Modular Arrangement Research Articles

Engineered intermodular and intramodular polyketide synthase fusions

Background: Modular polyketide synthases (PKSs) are very large multifunctional enzyme complexes that synthesize a number of medicinally important natural products. The modular arrangement of active sites has made these enzyme systems amenable to combinatorial manipulation for the biosynthesis of novel polyketides. Here, we investigate the involvement of subunit interactions in hybrid and artificially linked PKSs with several series of intermodular and intramodular fusions using the erythromycin (6-deoxyerythronolide B synthase; DEBS) and rapamycin (RAPS) PKSs. Results: Several two-module and three-module derivatives of DEBS were constructed by fusing module 6 to either module 2 or module 3 at varying junctions. Polyketide production by these intramodular fusions indicated that the core set of active sites remained functional in these hybrid modules, although the ketoreductase domain of module 6 was unable to recognize unnatural triketide and tetraketide substrates. Artificial trimodular PKS subunits were also engineered by covalently linking modules 2 and 3 of DEBS, thereby demonstrating the feasibility of constructing single-chain PKSs. Finally, a series of fusions containing DEBS and RAPS domains in module 2 of an engineered trimodular PKS revealed the structural and functional tolerance for hybrid modules created from distinct PKS gene clusters. Conclusions: The general success of the intermodular and intramodular fusions described here demonstrates significant structural tolerance among PKS modules and subunits and suggests that substrate specificity, rather than protein-protein interactions, is the primary determinant of molecular recognition features of PKSs. Furthermore, the ability to artificially link modules may considerably simplify the heterologous expression of modular PKSs in higher eukaryotic systems.

Uses of microchannel plate intensified detectors for imaging applications in the X-ray, EUV and visible wavelength regions

The Rutherford Appleton Laboratory Photon Counting Detector (RALPCD) has been refined to meet project requirements for a flexible imaging arrangement with applications at X-ray, EUV and visible wavelengths. The basic detector design comprises commercially available high gain microchannel plate intensifiers fibre optically coupled to CID or CCD cameras, to form a modular detector arrangement with the appropriate RAL detection and centroiding software. Frames of data from the cameras are detected and centroided in a Transputer or C40 parallel processor array where correction algorithms use look up tables to produce pattern free images at high resolution. Data from completed applications are used to illustrate the performance and future advances are discussed.

Transneuronal Labeling of a Nociceptive Pathway, the Spino-(Trigemino-)Parabrachio-Amygdaloid, in the Rat

Transneuronal tracing of a nociceptive pathway, the spino-(trigemino)-parabrachio-amygdaloid pathway, was performed using an alpha-herpes virus, the Bartha strain of pseudorabies virus (PRV). Microinjection of PRV into the central nucleus of the amygdala (Ce) resulted in progressive retrograde and transneuronal infection of a multisynaptic circuit involving neurons in the brainstem and spinal cord as detected immunocytochemically. At short survival (26 hr), retrogradely labeled neurons were concentrated in the external lateral nucleus of the parabrachial complex (elPB) but were absent from both the trigeminal nucleus caudalis (TNC) and the spinal cord. At longer survivals (52 hr), labeled cells were present in lamina I of both the TNC and spinal dorsal horn. Retrograde labeling from the Ce with Fluoro-gold demonstrated that elPB neurons have long dendrites extending laterally into the terminal field of spinal and trigeminal afferents, where transneuronal passage of PRV to these afferents could occur. Even longer survivals (76 hr) resulted in a columnar pattern of cell labeling in the TNC and spinal dorsal horn that extended from lamina I into lamina II. At this longest survival, primary sensory neurons became infected. Bilateral excitotoxic lesions of the elPB blocked almost all viral passage from the Ce to superficial laminae of the TNC and spinal dorsal horn. These results demonstrate that nociceptive input to the amygdala is relayed from neurons in lamina I through the elPB. We propose that this modular arrangement of lamina I and II neurons may provide the basis for spinal processing of peripheral input to the amygdala.

Meeting the 21st Century with SMART Mission Flexibility

ABSTRACT A revolution is underway in the design, production and installation of the Navy/Marine Corps team's command, control, communications, computers and intelligence (C4I) vision and strategies, which is significantly reducing costs, and dramatically improving the battle readiness of tomorrow's forward deployers. A strong emphasis on research and development has enabled the design, development and installation of the Shipboard Modular Arrangement Reconfiguration Technology (SMART) systems in ships.SMART is a methodology for installing equipment in shipboard spaces that will provide the fleet with enhanced mission flexibility. The heart of this technology is a track rail system, similar to that used by the aircraft industry, which enables equipment to be bolted to the deck, bulkheads or overhead, and meets all shipboard shock and vibration requirements. The fleet can reconfigure designated spaces to receive new systems, install equipment upgrades, position cross‐decked systems, or rearrange work areas with minimal industrial work (welding, grinding, lagging, painting, etc.), and maximum cost savings. Key C4I spaces such as Tactical Flag Command Center (TFCC), Joint Operations Center (JOC), Communication Centers, etc., can be reconfigured as required. Thereby, new technology insertion can enable rapid deployment of state‐of‐the‐art technologies much faster than the standard method of welding foundations in place to support various equipment installations.The SMART system includes a foundation track system, modular‐connectorized power and lighting, and modular workstations.

Engineered biosynthesis of peptide antibiotics

In certain bacteria and filamentous fungi, a wide variety of bioactive peptides are produced non-ribosomally on large protein templates, called peptide synthetases. Recently, significant progress has been made towards understanding the modular arrangement of these complex multifunctional enzymes and the mechanisms by which they generate their corresponding peptide products. It has now been established that the synthesis of bioactive peptides and the specification of their sequence are brought about by a protein template that contains the appropriate number and the correct order of activating units (domains). These advances have enabled the development of a technique that permits the construction of hybrid genes encoding peptide synthetases with specifically altered substrate specificities. A programmed alteration within the primary structure of a peptide antibiotic is achieved by the substitution of an amino acid-activating domain in the corresponding protein template at the genetic level by a two-step recombination method. It utilizes successive gene disruption and reconstitution and demonstrates, for the first time, the potential of genetic engineering in the biosynthesis of novel peptide antibiotics. Many organisms, for instance those that cause diseases like tuberculosis and pneumonia, have evolved potent mechanisms of drug resistance. Therefore, the targeted engineering of peptide antibiotics could be one potential strategy for the development of novel drugs that overcome this resistance.

Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.

The genes coding for the two type I collagen chains, which are active selectively in osteoblasts, odontoblasts, fibroblasts, and some mesenchymal cells, constitute good models for studying the mechanisms responsible for the cell-specific activity of genes which are expressed in a small number of discrete cell types. To test whether separate genetic elements could direct the activity of the mouse pro-alpha 1(I) collagen gene to different cell types in which it is expressed, transgenic mice were generated harboring various fragments of the proximal promoter of this gene cloned upstream of the Escherichia coli beta-galactosidase gene. During embryonic development, X-gal staining allows for the precise identification of the different cell types in which the beta-galactosidase gene is active. Transgenic mice harboring 900 bp of the pro-alpha 1(I) proximal promoter expressed the transgene at relatively low levels almost exclusively in skin. In mice containing 2.3 kb of this proximal promoter, the transgene was also expressed at high levels in osteoblasts and odontoblasts, but not in other type I collagen-producing cells. Transgenic mice harboring 3.2 kb of the proximal promoter showed an additional high level expression of the transgene in tendon and fascia fibroblasts. The pattern of expression of the lacZ transgene directed by the 0.9- and 2.3-kb pro-alpha 1(I) proximal promoters was confirmed by using the firefly luciferase gene as a reporter gene. The pattern of expression of this transgene, which can be detected even when it is active at very low levels, paralleled that of the beta-galactosidase gene. These data strongly suggest a modular arrangement of separate cell-specific cis-acting elements that can activate the mouse pro-alpha(I) collagen gene in different type I collagen-producing cells. At least three different types of cell-specific elements would be located in the first 3.2 kb of the promoter: (a) an element that confers low level expression in dermal fibroblasts; (b) a second that mediates high level expression in osteoblasts and odontoblasts; and (c) one responsible for high level expression in tendon and fascia fibroblasts. Our data also imply that other cis-acting cell-specific elements which direct activity of the gene to still other type I collagen-producing cells remain to be identified.

Modular arrangement of proteins as inferred from analysis of homology.

The structure of many proteins consists of a combination of discrete modules that have been shuffled during evolution. Such modules can frequently be recognized from the analysis of homology. Here we present a systematic analysis of the modular organization of all sequenced proteins. To achieve this we have developed an automatic method to identify protein domains from sequence comparisons. Homologous domains can then be clustered into consistent families. The method was applied to all 21,098 nonfragment protein sequences in SWISS-PROT 21.0, which was automatically reorganized into a comprehensive protein domain database, ProDom. We have constructed multiple sequence alignments for each domain family in ProDom, from which consensus sequences were generated. These nonreduntant domain consensuses are useful for fast homology searches. Domain organization in ProDom is exemplified for proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PEP:PTS) and for bacterial 2-component regulators. We provide 2 examples of previously unrecognized domain arrangements discovered with the help of ProDom.

Purification, cloning, and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops jararaca venom. Insights into the disintegrin gene family

A large hemorrhagin, jararhagin, has been cloned from a Bothrops jararaca venom gland cDNA expression library. The cDNA sequence predicts a 421-amino acid residue molecule with strong amino acid sequence homology and similar domain structure to HR1B, a high molecular weight hemorrhagic metalloprotease isolated from Trimeresurus flavoviridis (Habu) venom. Like HR1B, jararhagin contains enzyme, disintegrin, and cysteine-rich carboxyl-terminal regions. In the disintegrin region, the Arg-Gly-Asp sequence is replaced by Glu-Cys-Asp, as found in non-Arg-Gly-Asp disintegrin regions of HR1B and a guinea pig sperm fusion protein PH-30 beta. The cDNA sequence of jararhagin predicts a precursor protein (proprotein) with striking similarity to cryptic regions in precursors of the disintegrin peptides trigramin and rhodostomin. Comparison of jararhagin with disintegrin precursors highlights the modular arrangement of proprotein, metalloprotease, and disintegrin domains in the metalloprotease/disintegrin family and provides an insight into their biosynthesis and evolution.

A multipurpose instrument for quantitative intravital microscopy.

An in vivo microscope system has been developed that can measure fluorescence emission and/or light absorption at up to five wavelengths in a tissue area of 18-30 microns diam while imaging adjacent microcirculatory vessels with a video system. The system also incorporates a computer-controlled stage and data acquisition system for rapid and repeated measurements from a number of tissue sites. The tissue area monitored for fluorescence or absorption can be defined further by a confocal arrangement of the microscope optics. Tests of the system for NADH fluorescence measurements show good agreement between the fluorescence at 450 nm and NADH concentration in vitro and in skeletal muscle. The instrument can also be used simultaneously for spectrophotometric determination of O2 saturation and hematocrit in microcirculatory vessels. In vitro tests indicate suitable accuracy for such measurements. The open architecture and modular arrangement of the instrument facilitates its use for a variety of simultaneous measurements of parenchymal cell and microcirculatory function.

Identification of chromophore binding domains of yeast DNA photolyase.

Photolyases contain two chromophores, flavin plus either methenyltetrahydrofolate (MTHF) or 8-OH-5-deazaflavin (HDF). Amino acid sequence comparison reveals that all photolyases sequenced to date have extensive sequence homology in the carboxyl-terminal half; in the amino-terminal region the folate and deazaflavin class enzymes are more homologous to other members of the same class. This modular arrangement of sequence homologies suggests that the amino-terminal half of photolyase is involved in MTHF or HDF binding whereas the carboxyl-terminal half carries the flavin binding site. In this study we attempted to identify such structural domains of yeast photolyase by partial proteolysis and gene fusion techniques. Partial digestion with chymotrypsin yielded an amino-terminal 34-kDa fragment containing tightly bound MTHF and a carboxyl-terminal 20-kDa polypeptide which lacked chromophore or DNA binding activity. However, a fusion protein carrying the carboxyl-terminal 275 amino acids of yeast photolyase bound specifically to FAD but not to MTHF or DNA. We conclude that the amino-terminal half of yeast photolyase constitutes the folate binding domain and that the carboxyl-terminal half carries the flavin binding site.

The αA-crystallin gene: Conserved features of the 5′-flanking regions in human, mouse, and chicken

Approximately 2 kb of 5'-flanking sequences of the lens-specific alpha A-crystallin genes from human and mouse are presented and compared with similar regions of the chicken gene. A repetitive element was found approximately 1 kb upstream from the coding sequences of the alpha A-crystallin gene in all three species (Alu in human, B2 in mouse, and CR1 in chicken), suggesting that they may have an important functional or structural role. Despite the ability of alpha A-crystallin promoters to function across species, dot matrix analyses show only limited similarity among the 600 bp 5' to the structural genes of these three species. The human 5'-flanking sequence is more similar to that of the mouse and chicken than the mouse and chicken are to each other. Numerous short sequences (8-13 bp) are common to all three genes but are distributed differently in each species. The locations and conservation of these sequence motifs suggest functional roles, possibly as cis-regulatory elements of transcription. One motif is similar to the alpha A-CRYBP1 binding site implicated earlier in the transcriptional regulation of the mouse alpha A-crystallin gene, and other motifs correspond to sites previously mapped by methylation interference studies in the mouse alpha A-crystallin promoter. The modular arrangement of conserved sequence motifs is consistent with evolutionary changes occurring at the level of gene regulation.

SIMLYS — a software package for trajectory analysis of molecular dynamics simulations

SIMLYS is a tool to aid in the analysis of molecular dynamics, Monte Carlo and other simulations. Its purpose is twofold: it is a system performing the actual analysis and it serves as a shell to integrate new analysis functions. SIMLYS allows one to analyse the results from various simulations, as for example from proteins or polymers, by using the trajectories. The program is separated into modules performing the input/output, building the interface to the user, preparing the coordinates and performing the calculations. The modular arrangement allows the user to read the coordinates from various simulation systems, to extend the available commands, to select atoms or steps for different classes of molecules and to add any supplementary analysis function. A preprocessor program provides functions to adapt the program to the current machine or to modify the modules.

Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains

trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression.

The aquiferous systems ofSpongia officinalisand(Cliona viridis(Porifera) based on corrosion cast analysis

Abstract The three‐dimensional architecture of the aquiferous systems of Spongia officinalis and Cliona viridis has been studied by corrosion cast analysis. The two species present similar multi‐scale, modular canal arrangements. The incurrent systems consist of modular units with superficial small vessels, sub superficial cavities, and ducts penetrating into the sponge body. Choanocyte chambers are distributed in clumps generated by modular, branched, small canals. Canals which drain the choanocyte chambers form an anastomosed framework which converges on large vessels leading to oscules. Some canals directly connect incurrent and excurrent systems, allowing water to bypass the choanocyte chambers. Comparison with previous sponge cast analysis of Petrosia ficiformis and Chondrosia reniformis reveals a basic anatomical design which could be widespread among demo‐sponges.

Computerized predetermined motion-time systems in manufacturing industries

In recent years, a number of computerized work measurement systems have been developed. In particular, attempts have been made to computerize the manual versions of existing predetermined motion-time systems (PMTS). This can be attributed to the spread of computer technology in the last decade which may further the application of PMTS in manufacturing industries in terms of speed, ease of use, and minimization of degree of judgement on the part of the analyst in establishing time standards. The main objective of this paper is to survey the various computerized PMTS. In particular, this study will examine the computerized systems of Methods-Time Measurement (MTM), Modular Arrangement of Predetermined Time Standards (MODAPTS), and Maynard Operation Sequence Technique (MOST) due to their popular and worldwide support. The background and computer application of the aforementioned systems are discussed. Furthermore, comparative examples are given to illustrate the advantages and limitations of each system.

A comparison of computerized predetermined time systems

With the increased availability and use of personal computers by Industrial Engineers most of the widely used predetermined time systems are now beign offered with computerized versions to assist in analysis and documentation. The purpose of this paper is to compare three different systems, Methods Time Measurement (MTM), Maynard Operation Sequence Technique (MOST), and Modular Arrangement of Predetermined Tiem Standards (MODAPTS).

A modular arrangement of neuronal processes in human cortex: disruption with aging and in Alzheimer's disease.

Studies were undertaken to assess whether or not neuron-specific immunostaining of the human brain can reveal unique cytoarchitectural features that may be affected by healthy aging and Alzheimer's disease (AD). Human prefrontal cortex (PFC) and anterior cingulate cortex (ACC) were stained with an antibody raised against the neurofilament protein 200,000 molecular weight subunit (NFP-200) using an avidin-biotin immunolocalization procedure. Immunostaining of cortex was neuron-specific and highlighted axons in particular. This staining has revealed an orderly arrangement of horizontal and vertical axon bundles which form latticelike compartments or modules throughout most of the matrix of the two cortical areas studied. The ACC shows this pattern to be intact in individuals through the ninth decade, while the PFC there was blurring of the modularity beyond the fifth decade. Irrespective of the age-related blurring of the latticelike arrangement of axons in PFC, neurologically normal elderly individuals nevertheless showed a highly organized appearance to the cortical matrix. By contrast, patients with AD showed a marked disruption of the modular arrangement of fibers in PFC, but not ACC. Differences between PFC fibers in controls and AD patients were also demonstrated with an axon-specific monoclonal antibody that reacted with phosphorylated epitopes of the NFP-200. The chaotic appearance of fiber staining in PFC seen in patients with AD was noted to be present in one subject who died in an early stage of the disease. The possible significance of this previously unknown aspect of cortical cytoarchitecture for normal cognitive functioning in humans is discussed.

Modular growth and form of corals: a matter of metamers?

Following a lead from botanists, this paper offers a re-interpretation of the morphology and growth of scleractinian, rugosan and tabulate corals in terms of iterated morphological units (modules), with an emphasis on the shape and organization of colonies. It presents a new theoretical basis for modules, a new descriptive framework for corals above the zooidal level, a review of practical applications and a comparison of coral modules with other kinds of organic iteration. Consideration of cloning is a prerequisite and leads to a revival of the hypothesis that colonies have arisen by arrested budding (clonoteny), where zooids are paedomorphic to varying degrees with respect to the primitive state of clonally produced, detached (clonoparous) individuals. A second hypothesis follows, that the mouth structure is universally homologous in all corals. R. Riedl’s work (Order in living organisms. Chichester: John Wiley (1978)) on morphological organization and hierarchy can therefore be applied to corals to obtain a non-arbitrary theoretical basis for recognizing modules and distinguishing them from other kinds of repetition of parts. There are five criteria for modularity, four topological and one homological: (i) a modular structure is a three-dimensional tesselation; (ii) modules correspond to homogeneous units of hierarchical subdivision; (iii) an organism is modular if subdivision at the highest (or very high) level reveals homogeneous units; (iv) if homogeneous units occur in an unbroken series of subdivisions, they are all modules at their respective levels (hence modules of modules, that is, cormidia), and the finest unit is the fundamental module; (v) homogeneous units are those which are homologous with each other (homonoms). On this basis, and allowing for interzooidal connective structures, the fundamental module of corals is the zooid (not the polyp alone). Differences between zooids, as suggested by other authors, are of degree, not kind. Colony form can therefore be specified by using a scheme that starts with zooids, and is based on (i) organization (component morphology and modular arrangement), and (ii) shape, for every modular level present. The basis for higher level modules is reiterative, either homomodular, or heteromodular (subdivided into polymorphic or polystatic). Branching organization and branching shape should be distinguished, and either can occur at any modular level, or even not at all. Modules bring potentially greater precision to growth studies, statistically and topologically, either heuristically or to test hypotheses about fecundity, senescence, determinate growth, variation and inherited architecture. Higher level modularity has previously been largely ignored. Density banding, dye markers and computer modelling, ideally in combination, are likely to produce the most useful results in the near future, and should lead to advances in taxonomy and phylogenetics, in the understanding of the morphological consequences of the coral-zooxanthellae symbiosis, and in the inference of past conditions from fossil corals. In a wider phyletic context, zooidal ( = fundamental) modules are meristematic and broadly metameric, though it follows from R. B. Clark (Zool. Jb. 103, 169-195(1980)) that this does not automatically signify a close phylogenetic relationship with annelids, or with any other metameric organisms. Coral ‘metamers’ are probably non-serial and clonal because the coral mode of life is usually vegetative. They exist at a higher organizational level than these other metamers. ‘What is not identically repeated, we do not understand.’ (Riedl 1978) ‘The actinozooid is a living thing which knows no time of youthful vigour, no waxing to a period of adult life, no waning to senility — it knows no age — it practically knows no natural death. ’ (Wood-Jones 1907, 1910)

A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus.

Using the simian virus 40 "enhancer trap" approach, we have identified a transcription enhancer located just upstream of the major immediate early gene of murine cytomegalovirus. This enhancer has several striking properties. (i) Together with the enhancer of human cytomegalovirus, it is the strongest transcription enhancer found to date. (ii) It is an extremely long enhancer, spanning greater than 700 base pairs. (iii) It consists of a rather complex pattern of sequence repeats, the longest of which is 181 base pairs. Also, several types of short sequence motifs are scattered throughout the enhancer in monomeric, heterodimeric, or homodimeric (palindromic) form. These motifs have been identified to be components of other enhancers and promoters, and they are presumably binding sites for specific nuclear factors. Our analysis suggests that enhancers are composed of a modular arrangement of short conserved sequence motifs and that enhancer strength is correlated with the redundancy of these motifs.

A versatile low-cost automation system for thermal ionization mass spectrometers

An IBM personal computer was interfaced to a magnetic sector mass spectrometer, via a Keithley DAS Series 500 data acquisition system, to provide versatile automation and data processing. The DAS contained a modular arrangement of high resolution (14, 16 bit) A/D and D/A converters and a large number of digital input and output channels, all of which could be software controlled or monitored by BASIC programs in the IBM PC. The system was arranged to enable software selection of four different modes of operation: magnetic field stepping with either ion current or pulse counting detection, and high voltage stepping with either of the above two detection modes. In any of these modes the system could be readily calibrated to precisely centre each isotope of interest on the detector, sequentially. The convenient selection of operation modes allowed comparison of them during isotopic analyses. For example, pulse counting detection, compared with ion current detection, provided enhanced sensitivity and precision for minor isotopes. High voltage stepping yielded comparable precision to magnetic field stepping and was significantly faster. The overall system was easy to install and quite practical for retrofitting an older mass spectrometer.