The Speciation and Coordination of a Deep Earth Carbonate-Silicate-Metal Melt.

Ab initio molecular dynamics calculations on a carbonate-silicate-metal melt were performed to study speciation and coordination changes as a function of pressure and temperature. We examine in detail the bond abundances of specific element pairs and the distribution of coordination environments over conditions spanning Earth's present-day mantle. Average coordination numbers increase continuously from 4 to 8 for Fe and Mg, from 4 to 6 for Si, and from 2 to 4 for C from 1 to 148 GPa (4,000 K). Speciation across all pressure and temperature conditions is complex due to the unusual bonding of carbon. With the increasing pressure, C-C and C-Fe bonding increase significantly, resulting in the formation of carbon polymers, C-Fe clusters, and the loss of carbonate groups. The increased bonding of carbon with elements other than oxygen indicates that carbon begins to replace oxygen as an anion in the melt network. We evaluate our results in the context of diamond formation and of metal-silicate partitioning behavior of carbon. Our work has implications for properties of carbon and metal-bearing silicate melts, such as viscosity, electrical conductivity, and reactivity with surrounding phases.

Band structure and density of States effects in co-based magnetic tunnel junctions.

Utilizing Co/Al(2)O(3)/Co magnetic tunnel junctions with Co electrodes of different crystalline phases, a clear relationship between electrode crystal structure and junction transport properties is presented. For junctions with one fcc(111) textured and one polycrystalline (polyphase and polydirectional) Co electrode, a strong asymmetry is observed in the magnetotransport properties, while when both electrodes are polycrystalline the magnetotransport is essentially symmetric. These observations are successfully explained within a model based on ballistic tunneling between the calculated band structures (density of states) of fcc-Co and hcp-Co.

Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites.

Schistosoma mansoni uses a variety of proteases termed hemoglobinases to obtain nutrition from host globin. Previous reports have characterized cDNAs encoding 1 of these enzymes. However, these sequences did not define the primary structures of the mRNA and protein. The complete sequence of the 1390 base mRNA has now been determined. It encodes a 50 kDa primary translation product. In vitro translations coupled with immunoprecipitations and Western blots of parasite lysates allowed visualization of the 50 kDa form. Production of the 31 kDa mature hemoglobinase from the 50 kDa species involves removal of both NH2 and COOH terminal residues from the primary translation product. Expression of hemoglobinase mRNA and protein was examined during larval parasite development. Low levels were observed in young schistosomula. After 6-9 days in culture, high hemoglobinase levels were seen which correlated with the onset of red blood cell feeding. Immunoelectron microscopy was employed to examine hemoglobinase location and function. In adult worms the enzyme was associated with the gut lumen and gut epithelium. In cercariae, the protease was observed in the head gland, suggesting new roles for the protease.

Cloned Schistosoma mansoni proteinase (hemoglobinase) as a putative serodiagnostic reagent.

Expressed cDNA encoding a proteolytic enzyme from Schistosoma mansoni has been cloned recently. Circulating antibodies reacting with the recombinant protein have been detected in the blood of mice and humans infected with S. mansoni, S. japonicum, or S. haematobium. S. mansoni and S. haematobium infection can be distinguished by antibody titer.

Localization and pattern of expression of a female specific mRNA in Schistosoma mansoni.

To understand mechanisms involved in sex-specific gene expression in Schistosoma mansoni, a cDNA (fs800) was isolated that hybridized to an 800 nucleotide mRNA present in high levels only in mature female worms. The fs800 cDNA sequence was characterized by two long open reading frames and central stretches of repeated amino acids. Fs800 did not share similarities with other known sequences in computer searches. In situ hybridization, however, revealed that the mRNA corresponding to fs800 was found only in female vitelline cells, suggesting that the product of this gene may be involved in the production or function of eggs. Fs800 is developmentally regulated as expression of this gene is dependent on the maturity of female worms. Furthermore, during in vitro culture, when female worms are known to stop egg production, expression of fs800 selectively ceased.

Tandemly repeated exons encode 81-base repeats in multiple, developmentally regulated Schistosoma mansoni transcripts.

The adult Schistosoma mansoni cDNA clone 10-3 encodes an antigen that is recognized by sera from infected humans. We characterized multiple developmentally regulated transcripts homologous to the 10-3 cDNA and portions of the complex genomic loci encoding those transcripts. Transcripts of approximately 950, 870, and 780 nucleotides were expressed in adults, whereas only the 780-nucleotide transcript was observed in the larval stage. These transcripts were highly similar, containing variable numbers of identical direct tandem repeats of 81 bases. Although the sequence of the repeating elements and sequences 3' to them were identical in all the transcripts, sequences 5' of the repeating elements exhibited variations, including a 27-base insertion, alternative start sites for transcription, and alternate 5' exon usage. These transcripts appeared to be derived in part by the developmentally controlled alternative splicing of small exons and the use of alternative transcription initiation sites from the one or two complex loci of at least 40 kilobase pairs. Each 81-base repeat in the transcripts was encoded by three dispersed 27-base-pair exons. These 27-base-pair exons were contained within highly conserved, reiterated 3-kilobase-pair genomic tandem arrays.

Cloning and gene expression of Schistosoma mansoni protease.

Schistosomes utilize proteases (termed hemoglobinases) for degradation of host globin. cDNA clones encoding Schistosoma mansoni protease were isolated by immunologically screening an expression cDNA library with antisera raised against purified hemoglobinase. Confirmation of the identities of the clones was obtained immunologically and biochemically. The bacterially produced fusion protein encoded by one clone, lambda Hb2, degraded hemoglobin in vitro. The sequence of this clone suggested that this S. mansoni protease is synthesized in a precursor form in vivo. Gene titrations indicated that S. mansoni contains multiple genes corresponding to this cDNA. The expression of these genes may be regulated during the organism's life cycle since adult, female worms contained the highest abundances of homologous mRNA and protein compared to other stages.

Fibronectin is stored but not synthesized in mature human peripheral blood granulocytes.

To investigate whether peripheral blood granulocytes can synthesize the adhesive glycoprotein, fibronectin, we sought to demonstrate the presence of messenger RNA coding for fibronectin within mature circulating granulocytes. Polyadenylated-enriched RNA was isolated from human peripheral blood granulocytes, human skin fibroblasts (synthesize fibronectin) and HeLa cells (lack fibronectin) and probed with a cDNA clone coding for the cell attachment domain of fibronectin. Hybridization of a fibronectin cDNA fragment occurred with fibroblast RNA but did not occur with granulocyte RNA despite a 100 fold excess granulocyte RNA. Incubation of granulocytes with n-formyl methionyl leucyl phenylalanine, a chemotactic peptide known to augment the release of fibronectin from granulocytes, failed to induce detectable levels of mRNA for fibronectin in granulocytes. There was no difference in the quantity of fibronectin released from chemotactic peptide-stimulated granulocytes pre-incubated in the presence or absence of the protein synthesis inhibitor, cycloheximide, suggesting that fibronectin exists in a stored form in granulocytes. These data suggest that fibronectin in mature granulocytes is the product of synthesis during early myeloid maturation.

Isolation of cDNA clones for differentially expressed genes of the human parasite Schistosoma mansoni.

Little is known about the mechanisms that control transformations during the life cycle of Schistosoma mansoni. To enable isolation of DNA sequences encoding developmentally regulated antigens a cDNA expression library in the vector lambda gt11 amp3 was constructed from adult mRNA and immunologically screened with sera from infected individuals. We report here on the properties of three recombinant clones that derive from developmentally regulated genes. Clone 10-3 encoded a beta-galactosidase fusion protein present in high abundance in infected Escherichia coli. Clones 7-2 and 8-2 also produced immunologically recognized proteins; however, the peptides did not appear to be beta-galactosidase fusion proteins. The expression of mRNAs hybridizing to these cDNAs was examined in the different stages of the parasite life cycle. Messenger RNA corresponding to clone 10-3, approximately equal to 1000 bases in length, was present in higher abundance in male worms than in females but was not detected in schistosome eggs. A 900-base mRNA hybridizing to clone 7-2 was observed in adult worms and eggs. Both clone 10-3 and clone 7-2 hybridized to smaller mRNAs in cercariae and freshly transformed schistosomula than in adult worms. Clone 8-2 contained tandem cDNA inserts. One cDNA hybridized to a 1700-base mRNA present in all stages, while the second hybridized to an 800-base mRNA specific to adult female worms.

Stage and sex specific differences in actin gene expression in Schistosoma mansoni.

We have characterized actin gene expression in Schistosoma mansoni at the RNA and protein levels. Northern blot analyses showed two size classes of actin mRNA in eggs, cercariae and adult worms of both sexes, approximately 1 900 and 1 400 bases in length. A higher abundance of actin mRNA of both size classes was demonstrated in male worms than in eggs, cercariae, and females. Using a phalloidin-rhodamine conjugate, male worms were observed to contain more actin protein than females. Southern blot-hybridization indicated that the sexual differences in actin mRNA and protein levels were not related to some S. mansoni actin genes being sex linked. In addition, two other trematodes, Schistosoma japonicum and Fasciola hepatica and a cestode, Taenia pisiformis contained two classes of actin mRNA similar in size as those in S. mansoni. In contrast, a turbellarian, Dugesia tigrina contained only a single short actin message size class approximately 1 400 bases in length.

Varigated chromatin structures of mouse ribosomal RNA genes.

We have employed a chromatin fractionation procedure on micrococcal nuclease-digested nuclei to examine the chromatin structure of mouse ribosomal RNA genes in two systems that differ by at least 14-fold in the level of ribosomal RNA transcription. In a cultured cell line enriched in transcriptionally active ribosomal chromatin, most ribosomal sequences are preferentially sensitive to digestion by micrococcal nuclease, reside in an insoluble chromatin fraction, and lack typical nucleosomal packaging; only minor amounts of ribosomal sequences are packaged into soluble, nucleosomal chromatin. By contrast, in adult liver, which is enriched in transcriptionally inactive ribosomal chromatin, the majority of ribosomal genes are packaged into soluble, nucleosomal chromatin. However, a significant fraction of liver ribosomal chromatin is insoluble and possesses a non-nucleosomal structure. Therefore, within a single cell population or tissue, mouse ribosomal RNA genes are organized into both nucleosomal and non-nucleosomal chromatin structures. We suggest that these structures have functional significance.

Transferring DNA from electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose: properties of nucleosomes along mouse satellite DNA.

Electrophoresis fractionates nucleosomes which possess different protein compositions. We report here a procedure for transferring the DNA components of electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose (DBM-paper). Histones are first removed from nucleosome components by electrophoresis in the presence of cetyltrimethylammonium bromide (CTAB), leaving DNA fragments fixed within the original gel as the CTAB salts. The DNA is then converted to the sodium salt, denatured, and electrophoretically transferred to DBM-paper. The overall pattern of DNA on the resulting blot is visualized either by fluorography or by immunoautoradiography. This DNA pattern is then compared with autoradiograms obtained after hybridizing the same blot with specific 32P-labeled probes. Using mouse satellite DNA as a hybridization probe, we illustrate the above techniques and demonstrate that nucleosomes carrying satellite sequences are compositionally heterogeneous. The procedures described here should also be useful in the analysis of the nucleic acid components associated with other nucleoprotein complexes.