RNA recombination in brome mosaic virus: effects of strand-specific stem-loop inserts

A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3' noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.     

Phage display selects for amylases with improved low pH starch-binding

Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display we selected alpha-amylase variants, which have the ability to bind starch substrate at industrially preferred low pH conditions. First we displayed active alpha-amylase on the surface of phage fd. Secondly we developed a selection system that is based on the ability of alpha-amylase displaying phages to bind to cross-linked starch. This system was used to probe the involvement of specific beta-strands in substrate interaction. Finally, a saturated library of alpha-amylase mutants with one or more amino acid residues changed in their Cbeta4 starch-binding domain was subjected to phage display selection. Mutant molecules with good starch-binding and hydrolytic capacity could be isolated from the phage library by repeated binding and elution of phage particles at lowered pH value. Apart from the wild type alpha-amylase a specific subset of variants, with only changes in three out of the seven possible positions, was selected. All selected variants could hydrolyse starch and heptamaltose at low pH. Interestingly, variants were found with a starch hydrolysis ratio at pH 4.5/7.5 that is improved relative to the wild type alpha-amylase. These data demonstrate that useful alpha-amylase mutants can be selected via surface display on the basis of their binding properties to starch at lowered pH values.     

Functional analysis of the SRV-1 RNA frameshifting pseudoknot

Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located 12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A., Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol., 310, 1109–1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove of stem 1 involving base–base and base–sugar contacts. A mutational analysis was performed to test the functional importance of the triple helix for −1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of the triple helix in −1 frameshifting.     

Idiosyncratic behaviour of tRNA-like structures in translation of plant viral RNA genomes

Tobacco mosaic virus (TMV) and Nemesia ring necrosis virus (NeRNV) belong to the Tobamoviridae and Tymoviridae families, respectively. Although their RNAs present different 5'-untranslated regions and different family-specific genomic organizations, they share common 3'-ends organized into three consecutive pseudoknot structures followed by a histidylatable tRNA-like structure (TLS). We investigate here whether the histidine residue becomes incorporated into viral proteins and if the TLSs of TMV and NeRNV play a role in viral translation. Our results indicate that, regardless of the genomic context, the histidine moiety does not become incorporated in proteins via ribosomal translation, and that disruption of the TLS in either viral RNA does not perturb the viral translation patterns. In the light of the present data and of previous results on tymoviral TLSVal and bromoviral TLSTyr showing differential effects on translation, we suggest that the key role for the TLS in promoting translation initiation appears to be dictated by the TLS architecture and identity.     

A conformational switch at the 3' end of a plant virus RNA regulates viral replication.

3' untranslated regions of alfamo- and ilar-virus RNAs fold into a series of stem-loop structures to which the coat protein binds with high affinity. This binding plays a role in initiation of infection ('genome activation') and has been thought to substitute for a tRNA-like structure that is found at the 3' termini of related plant viruses. We propose the existence of an alternative conformation of the 3' ends of alfamo- and ilar-virus RNAs, including a pseudoknot. Based on (i) phylogenetic comparisons, (ii) in vivo and in vitro functional analyses of mutants in which the pseudoknot has been disrupted or restored by compensatory mutations, (iii) competition experiments between coat protein and viral replicase, and (iv) investigation of the effect of magnesium, we demonstrate that this pseudoknot is required for replication of alfalfa mosaic virus. This conformation resembles the tRNA-like structure of the related bromo- and cucumo-viruses. A low but specific interaction with yeast CCA-adding enzyme was found. The existence of two mutually exclusive conformations for the 3' termini of alfamo- and ilar-virus RNAs could enable the virus to switch from translation to replication and vice versa. The role of coat protein in this modulation and in genome activation is discussed.     

The relation between fine root density and proximity of stems in closed Douglas-fir plantations on homogenous sandy soils: implications for sampling design

Studies have been carried out in two fully stocked, fast growing Douglas-fir plantations of the Dutch ACIFORN project in three consecutive years, to obtain information on fine root densities (Olsthoorn 1991). For the present paper, data collected in early summer 1987 were used to study the relation of fine root density and proximity to the nearest tree or the dominant tree. A large number of samples (37 in one site and 55 in another) was collected in a small plot (10 × 11 m). Two distances were measured at each sampling point: the distance to the nearest tree and the distance to the tree with a dominant crown above that point. There was large variability in fine root density in the samples. Tests with different regression models showed a distinct rooting pattern for one of the two locations. It is concluded that systematic errors in the assessment of fine root density can arise when sampling points are chosen at a constant distance from trees. For Douglas-fir, this systematic error could have been an overestimation of the fine root density by up to 10%. These systematic errors can be avoided easily, using a stratified random design or a random sampling design. When trees are spaced irregularly, a grid sampling design is also appropriate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Heterologous protein production in the yeast Kluyveromyces lactis

Kluyveromyces lactis is both scientifically and biotechnologically one of the most important non-Saccharomyces yeasts. Its biotechnological significance builds on its history of safe use in the food industry and its well-known ability to produce enzymes like lactase and bovine chymosin on an industrial scale. In this article, we review the various strains, genetic techniques and molecular tools currently available for the use of K. lactis as a host for protein expression. Additionally, we present data illustrating the recent use of proteomics studies to identify cellular bottlenecks that impede heterologous protein expression.