Large mitochondrial repeats multiplied during the polymerase chain reaction

The number of repeats in repetitive DNA like micro- and minisatellites is often determined by polymerase chain reaction (PCR). When we counted repeats in an array of mitochondrial repeats in the cattle tick (Boophilus microplus) we found that the number of repeats increased during PCR. Multiplication of the repeats was independent of the primers used to amplify the region, the PCR annealing temperature and the length of the PCR product. The use of PCR to determine the number of repeats in arrays needs to be reassessed. For long repeats, a subset of samples should always be analysed by Southern blot hybridization to confirm the PCR results.

Intragenomic variation in ITS2 rDNA in the louse of humans, Pediculus humanus: ITS2 is not a suitable marker for population studies in this species

The two internal transcribed spacers (ITS) of ribosomal DNA are often used as markers of populations of insects. We studied the ITS2 of the head lice and body lice of humans, to determine whether this gene is a suitable marker of populations of these insects. ITS2 sequences were amplified by PCR from lice from four different countries: Australia, China, Japan and the USA. Direct cycle-sequencing of some of these PCR products gave equivocal nucleotide chromatograms. This indicated that some lice had more than one ITS2 sequence, so we cloned PCR products from these lice. Temperature gradient gel electrophoresis (TGGE) revealed that 50 of the 67 clones we screened had different nucleotide sequences. All lice had several ITS2 types, including those with unequivocal chromatograms. A phylogenetic tree of 15 different ITS2 sequences showed that the sequences from individual lice were not monophyletic. We conclude that the ITS2 is not a useful marker of populations for Pediculus humanus.

Application of biotechnology in breeding lentil for resistance to biotic and abiotic stress

Lentil is a self-pollinating diploid (2n = 14 chromosomes) annual cool season legume crop that is produced throughout the world and is highly valued as a high protein food. Several abiotic stresses are important to lentil yields world wide and include drought, heat, salt susceptibility and iron deficiency. The biotic stresses are numerous and include: susceptibility to Ascochyta blight, caused by Ascochyta lentis; Anthracnose, caused by Colletotrichum truncatum; Fusarium wilt, caused by Fusarium oxysporum; Sclerotinia white mold, caused by Sclerotinia sclerotiorum; rust, caused by Uromyces fabae; and numerous aphid transmitted viruses. Lentil is also highly susceptible to several species of Orabanche prevalent in the Mediterranean region, for which there does not appear to be much resistance in the germplasm. Plant breeders and geneticists have addressed these stresses by identifying resistant/tolerant germplasm, determining the genetics involved and the genetic map positions of the resistant genes. To this end progress has been made in mapping the lentil genome and several genetic maps are available that eventually will lead to the development of a consensus map for lentil. Marker density has been limited in the published genetic maps and there is a distinct lack of co-dominant markers that would facilitate comparisons of the available genetic maps and efficient identification of markers closely linked to genes of interest. Molecular breeding of lentil for disease resistance genes using marker assisted selection, particularly for resistance to Ascochyta blight and Anthracnose, is underway in Australia and Canada and promising results have been obtained. Comparative genomics and synteny analyses with closely related legumes promises to further advance the knowledge of the lentil genome and provide lentil breeders with additional genes and selectable markers for use in marker assisted selection. Genomic tools such as macro and micro arrays, reverse genetics and genetic transformation are emerging technologies that may eventually be available for use in lentil crop improvement.

Synchronization of mutually coupled chaotic systems

We report on the experimental observation of both basic frequency locking synchronization and chaos synchronization between two mutually coupled chaotic subsystems. We show that these two kinds of synchronization are two stages of interaction between coupled chaotic systems. in particular the chaos synchronization could be understood as a state of phase locking between coupled chaotic oscillations.

Immunolocalization of the 38.3 kDa calponin-like protein in stratified muscles of the tail of Schistosoma japonicum cercariae

Calponins are proteins present in vertebrate smooth musculature where they occur in association with thin myofilaments. Calponins are not present in vertebrate or invertebrate striated muscles. The blood fluke Schistosoma japonicum expresses a 38.3-kDa protein that bears substantial homology with vertebrate calponin and occurs entirely within smooth musculature of adults. Calponin-like immunoreactivity has been demonstrated in smooth muscles of many invertebrate phyla. The Schistosoma japonicum calponin has been localised in smooth myofibrils of adults where it is associated with myofilaments and sarcoplasmic reticulum. In this study, the ultrastructural localisation of the protein in muscles of S. japonicum cercariae is described. The protein is present in smooth muscles of the forebody and the stratified muscle of the tail. Within the stratified layer, the protein occurs predominantly in transverse arrays of sarcoplasmic reticulum. The localisation data suggest that the calponin-like protein of S. japonicum is involved in contraction of the stratified tail muscle. Furthermore, the presence of a calponin system in the stratified muscle suggests that this muscle is simply a superior form of muscle, closely related to smooth muscles that use a caldesmin-calponin system in contraction. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Structural and morphological anomalies in magnetosomes: possible biogenic origin for magnetite in ALH84001

We report biogenic magnetite whiskers, with axial ratios of 6: 1, elongated in the [1 1 1]. [1 1 2] and [1 0 0] directions, resembling the magnetite whiskers detected in the Martian meteorite ALH84001 by Bradley ct nl., and interpreted by those authors as evidence of vapour-phase (abiogenic) growth. Magnetosomal whiskers with extended defects consistent with screw dislocations and magnetosomes resembling flattened twinned platelets, as well as other twinning phenomena and other structural defects, are also reported here. Magnetosomes with teardrop-shaped. cuboidal. irregular and jagged structures similar to those detected in ALH84001 by McKay et al.. coprecipitation of magnetite possibly with amorphous calcium carbonate, coprecipitation of magnetite possibly with amorphous silica, the incorporation of titanium in volutin inclusions and disoriented arrays of magnetosomes are also described. These observations demonstrate that the structures of the magnetite particles in ALH84001. their spatial arrange ment and coprecipitation with carbonates and proximity to silicates are consistent with being biogenic. Electron-beam-induced flash-melting of magnetosomes produced numerous screw dislocations in the (1 1 1). (1 0 0) and (1 1 0) lattice planes and induced fusion of platelets. From this, the lack of screw dislocations reported in the magnetite particles in ALH84001 (McKay et al.. and Bradley et al.) indicates that they have a low-temperature origin.

Novel colloidal materials for high-throughput screening applications in drug discovery and genomics

Tracking the reaction history is the means of choice to identify bioactive compounds in large combinatorial libraries. The authors describe two approaches to synthesis on silica beads: a) addition of a reporter dye tag during each synthesis step (see Figure), which attaches itself to the bead by colloidal forces, and b) encapsulating arrays of fluorescent dyes into the beads to encode them uniquely, for recognition with a flow cytometer after each reaction step.

Optical encoding of microbeads for gene screening: alternatives to microarrays

Rapid access to genetic information is central to the revolution presently occurring in the pharmaceutical industry, particularly In relation to novel drug target identification and drug development. Genetic variation, gene expression, gene function and gene structure are just some of the important research areas requiring efficient methods of DNA screening. Here, we highlight state-of-the-art techniques and devices for gene screening that promise cheaper and higher-throughput yields than currently achieved with DNA microarrays. We include an overview of existing and proposed bead-based strategies designed to dramatically increase the number of probes that can be interrogated in one assay. We focus, in particular, on the issue of encoding and/or decoding (bar-coding) large bead-based libraries for HTS.

Fitting a mixture model to three-mode three-way data with missing information

When the data consist of certain attributes measured on the same set of items in different situations, they would be described as a three-mode three-way array. A mixture likelihood approach can be implemented to cluster the items (i.e., one of the modes) on the basis of both of the other modes simultaneously (i.e,, the attributes measured in different situations). In this paper, it is shown that this approach can be extended to handle three-mode three-way arrays where some of the data values are missing at random in the sense of Little and Rubin (1987). The methodology is illustrated by clustering the genotypes in a three-way soybean data set where various attributes were measured on genotypes grown in several environments.

Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively

The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER), Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CIM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.

A beam-forming network for a circular switched-beam phased array antenna

This paper reports on the design and development of a dividing/phasing network for a compact switched-beam array antenna for Land-vehicle mobile satellite communications, The device is formed by a switched radial divider/combiner and 1-bit phase shifters and generates a sufficient number of beams for the proper satellite tracking.

Transmit array of transistor amplifiers illuminated by a patch array in the reactive near-field region

In this paper, a small transmit array of transistor amplifiers illuminated by a passive array of microstrip patches in the reactive near-field region is investigated as a power-combining structure. The two cases considered are when the transmit array radiates in a free space and when a passive array similar to the one used for illumination collects the radiated power. A comparison of the performance of the proposed structure against the alternative one, which uses a conventional horn antenna as a power-launching/receiving device, is also presented.

The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

Protein targeting to the plasma membrane of adult skeletal muscle fiber: An organized mosaic of functional domains

The plasma membrane of differentiated skeletal muscle fibers comprises the sarcolemma, the transverse (T) tubule network, and the neuromuscular and muscle-tendon junctions. We analyzed the organization of these domains in relation to defined surface markers, beta -dystroglycan, dystrophin, and caveolin-3, These markers were shown to exhibit highly organized arrays along the length of the fiber. Caveolin-3 and beta -dystroglycan/dystrophin showed distinct, but to some extent overlapping, labeling patterns and both markers left transverse tubule openings clear. This labeling pattern revealed microdomains over the entire plasma membrane with the exception of the neuromuscular and muscle-tendon junctions which formed distinct demarcated macrodomains. Our results suggest that the entire plasma membrane of mature muscle comprises a mosaic of T tubule domains together with sareolemmal caveolae and beta -dystroglycan domains. The domains identified with these markers were examined with respect to targeting of viral proteins and other expressed domain-specific markers, We found that each marker protein was targeted to distinct microdomains, The macrodomains were intensely labeled with all our markers. Replacing the cytoplasmic tail of the vesicular stomatitis virus glycoprotein with that of CD4 resulted in retargeting from one domain to another. The domain-specific protein distribution at the muscle cell surface may be generated by targeting pathways requiring specific sorting information but this trafficking is different from the conventional apical-basolateral division. (C) 2001 Academic Press.

Alterations in gene expression and activity during squamous cell carcinoma development

This study focuses on characterizing the genetic and biological alterations associated with squamous cell carcinoma development. Normal human epidermal keratinocytes (HEKs), cells isolated from a preneoplastic lesion (IEC-1), and two neoplastic cell lines, SCC-25 and COLD-16, were grown as raft cultures, and their gene expression profiles were screened using cDNA arrays. Our data indicated that the expression levels of at least 37 genes were significantly (P less than or equal to 0.05; 1.9% of genes screened) altered in neoplastic cells compared with normal cells. Of these genes, 10 genes were up-regulated and 27 genes were down-regulated in the neoplastic cells. In addition, 51% of the genes altered in the neoplastic cells were already altered in the preneoplastic IEC-1 cells. Immunohistochemical staining of patient tumors was used to verify the cDNA array analysis. Our analysis indicated that alterations in genes associated with extracellular matrix production and apoptosis are disrupted in preneoplastic cells, whereas later stages of neoplasia are associated with alterations in gene expression for genes involved in DNA repair or epidermal growth factor (EGF) receptor/mitogen-activated protein kinase kinase (MAPKK)/MAPK/activator protein-1 (AP-1) signaling. Subsequent functional analysis of the alterations in expression of the EGF receptor/MAPKK/MAPK/AP-1 genes suggested they did not contribute to the neoplastic phenotype.