Alternative exon usage in the single CPT1 gene of Drosophila generates functional diversity in the kinetic properties of the enzyme:differential expression of alternatively spliced variants in drosophila tissues

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A.Weperformed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1mRNAwere found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K 0.5) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of the MAX randomisation technique

During the last decade the use of randomised gene libraries has had an enormous impact in the field of protein engineering. Such libraries comprise many variations of a single gene in which codon replacements are used to substitute key residues of the encoded protein. The expression of such libraries generates a library of randomised proteins which can subsequently be screened for desired or novel activities. Randomisation in this fashion has predominantly been achieved by the inclusion of the codons NNN or NNGCor T, in which N represents any of the four bases A,C,G, or T. The use of thesis codons however, necessities the cloning of redundant codons at each position of randomisation, in addition to those required to encode the twenty possible amino acid substitutions. As degenerate codons must be included at each position of randomisation, this results in a progressive loss of randomisation efficiency as the number of randomised positions is increased. The ratio of genes to proteins in these libraries rises exponentially with each position of randomisation, creating large gene libraries, which generate protein libraries of limited diversity upon expression. In addition to these problems of library size, the cloning of redundant codons also results in the generation of protein libraries in which substituted amino acids are unevenly represented. As several of the randomised codons may encode the same amino acid, for example serine which is encoded six time using the codon NNN, an inherent bias may be introduced into the resulting protein library during the randomisation procedure. The work outlined here describes the development of a novel randomisation technique aimed at a eliminating codon redundancy from randomised gene libraries, thus addressing the problems of library size and bias, associated with the cloning of redundant codons.

Seventeen a-subunit isoforms of paramecium V-ATPase provide high specialization in localization and function

In the Paramecium tetraurelia genome, 17 genes encoding the 100-kDa-subunit (a-subunit) of the vacuolar-proton-ATPase were identified, representing by far the largest number of a-subunit genes encountered in any organism investigated so far. They group into nine clusters, eight pairs with >82% amino acid identity and one single gene. Green fluorescent protein-tagging of representatives of the nine clusters revealed highly specific targeting to at least seven different compartments, among them dense core secretory vesicles (trichocysts), the contractile vacuole complex, and phagosomes. RNA interference for two pairs confirmed their functional specialization in their target compartments: silencing of the trichocyst-specific form affected this secretory pathway, whereas silencing of the contractile vacuole complex-specific form altered organelle structure and functioning. The construction of chimeras between selected a-subunits surprisingly revealed the targeting signal to be located in the C terminus of the protein, in contrast with the N-terminal targeting signal of the a-subunit in yeast. Interestingly, some chimeras provoked deleterious effects, locally in their target compartment, or remotely, in the compartment whose specific a-subunit N terminus was used in the chimera.