnoea, cardiac and hepatic involvement and coagulation anomalies. Compound heterozygosity for the mutations c.1145T>C (M382T) and c.1312C>T (R438W) was detected in the patient's ALG1-coding sequence. In contrast to a previously reported speculation on R438W we confirmed both mutations as disease-causing in ALG1-CDG.

The respiratory defects associated with mutations in human mitochondrial tRNA genes can be mimicked in yeast, which is the only organism easily amenable to mitochondrial transformation. This approach has shown that overexpression of several nuclear genes coding for factors involved in mitochondrial protein synthesis can alleviate the respiratory defects both in yeast and in human cells. The present paper analyzes in detail the effects of overexpressed yeast and human mitochondrial translation elongation factors EF-Tu. We studied the suppressing activity versus the function in mt translation of mutated versions of this factor and we obtained indications on the mechanism of suppression. Moreover from a more extended search for suppressor genes we isolated factors which might be active in mitochondrial biogenesis. Results indicate that the multiplicity of mitochondrial factors as well as their high variability of expression levels can account for the variable severity of mitochondrial diseases and might suggest possible therapeutic approaches.

The dual oxidases (DUOX1 and DUOX2) are NADPH-dependent hydrogen peroxide-producing enzymes that are reported to function in a physiological capacity and as a component of the mucosal immune response. We have previously reported increased expression of the DUOX2 gene in the gut mucosa of sheep in response to gastrointestinal nematode (GIN) challenge. In this paper, we report the cloning of the full-length ovine DUOX2 transcript, using a PCR based strategy. The ovine DUOX2 transcript includes an ORF of 4644 bases, and encodes a protein with 97% identity to the bovine sequence. We also cloned a fragment of DUOX1 (encompassing nucleotides 2692-2829), and the proximal promoter sequence of DUOX2. Through analysis of sequence data we have confirmed that DUOX1 and DUOX2 are co-located in a head to tail arrangement conserved across many species. Alignment of the sequences to the ovine genome predicts a location of this gene cluster on ovine chromosome 7. We quantified the expression of ovine DUOX1 and DUOX2 transcripts in 24 different sheep tissues, and discovered tissue specific expression signatures. DUOX2 was found to be most highly expressed in tissues of the gastrointestinal tract, while expression of DUOX1 predominated in the bladder. Rapid amplification of cDNA ends (RACE) analysis identified the existence of multiple 5' UTR variants in DUOX2, ranging in size from 32 to 242 nucleotides, with 3 distinct transcribed regions. Real time PCR quantification of the DUOX2 UTR variants revealed that these were differentially expressed between tissues, and at various stages of the response to GIN parasite infection. The collective evidence suggested a complex regulation of DUOX2, prompting a bioinformatic analysis of the proximal promoter regions of ovine DUOX2 to identify potential transcription factor binding sites (TFBS) that may explain the differences in the observed expression of the transcript variants of DUOX2. Possible transcription factor families that may regulate this process were identified as Kruppel-like factors (KLF), ETS-factors, erythroid growth receptor factors (EGRF) and myogenic differentiation factors (MYOD).

Arginine decarboxylase (ADC), one of the enzymes responsible for putrescine (Put) biosynthesis, has been shown to be implicated in stress response. In the current paper attempts were made to clone and characterize a gene encoding ADC from peach ( Prunus persica (L.) Batsch, ‘Akatsuki’). Rapid amplification of cDNA ends (RACE) gave rise to a full-length ADC cDNA ( PpADC ) with a complete open reading frame of 2178 bp, encoding a 725 amino acid polypeptide. Homology search and sequence multi-alignment demonstrated that the deduced PpADC protein sequence shared a high identity with ADCs from other plants, including several highly conservative motifs and amino acids. Southern blotting indicated that PpADC existed in peach genome as a single gene. Expression levels of PpADC in different tissues of peach ( P . persica ‘Akatsuki’) were spatially and developmentally regulated. Treatment of peach shoots from ‘Mochizuki’ with exogenous 5 mM Put, an indirect product of ADC, remarkably induced accumulation of PpADC mRNA. Transcripts of PpADC in peach leaves from ‘Mochizuki’ were quickly induced, either transiently or continuously, in response to dehydration, high salinity (200 mM NaCl), low temperature (4 °C) and heavy metal (150 µM CdCl 2 ), but repressed by high temperature 37 °C) during a 2-day treatment, which changed in an opposite direction when the stresses were otherwise removed with the exception of CdCl 2 treatment. In addition, steady-state of PpADC mRNA could be also transiently up-regulated by abscisic acid (ABA) in ‘Mochizuki’ leaves. All of these, taken together, suggest that PpADC is a stress-responsive gene and can be considered as a potential target that is genetically manipulated so as to create novel germplasms with enhanced stress tolerance in the future.

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported that two binding sites for transcription factor NRF-2 in the promoter region of the human TOMM70 gene are essential in activating transcription (Blesa et al., Mitochondrion 2004; 3:251-59. Blesa et al., Biochem Cell Biol 2006; 84:813-22). This region contains thirteen CpG methylation sites, three of which occur in the sequence 5'-CCGG-3' that is specifically recognized by HpaII methylase which modifies the internal cytosine residue. Interestingly, each NRF-2 site contains one CCGG sequence, allowing specific methylation of the NRF-2 sites and, therefore, providing an ideal model to study how methylation of these sites affects promoter activity. In this paper we report that site-specific methylation of the NRF-2 binding sites in the TOMM70 promoter down-regulated expression of a luciferase reporter in HeLa S3 cells. Electrophoretic mobility shift assays confirmed abrogation of NRF-2 binding at the methylated sites. These results suggest that methylation of the TOMM70 promoter in mammalian cells may silence TOMM70 expression. However, studies of methylation degree on DNAs from different sources found no methylation in the promoter regions of TOMM70 and other TOMM/TIMM family genes. Thus, although in vitro methylation inactivates the expression of TOMM70, our results suggest that this is not the mechanism modulating its expression in vivo. Since a number of nuclear genes encoding mitochondrial translocases have NRF-2 binding sequences containing CpG methylation sites, a possible role of methylation as a regulatory mechanism of mitochondrial biogenesis can be ruled out. (C) 2008 Elsevier B.V. All rights reserved.

Antimicrobial peptides (AMPs) include a diverse group of gene-encoded molecules that play a role in innate defense in many organisms. Evolutionary analyses of the AMP genes can be challenging because of gene duplication and diversification. Recently discovered, hepcidins are small, cysteine-rich antimicrobial peptides that also function as hormonal regulators of iron homeostasis. In this paper we investigated the organization, expression and molecular evolution of hepcidin. From searches of the literature and public genomic databases we collected 68 different hepcidin gene products from 51 different species, all among the vertebrates. Although some species have multiple hepcidin homologues, we suggest that each contains only one copy that functions as an iron regulator. Despite the recent report of hepcidin sequences in the pigeon (Fu, Y.M., Li, S.P., Wu, Y.F., Chang, Y.Z., 2007. Identification and expression analysis of hepcidin-like cDNAs from pigeon (Columba livia). Mol. Cell. Biochem. 305, 191-197.), searches of the chicken genomic, EST, and HTGS databases did not reveal any evidence of the presence of this gene in birds. This, along with the absence of reported avian transferrin receptor 2 and hemojuvelin sequences, suggests that iron homeostasis in birds may be regulated by an alternative mechanism.

Pseudomonas aeruginosa, an important opportunistic pathogen, has a single polar flagellum which is an important virulence and colonization factor by providing swimming motility. This paper describes the functional characterization of a novel gene pfm (PA2950) of P. aeruginosa. The pfm encodes a protein that is similar to a number of short-chain alcohol dehydrogenases of other bacterial species. Mutation of this gene results in a defect in swimming motility which can be completed back to that of wild type by a plasmid containing the pfm. Interestingly, the pfm mutant possesses an intact flagellum which does not rotate, thus giving rise to a non-motile phenotype. The pfm gene is encoded on an operon together with two upstream genes which code for electron transfer flavoprotein (ETF). Yeast two-hybrid tests indicated that the PFM interacts with the ETF, suggesting that the putative dehydrogenase (PFM) is involved in energy metabolism that is critical for the rotation of flagellum in P. aeruginosa.

Transferrins, found in invertebrates and vertebrates, form a physiologically important family of proteins playing a major role in iron acquisition and transport, defense against microbial pathogens, growth and differentiation. These proteins are bilobal in structure and each lobe is composed of two domains divided by a cleft harboring an iron atom. Vertebrate transferrins comprise of serotransferrins, lactoferrins and ovotransferrins. In mammals serotransferrins transport iron in physiological fluids and deliver it to cells, while lactoferrins scavenge iron, limiting its availability to invading microbes. In oviparous vertebrates there is only one transferrin gene, expressed either in the liver to be delivered to physiological fluids as serotransferrin, or in the oviduct with a final localization in egg white as ovotransferrin. Being products of one gene sero- and ovotransferrin are identical at the amino-acid sequence level but with different, cell specific glycosylation patterns. Our knowledge of the mechanisms of transferrin iron binding and release is based on sequence and structural data obtained for human serotransferrin and hen and duck ovotransferrins. No sequence information about other ovotransferrins was available until our recent publication of turkey, ostrich, and red-eared turtle (TtrF) ovotransferrin mRNA sequences [Ciuraszkiewicz, J., Olczak, M., Watorek, W., 2006. Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich and turkey. Comp. Biochem. Physiol. 143 B, 301-310]. In the present paper, ten new reptilian mRNA transferrin sequences obtained from the Nile crocodile (NtrF), bearded dragon (BtrF), Cuban brown anole (AtrF), veiled and Mediterranean chameleons (VtrF and KtrF), sand lizard (StrF), leopard gecko (LtrF), Burmese python (PtrF), African house snake (HtrF), and grass snake (GtrF) are presented and analyzed. Nile crocodile and red-eared turtle transferrins have a disulphide bridge pattern identical to known bird homologues. A partially different disulphide bridge pattern was found in the Squamata (snakes and lizards). The possibility of a unique interdomain disulphide bridge was predicted for LtrF. Differences were found in iron-binding centers from those of previously known transferrins. Substitutions were found in the iron-chelating residues of StrF and TtrF and in the synergistic anion-binding residues of NtrF. In snakes, the transferrin (PtrF, HtrF and GtrF) N-lobe "dilysine trigger" occurring in all other known transferrins was not found, which indicates a different mechanism of iron release.

Given the fact that NF-κB stays as a dormant molecule in the cytoplasm in steady state, one common step in all the metabolic activities comprising NF-κB is its activation. Consequently there are two pathways of interest related to NF-κB activation: Canonical and alternate. Both the pathways involve ubiquitination of its repressors, that is to say ubiquitination of I-κB by NEMO/IKK-α/IKK-β complex in case of NF-κB1 and that of p100 by IKK-α homodimer in case of NF-κB2. This paper attempts to figure out the ubiquitination sites in alternate pathway of NF-κB activation using a purely computational approach. We initiated the work by acquiring the genes involved in NF kappa B alternate pathway through Agilent literature search. For this we employed the Cytoscape and STRING database. Secondly, the MSA was built using the sequences obtained through BLAST search, and the results were used to update the original sequence list, which was further refined using HMMER. Structural alignment was achieved via Modeller libraries. The final model has been refined using loop_model and asses_dope functions of Modeller. Ubiquitination site is predicted to be comprised of residues 'SPECLDLLVDS' between sites 178 and 188, both positions inclusive. Unlike the classical pathway, due to absence of parallel studies for p100/RelB, a quality match could not be performed, but future studies are in pipeline to replicate the methodology for NF-κB1 activation and compare the results with existing observations. The study can be used to understand the cofactors involved and ubiquitination sites employed during the activation process during drug designing activities. The methodology can be easily scaled and adapted for classical pathway as well.

Ubiquitin (Ub) is a small 76-amino acid protein that is engaged in many different pathways within the cell, including protein turnover. During proteotoxic stress, when the demand of clearing damaged/misfolded proteins strongly increases, cells activate Ub gene transcription to face the need of extra ubiquitin. This paper shows the contribution of the four ubiquitin coding genes (UBB, UBC, UBA52, RPS27A) to the ubiquitin RNA pool under basal and stressful conditions. Our results reveal that UBC and RPS27A represent the major fraction of the Ub transcriptome in different cell lines, but when converted to the coding potential, polyubiquitin genes UBC and UBB mainly contribute to determine the intracellular ubiquitin content under basal conditions. Both the polyubiquitin genes UBB and UBC are upregulated upon proteasome inhibition and oxidative stress, with markedly higher responses from the UBC promoter. A similar output, with lower fold-inductions, is detected in heat-stressed cells, with UBC acting as the main contributor to thermotolerance. By contrast, upon these stressors, the levels of UBA52 and RPS27A mRNAs remain unchanged. Remarkably, UV irradiation fails to induce Ub gene transcription, but rather seems to act at the post-transcriptional level, by stabilizing ubiquitin mRNAs at UV doses which induce rapid degradation of other RNA molecules. Moreover, the evidence that the UBC core promoter contains multiple transcription start sites and their responsiveness to stress, is here reported for the first time.

Euryhaline teleosts possess the capacity to osmoregulate under various environmental conditions (freshwater to hypersaline water). This physiological capacity is generally monitored using enzyme activity assays (Na+/K+-ATPase...), hormones quantification (prolactine, growth hormone) or their mRNAs expression. To date, few studies addressed the genetic correlates of adaptation to varying salinity at a molecular level in such fish. In the sea bass Dicentrarchus labrax, genetic differentiation was observed at specific allozyme loci between lagoon- and open-sea populations. In the present study, we investigated transcriptomic response of D. labrax to salt- and freshwater acclimation in two organs involved in osmoregulation, gill and intestine. By using suppression subtractive hybridisation, we characterised 586 partial cDNA sequences encoding proteins potentially involved in the metabolism of sea bass acclimated to salt- or freshwater under experimental conditions. Using these results, we first characterised complete genomic sequence of a carbonic anhydrase and then analysed mRNA expression of genes potentially involved in osmoregulation mechanisms (Na+/K+-ATPase, carbonic anhydrase, angiotensin-converting enzyme and claudin-3), cell-cycle regulation (secretagogin) and immune system (nephrosin) in gill and intestine of wild fish from open sea and lagoons. Our analyses indicate a strong tissue- and environmental-dependant expression pattern for all the genes studied. A transcriptomic approach such as described in the present paper provides thus a first description of genes involved in metabolic or structural functions important for coping with environmental salinity variations in a euryhaline fish like the common sea bass D. labrax. It should be supplemented by proteomics to check the direct involvement of the gene products at the protein level, and by polymorphism analyses if one is to understand population or individual fluctuations in acclimation to salinity variation. (c) 2006 Elsevier B.V. All rights reserved.

Angiotensin-converting enzyme 2 (ACE2), a newly identified member in the renin-angiotensin system (RAS), acts as a negative regulator of ACE. It is mainly expressed in cardiac blood vessels and the tubular epithelia of kidneys and abnormal expression has been implicated in diabetes, hypertension and heart failure. The mechanism and physiological function of this zinc metallopeptidase in mammals are not yet fully understood. Non-mammalian vertebrate models offer attractive and simple alternatives that could facilitate the exploration of ACE2 function. In this paper we report the in silico analysis of Ace2 genes from the Gallus (chicken), Xenopus (frog), Fugu and Tetraodon (pufferfish) genome assembly databases, and from the Danio (zebrafish) cDNA library. Exon ambiguities of Danio and Xenopus Ace2s were resolved by RT-PCR and 3'RACE. Analyses of the exon-intron structures, alignment, phylogeny and hydrophilicity plots, together with the conserved synteny among these vertebrates, support the orthologous relationship between mammalian and non-mammalian ACE2s. The putative promoters of Ace2 from human, Tetraodon and Xenopus tropicalis drove the expression of enhanced green fluorescent protein (EGFP) specifically in the heart tissue of transgenic Xenopus thus making it a suitable model for future functional genomic studies. Additionally, the search for conserved cis-elements resulted in the discovery of WGATAR motifs in all the putative Ace2 promoters from 7 different animals, suggesting a possible role of GATA family transcriptional factors in regulating the expression of Ace2.

PKD1 is the gene responsible for autosomal dominant polycystic kidney disease (ADPKD) type 1 in humans. The PKD1 gene product is likely to be a calcium channel regulator. In this paper, we describe the isolation and characterization of the Xenopus homologue of the human PKD1 gene. We isolated and cloned genomic fragments corresponding to the amphibian homologue of PKD1 from a BAC library, and after sequencing the clones, we designed primers for the amplification of the transcript and sequenced 10 kb of ORF. The sequence of the putative protein clearly demonstrated that this gene is the homologue of human PKD1. Analysis of the tissue expression patterns of xPKD1 demonstrated a high level of expression in the kidney. A similar analysis in developing embryos and in an in vitro nephrogenic system suggests that xPKD1 is associated with, and probably involved in, the development of the amphibian pronephros.

DNA polymerase gamma is responsible for replication and repair of the mitochondrial genome. Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a 55-kDa accessory subunit. Mutations in the gene for the catalytic subunit (POLG) have been shown to be a frequent cause of mitochondrial disorders. To date over 40 disease mutations and 9 nonsynonymous polymorphisms in POLG have been found to be associated with autosomal recessive and dominant progressive external ophthalmoplegia (PEO), Alpers syndrome, sensory ataxia, neuropathy, dysarthria and ophthalmoparesis (SANDO), Parkinsonism, and male infertility. In this paper we review the literature of POLG mutations and discuss their impact on mitochondrial diseases. We also describe a public access web database to annotate POLG mutations for the research community.

SRY-related HMG-box genes (Sox genes) constitute a large family of developmentally regulated genes involved in the decision of cell fates during development and implicated in the control of diverse developmental processes. Sox3, an X-linked member of the family, is expressed in the central nervous system (CNS) from the earliest stages of development. It is considered to be one of the earliest neural markers in vertebrates playing the role in specifying neuronal fate. The aim of this study has been to determine and characterize the promoter of the human SOX3 gene and to elucidate molecular mechanisms underlying the regulation of its expression. In this study, we have isolated and performed the first characterization of the human SOX3 promoter. We have identified the transcription start point (tsp) and carried out the structural and functional analysis of the regulatory region responsible for SOX3 expression in NT2/D1 cell line. Using promoter–reporter constructs, we have determined the minimal SOX3 promoter region that confers the basal promoter activity, as well as two regulatory elements which have positive effects on the promoter activity. We have investigated in detail the functional properties of three conserved motifs within the core promoter sequence that bind transcription factors specificity protein 1 (Sp1), upstream stimulatory factor (USF) and nuclear factor Y (NF-Y). By mutational analysis, we have shown that all three sites are of functional relevance for constitutive SOX3 expression in NT2/D1 cells. We have also shown that, besides the TATA motif, at least one other essential regulatory element is required for the basal transcription of the human SOX3. Taken together, data presented in this paper suggest that transcription factors such as Sp1, USF and NF-Y could function as key regulators for the basal activation of the human SOX3 gene.

Modern technologies may improve fish production and quality and, at the same time, reduce environmental impact with benefits on the public perception of the industry. To be economically profitable, these modern technologies request an increase of rearing density that, however, could affect fish welfare. With the aim to search for molecular biomarkers to describe fish welfare, we have recently compared gene expression of sea bass farmed at different population densities by differential display obtaining six bands differentially expressed. In this paper, we have cloned the mRNA corresponding to one of those differentially expressed bands obtaining a 3860-bp sequence with an ORF of 2664 bp. Its virtual translation originated a 887-aa polypeptide that, by comparison with the other sequences available in the public data bases, resulted to be the 3-hydroxil-3-methyl-glutaryl coenzyme A reductase (HMGCR). In sea bass, as for the other species, the N- and C-terminus portions are the most conserved and are linked by an hydrophilic region that appears to be quite variable. Due to its role in the synthesis of cholesterol, HMGCR mRNA could be a good biomarker for detecting fish welfare. For this reason, we also followed, by real-time PCR, its expression after crowding stress comparing it with mRNA levels of HSP 70 and 90: HMGCR mRNA resulted highly expressed in the fishes farmed at 100 kg/m3.

The venom gland of the snake Bitis gabonica (Gaboon viper) was used for the first time to construct a unidirectional cDNA phage library followed by high-throughput sequencing and bioinformatic analysis. Hundreds of cDNAs were obtained and clustered into contigs. We found mostly novel full-length cDNA coding for metalloproteases (P-II and P-III classes), Lys49-phospholipase A2, serine proteases with essential mutations in the active site, Kunitz protease inhibitors, several C-type lectins, bradykinin-potentiating peptide, vascular endothelial growth factor, nucleotidases and nucleases, nerve growth factor, and L-amino acid oxidases. Two new members of the recently described short coding region family of disintegrin, displaying RGD and MLD motifs are reported. In addition, we have identified for the first time a cytokine-like molecule and a multi-Kunitz protease inhibitor in snake venoms. The CLUSTAL alignment and the unrooted cladograms for selected families of B. gabonica venom proteins are also presented. A significant number of sequences were devoid of database matches, suggesting that their biologic function remains to be identified. This paper also reports the N-terminus of the 15 most abundant venom proteins and the sequences matching their corresponding transcripts. The electronic version of this manuscript, available on request, contains spreadsheets with hyperlinks to FASTA-formatted files for each contig and the best match to the GenBank and Conserved Domain Databases, in addition to CLUSTAL alignments of each contig. We have thus generated a comprehensive catalog of the B. gabonica venom gland, containing for each secreted protein: (i) the predicted molecular weight, (ii) the predicted isoelectric point, (iii) the accession number, and (iv) the putative function. The role of these molecules is discussed in the context of the envenomation caused by the Gaboon viper.

We isolated Acanthogobius hasta mitochondrial DNA by long-polymerase chain reaction (long-PCR) with conserved primers, and sequenced this mitogenome with primer walking. The resultant A. hasta mitochondrial DNA sequence was found to consist of 16,663 bp with a structural organization conserved relative to that of other fish. In this paper, we report the basic characteristics of the A. hasta mitochondrial genome including structural organization, base composition of rRNAs and the tRNAs and protein-encoding genes, and characteristics of mitochondrial tRNAs. These findings are applicable to molecular phylogenetics in the suborder Gobioidei.

In mammals, thyroid hormone responsive Spot 14 (THRSP) is a small acidic protein that is predominately expressed in lipogenic tissue (i.e., liver, abdominal fat and the mammary gland). This gene has been postulated to play a role in lipogenesis, since it responds to thyroid hormone stimulation, high glucose levels and it is localized to a chromosomal region implicated in obesity. In this paper, we report the identification and characterization of duplicated polymorphic paralogs of Spot 14 in the chicken, THRSPα and THRSPβ . Despite low similarity in amino acid (aa) sequence between chickens and mammals, other properties of Spot 14 (i.e., pI, subcellular localization, transcriptional control and functional domains) appear to be highly conserved. Furthermore, a synteny group of THRSP and its flanking genes [NADH dehydrogenase ( NDUFC2 ) and glucosyltransferase ( ALG8 )] appears to be conserved among chickens, humans, mice and rats. Polymorphic alleles, involving a variable number of tandem repeats (VNTR), were discovered in the putative protein coding region of the duplicated chicken THRSPα (9 bp) and THRSPβ (6 or 12 bp) genes. Our study shows that the THRSPα locus is associated with abdominal fat traits in a broiler×Leghorn resource population.

Lysosomal sialidase is required for the catabolism of sialoglycoconjugates such as gangliosides and deficiency in this enzyme results in the autosomal recessive disease sialidosis. Furthermore, we have shown that overexpression of human sialidase is sufficient to clear accumulated ganglioside in Tay–Sachs neuroglia [Hum. Mol. Genet. 8 (1999) 1111]. In this paper, we have characterized the 5′ regulatory region of the mouse lysosomal sialidase gene in order to understand the molecular mechanisms regulating its expression. We used bioinformatic approaches to identify a transcriptional initiation site at −45 bp relative to the ATG and significant sequence homology with the rat and human promoters. Expression by the promoter was found to be cell-type restricted and required at least 750 bp upstream of the ATG for high-level expression. DNAse I footprinting analysis and reporter gene assays indicated that the promoter is responsive to Sp-1. We discovered a CCAAT box and four E-boxes within the mouse upstream region and demonstrated that CCAAT displacement protein as well as the muscle regulatory factors MyoD and Myf-5 influence sialidase expression. Taken together, these results identify cis- and trans-acting factors involved in the regulation of sialidase and point to mechanisms of gene upregulation.

The ascidian nervous system is extremely simple, although the structure of it is comparable with the complex vertebrate nervous system. This simplicity makes the ascidian nervous system a good model to understand how the neuronal circuit is built up in the chordate nervous system. In order to study the formation of the neuronal circuit at the single cell level, molecular markers to characterize specific single cells are desired. In the present paper, we describe the gene expression pattern of CIGL: an ascidian homologue of Goliath, a Drosophila RING-finger gene. In the early embryonic stage, CiGl is expressed in the lateral part of the neural tube and in several peripheral nerve cells. Later in the larval stage, CiGl specifically marks ocellus: one of the pigment cells in the ascidian brain, which is involved in the photoreceptive system. CiGl will be useful to understand the differentiation mechanism of ocellus, and especially to test the model proposed by. In addition, the finding of this single cells specific gene expression pattern at a certain developmental stage encourages us to look for more genes which mark single cells, especially those that have not been well characterized.

Human calcium-binding tyrosine-phosphorylation regulated protein (CABYR) is a polymorphic, testis-specific, calcium binding protein that undergoes tyrosine phosphorylation during in vitro capacitation. A protein kinase A (PKA) regulatory subunit type II alpha (RII-alpha) homologous domain in the N-terminus, phosphorylation dependent Ca(++) binding isoforms, and localization to the principal piece of the human sperm tail suggest that CABYR may be involved in sperm motility. In this paper, four mouse orthologous cDNAs and the genomic DNA of CABYR were cloned, nucleotide and protein sequences of mouse and humans were compared, and the genomic organization of the mCABYR gene was analyzed. Human and mouse CABYR conserve potential functional motifs including a domain homologous to the dimerization interface of cyclic adenosine monophosphate dependent PKA RII-alpha, 14 PXXP motifs, and regions of homology with extensins and src homology-3-binding protein 1. mCABYR is arranged into six exons spanning about 14 kb of DNA. Mouse CABYR showed several similarities with human CABYR: (1) the protein was localized to the principal piece of mouse epididymal spermatozoa; (2) mouse CABYR has two coding regions (CR-A and CR-B), with 66 and 82% identity, respectively to human; and (3) mCABYR showed the presence of two testis-specific transcripts of approximately 1.4 and approximately 2.4 kb. Three murine splice variants were identified, two of which spliced into CR-B. Exon 4, present in all human and mouse variants and comprising 85% of CR-A appears suitable for targeted deletion. The overall 81% nucleotide identity between mouse and human CABYR, the common genomic organization, presence of similar testis-specific transcripts, localization in the principal piece of tail and occurrence of homologous splice variants indicate an authentic murine orthologue of CABYR has been identified.

Latent transforming growth factor β binding protein-1 (LTBP-1) is important in regulating the localization and activation of transforming growth factor β. In this paper is reported the isolation of the full-length Xenopus LTBP-1 cDNA from screening a neurula embryo cDNA library. Sequence analysis of XLTBP-1 cDNA revealed an open reading frame of 4518 bp encoding a 1398 amino acid protein with a molecular mass of 154.1 kDa and an isoelectric point of 4.65. The Xenopus XLTBP-1 shares 61 and 65% amino acid identity with the mouse and human LTBP-1, respectively. It contains 17 epidermal growth factor-like motifs and four eight-cysteine repeats (8-Cys). RNase protection assay revealed that XLTBP-1 is a maternal and zygotic gene, while whole-mount in situ hybridization analysis performed on embryos at different stages showed that during early Xenopus development, XLTBP-1 mRNA is expressed in the Spemann organizer, prechordal and chordal mesoderm, and later on in the organizer derived tissues. These findings suggest an important role for XLTBP-1 in embryo axis formation.

Mitochondrial transcription factor A (mtTFA or Tfam) is a 25 kDa protein encoded by a nuclear gene and imported to mitochondria, where it functions as a key regulator of mammalian mitochondrial (mt) DNA transcription and replication. The coding sequence of the human mtTFA gene is reported in the literature and the sizes of few introns are known. In this paper we present the genomic structure of the human mtTFA gene along with the complete sequence of its six intronic regions. Three of the introns (I, III, VI) have been found to be less than 600 bp, while the other three were greater than 1.8 kb. In the course of this work, we discovered that, in addition to the active copy, different homologous sequences identified as processed pseudogenes ψh-mtTFA have been isolated and sequenced. Using an ‘in silico’ mapping approach we determined their locations on chromosomes 7, 11 and X. ψh-mtTFA locations are different from that of the gene, previously reported on chromosome 10. Transcription analysis by means of reverse transcriptase-polymerase chain reaction has shown that other than the RNA corresponding to the full-length transcript, an isoform lacking 96 bp is also present. Among the three sequenced pseudogenes only one of them located on chromosome 11 has been found to be transcribed in Jurkat cells under these culture conditions, even though transcription initiation and binding sites for different transcription factors have also been found upstream from the other two pseudogenes.

Allantoicase is one of the enzymes involved in uricolysis. The enzymes of this catabolic pathway (i.e. allantoinase, allantoicase, ureidoglycolate lyase and urease) were lost during vertebrate evolution and the causes for this loss are still unclear. In mammals, as well as in birds and reptiles, the activity of allantoicase is absent; notwithstanding, we recently cloned human and mouse cDNA sequences with high similarity with previously characterized allantoicases. In the present paper, we report the genomic organization of the allantoicase gene in mouse and in man. Both genes are constituted by 11 exons that appear to be very conserved; introns are more variable in length while maintain the same phase but for intron 4. We have also detected a second transcript of the human allantoicase gene in which exon 1 is absent. Moreover, the mouse gene maps in chromosome 12 at 13.0 cM from the centromere.

The microtubules of the mammalian nervous system are stabilised by several microtubule-associated proteins (MAPs), including the tau and MAP-2 protein families. The most prominent feature of mammalian tau and MAP-2 proteins is a common and highly homologous microtubule-binding region consisting of three or four imperfect tandem repeats. In this paper we report the cloning and characterisation of a Xenopus laevis tau-like protein (XTP) from tadpole tails. This protein encompasses two isoforms of 673 or 644 amino acids with four tandem repeats that are highly homologous to mammalian tau repeats. Both isoforms share a common amino terminal half, whereas the carboxyl terminus downstream of the repeat region is unique for each isoform. Northern blot analysis revealed that both isoforms are preferentially expressed in the tail of X. laevis tadpoles, whereas a shorter version of XTP is expressed in the head. Recombinant proteins of both XTP isoforms were able to bind microtubules. The longest isoform, however, was more effective at promoting tubulin polymerisation, indicating that sequences downstream of the repeat region affect the microtubule assembling capacity. These results demonstrate that tau-like proteins are found in non-mammalian vertebrate species, where they may support the stability of microtubules.

Fusobacterium necrophorum is a Gram-negative obligate anaerobe associated with several diseases in humans and animals. Despite its increasing clinical significance, there is little or no data on the relationship between its metabolism and virulence. Previous studies have shown that bacteria grown under iron-limitation express immunogenic antigens similar to those generated in vivo. Thus, this paper describes the relationship between F. necrophorum subsp. necrophorum (Fnn) metabolism and the expression of the encoded putative virulence factors under iron-restricted conditions. At the midlog phase, iron limitation reduced Fnn growth but the cell density was dependent on the size of the inoculum. Preferential utilization of glucose-1-phosphate, d-mannitol and l-phenylalanine; production of 2-hydroxycaproic acid and termination of dimethyl sulphide production were major Fnn response-factors to iron limitation. Ultimately, iron restriction resulted in an increased ability of Fnn to metabolize diverse carbon sources and in the expression of stress-specific virulence factors. Iron starvation in low Fnn cell density was associated with the up-regulation of haemagglutinin (HA) and leukotoxin (lktA) genes (2.49 and 3.72 fold change respectively). However, Fnn encoded Haemolysin (Hly), yebN homologue (febN) and tonB homologue, were down-regulated (0.15, 0.79 and 0.33, fold changes respectively). Interestingly, cell density appeared to play a regulatory role in the final bacteria cell biomass, induction of a metabolic gene expression and the expression pattern virulence factors in Fnn suggesting the role of a cell density-associated regulatory factor. This report suggest that future studies on differential expression of bacterial genes under altered environmental condition(s) should consider testing the effect of cell concentrations as this is often neglected in such studies. In conclusion, iron restriction induces preferential utilization of carbon sources and altered metabolism in Fnn with associated changes in the expression pattern of the virulence factors.

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.

Coenzymes derived from vitamin B 12 (cyanocobalamin) are particularly important for core metabolism in ruminant animals. Selenomonas ruminantium , a Gram-positive obligate anaerobe isolated from cattle, is the main contributor of vitamin B 12 to such ruminant animals. In nature, there are both aerobic and anaerobic pathways for B 12 synthesis – the latter is only partly elucidated. Until now, there has been no investigation of B 12 synthesis in S. ruminantium , which must use an anaerobic pathway. This paper reports the cloning of the chromosomal operon from S. ruminantium which is responsible for the first committed steps in corrinoid synthesis. Five open reading frames were found in the cloned fragment. All deduced amino acid sequences had similarity to defined proteins in the databases that are involved in porphyrin and corrin synthesis. Of particular interest is the gene designated cobA + hemD , which encodes a single polypeptide possessing two catalytic functions – uroporphyrinogen III synthase and uroporphyrinogen III 2,7-methyltransferase. This enzyme converts hydroxymethylbilane to precorrin-2. The functions of the protein coded by cobA + hemD were established by heterologous expression in Escherichia coli . The CobA activity has been demonstrated for three distinct types of proteins – monofunctional, bifunctional with siroheme formation and, this report, bifunctional with uroporphyrinogen III synthesis. The type found in S. ruminantium ( cobA + hemD ) is probably restricted to obligately anaerobic fermentative bacteria.

The present paper reports the full nucleotide sequence of a cloned cDNA prepared from RNA of lizard ovaries. The open reading frame consists of 2019 nucleotides, which encodes a protein of 673 amino acids belonging to the G protein-coupled receptor superfamily with a large extracellular N-terminal domain involved in hormone recognition. The transmembrane domain ends with a short intracytoplasmic COOH-terminal domain involved in effector activation. Phylogenetic analysis showed that the lizard receptor belongs to the family of follicle-stimulating hormone (FSH) receptors. The hydrophobicity profile is similar to that observed for mammalian and avian FSH receptors. Northern blot analysis of total RNA revealed that the FSH receptor is expressed at high levels in the ovary. In situ hybridization experiments demonstrate that FSH receptor mRNA is specifically localized within the small cells of the follicular epithelium surrounding the oocyte.

A large proportion of infection-causing isolates of the yeast Candida albicans belong to a general-purpose genotype, identifiable by fingerprinting with the moderately repetitive sequence Ca3. The high prevalence of this group – up to 70% in some patient categories – suggests that its members possess genetic determinants, which enhance their success as pathogens compared to other strains. To find such determinants we are comparing the genomes of representatives of the general-purpose genotype cluster with the genomes of other strains. In this paper we describe the identification of a 985 bp HpaII fragment (MU13-4) specific to general-purpose genotype strains. The fragment was present in 90% of these strains, but only in 10% of other strains. The fragment did not hybridize with probe Ca3, used to define the general-purpose cluster. It contains elevated levels of repetitive DNA. Sequences homologous to MU13-4 are dispersed throughout the chromosomes of general-purpose strains but are rarer or absent in other strains, as judged by Southern hybridization. Using the Stanford C. albicans genome database, we have placed the MU13-4 fragment next to a CARE-1 element. We also found 79 significant homologies between parts of MU13-4 and 19 other contigs. Attempts to amplify the region surrounding the polymorphic fragment in non-general-purpose genotype strains suggest, as do the hybridization data, that the polymorphism is created by a deletion in non-cluster strains. These results show that it is possible to identify polymorphisms specific to general-purpose genotype strains. Primers against the fragment will allow PCR-based discrimination between general-purpose genotype strains and other strains, facilitating investigations aimed at determining morbidity and mortality caused by general-purpose genotype strains compared to other strains.

This paper describes the cloning and characterization of the canine matrix metalloproteinase-9 (MMP-9) gene promoter. The 5′ untranslated region was obtained by genome walking upstream of the canine MMP-9 translation start site using canine genomic DNA as template. A DNA fragment of 1894 bp was isolated and on analysis demonstrated regions of sequence homology with the MMP-9 promoter sequences already determined for other species. In general, conserved regions correlated with DNA binding motifs such as a TATA-like box, AP-1 sites, GC boxes and a nuclear factor-κB binding domain. The DNA promoter fragment was sufficient to drive basal expression of a luciferase reporter gene in Madin Darby canine kidney (MDCK) cells and to a lesser extent in feline embryonic fibroblast (FEA) cells. Activity of the promoter was enhanced by the treatment of transfected MDCK cells with phorbol 12-myristate 13-acetate but no effect was observed in the FEA cells. Promoter deletion studies revealed that regions of promoter were necessary for induction of reporter gene expression.

This paper reports the first full-length cDNA sequence of a growth hormone receptor (GHR) from a teleost fish and its functional expression in cultured eukaryotic cells. The cDNA sequence, from Carassius auratus (goldfish), encodes a protein of 602 amino acids (aa) akin in architecture to the GHRs of other species. Despite the presence of motifs characteristic of GHR, the overall homology between the goldfish GHR and other GHRs is very low ( approximately 40% aa identity). CHO cells transfected with this receptor cDNA can be stimulated to proliferate by human growth hormone (hGH). In addition, the transfected cells can transactivate a co-expressed mammalian serine protease inhibitor (Spi) 2.1 promoter upon stimulation by hGH, indicating the successful interaction of the fish receptor with the mammalian ligand to evoke the down-stream post-receptor events. Tissue distribution studies indicated that the receptor is mostly expressed in the liver and hypothalamus of goldfish. A single mRNA transcript of a size of about 4 kb was found in the goldfish liver.

The whey acidic protein (WAP) is the major whey protein of rodent, rabbit and camel. Recently, it was identified in the milk of swine ( Simpson et al., 1998 . J. Mol. Endocrinol. 20, 27–35). In this paper, the cloning of the pig WAP cDNA and of bacterial artificial chromosome (BAC) construct containing the entire porcine WAP gene is reported. The comparison of the coding sequence of the pig WAP gene to rodent or lagomorph WAP sequence already published demonstrated that only exon sequences are partially conserved. The porcine WAP gene was localized on the subtelomeric region of the chromosome 18. The estimation of the expression of the swine WAP gene in the mammary gland from lactating animals revealed a high level of expression. In order to compare the expression level of the porcine WAP gene from the large genomic fragment which contained 70 kb downstream and 50 kb upstream the pig WAP gene or the smaller one (1 kb downstream and 2.4 kb upstream), these two genomic fragments were transfected in HC11 cell line. The BAC construct was expressed 15 times higher than the plasmid when reported to the integrated copy number. This report suggests that the HC11 cell line is a useful tool to identify the regulatory sequences of milk protein genes.

Several chemokine receptors (CKRs) act as coreceptors of human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and simian immunodeficiency virus (SIV). These CKRs interact with the V3 domain of the envelope (env) protein of HIV/SIV. In this study, we found that the amino acid sequences of two chemokines (SDF-1beta and RANTES), whose receptors (CXCR4 and CCR5) act as major coreceptors for HIV-1, HIV-2 or SIV, showed statistically significant similarity to those of the region containing the third variable (V3) and the third conserved (C3) domains (the V3--C3 domain) of the env protein of HIV-1 and HIV-2. We made a multiple alignment of amino acid sequences for 24 chemokines and the region encompassing the second conserved (C2), V3 and C3 domains (the C2--V3--C3 region) of 10 strains of HIV/SIV. Surprisingly, the hydropathic profile and several important amino acids for protein conformation, such as cysteine and tryptophan, are remarkably conserved between chemokines and the V3--C3 region of HIV/SIV. Moreover, hydrophobic amino acids, such as leucine, isoleucine and valine, are found to be clustered both in the amino-terminal region of chemokines and the C2 domain of HIV/SIV. Thus, chemokines have significantly similar profiles of amino acid properties to those of the C2--V3--C3 region of the env protein of HIV/SIV. These findings raise a hypothesis that chemokines and the C2--V3--C3 region have a common origin. Namely, the HIV/SIV ancestor incorporated a chemokine gene into its env gene. The captured chemokine gene has rapidly diverged by frequent mutations specific to the retroviral genome, and thereby obtained the ability to interact with various CKRs in a short period of time. This paper proposes that the capture of a ligand gene of the host cells into the viral genome may be one of the important mechanisms of viral evolution to expand its host range and generate new viral species.

This paper reports the characterization of the human tubulin tyrosine ligase-like 1 gene (TTLL1), which maps to the chromosome region 22q13.1 and has been partially duplicated on three other acrocentric chromosomes: 13, 15 and 21. We describe the complete cDNA, TTLL1a, coding for the putative 423 amino acid long TTLL1 and alternative transcripts coding for truncated TTLL1. Likely TTLL1a corresponds to the 1.8 kb transcript that was detected in a wide range of tissues and has a stronger expression in heart, brain and testis. A 4.8 kb transcript was found only in brain tissues. We present an interspecies sequence comparison, revealing three conserved domains, named TTLD1, TTLD2 and TTLD3, that are specific to the TTLs and TTL-like proteins.