The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of ...brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-L-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l(-1)) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.
With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, ...the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the ∼13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5′ regulatory sequences.
We have developed a class of binding proteins, called avimers, to overcome the limitations of antibodies and other immunoglobulin-based therapeutic proteins. Avimers are evolved from a large family ...of human extracellular receptor domains by in vitro exon shuffling and phage display, generating multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains creates avidity and results in improved affinity and specificity compared with conventional single-epitope binding proteins. Other potential advantages over immunoglobulin domains include simple and efficient production of multitarget-specific molecules in Escherichia coli, improved thermostability and resistance to proteases. Avimers with sub-nM affinities were obtained aganist five targets. An avimer that inhibits interleukin 6 with 0.8 pM IC50 in cell-based assays is biologically active in two animal models.
We demonstrate that the B chain of ricin toxin preserves its lectin activity when expressed as a fusion protein on the surface of fd phage. Moreover, B chain, which folds into two topologically ...similar globular domains, can be dissected into amino-terminal and carboxyl-terminal domains to form single binding domains (SBDs) of B chain, each of which displays specificity for complex galactosides. The specific binding exhibited by the fusion protein of these SBDs was eliminated when amino acid substitutions Gly-46 in SBD1 or Gly-255 in SBD2 for native asparagine were introduced to alter key residues implicated in hydrogen bonding with substrate. These data demonstrate that it is possible to use a prokaryotic expression system to stably express and screen ricin B chain and its SBDs for sugar-binding mutants. Expression of ricin B chain on the surface of fd phage provides a method that can be used to efficiently select mutants with altered binding activities from a randomly generated library.
We have used low stringency screening with the Drosophila melanogaster s36 chorion gene to recover its homologue from genomic and cDNA libraries of the medfly, Ceratitis capitata. The same gene has ...also been recovered from a genomic library of D. virilis. The medfly s36 gene shows similar developmental specificity as in Drosophila (early choriogenesis). It is also specifically amplified in ovarian follicles; this is the first report of chorion gene amplification outside the genus Drosophila. Alignments of s36 sequences from three species show that, in addition to its regulatory conservation, the s36 gene is extensively conserved in sequence, in a region corresponding to a central protein domain, and in short regions of 5' flanking DNA that might correspond to cis-regulatory elements.
A method is described which allows selection of sequences which can substitute for the normal AATAAA hexanucleotide involved in polyadenylation of SV40 late mRNAs. Plaques were generated from viral ...DNA lacking the motif, forcing acquisition of substitute sequences. Four variants wre characterized. All displayed wild-type growth kinetics and produced normal levels of late mRNAs and proteins. Two variants had reacquired AATAAA elements and one acquired an ATTAAA sequence. The last variant carried an ATTTTTTAAA segment, suggesting this novel sequence, or some portion of it, can also signal poly A addition.
A segment comprising the transcriptional enhancer elements was deleted from a recombinant plasmid carrying the simian virus 40 genome. The mutated viral chromosome was excised from the plasmid and ...propagated through several cycles of growth in monkey kidney cells. A variant was obtained that carried reiterations of sequences that span both sides of the deleted enhancer region. The mutant virus, dup 1495, displays a lag in its growth kinetics as compared to its parent, but it ultimately generates wild-type yields. The mutant virus expresses early mRNAs at near-normal levels, and the reiterated sequence functioned in cis to enhance transformation of mouse cells by the herpesvirus thymidine kinase gene. Thus dup 1495 reiterated segments encode enhancer activity even though their primary sequence is radically different from that of the normal simian virus 40 enhancer.
We have further characterized the cis-acting elements that control the amplification of the third chromosomal cluster of chorion genes in Drosophila melanogaster; these include the ...amplification-control element ACE-3 and four amplification-enhancing regions (AER-a to -d). We have used two types of deletions in the chorion cluster: the first was in vitro generated deletions of the ACE-3 region that were subsequently introduced into the germ line, and the second was deletions induced in vivo within a transposon at a preexisting chromosomal location, thus avoiding the complication of position effects. Some of the lines bearing deletions of either type showed amplification, albeit at drastically reduced levels. These unexpected results indicate that, despite its importance, ACE-3 is not essential for low-level amplification and that cis-acting amplification elements are functionally redundant within the autosomal chorion replicon.
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