Effects of PRE and POST therapy drug-pressure selected mutations on HIV-1 protease conformational sampling

Conformational sampling of pre- and post-therapy subtype B HIV-1 protease sequences derived from a pediatric subject infected via maternal transmission with HIV-1 were characterized by double electron–electron resonance spectroscopy. The conformational ensemble of the PRE construct resembles native-like inhibitor bound states. In contrast, the POST construct, which contains accumulated drug-pressure selected mutations, has a predominantly semi-open conformational ensemble, with increased populations of open-like states. The single point mutant L63P, which is contained in PRE and POST, has decreased dynamics, particularly in the flap region, and also displays a closed-like conformation of inhibitor-bound states. These findings support our hypothesis that secondary mutations accumulate in HIV-1 protease to shift conformational sampling to stabilize open-like conformations, while maintaining the predominant semi-open conformation for activity.     

Alternative Acetate Production Pathways in Chlamydomonas reinhardtii during Dark Anoxia and the Dominant Role of Chloroplasts in Fermentative Acetate Production

Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.     

mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence.

We identified and produced antibodies to the major proteins that interact with poly(A)+ RNAs in the yeast Saccharomyces cerevisiae. The major proteins which were cross-linked by UV light to poly(A)+ RNA in intact yeast cells had apparent molecular weights of 72,000, 60,000, and 50,000. The poly(A) segment of the RNA was selectively cross-linked to the 72,000-molecular-weight protein (72K protein). Mice immunized with purified UV-cross-linked RNA-protein (RNP) complexes produced antibodies to the three major RNP proteins. A yeast genomic DNA library constructed in the lambda gt11 expression vector was screened with the anti-RNP serum, and recombinant bacteriophage clones were isolated. One recombinant phage, lambda YPA72.1, bearing a 2.5-kilobase insert, produced a large beta-galactosidase-RNP fusion protein. Affinity-selected antibodies from the anti-RNP serum on this fusion protein recognized a single 72K protein which was cross-linked to the poly(A) segment of RNA in the intact cell. Furthermore, the fusion protein of lambda YPA72.1 had specific poly(A)-binding activity. Therefore, lambda YPA72.1 encodes the 72K poly(A)-binding protein. Immunofluorescence microscopy showed that this protein was localized in the cytoplasm. Hybrid-selected mRNA translated in vitro produced the 72K poly(A)-binding protein, and mRNA blot analysis detected a single 2.1-kilobase mRNA. DNA blot analysis suggested a single gene for the poly(A)-binding protein. DNA sequence analysis of genomic clones spanning the entire gene revealed a long open reading frame encoding a 64,272-molecular-weight protein with several distinct domains and repeating structural elements. A sequence of 11 to 13 amino acids is repeated three times in this protein. Strikingly, this repeated sequence (RNP consensus sequence) is highly homologous to a sequence that is repeated twice in a major mammalian heterogeneous nuclear RNP protein, A1. The conservation of the repetitive RNP consensus sequence suggests an important function and a common evolutionary origin for messenger RNP and heterogeneous nuclear RNP proteins.     

Homogeneous amplification nucleobase quenching assay to detect the E474Q LCHAD deficiency mutation

Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency is a rare and potentially fatal autosomal recessive disorder of fatty acid metabolism. Early institution of dietary therapy is essential and places a premium on rapid diagnosis. Pregnancy with an LCHAD-deficient fetus is often complicated in the third trimester by liver disease, particularly acute fatty liver of pregnancy. All cases of isolated LCHAD deficiency have at least one copy of the E474Q mutation in the gene encoding the alpha-subunit of the mitochondrial trifunctional protein. Previously published methods for detecting this mutation are based upon allele-specific restriction enzyme digestion of a DNA fragment generated by PCR, followed by gel electrophoresis to resolve the products. We have developed a faster and less expensive assay for the E474Q mutation using PCR followed directly by differential melting of a fluorescently labeled oligodeoxyribonucleotide probe, using nucleobase quenching to detect probe hybridization.     

Structural and evolutionary division of phosphotyrosine binding (PTB) domains

Proteins encoding phosphotyrosine binding (PTB) domains function as adaptors or scaffolds to organize the signaling complexes involved in wide-ranging physiological processes including neural development, immunity, tissue homeostasis and cell growth. There are more than 200 proteins in eukaryotes and nearly 60 human proteins having PTB domains. Six PTB domain encoded proteins have been found to have mutations that contribute to inherited human diseases including familial stroke, hypercholesteremia, coronary artery disease, Alzheimer's disease and diabetes, demonstrating the importance of PTB scaffold proteins in organizing critical signaling complexes. PTB domains bind both peptides and headgroups of phosphatidylinositides, utilizing two distinct binding motifs to mediate spatial organization and localization within cells. The structure of PTB domains confers specificity for binding peptides having a NPXY motif with differing requirements for phosphorylation of the tyrosine within this recognition sequence. In this review, we use structural, evolutionary and functional analysis to divide PTB domains into three groups represented by phosphotyrosine-dependent Shc-like, phosphotyrosine-dependent IRS-like and phosphotyrosine-independent Dab-like PTBs, with the Dab-like PTB domains representing nearly 75% of proteins encoding PTB domains. In addition, we further define the binding characteristics of the cognate ligands for each group of PTB domains. The signaling complexes organized by PTB domain encoded proteins are largely unknown and represents an important challenge in systems biology for the future.     

Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors

We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completely inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport.     

Above-ground biomass estimation in closed canopy Neotropical forests using lidar remote sensing: factors affecting the generality of relationships

Aim Previous studies have developed strong, site‐specific relationships between canopy metrics from lidar (light detecting and ranging) remote sensing data and forest structural characteristics such as above‐ground biomass (AGBM), but the generality of these relationships is unknown. In this study, we examine the generality of relationships between lidar metrics and forest structural characteristics, including AGBM, from two study areas in Central America with different precipitation patterns. Location A series of tropical moist forest sites in Panama and a tropical wet forest in Costa Rica. Methods Canopy metrics (e.g. canopy height) were calculated from airborne lidar data. Basal area, mean stem diameter and AGBM were calculated from measurements taken as a part of ongoing forest dynamics studies in both areas. We examined the generality of relationship between lidar metrics and forest structure, and possible environmental effects (e.g. leaf phenology). Results We found that lidar metrics were strongly correlated (R²: 0.65–0.92) with mean stem diameter, basal area and AGBM in both regions. We also show that the relationships differed between these regions. Deciduousness of canopy trees in the tropical moist forest area accounted for the differences in predictive equations for stem diameter and basal area. The relationships between lidar metrics and AGBM, however, remained significantly different between the two study areas even after adjusting for leaf drop. We attribute this to significant differences in the underlying allometric relationships between stem diameter and AGBM in tropical wet and moist forests. Conclusions Important forest structural characteristics can be estimated reliably across a variety of conditions sampled in these closed‐canopy tropical forests. Environmental factors such as drought deciduousness have an important influence on these relationships. Future efforts should continue to examine climatic factors that may influence the generality of the relationships between lidar metrics and forest structural characteristics and assess more rigorously the generality of field‐derived allometric relationships.     

Sequence and structure of the extrachromosomal palindrome encoding the ribosomal RNA genes in Dictyostelium

Ribosomal RNAs (rRNAs) are encoded by multicopy families of identical genes. In Dictyostelium and other protists, the rDNA is carried on extrachromosomal palindromic elements that comprise up to 20% of the nuclear DNA. We present the sequence of the 88 kb Dictyostelium rDNA element, noting that the rRNA genes are likely to be the only transcribed regions. By interrogating a library of ordered YAC clones, we provide evidence for a chromosomal copy of the rDNA on chromosome 4. This locus may provide master copies for the stable transmission of the extrachromosomal elements. The extrachromosomal elements were also found to form chromosome-sized clusters of DNA within nuclei of nocodazole-treated cells arrested in mitosis. These clusters resemble true chromosomes and may allow the efficient segregation of the rDNA during mitosis. These rDNA clusters may also explain the cytological observations of a seventh chromosome in this organism.