The complete sequence and coding content of snowshoe hare bunyavirus small (S) viral RNA species.

The complete sequence of the small (S) viral RNA species of snowshoe hare (SSH) bunyavirus has been determined, principally from a DNA copy of the RNA cloned in the E.coli plasmid pBr322. The viral S RNA (negative sense strand) is 982 nucleotides long (3.3 x 10(5) daltons) with complementary 5' and 3' end sequences. It has a base composition of 30.5%U, 25.8%A, 24.9%C and 18.7%G. In the viral complementary (plus sense) strand there are two overlapping open reading frames initiated by methionine codons. One reading frame codes for a 26.8 x 10(3) dalton protein, the other for a 10.5 x 10(3) dalton protein. The larger gene product is presumably related to the viral nucleoprotein (N) that is coded by the S RNA (Gentsch and Bishop (1978) J. Virol. 28, 417-419). The smaller gene product is probably related to the recently identified S RNA coded nonstructural protein (NSS) induced in virus infected cells (Fuller and Bishop (1982) J. Virol. 41, 643-648).     

Characterization of the exopolysaccharide produced by Streptococcus thermophilus 8S containing an open chain nononic acid.

The exopolysaccharide produced by Streptococcus thermophilus 8S in reconstituted skimmed milk is a heteropolysaccharide containing d-galactose, d-glucose, d-ribose, and N-acetyl-d-galactosamine in a molar ratio of 2 : 1 : 1 : 1. Furthermore, the polysaccharide contains one equivalent of a novel open chain nononic acid constituent, 3,9-dideoxy-d-threo-d-altro-nononic acid, ether-linked via C-2 to C-6 of an additional d-glucose per repeating unit. Methylation analysis and 1D/2D NMR studies (1H and 13C) performed on the native polysaccharide, and mass spectrometric and NMR analyses of the oligosaccharide obtained from the polysaccharide by de-N-acetylation followed by deamination and reduction demonstrated the 'hepta'saccharide repeating unit to be: -->4)-alpha-D-Galp-(1-->2)-beta-D-Ribf-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1--7')-Sub-(1-->4)-beta-D-GalpNAc-(1--> in which Sug is 6-O-(3',9'-dideoxy-d-threo-d-altro-nononic acid-2'-yl)-alpha-d-glucopyranose.     

Reinvestigation of the steady-state kinetics and physiological function of the soluble NiFe-hydrogenase I of Pyrococcus furiosus

Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H₂ evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H₂ production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low K_m for H₂ (~20 μM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 μM dissolved CO). Since the K_m for NADP⁺ is ~37 μM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.     

Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells

When P19 embryonal carcinoma (EC) cells were cocultured with cells from one of several established visceral-endoderm-like cell lines, the EC cells were rapidly induced to aggregate and differentiate, into cell types including mesoderm-derived cardiac and skeletal muscle. Neither parietal-endoderm- nor mesoderm-like cell lines induced aggregation or differentiation of EC cells in coculture, although a cell line with both parietal and visceral endoderm characteristics induced aggregation but not differentiation. Also, without the feeder cells aggregates of P19 failed to differentiate, provided that serum in the culture medium had been previously passed over dextran-coated charcoal to remove lipophilic substances, which may include endogenous retinoids. All experiments were carried out using serum treated in this way. Taken together, the results demonstrated that aggregation was necessary, but not sufficient, to make P19 EC cells differentiate. Direct contact between the two cell types was not necessary, since even when separated by an agar layer in cocultures, aggregates of P19 still differentiated. Medium conditioned by cells of the END-2 line, a visceral-endoderm-like derivative of P19, was particularly potent in inducing endodermal and mesodermal differentiation of single P19 aggregates, confirming the involvement of a diffusible factor secreted specifically by visceral-endoderm-like cells in this process.     

Nucleotide Sequence Analyses and Predicted Coding of Bunyavirus Genome RNA Species

We performed 3′ RNA sequence analyses of [³²P]pCp-end-labeled La Crosse (LAC) virus, alternate LAC virus isolate L74, and snowshoe hare bunyavirus large (L), medium (M), and small (S) negative-stranded viral RNA species to determine the coding capabilities of these species. These analyses were confirmed by dideoxy primer extension studies in which we used a synthetic oligodeoxynucleotide primer complementary to the conserved 3′-terminal decanucleotide of the three viral RNA species (Clerx-van Haaster and Bishop, Virology 105:564-574, 1980). The deduced sequences predicted translation of two S-RNA gene products that were read in overlapping reading frames. So far, only single contiguous open reading frames have been identified for the viral M- and L-RNA species. For the negative-stranded M-RNA species of all three viruses, the single reading frame developed from the first 3′-proximal UAC triplet. Likewise, for the L-RNA of the alternate LAC isolate, a single open reading frame developed from the first 3′-proximal UAC triplet. The corresponding L-RNA sequences of prototype LAC and snowshoe hare viruses initiated open reading frames; however, for both viral L-RNA species there was a preceding 3′-proximal UAC triplet in another reading frame that was followed shortly afterward by a termination codon. A comparison of the sequence data obtained for snowshoe hare virus, LAC virus, and the alternate LAC virus isolate showed that the identified nucleotide substitutions were sufficient to account for some of the fingerprint differences in the L-, M-, and S-RNA species of the three viruses. Unlike the distribution of the L- and M-RNA substitutions, significantly fewer nucleotide substitutions occurred after the initial UAC triplet of the S-RNA species than before this triplet, implying that the overlapping genes of the S RNA provided a constraint against evolution by point mutation. The comparative sequence analyses predicted amino acid differences among the corresponding L-, M-, and S-RNA gene products of snowshoe hare virus and the two LAC virus isolates.     

RADAR,a relational archaeobotanical database for advanced research

The structure of the Dutch Relational Archaeobotanical Database (RADAR) is presented. RADAR is a rather compact archaeobotanical database that is controlled centrally, but can be distributed to individual scientists. For this reason RADAR contains only the most important archaeobotanical data. For detailed archaeological, botanical and regional palaeoenvironmental information, links can be established with the national archaeological database (ARCHIS), the national botanical database (BBR) and the European Pollen Database (EPD). The software used for manipulation of the database is PARADOX for reasons of its highly visible nature, its control facilities for data entry and the ease of importing and exporting data from and to many other programs. The potential of the database is demonstrated with query examples.     

The embalming of the ancestors of the Dutch royal family

During the restoration of the main church in Breda (the Netherlands), the city archaeologists rediscovered a tomb with remains of the ancestors of the Dutch royal family. Research was carried out prior to the reburial of the remains. The combination of historical, osteological, dendrochronological and ¹⁴C data provided names and dates for 7 of the 8 embalmed bodies. The tomb was in use between A.D. 1475 and 1526. Archaeobotanical results could be compared with several recipes for embalming from the same period. Strikingly many macroremains were found where mainly pollen was expected. This could mean the bodies were resting on a bed of herbs and spices, but it could also have been due to the lack of confidence of the embalmers; they may have used all the available aromatics to conceal the smell of the corpse. At least some of the species were imported from the Mediterranean and/or subtropical regions, but others were probably locally cultivated. Received August 27, 2001 / Accepted April 1, 2002