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PubMed1999

Simulation of biomimetic recognition between polymers and surfaces.

Many biological processes, such as transmembrane signaling and pathogen-host interactions, are initiated by a protein recognizing a specific pattern of binding sites on part of a membrane or cell surface. By recognition, we imply that the polymer quickly finds and then adsorbs strongly on the pattern-matched region and not on others. The development of synthetic systems that can mimic such recognition between polymers and surfaces could have significant impact on advanced applications such as the development of sensors, molecular-scale separation processes, and synthetic viral inhibition agents. Attempting to affect recognition in synthetic systems by copying the detailed chemistries to which nature has been led over millenia of evolution does not seem practical for most applications. This leads us to the following question: Are there any universal strategies that can affect recognition between polymers and surfaces? Such generic strategies may be easier to implement in abiotic applications. We describe results that suggest that biomimetic recognition between synthetic polymers and surfaces is possible by exploiting certain generic strategies, and we elucidate the kinetic mechanisms by which this occurs. Our results suggest convenient model systems for experimental studies of dynamics in free energy landscapes characteristic of frustrated systems.

Algorithms↗
PubMed1999

Intracellular gene transfer in action: dual transcription and multiple silencings of nuclear and mitochondrial cox2 genes in legumes.

The respiratory gene cox2, normally present in the mitochondrion, was previously shown to have been functionally transferred to the nucleus during flowering plant evolution, possibly during the diversification of legumes. To search for novel intermediate stages in the process of intracellular gene transfer and to assess the evolutionary timing and frequency of cox2 transfer, activation, and inactivation, we examined nuclear and mitochondrial (mt) cox2 presence and expression in over 25 legume genera and mt cox2 presence in 392 genera. Transfer and activation of cox2 appear to have occurred during recent legume evolution, more recently than previously inferred. Many intermediate stages of the gene transfer process are represented by cox2 genes in the studied legumes. Nine legumes contain intact copies of both nuclear and mt cox2, although transcripts could not be detected for some of these genes. Both cox2 genes are transcribed in seven legumes that are phylogenetically interspersed with species displaying only nuclear or mt cox2 expression. Inactivation of cox2 in each genome has taken place multiple times and in a variety of ways, including loss of detectable transcripts or transcript editing and partial to complete gene loss. Phylogenetic evidence shows about the same number (3-5) of separate inactivations of nuclear and mt cox2, suggesting that there is no selective advantage for a mt vs. nuclear location of cox2 in plants. The current distribution of cox2 presence and expression between the nucleus and mitochondrion in the studied legumes is probably the result of chance mutations silencing either cox2 gene.

Cell Nucleus↗
PubMed2001

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation.

Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.

Collagen↗
PubMed1995

SAM: a system for iteratively building marker maps.

SAM (system for assembling markers) is a system which supports man-machine problem solving for iteratively ordering a set of markers. SAM aids the user in partially ordering a set of markers based on incomplete and uncertain data. As data is added and modified, SAM aids the user in updating the previously assembled maps. The input is a file of clones and for each clone, a list of the markers contained within it. The objective is to order the set of markers such that the markers contained in each clone are consecutive. The user directs the map building by selecting functions to assemble a region of markers, order the clones to fit the order of the markers and position new markers within an ordered set of markers. The user can edit the input data, edit the assembled map and add clones to the map based on their marker content. The results are displayed graphically and can be saved in a solution file. Based on the partial map, the user designs new experiments or edits the existing data to fill gaps and resolve ambiguities. When a previously assembled map is loaded into SAM, it is automatically updated with the new or altered data. SAM treats all markers as points, but has special features for multiple copy and long markers so that they can be used in the map building process. This system has supported the building of a YAC map of human chromosome 22 at the Sanger Centre, where use of Alu-PCR product markers is a major component in determining clone overlap and where we have an on-going effort to accumulate data from various sources. SAM is also being used at various other laboratories.

Algorithms↗
PubMed2004

BLAST2GENE: a comprehensive conversion of BLAST output into independent genes and gene fragments.

SUMMARY: BLAST2GENE is a program that allows a detailed analysis of genomic regions containing completely or partially duplicated genes. From a BLAST (or BL2SEQ) comparison of a protein or nucleotide query sequence with any genomic region of interest, BLAST2GENE processes all high scoring pairwise alignments (HSPs) and provides the disposition of all independent copies along the genomic fragment. The results are provided in text and PostScript formats to allow an automatic and visual evaluation of the respective region. AVAILABILITY: The program is available upon request from the authors. A web server of BLAST2GENE is maintained at http://www.bork.embl.de/blast2gene

Algorithms↗
PubMed2003

Genome-level evolution of resistance genes in Arabidopsis thaliana.

Pathogen resistance genes represent some of the most abundant and diverse gene families found within plant genomes. However, evolutionary mechanisms generating resistance gene diversity at the genome level are not well understood. We used the complete Arabidopsis thaliana genome sequence to show that most duplication of individual NBS-LRR sequences occurs at close physical proximity to the parent sequence and generates clusters of closely related NBS-LRR sequences. Deploying the statistical strength of phylogeographic approaches and using chromosomal location as a proxy for spatial location, we show that apparent duplication of NBS-LRR genes to ectopic chromosomal locations is largely the consequence of segmental chromosome duplication and rearrangement, rather than the independent duplication of individual sequences. Although accounting for a smaller fraction of NBS-LRR gene duplications, segmental chromosome duplication and rearrangement events have a large impact on the evolution of this multigene family. Intergenic exchange is dramatically lower between NBS-LRR sequences located in different chromosome regions as compared to exchange between sequences within the same chromosome region. Consequently, once translocated to new chromosome locations, NBS-LRR gene copies have a greater likelihood of escaping intergenic exchange and adopting new functions than do gene copies located within the same chromosomal region. We propose an evolutionary model that relates processes of genome evolution to mechanisms of evolution for the large, diverse, NBS-LRR gene family.

Arabidopsis↗
PubMed1986

The mRNA and RNA-copy pseudogenes encoding TM30nm, a human cytoskeletal tropomyosin.

We have determined the sequence of a 2.5 kb mRNA in human fibroblasts encoding a 248 amino acid cytoskeletal tropomyosin. The protein product of this mRNA is TM30nm, one of five tropomyosin-like proteins in human fibroblasts. The structural gene encoding this mRNA can also produce a 1.3 kb mRNA encoding a 285 amino acid skeletal muscle alpha-tropomyosin by tissue-specific alternative mRNA splicing. However, the multiple RNA-copy pseudogenes of this gene family are derived largely if not exclusively from transcripts processed according to the pattern observed in non-muscle cells.

Amino Acid Sequence↗
PubMed1997

The minute virus of mice (MVM) nonstructural protein NS1 induces nicking of MVM DNA at a unique site of the right-end telomere in both hairpin and duplex conformations in vitro.

The right-end telomere of replicative form (RF) DNA of the autonomous parvovirus minute virus of mice (MVM) consists of a sequence that is self-complementary except for a three nucleotide loop around the axis of symmetry and an interior bulge of three unpaired nucleotides on one strand (designated the right-end 'bubble'). This right-end inverted repeat can exist in the form of a folded-back strand (hairpin conformation) or in an extended form, base-paired to a copy strand (duplex conformation). We recently reported that the right-end telomere is processed in an A9 cell extract supplemented with the MVM nonstructural protein NS1. This processing is shown here to result from the NS1-dependent nicking of the complementary strand at a unique position 21 nt inboard of the folded-back genomic 5' end. DNA species terminating in duplex or hairpin configurations, or in a mutated structure that has lost the right-end bulge, are all cleaved in the presence of NS1, indicating that features distinguishing these structures are not prerequisites for nicking under the in vitro conditions tested. Cleavage of the hairpin structure is followed by strand-displacement synthesis, generating the right-end duplex conformation, while processing of the duplex structure leads to the release of free right-end telomeres. In the majority of molecules, displacement synthesis at the right terminus stops a few nucleotides before reaching the end of the template strand, possibly due to NS1 which is covalently bound to this end. A fraction of the right-end duplex product undergoes melting and re-folding into hairpin structures (formation of a 'rabbit-ear' structure).

Animals↗
PubMed1996

Selection of axial growth sites in yeast requires Axl2p, a novel plasma membrane glycoprotein.

Spa2p and Cdc10p both participate in bud site selection and cell morphogenesis in yeast, and spa2delta cdc10-10 cells are inviable. To identify additional components important for these processes in yeast, a colony-sectoring assay was used to isolate high-copy suppressors of the spa2delda cdc10-10 lethality. One such gene, AXL2, has been characterized in detail. axl2 cells are defective in bud site selection in haploid cells and bud in a bipolar fashion. Genetic analysis indicates that AXL2 falls into the same epistasis group as BUD3. Axl2p is predicted to be a type I transmembrane protein. Tunicamycin treatment experiments, biochemical fractionation and extraction experiments, and proteinase K protection experiments collectively indicate that Axl2p is an integral membrane glycoprotein at the plasma membrane. Indirect immunofluorescence experiments using either Axl2p tagged with three copies of a hemagglutinin epitope or high-copy AXL2 and anti-Axl2p antibodies reveal a unique localization pattern for Axl2p. The protein is present as a patch at the incipient bud site and in emerging buds, and at the bud periphery in small-budded cells. In cells containing medium-sized or large buds, Axl2p is located as a ring at the neck. Thus, Axl2p is a novel membrane protein critical for selecting proper growth sites in yeast. We suggest that Axl2p acts as an anchor in the plasma membrane that helps direct new growth components and/or polarity establishment components to the cortical axial budding site.

Amino Acid Sequence↗
PubMed2003

Site2 binding energetics of the regulatory step of growth hormone-induced receptor homodimerization.

Receptor signaling in the growth hormone (GH)-growth hormone receptor (GHR) system is controlled through a sequential two-step hormone-induced dimerization of two copies of the extracellular domain (ECD) of the receptor. The regulatory step of this process is the binding of the second ECD (ECD2) to the stable preassociated 1 : 1 GH/ECD1 complex on the cell surface. To determine the energetics that governs this step, the binding kinetics of 38 single- and double-alanine mutants in the hGH Site2 contact with ECD2 were measured by using trimolecular surface plasmon resonance (TM-SPR). We find that the Site2 interface of hGH does not have a distinct binding hot-spot region, and the most important residues are not spatially clustered, but rather are distributed over the whole binding surface. In addition, it was determined through analysis of a set of pairwise double alanine mutations that there is a significant degree of negative cooperativity among Site2 residues. Residues that show little effect or even improved binding on substitution with alanine, when paired with D116A-hGH, display significant negative cooperativity. Because most of these pairwise mutated residues are spatially separated by >or=10 A, this indicates that the Site2 binding interface of the hGH-hGHR ternary complex displays both structural and energetic malleability.

Alanine↗
PubMed2004

Plasmid partitioning and the spreading of P1 partition protein ParB.

Bacterial plasmids of low copy number, P1 prophage among them, are actively partitioned to nascent daughter cells. The process is typically mediated by a pair of plasmid-encoded proteins and a cis-acting DNA site or cluster of sites, referred to as the plasmid centromere. P1 ParB protein, which binds to the P1 centromere (parS), can spread for several kilobases along flanking DNA. We argue that studies of mutant ParB that demonstrated a strong correlation between spreading capacity and the ability to engage in partitioning may be misleading, and describe here a critical test of the dependence of partitioning on the spreading of the wild-type protein. Physical constraints imposed on the spreading of P1 ParB were found to have only a minor, but reproducible, effect on partitioning. We conclude that, whereas extensive ParB spreading is not required for partitioning, spreading may have an auxiliary role in the process.

Bacterial Proteins↗
PubMed1991

Physical mapping of the mec region of an American methicillin-resistant Staphylococcus aureus strain.

We mapped part of the mec region of a locally prevalent strain of Staphylococcus aureus. The mec region was found to harbor an insert of the transposon Tn554, which encodes spectinomycin and macrolide-lincosamide-streptogramin B resistance, and a 4.6-kb segment of DNA that contains the kanamycin resistance gene aadD. This 4.6-kb segment appears to be an integrated form of a previously described plasmid, pUB110, and is flanked by copies of the insertion sequence IS257. The integration event may be an example of processes that have led to accretion of resistance determinants in the mec region of S. aureus.

Base Sequence↗
PubMed1985

Reverse transcription of 7S L RNA by an avian retrovirus.

Linial et al. isolated a quail cell line, SE21Q1b, that is transformed by a single integrated provirus of Rous sarcoma virus. Virus particles are released from these cells, but because of a provirus defect, cellular rather than viral RNA is packaged. When these virus particles are disrupted with melittin in the presence of an appropriate reaction mixture containing actinomycin D, there is significant reverse transcription of packaged cellular RNA species. We have shown that (i) cellular 7S L RNA is an efficient template; (ii) initiation is on a unique tRNA-like primer; (iii) synthesis produces a 135-base strong-stop DNA product; and (iv) after synthesis, RNase H acts to remove the 135 bases of the 7S L RNA which acted as the template. A possible facilitator of such specific transcription may be that, in the virus particles but not in the cell, the majority of the 7S L RNA species exist complexed with the tRNA, even before the disruption of the virus. From the size and sequence features of the reverse transcript of 7S L RNA, we speculate that such events may have participated in the process by which animal cell genomes have, in the course of evolution, accumulated multiple copies of Alu-like elements.

Avian Sarcoma Viruses↗
PubMed2004

Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays.

BACKGROUND: The recent development of array based comparative genomic hybridization (CGH) technology provides improved resolution for detection of genomic DNA copy number alterations. In array CGH, generating spotting solution is a multi-step process where bacterial artificial chromosome (BAC) clones are converted to replenishable PCR amplified fragments pools (AFP) for use as spotting solution in a microarray format on glass substrate. With completion of the human and mouse genome sequencing, large BAC clone sets providing complete genome coverage are available for construction of whole genome BAC arrays. Currently, Southern hybridization, fluorescent in-situ hybridization (FISH), and BAC end sequencing methods are commonly used to identify the initial BAC clone but not the end product used for spotting arrays. The AFP sequencing technique described in this study is a novel method designed to verify the identity of array spotting solution in a high throughput manner. RESULTS: We show here that Southern hybridization, FISH, and AFP sequencing can be used to verify the identity of final spotting solutions using less than 10% of the AFP product. Single pass AFP sequencing identified over half of the 960 AFPs analyzed. Moreover, using two vector primers approximately 90% of the AFP spotting solutions can be identified. CONCLUSIONS: In this feasibility study we demonstrate that current methods for identifying initial BAC clones can be adapted to verify the identity of AFP spotting solutions used in printing arrays. Of these methods, AFP sequencing proves to be the most efficient for large scale identification of spotting solution in a high throughput manner.

Animals↗
PubMed2006

Homologous gene sequences mediate transcription-domain formation.

The organisation of transcription in the mammalian nucleus is a topic of particular interest because of its relevance to gene regulation. RNA polymerase II transcription occurs at hundreds of sites throughout the nucleoplasm. Recent data indicate that coordinately regulated genes can localise to shared transcription sites. Other transcribed sequences have also been shown to cluster in the nucleus. The ribosomal RNA genes cluster in the nucleoli. Similarly, transiently transfected plasmids and dsDNA viruses form transcription domains (TDs) containing multiple templates. Intriguingly, plasmids expressing beta-globin gene sequences recruit the endogenous beta-globin loci to their TDs. In light of this observation, we have investigated plasmid TDs as a model for gene recruitment. We find that TD formation is dependent on the presence of homologous gene sequences. Plasmids containing non-homologous gene sequences form separate TDs, independent of homology in the backbone or promoter sequences. TD formation is also favoured by low plasmid concentrations. This effect is sequence-specific and high concentrations of one plasmid do not disrupt domain formation by non-homologous plasmids in the same cell. We conclude that recruitment into TDs is an active process that is driven by homologies between transcribed sequences and becomes saturated at high copy numbers.

Base Sequence↗
PubMedDate not supplied

[Evaluation of the teaching of nursing within a new liberating idea].

Intending to offer new teaching technologies the Nursing Master Course at the Federal University of Ceará have been integrating the graduation and postgraduation courses. The study aimed at evaluating a subject in Nursing Graduation Course in a critical and transforming point of view. The used methodology involves SAUL's (1995) emancipating evaluation paradigm contemplating three moments: reality description, reality criticism and collective construction. The most significant results were: the constaint be compatible with the schedule, with methodological renewal and evaluation decentralization. The study has been concluded perceiving the methodological deficiency adopted by the subject as a critical point. It is certain that this investigation brings contribution for teaching-learning relationship where professor and students are process subjects, eliminating, so, the student position of a mere system manipulated object and copy copiers.

Brazil↗
PubMed2001

Identification of a differentially expressed mRNA in axenic Leishmania panamensis amastigotes.

Differential display technique was applied in order to identify transcripts which are present in axenic amastigotes but not in promastigotes of the Leishmania panamensis parasites. One of them was cloned and the sequence reveals an open reading frame of 364 amino acids (approximately 40 kDa). The deduced protein is homologous to the serine/threonine protein kinases and specially to the mitogen activates protein kinases from eukaryotic species. Southern blot analysis suggest that this transcript, named lpmkh, is present in the genome of the parasite as a single copy gene. These results could imply that lpmkh could be involved in the differentiation process or the preservation of amastigotes in axenic conditions.

Amino Acid Sequence↗
PubMed2006

Genomic profiling of hormone-naïve lymph node metastases in patients with prostate cancer.

The progression of organ-confined prostate cancer to metastatic cancer is inevitably fatal. Consequently, identification of structural changes in the genome and associated transcriptional responses that drive this progression is critical to understanding the disease process and the development of biomarkers and therapeutic targets. In this study, whole genome copy number changes in genomes of hormone-naïve lymph node metastases were profiled using array comparative genomic hybridization, and matched primaries were included for a subset. Matched primaries and lymph node metastases showed very similar copy number profiles that are distinct from primary tumors that fail to metastasize.

Biomarkers, Tumor↗