Non-Fickian diffusion and the accumulation of methane bubbles in deep-water sediments.

In the absence of fractures, methane bubbles in deep-water sediments can be immovably trapped within a porous matrix by surface tension. The dominant mechanism of transfer of gas mass therefore becomes the diffusion of gas molecules through porewater. The accurate description of this process requires non-Fickian diffusion to be accounted for, including both thermal diffusion and gravitational action. We evaluate the diffusive flux of aqueous methane considering non-Fickian diffusion and predict the existence of extensive bubble mass accumulation zones within deep-water sediments. The limitation on the hydrate deposit capacity is revealed; too weak deposits cannot reach the base of the hydrate stability zone and form any bubbly horizon.

Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.

Phosphorylation of Enabled by the Drosophila Abelson tyrosine kinase regulates the in vivo function and protein-protein interactions of Enabled.

Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila ena were discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins.

Creation of genome-wide protein expression libraries using random activation of gene expression.

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.

Primary care for the hearing impaired: a changing picture.

Fortunately, technologic improvements such as these have vastly increased acceptability: miniaturization, cosmetic improvements, and increased amplification ability for modest hearing losses earlier thought unaidable. The cochlear implant, approved by the FDA in 1984, is a major breakthrough for adults with profound hearing losses who are unable to be helped by conventional hearing aids.

A method for mapping intranuclear protein-DNA interactions and its application to a nuclease hypersensitive site.

We have devised a method for mapping sites on DNA within the nucleus that are protected against nuclease attack by interaction with bound protein or other factors. This "footprinting" method uses an end-labeled sequence-specific DNA probe, which is annealed to the DNA from nuclear digests under carefully controlled conditions. The annealed complexes are treated with single-strand-specific nuclease, the resulting duplex molecules are electrophoresed on gels, and the gels are autoradiographed. The high sensitivity and resolution of the method have made it possible to obtain a detailed map of DNase I cutting patterns in the 5' flanking sequence of the chicken adult beta (beta A)-globin gene within nuclei from various tissues. In nuclei from adult erythrocytes, this domain is hypersensitive to nucleases. However, we detect within the domain two well-defined regions that are protected against attack, indicating the presence of one or more bound factors. Nuclei from oviduct or 5-day-old embryonic erythrocytes, in which the domain is not hypersensitive, show limited and different patterns of protection.

Embryonic expression patterns of the Drosophila decapentaplegic gene: separate regulatory elements control blastoderm expression and lateral ectodermal expression.

Patterns of decapentaplegic (dpp) transcripts derived from the intact gene were compared to the patterns of transcripts generated by partial dpp transgenes in Drosophila embryos. Sequences closest to the dpp coding regions, the dpp hin region, were sufficient to express lacZ-tagged mRNA in patterns indistinguishable from the patterns of endogenous dpp expression in the dorsal and terminal cells at the blastoderm stage, in the dorsal ectoderm during germ band elongation, and in narrow stripes of ectodermal cells along the dorsal edge of the ectoderm and at the boundary between the lateral and ventral neurogenic regions during germ band shortening. The latter pattern of expression responded to the segment polarity genes naked and wingless. However, these dpp sequences were not sufficient to drive lacZ-tagged mRNA expression in other cells normally expressing dpp, including cells in the gnathal segments, the clypeolabrum, the foregut, the midgut visceral mesoderm, and the hindgut. Two separate regulatory regions were found in the dpp hin region. A 479 bp region upstream of the promoter was necessary for the segmented pattern of expression in the lateral ectoderm and for expression in the midgut endoderm. Cis-acting elements in the 2 kbp second intron directed expression in the dorsal and terminal regions of the blastoderm, acted on a heterologous promoter, the P-element promoter, and responded to pattern information derived from the maternal effect dorsal/ventral patterning genes.

Sequence and function of the aas gene in Escherichia coli.

2-Acylglycerophosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthase activities are encoded by the aas gene in Escherichia coli. The aas gene was cloned, and the DNA sequence of the aas gene and the region between aas and galR established the clockwise gene order in the 61.2 min of the E. coli chromosome as aas-orf-galR-lysA-lysR-orf-araE. The aas gene consists of a single open reading frame of 2,157 base pairs predicted to encode a protein of 80.6 kDa. Strains harboring multiple copies of the aas gene overproduced both 2-acyl-GPE acyltransferase and acyl-ACP synthetase activities in vitro and had higher specific activities for the incorporation of exogenous fatty acids and lysophospholipids into the membrane in vivo. Specific expression of the aas gene yielded a protein with an apparent molecular mass of 81 kDa. Comparison of the predicted amino acid sequence of the aas gene with mammalian, yeast, and bacterial long chain acyl-coenzyme A synthetases revealed three domains of high similarity which are postulated to form the acyl-AMP binding pocket. These data verify that 2-acyl-GPE acyltransferase and acyl-ACP synthetase are reactions carried out by the same gene product, verify the role of 2-acyl-GPE acyltransferase/acyl-ACP synthetase in the acylation of endogenous 2-acyl-GPE, and establish the product of the aas gene as the rate-limiting enzyme in the uptake and incorporation of exogenous 2-acyllysophospholipids.

Analysis of the tissue-specific enhancer at the 3' end of the chicken adult beta-globin gene.

In an earlier paper we identified a tissue-specific enhancer in the 3' flanking region of the chicken adult beta-globin gene. In this paper we analyze the properties of this enhancer. Deletion analysis and transient expression assays show that the domain responsible for activation of transcription is at most 136 base pairs long. Specific factors that bind to discrete sequences within the enhancer DNA are found in extracts of embryonic and adult erythrocytes and in brain. These factors are specific for the tissue or the erythrocyte developmental stage and protect at least five discrete regions in or near the enhancer against DNase I digestion in "footprinting" experiments. Four of these regions reside wholly within the 136-base-pair functional enhancer domain, which also comprises a site in chromatin that is hypersensitive to nucleases. The nature of the binding sites and the program of appearance of the factors during development suggest that a subset of these interactions may be responsible for the developmental specificity of the enhancer.

Developmental modulation of protein binding to beta-globin gene regulatory sites within chicken erythrocyte nuclei.

We describe the interaction of two adjacent binding sites in the chicken beta-globin gene promoter with regulatory factors present in erythroid cells. One of these sites is a palindromic sequence (Pal) that binds a member of the nuclear factor 1 family; the other is the CACCC sequence found in most adult beta-globin promoters. Transfection of primary erythrocytes with plasmids carrying the gene coupled to truncated promoters reveals that the Pal site inhibits and the CACCC site stimulates expression. Nuclease protection experiments on intact nuclei show that at early stages of embryonic development, the CACCC site is occupied and the Pal site is vacant, but as development progresses, the Pal site is filled gradually and the CACCC site loses its bound protein. Beyond day 15 of development, Pal is completely occupied and CACCC is empty in vivo. Parallel DNase I footprinting and gel retardation studies in vitro show that nuclear extracts contain sharply increasing Pal-binding activity as development proceeds, but CACCC-binding activity falls off only slightly. We show that the two factors bind to their sites in vitro in an anticooperative manner and conclude that this could account for the observed changes in site occupancy in vivo. Our results suggest that the Pal factor may play a role in the shutdown of adult beta-globin expression late in erythroid development.

The role of CDP-diacylglycerol synthetase and phosphatidylinositol synthase activity levels in the regulation of cellular phosphatidylinositol content.

The regulation of phosphatidylinositol synthesis was examined by cloning and expressing in COS-7 cells the human cDNAs encoding the two enzymes in the biosynthetic pathway. Human CDP-diacylglycerol synthetase (cds1) and phosphatidylinositol synthase (pis1) clones were identified in the human expressed sequence-tagged (EST) data base, and full-length cDNAs were obtained by library screening. The cds1 cDNA did not possess a recognizable mitochondrial import signal, and the activity of the expressed Cds1 protein was stimulated by nucleoside triphosphates in vitro, indicating that cds1 did not encode the mitochondrial-specific isozyme. There were two mRNA species (3.9 and 5.6 kilobases) detected on Northern blots hybridized with the cds1 probe that were expressed at distinctly different levels in various human tissues. Consistent with the presence of the two mRNAs, a cDNA predicted to encode a second human CDP-diacylglycerol synthetase (cds2) was also uncovered in the EST data base. In contrast to the two cds mRNAs, a single, 2.1-kilobase pis1 mRNA was uniformly expressed in all human tissues examined. Expression of the pis1 gene led to the overproduction of both phosphatidylinositol synthase and phosphatidylinositol:inositol exchange reactions, indicating that the Pis1 polypeptide catalyzed both of these activities. Phosphatase treatment of cell extracts abolished the CMP-independent phosphatidylinositol:inositol exchange reaction, and exchange activity was completely restored by the addition of CMP. Overexpression of cds1 or pis1 alone or in combination did not enhance the rate of phosphatidylinositol biosynthesis. Also, overexpression did not result in a significant proportional increase in the cellular levels of CDP-diacylglycerol or phosphatidylinositol. These data illustrate that the levels of Cds1 and Pis1 protein expression are not critical determinants of cellular PtdIns content and argue against a determining role for the activity of either of these enzymes in the regulation of PtdIns biosynthesis.

Sequence, biochemical characterization, and developmental expression of a new member of the TGF-beta superfamily in Drosophila melanogaster.

More than 20 members of the transforming growth factor-beta (TGF-beta) superfamily of growth and differentiation factors have been implicated in development. One member of the TGF-beta family has been previously reported from Drosophila, the decapentaplegic (dpp) gene which is involved in embryonic dorsal/ventral polarity, embryonic gut formation, and imaginal disk development. Using PCR methods, we have identified a second Drosophila gene in the TGF-beta family. It encodes a protein product that is more similar to the TGF-beta-related human bone morphogenetic proteins (BMPs) 5, 6, and 7 than it is to the Drosophila dpp gene product. Because of its localization on the polytene chromosome map, we refer to this gene as 60A. Expression of a 60A cDNA in Drosophila S2 cells was used to determine that 60A encodes a preproprotein that is processed to yield secreted amino- and carboxy-terminal polypeptides. The carboxy-terminal peptides are recovered as disulfide-linked homodimers. The 60A transcripts and protein are first detected at the onset of gastrulation, primarily in the mesoderm of the extending germ band. As the germ band retracts, and throughout later stages of embryonic development, the 60A transcript and protein are most readily detected in cells of the developing foregut and hindgut.

Specificity of bone morphogenetic protein-related factors: cell fate and gene expression changes in Drosophila embryos induced by decapentaplegic but not 60A.

Reported assays of the bone morphogenetic proteins (BMPs) have not in general revealed specific functions for the different proteins, belying the specificity implied by the evolutionary conservation and distinct expression patterns of the genes encoding BMPs. We have used assays of developmental function to show that the two Drosophila homologues of the BMPs, decapentaplegic (dpp) and 60A, that both induce ectopic bone formation in mammalian assay systems, have distinct effects in Drosophila development. A binary expression system using the yeast transcriptional activator GAL4 directed identical patterns of tissue and temporally specific dpp and 60A expression. When dpp enhancer elements drove GAL4 expression, GAL4-responsive dpp transgenes rescued dpp mutant phenotypes, but GAL4-responsive 60A transgenes did not. Ectopic ectodermal expression of dpp during gastrulation respecified the dorsal/ventral pattern of the embryo. In contrast, ectopic 60A expression had no detectable effects on embryonic development but led to defects in adult structures or lethality during metamorphosis. Expression of 60A in cells expressing dpp did not interfere with dpp functions, indicating that dysfunctional heterodimers did not form at sufficient levels to inhibit dpp. These specific developmental responses in Drosophila indicate that in vivo functions of BMP-like factors can be more specific than indicated by the ectopic bone formation assays and that the Drosophila embryo provides an assay system sensitive to the structural differences that contribute to BMP specificity in vivo.

A conserved system for dorsal-ventral patterning in insects and vertebrates involving sog and chordin.

Dorsal-ventral patterning within the ectoderm of the Drosophila embryo requires seven zygotic genes, including short gastrulation (sog). Here we demonstrate that sog, which is expressed in the ventrolateral region of the embryo that gives rise to the nerve cord, is functionally homologous to the chordin gene of Xenopus, which is expressed in the dorsal blastopore lip of the embryo and in dorsal mesoderm, in particular the notochord. We show by injections of messenger RNA that both sog and chordin can promote ventral development in Drosophila, and that sog, like chordin, can promote dorsal development in Xenopus. In Drosophila, sog antagonizes the dorsalizing effects of decapentaplegic (dpp), a member of the transforming growth factor-beta family. One of the dpp homologues in vertebrates, bmp-4, is expressed ventrally in Xenopus and promotes ventral development. We show that dpp can promote ventral fates in Xenopus, and that injection of sog mRNA counteracts the ventralizing effects of dpp. These results suggest the molecular conservation of dorsoventral patterning mechanisms during evolution.