Biomedical subjects

R A Cameron

Publications and source records attributed to R A Cameron.

At least 19 recordsLinked to original sources

PubMed2004

Metagenomics, gene discovery and the ideal biocatalyst.

With the rapid development of powerful protein evolution and enzyme-screening technologies, there is a growing belief that optimum conditions for biotransformation processes can be established without the constraints of the properties of the biocatalyst. These technologies can then be applied to find the "ideal biocatalyst" for the process. In identifying the ideal biocatalyst, the processes of gene discovery and enzyme evolution play major roles. However, in order to expand the pool genes for in vitro evolution, new technologies, which circumvent the limitations of microbial culturability, must be applied. These technologies, which currently include metagenomic library screening, gene-specific amplification methods and even full metagenomic sequencing, provide access to a volume of "sequence space" that is not addressed by traditional screening.

Bacterial Proteins↗

PubMed2001

A large-scale analysis of mRNAs expressed by primary mesenchyme cells of the sea urchin embryo.

The primary mesenchyme cells (PMCs) of the sea urchin embryo have been an important model system for the analysis of cell behavior during gastrulation. To gain an improved understanding of the molecular basis of PMC behavior, a set of 8293 expressed sequenced tags (ESTs) was derived from an enriched population of mid-gastrula stage PMCs. These ESTs represented approximately 1200 distinct proteins, or about 15% of the mRNAs expressed by the gastrula stage embryo. 655 proteins were similar (P<10(-7) by BLAST comparisons) to other proteins in GenBank, for which some information is available concerning expression and/or function. Another 116 were similar to ESTs identified in other organisms, but not further characterized. We conservatively estimate that sequences encoding at least 435 additional proteins were included in the pool of ESTs that did not yield matches by BLAST analysis. The collection of newly identified proteins includes many candidate regulators of primary mesenchyme morphogenesis, including PMC-specific extracellular matrix proteins, cell surface proteins, spicule matrix proteins and transcription factors. This work provides a basis for linking specific molecular changes to specific cell behaviors during gastrulation. Our analysis has also led to the cloning of several key components of signaling pathways that play crucial roles in early sea urchin development.

Animals↗

PubMed2000

A sea urchin genome project: sequence scan, virtual map, and additional resources.

Results of a first-stage Sea Urchin Genome Project are summarized here. The species chosen was Strongylocentrotus purpuratus, a research model of major importance in developmental and molecular biology. A virtual map of the genome was constructed by sequencing the ends of 76,020 bacterial artificial chromosome (BAC) recombinants (average length, 125 kb). The BAC-end sequence tag connectors (STCs) occur an average of 10 kb apart, and, together with restriction digest patterns recorded for the same BAC clones, they provide immediate access to contigs of several hundred kilobases surrounding any gene of interest. The STCs survey >5% of the genome and provide the estimate that this genome contains approximately 27,350 protein-coding genes. The frequency distribution and canonical sequences of all middle and highly repetitive sequence families in the genome were obtained from the STCs as well. The 500-kb Hox gene complex of this species is being sequenced in its entirety. In addition, arrayed cDNA libraries of >10(5) clones each were constructed from every major stage of embryogenesis, several individual cell types, and adult tissues and are available to the community. The accumulated STC data and an expanding expressed sequence tag database (at present including >12, 000 sequences) have been reported to GenBank and are accessible on public web sites.

Aging↗

PubMed2000

Quantitative assessment of Hox complex expression in the indirect development of the polychaete annelid Chaetopterus sp.

A prediction from the set-aside theory of bilaterian origins is that pattern formation processes such as those controlled by the Hox cluster genes are required specifically for adult body plan formation. This prediction can be tested in animals that use maximal indirect development, in which the embryonic formation of the larva and the postembryonic formation of the adult body plan are temporally and spatially distinct. To this end, we quantitatively measured the amount of transcripts for five Hox genes in embryos of a lophotrochozoan, the polychaete annelid Chaetopterus sp. The polychaete Hox complex is shown not to be expressed during embryogenesis, but transcripts of all measured Hox complex genes are detected at significant levels during the initial stages of adult body plan formation. Temporal colinearity in the sequence of their activation is observed, so that activation follows the 3'-5' arrangement of the genes. Moreover, Hox gene expression is spatially localized to the region of teloblastic set-aside cells of the later-stage embryos. This study shows that an indirectly developing lophotrochozoan shares with an indirectly developing deuterostome, the sea urchin, a common mode of Hox complex utilization: construction of the larva, whether a trochophore or dipleurula, does not involve Hox cluster expression, but in both forms the complex is expressed in the set-aside cells from which the adult body plan derives.

Animals↗

PubMed2000

Bilaterian origins: significance of new experimental observations.

Several recent laboratory observations that bear on the origin of the Bilateria are reviewed and interpreted in light of our set-aside cell theory for bilaterian origins. We first discuss new data concerning the phylogeny of bilaterian phyla. Next, we use systematic, molecular, and paleontological lines of evidence to argue that the latest common ancestor of echinoderms plus hemichordates used a maximal indirect mode of development. Furthermore, the latest common ancestor of molluscs and annelids was also indirectly developing. Finally, we discuss new data on Hox gene expression patterns which suggest that both sea urchins and polychaete annelids use Hox genes in a very similar fashion. Neither utilizes the complete Hox complex in the development of the larva per se, while the Hox complex is expressed in the set-aside cells from which the adult body plan is formed. Our current views on the ancestry of the bilaterians are summarized in phylogenetic terms, incorporating the characters discussed in this paper.

Animals↗

PubMed1999

Microsatellite loci in wild-type and inbred Strongylocentrotus purpuratus.

Strongylocentrotus purpuratus, a major research model in developmental molecular biology, has been inbred through six generations of sibling matings. Though viability initially decreased, as described earlier, the inbred line now consists of healthy, fertile animals. These are intended to serve as a genomic resource in which the level of polymorphism is decreased with respect to wild S. purpuratus. To genotype the inbred animals eight simple sequence genomic repeats were isolated, in context, and PCR primers were generated against the flanking single-copy sequences. Distribution and polymorphism of these regions of the genome were studied in the genomes of 27 wild individuals and in a sample of the inbred animals at F2 and F3 generations. All eight regions were polymorphic, though to different extents, and their homozygosity was increased by inbreeding as expected. The eight markers suffice to identify unambiguously the cellular DNA of any wild or F3 S. purpuratus individual.

Animals↗

PubMed1999

Expression pattern of Brachyury and Not in the sea urchin: comparative implications for the origins of mesoderm in the basal deuterostomes.

This work concerns the expression of two transcription factors during the development of the sea urchin Strongylocentrotus purpuratus: SpNot, the orthologue of the vertebrate Not gene, and SpBra, the orthologue of the vertebrate Brachyury gene. SpNot transcripts are detected by in situ hybridization in the vegetal plate at the mesenchyme-blastula stage. Later the gene is expressed in the secondary mesenchyme, but expression is no longer detectable after gastrulation. SpNot is upregulated during larval development, in the invaginating vestibule of the adult rudiment. Transcripts are also found in several larva-specific tissues, including the epaulets, blastocoelar cells, and pigment cells. SpBra also displays a discontinuous pattern of expression. Much like SpNot, this gene is expressed during embryogenesis in the embryonic vegetal plate and secondary mesenchyme founder cells, and expression is then extinguished. The gene is upregulated over a week later in the feeding larva, in the vestibule of the adult rudiment. In contrast to SpNot, SpBra is also expressed in the mesoderm of both left and right hydrocoels, and it is not expressed in any larva-specific tissues. We compare the spatial expression profile determined in this study with that of the orthologous Brachyury gene in an indirectly developing enteropneust hemichordate, a representative of the sister group to the echinoderms within the deuterostomes. These observations illuminate the genetic basis underlying the process of maximal indirect development in basal deuterostomes. Finally, Brachyury appears to be an excellent marker for the progeny of the set-aside cells of the sea urchin embryo.

Amino Acid Sequence↗

PubMed1999

A comparative molecular approach to mesodermal patterning in basal deuterostomes: the expression pattern of Brachyury in the enteropneust hemichordate Ptychodera flava.

This work concerns the formation of mesoderm in the development of an enteropneust hemichordate, Ptychodera flava, and the expression of the Brachyury gene during this process. Brachyury expression occurs in two distinct phases. In the embryo, Brachyury is transcribed during gastrulation in the future oral and anal regions of the gut, but transcripts are no longer detected by 2 weeks of development. Brachyury expression is not detected during the 5 months of larval planktonic existence. During this time, the adult coeloms begin to develop, originating as coalescences of cells that appear to delaminate from the wall of the gut. Brachyury expression cannot be detected again until metamorphosis, when transcripts appear in the mesoderm of the adult proboscis, collar and the very posterior region of the trunk. It is also expressed in the posterior end of the gut. At no time is Brachyury expressed in the stomochord, the putative homologue of the chordate notochord. These observations illuminate the process of maximal indirect development in Ptychodera and, by comparison with patterns of Brachyury expression in the indirect development of echinoderms, their sister group, they reveal the evolutionary history of Brachyury utilization in deuterostomes.

Animals↗

PubMed1999

EST analysis of gene expression in early cleavage-stage sea urchin embryos.

A set of 956 expressed sequence tags derived from 7-hour (mid-cleavage) sea urchin embryos was analyzed to assess biosynthetic functions and to illuminate the structure of the message population at this stage. About a quarter of the expressed sequence tags represented repetitive sequence transcripts typical of early embryos, or ribosomal and mitochondrial RNAs, while a majority of the remainder contained significant open reading frames. A total of 232 sequences, including 153 different proteins, produced significant matches when compared against GenBank. The majority of these identified sequences represented 'housekeeping' proteins, i.e., cytoskeletal proteins, metabolic enzymes, transporters and proteins involved in cell division. The most interesting finds were components of signaling systems and transcription factors not previously reported in early sea urchin embryos, including components of Notch and TGF signal transduction pathways. As expected from earlier kinetic analyses of the embryo mRNA populations, no very prevalent protein-coding species were encountered; the most highly represented such sequences were cDNAs encoding cyclins A and B. The frequency of occurrence of all sequences within the database was used to construct a sequence prevalence distribution. The result, confirming earlier mRNA population analyses, indicated that the poly(A) RNA of the early embryo consists mainly of a very complex set of low-copy-number transcripts.

Animals↗

PubMed1998

Expression of the Hox gene complex in the indirect development of a sea urchin.

Hox complex genes control spatial patterning mechanisms in the development of arthropod and vertebrate body plans. Hox genes are all expressed during embryogenesis in these groups, which are all directly developing organisms in that embryogenesis leads at once to formation of major elements of the respective adult body plans. In the maximally indirect development of a large variety of invertebrates, the process of embryogenesis leads only to a free-living, bilaterally organized feeding larva. Maximal indirect development is exemplified in sea urchins. The 5-fold radially symmetric adult body plan of the sea urchin is generated long after embryogenesis is complete, by a separate process occurring within imaginal tissues set aside in the larva. The single Hox gene complex of Strongylocentrotus purpuratus contains 10 genes, and expression of eight of these genes was measured by quantitative methods during both embryonic and larval developmental stages and also in adult tissues. Only two of these genes are used significantly during the entire process of embryogenesis per se, although all are copiously expressed during the stages when the adult body plan is forming in the imaginal rudiment. They are also all expressed in various combinations in adult tissues. Thus, development of a microscopic, free-living organism of bilaterian grade, the larva, does not appear to require expression of the Hox gene cluster as such, whereas development of the adult body plan does. These observations reflect on mechanisms by which bilaterian metazoans might have arisen in Precambrian evolution.

Animals↗

PubMed1998

Computer tomography determined intracranial volume of infants with deformational plagiocephaly: a useful "normal"?

Over the last 10 years, children with deformational (nonsynostotic) plagiocephaly have undergone computed tomography scans using the same protocols as children with lambdoid craniosynostosis because of their similarly distorted head shapes. These children are believed to have normal intracranial volume. Given the recent questioning of what is the normal range of intracranial volume in human populations, the authors have undertaken a comparison of the intracranial volume of children with deformational plagiocephaly and Lichtenberg's normal population (Lichtenberg R. Radiographie du crane de 226 enfants normaux de la naissance a 8 ans: Impressions digitiformes, capacite, angles et indices [thesis]. Paris: University of Paris, 1960). The intracranial volume was determined for 20 females and 46 males with deformational plagiocepahly ranging in age from 2.5 to 20.7 months using computed tomography scan data. Although no significant differences were found for the females, the authors found that the intracranial volume of the males with deformational plagiocephaly were significantly larger than Lichtenberg's population. The authors conclude that this is because Lichtenberg's data do not adequately reflect the normal range of intracranial volume for males ranging in age from 2.5 to 20.7 months, rather than gender differences associated with deformational plagiocephaly. Further, until a more suitable normal becomes available, the deformational (nonsynostotic) plagiocephaly data could be used as substitute normal reference material in the measured age range for assessment of the intracranial volumes of children with craniosynostosis.

Analysis of Variance↗

PubMed1998

Specification of cell fate in the sea urchin embryo: summary and some proposed mechanisms.

An early set of blastomere specifications occurs during cleavage in the sea urchin embryo, the result of both conditional and autonomous processes, as proposed in the model for this embryo set forth in 1989. Recent experimental results have greatly illuminated the mechanisms of specification in some early embryonic territories, though others remain obscure. We review the progressive process of specification within given lineage elements, and with reference to the early axial organization of the embryo. Evidence for the conditional specification of the veg2 lineage subelement of the endoderm and other potential interblastomere signaling interactions in the cleavage-stage embryo are summarized. Definitive boundaries between mesoderm and endoderm territories of the vegetal plate, and between endoderm and overlying ectoderm, are not established until later in development. These processes have been clarified by numerous observations on spatial expression of various genes, and by elegant lineage labeling studies. The early specification events depend on regional mobilization of maternal regulatory factors resulting at once in the zygotic expression of genes encoding transcription factors, as well as downstream genes encoding proteins characteristic of the cell types that will much later arise from the progeny of the specified blastomeres. This embryo displays a maximal form of indirect development. The gene regulatory network underlying the embryonic development reflects the relative simplicity of the completed larva and of the processes required for its formation. The requirements for postembryonic adult body plan formation in the larval rudiment include engagement of a new level of genetic regulatory apparatus, exemplified by the Hox gene complex.

Animals↗

PubMed1997

LiCl perturbs ectodermal veg1 lineage allocations in Strongylocentrotus purpuratus embryos.

In normal development the veg1 tier of the sixth cleavage Strongylocentrotus purpuratus embryo contributes progeny to both ectodermal lineages and portions of the archenteron. Treatment with 18 mM LiCl specifically affects this lineage allocation, reducing or eliminating the veg1 contribution to ectoderm. Less frequently this concentration of lithium causes the progeny of animal blastomeres to contribute to the archenteron.

Animals↗

PubMed1997

Set-aside cells in maximal indirect development: evolutionary and developmental significance.

In the maximal form of indirect development found in many taxa of marine invertebrates, embryonic cell lineages of fixed fate and limited division capacity give rise to the larval structures. The adult arises from set-aside cells in the larva that are held out from the early embryonic specification processes, and that retain extensive proliferative capacity. We review the locations and fates of set-aside cells in two protostomes, a lophophorate and a deuterostome. The distinct adult body plans of many phyla develop from homologous set-aside cells within homologous larvae. We argue that the stocks from which these phyla arose utilized these respective larvae, and the diversity of their adult body plans reflects diverse pattern formation processes executed in their set-aside cell populations. Chordates and arthropods develop directly, but share adult characters with indirectly developing phyla. Thus the deuterostome and protostome stocks that were ancestral to chordates and arthropods, respectively, also utilized maximal indirect development.

Animals↗

PubMed1997

Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae.

The use of Green Fluorescent Protein (GFP) as a reporter for expression transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development. A GFP coding sequence was associated with three different previously studied cis-regulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic and secondary mesenchyme, and the Endo16 gene, expressed in vegetal plate, archenteron and midgut. We demonstrate that the sensitivity with which expression can be detected is equal to or greater than that of whole-mount in situ hybridization applied to detection of CAT mRNA synthesized under the control of the same cis-regulatory systems. However, in addition to the important feature that it can be visualized nondestructively in living embryos, GFP has other advantages. First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connections between cells, which in sea urchin embryos is particularly useful for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme. Second, GFP expression can be dramatically visualized in postembryonic larval tissues. This brings postembryonic larval developmental processes for the first time within the easy range of gene transfer analyses. Third, GFP permits identification and segregation of embryos in which the clonal incorporation of injected DNA has occurred in any particular desired region of the embryo. Thus, we show explicitly that, as expected, GFP transgenes are incorporated in the same nuclei together with other transgenes with which they are co-injected.

Animals↗

PubMed1996

Specific in vitro interaction between recombinant Strongylocentrotus purpuratus bindin and a recombinant 45A fragment of the putative bindin receptor.

The interaction between recombinant Strongylocentrotus purpuratus sperm bindin and a recombinant fragment of the putative egg bindin receptor of the same species was measured in vitro. In solution these molecules interact with simple bimolecular kinetics, displaying an equilibrium dissociation constant of about 0.1 microM. Thus, as implied by many observations in vivo and in vitro, bindin and the putative egg receptor display a specific affinity for one another.

Animals↗

PubMed1996

Twins raised from separated blastomeres develop into sexually mature Strongylocentrotus purpuratus.

The first two blastomeres of Strongylocentrotus purpuratus embryos were separated and the resulting twins raised in pairs through larval life and to sexual maturity. We derive two conclusions from this study: changes in the pattern of specification of embryonic cell fates following blastomere separation result in the creation of two sets of fully functional coelomic pouches and imaginal rudiments, and sex determination in sea urchins is chromosomal, since the pairs of twins were always of the same sex.

Animals↗

PubMed1996

Postembryonic segregation of the germ line in sea urchins in relation to indirect development.

The four small micromeres of the sea urchin embryo contribute only to the coelomic sacs, which produce major components of the adult body plan during postembryonic development. To test the proposition that the small micromeres are the definitive primordial germ cell lineage of the sea urchin, we deleted their 4th cleavage parents, and raised the deleted embryos through larval life and metamorphosis to sexual maturity. Almost all of the experimental animals produced functional gametes, excluding the possibility that the germ cell lineage arises exclusively and obligatorily from descendants of the small micromeres; rather, the germ cell lineage arises during the postembryonic development of the rudiment. A survey of the literature indicates that there is no known case of an embryonic primordial germ cell lineage in a bilaterian species that displays maximal indirect development.

Animals↗