Gene expression profiles in murine hematopoietic stem cells revisited: Analysis of cDNA libraries reveals high levels of translational and metabolic activities

Gene expression studies from hematopoietic stem cell (HSC) populations purified to variable degrees have defined a set of sternness genes. Unexpectedly, results also hinted toward a HSC chromatin poised in a wide-open state. With the aim of providing a robust tool for further studies into the molecular biology of HSCs, the studies herein describe the construction and comparative molecular analysis of A-phage cDNA libraries from highly purified HSCs that retained their long-term repopulating activities (long-term HSCs [LT-HSCs]) and from short-term repopulating HSCs that were largely depleted of these activities. Microarray analysis of the libraries confirmed the previous results but also revealed an unforeseen preferential expression of translation- and metabolism-associated genes in the LT-HSCs. Therefore, these data indicate that HSCs are quiescent only in regard of proliferative activities but are in a state of readiness to provide the metabolic and translational activities required after induction of proliferation and exit from the HSC pool.

Comparison of selected methods for measuring the virulence properties of Listeria spp.

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.

Evolutionary turnover of mammalian transcription start sites

Alignments of homologous genomic sequences are widely used to identify functional genetic elements and study their evolution. Most studies tacitly equate homology of functional elements with sequence homology. This assumption is violated by the phenomenon of turnover, in which functionally equivalent elements reside at locations that are nonorthologous at the sequence level. Turnover has been demonstrated previously for transcription-factor-binding sites. Here, we show that transcription start sites of equivalent genes do not always reside at equivalent locations in the human and mouse genomes. We also identify two types of partial turnover, illustrating evolutionary pathways that could lead to complete turnover. These findings suggest that the signals encoding transcription start sites are highly flexible and evolvable, and have cautionary implications for the use of sequence-level conservation to detect gene regulatory elements.

Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome

Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did flat contain ail apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-rnRNA contamination during library construction, or alternatively represent nonspecific transcriptional noise. Here we Show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts Of unknown function whose expression appears to be regulated. The ncRNA transcripts have larger exons and fewer introns than protein-coding transcripts. Analysis of the genomic landscape around these sequences indicates that some cDNA clones were produced not from terminal poly(A) tracts but internal priming sites within longer transcripts, only a minority of which is encompassed by known genes. A significant proportion of these transcripts exhibit tissue-specific expression patterns, as well as dynamic changes in their expression in macrophages following lipopolysaccharide Stimulation. Taken together, the data provide strong support for the conclusion that ncRNAs are an important, regulated component of the mammalian transcriptome.

Mimicking the action of GroEL in molecular dynamics simulations: Application to the refinement of protein structures

Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL. GroES then encapsulates the substrate and triggers its release into the central cavity of the GroEL/ES complex for folding. In this work, we investigate the possibility to facilitate protein folding in molecular dynamics simulations by mimicking the effects of GroEL/ES namely, repeated binding and release, together with spatial confinement. During the binding stage, the (metastable) partially folded proteins are allowed to attach spontaneously to a hydrophobic surface within the simulation box. This destabilizes the structures, which are then transferred into a spatially confined cavity for folding. The approach has been tested by attempting to refine protein structural models generated using the ROSETTA procedure for ab initio structure prediction. Dramatic improvements in regard to the deviation of protein models from the corresponding experimental structures were observed. The results suggest that the primary effects of the GroEL/ES system can be mimicked in a simple coarse-grained manner and be used to facilitate protein folding in molecular dynamics simulations. Furthermore, the results Sur port the assumption that the spatial confinement in GroEL/ES assists the folding of encapsulated proteins.

Molecular crowding effects of linear polymers in protein solutions

Measurement of protein-polymer second virial coefficients (B-AP) by sedimentation equilibrium studies of carbonic anhydrase and cytochrome c in the presence of dextrans (T10-T80) has revealed an inverse dependence of B-AP upon dextran molecular mass that conforms well with the behaviour predicted for the excluded-volume interaction between a spherical protein solute A and a random-flight representation of the polymeric cosolute P. That model of the protein-polymer interaction is also shown to provide a reasonable description of published gel chromatographic and equilibrium dialysis data on the effect of polymer molecular mass on BAP for human serum albumin in the presence of polyethylene glycols, a contrary finding from analysis of albumin solubility measurements being rejected on theoretical grounds. Inverse dependence upon polymer chainlength is also the predicted excluded-volume effect on the strength of several types of macromolecular equilibria-protein isomerization, protein dimerization, and 1 : 1 complex formation between dissimilar protein reactants. It is therefore concluded that published experimental observations of the reverse dependence, preferential reaction enhancement within DNA replication complexes by larger polyethylene glycols, must reflect the consequences of cosolute chemical interactions that outweigh those of thermodynamic nonideality arising from excluded-volume effects. (c) 2005 Elsevier B.V. All rights reserved.

Molecular determinants of ginkgolide binding in the glycine receptor pore

Ginkgolides are potent blockers of the glycine receptor Cl- channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on alpha 1, alpha 2, alpha 1 beta and alpha 2 beta GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the beta subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by beta subunit expression. However, ginkgolide A sensitivity was increased in the alpha 2 beta GlyR relative to the alpha 2 GlyR but not in the alpha 1 beta GlyR relative to the alpha 1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2' and 6' pore-lining positions. The increased ginkgolide A sensitivity of the alpha 2 beta GlyR was transferred to the alpha 1 beta GlyR by the G2'A (alpha 1 to alpha 2 subunit) substitution. In addition, the alpha 1 subunit T6'F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2' residues of both alpha 1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position.

Identification of the dominant translation start site in the attB1 sequence of the pET-DEST42 Gateway vector

Gateway technology is a powerful system for converting a single entry vector into a wide variety of expression vectors. We expressed recombinant influenza matrix protein M1 (FMP), a potent antigen for cytotoxic T cells, using the Gateway vector pET-DEST42 containing the FMP cDNA, and purified the expressed FMP as a single 32 kDa recombinant protein. N-terminal and internal protein sequencing, however, showed that the recombinant FMP contained an extra 10 amino acids fused to the N-terminal of native FMP. Further investigation of the DNA sequence adjacent to the 5'-FMP cDNA indicated that the TTG in the attB1 site (30bp upstream of the ATG in the 5'-FMP cDNA) behaved as a dominant translation start site, resulting in a 10 amino acid extension of the recombinant FMP. Thus, it is possible that recombinant proteins produced by this Gateway vector contain unexpected vector-derived peptides, which may affect experimental outcomes. (c) 2006 Elsevier Inc. All rights reserved.

Molecular diversity in venom from the Australian Brown Snake, Pseudonaja textilis

Venom from the Australian elapid Pseudonaja textilis (Common or Eastern Brown snake), is the second most toxic snake venom known and is the most common cause of death from snake bite in Australia. This venom is known to contain a prothrombin activator complex, serine proteinase inhibitors, various phospholipase A(2)s, and pre-and postsynaptic neurotoxins. In this study, we performed a proteomic identification of the venom using two- dimensional gel electrophoresis, mass spectrometry, and de novo peptide sequencing. We identified most of the venom proteins including proteins previously not known to be present in the venom. In addition, we used immunoblotting and post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Modifications observed include phosphorylation, gamma-carboxylation, and glycosylation. Glycoproteins were further characterized by enzymatic deglycosylation and by lectin binding specificity. The venom contains an abundance of glycoproteins with N-linked sugars that include glucose/mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acids. Additionally there are multiple isoforms of mammalian coagulation factors that comprise a significant proportion of the venom. Indeed two of the identified proteins, a procoagulant and a plasmin inhibitor, are currently in development as human therapeutic agents.

Molecular dynamics characterization of n-octyl-beta-D-glucopyranoside micelle structure in aqueous solution

n-Octyl-beta-D-glueopyranoside (OG) is a non-ionic glycolipid, which is used widely in biotechnical and biochemical applications. All-atom molecular dynamics simulations from two different initial coordinates and velocities in explicit solvent have been performed to characterize the structural behaviour of an OG aggregate at equilibrium conditions. Geometric packing properties determined from the simulations and small angle neutron scattering experiment state that OG micelles are more likely to exist in a non-spherical shape, even at the concentration range near to the critical micelle concentration (0.025 M). Despite few large deviations in the principal moment of inertia ratios, the average micelle shape calculated from both simulations is a prolate ellipsoid. The deviations at these time scales are presumably the temporary shape change of a micelle. However, the size of the micelle and the accessible surface areas were constant during the simulations with the micelle surface being rough and partially elongated. Radial distribution functions computed for the hydroxyl oxygen atoms of an OG show sharper peaks at a minimum van der Waals contact distance than the acetal oxygen, ring oxygen, and anomeric carbon atoms. This result indicates that these atoms are pointed outwards at the hydrophilic/hydrophobic interface, form hydrogen bonds with the water molecules, and thus hydrate the micelle surface effectively. (c) 2005 Elsevier Inc. All rights reserved.

Molecular dynamics simulations of the hydrophobin SC3 at a hydrophobic/hydrophilic interface

Hydrophobins are small (similar to 100 aa) proteins that have an important role in the growth and development of mycelial fungi. They are surface active and, after secretion by the fungi, self-assemble into amphipathic membranes at hydrophobic/hydrophilic interfaces, reversing the hydrophobicity of the surface. In this study, molecular dynamics simulation techniques have been used to model the process by which a specific class I hydrophobin, SC3, binds to a range of hydrophobic/ hydrophilic interfaces. The structure of SC3 used in this investigation was modeled based on the crystal structure of the class II hydrophobin HFBII using the assumption that the disulfide pairings of the eight conserved cysteine residues are maintained. The proposed model for SC3 in aqueous solution is compact and globular containing primarily P-strand and coil structures. The behavior of this model of SC3 was investigated at an air/water, an oil/water, and a hydrophobic solid/water interface. It was found that SC3 preferentially binds to the interfaces via the loop region between the third and fourth cysteine residues and that binding is associated with an increase in a-helix formation in qualitative agreement with experiment. Based on a combination of the available experiment data and the current simulation studies, we propose a possible model for SC3 self-assembly on a hydrophobic solid/water interface.

Molecular mechanisms for the variation of mitochondrial gene content and gene arrangement among chigger mites of the genus Leptotrombidium (Acari : Acariformes)

The gene content of a mitochondrial (mt) genome, i.e., 37 genes and a large noncoding region (LNR), is usually conserved in Metazoa. The arrangement of these genes and the LNR is generally conserved at low taxonomic levels but varies substantially at high levels. We report here a variation in mt gene content and gene arrangement among chigger mites of the genus Leptotrombidium. We found previously that the mt genome of Leptotrombidium pallidum has an extra gene for large-subunit rRNA (rrnL), a pseudo-gene for small-subunit rRNA (PrrnS), and three extra LNRs, additional to the 37 genes and an LNR typical of Metazoa. Further, the arrangement of mt genes of L. pallidum differs drastically from that of the hypothetical ancestor of the arthropods. To find to what extent the novel gene content and gene arrangement occurred in Leptotrombidium, we sequenced the entire or partial mt genomes of three other species, L. akamushi, L. deliense, and L. fletcheri. These three species share the arrangement of all genes with L. pallidum, except trnQ (for tRNA-glutamine). Unlike L. pallidum, however, these three species do not have extra rrnL or PrrnS and have only one extra LNR. By comparison between Leptotrombidium species and the ancestor of the arthropods, we propose that (1) the type of mt genome present in L. pallidum evolved from the type present in the other three Leptotrombidium species, and (2) three molecular mechanisms were involved in the evolution of mt gene content and gene arrangement in Leptotrombidium species.

Molecular recognition of an RNA trafficking element by heterogeneous nuclear ribonucleoprotein A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multitasking protein involved in RNA packaging, alternative splicing of pre-mRNA. telomere maintenance, cytoplasmic RNA trafficking, and translation. It binds short segments of single-stranded nucleic acids, including the A2RE11 RNA element that is necessary and sufficient for cytoplasmic transport of a subset of rnRNAs in oligodendrocytes and neurons. We have explored the structures of hnRNP A2, its RNA recognition motifs (RRMs) and Gly-rich module, and the RRM complexes with A2RE11. Circular dichroism spectroscopy showed that the secondary structure of the first 189 residues of hnRNP A2 parallels that of the tandem beta alpha beta beta alpha beta RRMs of its paralogue, hnRNP A1, previously deduced from X-ray diffraction studies. The unusual GRD was shown to have substantial beta-sheet and beta-turn structure. Sedimentation equilibrium and circular dichroism results were consistent with the tandem RRM region being monomeric and supported earlier evidence for the binding of two A2RE11 oligoribonucleotides to this domain, in contrast to the protein dimer formed by the complex of hnRNP A1 with the telomeric ssDNA repeat. A three-dimensional structure for the N-terminal, two-RRM-containing segment of hnRNP A2 was derived by homology modeling. This structure was used to derive a model for the complex with A2RE11 using the previously described interaction of pairs of stacked nucleotides with aromatic residues on the RRM beta-sheet platforms, conserved in other RRM-RNA complexes, together with biochemical data and molecular dynamics-based observations of inter-RRM mobility.

Regulation of bone biology by prostaglandin endoperoxide H synthases (PGHS): A rose by any other name...

It is well established that prostaglandins are essential mediators of bone resorption and formation. In the early 1990s, it was discovered that enzymatic reactions producing prostaglandins were regulated by two cyclooxygenase enzymes, one producing prostaglandins constitutively in tissues like the stomach, prostaglandin endoperoxide H synthase-1 (PGHS-1 or COX-1), and another induced by mitogens or inflammatory mediators (PGHS-2 or COX-2). This neat distinction has not been maintained because both enzymes act in different cell systems to provide physiological signaling, constitutively or by induction under certain conditions. For example, the regulation patterns of PGHS-1 and PGHS-2 are distinct, but the evidence shows that PGHS-2 functions constitutively in the skeleton. PGHS-2 hits quickly been established, therefore, as a key regulator of bone biology, capable of rapid and transient expression in bone cells, and mediating osteoclastogenesis, mechanotransduction, bone formation and fracture repair. The goal of this review is to Summarize the current state of our knowledge of PGHS regulation of bone metabolism and to identify some of the key unresolved challenges and questions that require further study. (c) 2006 Elsevier Ltd. All rights reserved.

The molecular basis of temperature compensation in the Arabidopsis circadian clock

Circadian clocks maintain robust and accurate timing over a broad range of physiological temperatures, a characteristic termed temperature compensation. In Arabidopsis thaliana, ambient temperature affects the rhythmic accumulation of transcripts encoding the clock components TIMING OF CAB EXPRESSION1 (TOC1), GIGANTEA (GI), and the partially redundant genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). The amplitude and peak levels increase for TOC1 and GI RNA rhythms as the temperature increases (from 17 to 27 degrees C), whereas they decrease for LHY. However, as temperatures decrease ( from 17 to 12 degrees C), CCA1 and LHY RNA rhythms increase in amplitude and peak expression level. At 27 degrees C, a dynamic balance between GI and LHY allows temperature compensation in wild-type plants, but circadian function is impaired in Ihy and gi mutant plants. However, at 12 degrees C, CCA1 has more effect on the buffering mechanism than LHY, as the cca1 and gi mutations impair circadian rhythms more than Ihy at the lower temperature. At 17 degrees C, GI is apparently dispensable for free-running circadian rhythms, although partial GI function can affect circadian period. Numerical simulations using the interlocking-loop model show that balancing LHY/CCA1 function against GI and other evening-expressed genes can largely account for temperature compensation in wild-type plants and the temperature-specific phenotypes of gi mutants.