Informing the management of multiple stressors on estuarine ecosystems using an expert-based Bayesian Network model.

The approach of applying stressor load limits or thresholds to aid estuarine management is being explored in many global case studies. However, there is growing concern regarding the influence of multiple stressors and their cumulative effects on the functioning of estuarine ecosystems due to the considerable uncertainty around stressor interactions. Recognising that empirical data limitations hinder parameterisation of detailed models of estuarine ecosystem responses to multiple stressors (suspended sediment, sediment mud and metal content, and nitrogen inputs), an expert driven Bayesian network (BN) was developed and validated. Overall, trends in estuarine condition predicted by the BN model were well supported by field observations, including results that were markedly higher than random (71-84% concordance), providing confidence in the overall model dynamics. The general BN framework was then applied to a case study estuary to demonstrate the model's utility for informing management decisions. Results indicated that reductions in suspended sediment loading were likely to result in improvements in estuarine condition, which was further improved by reductions in sediment mud and metal content, with an increased likelihood of high abundance of ecological communities relative to baseline conditions. Notably, reductions in suspended sediment were also associated with an increased probability of high nuisance macroalgae and phytoplankton if nutrient loading was not also reduced (associated with increased water column light penetration). Our results highlight that if stressor limit setting is to be implemented, limits must incorporate ecosystem responses to cumulative stressors, consider the present and desired future condition of the estuary of interest, and account for the likelihood of unexpected ecological outcomes regardless of whether the experts (or empirical data) suggest a threshold has or has not been triggered.

The development of a national approach to monitoring estuarine health based on multivariate analysis.

New Zealand has a complex coastal environment spanning a large latitudinal gradient and three water masses. Here we assess whether multivariate analyses of benthic macrofaunal community composition can be a sensitive approach to assessing relative estuarine health across the country, negating the need for regional indices and reducing reliance on reference sites. Community data were used in separate canonical analyses of principal coordinates to create multivariate models of community responses to gradients in mud content and heavy metal contamination. Both models performed well (R2 = 0.81, 0.71), and were unaffected by regional and estuarine typology differences. The models demonstrate a sensitive and standardized approach to assessing estuarine health that allowed separation of the two stressors. This approach could be applied to other stressors, countries or regions.

FTIR imaging detects diet and genotype-dependent chemical composition changes in wild type and mutant C. elegans strains.

This study focuses on the use of Fourier Transform Infrared (FTIR) microspectroscopy to determine chemical changes induced in the nematode Caenorhabditis elegans by supplementation of C. elegans maintenance media (CeMM) by Eicosapentaenoic acid (EPA). Wild-type C. elegans (N2) and mutant strains (tub-1 and fat-3) were grown in CeMM alone, and CeMM supplemented with EPA at 25 or 100 µM. Feeding was performed for 72 h. FTIR imaging was performed in transmission mode on individual worms. The FTIR imaging analysis of wild-type animals revealed the presence of vibrations assigned to unsaturated fatty acids, specifically bands at 3008 cm-1 ([double bond, length as m-dash]C-H, olefinic stretch) and 1744 cm-1 (C[double bond, length as m-dash]O, unsaturated fatty acids). It confirmed previously reported synthesis of unsaturated fatty acids in wild-type C. elegans. For the FTIR spectra of mutant strains, these vibrations were absent or present only as very small shoulder, which indicates that tub-1 and fat-3 synthesize essentially saturated fatty acids as indicated by the presence of -CH2 and C[double bond, length as m-dash]O vibrations. These results are in agreement with previous studies which reported that these mutants have altered lipid compositions. Principal component analysis showed differences in chemical composition between wild-type and mutant strains as well as between mutant strains cultured in normal CeMM and those cultured in CeMM supplemented with EPA. This study demonstrated the usefulness of FTIR microspectroscopy to investigate fat metabolism in C. elegans.

Interaction networks in coastal soft-sediments highlight the potential for change in ecological resilience.

Recent studies emphasize the role of indirect relationships and feedback loops in maintaining ecosystem resilience. Environmental changes that impact on the organisms involved in these processes have the potential to initiate threshold responses and fundamentally shift the interactions within an ecosystem. However, empirical studies are hindered by the difficulty of designing appropriate manipulative experiments to capture this complexity. Here we employ structural equation modeling to define and test the architecture of ecosystem interaction networks. Using survey data from 19 estuaries we investigate the interactions between biological (abundance of large bioturbating macrofauna, microphytobenthos, and detrital matter) and physical (sediment grain size) processes. We assess the potential for abrupt changes in the architecture of the network and the strength of interactions to occur across environmental gradients. Our analysis identified a potential threshold in the relationship between sediment mud content and benthic chlorophyll a, at -12 microg/g, using quantile regression. Below this threshold, the interaction network involved different variables and fewer feedbacks than above. This approach has potential to improve our empirical understanding of thresholds in ecological systems and our ability to design manipulative experiments that test how and when a threshold will be passed. It can also be used to indicate to resource managers that a particular system has the potential to exhibit threshold responses to environmental change, emphasizing precautionary management and facilitating a better understanding of how persistent multiple stressors threaten the resilience and long-term use of natural ecosystems.

Effective long-term ecological monitoring using spatially and temporally nested sampling.

Effective environmental management requires documentation of ecosystem status and changes to that status. Without long-term data, short-term natural variability can mask chronic and/or cumulative impacts, often until critical levels are reached. However, a trade-off generally occurs between sampling in space and time. This study analyses a spatially and temporally nested long-term (12 years) monitoring programme conducted on benthic macrofauna in a large harbour. Sampling was carried out at six sites for 5.5 years, after which only two sites were sampled for the next 5 years. After this period, all six sites were sampled for another 2 years. While ecology is frequently thought of being highly variable, this design was able to detect trends, and cycles, in abundance, with only around 10% of species at each site exhibiting unpredictable temporal variability. Sites exhibiting similar trends in the abundance of a species over the 12.5-year period were generally spatially contiguous, and the spatial scale of change could be assessed. Continuous sampling at two sites identified whether changes in unsampled sites were related to long-term cycles. Moreover, this sampling provided a long-term background of temporal fluctuations against which to assess the ecological significance of observed changes.

FRG2, an FSHD candidate gene, is transcriptionally upregulated in differentiating primary myoblast cultures of FSHD patients.

BACKGROUND: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with partial deletion of the subtelomeric D4Z4 repeat array on chromosome 4qter. This chromosomal rearrangement may result in regional chromatin relaxation and transcriptional deregulation of genes nearby. METHODS AND RESULTS: Here we describe the isolation and characterisation of FRG2, a member of a chromosomally dispersed gene family, mapping only 37 kb proximal to the D4Z4 repeat array. Homology and motif searches yielded no clues to the function of the predicted protein. FRG2 expression is undetectable in all tissues tested except for differentiating myoblasts of FSHD patients, which display low, yet distinct levels of FRG2 expression, partly from chromosome 4 but predominantly originating from its homologue on chromosome 10. However, in non-FSHD myopathy patients only distantly related FRG2 homologues are transcribed, while differentiating myoblasts from healthy controls fail to express any member of this gene family. Moreover, fibroblasts of FSHD patients and control individuals undergoing forced Ad5-MyoD mediated myogenesis show expression of FRG2 mainly originating from chromosome 10. Luciferase reporter assays show that the FRG2 promoter region can direct high levels of expression but is inhibited by increasing numbers of D4Z4 repeat units. Transient transfection experiments with FRG2 fusion-protein constructs reveal nuclear localisation and apparently FRG2 overexpression causes a wide range of morphological changes. CONCLUSION: The localisation of FRG2 genes close to the D4Z4 repeats on chromosome 4 and 10, their transcriptional upregulation specifically in FSHD myoblast cultures, potential involvement in myogenesis, and promoter properties qualify FRG2 as an attractive candidate for FSHD pathogenesis.

Glycosylation defects in inherited muscle disease.

The gene mutated in the myodystrophy mouse, a model of muscular dystrophy, encodes a putative glycosyltransferase, Large. Mutations in genes encoding proteins thought to be involved in glycosylation have now been identified in six human forms of muscular dystrophy. Hereditary inclusion body myopathy and Nonaka myopathy result from defects in sialic acid production. Two forms of congenital muscular dystrophy, Fukuyama-type and MDC1C, result from mutations in members of the fukutin family. MDC1C and limb girdle muscular dystrophy type 2I are allelic, as they are both associated with mutations in the FKRP gene. Mutations in POMGnT, which encodes an enzyme involved in the synthesis of O-mannosyl glycans, result in muscle-eye-brain disease--another congenital form of muscular dystrophy. Abnormal alpha-dystroglycan has been reported in the myodystrophy mouse, and in the congenital and limb girdle muscular dystrophies. Recent data have shown that there is altered glycosylation of the protein and that this reduces its ability to bind to extracellular matrix ligands such as laminin and agrin.

Intron loss in the SART1 genes of Fugu rubripes and Tetraodon nigroviridis.

The human SART1 gene was initially identified in a screen for proteins recognised by IgE, which may be implicated in atopic disease. We have examined the genomic structure and cDNA sequence of the SART1 gene in the compact genomes of the pufferfish Fugu rubripes and Tetraodon nigroviridis. The entire coding regions of both the Fugu and Tetraodon SART1 genes are contained within single exons. The Fugu gene contains only one intron located in the 5' untranslated region. Southern blot hybridisation of Fugu genomic DNA confirmed the SART1 gene to be single copy. Partial genomic structures were also determined for the human, mouse, Drosophila and C. elegans SART1 homologues. The human and mouse genes both contain many introns in the coding region, the human gene possessing at least 20 exons. The Drosophila and C. elegans homologues contain 6 and 12 exons, respectively. This is only the second time such a difference in the organization of homologous Fugu and human genes has been reported. The Fugu and Tetraodon SART1 genes encode putative proteins of 772 and 774 aa, respectively, each having 65% amino acid identity to human SART1. Leucine zipper and basic motifs are conserved in the predicted Fugu and Tetraodon proteins.

Mutant glycosyltransferase and altered glycosylation of alpha-dystroglycan in the myodystrophy mouse.

Spontaneous and engineered mouse mutants have facilitated our understanding of the pathogenesis of muscular dystrophy and they provide models for the development of therapeutic approaches. The mouse myodystrophy (myd) mutation produces an autosomal recessive, neuromuscular phenotype. Homozygotes have an abnormal gait, show abnormal posturing when suspended by the tail and are smaller than littermate controls. Serum creatine kinase is elevated and muscle histology is typical of a progressive myopathy with focal areas of acute necrosis and clusters of regenerating fibers. Additional aspects of the phenotype include sensorineural deafness, reduced lifespan and decreased reproductive fitness. The myd mutation maps to mouse chromosome 8 at approximately 33 centimorgans (cM) (refs. 2, 4-7). Here we show that the gene mutated in myd encodes a glycosyltransferase, Large. The human homolog of this gene (LARGE) maps to chromosome 22q. In myd, an intragenic deletion of exons 4-7 causes a frameshift in the resultant mRNA and a premature termination codon before the first of the two catalytic domains. On immunoblots, a monoclonal antibody to alpha-dystroglycan (a component of the dystrophin-associated glycoprotein complex) shows reduced binding in myd, which we attribute to altered glycosylation of this protein. We speculate that abnormal post-translational modification of alpha-dystroglycan may contribute to the myd phenotype.

Identification of a novel beta-tubulin subfamily with one member (TUBB4Q) located near the telomere of chromosome region 4q35.

The human beta-tubulin supergene family consists of several isotypes with many associated pseudogenes. Here we report the identification of yet another beta-tubulin sequence designated TUBB4Q. This tubulin maps 80 kb proximal to the facioscapulohumeral muscular dystrophy (FSHD1) associated D4Z4 repeats on chromosome 4q35. The genomic structure contains four exons encoding a putative protein of 434 amino acids. The TUBB4Q nucleotide and protein sequence show 87% and 86% homology to beta2-tubulin, respectively. Although the genomic structure shows all functional aspects of a genuine gene, no transcript could be detected. TUBB4Q-related sequences were identified on multiple chromosomes. Since these sequences mutually exhibit a high nucleotide sequence homology, they presumably belong to a novel subfamily of beta-tubulin genes. Although the chromosome 4q35 tubulin-member probably represents a pseudogene, ectopic expression due to a postulated position effect variegation (PEV), makes TUBB4Q an ideal dominant-negative candidate gene for FSHD1.

De novo facioscapulohumeral muscular dystrophy: frequent somatic mosaicism, sex-dependent phenotype, and the role of mitotic transchromosomal repeat interaction between chromosomes 4 and 10.

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is caused by deletion of most copies of the 3.3-kb subtelomeric D4Z4 repeat array on chromosome 4q. The molecular mechanisms behind the deletion and the high proportion of new mutations have remained elusive. We surveyed 35 de novo FSHD families and found somatic mosaicism in 40% of cases, in either the patient or an asymptomatic parent. Mosaic males were typically affected; mosaic females were more often the unaffected parent of a nonmosaic de novo patient. A genotypic-severity score, composed of the residual repeat size and the degree of somatic mosaicism, yields a consistent relationship with severity and age at onset of disease. Mosaic females had a higher proportion of somatic mosaicism than did mosaic males. The repeat deletion is significantly enhanced by supernumerary homologous repeat arrays. In 10% of normal chromosomes, 4-type repeat arrays are present on chromosome 10. In mosaic individuals, 4-type repeats on chromosome 10 are almost five times more frequent. The reverse configuration, also 10% in normal chromosomes, was not found, indicating that mutations may arise from transchromosomal interaction, to which the increase in 4-type repeat clusters is a predisposing factor. The somatic mosaicism suggests a mainly mitotic origin; mitotic interchromosomal gene conversion or translocation between fully homologous 4-type repeat arrays may be a major mechanism for FSHD mutations.

Cloning of the murine unconventional myosin gene Myo9b and identification of alternative splicing.

We report the cloning of a cDNA for the mouse unconventional myosin Myo9b, the orthologue of the rat myr5 and human MYOIXb genes. A full-length spleen cDNA of 7087bp encoding a protein of 1961 amino acids was isolated. By RT-PCR, we show that Myo9b is expressed in a wide range of tissues, including heart, brain, muscle and inner ear. In addition, we have identified two alternatively spliced exons. Equivalent exons have not been previously reported for either the human or rat homologues. These exons are located in the Myo9b specific actin-binding site insert of the head domain and in the tail region. A third splice form utilizing an alternative reading frame within the 3'UTR is also described. Several polymorphisms within the coding region were identified; of interest is an in-frame 33bp imperfect duplication within the tail region that was observed only in the C57Bl/6 strain. Myo9b has been previously mapped to mouse chromosome 8 and is a candidate for the mouse mutations myodystrophy and quinky.

A new dosage test for subtelomeric 4;10 translocations improves conventional diagnosis of facioscapulohumeral muscular dystrophy (FSHD).

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the size reduction of a polymorphic repeat array on 4q35. Probe p13E-11 recognises this chromosomal rearrangement and is generally used for diagnosis. However, diagnosis of FSHD is complicated by three factors. First, the probe cross hybridises to a highly homologous repeat array locus on chromosome 10q26. Second, although a BlnI polymorphism allows discrimination between the repeat units on chromosomes 4 and 10 and greatly facilitates FSHD diagnosis, the occurrence of translocations between chromosomes 4 and 10 further complicates accurate FSHD diagnosis. Third, the recent identification of deletions of p13E-11 in both control and FSHD populations is an additional complicating factor. Although pulsed field gel electrophoresis is very useful and sometimes necessary to detect these rearrangements, this technique is not operational in most FSHD diagnostic laboratories. Moreover, repeat arrays >200 kb are often difficult to detect and can falsely suggest a deletion of p13E-11. Therefore, we have developed an easy and reliable Southern blotting method to identify exchanges between 4 type and 10 type repeat arrays and deletions of p13E-11. This BglII-BlnI dosage test addresses all the above mentioned complicating factors and can be carried out in addition to the standard Southern blot analysis for FSHD diagnosis as performed in most laboratories. It will enhance the specificity and sensitivity of conventional FSHD diagnosis to the values obtained by PFGE based diagnosis of FSHD. Moreover, this study delimits the FSHD candidate gene region by mapping the 4;10 translocation breakpoint proximal to the polymorphic BlnI site in the first repeat unit.

The FSHD region on human chromosome 4q35 contains potential coding regions among pseudogenes and a high density of repeat elements.

The distal end of chromosome 4q contains the locus involved in facioscapulohumeral muscular dystrophy (FSHD1). Specific genomic deletions within a tandem DNA repeat (D4Z4) are associated with the disease status, but no causal genes have yet been discovered. In a systematic search for genes, a 161-kb stretch of genomic DNA proximal to D4Z4 was sequenced, analyzed for homologies, and subjected to gene prediction programs. A major fraction (45%) of the subtelomeric region is composed of repeat sequences attributable mainly to LINE-1 elements. Apart from the previously identified FRG1 and TUB4q sequences, several additional potential coding regions were identified by analyzing the sequence with exon prediction programs. So far, we have been unable to demonstrate transcripts by RT-PCR or cDNA library hybridization. However, several retrotransposed pseudogenes were identified. The high density of pseudogenes and repeat elements is consistent with the subtelomeric location of this region and explains why previous transcript identification studies have been problematic.

Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the polymorphic D4Z4 locus on chromosome 4q35. In non-affected individuals, this locus comprises 10-100 tandem copies of members of the 3.3kb dispersed repeat family. Deletions leaving 1-8 such repeats have been associated with FSHD, for which no candidate gene has been identified. We have determined the complete nucleotide sequence of a 13.5kb EcoRI genomic fragment comprising the only two 3.3kb elements left in the affected D4Z4 locus of a patient with FSHD. Sequence analyses demonstrated that the two 3.3kb repeats were identical. They contain a putative promoter that was not previously detected, with a TACAA instead of a TATAA box, and a GC box. Transient expression of a luciferase reporter gene fused to 191bp of this promoter, demonstrated strong activity in transfected human rhabdomyosarcoma TE671 cells that was affected by mutations in the TACAA or GC box. In addition, these 3.3kb repeats include an open reading frame (ORF) starting 149bp downstream from the TACAA box and encoding a 391 residue protein with two homeodomains (DUX4). In-vitro transcription/translation of the ORF in a rabbit reticulocyte lysate yielded two (35)S Cys/ (35)S Met labeled products with apparent molecular weights of 38 and 75kDa on SDS-PAGE, corresponding to the DUX4 monomer and dimer, respectively. In conclusion, we propose that each of the 3.3kb elements in the partially deleted D4Z4 locus could include a DUX4 gene encoding a double homeodomain protein.

Recent amplification of the human FRG1 gene during primate evolution.

There is evidence of multiple copies of the FSHD Region Candidate Gene 1 (FRG1) in humans. Analysis of human FRG1 ESTs showed many of them to be non-processed pseudogenes dispersed throughout the genome. To determine when the amplification of FRG1 occurred, we used a PCR-based approach to identify FRG1 sequences from great apes, chimpanzee, gorilla and orang-utan, and an Old World monkey, Macaca mulatta. In common with humans, multiple copies of FRG1 were detected in the great apes. However, in Macaca mulatta, only two FRG1 loci were identified, one presumed to be the homologue of the human chromosome 4q gene. This is strikingly similar to the distribution of a dispersed 3.3-kb repeat family in primates. A member of this family, D4Z4, maps to the subtelomeric region of 4q, in close proximity to FRG1. We propose that an ancestral duplication of distal 4q included FRG1. This duplication is present in Macaca mulatta whose divergence from hominoids is thought to have occurred at least 33 million years ago. We propose that this telomeric region then underwent further amplification and dispersion events in the great ape lineage, with copies of FRG1 and the 3.3-kb repeats being localized in heterochromatic regions.

FRG1, a gene in the FSH muscular dystrophy region on human chromosome 4q35, is highly conserved in vertebrates and invertebrates.

The human FRG1 gene maps to human chromosome 4q35 and was identified as a candidate for facioscapulohumeral muscular dystrophy. However, FRG1 is apparently not causally associated with the disease and as yet, its function remains unclear. We have cloned homologues of FRG1 from two additional vertebrates, the mouse and the Japanese puffer fish Fugu rubripes, and investigated the genomic organization of the genes in the two species. The intron/exon structure of the genes is identical throughout the protein coding region, although the Fugu gene is five times smaller than the mouse gene. We have also identified FRG1 homologues in two nematodes; Caenorhabditis elegans and Brugia malayi. The FRG1 protein is highly conserved and contains a lipocalin sequence motif, suggesting it may function as a transport protein.

High-resolution mapping of mouse chromosome 8 identifies an evolutionary chromosomal breakpoint.

The central region of mouse Chromosome (Chr) 8, containing the myodystrophy (myd) locus, is syntenic with human Chr 4q28-qter. The human neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) maps to Chr 4q35, and myd has been proposed as a mouse homolog of FSHD. We have employed a comparative mapping approach to investigate this relationship further by extending the mouse genetic map of this region. We have ordered 12 genes in a single cross, 8 of which have human homologs on 4q28-qter. The results confirm a general relationship between the most distal genes on human 4q and the most proximal genes in the mouse 8 syntenic region. Despite chromosomal rearrangements of syntenic groups in this region, conservation of gene order is maintained between the group of genes in the human telomeric region of 4q35 and MMU8. Furthermore, this conserved telomeric HSA4q35 syntenic group maps proximal to the myd mutation and is flanked by genes with homologs on HSA8p22. At the proximal boundary of the MMU8 linkage group we have identified a single 300-kb YAC containing the genes Frgl and Pcml, which have human homologs on 4q35 and 8p22, respectively. Thus, this YAC spans an evolutionary chromosomal breakpoint. As well as providing clues about chromosomal evolution, this map of the FSHD syntenic mouse region should prove invaluable in the isolation of candidate genes for this disease.

The mouse homolog of FRG1, a candidate gene for FSHD, maps proximal to the myodystrophy mutation on chromosome 8.

The human autosomal dominant neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) is associated with deletions within a complex tandem DNA repeat (D4Z4) on Chromosome (Chr) 4q35. The molecular mechanism underlying this association of FSHD with DNA rearrangements is unknown, and, thus far, no gene has been identified within the repeat. We isolated a gene mapping 100 kb proximal to D4Z4 (FSHD Region Gene 1:FRG1), but were unable to detect any alterations in total or allele-specific mRNA levels of FRG1 in FSHD patients. Human Chr 4q35 exhibits synteny homology with the region of mouse Chr 8 containing the gene for the myodystrophy mutation (myd), a possible mouse homolog of FSHD. We report the cloning of the mouse gene (Frg1) and show that it maps to mouse Chr 8. Using a cross segregating the myd mutation and the European Collaborative Interspecific Backcross, we showed that Frg1 maps proximal to the myd locus and to the Clc3 and Ant1 genes.