Tom Humphreys

An analysis of the partial metabolic derepression of sea urchin eggs by ammonia: The existence of independent pathways☆

Incubation of unfertilized eggs in ammonia has been reported to initiate such late responses to fertilization as K+-conductance, DNA synthesis, chromosome condensation and cytoplasmic mRNA polyadenylation. It does not initiate such early responses as Na+-influx and the cortical reactions. We have further analyzed this metabolic derepression and find that ammonia activation does not result in the early respiratory burst and also does not initiate the late activation of Na+-dependent amino acid transport. Protein synthesis is increased, similar to that following normal fertilization. This indicates that augmentation of protein synthesis is causally linked neither to the earlier Na+-influx, cortical reactions, and respiratory burst nor to the later activation of amino acid transport. The temporal correlation between activation of transport and increased protein synthesis is therefore coincidental. The association between increased protein synthesis and establishment of K+-conductance was analyzed by abolishing K+-conductance through acidification of the sea water. This did not affect protein synthesis, indicating that K+-conductance and protein synthesis are also not causally linked. There is also no obligate link between protein synthesis and chromosome condensation. Incubation in low concentrations of ammonia results in increased protein synthesis but not chromosome condensation. Higher ammonia concentrations cause chromosome condensation but with no further increase in rate of protein synthesis. This suggests a concentration-dependent hierarchy of activation. These results are consistent with the concept that the late fertilization changes are not causally linked and proceed independently of each other. As we have not been able to disassociate the early changes, they may be obligately linked and dependent on each other.

Development of a recombinant tetravalent dengue virus vaccine: immunogenicity and efficacy studies in mice and monkeys.

Truncated recombinant dengue virus envelope protein subunits (80E) are efficiently expressed using the Drosophila Schneider-2 (S2) cell expression system. Binding of conformationally sensitive antibodies as well as X-ray crystal structural studies indicate that the recombinant 80E subunits are properly folded native-like proteins. Combining the 80E subunits from each of the four dengue serotypes with ISCOMATRIX adjuvant, an adjuvant selected from a set of adjuvants tested for maximal and long lasting immune responses, results in high titer virus neutralizing antibody responses. Immunization of mice with a mixture of all four 80E subunits and ISCOMATRIX adjuvant resulted in potent virus neutralizing antibody responses to each of the four serotypes. The responses to the components of the tetravalent mixture were equivalent to the responses to each of the subunits administered individually. In an effort to evaluate the potential protective efficacy of the Drosophila expressed 80E, the dengue serotype 2 (DEN2-80E) subunit was tested in both the mouse and monkey challenge models. In both models protection against viral challenge was achieved with low doses of antigen in the vaccine formulation. In non-human primates, low doses of the tetravalent formulation induced good virus neutralizing antibody titers to all four serotypes and protection against challenge with the two dengue virus serotypes tested. In contrast to previous reports, where subunit vaccine candidates have generally failed to induce potent, protective responses, native-like soluble 80E proteins expressed in the Drosophila S2 cells and administered with appropriate adjuvants are highly immunogenic and capable of eliciting protective responses in both mice and monkeys. These results support the development of a dengue virus tetravalent vaccine based on the four 80E subunits produced in the Drosophila S2 cell expression system.

Novel pattern of Brachyury gene expression in hemichordate embryos

Together with echinoderms and chordates, hemichordates constitute the third major group of the deuterostomes, which share a number of common developmental features. The Brachyury gene is responsible for the formation of notochord, the most defining feature of chordates. Therefore, isolation and characterization of the hemichordate homolog of Brachyury is key to understand the origin and evolution of chordates. Here we show that the hemichordate Brachyury gene (PfBra) is expressed in two regions of the gastrula and young tornaria larva, the archenteron invagination region and the stomodeum invagination region.

Regulation of DNA-like RNA and the apparent activation of ribosomal RNA synthesis in sea urchin embryos: Quantitative measurements of newly synthesized RNA

Summary RNA synthesis was analyzed in sea urchin embryos in order to determine the contribution which changes in synthesis of heterogeneous DNA-like RNA and of ribosomal RNA make to the changes in TNA metabolism which occur during embryogenesis. The base composition and sedimentation behavior of RNA molecules incorporating radioactivity during a short incubation of embryos with radioactive RNA precursors indicate that heterogeneous, DNA-like RNA was the predominant class of RNA synthesized in embryos at all stages of development. During longer incubations with radioactive precursors, radioactivity preferentially accumulated in ribosomal RNA. This preferential accumulation of newly synthesized rRNA was much more extensive at later than at early stages of development. However, accumulation of newly synthesized 28 S ribosomal RNA could be demonstrated as early as the first half of blastula stage when rRNA was extensively purified from DNA-like RNA by sucrose gradient sedimentation and MAK chromatography. In order to interpret these developmental changes in the relative accumulation of ribosomal and DNA-like RNA, the amounts of newly synthesized RNA accumulated during cleavage, blastula, and pluteus stages were quantitatively estimated by measurement of the incorporation of radioactive precursors into RNA and simultaneous determination of the specific activity of the nucleotide triphosphate precursor pools. The results indicated that the accumulation of newly synthesized DNA-like RNA per nucleus was quantitatively much greater at cleavage and blastula stages than at pluteus stage, whereas the accumulation of newly synthesized rRNA per nucleus at blastula and pluteus stages were similar. Therefore, decreases in the quantitative accumulation of DNA-like RNA account entirely for the change in the relative accumulation of ribosomal RNA and DNA-like RNA which occurs during development of sea urchin embryos.

Efficiency of translation of messenger-RNA before and after fertilization in sea urchins☆

Abstract The efficiency of translation, defined as the number of protein molecules produced per mRNA molecule per unit time, was compared before and after the acceleration of protein synthesis which occurs upon fertilization of sea urchin eggs. Examination of protein turnover, extent of protein synthesis on ribosomes, and the size distribution of newly synthesized protein before and after fertilization showed that this acceleration of protein synthesis involved the synthesis of 10 to 15 times more protein molecules on 10 to 15 times as many active ribosomes. To determine whether these increased numbers of active ribosomes were interacting more efficiently with an unchanged number of mRNA molecules or at the same efficiency with an increased number of mRNA molecules, the size of the polysomes and the time a ribosome stayed on the polysomes was compared before and after fertilization. The latter time interval was determined by measuring the average time a newly incorporated amino acid remained in the nascent chains and the size distribution of the nascent chains. New methods giving essentially 100% yield of active polysomes from sea urchin cells made these measurements possible. The results of the experiments show that efficiency of translation is similar in egg and embryos. Protein synthesis, thus, is accelerated at fertilization by the translation of additional mRNA molecules. This translation level control of protein synthesis therefore cannot be a general change in activity of some component in the cellular synthetic machinery such as ribosomes, but must specifically control the activity of a defined population of mRNA molecules.

Similarity of hnRNA sequences in blastula and pluteus stage sea urchin embryos

Abstract We have compared the total single-copy sequences transcribed as nuclear RNA in blastula and pluteus stage embryos of the sea urchin Tripneustes gratilla by hybridization of excess nuclear RNA with purified radioactive single-copy DNA. The kinetics of hybridization of either blastula or pluteus nuclear RNA with single-copy DNA show a single pseudo-first-order reaction with 34% of the single-copy genome. From the rate of the reaction and the purity of the nuclear RNA, it can be estimated that the reacting RNAs are present on the average at a concentration of one molecule per 14 nuclei. A mixture of blastula and pluteus RNA also hybridizes with 34% of the single-copy genome, indicating that the total complexity of RNAs transcribed at both stages is no greater than transcribed at each stage alone. The identity of the sequences transcribed by blastula and pluteus embryos was further examined by fractionation of the labeled DNA into sequences complementary and not complementary to pluteus RNA. This was achieved by hybridization of single-copy DNA to high pluteus RNA Cot, and separation of the hybridized and nonhybridized DNA on hydroxylapatite. Using either the DNA complementary or noncomplementary with pluteus RNA, essentially identical amounts of RNA:DNA hybrids are formed at high RNA Cot with blastula or pluteus RNA. Gross changes in the total RNA sequences transcribed do not appear to be involved in the developmental changes between blastula and pluteus, even though 45% of the mRNA sequences change between these two stages (Galau et al., 1976).

Hemichordate genomes and deuterostome origins

Acorn worms, also known as enteropneust (literally, ‘gut-breathing’) hemichordates, are marine invertebrates that share features with echinoderms and chordates. Together, these three phyla comprise the deuterostomes. Here we report the draft genome sequences of two acorn worms, Saccoglossus kowalevskii and Ptychodera flava. By comparing them with diverse bilaterian genomes, we identify shared traits that were probably inherited from the last common deuterostome ancestor, and then explore evolutionary trajectories leading from this ancestor to hemichordates, echinoderms and chordates. The hemichordate genomes exhibit extensive conserved synteny with amphioxus and other bilaterians, and deeply conserved non-coding sequences that are candidates for conserved gene-regulatory elements. Notably, hemichordates possess a deuterostome-specific genomic cluster of four ordered transcription factor genes, the expression of which is associated with the development of pharyngeal ‘gill’ slits, the foremost morphological innovation of early deuterostomes, and is probably central to their filter-feeding lifestyle. Comparative analysis reveals numerous deuterostome-specific gene novelties, including genes found in deuterostomes and marine microbes, but not other animals. The putative functions of these genes can be linked to physiological, metabolic and developmental specializations of the filter-feeding ancestor.

Molecular studies of hemichordate development : a key to understanding the evolution of bilateral animals and chordates

SUMMARY Using the Hawaiian acorn worm, Ptychodera flava, we began molecular studies on the development of hemichordates, a phylum previously unstudied at this level. Here we review results garnered from the examination of a few specific genes selected to help understand the evolution of vertebrate structures. These studies suggest new ideas about the evolution of developmental mechanisms in the deuterostomes. In a seminal observation, we noted an unexpected zone of expression of the Brachyury gene in the early anterior embryonic ectoderm where the mouth will form. Typically, the Brachyury gene is closely linked to development of the notochord and is expressed around the blastopore and in the posterior mesoderm in most animals. This first expression of Brachyury at the blastopore may represent a regulatory program associated with organizing the original animal head and gut opening, as suggested by the expression of Brachyury during hypostome formation in hydra. We believe that the anterior expression of Brachyury in deuterostomes represents the cooption of the program for organizing the original animal gut opening to form the deuterostome mouth. Recent data from the trochophore larva of a polychaete show that an anterior zone of expression of Brachyury is produced in this protostome by splitting of the Brachyury field during the formation of a gut with a mouth and anus by the lateral fusion of the sides of the blastopore. The ability to initiate independently a secondary regulatory program to organize the new mouth leading to an anterior field of Brachyury expression may be a signal event in the evolution of the deuterostomes. We also noted that the P. flava homolog of T‐brain/Eomes, a gene closely related by sequence and expression around the blastopore to Brachyury and associated with development of the vertebrate brain, also exhibits early posterior expression around the blastopore and a field of de novo anterior ectoderm expression during later embryogenesis. The tissue in the zone of de novo anterior ectoderm expression of Pf‐Tbrain produces the apical organ, a larval neural structure that has been touted as an evolutionary precursor of the chordate dorsal brain. The gene regulatory mechanisms responsible for initiating the anterior zone of de novo expression of T‐brain may represent a cooption to specify early neuroectoderm of the regulatory program evolved first to drive anterior Brachyury expression for deuterostome mouth formation. It will be interesting to examine the possibilities that an ability to initiate the de novo anterior expression of the program that includes T‐brain may be a key event in the evolution of the developmental mechanisms leading to the chordate dorsal nervous system.

T-Brain expression in the apical organ of hemichordate tornaria larvae suggests its evolutionary link to the vertebrate forebrain.

T-box genes encode a novel family of sequence-specific activators that appear to play crucial roles in various processes of animal development. Although most of the T-box genes are involved in the mesoderm formation of chordate embryos, mammalian T-Brain is expressed in the developing central nervous system, and defines molecularly distinct domains within the cerebral cortex. Here we report the first invertebrate T-Brain homologue from the hemichordate acorn worm, Ptychodera flava, which we designate Pf-Tbrain. Developmental expression of Pf-Tbrain was examined by whole mount in situ hybridization to various stages of P. flava embryos. A weak, broad in situ hybridization signal of the Pf-Tbrain transcript is first detected during gastrulation in cells around the archenteron, but this signal disappears as gastrulation proceeds. At mid-gastrula an intense signal appears in several apical ectoderm cells of the gastrula. This signal becomes restricted to the apical region, where the eyespots or the light-sensory organ of the tornaria larva form. Expression of Pf-Tbrain in the apical sensory organ of the tornaria and vertebrate T-Brain in the forebrain suggests an evolutionary relationship between the non-chordate deuterostome larval apical sensory organ and the chordate forebrain.

Developmental expression of the hemichordate otx ortholog.

The phylogenetic location of hemichordates is unique because they seem to fill an evolutionary gap between echinoderms and chordates. We report here characterization of Pf-otx, a hemichordate ortholog of otx, with its embryonic and larval expression pattern. Pf-otx is initially expressed in the vegetal plate of the blastula. Expression remains evident in the archenteron through gastrulation and then disappears. A new expression domain appears near the mouth along the preoral and postoral ciliated bands in the early tornaria larva.