Printing protein arrays from DNA arrays

We describe a method, DNA array to protein array (DAPA), which allows the 'printing' of replicate protein arrays directly from a DNA array template using cell-free protein synthesis. At least 20 copies of a protein array can be obtained from a single DNA array. DAPA eliminates the need for separate protein expression, purification and spotting, and also overcomes the problem of long-term functional storage of surface-bound proteins.     

Analysis of chromatin-associated fiber arrays

The distribution of constitutive heterochromatin has been investigated in four chromosomal races of the grasshopper Caledia captiva (2n= 23 /24 ) by the C-banding technique. Each of the four races was found to have a distinctive banding pattern which is associated with the inter-racial differences in chromosomal rearrangements. — The Ancestral race has a telocentric chromosome complement with large procentric C-bands which are structurally double on six pairs of chromosomes. The centromeres are unstained. — The General Purpose race has a C-banding pattern very similar to that seen in other Acridine grasshoppers with the majority of its chromosomes showing a centromeric localisation of the bands. — The two southern races, which show a complex polymorphism for presumed pericentric inversions on all twelve chromosomes, also show an unusually high level of interstitial and terminal C-bands. The different locations and numbers of these bands allow unambiguous identification of all the chromosome pairs within the complement. — In two cases, there is good evidence to indicate that a C-band redistribution between acrocentric and metacentric chromosomes has occurred by pericentric inversion. Furthermore, C-band variation on the long arm of the metacentric X-chromosome indicates the presence of a large paracentric inversion. This double inversion system has involved over 95% of the X-chromosome. — The interstitial and terminal C-bands probably have not resulted from heterochromatin movement within the complement but, more likely, have arisen by saltatory duplication of pre-existing sequences on the chromosome. — A new nomenclature system for banded chromosomes is proposed which allows most kinds of chromosomal restructuring and rearrangement to be adequately enumerated.     

Microbial sensor cell arrays

Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in incorporating multiple reporter cells into a single platform; the technology now allows the simultaneous scrutiny of a large collection of sensor strains. We review current aspects in cell array technology with emphasis on microbial sensor arrays. We consider various techniques for patterning live cells on solid surfaces, describe different array-based applications and devices, and highlight recent efforts for live cell storage. We review mathematical approaches for deciphering the data emanating from bioreporter collections, and discuss the future of single cell arrays. Innovative technologies for cell patterning, preservation and interpretation are continuously being developed; when they all mature, cell arrays may become an efficient analytical tool, in a scope resembling that of DNA microarray biochips.     

Twist propagation in dinucleosome arrays

We present a Monte Carlo simulation study of the distribution and propagation of twist from one DNA linker to another for a two-nucleosome array subjected to externally applied twist. A mesoscopic model of the array that incorporates nucleosome geometry along with the bending and twisting mechanics of the linkers is employed and external twist is applied in stepwise increments to mimic quasistatic twisting of chromatin fibers. Simulation results reveal that the magnitude and sign of the imposed and induced twist on contiguous linkers depend strongly on their relative orientation. Remarkably, the relative direction of the induced and applied twist can become inverted for a subset of linker orientations—a phenomenon we refer to as “twist inversion”. We characterize the twist inversion, as a function of relative linker orientation, in a phase diagram and explain its key features using a simple model based on the geometry of the nucleosome/linker complex. In addition to twist inversion, our simulations reveal “nucleosome flipping”, whereby nucleosomes may undergo sudden flipping in response to applied twist, causing a rapid bending of the linker and a significant change in the overall twist and writhe of the array. Our findings shed light on the underlying mechanisms by which torsional stresses impact chromatin organization.     

Redundancy in genotyping arrays

Despite their unprecedented density, current SNP genotyping arrays contain large amounts of redundancy, with up to 40 oligonucleotide features used to query each SNP. By using publicly available reference genotype data from the International HapMap, we show that 93.6% sensitivity at <5% false positive rate can be obtained with only four probes per SNP, compared with 98.3% with the full data set. Removal of this redundancy will allow for more comprehensive whole-genome association studies with increased SNP density and larger sample sizes.     

Two-hybrid arrays

The two-hybrid system is a genetic method for detecting protein-protein interactions. The assay can be applied to random libraries or arrays of colonies that express defined pairs of proteins. Arrays enable the testing of all possible protein pairs for interactions in a systematic fashion. The array format makes a large number of individual assays comparable and thus greatly simplifies the identification of false positives. Two-hybrid arrays have been used to study interactions among the proteins of yeast, hepatitis C virus, vaccinia virus, Drosophila, Caenorhabditis elegans, mouse and other species, and have already identified thousands of interactions.     

Antibody arrays in cancer research

Antibody arrays have valuable applications in cancer research. Many different antibody array technologies have been developed, each with particular advantages, disadvantages, and optimal applications. The methods have been demonstrated on various sample types, such as serum, plasma, and other bodily fluids; cell culture supernatants; tissue culture lysates; and resected tumor specimens. The applications to cancer research have included profiling proteins to identify candidate biomarkers, characterizing signaling pathways, and the measurement of changes in modification or expression level of cancer-related proteins. Further innovations in the methods and experimental strategies are broadening the scope of the applications and the type of information that can be gathered. These alternate formats and uses of antibody arrays include arrays to measure whole cells, arrays to measure enzyme activities, reverse phase arrays, and bead-based arrays. This article reviews the various types of antibody array methods and their applications to cancer research.     

The construction of customized nucleosomal arrays

A simple, efficient, and reliable method is demonstrated for cloning long tandem arrays of the 601 nucleosomal positioning sequence. In addition, it is shown that such long arrays can be ligated together in vitro with high efficiency. By combining these two procedures it becomes straightforward to synthesize customized arrays that contain different (or variable) nucleosomal repeat lengths (NRLs) and monosome units bearing chemical modifications such as fluorophores, methyl groups, and reaction sites. This is, therefore, an enabling technology for the in vitro study of chromatin structure and function.     

Heterochromatin protein 1 binds transgene arrays

Heterochromatin protein 1 (HP1) of Drosophila and its homologs in vertebrates are key components of constitutive heterochromatin. Here we provide cytological evidence for the presence of heterochromatin within a euchromatic chromosome arm by immunolocalization of HP1 to the site of a silenced transgene repeat array. The amount of HP1 associated with arrays in polytene chromosomes is correlated with the array size. Inverted transposons within an array or increased proximity of an array to blocks of naturally occurring heterochromatin may increase transgene silencing without increasing HP1 labeling. Less dense anti-HP1 labeling is found at transposon arrays in which there is no transgene silencing. The results indicate that HP1 targets the chromatin of transposon insertions and binds more densely at a site with repeated sequences susceptible to heterochromatin formation. Received: 26 June 1998; in revised form: 6 July 1998 / Accepted: 12 July 1998     

A comparison of Affymetrix gene expression arrays

Background  

Affymetrix GeneChips™ are an important tool in many facets of biological research. Recently, notable design changes to the chips have been made. In this study, we use publicly available data from Affymetrix to gauge the performance of three human gene expression arrays: Human Genome U133 Plus 2.0 (U133), Human Exon 1.0 ST (HuEx) and Human Gene 1.0 ST (HuGene).     

Discrete membrane arrays

This review describes various methods for the attachment of phospholipid bilayers to solid supports. The simplest approach involves vesicle unrolling onto a surface that has been previously modified with a continuous self-assembled monolayer (SAM). The choice of a suitable SAM can lead to the formation of attached bilayers that have the desired biomimetic properties and are suitable for studying transmembrane proteins. However, there are intrinsic problems associated with this approach if one is interested in studying ion transport phenomena. In particular, the relatively low resistance values found for such bilayers do not permit studies of single ion channels. For such studies to be carried out the background leakage through the lipid film must be greatly reduced. In an attempt to reduce the problems of leakage we have formed patterned SAMs in which a blocking, hydrophobic, layer covers 90% of the electrode surface. The remaining portion of the surface, which is hydrophilic, supports the formation of a bilayer. This approach has led to an improvement in the quality of the bilayers formed but has still not provided bilayers with sufficiently high specific resistances to study single ion channels. Finally, we describe new approaches based on the formation of bilayers suspended over small apertures. These ‘suspended’ bilayers are similar in structure to those used in black lipid membrane experiments and give rise to highly blocking bilayer membranes. Unfortunately, this approach requires the use of solvents to create the suspended bilayer and they are relatively fragile.     

Single molecular functional assay of ferritin arrays

In situ functional assay of each ferritin molecule in single-layer 2D arrays for horse spleen apoferritin and recombinant horse L- and human H-apoferritins was conducted by observing the iron-cores formed in the arrays by TEM. The study of the time-course, pH-dependence, and temperature-dependence of the function confirmed the iron-core formation to be due to the native function of apoferritins in array. Dark-field TEM imaging revealed that there was crystallinity in the cores in the array of recombinant human H-apoferritin. This iron-core formation was perfectly preserved in the array even after 3 months of storage at room temperature and low humidity. Moreover, about 50% of the function was found to remain in the array after it was exposed to 150 ° C in vacuum for 1 hr.     

Graphene microelectrode arrays for neural activity detection

We demonstrate a method to fabricate graphene microelectrode arrays (MEAs) using a simple and inexpensive method to solve the problem of opaque electrode positions in traditional MEAs, while keeping good biocompatibility. To study the interface differences between graphene–electrolyte and gold–electrolyte, graphene and gold electrodes with a large area were fabricated. According to the simulation results of electrochemical impedances, the gold–electrolyte interface can be described as a classical double-layer structure, while the graphene–electrolyte interface can be explained by a modified double-layer theory. Furthermore, using graphene MEAs, we detected the neural activities of neurons dissociated from Wistar rats (embryonic day 18). The signal-to-noise ratio of the detected signal was 10.31 ± 1.2, which is comparable to those of MEAs made with other materials. The long-term stability of the MEAs is demonstrated by comparing differences in Bode diagrams taken before and after cell culturing.     

Discrete membrane arrays

This review describes various methods for the attachment of phospholipid bilayers to solid supports. The simplest approach involves vesicle unrolling onto a surface that has been previously modified with a continuous self-assembled monolayer (SAM). The choice of a suitable SAM can lead to the formation of attached bilayers that have the desired biomimetic properties and are suitable for studying transmembrane proteins. However, there are intrinsic problems associated with this approach if one is interested in studying ion transport phenomena. In particular, the relatively low resistance values found for such bilayers do not permit studies of single ion channels. For such studies to be carried out the background leakage through the lipid film must be greatly reduced. In an attempt to reduce the problems of leakage we have formed patterned SAMs in which a blocking, hydrophobic, layer covers 90% of the electrode surface. The remaining portion of the surface, which is hydrophilic, supports the formation of a bilayer. This approach has led to an improvement in the quality of the bilayers formed but has still not provided bilayers with sufficiently high specific resistances to study single ion channels. Finally, we describe new approaches based on the formation of bilayers suspended over small apertures. These 'suspended' bilayers are similar in structure to those used in black lipid membrane experiments and give rise to highly blocking bilayer membranes. Unfortunately, this approach requires the use of solvents to create the suspended bilayer and they are relatively fragile.     

Living microlens arrays

Both individual cells and sheets of cells exert traction forces on the substrate and these forces have been investigated using a wide range of methods. Here we compare the mechanical properties of fibroblasts and epithelial cells using a novel surface geometry. Living cells are added to a thin film of polystyrene [PS] attached to a substrate of crosslinked poly(dimethyl siloxane) [PDMS] microwells. The contractile nature of the cells attached to the surface and the compliance of the PDMS surface geometry allows the PS thin film to buckle, forming arrays of convex microlenses. The resulting curvature of the microlenses allows us to determine the applied strain of growing cell sheets. We report that a monolayer of epithelial cells exerts more stress on the substrate than fibroblasts and attribute this to the collective behavior of the epithelium. By subsequently adding different chemical triggers to the system, the contractile nature of the cells changes, thus modifying the focal length of the microlenses. Together, these findings demonstrate the importance of studying the mechanics of cell sheets and also introduce a new design paradigm for advanced materials, offering great promise for a range of applications.     

RAID level selection for heterogeneous disk arrays

Heterogeneous Disk Arrays (HDAs) allow resource sharing of their hardware by multiple RAID levels. RAID1 (mirrored disks) and RAID5 (distributed parity arrays) are the two RAID levels considered in this study. They are both single disk failure tolerant (1DFT), but differ significantly in their efficiency in processing database workloads. The goal of the study is to maximize the number of Virtual Array (VA) allocations in HDA. We develop an analysis to estimate the load per VA based on a few parameters: the fraction of accesses to small versus large blocks and the fraction of updates versus reads. A VA is allocated according to the RAID level, which minimizes the anticipated load based on input parameters. Operation in normal and degraded mode is considered for comparison purposes, but in fact allocations are carried out using the higher load in degraded mode to ensure that single disk failures will not result in overload. We report on parametric studies to gain insight into circumstances leading to a RAID1 or RAID5 classification. An allocation experiment with a synthetic workload is used to demonstrate the superiority of HDA with respect to purely RAID1 or RAID5 disk arrays. This analytic study can be extended to 2DFT arrays, namely RAID6 versus 3-way replication.