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Abstract: Mycobacteria contain a large number of redundant genes whose functions are difficult to analyze in mutants, because there are only two efficient resistance markers available for allelic exchange experiments. We have established a system based on the Flp recombinase of the yeast Saccharomyces cerevisiae for use in the nonpathogenic model organism Mycobacterium smegmatis. This system consists of a hygromycin resistance cassette flanked by two Flp recognition targets (FRT) in direct orientation and a curable plasmid for expression of the flp gene. The FRT-hyg-FRT cassette was used on a suicide plasmid and on a conditionally replicating plasmid to delete two of the four known porin genes of M. smegmatis, mspA and mspC, respectively, by homologous recombination. The hyg gene was specifically removed from the chromosome of both mutants upon expression of the flp gene. Based on the marker-less mspC mutant strain, a double knock-out mutant lacking also mspA was obtained using the same strategy. Thus, by a fast and efficient two-step procedure, each of the porin genes was replaced by a single FRT site, which can be further used for site-specific integration. These results show that the Flp/FRT system is a suitable genetic tool for constructing unmarked mutations and for the analysis of redundant genes by consecutive gene deletions in M. smegmatis. [Copyright &y& Elsevier]

Abstract: Calcitonin inhibits bone resorption by acting on osteoclasts via a specific receptor. The calcitonin receptor (CTR) is also found in many other normal and malignant tissues and cell lines. It has been cloned and sequenced in several species including humans. It belongs to a subclass of seven-transmembrane G protein-coupled receptors. Four human CTR (H-CTR) isoforms generated by alternatively spliced mRNA have previously been described. Two H-CTR encoding DNAs containing an unidentified 50-bp insert are now reported from T47D cells. The 50-bp insert corresponds to a DNA region located between exon 9 and exon 10, and appears to originate from an alternative splicing process. The two H-CTR cDNAs encode 274 and 290 aa long isoforms. Both are deleted from the putative fourth transmembrane domain to C-tail. They differ by the presence (H-CTR5) or absence (H-CTR6) of a previously known 16-aa insert in the putative first intracellular loop. Cell- and tissue-distribution analysis using RT-PCR demonstrates that the shorter one, HCTR6, is more prevalent. The mRNA of both isoforms was detected in giant cell tumor, whereas only H-CTR6 mRNA was detected in TT cells and kidney tissue. Neither H-CTR5 nor H-CTR6 could be detected in peripheral blood mononuclear cells cultured in the presence of RANKL, in MCF7 cells, and in cortical brain and ovarian tissues. When H-CTR6 was transiently expressed in HEK293 cells, CT failed to induce production of cAMP or to bind to the receptor. These suggest either an intrinsic loss of ligand binding function, or an altered intracellular trafficking. Our findings therefore indicate the existence of two novel splice variants of the H-CTR and confirm that multiple splicing patterns could be involved in the post-transcriptional regulation of the gene. [Copyright &y& Elsevier]

Abstract: The present study was conducted to clarify what occurs during the metamorphosis of the imaginal disc in insects. To understand the metamorphosis on a molecular level, the changes in expression profiles in the imaginal disc during metamorphosis were investigated. For this purpose, we constructed cDNA libraries from four different stages of wing discs of Bombyx mori, sequenced about 1000 cDNAs randomly collected from each library, and constructed a database of expressed sequence tags (EST). The morphological changes and expression profiles from EST were compared during those four stages. Microarray analysis was applied to quantify the expression of each gene in each stage in order to confirm whether the expression of the genes identified from EST was induced by 20-hydroxyecdysone (20E) in a stage-specific manner. Wing discs showed dynamic morphogenesis in 4–5 days during the preparatory stage of metamorphosis, which was under the control of an ecdysteroid. Different expressed profiles were observed in each of the four different stages by comparison of each EST clone. These profiles reflected the morphological changes of the Bombyx wing disc during metamorphosis. The results of expression profiles from the four stages suggested that the V4 stage was cell proliferating; W0, proliferating and the beginning of differentiation; W2, morphologically changing; W3, cuticle secreting. Microarray analysis showed the effectiveness of its application on 20E induction of genes in wing discs. The wing disc of B. mori is an exceptionally suitable system for understanding the relationship between morphological changes and the distribution of mRNA. [Copyright &y& Elsevier]

Abstract: Amphotericin B is a medically important antifungal antibiotic that is produced by Streptomyces nodosus. Genetic manipulation of this organism has led to production of the first amphotericin analogues by engineered biosynthesis. Here, these studies were extended by sequencing the chromosomal regions flanking the amphotericin polyketide synthase genes, and by refining the phage KC515 transduction method for disruption and replacement of S. nodosus genes. A hybrid vector was constructed from KC515 DNA and the Escherichia coli plasmid pACYC177. This vector replicated as a plasmid in E. coli and the purified DNA yielded phage plaques on Streptomyces lividans after polyethylene glycol (PEG)-mediated transfection of protoplasts. The left flank of the amphotericin gene cluster was found to include amphRI, RII, RIII and RIV genes that are similar to regulatory genes in other polyene biosynthetic gene clusters. One of these regulatory genes, amphRI, was found to have a homologue, amphRVI, located in the right flank at a distance of 127 kbp along the chromosome. However, disruption of amphRVI using the hybrid vector had no effect on the yield of amphotericin obtained from cultures grown on production medium. The hybrid vector was also used for precise deletion of the DNA coding for two modules of the AmphC polyketide synthase protein. Analysis by UV spectrophotometry revealed that the deletion mutant produced a novel pentaene, with reduced antifungal activity but apparently greater water-solubility than amphotericin B. This shows the potential for use of the new vector in engineering of this and other biosynthetic pathways in Streptomyces. [Copyright &y& Elsevier]

Abstract: Bex3 expression and possible function in growth control were studied. It was expressed in a limited number of organs, including gonads and hippocampal regions of the brain. Visualized by deconvolution microscopy as a GFP-fusion protein in F9 teratocarcinoma cells, Bex3 localized, along with concentrations of actin, at perinuclear mitochondria that were undergoing active DNA replication. Bex3 association with mitochondria required a nuclear export signal (NES) and the C-terminal four amino acids (CaaX box), and siRNA reduction of Bex3 levels led to slow or negligible growth rates of the F9 cells. Thus, Bex3 may be required in target tissues for mitochondrial function at a distinct phase of the cellular growth cycle. [Copyright &y& Elsevier]

Six acyl-CoA desaturase-encoding cDNAs from mRNA isolated from the spotted fireworm moth, Choristoneura parallela (Lepidoptera: Tortricidae) were characterized and assayed for functionality. The expression levels of these cDNAs were determined in the pheromone gland and fat body by real-time PCR and the resulting patterns are in line with results from published studies on other moth sex pheromone desaturases. The cDNAs were found to correspond to six genes. Using both biochemical and phylogenetic analyses, four of these were found to belong to previously characterized desaturase functional groups [the Δ10,11, the Δ9 (16>18) and the Δ9 (18>16) groups]. A desaturase highly expressed in the pheromone gland was a novel E11 desaturase that was specific to 14-carbon precursor acids. The fifth gene [CpaZ9(14–26)] was found to display a novel Z9 activity indicating that it belongs to a new Δ9 functional group, whereas the sixth gene was determined to be nonfunctional with respect to desaturase activity. In accordance with previous studies, we find that desaturases of the Δ10,11 and Δ14 groups, which are the fastest evolving desaturases and possess the novel pheromone biosynthetic function, are expressed primarily in the pheromone gland whereas all other desaturases, which do not possess the novel reproductive function, evolve more slowly and display the ancestral metabolic function and pattern of gene expression. [Copyright &y& Elsevier]

The genome of marine sponge Suberites domuncula, a member of the most ancient and most simple metazoan phylum Porifera, encodes at least five genes for Src-type proteins, more than, i.e., Caenorhabditis elegans or Drosophila melanogaster (two in each). Three proteins, SRC1SD, SRC2SD and SRC3SD, were fully characterized. The overall homology (identity+similarity) among the three S. domuncula Srcs (68–71%) is much lower than the sequence conservation between orthologous Src proteins from freshwater sponges (82–85%). It is therefore very likely that several src genes/proteins were already present in the genome of Urmetazoa, the hypothetical metazoan ancestor.We have identified in the S. domuncula expressed sequence tags (ESTs) database further Src homology 2 (SH2) and 3 (SH3) domains that are unrelated to protein tyrosine kinases (PTKs). Src-related SH2 and SH3 domains from different species are much more conserved than SH2 and SH3 domains from different proteins in the same organism (S. domuncula), supporting the view that the common, ancestral src gene was already a multidomain protein composed of SH3, SH2 and tyrosine kinase (TK) domains.Two S. domuncula src genes were fully sequenced: src1SD gene has six and src2SD gene only one intron in front of SH2 domain, located at the same position in both genes. All vertebrate src genes, from fish to human, originated from the same ancestral gene, because they all have 10 introns at conserved positions. However, src genes in invertebrates have fewer introns that are located at different positions. Only the intron in front of the SH2 domain is present at the absolutely conserved position (and phase) in all known src genes, indicating that at least this intron was already present in the ancestral gene, common to all Metazoa. Our results also suggest that TK domain in this ancestral src was encoded on a single exon. [Copyright &y& Elsevier]

Fibrinogen-related proteins (FREPs) are found in the hemolymph of the freshwater snail Biomphalaria glabrata, are up-regulated following exposure to digenetic trematode parasites, and bind to trematode larval surfaces, suggestive of a role in internal defense. Southern blot and degenerate-polymerase chain reaction (PCR) analyses were undertaken to better understand the diversity of the FREP-encoding gene family. Probes corresponding to the N-terminal IgSF domains of specific FREP gene subfamilies (FREPs 2, 3, 4, 7, 12 and 13) revealed between 1 to 8 loci per subfamily on Southern blots. Probes representing the relatively conserved C-terminal fibrinogen domain of FREPs bound many sequences in Southern blots of genomic DNA from B. glabrata, and from two related gastropod species, Biomphalaria pfeifferi and Helisoma trivolvis. Using degenerate-PCR, we obtained 42 unique fibrinogen-encoding sequences from 180 clones derived from a single individual of the M-line strain of B. glabrata, further supporting the notion of their abundant representation in the B. glabrata genome. The fibrinogen-encoding sequences of FREPs encoding one or two IgSF domains tended to separate into distinct clades, but bootstrap support for this separation was low. A novel category of fibrinogen-encoding sequence was also revealed. This study provides the approximate number of gene copies in several FREP subfamilies, confirms the existence of a diverse FREP gene family, reports additional unusual sequences encoding fibrinogen-like molecules, and provides further justification to explore the functional roles of FREPs in both B. glabrata and B. pfeifferi, both important intermediate hosts of the human pathogen, Schistosoma mansoni. [Copyright &y& Elsevier]

There is a growing family of novel GTPases conserved among higher plants and vertebrates, abbreviated as AIG1, IAP, IMAP, and IAN, respectively. Here, we comparatively analyze the human gene family encoding GTPases of the immunity-associated protein family recently re-termed GIMAP. Chromosome 7q36.1 contains, within 300 kb, a gimap gene cluster with seven functional genes and one pseudogene (hgimap3). The six genes hgimap1, hgimap2, hgimap4, hgimap5, hgimap6, and hgimap7 encode 33–46 kDa proteins with one GTP-binding domain, whereas hgimap8 encodes a very unusual 75-kDa protein with three GTP-binding domains. All hgimap genes except hgimap2 have orthologs in the mouse. Major expression sites of hgimap mRNAs are the spleen and lymph nodes, but also other organs such as muscle, heart, placenta, and digestive tract display detectable hgimap mRNA levels. The proteins hGIMAP4 and hGIMAP7 can be localized at ER and Golgi apparatus, but not in mitochondria, lysosomes and nuclei. All hgimap genes were expressed at very low levels—if at all—in diverse cancer cell lines. Our data support the view that the GIMAP proteins are involved in the control of cell survival not only in cells of the immune system as commonly anticipated. [Copyright &y& Elsevier]

During the complex life cycle of Plasmodium falciparum, through mosquito and human, the erythrocytic cycle is responsible for malarial disease and transmission. The regulation of events that occur during parasite development, such as proliferation and differentiation, implies a fine control of transcriptional activities that in turn governs the expression profiles of sets of genes. Pathways that underline gametocyte commitment are yet poorly understood even though kinases and transcription factors have been assumed to play a crucial role in this event. In order to understand the molecular mechanisms controlling the variation of gene expression profiles that might participate in early gametocytogenesis, the transcriptome of two clones, 3D7 and its gametocyte-less derivative F12, was compared at five time points of the erythrocytic asexual development. We have used a thematic DNA microarray containing 150 PCR fragments, representative of P. falciparum genes involved in signal transduction, cell cycle and transcriptional regulation. We identified several genes eliciting different expression profiles among which some implicated in gene regulation or encoding putative transcription factors. The differential expression of transcription factor and kinase transcripts observed in the two clones may enlighten genes that might have a role in impairment of the early gametocytogenesis of the F12 clone. [Copyright &y& Elsevier]

Rac2 is a Rho family GTPase expressed specifically in hematopoietic cells. The 4.5 kb proximal Rac2 gene promoter exhibits strong but promiscuous activity following either transient or stable transfection into tissue culture cells, indicating that additional cis-elements are required to silence Rac2 expression in non-hematopoietic cells. A bacterial artificial chromosome (BAC) containing the human Rac2 gene, including as little as 1.6 kb of upstream and 8 kb of downstream sequence, exhibits hematopoietic-restricted expression in transgenic mice. The Rac2 genomic locus exhibits distinct patterns of DNA methylation in expressing versus non-expressing cells. Cells that lack Rac2 expression exhibit increased cytosine methylation in the sequences flanking the gene, whereas cells that express Rac2 exhibit increased cytosine methylation within the body of the Rac2 gene. Treatment of non-expressing cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine reduces cytosine methylation of the Rac2 gene locus and is sufficient to induce Rac2 gene expression. Conversely, treatment with the histone deacetylase (HDAC) inhibitor trichostatin A fails to induce Rac2 gene expression. These findings define a genomic fragment sufficient to direct hematopoietic-specific expression of Rac2, and reveal the importance of cytosine methylation in the repression of Rac2 expression in non-hematopoietic cells. [Copyright &y& Elsevier]

Filamentous tau deposits are a defining feature of a number of human neurodegenerative diseases. Apes and monkeys have been reported to be differentially susceptible to developing tau pathology. Despite this, only little is known about the organisation and sequence of Tau from nonhuman primates. Here we have sequenced Tau exons 1–13, including flanking intronic regions, and the region in intron 9 that contains Saitohin in chimpanzees, gorillas, and gibbons. Partial sequences were obtained for cynomolgus macaque and green monkey. Chimpanzee brain tau was 100% identical to human tau. Identities were 99.5% for gorilla tau and 99.0% for gibbon tau. Chimpanzee DNA was polymorphic for a repeat in intron 9, which was present in human and gorilla tau, and for the nucleotide at position +29 of the intron that follows exon 10. As was the case of the other nonhuman primates examined, chimpanzee DNA was homozygous for nucleotides used to define the H2 haplotype in human Tau. These differences between human and chimpanzee Tau may contribute to the apparent resistance of chimpanzee brain to developing tau pathology. Sequencing of Saitohin revealed an intact open reading frame in chimpanzee and gorilla, but not in gibbon or macaque. [Copyright &y& Elsevier]

Op18 (Oncoprotein 18, Stathmin) is an important mitotic regulator that is highly expressed in many cancers including the metastatic prostate carcinoma cell line PC-3-M. Recent studies indicate that antisense-mediated down-regulation of Op18 can inhibit cellular proliferation. However, the transcriptional mechanisms responsible for its normal regulation and for its high level of expression in proliferating cells remain poorly understood. In the study presented here, we have characterized transcription factor binding sites that together account for nearly 80% of the Op18 expression in PC-3-M cells. The 5′ flanking region of the Op18 gene contains four putative E2F sites located at −700 (site 1), −28 (site 2), −19 (site 3), and +720 (site 4) relative to the transcriptional start site. E2F has been implicated in both the c-Jun-mediated up-regulation and the doxorubicin-induced repression of Op18 expression. We have used promoter–reporter assays and mobility shift assays to functionally examine each of these E2F sites. Mutagenesis studies indicate that all sites contribute to the basal expression of Op18. Mutagenesis of either site 1 or 4 reduced the reporter activity by 40%, mutagenesis of site 2 reduced reporter activity by 20%, and mutations in site 3 had no effect on reporter activity. Combinatorial mutagenesis indicates that site 1 and 4 function independently, whereas site 2 functions synergistically with either site 3 or 4. Mobility shift assays indicate that all elements bind factors in the nuclear extracts of PC-3-M cells. Characterization of the sites show that site 1, though a positive element, is not E2F specific; sites 2 and 3 may contain an overlapping binding site for E2F and NF1; and site 4, which resides in intron 1, is the only site shown to interact exclusively with E2F. These studies suggest that the overexpression of Op18 in PC-3-M cells is mediated predominately through the E2F family of transcription factors. [Copyright &y& Elsevier]

The human transcription factor Sp3 has been widely studied at the translational level and has been described as a regulatory factor for a number of genes. Sp3 is currently characterized as a bifunctional transcription factor having the ability to behave as both an activator and/or a repressor in various promoter regions. Previous translational studies have attempted to determine the basis for these diverse functions with mostly contradictory evidence to date. Little data are available, however, concerning genomic structure, full-length cDNA, potential transcript variants, or location of translation initiation sites for the large isoform of the Sp3 gene. In this study, bacterial artificial chromosome (BAC) sequencing, reverse transcription-polymerase chain reaction (RT-PCR), genomic PCR, and database mining indicate that the Sp3 gene encompasses seven exons spanning more than 55 kb of genomic DNA on Chromosome 2. The 5′ end of this sequence contains a large CpG island. This work also detected a processed pseudogene, ψSp3, located on Chromosome 13, spanning approximately 4.0 kb. Northern blot analysis detected three predominant transcripts at 4.0, 6.0 and 2.5 kb. Sequence analysis indicated that alternative splicing of exon 3 allows for multiple transcripts of Sp3. Each sequenced transcript possesses three to five potential translation initiation sites. This diversity at the level of gene expression will likely be key to understanding the diverse functions of Sp3. [Copyright &y& Elsevier]

Lecithin:retinol acyltransferase (LRAT) catalyzes the synthesis of retinyl esters in many tissues and is crucial for the transport and intracellular storage of vitamin A. LRAT expression is highly regulated in the liver. In this study, we have cloned and sequenced the full-length LRAT mRNA from human liver and identified its 5′- and 3′-ends. Full-length LRAT mRNA comprises 5023 nt with a predicted ORF of 230 amino acids, a short 5′UTR, and a relatively long 3′UTR of 4 kb containing several polyadenylation signals and AU-rich regions. Based on alignment of this mRNA with human genomic DNA in the GenBank database, the human LRAT gene spans about 9.1 kbp and consists of two exons and a relatively long 4-kbp intron. Further analysis of normal liver revealed a minor alternative splicing variant which lacks a 103 nt polynucleotide contained in the 5′UTR of the full-length LRAT transcript. This variant predicts that the LRAT gene is organized into three exons and two introns, as reported for LRAT cloned from retinal pigment epithelium (RPE) cells. These two LRAT mRNA variants are also present in testis, which is known to express LRAT and contain retinyl esters. Major and minor transcription start sites for human liver LRAT mRNA were identified and the sequence of the upstream proximal promoter region was retrieved from the GenBank database and physically analyzed for the presence of putative cis-acting elements essential for basal transcription. This region contains a TATA box, CCAAT box and Sp1 site, which are apparently conserved in mouse and rat LRAT genes. Our results provide evidence that multiple LRAT mRNA transcripts, which are expressed in a tissue-specific manner, may result from several mechanisms including differential splicing of the 5′UTR region and the use of multiple polyadenylation signals in the 3′UTR. [Copyright &y& Elsevier]

Ras-related C3 toxin substrate 1 (Rac1) is a small Rho-GTPase with important functions in fundamental cellular processes such as cytoskeleton rearrangements, signal transduction, cell cycle progression and malignant transformation. Using Rac1 primer, we identified a 5.5-kb DNA sequence on chromosome 4 (Chr. 4) in the human genome, containing the intronless protein coding sequence of Rac1. Sequence analysis revealed features of a processed pseudogene, which we named ψ1Rac1, that could be detected by Southern blot and polymerase chain reaction (PCR) on genomic DNA. A ψ1Rac1 pseudogene transcript was not detected by reverse transcription-polymerase chain reaction (RT-PCR), nor had the ψ1Rac1 promoter any transcriptional activity. In addition, three other intronless pseudogenes of Rac1 on chromosomes 4, 13 and X were identified (ψ1Rac1–ψ4Rac1) sharing an 86–96% sequence similarity with Rac1. Neither RT-PCR with pseudogene specific restriction enzymes, nor the sequencing of 130 cDNA clones from benign and malignant breast tissue and cell lines, detected the transcription of any of the Rac1 pseudogenes (ψ2Rac1–ψ4Rac1). Existence of Rac1 pseudogenes should be taken into consideration when analyzing genomic alterations of the human Rac1 gene. [Copyright &y& Elsevier]

The ruminant-specific p97bcnt gene (bcntp97) is a paralogous gene that includes a region derived from a retrotransposable element 1 (RTE-1). The region comprises an exon (RTE-1 exon) encoding 325 amino acids in the middle of the p97bcnt protein. To understand how the bcntp97 paralog evolved, we examined its organization in several ruminants. We found a 700-base pair (bp) insert in the 5′ intron of the RTE-1 exon in giraffe bcntp97. This insert is missing in the corresponding regions of bovine and sika deer. Furthermore, the sequence of the insert is interspersed in the genome of giraffe but not bovine and also contains a (GA)n microsatellite. A highly homologous insert harboring significantly different (GA)n microsatellite was detected in the corresponding region of okapi bcntp97. Therefore, the interspersed fragments with (GA)n microsatellite might serve as a marker for tracking how duplicated genes evolve in a family-specific manner. [Copyright &y& Elsevier]

The Brachyury family of T-domain containing transcription factor has been recently the subject of a number of Evo-Devo studies, with expression data obtained from a wide sampling of eumetazans, pointing to a possible conserved role in the formation of the blastopore and the extremities of the digestive tract. Here we present a comparative analysis of Brachyury sequences at the metazoan scale, using published data and two new sponge Brachyury sequences. Alignment features, gene phylogeny, and the evolution of variable positions within the T-domain are discussed in the light of available data about functional constraints on the residues. Interestingly, the high sequence divergence observed in Brachyury T-domains from sponges appears to be mostly the consequence of autapomorphic changes within the sponge lineages, rather than the retention of primitive character states. [Copyright &y& Elsevier]

We have examined the protocadherin (Pcdh) gene clusters of the zebrafish (Danio rerio). At least three sets of the Pcdh gene cluster were found in the zebrafish genome. Here, we describe the complete organization of the DrPcdh2 gene clusters. Classification by phylogenetic and transcript analyses revealed 7 DrPcdh2ο, 20 DrPcdh2αa, 12 DrPcdh2αb, and 1 DrPcdh2αc variable exons upstream of the DrPcdh2α constant region exons in the DrPcdh2 gene cluster. The constant regions of the DrPcdh1α  and DrPcdh2α genes in zebrafish were orthologs of those of the mammalian Pcdhα. These exons all encoded plural PXXP motifs in their cytoplasmic tails. The sequences of the variable exons were highly conserved within each family: DrPcdh2ο, DrPcdh2αa, and DrPcdh2αb. Transcript analysis revealed that zebrafish Pcdhs had alternatively spliced variants in the constant region that were not found in mammals. More gene clusters, more variable exons, and more alternative splicing variants were found in zebrafish than in mammals. Thus, although the Pcdhα families were common to diverse vertebrates, their gene number, structure, and transcripts were different between teleosts and mammals. [Copyright &y& Elsevier]

The most abundant organic compound produced by plants is cellulose; however, it has long been accepted that most animals do not produce endogenous enzymes required for its degradation, but rely instead on symbiotic relationships with microbes that produce the necessary enzymes. Here, we present the genomic organisation of an endogenous glycosyl hydrolase family (GHF) 9 gene in redclaw crayfish (Cherax quadricarinatus), consolidated from a cDNA sequence determined by Byrne et al. [Gene 239 (1999) 317–324.]. Comparison with several other invertebrate GHF9 genes reveals the conservation of both intron position/phase and splice sequence, which adds support to an argument for an ancestral animal cellulase gene. Furthermore, two introns in plant GHF9 genes are also identical in position, implying a more ancient origin for this class of animal cellulase.Protein purification from redclaw gastric fluid via fast performance liquid chromatography (FPLC) indicated the presence of two endoglucanase enzymes. The molecular weights of these components were determined by matrix-assisted laser desorption/ionisation—time-of-flight (MALDI-TOF) to be 47,887 Da (Cel1) and 50,295 Da (Cel2). Cel1 is possibly the functional product of the described cellulase gene, with N-terminal amino acid residues identical to the translated amino acid sequence from the corresponding gene region. Cel2 was identical to Cel1 for 7 of 11 N-terminal residues and likely to be the product of a paralogous endoglucanase gene. These results suggest that redclaw crayfish possess at least one and possibly two functional, endoglucanase enzymes, although further work is required to confirm their origin and attributes. [Copyright &y& Elsevier]

Production of recombinant canine (Canis familiaris) growth hormone (rCFGH) by two expression systems, methanol utilization slow (Muts) and methanol utilization plus (Mut+) based on Pichia pastoris. Led by the Saccharomyces cerevisiae α-mating type signal sequence (SS), the hormone was secreted into the culture medium in its mature and active form. The level of total proteins secreted into the medium achieved at 25 ml working volume using Erlenmeyer flasks was approximately 40 and 15 μg/ml for Muts and Mut+ constructs, respectively. As judged by densitometry of proteins resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the hormone produced by the fermented Muts strain upon induction with methanol reached 24 μg/ml, representing around 60% of the total secreted proteins and being eight times more abundant than in its Mut+ counterpart. Finally, the recombinant hormone showed activity when tested in the Nb2 cell proliferation assay. [Copyright &y& Elsevier]

Despite longstanding efforts, the juvenile hormone (JH) signaling pathway remains unknown. In Drosophila melanogaster (Diptera), JH activates expression of the E75A nuclear receptor. The E75 gene encodes a family of related proteins. A homologue of Drosophila E75 was previously identified and two isoforms, mE75A and mE75B, were reported in Manduca sexta (Lepidoptera). Here, we describe the identification of two additional isoforms, mE75C and mE75D, and the hormonal regulation of mE75 gene expression in Manduca CH1 cultured cells. mE75A and mE75B isoforms are specifically induced by ecdysone in CH1 cells. One isoform, mE75C, shows constitutive expression. The mE75D isoform exhibits dual hormonal regulation; it can be activated by either ecdysone or the JH analog, methoprene. E75-encoded proteins represent the first example of transcription factors directly induced by JH. E75 activation by JH, in both Diptera and Lepidoptera, suggests a conserved function in the JH signaling pathway. [Copyright &y& Elsevier]

Recent marsupials include about 280 species divided into 18 families and seven orders. Approximately 200 species live in Australia/New Guinea. The remaining species inhabit South America with some of these secondarily ranging into North America. In this study, we examine marsupial relationships and estimate their divergences times using complete mitochondrial (mt) genomes. The sampling, which includes nine new mtDNAs and a total number of 19 marsupial genomes, encompasses all extant orders and 14 families. The analysis identified a basal split between Didelphimorphia and remaining orders about 69 million years before present (MYBP), while other ordinal divergences were placed in Tertiary times. The monotypic South American order Microbiotheria (Dromiciops gliroides, Monito del Monte) was solidly nested among its Australian counterparts. The results suggest that marsupials colonized Australia twice from Antarctica/South America and that the divergence between Microbiotheria and its Australian relatives coincided with the geological separation of Antarctica and Australia. Within Australia itself, several of the deepest divergences were estimated to have taken place close to the Eocene/Oligocene transition. [Copyright &y& Elsevier]

We have analysed a centromeric satellite DNA family that is conserved in several commercial and non-commercial oyster species (Ostrea edulis, O. stentina, Crassostrea angulata, C. gigas, C. gasar, C. ariakensis, C. virginica and C. sikamea). This satellite DNA family is composed of AT-rich repeat sequences of 166±2 bp and presents a 9-bp motif similar to the mammalian CENP-B box. The homology of oyster HindIII satellite DNA with satellite DNAs from other bivalves and its relation to a part of a mobile element suggest the existence of an ancient transposable element as a generating unit of satellite DNA in bivalve molluscs. Taking advantage of its degree of conservation in oyster species, we have used this element as a taxonomic marker. This marker clearly supports a high degree of differentiation between O. edulis and O. stentina, and, conversely, upholds the contention that C. gigas and C. angulata are the same species. Finally, we have used HindIII satellite DNA as a phylogenetic marker between these species, revealing two clades, one formed by Asiatic species (C. angulata, C. gigas and C. ariakensis) and another by the European, American and African species (O. edulis, C. virginica and C. gasar, respectively). [Copyright &y& Elsevier]

Three ASF/SF2-like alternative splicing genes from maize were identified, cloned, and analyzed. Each of these genes (zmSRp30, zmSRp31, and zmSRp32) contains two RNA binding domains, a signature sequence SWQDLKD, and a characteristic serine/ariginine-rich domain. There is a strong structural similarity to the human ASF/SF2 splicing factor and to the Arabidopsis atSRp34/p30 proteins. Similar to ASF/SF2-like genes in other organisms, the maize pre-mRNA messages are alternatively spliced. They are differentially expressed in maize tissues with relatively uniform levels of zmSRp30 and zmSRp31 messages being observed throughout the plant, while zmSRp32 messages preferentially accumulated in the meristematic regions. Overexpression of zmSRp32 in maize cells leads to the enhanced selection of weak 5′ intron splice sites during the processing of pre-mRNA molecules. Overexpression of the zmSRp31 or zmSRp32 gene affects regulation of wheat dwarf virus rep gene pre-mRNA splicing, presumably by interacting with the weak 5′ splice site, CCGU. Our results suggest that the described genes are functional homologues of the human ASF/SF2 alternative splicing factor and they indicate a diversity of the ASF/SF2-like alternative splicing factors in monocot plant cells. [Copyright &y& Elsevier]

Pxmp2 is the most abundant peroxisomal membrane protein in higher eukaryotes. Its expression is tissue-specific with highest levels of expression in liver, kidney and heart tissue. We have analysed the 5′-flanking genomic region of the murine Pxmp2 gene and we found, that the first exon of the gene encoding the DNA polymerase ϵ (PoleI) was localized adjacent to the first exon of the Pxmp2 gene in head to head orientation. Both genes were separated by only 393 bp containing a CpG island with numerous binding sites for Sp1. A TATA box, however, was lacking. Northern blot analysis revealed that both genes were expressed differently, indicating that their expression was regulated independently. We have analysed the promoter activity of the small genomic fragment separating the Pxmp2 and PoleI genes using luciferase as a reporter molecule in transient transfection assays. The small genomic fragment was a functional promoter, controlling gene expression regardless of its orientation. Promoter activity was 60-70% compared with the activity of the strong CMV promoter. The Pxmp2 and PoleI genes were also linked on the human and rat genome. Furthermore, the sequence of the intergenic fragment was highly conserved among these species. Thus, the small intergenic fragment is probably the common basic element of two independently regulated promoters. [Copyright &y& Elsevier]

The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence. [Copyright &y& Elsevier]

Rhodopsin genes in most vertebrate species, with the exception of teleost fishes, contain introns. Despite differences in the gene structure, similar regulatory motifs have been identified in fish, amphibian and mammalian rhodopsin promoters, suggesting that rhodopsin gene regulation may be conserved in vertebrates. However, there is no direct evidence to support this notion. To address this, the rhodopsin promoter from the pufferfish, Fugu rubripes, was isolated and tested in transgenic mice and frogs. A 6.5 kb Fugu genomic fragment containing the rhodopsin gene and 4.5 kb 5′ flanking region was able to direct expression of the Fugu rhodopsin gene to the retina of transgenic mice. In transgenic tadpoles, photoreceptor rod cell-specific expression of a reporter gene was achieved using only 500 bp Fugu rhodopsin promoter fragment. Mutagenesis of this promoter fragment revealed that a conserved NRE-like motif is crucial for the retina-specific expression. Our investigation suggests that the regulation of retinal specific expression is conserved in the pufferfish, frog and mouse and that the ancestral intron-containing rhodopsin gene has been displaced by an intronless copy in teleosts. [Copyright &y& Elsevier]

Expression of myelin basic protein in differentiating oligodendrocytes is mainly regulated at the transcriptional level. To better understand the regulation of myelin basic protein gene expression in mammalian cells, we cloned and characterized the rat myelin basic protein promoter by a genome walking technique. Extensive sequence homology has been found among mouse, rat and human MBP promoters. Alignment of the proximal core promoter of mouse and rat reveals highly conserved cis-elements that are important for regulating myelin basic protein gene transcription. One major transcription start site along with two minor sites have been identified in both mouse and rat myelin basic protein gene promoters using RNA ligase-mediated rapid amplification of 5′ cDNA ends. The amplified rat myelin basic protein promoter was cloned into a luciferase reporter construct. Transient transfection experiments show that both mouse and rat myelin basic protein promoters yield increased expression when oligodendrocytes differentiate. The sequence and characterization of the rat MBP promoter provide a useful tool to investigate MBP gene regulation in mammalian cells. [Copyright &y& Elsevier]

Molecular genetics are difficult to perform in Bartonella henselae, the causative agent of cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and bacillary peliosis. To elucidate the underlying bacterial pathogenic mechanisms, genetic manipulation of B. henselae is the method of choice. We describe how to perform transposon mutagenesis in B. henselae using transposome technology. B. henselae mutants revealed by this technique showed random transpositional insertion into the chromosome. In contrast to transposon mutagenesis by conjugational transfer, transposome technology allows transposon mutagenesis of early passaged Bartonella spp. with approximately 100-fold higher efficiency. The results show that transposome technique is a rapid, efficient and simple method to generate transposon mutants of B. henselae. [Copyright &y& Elsevier]

A macroarray approach used to list genes differentially expressed in goat mammary gland (gestation vs. lactation), other than milk protein genes, allowed us to detect the Glycosylation-dependent Cell Adhesion Molecule 1 (GLYCAM1) gene. GLYCAM1, a member of the glycoprotein mucin family, is a component of the milk fat globule membrane (MFGM). Its complete cDNA and gene sequences were determined and it was mapped by fluorescent in situ hybridization (FISH) on goat and cattle chromosome 5 (CHI5q21 and BTA5q21), and on sheep chromosome 3 (OAR3q21). Northern blot analyses confirmed its differential expression during the development and differentiation of the mammary gland of ruminants with a significantly higher mRNA amount during lactation than during pregnancy. An experimental in vivo induction model for lactation, developed by Kann et al. [J. Endocrinol. 160 (1999) 265], showed that the expression of GLYCAM1 is hormonally regulated in the mammary gland of ewes. Interspecies comparison of the gene promoter revealed the evolutionary conservation of a short proximal nucleotide sequence encompassing several transcription factor binding sites that could mediate the above-mentioned hormonal regulation. [Copyright &y& Elsevier]

Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyses sterol side-chain oxidation of bile acid synthesis from cholesterol, and the first reaction of the acidic bile acid biosynthetic pathway. Hydrophobic bile acids suppress human CYP27A1 gene reporter activity when assayed in human hepatocellular blastoma HepG2 cells. Bile acids also inhibit CYP27A1 reporter activity in human embryonic kidney 293 cells. A putative bile acid response element (BARE) was mapped to a region downstream of nt −147 of the human CYP27A1 gene, within which a binding site for a liver-specific nuclear receptor, HNF4α, is identified. HNF4α strongly stimulates CYP27A1 gene transcription and mutation of its binding site markedly reduced promoter activity. Results suggest that human CYP27A1 gene transcription is suppressed by bile acids and HNF4α plays a pivotal role in transcriptional regulation of this gene. [Copyright &y& Elsevier]

Some clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans are naturally competent for DNA uptake. In this study, we examined the sequence and the function of a type IV pilus-like pilABCD gene cluster and its downstream region in a naturally transformable A. actinomycetemcomitans strain D7S. Specific knockout mutants of pilABCD of strain D7S were constructed by replacing individual genes with an antibiotic resistance cassette. The transformation frequency of chromosome markers in the wildtype strain D7S was ∼10−3 per CFU. In contrast, the ΔpilA, ΔpilB, ΔpilC, ΔpilBC or ΔpilD mutants were non-transformable (transformation frequency <10−8). Disruption of an ORF downstream of pilD had no apparent effect on the transformability of this bacterium. The pilA or pilBC deletion did not seem to affect fimbria expression or cell surface structure in either rough or smooth strains as determined by scanning and transmission electron microscopy examinations. RT-PCR analysis showed that pilA was expressed in strain D7S under a competence-inducing growth condition. The expression of pilA was barely detectable in strain D7S cultured under a non-competence-inducing condition or in the non-transformable strain JP-2. The results indicate that pilABCD are required for competence but are apparently not involved in fimbria expression of A. actinomycetemcomitans. [Copyright &y& Elsevier]

The group II intron (GBSi1) identified downstream of the C5a-peptidase gene (scpB) in a subpopulation of Streptococcus agalactiae isolates is a suggested marker for a separate genetic lineage of serotype III isolates. In the present study two additional copies of GBSi1, one of which not previously described, were identified among serotype III isolates. All intron copies shared a common target site motif. A single copy of GBSi1 was found in a subgroup of serotype II and V isolates. In these isolates, the intron had inserted downstream of scpB, which suggests that this is the primary insertion site for GBSi1. Most bacterial group II introns described to date reside in transposable elements. The scpB locus was found to be flanked by insertion sequences similar to what has been described in an intronless serotype Ia isolate. However, this region contained an additional 2.1 kb DNA fragment present only in intron carrying isolates. This DNA fragment contained a partial transposase and putative plasmid related proteins. This may suggest that GBSi1 once was brought into the S. agalactiae genome by an integrated plasmid. [Copyright &y& Elsevier]

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) produces isofunctional enzymes of the gentisate pathway that enables the degradation of xylenols and cresols via gentisate. Previous reports had indicated that one set of enzymes is constitutively expressed whereas the other set is strictly inducible by aromatic hydrocarbon substrates. The gene encoding gentisate 1,2-dioxygenase (GDO), the enzyme that catalyzes the cleavage of the gentisate aromatic ring, was cloned from strain P25X. The GDO gene, designated xlnE, is 1,044 bp, and is part of a 5.4 kb operon which consists of six genes, xlnEFGHID. Transcription of this operon was driven by a σ70-type promoter, PxlnE, located 123 bp upstream of the xlnE start codon. Primer extension analysis showed that the xlnE transcription start point is located at the −87 adenine residue. In a P25X xlnE knockout mutant, GDO activity could only be detected when cells were grown in the presence of aromatic substrates, suggesting that xlnE encodes for the constitutive copy of GDO. This was verified by constructing a P25X strain with xlnE transcriptionally fused to a promoterless catechol 2,3-dioxygenase gene. In this strain, catechol 2,3-dioxygenase activity was detected in cells that were grown in the absence of aromatic inducers. However, catechol 2,3-dioxygenase activity increased up to four fold when these cells were grown in the presence of aromatic substrates, in particular 3-hydroxybenzoate. Thus, xlnE is in fact, inducible and the constitutive activity observed under non-inducing conditions was due to its relatively high basal levels of expression. [Copyright &y& Elsevier]

The tuberactinomycins are a family of basic cyclic peptides that exhibit potent antitubercular activity. These peptides are characterized by the presence of an amino acid with a 6-membered cyclic guanidine side chain (capreomycidine) and two or more 2,3-diaminopropionate residues. Viomycin (tuberactinomycin B) is a well-studied member of the family, was once prescribed for the treatment of tuberculosis, and has been shown to block translocation during protein biosynthesis. The gene cluster encoding viomycin biosynthesis was identified and cloned from Streptomyces vinaceus. The cluster was identified by screening genomic libraries with the viomycin phosphotransferase self-resistance gene (vph) and non-ribosomal peptide synthetase (NRPS) gene probes amplified from S. vinaceus genomic DNA. The viomycin cluster was localized to ca. 120 kb of contiguous DNA defined by four overlapping cosmid inserts. Each cosmid hybridized with one or more peptide synthetase gene probes and two also hybridized with vph. Confirmation that the cluster encoded viomycin biosynthesis was obtained from the disruption of two NRPS adenylation domains. Partial sequence analysis revealed an ORF (svox) predicted to encode a rare non-heme iron, α-ketoglutarate dependent oxygenase proposed to function in the oxidative cyclization of arginine to the capreomycidine residue. Insertional disruption of svox resulted in complete loss of viomycin production, confirming its involvement in the pathway. [Copyright &y& Elsevier]

We isolated a novel cytokeratin gene of zebrafish (Danio rerio), DAPK-1 closely related to other vertebrate type I cytokeratin genes. Zygotic transcription starts at the sphere stage. After the mid-blastula stage, DAPK-1 is expressed in all surface cells, notably in those of the outer enveloping layer. DAPK-1 messages are also present specifically during the segmentation, pharyngula, and hatching periods. In particular, after 24 h post-fertilization, its expression is restricted to the developing eye region, otic vesicle, pectoral fin, dorsal aorta, and pronephric duct. In the mindbomb mutant embryo that has defects in the dorsal aorta development, DAPK-1 transcripts are not detected in the dorsal aorta and pronephric duct. The characteristic expression pattern of DAPK-1 may facilitate more detailed studies related to the morphogenesis of dorsal aorta and pronephric duct. [Copyright &y& Elsevier]

We isolated a 918 bp long full-length rice HMGB1 cDNA, which has an open reading frame of 471 bp encoding 157 amino acids, with a central domain of high sequence similarity to the HMG-box domain of other plant HMGB1 proteins. RNA gel blot analysis indicated that rice HMGB1 gene is constitutively expressed in various tissues and organs. Southern hybridization and sequence analyses suggested that a single copy of the HMGB1 gene composed of seven exons and six introns exists in rice. We have also cloned a 1755 bp long 5′ flanking region of the rice HMGB1 gene, which can be regarded as its promoter. 5′ deletion analysis of this promoter indicated that positive cis-elements residing between −1400 and −1115 are important to enhance quantitative expression, whereas negative cis-elements between −1755 and −1400 and between −1115 and −351 inhibit expression. [Copyright &y& Elsevier]

Histones are the major protein component of chromatin. Except H4, all histone classes consist of several subtypes. The H3 family includes two replacement histone genes, H3.3A and H3.3B, which both encode the same protein and are expressed independently from the cell cycle. Since the two genes encode an identical protein, we analyzed whether they are differentially expressed. Therefore we cloned, sequenced and characterized the regulatory structures of the H3.3A gene and compared these with the corresponding regions in the H3.3B gene. In contrast to the H3.3B promoter, the promoter region of the H3.3A gene revealed neither a TATA nor any CCAAT boxes but an initiator element and several SP1 binding sequence motifs within an overall GC-rich sequence. Northern blot analysis of RNA from six human cell lines revealed that every cell line expressed each of the H3 isoform genes H3.1, H3.3A and H3.3B. In contrast, analysis of total RNA from human tissues showed a differential expression of the H3 isoform genes. The H3.3 genes are essentially only expressed in adult tissue, whereas the H3.1 gene is transcribed just in fetal tissue. The functional relevance of the elements identified by sequence analysis was established using a reporter gene assay with deletion constructs of the H3.3A promoter. In this assay a 256 bp fragment was sufficient for the full promoter activity and three promoter segments, each containing SP1 binding motifs, contribute to the H3.3A gene expression. The possible functional relevance of the differences between the two H3.3 genes in structure and expression is discussed. [Copyright &y& Elsevier]

We have recently described a novel gene on human chromosome 20q 12-13.2 called Eppin (Epididymal protease inhibitor) that expresses three mRNAs encoding two isoforms of a cysteine-rich protein containing both Kunitz-type and WAP-type (four disulfide core) consensus sequences (). To further our studies on Eppin, we have cloned, sequenced and characterized mouse Eppin and report that it lies within a 200 Kb cluster of putative Eppin-like genes on mouse chromosome 2. Analysis of the homologies between the genes in the human and mouse Eppin clusters indicates that the first part of the cluster immediately surrounding Eppin represents a conserved linkage because the order of homologous genes is conserved. Sequencing of reverse transcription polymerase chain reaction (RT-PCR) products confirmed the expression of five of these novel Eppin-like genes in the mouse, which include the mouse homologue of HE-4. These genes are characterized by having either one or both of the Kunitz-type and WAP-type consensus sequences. Additional RT-PCR experiments revealed that expression of some of the Eppin-like genes is restricted to epididymis and testis while others are expressed in several somatic tissues. Northern blot analysis of 22 different mouse tissues identified Eppin transcripts only in the epididymis and testis. Immunostaining of Eppin with anti-recombinant mouse Eppin demonstrated Eppin predominantly on the postacrosomal region of mouse spermatozoa, in Sertoli cells, Leydig cells, and round spermatids in the testis, and in the principal cells of the cauda epididymidis epithelium. Eppin is first expressed by Sertoli cells of 12-day-old mice and subsequently in round spermatids, which is consistent with androgen regulation. Our results demonstrate that mouse chromosome 2 contains a conserved linkage of Eppin-like protease inhibitor genes that are expressed in the epididymis. [Copyright &y& Elsevier]

Satellite DNA sequence evolution has been studied in several insect species from the genus Pimelia (Tenebrionidae, Coleoptera). Low-copy number homologs of the previously characterized major satellite DNA from P. monticola (PMON) have been cloned and sequenced from six congeneric species belonging to two species groups: Ibero-Balearic and Moroccan. Sequence analysis of a sample of low-copy number repeats revealed two subfamilies, differing on average 17.5% due to randomly spread single point mutations. Each subfamily is specific for a group of taxa in congruence with their biogeography. Within each group, there is no significant species-specific clustering of the sequences. These results suggest that the two satellite subfamilies arose after the split of an ancestral lineage into the North African and Ibero-Balearic Pimelia species-groups, but before their subsequent radiation. Rate heterogeneity tests suggest that PMON sequences have evolved faster in the lineage leading to the Moroccan group. Comparison of sequence divergences between minor PMON and the previously characterized major PIM357 satellite obtained from the same taxa, points to similar evolutionary dynamics. Both sequences are evolving in parallel accumulating mutations in a gradual manner irrespectively of significant differences in abundance. These data show that copy number of the sequence families does not necessarily affect the sequence change dynamics of satellite repeats. [Copyright &y& Elsevier]

In this study, we describe the characterisation of the complement component C4 gene in Fugu rubripes. The Fugu C4 gene, orthologous to the tetrapod C4 gene, encompasses a genomic span of 9702 base pairs and contains 41 exons, encoding the typical C4 three-chain polypeptide. The gene encodes a protein containing 1703 amino acids. The Fugu C4 protein demonstrates the presence of 25 conserved cysteine residues, as well as conservation of the functionally important thioester site. Complete sequencing of one cosmid and sequence scans from a cluster of 18 overlapping BAC clones, centering around the C4 gene, have identified the short-range linkage with five orthologous human genes mapping to the Major Histocompatibility Complex (MHC) including: tenascin X (TNX); cytochrome P450, subfamily XXIA, polypeptide 2 (CYP21A2); allograft inflammatory factor 1 (AIF1) and casein kinase 2, beta polypeptide (CSNK2B), all found in the MHC class III region; and retinoid X receptor, beta (RXRB),which resides in the MHC extended class II region. To date, this syntenic association of the Fugu C4 and other MHC class III region genes has not been observed in other teleost fish. Data from the recent whole-genome shotgun assemblies reveal the Fugu MHC-related cluster of genes to be flanked predominantly by genes mapping to human chromosomes 7 and 19. All of the six identified Fugu MHC-related genes have been characterised at the genomic level. [Copyright &y& Elsevier]

We have analysed the genomic structure and transcriptional activity of a 2.3-Mb genomic sequence in the juxtacentromeric region of human chromosome 21. Our work shows that this region comprises two different chromosome domains. The 1.5-Mb proximal domain: (i) is a patchwork of chromosome duplications; (ii) shares sequence similarity with several chromosomes; (iii) contains several gene fragments (truncated genes having an intron/exon structure) intermingled with retrotransposed pseudogenes; and (iv) harbours two genes (TPTE and BAGE2) that belong to gene families and have a cancer and/or testis expression profile. The TPTE gene family was generated before the branching of Old World monkeys from the great ape lineage, by intra- and interchromosome duplications of the ancestral TPTE gene mapping to phylogenetic chromosome XIII. By contrast, the 0.8-Mb distal domain: (i) is devoid of chromosome duplications; (ii) has a chromosome 21-specific sequence; (iii) contains no gene fragments and only one retrotransposed pseudogene; and (iv) harbours six genes including housekeeping genes. G-rich sequences commonly associated with duplication termini cluster at the boundary between the two chromosome domains. These structural and transcriptional features lead us to suggest that the proximal domain has heterochromatic properties, whereas the distal domain has euchromatic properties. [Copyright &y& Elsevier]

We determined the complete mitochondrial DNA sequence for a swimming crab Portunus trituberculatus (Miers, 1876) (Crustacea: Decapoda: Brachyura). The entire genome was amplified using a long PCR technique, and the products were subsequently used as templates for direct sequencing using a primer walking strategy. The genome (16,026 bp) contained the same 37 genes (two rRNAs, 22 tRNAs, and 13 proteins) plus the putative control region as found in other arthropods. The gene order of P. trituberculatus was largely identical to those so far obtained for other arthropods such as Drosophila yakuba, although the relative position of the tRNAHis gene differed from all other arthropods studied so far. While the tRNAHis gene in other arthropod mitochondrial genomes is located between the ND4 and ND5 genes, that of P. trituberculatus was found between the tRNAGlu and the tRNAPhe genes, which were located downstream of the ND5 gene. The present gene order could have resulted from tandem duplication occurring in the tRNAPhe-ND5-tRNAHis region (typical gene order in other arthropods) and following deletions of redundant genes. This is the first report of a mitochondrial genome of a brachyuran crab, one of the most diversified and economically important groups of crustaceans. [Copyright &y& Elsevier]

In an effort to identify novel genes relevant to tumor angiogenesis, we compared the genes expressed in a matched pair composed of vascularized breast tumor and its adjacent normal tissue obtained from the same cancer patient. Using differential display, we identified a cDNA fragment that was reproducibly upregulated in vascularized breast tumor. Up-regulation of this gene fragment in vascularized breast tumor was further verified by semi-quantitative PCR on the same RNA pair using gene-specific primers. The cDNA encoding the full-length ORF of that gene was then cloned by both 3′ and 5′ RACE. Sequence analysis showed that this gene encodes an ORF of 1353 bp having a hydrophobic N-terminal signal sequence and a cleavage site. We named this novel gene BNF-1 (breast tumor novel factor 1). The mature protein of this gene contains cysteine-rich repeats that are a specific feature of several extracellular matrix proteins including thrombospondin-1, thrombospondin-2, pro-collagen type 1, and von Willebrand Factor 1. PCR analysis of BNF-1 expression in a variety of human adult normal tissues revealed that BNF-1 is expressed predominantly in liver, heart, prostate, testis, and ovary. To further study the expression pattern of this novel gene in tumor tissues, we extended our analysis to additional matched pairs of tumor tissues obtained from breast, lung, and colon cancer patients. We show here that BNF-1 is over-expressed not only in breast tumors but also in lung and colon tumors. [Copyright &y& Elsevier]

The human elongation factor 1A-1 (eEF1A-1) gene is a member of the 5′ terminal oligopyrimidine tract (5′ TOP) gene family, and the number of thymidines (Ts) at the 5′ TOP of cDNAs corresponding to this gene is known to show variation. Here we determined the 5′-end sequences of 125 eEF1A-1 clones and the complete sequences of 19 eEF1A-1 clones from an oligo-capped cDNA library and showed that variation in the number of Ts is generated by an in vivo process, not by an in vitro artifact during the construction of the cDNA library. Moreover, using green fluorescent protein transgenic mice, we demonstrated that the variation in T number is probably generated during or after transcription. We also introduced various mutations in the mRNA start site of this gene, particularly in the T stretch at the 5′ TOP, and examined the effects on the promoter activity. The results showed that at least three Ts must exist at the 5′ TOP for the high transcriptional activity of the eEF1A-1 gene promoter. Many other housekeeping genes, including ribosomal protein genes, are also members of the 5′ TOP gene family, and the 5′ TOP sequence may be an important core-promoter element of these genes. [Copyright &y& Elsevier]

The mitochondrial genome of the Chinese giant salamander Andrias davidianus was isolated using the long-and-accurate polymerase chain reaction (LA PCR) method. The sequencing work adopted the shotgun strategy accompanying with seven internal primers to cover the gaps where overlapping clones were not available. The entire mtDNA sequence is 16,503 bp long, with a gene content of 13 protein-coding, two ribosomal RNA and 22 transfer RNA genes, and order identical to that observed in most other vertebrates except for an additional 318 bp non-coding sequence between tRNA-Thr and tRNA-Pro genes. In order to carry out molecular phylogenetic analyses, all 13 protein sequences deduced from whole mitochondrial genomes for eight vertebrate species (six amphibians, two lobe-finned fishes) were combined to a single data set. This data set was refined by the program Gblocks using a stringent parameter setting and then subjected to MP, ML and NJ analyses. Thus phylogenetic relationships among living amphibians were discussed. [Copyright &y& Elsevier]

An immunomodulatory membrane protein, CD200R displays an expression pattern restricted to myeloid cells in mice. It is the receptor for a ligand, CD200, expressed by a broad range of cell types. In this study, we describe the cloning and characterization of the human homologue of the CD200R gene. This gene maps closely to the CD200 gene on human chromosome 3q12-13. The human CD200R gene spans a region of 52 kb, consists of nine exons, and encodes a 348-amino-acid cell-surface protein consisting of two IgFF domains in a typical V/C2 arrangement. The 59-amino-acid cytoplasmic domain has two tyrosine residues, one of which is contained within a NPXY motif. In common with other IgSF genes, the CD200R gene can generate different protein isoforms through alternative splicing. An alternative spliceout form, which has not yet been described in mice, encodes a 188-amino-acid truncated soluble polypeptide containing only the V immunoglobulin domain. In contrast to murine CD200R protein, the human membrane-bound and soluble CD200R proteins have an insertion of 23 amino acids at position 23, encoded by exon 2, which generates a putative dihydroxyacid dehydratase domain. The splicing of exon 2 generates two new isoforms, encoding the membrane and soluble proteins but lacking the dyhydroxyacid dehydratase domain. Northern-blot analysis shows that both membrane-bound and soluble isoforms are expressed in the thymus, liver, spleen and placenta. By RT-PCR, we have analyzed the expression of the four transcript variants in human placenta, spleen, liver, brain and kidney. [Copyright &y& Elsevier]