Specification of endoderm in the sea urchin embryo.

Expression of the endoderm specific gene Endo16, was used to monitor endoderm specification in developmentally arrested and in dissociated embryos. beta-APN treatment halts gastrulation, however, in two species of sea urchins Endo16 mRNA is still expressed, suggesting that specification of the endodermal lineage has taken place in these developmentally arrested embryos. Endo16 mRNA is not expressed in embryos dissociated at the 4-8-cell stage unless they are reassociated shortly after the 16-cell stage. Interestingly, dissociation after the 16-cell stage also results in a lack of Endo16 expression. Lithium is unable to rescue Endo16 expression in these dissociated embryos. These results indicate that early signaling events mediated by cell-cell contact are required for the initial specification and maintenance of the endoderm in the developing embryo.

Histones in transit: cytosolic histone complexes and diacetylation of H4 during nucleosome assembly in human cells.

The organization and acetylation of nascent histones prior to their stable incorporation into chromatin were examined. Through sedimentation and immunoprecipitation analyses of HeLa cytosolic extracts, two somatic non-nucleosomal histone complexes were detected: one containing nascent H3 and H4, and a second containing H2A (and probably H2B) in association with the nonhistone protein NAP-1. The H3/H4 complex has a sedimentation coefficient of 5-6S, consistent with the presence of one or more escort proteins. H4 in the cytosolic H3/H4 complex is diacetylated, fully in accord with the acetylation state of newly synthesized H4 in chromatin. The diacetylation of nascent human H4 is therefore completed prior to nucleosome assembly. As part of our studies of the nascent H3/H4 complex, the cytoplasmic histone acetyltransferase most likely responsible for acetylating newly synthesized H4 was also investigated. HeLa histone acetyltransferase B (HAT B) acetylates H4 but not H3 in vitro, and maximally diacetylates H4 even in the presence of sodium butyrate. Human HAT B acetylates H4 exclusively on the lysine residues at positions 5 and 12, in complete agreement with the highly conserved acetylation pattern of nascent nucleosomal H4 (Sobel et al., 1995), and has a native molecular weight of approximately 100 kDa. Based on our findings a model is presented for the involvement of histone acetylation and NAP-1 in H2A/H2B deposition and exchange, during nucleosome assembly and chromatin remodeling in vivo.

Applied DC magnetic fields cause alterations in the time of cell divisions and developmental abnormalities in early sea urchin embryos.

Most work on magnetic field effects focuses on AC fields. The present study demonstrates that exposure to medium-strength (10 mT-0.1 T) static magnetic fields can alter the early embryonic development of two species of sea urchin embryos. Batches of fertilized eggs from two species of urchin were exposed to fields produced by permanent magnets. Samples of the continuous cultures were scored for the timing of the first two cell divisions, time of hatching, and incidence of exogastrulation. It was found that static fields delay the onset of mitosis in both species by an amount dependent on the exposure timing relative to fertilization. The exposure time that caused the maximum effect differed between the two species. Thirty millitesla fields, but not 15 mT fields, caused an eightfold increase in the incidence of exogastrulation in Lytechinus pictus, whereas neither of these fields produced exogastrulation in Strongylocentrotus purpuratus.

Alternative splicing of the Endo16 transcript produces differentially expressed mRNAs during sea urchin gastrulation.

The Endo16 gene codes for an RGD-containing calcium-binding protein that is found on the basal surfaces and in the extracellular matrix of cells of the invaginating archenteron during sea urchin gastrulation. Previously, we have shown that Endo16 is a single copy gene and we have determined the coding sequence and analyzed the temporal and spatial expression of a 6.6-kb mRNA. In this report we demonstrate that two additional longer Endo16 mRNAs are produced by differential splicing rather than alternative promoter usage. cDNA clones for two 8.5-kb mRNAs have been isolated and analyzed. The two 8.5-kb mRNAs are identical to each other in the coding region and differ only in their 3' UTRs. The extended open reading frame of the 8.5-kb mRNAs code for domains already identified in the 6.6-kb mRNA, including two different types of calcium-binding motifs and a region with a highly conserved cysteine pattern similar to that found in Ecm1 in the mouse. The 6.6- and 8.5-kb mRNAs show overlapping but distinct temporal as well as spatial expression patterns during gastrulation.

Applied AC and DC magnetic fields cause alterations in the mitotic cycle of early sea urchin embryos.

This study demonstrates that exposure to 60 Hz magnetic fields (3.4-8.8 mT) and magnetic fields over the range DC-600 kHz (2.5-6.5 mT) can alter the early embryonic development of sea urchin embryos by inducing alterations in the timing of the cell cycle. Batches of fertilized eggs were exposed to the fields produced by a coil system. Samples of the continuous cultures were taken and scored for cell division. The times of both the first and second cell divisions were advanced by ELF AC fields and by static fields. The magnitude of the 60 Hz effect appears proportional to the field strength over the range tested. The relationship to field frequency was nonlinear and complex. For certain frequencies above the ELF range, the exposure resulted in a delay of the onset of mitosis. The advance of mitosis was also dependent on the duration of exposure and on the timing of exposure relative to fertilization.

Familial recurrence of atypical symptoms in an extended pedigree with the syndrome of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS).

We report a clinically heterogeneous, multigenerational pedigree with the syndrome of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) associated with a mutation at nucleotide 3243 in the mitochondrial DNA tRNA(Leu)(UUR) gene. Our findings suggest that the mutation at nucleotide 3243 is not always associated with the classic MELAS phenotype and that other symptoms (notably cardiac and gastrointestinal abnormalities) should raise the suspicion of a mitochondrial disorder.

Endo16, a large multidomain protein found on the surface and ECM of endodermal cells during sea urchin gastrulation, binds calcium.

Endo16 encodes a developmentally regulated protein restricted to cells participating in the formation of the archenteron during sea urchin gastrulation and to the stomach of the pluteus. The 4680-nt coding region of the Endo16 gene has been sequenced from overlapping cDNAs. Sequence analysis revealed that Endo16 is a large multidomain protein starting with a putative signal sequence at its amino terminus which is followed by a cysteine-rich region, two potential heparin-binding regions, an acidic domain of 5 clustered repeats, an RGD cell binding motif, and a group of 12 additional acidic repeats. Immunolocalization by confocal and electron microscopy demonstrate that the Endo16 protein is in the extracellular matrix and associated with the surface of endodermal cells in the mid and hindgut of the archenteron. The two distinct acidic repeat regions are similar to known calcium-binding sequences. A recombinant Endo16 protein containing both putative calcium-binding repeat regions has been shown to bind radioactive calcium. Tryptic digests of gastrula stage protein extracts in the presence and the absence of calcium have established that calcium stabilizes Endo16 protein against proteolytic degradation.

Mitochondrial disorder associated with newborn cardiopulmonary arrest.

A female infant who died 2.5 d after birth with hypoglycemia, lactic acidosis, and sudden multisystem failure was studied. Biochemical studies showed complex III and IV deficiency in liver, kidney, and muscle, with muscle most severely affected. Southern blot analysis of the patient's mitochondrial DNA did not reveal any deletions. Denaturing gradient gel analysis, which detects single base changes by differences in melting behavior, showed an extra band that was not seen in mitochondrial DNA from the mother, the mother's identical twin sister, or an unrelated normal subject. This extra band indicated heteroplasmy for a restriction fragment containing the apocytochrome b and transfer RNA(thr) genes. Sequencing revealed an A to G mutation at nucleotide 15923, the last base of the anticodon loop of the transfer RNA(thr) gene. The mutation lengthens the anticodon stem by added pairing and reduces the anticodon loop size from 7 to 5 nucleotides, potentially compromising transfer RNA(thr) function in translation and/or in processing the polycistronic RNA transcript. The patient's mother previously had a male infant who also died at 1.5 d postnatal, and both the mother and her twin have had multiple miscarriages. Amniocentesis for a genetic screen was performed on the mother's twin sister during a recent pregnancy; some of the cultured cells were made available for this study. The mutation was not found in the amniocytes or in umbilical cord blood obtained at birth; the baby was normal at birth and remains healthy. It is concluded that the mutation at nucleotide 15923 was most likely the cause of the fatal disease in the index case.(ABSTRACT TRUNCATED AT 250 WORDS)

Histone H2A.F/Z mRNA is stored in the egg cytoplasm and basally regulated in the sea urchin embryo.

The sea urchin H2A.F/Z histone is a member of a subclass of highly conserved H2A variants. Sequence analysis confirms that H2A.F/Z mRNA is polyadenylated. In situ hybridization studies demonstrate that maternal H2A.F/Z message is stored in the egg cytoplasm and present at equal levels in all cells of the mesenchyme blastula-stage embryo, suggesting that H2A.F/Z is not coordinately regulated with DNA synthesis. When blastula-stage embryos were exposed to DNA synthesis inhibitors, no effect on the steady-state level of H2A.F/Z mRNA was observed, while the level of late class H2B mRNA decreased substantially. These results provide evidence that the basal mode of regulation of this unusual histone variant is conserved evolutionarily.

Altered cell fate in LiCl-treated sea urchin embryos.

Endo16 is a lineage-specific protein expressed during embryogenesis in the sea urchin, Strongylocentrotus purpuratus. We have examined the effects of various concentrations of lithium chloride, a well-known vegetalizing agent, on Endo16 expression in whole sea urchin embryos. Our results show that treatment with LiCl causes increased steady-state levels of Endo16 transcripts. An increase in the number of endodermal cells in treated embryos demonstrates a change in cell lineage. In addition, we observed a delay in development of lithium-treated embryos that is accompanied by a delay in the expression of a late class histone H2B gene.

Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA.

A denaturing gradient gel electrophoresis (DGGE) method is described that detects even single base pair changes in mitochondrial DNA (mtDNA). In this method, restriction fragments of mtDNA are electrophoresed in a urea/formamide gradient gel at 60 degrees C. Migration distance of each mtDNA fragment in the gel depends on melting behavior which reflects base composition. Fragments are located by Southern blotting with specific mtDNA probes. With just four carefully chosen restriction enzymes and as little as 50-100 ng of mtDNA, the method covers almost the entire human mitochondrial genome. To demonstrate the method, human mtDNA was analyzed. In six normal individuals, DGGE revealed melting behavior polymorphisms (MBPs) in mtDNA fragments that were not detected by restriction fragment length polymorphism (RFLP) analysis in agarose gels. Another individual, shown to have a melting behavior polymorphism in the cytochrome b coding region, was studied in detail. By mapping, the mutation was deduced to lie between nt 14905 and 15370. The affected fragment was amplified by PCR and sequenced. Specific base changes were identified in the region predicted by the gel result. This method will be especially useful as a diagnostic tool in mitochondrial disease for rapid localization of mtDNA mutations to specific regions of the genome, but DGGE also could complement RFLP analysis as a more sensitive method to follow maternal lineage in human and animal populations in a variety of research fields.

Photoperiod control of poplar bark storage protein accumulation.

Bark storage proteins (BSPs) accumulate in the inner bark parenchyma of many woody plants during autumn and winter. We investigated the effect of a short-day (SD) photoperiod on the accumulation of the 32-kilodalton bark storage protein of poplar (Populus deltoides Bart. ex Marsh.) under controlled environmental and natural growing conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein gel blot analysis revealed that 10 days of SD exposure (8 hours of light) resulted in a 20% increase in the relative abundance of the 32-kilodalton bark storage protein of poplar. After 17 days of SD exposure, the 32-kilodalton bark storage protein accounted for nearly one-half of the soluble bark proteins. In natural field conditions, accumulation of the 32-kilodalton bark storage protein was observed to start by August 18 (daylength 14.1 hours). Immunoprecipitation of in vitro translation products with anti-BSP serum revealed that the SD protein accumulation was correlated with changes in the pool of translatable mRNA. A survey of poplar clones from different geographic origins revealed the presence of the 32-kilodalton BSP in the dormant bark of all the clones tested. These results demonstrate that a SD photoperiod induces, whether directly or indirectly, rapid changes in woody plant gene expression, leading to the accumulation of BSP.

Mitochondrial tRNA(thr) mutation in fatal infantile respiratory enzyme deficiency.

The mitochondrial DNA (mtDNA) of two unrelated infants with lethal respiratory chain defects was studied using denaturing gradient gel analysis. This analysis revealed melting behavior differences suggesting a point mutation(s) in a restriction fragment containing the apocytochrome b and tRNA(thr) genes. Sequencing revealed that patient 1 had an A to G mutation at nt 15924 which is the last base pair of the anticodon stem adjacent to the anticodon loop of tRNA(thr). Patient 2 had an A to G mutation at nt 15923 which is the last base of the anticodon loop. The results suggest that mtDNA mutations affecting the anticodon loop structure of tRNA(thr) cause mitochondrial disease that is fatal in infancy.

Axillary shoot proliferation and growth of Populus deltoides shoot cultures.

Shoot cultures of four genotypes of Populus deltoides Bartr. ex Marsh. were established from adventitious shoots regenerated from internodal stem explants. Stable shoot cultures for all four genotypes were maintained in a continuous culture regime for over one year. The stable shoot cultures were used as explants to investigate the effects of zeatin concentration and genotype on axillary shoot production and growth. The concentration of zeatin significantly affected the production of axillary shoots, with 1.0 mgL(-1) zeatin producing the greatest number of shoots (31.0 shoots per culture vessel) while 0.25 mgL(-1) zeatin produced the greatest growth (5.9 mg per axillary shoot) when measured by dry weight accumulation per shoot. Genotypic differences were significant in the production and growth of axillary shoots.

Endo16, a lineage-specific protein of the sea urchin embryo, is first expressed just prior to gastrulation.

We have isolated and characterized a new endoderm-specific gene, designated Endo16, from a sea urchin gastrula stage cDNA library. Northern blot analysis and in situ hybridization experiments indicate that this gene is first expressed in the vegetal plate, a group of endodermal and mesenchymal precursor cells that are poised to invaginate in the first movement of gastrulation. Expression becomes progressively restricted to a subset of endodermal cells as development proceeds. To study the Endo16 gene product, a polyclonal antiserum was raised against bacterially expressed Endo16 protein. Indirect immunofluorescence experiments in midgastrula stage embryos reveal that the Endo16 protein is localized to the surface of endoderm and secondary mesenchyme cells. In Western blot experiments, the antiserum detects a small set of high molecular weight proteins ranging from 180 to greater than 300 kDa. Analysis of the nucleotide-derived amino acid sequence from a partial Endo16 cDNA clone reveals a highly repetitive, extremely acidic protein segment that includes the Arg-Gly-Asp (RGD) tripeptide known to be important in cell binding domains of a number of extracellular proteins. Taken together, these data suggest that the Endo16 protein may be an adhesion molecule involved in gastrulation of the sea urchin embryo.

Sea urchin oocytes possess elaborate cortical arrays of microfilaments, microtubules, and intermediate filaments.

Extensive arrays of microfilaments, microtubules and cytokeratin-type intermediate filaments were detected in the cortex of Strongylocentrotus droebachiensis oocytes using fluorescently labeled antibodies on both cortex and whole mount preparations. All three filament systems undergo dramatic structural reorganization during meiotic maturation of the egg. Microfilaments form a dense meshwork within the cortex of the oocyte. After meiosis, the filaments rearrange and shorten, resulting in a more loosely organized network. Both cortical microtubules and microtubules associated with a microtubule-organizing center are observed within the oocyte. After meiosis, the number and length of the cortical microtubules gradually diminish. A microtubule organizing center is found situated between the germinal vesicle and the plasma membrane in many oocytes. A network of filaments extends from the microtubule organizing center and radiates peripherally toward the germinal vesicle, presumably marking the animal pole. Cytokeratin-like intermediate filaments form a reticular network within the oocyte cortex, then solubilize during meiosis. In whole mounts of oocytes there is a single focal center of cytokeratin staining from which filaments radiate. Indirect immunofluorescence experiments, using anti-tubulin and anti-cytokeratin antibodies simultaneously, reveal the intermediate filament focal center to be localized within the microtubule organizing center. These results demonstrate the presence of a complex cortical cytoskeleton in premeiotic eggs of the sea urchin, Strongylocentrotus droebachiensis.

In vitro shoot regeneration of Populus deltoides: effect of cytokinin and genotype.

Treatment differences were observed in the in vitro adventitious shoot regeneration response from internodal explants of three genotypes of Populus deltoides cultured on media supplemented with five concentrations each of the cytokinins 6-benzyladenine, 2-isopentyladenine, and zeatin. For each of the three genotypes, the greatest number of shoots were consistently regenerated on media containing the cytokinin zeatin. Tissue necrosis resulted when explants from any of the three genotypes were cultured on media supplemented with 6-benzyladenine. A zeatin concentration by genotype interaction was also observed. Genotypic differences in shoot regeneration were observed for 16 genotypes of Populus deltoides when cultured on medium supplemented with 0.5 mgL(-1) zeatin. Six genotypes were highly recalcitrant and failed to regenerate shoots. The percent of explants regenerating was greater than 50% for four genotypes.

Characterization of a cDNA clone coding for a sea urchin histone H2A variant related to the H2A.F/Z histone protein in vertebrates.

A cDNA clone coding for a sea urchin histone H2A variant has been isolated. The coding region of the clone has been sequenced and the sequence found to be closely related to the H2A.F sequence in chickens. The nucleotide sequence of the sea urchin H2A.F/Z is 74% conserved when compared to chicken H2A.F and 51% conserved compared to sea urchin H2A early and 60% compared to sea urchin H2A late. The nucleotide-derived amino acid comparisons show that H2A.F/Z is 97% homologous with H2A.F in chickens and 57% and 56% homologous when compared to sea urchin H2A early and late respectively. There are between 3-6 copies of the H2A.F/Z sequence in the S. purpuratus genome. The H2A.F/Z gene sequence codes for the previously identified H2A.Z protein. All embryonic stages and adult tissues tested contain mRNA for H2A.F/Z. The mRNA appears in the poly A+ RNA fraction after chromatography over oligo dT cellulose.

Developmental time, cell lineage, and environment regulate the newly synthesized proteins in sea urchin embryos.

Strongylocentrotus purpuratus embryos were fractionated into two cell populations of defined lineages at times corresponding to two critical developmental events: determination (16-cell stage) and early differentiation (mesenchyme blastula). The 16-cell stage blastomeres, labeled with [35S]methionine, exhibited identical protein synthesis patterns by fluorography, and this pattern was not significantly altered by cell separation. In comparing the proteins of the mesenchyme blastula to the 16-cell stage, differences (increases and decreases) were seen by fluorography of newly synthesized proteins. The synthesis of 2.9% of the mesenchyme blastula proteins is specific to or enriched in primary mesenchyme cells and 8.2% is specific to or enriched in endoderm/ectoderm cells. Additionally, in contrast to the earlier stage, the pattern of protein synthesis in the mesenchyme blastula embryos is substantially altered by cell separation. The ability to alter protein synthesis in response to environmental factors may be a further demonstration of the differentiation of these cells.