Förster resonance energy transfer measurements are consistent with a helical bundle model for lipid-free apolipoprotein A-I

Apolipoprotein (apo) A-I mutants were constructed for FRET studies to distinguish between two possible lipid-free conformers, a globular helix bundle and an elongated helical hairpin. Mutants containing a single Trp at position 50 were prepared by replacing Trps at positions 8, 72, and 108 with Phe (W@50). Two mutants were constructed from W@50 by incorporating Cys at Arg83 (W@50R83C) or Arg173 (W@50R173C) for attachment of the fluorescent probe AEDANS. Secondary structure of the mutants is very similar to wild type (wt) apo A-I, and fluorescence emission indicates that W50 is protected from solvent. Thermal stabilities of the AEDANS-labeled mutants are also similar to wt. These results indicate that no discernible changes occur in structure or stability as a result of mutations or labeling. The FRET data from W@50 to AEDANS are well-represented by a single distance distribution function with a distance of approximately 22 A for W@50R83C and approximately 19 A for W@50R173C. These distances are consistent with theoretical values calculated from a helical bundle model but not from a helical hairpin. A probability distance distribution function yields significantly small half-width values of 5.6 and 3.7 A, respectively, suggesting low conformational dynamics in both mutants. Differential scanning calorimetry (DSC) was performed on wt and a C-terminal deletion mutant, Delta(187-243), to obtain information on domain architecture. Contrary to expectations, both proteins unfold cooperatively. The results are consistent with the presence of a single folded domain within residues 1-186. These results support the presence of a discrete globular bundle conformation for lipid-free apo A-I.     

Uncommon deletions of the Smith-Magenis syndrome region can be recurrent when alternate low-copy repeats act as homologous recombination substrates

Several homologous recombination "hotspots," or sites of positional preference for strand exchanges, associated with recurrent deletions and duplications have been reported within large low-copy repeats (LCRs). Recently, such a hotspot was identified in patients with the Smith-Magenis syndrome (SMS) common deletion of approximately 4 Mb or a reciprocal duplication within the KER gene cluster of the SMS-REP LCRs, in which 50% of analyzed strand exchanges resulting in deletion and 23% of those resulting in duplication occurred. Here, we report an additional recombination hotspot within LCR17pA and LCR17pD, which serve as alternative substrates for nonallelic homologous recombination that results in large (approximately 5 Mb) deletions of 17p11.2, which include the SMS region. Using polymerase-chain-reaction mapping of somatic cell hybrid lines, we refined the breakpoints of six deletions within these LCRs. Sequence analysis of the recombinant junctions revealed that all six strand exchanges occurred within a 524-bp interval, and four of them occurred within an AluSq/x element. This interval represents only 0.5% of the 124-kb stretch of 98.6% sequence identity between LCR17pA and LCR17pD. A search for potentially stimulating sequence motifs revealed short AT-rich segments flanking the recombination hotspot. Our findings indicate that alternative LCRs can mediate rearrangements, resulting in haploinsufficiency of the SMS critical region, and reimplicate homologous recombination as a major mechanism for genomic disorders.     

Elevated SNAP-25 is associated with fatty acid-induced impairment of mouse islet function

The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in insulin secretion following chronic exposure to non-esterified fatty acids (NEFAs) has not been extensively investigated. Here, we show that synaptosome-associated protein of 25 kDa (SNAP-25) levels were predominantly elevated in the soluble fraction of mouse islets exposed to palmitate. This coincided with an impairment of insulin secretion to glucose and non-glucose secretagogues, consistent with a defect at a distal regulatory step in exocytosis. Removal of palmitate from the media restored both SNAP-25 protein levels and insulin secretion to control levels. We conclude that increased expression of SNAP-25 is associated with NEFA-induced impairment of insulin secretion in mouse islets.     

ATP-dependent remodeling by SWI/SNF and ISWI proteins stimulates V(D)J cleavage of 5 S arrays

Control of V(D)J recombination is critical for the generation of a fully developed immune repertoire. The molecular mechanisms underlying the regulation of antigen receptor gene assembly are beginning to be revealed. Here we studied the influence of chromatin modifications on V(D)J cleavage of a polynucleosomal substrate, in which V(D)J cleavage is greatly reduced compared with naked DNA. ATP-dependent remodeling by human SWI/SNF (hSWI/SNF) in the presence of HMG1 led to a substantial increase of cleavage by the recombination activation gene (RAG) proteins. Either BRG1, the ATPase subunit of hSWI/SNF, or SNF2h, the ATPase of human ISWI complexes, was capable of stimulating V(D)J cleavage of the array, although these remodelers act by different mechanisms. No effect of histone hyperacetylation was detectable in this system. As is observed on naked DNA, in the presence of core RAG1, the full-length RAG2 protein proved to be more active than core RAG2 on these polynucleosomal arrays, reinforcing the importance of the RAG2 C-terminal domain for efficient recombination. Comparison of 5 S array cleavage by the RAG proteins or by the restriction enzyme HhaI after remodeling by hSWI/SNF suggested that RAG proteins and HhaI might have different requirements for maximal accessibility of the substrate.     

Ordered DNA release and target capture in RAG transposition

Following V(D)J cleavage, the newly liberated DNA signal ends can be either fused together into a signal joint or used as donor DNA in RAG-mediated transposition. We find that both V(D)J cleavage and release of flanking coding DNA occur before the target capture step of transposition can proceed; no coding DNA is ever detected in the target capture complex. Separately from its role in V(D)J cleavage, the DDE motif of the RAG1/2 active site is specifically required for target DNA capture. The requirement for cleavage and release of coding DNA prior to either physical target binding or functional target commitment suggests that the RAG1/2 transposase contains a single binding site for non-RSS DNA that can accommodate either target DNA or coding DNA, but not both together. Perhaps the presence of coding DNA may aid in preventing transpositional resolution of V(D)J recombination intermediates.     

Reconstructing atoll-like mounds from the Frasnian of Belgium

A succession of Frasnian mounds on the southern border of the Dinant Synclinorium (Belgium) was investigated for their facies architecture, sedimentary dynamics and palaeogeographic evolution. Seven mound facies were defined from the Arche (A) and Lion (L) members, each characterized by a specific range of textures and association of organisms (A2/L2: red or pink limestone with stromatactis, corals and crinoids; A3/L3: grey, pink or green limestone with stromatactis, corals and stromatoporoids; A4/L4: grey limestone with corals, peloids and dasycladaceens; A5/L5: grey microbial limestone; A6/L6: grey limestone with dendroid stromatoporoids; A7/L7: grey laminated limestone with fenestrae; and A8/L8: grey bioturbated limestone). Laterally equivalent sediments include substantial reworked material from the buildups and background sedimentation. Textures and fossils suggest that A2/L2 and A3/L3 facies developed close to storm wave base, in a subphotic environment. Facies A4/L4, occurring near fair weather wave base in the euphotic zone, includes lenses of A5/L5 with stromatolitic coatings and thrombolithes. A6/L6 corresponds to a slightly restricted environment and shows a progressive transition to fenestral limestone of A7/L7. This facies was deposited in a moderately restricted intertidal area. A8/L8 developed in a quiet lagoonal subtidal environment. The mounds started with A2/L2 or A3/L3 in which microbial lenses and algal facies A4/L4 became progressively more abundant upwards. Following 20 m of laterally undifferentiated facies, more restricted facies occur in the central part of the buildups. This geometry suggests the initiation of restricted sedimentation, sheltered by bindstone or floatstone facies. The facies interpretation shows that after construction of the lower part of the mounds during a transgression and a sea-level highstand, a lowstand forced reef growth to the margin of the buildups, initiating the development of atoll-like crowns during the subsequent transgressive stage. The persistence of restricted facies results from the balance between sea-level rise and reef growth.     

FunnyBase: a systems level functional annotation of Fundulus ESTs for the analysis of gene expression

Background  

While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes.