Identification and characterization of Uss1p (Sdb23p): a novel U6 snRNA-associated protein with significant similarity to core proteins of small nuclear ribonucleoproteins.

The SDB23 gene of Saccharomyces cerevisiae was isolated in a search for high copy-number suppressors of mutations in a cell cycle gene, DBF2, SDB23 encodes a 21,276 Da protein with significant sequence similarity to characterized mammalian snRNP core proteins. Examination of multiple sequence alignments of snRNP core proteins with Sdb23p indicates that all of these proteins share a number of highly conserved residues, and identifies a novel motif for snRNP core proteins. Sdb23p is essential for cell viability and is required for nuclear pre-mRNA splicing both in vivo and in vitro. Extracts prepared from Sdb23p-depleted cells are unable to support splicing and have vastly reduced levels of U6 snRNA. The stability of U1, U2, U4 and U5 spliceosomal snRNAs is not affected by the loss of Sdb23p. Antibodies raised against Sdb23p strongly coimmunoprecipitate free U6 snRNA and U4/U6 base-paired snRNAs. These results establish that SDB23 encodes a novel U6 snRNA-associated protein that is essential for the stability of U6 snRNA. We therefore propose the more logical name USS1 (U-Six SnRNP) for this gene.     

Temperature coefficients of peptides dissolved in hexafluoroisopropanol monitor distortions of helices

Temperature coefficients are widely used as an indication of solvent accessibility to amide protons. Low temperature coefficients are related to low accessibility and are often interpreted as evidence for intramolecular hydrogen bonding. Conformational shifts, i.e. the difference between chemical shifts of a particular residue in a structured and in a random-coil conformation, provide information on secondary structure. In particular, negative CH conformational shifts are often used to delineate the extent of helical stretches. NH conformational shifts show large oscillations within a helix that have been interpreted as the result of helix distortions affecting hydrogen bond lengths. In the course of the study of different peptides that adopt a helical structure in the presence of the structure-inducing solvent hexafluoroisopropanol (HFIP), we have found a strong correlation between temperature coefficients and amide conformational shifts. However, contrary to the initial expectations, lower temperature coefficients were associated to amide protons involved in longer, and presumably weaker, hydrogen bonds. The correlation can be explained, however, assuming that, in helical peptides dissolved in HFIP, temperature affects the chemical shift of amide protons mainly by changing the average length of intramolecular hydrogen bonds and changes in solvent accessibility play only a secondary role under these experimental conditions. The pattern of temperature coefficients in helical peptides can therefore be used to identify short or long hydrogen bonds causing bending of the helix axis.     

Regulation of inositol monophosphatase in Saccharomyces cerevisiae

Inositol monophosphatase is a key enzyme in the de novo biosynthesis of inositol and in the phosphoinositide second-messenger signalling pathway. Inhibition of this enzyme is a proposed mechanism for lithium's pharmacological action in bipolar illness (manic depression). Very little is known about how expression of this enzyme is regulated. Because the yeast Saccharomyces cerevisiae has been shown to be an excellent model system in which to understand the regulation of inositol metabolism, we characterized inositol monophosphatase in this yeast. Lithium inhibited monophosphatase activity in vitro . Growth in the presence of inositol resulted in increased expression of the enzyme in vivo , although inositol had no effect on enzyme activity in vitro . The inositol effect was apparent when cells were grown in glucose but not in glycerol/ethanol. Monophosphatase activity was derepressed as cells entered stationary phase. This effect was apparent only during growth in glucose plus inositol. The results demonstrate that S. cerevisiae monophosphatase is inhibited by lithium and regulated by factors affecting phospholipid biosynthesis.     

SOURCES OF INORGANIC CARBON FOR PHOTOSYNTHESIS BY THREE SPECIES OF MARINE DIATOM1

The utilization of inorganic carbon by three species of marine diatom, Skeletonema costatum (Grev.) Cleve. Ditylum brightwellii (West) Grun., and Chaetoceros calcitrans Paulsen was investigated using an inorganic carbon isotopic disequilibnum technique and inorganic carbon dose-response curves. Stable carbon isotope data of the diatoms are also presented. Observed rates of photosynthetic oxygen evolution were greater than could be accounted for by the theoretical rate of CO₂ supply from the uncatalyzed dehydration of HCO₃^? in the external medium, suggesting use of HCO₃^? as an inorganic carbon source. Data from the isotopic disequilibrium experiment demonstrate the use of both HCO₃^? and CO₂ for photosynthesis. Carbon isotope discrimination values support the use of HCO₃^? by the diatoms.     

Cloning and characterization of a gene from Pasteurella haemolytica A1 involved in lipopolysaccharide biosynthesis

Abstract A Pasteurella haemolytica A1 gene involved in the biosynthesis of a moiety on the core of the lipopolysaccharide molecule has been cloned and characterized. Escherichia coli clones which carry this gene showed an alteration of its lipopolysaccharide migration profile on tricine SDS-PAGE and exhibited resistance to the core-specific phage U3. In addition, lipopolysaccharide extracted from the E. coli clones was recognized by an anti-corespecific antiserum, but not by antiserum specific for the O antigen of P. haemolytica A1 lipopolysaccharide. Nucleotide sequence analysis of the cloned DNA identified an open reading frame ( lpsA ) coding for a protein of 263 amino acids which showed significant homology with a Haemophilus influenzae type b lipooligosaccharide biosynthesis gene. PCR amplification of genomic DNA, using primers based on the P. haemolytica A1 lpsA sequence, yielded products from only the A biotypes of P. haemolytica .     

Analysis of viability and cell types of macroalgal protoplasts using flow cytometry

The ability to rapidly distinguish viable sub-populations of cells within populations of macroalgal protoplast isolations was demonstrated using flow cytometry. Viable protoplasts from Ulva sp. and Porphyra perforata J. Ag. were distinguished from non-viable protoplasts based on differential fluorescein accumulation. The identities of cortical and epidermal protoplasts from Macrocystis pyrifera (L.) C. Ag. were inferred based on light-scattering and chlorophyll a autofluorescence. Three cell types could be distinguished among protoplasts released from thalli of P. perforata based on chlorophyll a and phycoerythrin autofluorescence. Mixed protoplast populations of Ulva sp. and P. perforata were also discernable based on relative chlorophyll a and phycoerythrin autofluorescence. The ability to screen heterogenous protoplast populations rapidly, combined with the cell sorting capabilities of many flow cytometers, should prove valuable for seaweed biotechnology.     

Salmonella typhimurium secreted invasion determinants are homologous to Shigella lpa proteins

Salmonella typhimurium secreted proteins (Ssp) were previously implicated in epithelial cell invasion. Here we describe four genes ( sspB , sspC , sspD , and sspA ), located between spaT and prgH , which encode proteins of 63, 42, 36, and 87 kDa, respectively. These Ssp are homologous to Shigella flexneri secreted proteins lpaB, lpaC, lpaD and lpaA. A non-invasive mutant with a transposon insertion in sspC lacks Ssp of 87,42 and 36 kDa. Complementation analyses show that sspC and sspD encode the 42 and the 36 kDa Ssp, while the 87 kDa Ssp is encoded by sspA . sspC and sspD , but not sspA are required for invasion. Amino-terminal sequencing shows that SspC and SspA are secreted without amino-terminal processing. We further demonstrate that Ssp secretion requires proteins encoded by prgHIJK , homologous to the Shigella lpa secretion system, since SspA is abundantly secreted by wild-type bacteria but is completely retained within the cellular fraction of a prgHIJK mutant. A precipitate containing abundant SspC and three other major Ssp of 63,59 and 22 kDa was isolated from culture supernatants of wild-type bacteria. These data indicate that major secreted invasion determinants of S. typhimurium are structurally and functionally homolgous to S. flexneri lpa proteins.     

The maintenance ofBdellovibrio at low prey density

A mathematical model for the interaction ofBdellovibrio and its prey predicted that a relatively high prey density (7×10⁵ cells ml^–1) would be required for the establishment of an equilibrium in a mixed population [8]. The present report shows thatBdellovibrio can be maintained in a continuous culture when the prey cell density is much lower (2–5×10⁴ cells ml^–1), and closer to that of naturally occurring bacterial populations in sea waters.     

Thermal dependence of contractile properties of single skinned muscle fibres from Antarctic and various warm water marine fishes including Skipjack Tuna (Katsuwonus pelamis) and Kawakawa (Euthynnus affinis)

Summary Single fast fibres and small bundles of slow fibres were isolated from the trunk muscles of an Antarctic (Notothenia neglecta) and various warm water marine fishes (Blue Crevally,Carangus melampygus; Grey Mullet,Mugil cephalus; Dolphin Fish,Coryphaena hippurus; Skipjack-tuna,Katsuwonus pelamis and Kawakawa,Euthynuus affinis). Fibres were chemically skinned with the nonionic detergent Brij 58.For warm water species, maximum Ca²⁺-activated tension (P ₀) almost doubled between 5–20°C with little further increase up to 30°C. However, when measured at their normal body temperatures,P ₀ values for fast fibres were similar for all species examined, 15.7–22.5 N · cm^–2. Ca²⁺-regulation of contraction was disrupted at temperatures above 15°C in the Antarctic species, but was maintained at up to 30°C for warm water fish.Unloaded (maximum) contraction speeds (V _max) of fibres were determined by the slacktest method. In general,V _max was approximately two times higher in white than red muscles for all species studied, except Skipjack tuna. For Skipjack tuna,V _max of superficial red and white fibres was similar (15.7 muscle lengths · s^–1 (L ₀ · s^–1)) but were 6.5 times faster than theV _max of internal red muscle fibres (2.4±0.2L ₀ · s^–1) (25°C). V _max forN. neglecta fast fibres at 0–5°C (2–3L ₀ · s^–1) were similar to that of warm water species measured at 10–20°C. However, when measured at their normal muscle temperatures, theV _max for the fast muscle fibres of the warm water species were 2–3 times higher than that forN. neglecta.In general,Q _{10(15–30°C)} values forV _max were in the range 1.8–2.0 for all warm water species studied except Skipjack tuna.V _max for the internal red muscle fibres of Skipjack tuna were much more temperature dependent (Q _{10(15–30°C)}=3.1) (P<0.01) than for superficial red or white muscle fibres. The proportion of slower red muscle fibres in tuna (28% for 1 kg Skipjack) is 3–10 times higher than for most teleosts and is related to the tuna's need to sustain high cruising speeds. We suggest that the 8–10°C temperature gradient that can exist in Skipjack tuna between internal red and white muscles allows both fibre types to contract at the same speed. Therefore, in tuna, both red and white muscle may contribute to power generation during high speed swimming.