Cloning and functional analysis of the Sry-related HMG box gene, Sox18

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii);ln intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor-2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue. (C) 2001 Elsevier Science B.V. All rights reserved.

A Hermeneutic Case Reconstruction of a published first-person narrative

Hermeneutic Case Reconstruction (Rosenthal, 1993) is a systematic method of analysing biographical self-presentations from an interpretivist perspective. The method consists of five major analytic steps. The first is an analysis of the biographical data that can stand independently of the narrator’s perspective. ‘Objective’ data is extracted from the text or interview transcript and ordered chronologically. Secondly, a thematic field analysis is undertaken in which the data is divided into separate units according to the type of text used, whilst keeping the sequence of these texts units intact. In this step, hypotheses are developed regarding the potential significance of the style and sequence of the events presented. The product of this second step is a reconstruction of the life story. A reconstruction of the life history then follows as the third step. The purpose of this step is to generate hypotheses about the meanings that biographical experiences might have had for the narrator at the time they occurred, given the sociocultural context in which they occurred. In the fourth step, microanalysis of individual text segments is undertaken, in which all hypotheses generated in the earlier steps are tested against the text for support or refutation. The final step consists of a contrastive comparison of the life history and life story. The life story and life history are compared to determine, for example, which aspects of the narrator’s experience have been emphasised or minimised. Through this comparison, the selective process is highlighted. This is referred to as the case structure. This paper describes an application of this method to a published first-person narrative of a woman’s experiences of sustaining a brain injury in a motor vehicle accident.

Spatial distribution of benthic microalgae on coral reefs determined by remote sensing

Understanding the ecological role of benthic microalgae, a highly productive component of coral reef ecosystems, requires information on their spatial distribution. The spatial extent of benthic microalgae on Heron Reef (southern Great Barrier Reef, Australia) was mapped using data from the Landsat 5 Thematic Mapper sensor. integrated with field measurements of sediment chlorophyll concentration and reflectance. Field-measured sediment chlorophyll concentrations. 2 ranging from 23-1.153 mg chl a m(2), were classified into low, medium, and high concentration classes (1-170, 171-290, and > 291 mg chl a m(-2)) using a K-means clustering algorithm. The mapping process assumed that areas in the Thematic Mapper image exhibiting similar reflectance levels in red and blue bands would correspond to areas of similar chlorophyll a levels. Regions of homogenous reflectance values corresponding to low, medium, and high chlorophyll levels were identified over the reef sediment zone by applying a standard image classification algorithm to the Thematic Mapper image. The resulting distribution map revealed large-scale ( > 1 km 2) patterns in chlorophyll a levels throughout the sediment zone of Heron Reef. Reef-wide estimates of chlorophyll a distribution indicate that benthic Microalgae may constitute up to 20% of the total benthic chlorophyll a at Heron Reef. and thus contribute significantly to total primary productivity on the reef.

A molecular phylogeny of rainbow skinks (Scincidae : Carlia): taxonomic and biogeographic implications

The phylogenetic relationships amongst 29 species of Carlia and Lygisaurus were estimated using a 726-base-pair segment of the protein-coding mitochondrial ND4 gene. Results do not support the recent resurrection of the genus Lygisaurus. Although most Lygisaurus species formed a single clade, this clade is nested within Carlia and includes Carlia parrhasius. Due to this new molecular evidence, and the paucity of diagnostic morphological characters separating the genera, Lygisaurus de Vis 1884 is re-synonymised with Carlia Gray 1845. Our analysis is also inconsistent with a previous suggestion that Lygisaurus timlowi should be removed to Menetia, a genus that is distantly related relative to outgroups used here. Intraspecific variation in Carlia is, in several instances, greater than interspecific distance. The most strikingly divergent lineages are found within C. rubrigularis, which appears to be paraphyletic, with southern populations more closely related to C. rhomboidalis than to northern populations of C. rubrigularis. The two C. rubrigularis-C. rhomboidalis lineages form part of a major polytomy at an intermediate level of divergence. Lack of resolution at this level, however, does not appear to be due to saturation or loss of phylogenetic signal. Rather, the polytomy probably reflects a period of relatively rapid diversification that occurred sometime during the Miocene.

Germline BRCA1 promoter deletions in UK and Australian familial breast cancer patients: Identification of a novel deletion consistent with BRCA1 :psi VBRCA1 recombination

Inherited susceptibility to breast cancer results from germline mutations in one of a number of genes including BRCA1. A significant number of BRCA1-linked familial breast cancer patients, however, have no detectable BRCA1 mutation. This could be due in part to the inability of commonly used mutation-detection techniques to identify mutations outside the BRCA1 coding region. This paper addresses the hypothesis that non coding region mutations, specifically in the BRCA1 promoter, account for some of these cases. We describe a new and detailed restriction map of the 5' region of the BRCA1 gene including the nearby NBR2, psiBRCA1, and NBR1 genes and the isolation of a number of new informative hybridization probes suitable for Southern analysis. Using this information we screened DNA from lymphoblastoid cell-lines made from 114 UK familial breast cancer patients and detected one large deletion in the 5' region of BRCA1. We show that the breakpoints for this deletion are in BRCA1 intron 2 and between NBR2 and exon 2 of psiBRCA1, raising the possibility that this deletion arose via a novel mechanism involving BRCA1:psiBRCA1 recombination. We have also screened 60 familial breast cancer patients from the Australian population, using an amplification refractory mutation system (ARMS) technique described previously by our group, and found one patient with a genotype consistent with a BRCA1 promoter deletion. These findings indicate that germline BRCA1 promoter deletions are a rare and yet significant mutation event and that they could arise via a novel genetic mechanism. Hum Mutat 19:435-442, 2002. (C) 2002 Wiley-Liss, Inc.

Characterization of mouse profilaggrin: Evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N-terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal anti-body raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

Characterization of the mouse hnRNP A2/B1/B0 gene and identification of processed pseudogenes

The mouse hnRNP A2/B1/B0 gene has been cloned using a PCR-based strategy and sequenced. Analysis of this sequence showed that the gene organization closely follows that of the human orthologue with 12 exons and 11 introns. The hnRNP A2/B1/B0 gene gives rise to four splice variants through alternative splicing of exons 2 and 9. RT-PCR assays indicated that all splice variants were expressed in mouse brain, skin, and stomach tissues of varying ages, although their ratios to one another varied with age and tissue type. We also identified a small subset of all polyadenylated splice variants that included intron 11, which shows 94% sequence identity between human and mouse. Several processed pseudogenes were identified in the mouse genome. A search of the mouse genome databases located five pseudogenes, four of. which are presumed to be non-functional because of the presence of premature stop codons, large deletions or rearrangements within the coding region. The fifth, which possesses putative promoter elements and has a coding sequence identical to that of the hnRNP A2 mRNA, variant, may be functional. (C) 2002 Elsevier Science B.V. All rights reserved.

The draft genome of Ciona intestinalis: Insights into chordate and vertebrate origins

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains similar to16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

Categorization and characterization of transcript-confirmed constitutively and alternatively spliced introns and exons from human

By spliced alignment of human DNA and transcript sequence data we constructed a data set of transcript-confirmed exons and introns from 2793 genes, 796 of which (28%) were seen to have multiple isoforms. We find that over one-third of human exons can translate in more than one frame, and that this is highly correlated with G+C content. Introns containing adenosine at donor site position +3 (A3), rather than guanosine (G3), are more common in low G+C regions, while the converse is true in high G+C regions. These two classes of introns are shown to have distinct lengths, consensus sequences and correlations among splice signals, leading to the hypothesis that A3 donor sites are associated with exon definition, and G3 donor sites with intron definition. Minor classes of introns, including GC-AG, U12-type GT-AG, weak, and putative AG-dependant introns are identified and characterized. Cassette exons are more prevalent in low G+C regions, while exon isoforms are more prevalent in high G+C regions. Cassette exon events outnumber other alternative events, while exon isoform events involve truncation twice as often as extension, and occur at acceptor sites twice as often as at donor sites. Alternative splicing is usually associated with weak splice signals, and in a majority of cases, preserves the coding frame. The reported characteristics of constitutive and alternative splice signals, and the hypotheses offered regarding alternative splicing and genome organization, have important implications for experimental research into RNA processing. The 'AltExtron' data sets are available at http://www.bit.uq.edu.au/altExtron/ and http://www.ebi.ac.uk/similar tothanaraj/altExtron/.

Mapping glider habitat in dry eucalypt forests for Montreal Process indicator 1.1: Fragmentation of forest types

Accurate habitat mapping is critical to landscape ecological studies such as required for developing and testing Montreal Process indicator 1.1e, fragmentation of forest types. This task poses a major challenge to remote sensing, especially in mixedspecies, variable-age forests such as dry eucalypt forests of subtropical eastern Australia. In this paper, we apply an innovative approach that uses a small section of one-metre resolution airborne data to calibrate a moderate spatial resolution model (30 m resolution; scale 1:50 000) based on Landsat Thematic Mapper data to estimate canopy structural properties in St Marys State Forest, near Maryborough, south-eastern Queensland. The approach applies an image-processing model that assumes each image pixel is significantly larger than individual tree crowns and gaps to estimate crown-cover percentage, stem density and mean crown diameter. These parameters were classified into three discrete habitat classes to match the ecology of four exudivorous arboreal species (yellowbellied glider Petaurus australis, sugar glider P. breviceps, squirrel glider P. norfolcensis , and feathertail glider Acrobates pygmaeus), and one folivorous arboreal marsupial, the greater glider Petauroides volans. These species were targeted due to the known ecological preference for old trees with hollows, and differences in their home range requirements. The overall mapping accuracy, visually assessed against transects (n = 93) interpreted from a digital orthophoto and validated in the field, was 79% (KHAT statistic = 0.72). The KHAT statistic serves as an indicator of the extent that the percentage correct values of the error matrix are due to ‘true’ agreement verses ‘chance’ agreement. This means that we are able to reliably report on the effect of habitat loss on target species, especially those with a large home range size (e.g. yellow-bellied glider). However, the classified habitat map failed to accurately capture the spatial patterning (e.g. patch size and shape) of stands with a trace or sub-dominance of senescent trees. This outcome makes the reporting of the effects of habitat fragmentation more problematic, especially for species with a small home range size (e.g. feathertail glider). With further model refinement and validation, however, this moderateresolution approach offers an important, cost eff e c t i v e advancement in mapping the age of dry eucalypt forests in the region.

Monitoring the composition and form of urban environments based on the vegetation-impervious surface-soil (VIS) model by sub-pixel analysis techniques

The majority of the world's population now resides in urban environments and information on the internal composition and dynamics of these environments is essential to enable preservation of certain standards of living. Remotely sensed data, especially the global coverage of moderate spatial resolution satellites such as Landsat, Indian Resource Satellite and Systeme Pour I'Observation de la Terre (SPOT), offer a highly useful data source for mapping the composition of these cities and examining their changes over time. The utility and range of applications for remotely sensed data in urban environments could be improved with a more appropriate conceptual model relating urban environments to the sampling resolutions of imaging sensors and processing routines. Hence, the aim of this work was to take the Vegetation-Impervious surface-Soil (VIS) model of urban composition and match it with the most appropriate image processing methodology to deliver information on VIS composition for urban environments. Several approaches were evaluated for mapping the urban composition of Brisbane city (south-cast Queensland, Australia) using Landsat 5 Thematic Mapper data and 1:5000 aerial photographs. The methods evaluated were: image classification; interpretation of aerial photographs; and constrained linear mixture analysis. Over 900 reference sample points on four transects were extracted from the aerial photographs and used as a basis to check output of the classification and mixture analysis. Distinctive zonations of VIS related to urban composition were found in the per-pixel classification and aggregated air-photo interpretation; however, significant spectral confusion also resulted between classes. In contrast, the VIS fraction images produced from the mixture analysis enabled distinctive densities of commercial, industrial and residential zones within the city to be clearly defined, based on their relative amount of vegetation cover. The soil fraction image served as an index for areas being (re)developed. The logical match of a low (L)-resolution, spectral mixture analysis approach with the moderate spatial resolution image data, ensured the processing model matched the spectrally heterogeneous nature of the urban environments at the scale of Landsat Thematic Mapper data.

A new joint coding and modulation scheme for channels with clipping

A major limitation in any high-performance digital communication system is the linearity region of the transmitting amplifier. Nonlinearities typically lead to signal clipping. Efficient communication in such conditions requires maintaining a low peak-to-average power ratio (PAR) in the transmitted signal while achieving a high throughput of data. Excessive PAR leads either to frequent clipping or to inadequate resolution in the analog-to-digital or digital-to-analog converters. Currently proposed signaling schemes for future generation wireless communications suffer from a high PAR. This paper presents a new signaling scheme for channels with clipping which achieves a PAR as low as 3. For a given linear range in the transmitter's digital-to-analog converter, this scheme achieves a lower bit-error rate than existing multicarrier schemes, owing to increased separation between constellation points. We present the theoretical basis for this new scheme, approximations for the expected bit-error rate, and simulation results. (C) 2002 Elsevier Science (USA).

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2)

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid D-Phe in aSvg-30 [D-Phe11,His12] Svg((11-40)) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl) benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[ His12] Svg was tested for its ability to displace [I-125-Tyr0] oCRF or [I-125-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 ( rCRF(1)) or mouse CRF receptor, type 2beta (mCRF(2beta)). Furthermore, the ability of ATB- [His12] Svg((12-40)) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF(1) (HEK-rCRF(1) cells) or mCRF(2beta) (HEK-mCRF(2beta) cells) was determined. Unlike astressin and photo astressin, ATB- [His12]Svg((12-40)) showed high selective binding to mCRF(2beta) (K-i = 3.1 +/- 0.2 nM) but not the rCRF(1) receptor (K-i = 142. 5 +/- 22.3 nM) and decreased Svg-stimulated cAMP activity in mCRF(2beta)-expressing cells in a similar fashion as aSvg-30. A66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB- [His12]Svg((12-40)) was covalently linked to mCRF(2beta) receptor. The specificity of the photoactivatable I-125-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).

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.

Mitochondrial genomes of parasitic flatworms

Complete or near-complete mitochondrial genomes are now available for 11 species or strains of parasitic flatworms belonging to the Trematoda and the Cestoda. The organization of these genomes is not strikingly different from those of other eumetazoans, although one gene (atp8) commonly found in other phyla is absent from flatworms. The gene order in most flatworms has similarities to those seen in higher protostomes such as annelids. However, the gene order has been drastically altered in Schistosoma mansoni, which obscures this possible relationship. Among the sequenced taxa, base composition varies considerably, creating potential difficulties for phylogeny reconstruction. Long non-coding regions are present in all taxa, but these vary in length from only a few hundred to similar to10 000 nucleotides. Among Schistosoma spp., the long non-coding regions are rich in repeats and length variation among individuals is known. Data from mitochondrial genomes are valuable for studies on species identification, phylogenies and biogeography.