Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin-containing enzymes

Dimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum . Dimethyl sulphide dehydrogenase was shown to contain bis (molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdop-terin-containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB , ddhD and ddhC . DdhB showed sequence homology to NarH, suggesting that it contains multiple iron-sulphur clusters. Analysis of the N-terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec-dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo-trophic growth on the same substrate. Mutational analysis showed that cytochrome c (2) mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.

Social structure in migrating humpback whales (Megaptera novaeangliae)

Although largely solitary, humpback whales exhibit a number of behaviours where individuals co-operate with one another, for example during bubble net feeding. Such cases could be due to reciprocal altruism brought on by exceptional circumstances, for example the presence of abundant shoaling fish. An alternative explanation is that these behaviours have evolved through kin selection. With little restriction to either communication or movement, diffuse groups of relatives could maintain some form of social organization without the need to travel in tight-nit units. To try to distinguish between these hypotheses, we took advantage of the fact that migrating humpback whales often swim together in small groups. If kin selection is important in humpback whale biology, these groups should be enriched for relatives. Consequently, we analysed biopsy samples from 57 groups of humpback whales migrating off Eastern Australia in 1992. A total of 142 whales were screened for eight microsatellite markers. Mitochondrial DNA sequences (371 bp) were also used to verify and assist kinship identification. Our data add support to the notion that mothers travel with their offspring for the first year of the calf's life. However, beyond the presence of mother-calf/yearling pairs, no obvious relatedness pattern was found among whales sampled either in the same pod or on the same day. Levels of relatedness did not vary between migratory phases (towards or away from the breeding ground), nor between the two sexes considered either overall or in the north or south migrations separately. These findings suggest that, if any social organization does exist, it is formed transiently when needed rather than being a constant feature of the population, and hence is more likely based on reciprocal altruism than kin selection.

In vivo DNA electrotransfer

The use of electrotransfer for DNA delivery to prokaryotic cells, and eukaryotic cells in vitro, has been well known and widely used for many years. However, it is only recently that electric fields have been used to enhance DNA transfer to animal cells in vivo, and this is known as DNA electrotransfer or in vivo DNA electroporation. Some of the advantages of this method of somatic cell gene transfer are that it is a simple method that can be used to transfer almost any DNA construct to animal cells and tissues in vivo; multiple constructs can be co-transfected; it is equally applicable to dividing and nondividing cells; the DNA of interest does not need to be subeloned into a specific viral transfer vector and there is no need for the production of high titre viral stocks; and, as no viral genes are expressed there is less chance of an adverse immunologic reaction to vector sequences. The ease with which efficient in vivo gene transfer can be achieved with in vivo DNA electrotransfer is now allowing genetic analysis to be applied to a number of classic animal model systems where transgenic and embryonic stem cell techniques are not well developed, but for which a wealth of detailed descriptive embryological information is available, or surgical manipulation is much more feasible. As well as exciting applications in developmental biology, in vivo DNA electrotransfer is also being used to transfer genes to skeletal muscle and drive expression of therapeutically active proteins, and to examine exogenous gene and protein function in normal adult cells situated within the complex environment of a tissue and organ system in vivo. Thus, in effect providing the in vivo equivalent of the in vitro transient transfection assay. As the widespread use of in vivo electroporation has really only just begun, it is likely that the future will hold many more applications for this technology in basic research, biotechnology and clinical research areas.

The phylogeography and connectivity of the latitudinally widespread scleractinian coral Plesiastrea versipora in the Western Pacific

Whereas terrestrial animal populations might show genetic connectivity within a continent, marine species, such as hermatypic corals, may have connectivity stretching to all corners of the planet. We quantified the genetic variability within and among populations of the widespread scleractinian coral, Plesiastrea versipora along the eastern Australian seaboard (4145 km) and the Ryukyu Archipelago (Japan, 681 km) using sequences of internal transcribed spacers (ITS1-2) from ribosomal DNA. Geographic patterns in genetic variability were deduced from a nested clade analysis (NCA) performed on a parsimony network haplotype. This analysis allowed the establishment of geographical associations in the distribution of haplotypes within the network cladogram, therefore allowing us to deduce phylogeographical patterns based under models of restricted gene flow, fragmentation and range expansion. No significant structure was found among Ryukyu Archipelago populations. The lack of an association between the positions of haplotypes in the cladogram with geographical location of these populations may be accounted for by a high level of gene flow of P. versipora within this region, probably due to the strong Kuroshio Current. In contrast, strong geographical associations were apparent among populations of P. versipora along the south-east coast of Australia. This pattern of restricted genetic connectivity among populations of P. versipora on the eastern seaboard of Australia seems to be associated with the present surface ocean current (the East Australian Current) on this side of the south-western Pacific Ocean.

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.

Cuticular hydrocarbons of Drosophila birchii and D-serrata: Identification and role in mate choice in D-serrata

The cuticular hydrocarbon compositions of two sympatric species of Australian Drosophila in the montium subgroup of the melanogaster group that use cuticular hydrocarbons in mate recognition have been characterized. Drosophila birchii has 34 components in greater than trace amounts, with a carbon number range of C-20 to C-33. Drosophila serrata has 21 components above trace level and a carbon number range of C-24 to C-31. These two species share eight hydrocarbon components, with all but two of them being monoenes. For both species, the (Z)-9-monoenes are the predominant positional isomer. The hydrocarbons of D. birchii are n-alkanes, n-alkenes (Z)-5-, (Z)-7-, (Z)-9-, and (Z)-11-), low to trace levels of homologous (Z,Z)-7,11- and (Z,Z)-9,13-dienes; and trace amounts of (Z,Z)-5,9- C-25:2, a major component of D. serrata. Only one methyl branched hydrocarbon was detected (2-methyl C-28), and it occurred at very low levels. The hydrocarbons of D. serrata are dominated by a homologous series of (Z,Z)-5,9-dienes, and notably, are characterized by the apparent absence of n-alkanes. Homologous series of (Z)-5-, (Z)-7-, and (Z)-9- alkenes are also present in D. serrata as well as 2-methyl alkanes. Drosophila serrata females display strong directional mate choice based on male cuticular hydrocarbons and prefer D. serrata males with higher relative abundances of the 2-methyl alkanes, but lower relative abundances of (Z,Z)-5,9- C-24:2 and (Z)-9-C-25:1.

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.

Oceanic interchange and nonequilibrium population structure in the estuarine dependent Indo-Pacific tasselfish, Polynemus sheridani

We assayed mtDNA haplotype [300 base pairs (bp) control region] geography and genealogy in the Indo-Pacific tasselfish, Polynemus sheridani from its contiguous estuarine distribution across northern Australia (n = 169). Eight estuaries were sampled from three oceanographic regions (Timor Sea, Gulf of Carpentaria and the Coral Sea) to assess the impact of Pleistocene sea level changes on the historical connectivity among P. sheridani populations. Specifically, we investigated the genetic consequences of disruption to Indian-Pacific Ocean connectivity brought about by the closure of the Torres Strait. Overall there was significant population subdivision among estuaries (F-ST = 0.161, (Phi(ST) = 0.187). Despite a linear distribution, P. sheridani did not show isolation by distance over the entire sampled range because of genetic similarity of estuaries greater than 3000 km apart. However, significant isolation by distance was detected between estuaries separated by less than 3000 km of coastline. Unlike many genetic studies of Indo-Pacific marine species, there was no evidence for an historical division between eastern and western populations. Instead, phylogeographical patterns were dominated by a starlike intraspecific phylogeny coupled with evidence for population expansion in both the Gulf of Carpentaria and the Coral Sea but not the Timor Sea. This was interpreted as evidence for recent west to east recolonization across of northern Australia following the last postglacial marine advance. We argue that although sufficient time has elapsed postcolonization for populations to approach gene flow-drift equilibrium over smaller spatial scales (< 3000 km), the signal of historical colonization persists to obscure the expected equilibrium pattern of isolation by distance over large spatial scales (> 3000 km).

Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.

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.

CRIM1 Regulates the Rate of Processing and Delivery of Bone Morphogenetic Proteins to the Cell Surface

The Crim1 gene is predicted to encode a transmembrane protein containing six von Willebrand-like cysteine-rich repeats (CRRs) similar to those in the BMP-binding antagonist Chordin (Chrd). In this study, we verify that CRIM1 is a glycosylated, Type I transmembrane protein and demonstrate that the extracellular CRR-containing domain can also be secreted, presumably via processing at the membrane. We have previously demonstrated Crim1 expression at sites consistent with an interaction with bone morphogenetic proteins (BMPs). Here we show that CRIM1 can interact with both BMP4 and BMP7 via the CRR-containing portion of the protein and in so doing acts as an antagonist in three ways. CRIM1 binding of BMP4 and -7 occurs when these proteins are co-expressed within the Golgi compartment of the cell and leads to (i) a reduction in the production and processing of preprotein to mature BMP, (ii) tethering of pre-BMP to the cell surface, and (iii) an effective reduction in the secretion of mature BMP. Functional antagonism was verified by examining the effect of coexpression of CRIM1 and BMP4 on metanephric explant culture. The presence of CRIM1 reduced the effective BMP4 concentration of the media, thereby acting as a BMP4 antagonist. Hence, CRIM1 modulates BMP activity by affecting its processing and delivery to the cell surface

Nerve growth factor overexpression and autocrine loop in breast cancer cells

We show here that nerve growth factor (NGF), the canonical neurotrophic factor, is synthesized and released by breast cancer cells. High levels of NGF transcript and protein were detected in breast cancer cells by reverse transcription-PCR, Western blotting, ELISA assay and immunohistochemistry. Conversely, NGF production could not be detected in normal breast epithelial cells at either the transcriptional or protein level. Confocal analysis indicated the presence of NGF within classical secretion vesicles. Breast cancer cell-produced NGF was biologically active, as demonstrated by its ability to induce the neuronal differentiation of embryonic neural precursor cells. Importantly, the constitutive growth of breast cancer cells was strongly inhibited by either NGF-neutralizing antibodies or K-252a, a pharmacological inhibitor of NGF receptor TrkA, indicating the existence of an NGF autocrine loop. Together, our data demonstrate the physiological relevance of NGF in breast cancer and its potential interest as a marker and therapeutic target.

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, intergrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations. (C) 2003 Elsevier Science Ltd. All rights reserved.