Hox genes in the echiuroid Urechis unicinctus

An echiuroid species, Urechis unicinctus, was surveyed for Hox genes using polymerase chain reaction with homeobox-specific degenerate primers. We identified nine distinct homeodomain-containing gene fragments. These nine fragments were classified by comparative analysis. This analysis revealed that this echiuroid possessed at least three Hox genes from the anterior group, five from the central group, and one from the posterior group.Sung-Jin and Dae-Hee Lee contributed equally to this work.An erratum to this article can be found at     

The ten Hox genes of the millipede Glomeris marginata

We have isolated the ten Hox genes from the pill millipede Glomeris marginata (Myriapoda:Diplopoda). All ten genes are expressed in characteristic Hox-gene-like expression patterns. The register of Hox gene expression borders is conserved and the expression profiles show that the anterior-most limb-bearing segment in arthropods (antennal/cheliceral segment) does not express any Hox gene, while the next segment (intercalary/second-antennal/premandibular/pedipalpal segment) does express Hox genes. The Hox expression patterns in this millipede thus support the conclusion that all arthropods possess a deuterocerebral segment. We find that there is an apparent posterior shift of Hox gene expression domains dorsally relative to their ventral patterns, indicating that the decoupling of dorsal and ventral segmentation is not restricted to the level of segment polarity genes but apparently includes the Hox genes. Although the mechanism for the decoupling of dorsal and ventral segmentation remains unsolved, the decoupling must be at a level higher in the hierarchy than that of the segment polarity and Hox genes. The expression patterns of Ultrabithorax and abdominal-A suggest a correlation between the function of these genes and the delayed outgrowth of posterior trunk appendages. This delay may be caused by an assumed repressor function of Ultrabithorax, which might partially repress the activation of the Distal-less gene. The Glomeris fushi tarazu gene is expressed in a Hox-like domain and in the developing central nervous system, but not in segmental stripes such as has been reported in another myriapod species, the centipede Lithobius. In contrast to the Lithobius fushi tarazu gene, there is no indication for a role in segment formation for the millipede fushi tarazu gene, suggesting that fushi tarazu first acquired its segmentation function in the lineage of the insects.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The duplication of the <Emphasis Type="Italic">Hox</Emphasis> gene clusters in teleost fishes

Higher teleost fishes, including zebrafish and fugu, have duplicated their Hox genes relative to the gene inventory of other gnathostome lineages. The most widely accepted theory contends that the duplicate Hox clusters orginated synchronously during a single genome duplication event in the early history of ray-finned fishes. In this contribution we collect and re-evaluate all publicly available sequence information. In particular, we show that the short Hox gene fragments from published PCR surveys of the killifish Fundulus heteroclitus, the medaka Oryzias latipes and the goldfish Carassius auratus can be used to determine with little ambiguity not only their paralog group but also their membership in a particular cluster. Together with a survey of the genomic sequence data from the pufferfish Tetraodon nigroviridis we show that at least percomorpha, and possibly all eutelosts, share a system of 7 or 8 orthologous Hox gene clusters. There is little doubt about the orthology of the two teleost duplicates of the HoxA and HoxB clusters. A careful analysis of both the coding sequence of Hox genes and of conserved non-coding sequences provides additional support for the “duplication early” hypothesis that the Hox clusters in teleosts are derived from eight ancestral clusters by means of subsequent gene loss; the data remain ambiguous, however, in particular for the HoxC clusters. Assuming the “duplication early” hypothesis we use the new evidence on the Hox gene complements to determine the phylogenetic positions of gene-loss events in the wake of the cluster duplication. Surprisingly, we find that the resolution of redundancy seems to be a slow process that is still ongoing. A few suggestions on which additional sequence data would be most informative for resolving the history of the teleostean Hox genes are discussed. Supplemental material is available at http://www.bioinf.uni-leipzig.de/Publications/SUPPLEMENTS/04-006/.     

同源盒基因(Hox)与哺乳动物生殖

哺乳动物的同源盒基因（Hox）与果蝇的同源异形基因是同源基因,该基因编码的DNA片段含183碱基对,转录由61个氨基酸残基组成的蛋白质保守结构域,称同源异型域.Hox基因碱基顺序及在染色体中的位置都是高度保守的.Hox基因在体节结构分化等空间信息调控中起着重要作用,按特异的空间模式赋予每一体节其自身的特点.近年来的研究表明,Hox基因不但影响胚胎发育,而且与成体生殖系统分化有关,在着床期子宫接受态的建立及子宫蜕膜反应的发生等生殖过程中起着重要的调节作用.     

Hox Genes in the Parasitic Platyhelminthes Mesocestoides corti, Echinococcus multilocularis, and Schistosoma mansoni: Evidence for a Reduced Hox Complement

Little is known about the Hox gene complement in parasitic platyhelminthes (Neodermata). With the aim of identifying Hox genes in this group we performed two independent strategies: we performed a PCR survey with degenerate primers directed to the Hox homeobox in the cestode Mesocestoides corti, and we searched genomic assemblies of Echinococcus multilocularis and Schistosoma mansoni. We identified two Hox genes in M. corti, seven in E. multilocularis, and nine in S. mansoni (including five previously reported). The affinities of these sequences, and other previously reported Hox sequences from flatworms, were determined according to phylogenetic analysis, presence of characteristic parapeptide sequences, and unusual intron positions. Our results suggest that the last common ancestor of triclads and neodermatans had a Hox gene complement of at least seven genes, and that this was probably derived by gene loss from a larger ancestral Hox complement in lophotrochozoans.     

Isolation and expression analysis of a poriferan <Emphasis Type="Italic">Antp</Emphasis>-class <Emphasis Type="Italic">Bar-/Bsh-</Emphasis>like homeobox gene

A novel non-Hox Antp-class gene (BarBsh-Hb) was isolated from the marine sponge Halichondria sp. This gene shares high sequence identity with eumetazoan genes from the Bsh and Bar gene families and can be distinguished from other non-Hox Antp-class genes by diagnostic residues. We also present an alignment of all known (full-length) poriferan non-Hox Antp-class genes. Maximum likelihood methods were employed to estimate phylogenetic relationships among non-Hox genes and BarBsh-Hb. We employed RT-PCR techniques to look at expression across different developmental stages (larval to rhagon). BarBsh-Hb product was present in newly released larvae, but expression was not detected 8–16 h post-release. Expression of BarBsh-Hb was detected in later-stage (>16 h post-release), free-swimming larvae until they settled and attached to the substratum, after which expression was down-regulated. In a separate set of experiments, low levels of expression were observed in normal adult tissue and disaggregated adult tissue, but BarBsh-Hb expression increased during tissue re-aggregation. These data increase the number of non-Hox homeobox genes identified in sponges and provide evidence of regulation of this non-Hox gene during sponge development. While the Bar and Bsh genes play important roles in the development of nervous tissue—especially visual systems—in metazoans, the specific role(s) BarBsh-Hb play(s) in sponge development is unclear and deserves greater attention.Edited by C. Desplan     

Vertebrate homeobox genes

In the former part of the review the principal available data aboutHox genes, their molecular organisation and their expression in vertebrate embryos, with particular emphasis for mammals, are briefly summarized.In the latter part we analysed the expression of four mouse homeobox genes related to twoDrosophila genes expressed in the developing head of the fly: Emx1 and Emx2, related toems, and Otx1 and Otx2, related tootd.     

红树植物杯萼海桑SAMS基因的克隆与生物信息学分析

红树植物杯萼海桑是最耐盐的红树植物之一。S-腺苷甲硫氨酸合成酶(S-adenosylmethionine synthetase,SAMS)是S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)生物合成途径的关键酶。SAMS作为一个逆境胁迫响应蛋白在植物的耐盐调控中发挥着极其重要的作用。本文结合杯萼海桑根的转录组注释,根据编码区序列设计引物,通过PCR克隆杯萼海桑SAMS基因的编码区cDNA,并对其进行生物信息分析,为研究杯萼海桑适应逆境的机制奠定理论基础。结果显示PCR扩增了一个长1 182 bp的基因片段,该片段编码由393个氨基酸组成的S-腺苷甲硫氨酸合成酶。同源性比对及进化树分析显示杯萼海桑的SAMS氨基酸序列进化上相对保守。本研究首次从红树林植物杯萼海桑中克隆S-腺苷甲硫氨酸合成酶基因,并获得其编码区序列,为进一步研究杯萼海桑应对逆境胁迫的分子生物学机制与胁迫相关基因调控网络奠定基础。     

Molecular cloning and expression of perhydrolase genes from Pseudomonas aeruginosa and Burkholderia cepacia in Escherichia coli

Two bacterial perhydrolase genes, perPA and perBC, were cloned from Pseudomonas aeruginosa and Burkholderia cepacia, respectively, using PCR amplification with primers designed to be specific for conserved amino acid sequences of the already-known perhydrolases. The amino acid sequence of PerPA was identical to a putative perhydrolase of P. aeruginosa PAO1 genome sequences, whereas PerBC of B. cepacia was a novel bacterial perhydrolase showing similarity of less than 80% with all other existing perhydrolases. Most importantly, the perPA gene was expressed as a soluble intracellular form to an extent of more than 50% of the total protein content in Escherichia coli. Two perhydrolase enzymes were confirmed to exhibit the halogenation activity towards Phenol Red and monochlorodimedone. These results suggested that we successfully obtained the newly identified members of the bacterial perhydrolase family, expanding the pool of available perhydrolases.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ Nucleotide Sequence Databanks and have been assigned the accession number X78413     

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resilied in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.     

<Emphasis Type="Italic">Hox</Emphasis> cluster duplication in the basal teleost <Emphasis Type="Italic">Hiodon alosoides</Emphasis> (Osteoglossomorpha)

Large-scale—even genome-wide—duplications have repeatedly been invoked as an explanation for major radiations. Teleosts, the most species-rich vertebrate clade, underwent a “fish-specific genome duplication” (FSGD) that is shared by most ray-finned fish lineages. We investigate here the Hox complement of the goldeye (Hiodon alosoides), a representative of Osteoglossomorpha, the most basal teleostean clade. An extensive PCR survey reveals that goldeye has at least eight Hox clusters, indicating a duplicated genome compared to basal actinopterygians. The possession of duplicated Hox clusters is uncoupled to species richness. The Hox system of the goldeye is substantially different from that of other teleost lineages, having retained several duplicates of Hox genes for which crown teleosts have lost at least one copy. A detailed analysis of the PCR fragments as well as full length sequences of two HoxA13 paralogs, and HoxA10 and HoxC4 genes places the duplication event close in time to the divergence of Osteoglossomorpha and crown teleosts. The data are consistent with—but do not conclusively prove—that Osteoglossomorpha shares the FSGD. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

RNA interference of β1 integrin subunit impairs development and immune responses of the beet armyworm, Spodoptera exigua

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2862 bp) of a β1 subunit integrin (βSe1) was cloned from the beet armyworm, Spodoptera exigua. Phylogenetic analysis showed that βSe1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (98.3%) to β1 of Spodoptera litura. Structural analysis of the deduced amino acid sequence indicated that βSe1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that βSe1 was expressed in all developmental stages and all tested tissues of S. exigua. Its expression was further upregulated in hemocytes by injections of various microbes from quantitative RT-PCR analysis. Injection of double-stranded βSe1 RNA (dsRNA^βSe1) into late instar S. exigua suppressed βSe1 expression and resulted in significant reduction in pupal weight. The dsRNA^βSe1 injection significantly impaired hemocyte-spreading and nodule formation of S. exigua in response to bacterial challenge. Furthermore, oral ingestion of dsRNA^βSe1 induced reduction of βSe1 expression in midgut and resulted in significant mortality of S. exigua during immature development. These results suggest that βSe1 plays crucial roles in performing cellular immune responses as well as larval development in S. exigua.     

The Planarian HOM/HOX Homeobox Genes (Plox) Expressed along the Anteroposterior Axis

In the freshwater planarian Dugesia japonica, five cDNAs for HOM/HOX homeobox genes were cloned and sequenced. Together with sequence data on HOM/HOX homeobox genes of platyhelminthes deposited in databases, comparison of the deduced amino acid sequences revealed that planarians have at least seven HOM/HOX homeobox genes, Plox1 to Plox7 ( anarian HOM/H homeobox genes). Whole-mount in situ hybridization and RT-PCR revealed that Plox4 and Plox5 were increasingly expressed along a spatial gradient in the posterior region of intact animals. During regeneration, Plox5 was expressed only in the posterior region of regenerating body pieces, suggesting that the gene is involved in the anteroposterior patterning in planarians. Plox5 was not found to be expressed in a blastema-specific manner, which contradicts a previous report (J. R. Bayascas, E. Castillo, A. M. Muños-Mármol, and E. Saló. Development 124, 141–148, 1997). X-ray irradiation experiments showed that Plox5 was expressed at least in some cells other than neoblasts, but that the induction of Plox5 expression during regeneration might require neoblasts.     

Flagellin (FliC) protein sequence diversity among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> does not correlate with H serotype diversity

In Escherichia coli, the fliC gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Here, the presence of the flagellin fliC gene was studied in 86 Bacillus thuringiensis strains encompassing 67 H serotypes. Nineteen strains from four additional species in the B. cereus sensu lato group, B. cereus, B. anthracis, B. mycoides, and B. weihenstephanensis, were added for comparison purposes. The fliC genes were amplified, cloned and their nucleotide sequences determined and translated into amino acid sequences. A bootstrapped neighbor-joining tree was generated from the alignment of the translated amino acid sequences of the amplicons. Although most B. thuringiensis H serotypes had different flagellin amino acid sequences, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. In addition, although serovars from the same H serotype were sometimes found clustered together, several serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. No correlations could be established between flagellin (FliC) protein sequence diversity among B. thuringiensis H serotypes and H serotype diversity. These suggest that the B. thuringiensis fliC gene does not code for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. In a previous study, the authors have shown that the B. thuringiensis hag gene codes for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. It is proposed that the B. thuringiensis fliC gene studied here be renamed and that the so-called hag gene studied before be renamed fliC, both in accordance with the E. coli nomenclature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

The applicability of hns and fimA primers for detecting Salmonella in bioaerosols associated with animal and municipal wastes

The ability to rapidly screen environmental samples for specific pathogens such as Salmonella spp., is of particular importance in molecular epidemiology. Although gene amplification reactions allow the rapid detection of microorganisms, the use of appropriate oligonucleotide primers targeted against specific microbial genes is critical for accurate detection specificity and sensitivity. Primers such as fimA and hns have been previously shown to be specific for pure cultures of Salmonella. However, the analysis of environmental samples requires post-amplification hybridizations to detect amplicons, since the presence of inhibitory environmental components reduces amplification efficiency of the target organism. These sensitive post-amplification approaches also enable the detection of spurious amplification from non-target sequences. Bioaerosols associated with animal facilities and municipal wastes contain a diverse array of pathogens including Clostridium spp. In our studies, hybridization sensor data revealed spurious amplification of clostridial species with Salmonella hns primers. Specificity checks using type cultures of Clostridium spp. revealed non-specific amplification by hns primers. These results suggest that fimA primers may be better suited to screen Salmonella-specific sequences in environmental samples, especially those obtained from animal and municipal waste facilities.