Geometry Regulates Traction Stresses in Adherent Cells

Cells generate mechanical stresses via the action of myosin motors on the actin cytoskeleton. Although the molecular origin of force generation is well understood, we currently lack an understanding of the regulation of force transmission at cellular length scales. Here, using 3T3 fibroblasts, we experimentally decouple the effects of substrate stiffness, focal adhesion density, and cell morphology to show that the total amount of work a cell does against the substrate to which it is adhered is regulated by the cell spread area alone. Surprisingly, the number of focal adhesions and the substrate stiffness have little effect on regulating the work done on the substrate by the cell. For a given spread area, the local curvature along the cell edge regulates the distribution and magnitude of traction stresses to maintain a constant strain energy. A physical model of the adherent cell as a contractile gel under a uniform boundary tension and mechanically coupled to an elastic substrate quantitatively captures the spatial distribution and magnitude of traction stresses. With a single choice of parameters, this model accurately predicts the cell’s mechanical output over a wide range of cell geometries.     

Multiple Replication Origins of Halobacterium sp. Strain NRC-1: Properties of the Conserved orc7-Dependent oriC1

The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and -200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.Archaea are of considerable interest due to their unusual phylogenetic position and the similarity of their information transfer system to that of eukaryotes. In particular, studies of DNA replication in archaea have revealed characteristics of both bacterial and eukaryotic systems (1). While genome sequencing has shown that archaeal and bacterial genomes are composed of a single or few circular chromosomes, comparative genomic studies have found that most components of the archaeal DNA replication machinery, such as the origin recognition proteins, DNA polymerases, helicases, and primases, are similar to eukaryotic proteins. The hybrid nature of archaeal DNA replication systems raises important questions regarding the mechanism by which they select an origin(s) for initiation and coordinate orderly DNA replication and segregation into daughter cells.Our understanding of DNA replication in archaea has thus far been based primarily on bioinformatic studies, with experimental analysis restricted to only a few tractable systems. An initial study of Pyrococcus species using GC (tetramer) skew analysis suggested that they use a single, unique origin of replication in their chromosomes. Subsequent [³H]uracil labeling analysis of Pyrococcus abyssi (21) showed that newly synthesized DNA mapped to the predicted replication origin region, which contained the only orc gene in the genome, a D family DNA polymerase gene, and a DNA sliding clamp loader subunit. In addition, two-dimensional gel analysis of replicating molecules confirmed the location of the DNA replication origin near the orc1 gene of P. abyssi, with predicted origin binding sequences and AT-rich DNA unwinding elements nearby (18). An investigation of DNA replication in Aeropyrum pernix used a combination of biochemical and two-dimensional gel electrophoresis and identified two potential sites of replication initiation, on opposite sides of the circular genome (14, 28). One of these sites (oriC1_Ap) contained four origin recognition boxes and an AT-rich region and was shown to be bound by the ORC1 gene. The other site (oriC2_Ap) contained repeat elements without an intervening AT-rich region and has been shown by two-dimensional gel electrophoresis to contain an active replication origin (28). An examination of replication in two Sulfolobus spp., Sulfolobus solfataricus and Sulfolobus acidocaldarius (16, 30), by use of a combination of bioinformatic and two-dimensional gel analysis and of marker frequency by use of DNA microarrays identified three well-separated replication origins per genome. Only two of the three origins were originally identified, due to their linkage to orc genes and conserved origin binding sequences, while the third was identified by marker frequency analysis (MFA). Using partially synchronized cells of S. acidocaldarius, the origins were shown to initiate DNA replication synchronously, indicating a highly coordinated and regulated process. Biochemical analysis has shown that either two or all three Orc proteins are able to bind to all Sulfolobus origins; however, binding at the third origin is considerably weaker (29). Replication origins were also recently identified in Methanothermobacter thermoautotrophicus (17).Our laboratory has been investigating DNA replication in a halophilic archaeon capable of growth at saturating NaCl concentrations. The model system, Halobacterium sp. strain NRC-1, was one of the earliest archaeal genomes to be sequenced (23) and provided a DNA knockout method, utilizing the selectable and counterselectable ura3 gene, for genetic analysis (25). The NRC-1 genome was found to be organized into a 2-Mbp chromosome and two large and partially redundant extrachromosomal elements, pNRC100 and pNRC200. The genome sequence showed that the orc gene family was highly expanded, with four genes (orc6, -7, -8, and -10) distributed in the chromosome and six genes (orc1, -2, -3, -4, -5, and -9) in pNRC200, one of which (orc9) was also present in pNRC100. Three rep genes thought to be important for replication initiation were present in one (repJ in pNRC100) or both (repH and repI) of the extrachromosomal elements. Regions near two of these genes, orc7 and repH, were shown to harbor autonomous replicating ability and to contain inverted repeat sequences (IRs) and an AT-rich presumptive DNA unwinding region detectable by χ² analysis (3, 22). Additionally, GC/oligomer skew analyses of Halobacterium sp. strain NRC-1 showed multiple inflection points in the chromosome, suggestive of multiple replication origins in this strain (15, 34).Halobacterium sp. strain NRC-1 is the only archaeal system where gene mutation analysis has established which predicted DNA replication genes are essential to cells (2). As expected, two DNA polymerases (one B family and one D family polymerase), the MCM DNA helicase, DNA primase (Pri1/Pri2), the sliding clamp (PCNA), and flap endonuclease (Rad2) were all found to be essential. However, one B family DNA polymerase gene and 8 of the 10 orc and cdc6 genes, including the orc7 gene, were found to be nonessential by deletion analysis. Only the orc2 gene in pNRC200 and the orc10 gene in the chromosome were found to be essential, suggesting a critical role(s) for these genes in DNA replication.In this study, we used a combination of MFA, employing whole-genome DNA microarrays, the ura3-based gene knockout method, and replication initiation point (RIP) analysis to further investigate DNA replication in Halobacterium sp. strain NRC-1. Our results indicate that initiation of DNA replication in NRC-1 is more complex than originally anticipated, with multiple origins likely present on the chromosome and the extrachromosomal elements.     

Saline Systems highlights for 2005

On the 4^th of July, 2005, the Saline Systems editorial group launched the new online open access journal, Saline Systems, with BioMed Central as the publisher. The scope of the journal includes both basic and applied research on halophilic organisms and saline environments, from gene systems to ecosystems. The stated goal of the journal is to meet publication needs for researchers working in coastal and inland saline environments and provide an interdisciplinary and readily accessible forum for scientists worldwide. The inaugural volume of the journal contains a significant number of high quality original research papers and reviews on a wide range of relevant topics. At the end of the launch period, from January 1, 2006 onwards, the journal will be introducing article-processing charges to cover the cost of publication. Charges will be partly or completely waived for authors from BioMed Central institutional subscribers and in cases of financial hardship.     

In silico study of rotavirus VP7 surface accessible conserved regions for antiviral drug/vaccine design

Background

Rotaviral diarrhoea kills about half a million children annually in developing countries and accounts for one third of diarrhea related hospitalizations. Drugs and vaccines against the rotavirus are handicapped, as in all viral diseases, by the rapid mutational changes that take place in the DNA and protein sequences rendering most of these ineffective. As of now only two vaccines are licensed and approved by the WHO (World Health Organization), but display reduced efficiencies in the underdeveloped countries where the disease is more prevalent. We approached this issue by trying to identify regions of surface exposed conserved segments on the surface glycoproteins of the virion, which may then be targeted by specific peptide vaccines. We had developed a bioinformatics protocol for these kinds of problems with reference to the influenza neuraminidase protein, which we have refined and expanded to analyze the rotavirus issue.

Results

Our analysis of 433 VP7 (Viral Protein 7 from rotavirus) surface protein sequences across 17 subtypes encompassing mammalian hosts using a 20D Graphical Representation and Numerical Characterization method, identified four possible highly conserved peptide segments. Solvent accessibility prediction servers were used to identify that these are predominantly surface situated. These regions analyzed through selected epitope prediction servers for their epitopic properties towards possible T-cell and B-cell activation showed good results as epitopic candidates (only dry lab confirmation).

Conclusions

The main reasons for the development of alternative vaccine strategies for the rotavirus are the failure of current vaccines and high production costs that inhibit their application in developing countries. We expect that it would be possible to use the protein surface exposed regions identified in our study as targets for peptide vaccines and drug designs for stable immunity against divergent strains of the rotavirus. Though this study is fully dependent on computational prediction algorithms, it provides a platform for wet lab experiments.     

Function and biotechnology of extremophilic enzymes in low water activity

ABSTRACT: Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology.