Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Expression, Purification, and Initial Characterization of the Recombinant Storage Protein Precursor ofTheobroma cacao

The gene encoding the 67-kDa cocoa storage protein precursor has been cloned fromTheobroma cacaoand expressed inEscherichia coliusing the pET expression system. The recombinant storage protein has been renatured from inclusion bodies at 30°C using 20 m glycine–NaOH buffer, pH 10.0, containing 1 m oxidized glutathione and 0.1% Brij. The renatured protein was purified and demonstrated to adopt a stable native conformation by optical spectroscopy. Secondary structure analysis from circular dichroism indicated the protein to be 23% α-helix and 38% β-sheet, in close agreement with values obtained using a secondary structure prediction program.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

Divergence in coloration and ecological speciation in the Anolis marmoratus species complex

Adaptive divergence in coloration is expected to produce reproductive isolation in species that use colourful signals in mate choice and species recognition. Indeed, many adaptive radiations are characterized by differentiation in colourful signals, suggesting that divergent selection acting on coloration may be an important component of speciation. Populations in the Anolis marmoratus species complex from the Caribbean island of Guadeloupe display striking divergence in the colour and pattern of adult males that occurs over small geographic distances, suggesting strong divergent selection. Here we test the hypothesis that divergence in coloration results in reduced gene flow among populations. We quantify variation in adult male coloration across a habitat gradient between mesic and xeric habitats, use a multilocus coalescent approach to infer historical demographic parameters of divergence, and examine gene flow and population structure using microsatellite variation. We find that colour variation evolved without geographic isolation and in the face of gene flow, consistent with strong divergent selection and that both ecological and sexual selection are implicated. However, we find no significant differentiation at microsatellite loci across populations, suggesting little reproductive isolation and high levels of contemporary gene exchange. Strong divergent selection on loci affecting coloration probably maintains clinal phenotypic variation despite high gene flow at neutral loci, supporting the notion of a porous genome in which adaptive portions of the genome remain fixed whereas neutral portions are homogenized by gene flow and recombination. We discuss the impact of these findings for studies of colour evolution and ecological speciation.     

The G matrix under fluctuating correlational mutation and selection

Theoretical quantitative genetics provides a framework for reconstructing past selection and predicting future patterns of phenotypic differentiation. However, the usefulness of the equations of quantitative genetics for evolutionary inference relies on the evolutionary stability of the additive genetic variance-covariance matrix (G matrix). A fruitful new approach for exploring the evolutionary dynamics of G involves the use of individual-based computer simulations. Previous studies have focused on the evolution of the eigenstructure of G. An alternative approach employed in this paper uses the multivariate response-to-selection equation to evaluate the stability of G. In this approach, I measure similarity by the correlation between response-to-selection vectors due to random selection gradients. I analyze the dynamics of G under several conditions of correlational mutation and selection. As found in a previous study, the eigenstructure of G is stabilized by correlational mutation and selection. However, over broad conditions, instability of G did not result in a decreased consistency of the response to selection. I also analyze the stability of G when the correlation coefficients of correlational mutation and selection and the effective population size change through time. To my knowledge, no prior study has used computer simulations to investigate the stability of G when correlational mutation and selection fluctuate. Under these conditions, the eigenstructure of G is unstable under some simulation conditions. Different results are obtained if G matrix stability is assessed by eigenanalysis or by the response to random selection gradients. In this case, the response to selection is most consistent when certain aspects of the eigenstructure of G are least stable and vice versa.     

Responses of genotypes from species of Trifolium, Ornithopus, Biserrula and Hedysarum to a highly virulent race of Phytophthora clandestina and new sources of resistance

Thirty‐six genotypes, including 15 cultivars and 10 breeding lines of Trifolium subterraneum, a single genotype of each of seven other species of Trifolium (viz. Trifolium dasyurum, Trifolium glanduliferum, Trifolium incarnatum, Trifolium michelanium, Trifolium purpureum, Trifolium spumosum and Trifolium vesiculosum), Biserrula pelecinus, Hedysarum coronarium, Ornithopus compressus and Ornithopus sativus were screened under controlled environmental conditions for resistance to root disease caused by the most pathogenic race of Phytophthora clandestina occurring in Australia, namely race 177. This is the first time any of these genera/species other than T.subterraneum has ever been screened for its response to P. clandestina. The root disease caused by P.clandestina is the first report of susceptibility to this pathogen for the seven other species of Trifolium and also for B.pelecinus, H.coronarium and O.sativus. Within T.subterraneum, a very high level of resistance was identified in cvs Denmark, Junee and Meteora [scores ≤1.5 (0–5 scale where 0 = no disease) across two separate screening tests] and in the breeding lines SL027 and SM023 (scores ≤1.3 across two separate screening tests). Six of the seven other species of Trifolium (viz. T.dasyurum, T.glanduliferum, T.incarnatum, T.michelanium, T.purpureum and T.spumosum) showed a high level of resistance (scores ≤0.8 across two separate screening tests), while T.vesiculosum showed a disease score of ≤1.2 across both screening tests. O.compressus showed no disease in either test, and O.sativus showed a disease score of ≤0.7 across both screening tests. H.coronarium was susceptible with a disease score of ≤2.8 across two separate screening tests, while B.pelecinus was highly susceptible with disease scores of 3.5 and 4.6 in these tests. The high levels of resistance identified against P.clandestina are useful sources of resistance that can be exploited commercially, either directly to minimise damage from this disease or as parents in breeding programs to develop cultivars within the genera/species tested with improved resistance to this highly pathogenic race of P.clandestina.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.