Characterization and expression of <Emphasis Type="Italic">DjPreb</Emphasis> gene in the planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

In this study, we report the expression and identification of a PREB-related gene from the planarian Dugesia japonica, DjPreb. The planarian DjPreb cDNA is comprised of 1101 bp and contains a 972 bp open reading frame corresponding to a deduced protein of 323 amino acids with a 69 bp 5’-UTR and a 60 bp 3’-UTR. Phylogenetic analysis shows that DjPreb is PREB/PREB-like members. We examined its spatial and temporal expression and distribution in both intact and regenerating planarians by Relative quantitative real-time PCR and Whole-mount in situ hybridization. The analysis indicates that DjPreb shows a gradient express with peak levels present in the anterior and posterior regions and progressively lower levels in central regions in intact and regenerating planarians. During regeneration the expression of DjPreb is upregulated. Strong expression of DjPreb is observed in the anterior and posterior blastemas. These results suggest that DjPreb may participate in head and tail formation.     

Evolution of <Emphasis Type="Italic">Hox3</Emphasis> and <Emphasis Type="Italic">ftz</Emphasis> in arthropods: insights from the crustacean <Emphasis Type="Italic">Daphnia pulex</Emphasis>

The Drosophila melanogaster genes zerknüllt (zen) and fushi tarazu (ftz) are members of the Hox gene family whose roles have changed significantly in the insect lineage and thus provide an opportunity to study the mechanisms underlying the functional evolution of Hox proteins. We have studied the expression of orthologs of zen (DpuHox3) and ftz (Dpuftz) in the crustacean Daphnia pulex (Branchiopoda), both of which show a dynamic expression pattern. DpuHox3 is expressed in a complex pattern in early embryogenesis, with the most anterior boundary of expression lying at the anterior limit of the second antennal segment as well as a ring of expression around the embryo. In later embryos, DpuHox3 expression is restricted to the mesoderm of mandibular limb buds. Dpuftz is first expressed in a ring around the embryo following the posterior limit of the mandibular segment. Later, Dpuftz is restricted to the posterior part of the mandibular segment. This is the first report of expression of a Hox3 ortholog in a crustacean, and together with Dpuftz data, the results presented here show that Hox3 and ftz have retained a Hox-like expression pattern in crustaceans. This is in accordance with the proposed model of Hox3 and ftz evolution in arthropods and allows a more precise pinpointing of the loss of ftz “Hox-like behaviour”: in the lineage between the Branchiopoda and the basal insect Thysanura.     

<Emphasis Type="Italic">Rab11</Emphasis> is required for myoblast fusion in <Emphasis Type="Italic">Drosophila</Emphasis>

Rab11, an evolutionarily conserved, ubiquitously expressed subfamily of small monomeric Rab GTPases, has been implicated in regulating vesicular trafficking through the recycling of endosomal compartment. In order to gain an insight into the role of this gene in myogenesis during embryonic development, we have studied the expression pattern of Rab11 in mesoderm during muscle differentiation in Drosophila embryo. When dominant-negative or constitutively active Drosophila Rab11 proteins are expressed or Rab11 is reduced via double-stranded RNA in muscle precursors, they cause partial failure of myoblast fusion and show anomalies in the shape of the muscle fibres. Our results suggest that Rab11 plays no role in cell fate specification in muscle precursors but is required late in the process of myoblast fusion. This work was supported by grants from the DST (to J.K.R.) and SRF from ICMR, New Delhi (to T.B.).     

<Emphasis Type="Italic">Drosophila</Emphasis> Bys is nuclear and shows dynamic tissue-specific expression during development

Although the bys-like family of genes has been conserved from yeast to humans, it is not apparent to what extent the function of Bys-like proteins has been conserved across phylogenetic groups. Human Bystin is thought to function in a novel cell adhesion complex involved in embryo implantation. The product of the yeast bys-like gene, Enp1, is nuclear and has a role in pre-ribosomal RNA (pre-rRNA) splicing and ribosome biogenesis. To gain insight into the function of the Drosophila melanogaster bys-like family member, termed bys, we examined bys mRNA expression and the localization of Bys protein. In embryos, bys mRNA is expressed in a tissue-specific pattern during gastrulation. In the larval wing imaginal disc, bys mRNA is expressed in the ventral and dorsal regions of the wing pouch, regions that give rise to epithelia that adhere to one another after the wing disc everts. The bys mRNA expression patterns could be interpreted as being consistent with a role for Bys in events requiring cell-cell interactions. However, embryonic bys mRNA expression patterns mirror those of genes that are potential targets of the growth regulator Myc and encode nucleolar proteins implicated in cell growth. Additionally, in Schneider line 2 (S2) cells, an epitope-tagged Bys protein is localized to the nucleus, suggesting that Drosophila Bys function may be conserved with that of yeast Enp1.Edited by D.A. Weisblat     

<Emphasis Type="Italic">Sex combs reduced</Emphasis> (<Emphasis Type="Italic">Scr</Emphasis>) regulatory region of <Emphasis Type="Italic">Drosophila</Emphasis> revisited

The Hox gene Sex combs reduced (Scr) is responsible for the differentiation of the labial and prothoracic segments in Drosophila. Scr is expressed in several specific tissues throughout embryonic development, following a complex path that must be coordinated by an equally complex regulatory region. Although some cis-regulatory modules (CRMs) have been identified in the Scr regulatory region (~75 kb), there has been no detailed and systematic study of the distinct regulatory elements present within this region. In this study, the Scr regulatory region was revisited with the aim of filling this gap. We focused on the identification of Initiator elements (IEs) that bind segmentation factors, Polycomb response elements (PREs) that are recognized by the Polycomb and Trithorax complexes, as well as insulators and tethering elements. To this end, we summarized all currently available information, mainly obtained from high throughput ChIP data projects. In addition, a bioinformatic analysis based on the evolutionary conservation of regulatory sequences using the software MOTEVO was performed to identify IE and PRE candidates in the Scr region. The results obtained by this combined strategy are largely consistent with the CRMs previously identified in the Scr region and help to: (i) delimit them more accurately, (ii) subdivide two of them into different independent elements, (iii) identify a new CRM, (iv) identify the composition of their binding sites and (v) better define some of their characteristics. These positive results indicate that an approach that integrates functional and bioinformatic data might be useful to characterize other regulatory regions.     

Harvest-inducibility of the promoter of alfalfa<Emphasis Type="Italic"> S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine:<Emphasis Type="Italic"> trans</Emphasis>-caffeoyl-CoA3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase gene

A major limitation on the expression of some foreign proteins in transgenic plants is the toxic effect of such proteins on the host plant resulting in inhibition of normal growth and development. A solution to this problem is to control the expression of genes for such proteins by means of inducible promoters, as is frequently done in microbial systems. A cDNA clone was obtained from subtractive hybridization of non-harvested and harvested alfalfa leaf tissue, named hi12. The hi12 cDNA was identified as part of the S-adenosyl-l-methionine: trans-caffeoyl-CoA3-O-methyltransferase gene of alfalfa, a gene encoding an essential key enzyme in lignin synthesis. The hi12 gene was strongly induced by harvesting and wounding but not by heat shock. The promoter of the hi12 gene, isolated by genomic walking, contained several stress response cis-elements. Transgenic plants of tobacco and Medicago truncatula containing the GUS gene driven by the promoter showed GUS expression following harvesting, demonstrating the activity of these regulatory regions in other plant species.     

<Emphasis Type="Italic">kitb</Emphasis>, a second zebrafish ortholog of mouse <Emphasis Type="Italic">Kit</Emphasis>

The large numbers of duplicated pairs of genes in zebrafish compared to their mammalian counterparts has lead to the notion that expression of zebrafish co-orthologous pairs in some cases can together describe the expression of their mammalian counterpart. Here, we explore this notion by identification and analysis of a second zebrafish ortholog of the mammalian Kit receptor tyrosine kinase (kitb). We show that in embryos, kitb is expressed in a non-overlapping pattern to that of kita, in the anterior ventral mesoderm, Rohon-beardRohon–Beard neurons, the otic vesicle, and trigeminal ganglia. The expression pattern of kita and kitb in zebrafish together approximates that of Kit in mouse, with the exception that neither zebrafish kit gene is expressed in primordial germ cells, a site of kit expression in the mouse embryo. In addition, zebrafish kita is expressed in a site of zebrafish primitive hematopoiesis but not required for blood development, and we fail to detect kitb expression in sites of zebrafish hematopoiesis. Thus, the expression and function of zebrafish kit genes cannot be described as a simple partition of the expression and function of mouse Kit. We discuss the possibility that these unaccounted for expression domains and functions are derived from more ancestral gene duplications and partitioning instead of the relatively recent teleost teleost-specific duplication. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

<Emphasis Type="Italic">Rab11</Emphasis> is required for embryonic nervous system development in <Emphasis Type="Italic">Drosophila</Emphasis>

In eukaryotes, membrane trafficking is regulated by the small monomeric GTPases of Rab protein family. Rab11, an evolutionary conserved, ubiquitously expressed subfamily of the Rab GTPases, has been implicated in the regulation of vesicular trafficking through the recycling of endosomes. To dissect out the role of this protein during embryonic nervous system development, we have studied the expression pattern of Rab11 in the ventral nerve cord during neuronal differentiation in the Drosophila embryo. When the dominant-negative or constitutively-active mutant DRab11 proteins are expressed in neurons, or when homozygous mutant Rab11 embryos are analyzed, defects are found in the developing central nervous system, along with disorganization and misrouting of embryonic axons. Our results provide the first in vivo evidence that Rab11 is involved in the development of the nervous system during Drosophila embryogenesis. This work was supported by the DST (to J.K.R.) and SRF from ICMR, New Delhi (to T.B.).     

Identification and expression analysis of rainbow trout <Emphasis Type="Italic">pumilio</Emphasis>-1 and <Emphasis Type="Italic">pumilio</Emphasis>-2

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.This work was in part supported by a grant from the Akiyama Foundation to E.I. Nucleotide sequence data for rainbow trout pumilio-1 and pumilio-2 have been deposited in the DDBJ/EMBL/GenBank databases.     

Molecular cloning and expression analysis of <Emphasis Type="Italic">Bmrbp1</Emphasis>, the <Emphasis Type="Italic">Bombyx mori</Emphasis> homologue of the <Emphasis Type="Italic">Drosophila</Emphasis> gene <Emphasis Type="Italic">rbp1</Emphasis>

RBP1 is an important splicing factor involved in alternative splicing of the pre-mRNA of Drosophila sex-determining gene dsx. In this work, the Bombyx mori homologue of the rbp1 gene, Bmrbp1, was cloned. The pre-mRNA of Bmrbp1 gene is alternatively spliced to produce four mature mRNAs, named Bmrbp1-PA, Bmrbp1-PB, Bmrbp1-PC and Bmrbp1-PD, with nucleotide lengths of 799 nt, 1,316 nt, 894 nt and 724 nt, coding for 142 aa, 159 aa, 91 aa and 117 aa, respectively. BmRBP1-PA and BmRBP1-PD contain a N terminal RNA recognization motif (RRM) and a C terminal arginine/serine-rich domain, while BmRBP1-PB and BmRBP1-PC only share a RRM. Amino acid sequence alignments showed that BmRBP1 is conserved with its homologues in other insects and with other SR family proteins. The RT-PCR showed that Bmrbp1-PA was strongly expressed in all examined tissues and development stages, but Bmrbp1-PB was weakly expressed in these tissues and stages. The expression of both Bmrbp1-PA and Bmrbp1-PB showed no obvious sex difference. While the Bmrbp1-PC and Bmrbp1-PD were beyond detection by RT-PCR very likely due to their tissue/stage specificity. These results suggested that Bmrbp1 should be a member of SR family splicing factors, whether it is involved in the sex-specific splicing of Bmdsx pre-mRNA needs further research.     

Expression of <Emphasis Type="Italic">hunchback</Emphasis> during oogenesis and embryogenesis in <Emphasis Type="Italic">Locusta migratoria manilensis</Emphasis> (Meyen)

hb (hunchback) is a contributing factor in anteroposterior axial patterning of insects. Although the hb function in Locusta migratoria manilensis has been investigated, its expression pattern remains unknown. Here, the mouse polyclonal antibody was produced against Hb fusion protein, and then its expression pattern during oogenesis and embryogenesis of L. migratoria manilensis was examined by immunohistochemical staining. Hb protein was detected in the oocyte nucleus which was positioned centrally within the developing oocyte. The oocyte nucleus gradually moved to the posterior end of the egg along with the oocyte maturing. In freshly laid eggs, Hb formed gradient at the posterior end of the egg, and then hb was expressed as a band in the middle of the blastodisc. As the blastodisc differentiated into the head and trunk, the expression region became wide, which would develop into spatial gnathal and thoracic segments. With abdominal segmentation, the expression domain in the gnathal and thoracic region became faint and eventually faded out, while the Hb expression domain appeared at the posterior growth zone in a discontinuous expression manner. The hb expression pattern of L. migratoria manilensis is greatly similar to that of other locusts, such as Schistocerca americana and another L. migratoria. Compared with other insects, hb expression is conserved in the gnathal and thoracic domains, while divergent in oogenesis and abdomen.     

Strong Expression and Conserved Regulation of <Emphasis Type="Italic">ACT2</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Arabidopsis ACT2 represents an ancient class of vegetative plant actins and is strongly and constitutively expressed in almost all Arabidopsis sporophyte vegetative tissues. Using the beta glucuronidase report system, the studies showed that ACT2 5′ regulatory region was significantly more active than CaMV 35S promoter in Arabidopsis seedlings and gametophyte vegetative tissues of Physcomitrella patens. Its activity was also observed in rice and maize seedlings. Thus, the ACT2 5′ regulatory region could potentially serve as a strong regulator to express a transgene in divergent plant species. ACT2 5′ regulatory region contained 15 conserved sequence elements, an ancient intron in its 5′ un-translated region (5′ UTR), and a purine-rich stretch followed by a pyrimidine-rich stretch (PuPy). Mutagenesis and deletion analysis illustrated that some of the conserved sequence elements and the region containing PuPy sequences played regulatory roles in Arabidopsis. Interestingly, mutation of the conserved elements did not lead a dramatic change in the activity of ACT2 5′ regulatory region. The ancient intron in ACT2 5′ UTR was required for its strong expression in both Arabidopsis and P. patens, but did not fully function as a canonical intron. Thus, it was likely that some of the conserved sequence elements and gene structures had been preserved in ACT2 5′ regulatory region over the course of land plant evolution partly due to their functional importance. The studies provided additional evidences that identification of evolutionarily conserved features in non-coding region might be used as an efficient strategy to predict gene regulatory elements.     

Gene expression during postembryonic segmentation in the centipede <Emphasis Type="Italic">Lithobius peregrinus</Emphasis> (Chilopoda,Lithobiomorpha)

Postembryonic segmentation (anamorphosis) is widespread among arthropods, but only partially known as for its developmental mechanics and control. Studies on developmental genetics of segmentation in anamorphic arthropods are mostly limited to the germ band stage, during early phases of embryonic development. This work presents the first data on the postembryonic expression of a segmentation gene in a myriapod. Using real-time PCR, we analyzed engrailed expression patterns during the anamorphic stages of the centipede Lithobius peregrinus. A variation pattern in en RNA level during anamorphosis suggests that gene expression is precisely modulated during this period of development and that engrailed is mainly expressed in the posterior part of the body, in the newly differentiating segments of each stage. As anamorphosis is possibly the primitive segmentation mode in arthropods, the postembryonic en expression pattern documented here provides evidence for a conservation of en role in ontogeny, across the embryonic/postembryonic boundary, as well as in phylogeny, across the same boundary, but in the opposite direction, from primitive postembryonic expression to the more derived expression in clades with exclusively embryonic segmentation.     

The <Emphasis Type="Italic">flhDC</Emphasis> gene affects motility and biofilm formation in <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

The flagella master regulatory gene flhDC of Yersinia pseudotuberculosis serotype III (YPIII) was mutated by deleting the middle region and replaced by a tetracycline resistant gene, and the subsequent mutant strain named YPIIIΔflhDC was obtained. Swimming assay showed that the swimming motility of the mutant strain was completely abolished. The promoter region of the flagella second-class regulatory gene fliA was fused with the lux box, and was conjugated with the mutant and the parent strains respectively for the first cross. LUCY assay result demonstrated that flhDC regulated the expression of fliA in YPIII as reported in E. coli. Biofilm formation of the mutant strain on abiotic and biotic surfaces was observed and quantified. The results showed that mutation of flhDC decreased biofilm formation on both abiotic and biotic surfaces, and abated the infection on Caenorhabdtis elegans. Our results suggest that mutation of the flagella master regulatory gene flhDC not only abolished the swimming motility, but also affected biofilm formation of YPIII on different surfaces. The new function of flhDC identified in this study provides a novel viewpoint for the control of bacterial biofilm formation.     

Characterization of the expression profile of <Emphasis Type="Italic">calpain</Emphasis>-<Emphasis Type="Italic">3</Emphasis> (<Emphasis Type="Italic">CAPN3</Emphasis>) gene in chicken

Calpain-3 is a skeletal muscle-specific protease and participates in the regulation of myogenesis. In this study, we quantified the expression of calpain-3 (CAPN3) mRNA in a Chinese local chicken breed (Sichuan Mountainous Black-boned chicken [MB]), to discern the tissue and ontogenic expression pattern. Meanwhile, we compared the CAPN3 mRNA expression pattern in MB chicken at 10 weeks with a commercial meat type chicken line (S01) of the same age to identify the unique expression pattern under different genetic background. A real time quantitative PCR (qRT-PCR) assay was developed for an accurate measurement of its expression in various tissues from chickens at different ages (0, 2, 4, 6, 8, 10, and 12 weeks). Expression of the CAPN3 mRNA was detected in the selected tissues, regardless of age. The breast muscle and leg muscle tissues had a significantly higher expression than the other tissues from the same individual (P < 0.01). Overall, the CAPN3 mRNA level exhibited a “rise-decline” developmental change in detected tissues except for brain. The S01 chicken had a higher expression of the CAPN3 mRNA in detected tissues than the MB chicken at 10 weeks. The present expression data of chicken CAPN3 gene may provide some information to shed light on the tissue and ontogenic expression pattern during chicken development.