The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA

RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs.     

Differential uptake of fumarate by Candida utilis and Schizosaccharomyces pombe

The dicarboxylic acid fumarate is an important intermediate in cellular processes and also serves as a precursor for the commercial production of fine chemicals such as l-malate. Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. In this study we have shown that the yeast Candida utilis effectively degraded extracellular fumarate and l-malate, but glucose or other assimilable carbon sources repressed the transport and degradation of these dicarboxylic acids. The transport of both dicarboxylic acids was shown to be strongly inducible by either fumarate or l-malate while kinetic studies suggest that the two dicarboxylic acids are transported by the same transporter protein. In contrast, Schizosaccharomyces pombe effectively degraded extracellular l-malate, but not fumarate, in the presence of glucose or other assimilable carbon sources. The Sch. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of l-malate. Received: 15 March 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates

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Occurrence and distribution of Clinostomum tilapiae Ukoli, 1966 and Euclinostomum heterostomum (Rudolphi, 1809) metacercarial infections of freshwater fish in Venda and Lebowa, southern Africa

During 1980 to 1982, parasitological surveys were carried out at large impoundments in Venda and Lebowa, southern Africa, coinciding with fish harvesting programmes. Of the various fish species examined, only Oreochromis mossambicus was found to be infected by Clinostomum tilapiae prevalent in the gills, and Euclinostomum heterostomum prevalent in the muscles. Distribution records and infection statistics are presented for these two parasite species.     

Binding and spectroscopic properties of ostrich neurophysins. Probing the role of residue 35 at the monomer-monomer interface.

Binding and spectroscopic properties of ostrich neurophysins were examined with emphasis on the behavior of Tyr-35, a residue that provides a potential probe of the monomer-monomer interface and of allosteric interrelationships between this region and the binding site. Mesotocin-associated ostrich neurophysin was found to bind oxytocin and related peptides with affinities comparable to the mammalian proteins, but induced a significantly different optical activity in bound peptides than the mammalian proteins. Gel-filtration studies indicated higher dimerization constants for the ostrich neurophysins than for the bovine neurophysins. Consistent with this, Tyr-35 was found to be largely buried, as monitored by tyrosine titration and lack of reactivity towards tetranitromethane under non-denaturing conditions. Reaction of Tyr-35 of the mesotocin-associated protein with tetranitromethane under denaturing conditions, followed by refolding, allowed isolation of an active product with an altered interface region as partially evidenced by its titration properties and consistent with its markedly altered CD spectrum. Comparison of the CD spectra of the modified and native proteins and analysis of pH effects indicated the contribution of Tyr-35 to an unusual 237 nm band in the mesotocin-associated protein. Small shifts in the 350 nm CD band of nitrated Tyr-35 on binding peptide and apparent effects of nitration on the induced optical activity in bound peptide provided evidence of at least weak structural communication between Tyr-35 and the binding site. However, no significant effect of nitration on binding affinity was observed, suggesting that, in the mesotocin-associated protein, the region around residue 35 is not a stringent modulator of the thermodynamic behavior of the binding site.     

FIRST RECORD OF THE SNAKE CATFISH CLARIAS THEODORAE (WEBER, 1897) (CLARIIDAE: PISCES) FROM THE TRANSVAAL,SOUTH AFRICA

SUMMARY

Two new distributional records of the snake catfish Clarias theodorae (Clariidae: Pisces) from Transvaal tributaries of the Limpopo River are documented. This is also the first record of the snake catfish from the Transvaal. The habitat preference of this species is briefly discussed and brief comment is made on probable migration routes.     

Purification and primary structure of glucagon from ostrich pancreas splenic lobes

Glucagon is a highly conserved polypeptide hormone which appears to play a more important role in regulation of glycaemia in birds than insulin. Ostrich glucagon was isolated and purified from ostrich pancreas splenic lobes using an adapted acid ethanol extraction procedure, gel filtration, ion exchanges, and HPLC steps. The purified glucagon fraction appeared to contain small quantities of a more acidic contaminant (polyacrylamide gel isoelectric focussing, PAGE) but appeared homogeneous on SDS-PAGE. Amino acid analysis and sequence analysis showed identity with the duck hormone. Identity with the duck hormone was confirmed by liquid phase as well as gas phase sequencing. The ostrich glucagon preparation seemed to have a higher Km than the porcine homologue in stimulating glycerol release from isolated chicken adipocytes.