Ancient DNA gives green light to Galápagos Land Iguana repatriation

Land Iguanas, Conolophus subcristatus,were extirpated from Isla Baltra, GalápagosArchipelago in the 1940s. Historical recordsindicate that some Baltra iguanas weretranslocated to nearby Isla Seymour Norte inthe 1930s. Plans to repatriate iguanas toBaltra were suspended when evidence suggestedthat iguanas on Seymour Norte may not beentirely of Baltra origin. Comparison of DNAfrom century-old museum specimens with extantiguanas has identified those individuals ofunambiguous Baltra origin on Seymour Norte. These results provide scientific criteria forthe ecological restoration of these endangeredreptiles.     

Identification of a subunit interface in transthyretin amyloid fibrils: evidence for self-assembly from oligomeric building blocks

Amyloid and prion diseases appear to stem from the conversion of normally folded proteins into insoluble, fiber-like assemblies. Despite numerous structural studies, a detailed molecular characterization of amyloid fibrils remains elusive. In particular, models of amyloid fibrils proposed thus far have not adequately defined the constituent protein subunit interactions. To further our understanding of amyloid structure, we employed thiol-specific cross-linking and site-directed spin labeling to identify specific protein-protein associations in transthyretin (TTR) amyloid fibrils. We find that certain cysteine mutants of TTR, when dimerized by chemical cross-linkers, still form fibers under typical in vitro fibrillogenic conditions. In addition, site-directed spin labeling of many residues at the natural dimer interface reveals that their spatial proximity is preserved in the fibrillar state even in the absence of cross-linking constraints. Here, we present the first view of a subunit interface in TTR fibers and show that it is very similar to one of the natural dimeric interchain associations evident in the structure of soluble TTR. The results clarify varied models of amyloidogenesis by demonstrating that transthyretin amyloid fibrils may assemble from oligomeric protein building blocks rather than structurally rearranged monomers.     

Construction and evaluation of cDNA libraries for large-scale expressed sequence tag sequencing in wheat (Triticum aestivum L.)

A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.     

Mutational analysis of the active center of plant fructosyltransferases: Festuca 1-SST and barley 6-SFT

The active center of the glycoside hydrolase family 32 contains the three characteristic motifs (N/S)DPNG, RDP, and EC. We replaced the N-terminal region including the (N/S)DPNG motif of barley 6-SFT (sucrose:fructan 6-fructosyltransferase) by the corresponding region of Festuca 1-SST (sucrose:sucrose 1-fructosyltransferase). The chimeric enzyme, expressed in Pichia, retained the specificity of 6-SFT. Attempts to replace a larger piece at the N-terminus including also the RDP motif failed. A point mutation introduced in the RDP motif of 1-SST abolished enzymatic activity. Interestingly, point mutations of the EC-motif resulted in an enzyme which had lost the capability to form 1-kestose and glucose from sucrose but still accepted 1-kestose, producing fructose and sucrose as well as nystose.     

Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine

The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.     

Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich protein in transgenic plants

We have constructed a chimeric gene encoding a Brazil nut methionine-rich seed protein which contains 18% methionine. This gene has been transferred to tobacco and expressed in the developing seeds. Tobacco seeds are able to process the methionine-rich protein efficiently from a larger precursor polypeptide of 17 kDa to the 9kDa and 3 kDa subunits of the mature protein, a procedure which involves three proteolytic cleavage steps in the Brazil nut seed. The accumulation of the methionine-rich protein in the seeds of tobacco results in a significant increase (30%) in the levels of the methionine in the seed proteins of the transgenic plants. Our data indicate that the introduction of a chimeric gene encoding a methionine-rich seed protein into crop plants, particularly legumes whose seeds are deficient in the essential sulfur-containing amino acids, represents a feasible method for improving the nutritional quality of seed proteins.     

Conformation of spin-labeled melittin at membrane surfaces investigated by pulse saturation recovery and continuous wave power saturation electron paramagnetic resonance.

Melittin spin-labeled specifically with a nitroxide at positions 7, 21, 23, or the amino terminus was bound to phospholipid membranes, and the exposure of the spin label to the aqueous phase was investigated by measurement of Heisenberg exchange with chromium oxalate in the solution. The exchange frequency was determined by saturation recovery electron paramagnetic resonance (EPR) using a loop-gap resonator. This method allows use of very low concentrations (less than 1 mM) of chromium oxalate compared with conventional measurements of EPR line broadening (typically 50 mM), thus avoiding problems associated with high metal ion concentration. Differences in exchange frequency between the various positions were also estimated by continuous wave power saturation methods. In either approach, the spin label at lysine 7 was found to be the most exposed to chromium oxalate whereas that at lysine 23 was found to be the least exposed. This is consistent with a model for the membrane bound peptide in which an amphiphilic helix lies with its axis parallel to the bilayer surface and the hydrophobic moment points toward the bilayer interior.     

High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein

We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements.     

Single-cysteine substitution mutants at amino acid positions 306-321 in rhodopsin, the sequence between the cytoplasmic end of helix VII and the palmitoylation sites: sulfhydryl reactivity and transducin activation reveal a tertiary structure.

As sensors for structure at the cytoplasmic face of rhodopsin, single-cysteine substitution mutants have been previously studied in the regions connecting helices III and IV and helices V and VI. In this paper we report on single-cysteine substitution mutants at amino acid positions 306-321, comprising the cytoplasmic sequence between helix VII and the palmitoylation sites in rhodopsin. The cysteine opsin mutants were expressed in COS-1 cells and on treatment with 11-cis-retinal all formed the characteristic rhodopsin chromophore. Cysteines at positions 306-316 and 319 reacted in the dark with the thiol-specific reagent 4, 4'-dithiodipyridine (4-PDS) but showed a wide variation in reactivity. Cysteines at positions 317, 318, 320, and 321 showed no reaction with 4-PDS. The mutants on illumination also showed wide variations in activating GT. The mutant Y306C showed almost no GT activation, I307C and N310C were poor, and the activity of the mutants M309C, F313C, and M317C was also reduced relative to WT. The results suggest that the region comprising amino acids 306-321 is a part of a tertiary structure and that specific amino acids in this region on light-activation participate in the interaction with GT.