A family of textilinin genes, two of which encode proteins with antihaemorrhagic properties

Two peptides, textilinins 1 and 2, isolated from the venom of the Australian common brown snake, Pseudonaja textilis textilis, are effective in preventing blood loss. To further investigate the potential of textilinins as anti-haemorrhagic agents, we cloned cDNAs encoding these proteins. The isolated full-length cDNA (430 bp in size) was shown to code for a 59 amino acid protein, corresponding in size to the native peptide, plus an additional 24 amino acid propeptide. Six such cDNAs were identified, differing in nucleotide sequence in the coding region but with an identical propeptide. All six sequences predicted peptides containing six conserved cysteines common to Kunitz-type serine protease inhibitors. When expressed as glutathione S-transferase (GST) fusion proteins and released by cleavage with thrombin, only those peptides corresponding to textilinin 1 and 2 were active in inhibiting plasmin with K-i values similar to those of their native counterparts and in binding to plasmin less tightly than aprotinin by two orders of magnitude. Similarly, in the mouse tail vein blood loss model only recombinant textilinin 1 and 2 were effective in reducing blood loss. These recombinant textilinins have potential as therapeutic agents for reducing blood loss in humans, obviating the need for reliance on aprotinin, a bovine product with possible risk of transmissible disease, and compromising the fibrinolytic system in a less irreversible manner.

Novel genes regulated by Sonic Hedgehog in pluripotent mesenchymal cells

Sonic Hedgehog is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. Disruption of the Hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. The identification of genes regulated by Hedgehog is crucial to understanding how disruption of this pathway leads to neoplastic transformation. We have used a Sonic Hedgehog (Shh) responsive mouse cell line, C3H/10T1/2, to provide a model system for hedgehog target gene discovery. Following activation of cell cultures with Shh, RNA was used to interrogate microarrays to investigate downstream transcriptional consequences of hedgehog stimulation. As a result 11 target genes have been identified, seven of which are induced (Thrombomodulin, GILZ, BF-2, Nr4a1, IGF2, PMP22, LASP1) and four of which are repressed (SFRP-1, SFRP-2, Mip1-gamma, Amh) by Shh. These targets have a diverse range of putative functions and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery.

Conserved modularity and potential for alternate splicing in mouse and human Slit genes

The vertebrate Slit gene family currently consists of three members;Slit1,Slit2 and Slit3. Each gene encodes a protein containing multiple epidermal growth factor and leucine rich repeat motifs, which are likely to have importance in cell-cell interactions. In this study, we sought to fully define and characterise the vertebrate Slit gene family. Using long distance PCR coupled with in silico mapping, we determined the genomic structure of all three Slit genes in mouse and man. Analysis of EST and genomic databases revealed no evidence of further Slit family members in either organism. All three Slit genes were encoded by 36 (Slit3) or 37 (Slit1 and Slit2) exons covering at least 143 kb or 183 kb of mouse or human genomic DNA respectively. Two additional potential leucine-rich repeat encoding exons were identified within intron 12 of Slit2. These could be inserted in frame, suggesting that alternate splicing may occur in Slit2 A search for STS sequences within human Slit3 anchored this gene to D5S2075 at the 5' end (exon 4) and SGC32449 within the 3' UTR, suggesting that Slit3 may cover greater than 693 kb. The genomic structure of all Slit genes demonstrated considerable modularity in the placement of exon-intron boundaries such that individual leucine-rich repeat motifs were encoded by individual 72 by exons. This further implies the potential generation of multiple Slit protein isoforms varying in their number of repeat units. cDNA library screening and EST database searching verified that such alternate splicing does occur.

Macropodid herpesvirus 1 encodes genes for both thymidylate synthase and ICP34.5

Macropodid herpesvirus 1 (MaHV-1) is an unclassified alphaherpesvirus linked with the fatal infections of kangaroos and other marsupials. During the characterisation of the internal repeat region of MaHV-1, an open reading frame (ORF) encoding for thymidylate synthase (TS) gene was identified and completely sequenced. Southern blot analysis confirmed the presence of two copies of the TS gene in the MaHV-1 genome as expected. Computer analysis of the TS ORF showed it was 948 nucleotides in length. A putative polyadenylation signal was identified 17-22 bp inside the ORF implying a minimal or absent 3' untranslated region. The predicted polypeptide was 316 amino acid residues in length and contained the highly conserved motifs for folate binding and F-dUMP binding, typical of all TS enzymes. Interestingly, MaHV-1 TS polypeptide had highest similarity to the human TS polypeptide (81%) compared to the TS polypeptides of other herpesviruses (72-75%). Immediately upstream of the TS gene, a second ORF of 510 bp, encoding a polypeptide with 170 amino acid residues, was identified. The carboxyl domain of this MaHV-1 polypeptide shared 68% similarity to a 59 amino acid motif of human herpesvirus 1 ICP34.5, identifying it as the MaHV-1 ICP34.5 homologue. This is the first report of a herpesvirus that encodes for both TS and ICP34.5.

Genes induced by growth hormone in a model of adipogenic differentiation

A substantial number of GH regulated genes have been reported in mature hepatocytes. but genes involved in GH-initiated cell differentiation have not yet been identified. Here we have studied a, ell-characterised model of GH-dependent differentiation, adipogenesis of 3T3-F442A preadipocytes, to identify genes rapidly induced by GH. Using the suppression subtractive hybridisation technique, we have identified eight genes induced within 60 min of GH treatment, and verified these by northern analysis. Six were identifiable as Stat 2. Stat 3, thrombospondin-1. oncostatin M receptor beta chain. a DEAD box RNA helicase. and muscleblind. a developmental transcription factor. Bioinformatic approaches assigned one of the two remaining unknown genes as a novel 436 residue serine,threonine kinase. As each of the identified genes hake important developmental roles. they may be important in initiating GH-induced adipogenesis. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea

Shoot branching is inhibited by auxin transported down the stem from the shoot apex. Auxin does not accumulate in inhibited buds and so must act indirectly. We show that mutations in the MAX4 gene of Arabidopsis result in increased and auxin-resistant bud growth. Increased branching in max4 shoots is restored to wild type by grafting to wild-type rootstocks, suggesting that MAX4 is required to produce a mobile branch-inhibiting signal, acting downstream of auxin. A similar role has been proposed for the pea gene, RMS1. Accordingly, MAX4 and RMS1 were found to encode orthologous, auxin-inducible members of the polyene dioxygenase family.

A genomewide survey of developmentally relevant genes in Ciona intestinalis - IV. Genes for HMG transcriptional regulators, bZip and GATA/Gli/Zic/Snail

Many kinds of transcription factors and regulators play key roles in a variety of developmental processes. In the present survey, genes encoding proteins with conserved HMG-box, bZip domains, and some types of zinc finger motifs were surveyed in the completely sequenced genome of Ciona intestinalis. In the present analysis, 21 HMG-box-containing genes and 26 bZip genes were identified as well as four small groups of zinc finger genes in the Ciona genome. The results also showed that a less redundant set of genes is present in the Ciona genome compared with vertebrate genomes. In addition, cDNA clones for almost all genes identified have been cloned and distributed as a Ciona intestinalis Gene Collection Release I. The present comprehensive analysis therefore provides a means to study the role of these transcription factors in developmental processes of basal chordates.

A genomewide survey of developmentally relevant genes in Ciona intestinalis - V. Genes for receptor tyrosine kinase pathway and Notch signaling pathway

In the present survey, we identified most of the genes involved in the receptor tyrosine kinase (RTK), mitogen activated protein kinase (MAPK) and Notch signaling pathways in the draft genome sequence of Ciona intestinalis, a basal chordate. Compared to vertebrates, most of the genes found in the Ciona genome had fewer paralogues, although several genes including ephrin, Eph and fringe appeared to have multiplied or duplicated independently in the ascidian genome. In contrast, some genes including kit/flt, PDGF and Trk receptor tyrosine kinases were not found in the present survey, suggesting that these genes are innovations in the vertebrate lineage or lost in the ascidian lineage. The gene set identified in the present analysis provides an insight into genes for the RTK, MAPK and Notch signaling pathways in the ancient chordate genome and thereby how chordates evolved these signaling pathway.

Expression of anterior Hox genes during larval development of the gastropod Haliotis asinina

We report the spatial expression patterns of five anterior Hox genes during larval development of the gastropod mollusc Haliotis asinina, an unsegmented spiralian lophotrochozoan. Molecular alignments and phylogenetic analysis indicate that these genes are homologues of Drosophila HOM-C genes labial, proboscipedia, zen, Deformed, and Sex combs reduced, the abalone genes are named Has-Hox1, -Hox2, -Hox3, -Hox4, and -Hox5. Has-Hox transcripts are first detected in the free-swimming trochophore larval stage- and restricted to the posttrochal ectoderm. Has-Hox2, -Hox3, and -Hox4 are expressed in bilaterally symmetrical and overlapping patterns in presumptive neuroectodermal cells on the ventral side of the trochophore. Has-Hox1 expression is restricted to a ring of cells on the dorsoposterior surface, corresponding to the outer mantle edge where new larval shell is being synthesized. There appears to be little change in the expression domains of these Has-Hox genes in pre- and posttorsional veliger larvae, with expression maintained in ectodermal and neuroectodermal tissues. Has-Hox2, -Hox3, -Hox4, and-Hox5 appear to be expressed in a colinear manner in the ganglia and connectives in the twisted nervous system. This pattern is not evident in older larvae. Has-Hox1 and-Hox4 are expressed in the margin of the mantle in the posttorsional veliger, suggesting that Hox genes play a role in gastropod shell formation.

Phytophthora sojae avirulence genes Avr4 and Avr6 are located in a 24kb, recombination-rich region of genomic DNA

A cross between two different races (race 7 x race 25) of the soybean root and stem rot pathogen Phytophthora sojae was analyzed to characterize the genomic region flanking two cosegregating avirulence genes, Anur4 and Anur6. Both genes cosegregated in the ratio of 82:17 (avirulent:virulent) in an F-2 population, suggestive of a single locus controlling both phenotypes. A chromosome walk was commenced from RAPD marker OPE7.1C, 2.0 cM distant from the Anur4/6 locus. Three overlapping cosmids were isolated which included genetic markers that flank the Anur4/6 locus. The chromosome walk spanned a physical distance of 67 kb which represented a genetic map distance of 22.3cM, an average recombination frequency of 3.0kb/cM and 11.7-fold greater than the predicted average recombination frequency of 35.3 kb/cM for the entire P. sojae genome. Six genes (cDNA clones) expressed from the Anur4/6 genomic region encompassed by the cosmid contig were identified. Single nucleotide polymorphisms and restriction fragment length polymorphisms showed these six genes were closely linked to the Anur4/6 locus. Physical mapping of the cDNA clones within the cosmid contig made it possible to deduce the precise linkage order of the cDNAs. None of the six cDNA clones appear to be candidates for Anur4/6. We conclude that two of these cDNA clones flank a physical region of approximately 24 kb and 4.3 cM that appears to include the Anur4/6 locus. (C) 2003 Elsevier Inc. All rights reserved.

Genomic screen for genes involved in mammalian craniofacial development

Using a subtractive hybridisation approach, we enriched for genes likely to play a role in embryonic development of the mammalian face and other structures. This was achieved by subtracting cDNA derived from adult mouse liver from that derived from 10.5 dpc mouse embryonic branchial arches 1 and 2. Random sequencing of clones from the resultant library revealed that a high percentage correspond to genes with a previously established role in embryonic development and disease, while 15% represent novel or uncharacterised genes. Whole mount in situ hybridisation analysis of novel genes revealed that approximately 50% have restricted expression during embryonic development. In addition to expression in branchial arches, these genes showed a range of expression domains commonly including neural tube and somites. Notably, all genes analysed were found to be expressed not only in the branchial arches but also in the developing limb buds, providing support for the hypothesis that development of the limbs and face is likely to involve analogous molecular processes. (C) 2003 Wiley-Liss, Inc.

Linkage and Association Analysis of Radiation Damage Repair Genes XRCC3 and XRCC5 with Nevus Density in Adolescent Twins

Previous studies have shown that a deficiency in DNA damage repair is associated with increased cancer risk, and exposure to UV radiation is a major risk factor for the development of malignant melanoma. High density of common nevi (moles) is a major risk factor for cutaneous melanoma. A nevus may result from a mutation in a single UV-exposed melanocyte which failed to repair DNA damage in one or more critical genes. XRCC3 and XRCC5 may have an effect on nevus count through their function as components of DNA repair processes that may be involved directly or indirectly in the repair of DNA damage due to UV radiation. This study aims to test the hypothesis that the frequency of flat or raised moles is associated with polymorphism at or near these DNA repair genes, and that certain alleles are associated with less efficient DNA repair, and greater nevus density. Twins were recruited from schools in south eastern Queensland and were examined close to their 12th birthday. Nurses examined each individual and counted all moles on the entire body surface. A 10cM genome scan of 274 families (642 individuals) was performed and microsatellite polymorphisms in XRCC3 and adjacent to XRCC5 were also typed. Linkage and association of nevus count to these loci were tested simultaneously using a structural-equation modeling approach implemented in MX. There is weak evidence for linkage of XRCC5 to a QTL influencing raised mole count, and also weak association. There is also weak evidence for association between flat mole count and XRCC3. No tests were significant after correction for testing multiple alleles, nor were any of the tests for total association significant. If variation in XRCC3 or XRCC5 influences UV sensitivity, and indirectly affects nevus density, then the effects are small.

Analysis of the mouse transcriptome for genes involved in the function of the nervous system

We analyzed the mouse Representative Transcript and Protein Set for molecules involved in brain function. We found full-length cDNAs of many known brain genes and discovered new members of known brain gene families, including Family 3 G-protein coupled receptors, voltage-gated channels, and connexins. We also identified previously unknown candidates for secreted neuroactive molecules. The existence of a large number of unique brain ESTs suggests an additional molecular complexity that remains to be explored. A list of genes containing CAG stretches in the coding region represents a first step in the potential identification of candidates for hereditary neurological disorders.

The highly rearranged mitochondrial genome of the plague thrips, Thrips imaginis (Insecta : thysanoptera): Convergence of two novel gene boundaries and an extraordinary arrangement of rRNA genes

To help understand the mechanisms of gene rearrangement in the mitochondrial (mt) genomes of hemipteroid insects, we sequenced the mt genome of the plague thrips, Thrips imaginis (Thysanoptera). This genome is circular, 15,407 by long, and has many unusual features, including (1) rRNA genes inverted and distant from one another, (2) an extra gene for tRNA-Ser, (3) a tRNA-Val lacking a D-arm, (4) two pseudo-tRNA genes, (5) duplicate control regions, and (6) translocations and/or inversions of 24 of the 37 genes. The mechanism of rRNA gene transcription in T. imaginis may be different from that of other arthropods since the two rRNA genes have inverted and are distant from one another. Further, the rRNA genes are not adjacent or even close to either of the two control regions. Tandem duplication and deletion is a plausible model for the evolution of duplicate control regions and for the gene translocations, but intramitochondrial recombination may account for the gene inversions in T. imaginis. All the 18 genes between control regions #1 and #2 have translocated and/or inverted, whereas only six of the 20 genes outside this region have translocated and/or inverted. Moreover, the extra tRNA gene and the two pseudo-tRNA genes are either in this region or immediately adjacent to one of the control regions. These observations suggest that tandem duplication and deletion may be facilitated by the duplicate control regions and may have occurred a number of times in the lineage leading to T. imaginis. T. imaginis shares two novel gene boundaries with a lepidopsocid species from another order of hemipteroid insects, the Psocoptera. The evidence available suggests that these shared gene boundaries evolved by convergence and thus are not informative for the interordinal phylogeny of hemipteroid insects. We discuss the potential of hemipteroid insects as a model system for studies of the evolution of animal rut genomes and outline some fundamental questions that may be addressed with this system.