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PubMed1995

Schizosaccharomyces pombe RNase MRP RNA is homologous to metazoan RNase MRP RNAs and may provide clues to interrelationships between RNase MRP and RNase P.

RNase MRP and RNase P ribonucleoproteins are structurally and functionally similar across a large evolutionary distance. To better characterize possible complex interrelationships between these two enzymes, we have employed the fission yeast Schizosaccharomyces pombe. Unlike Saccharomyces cerevisiae, S. pombe is believed to harbour only one genetic locus for the RNA component of RNase P and does not contain a known mitochondrially encoded RNase P RNA. We have identified the single nuclear gene for the RNA component of RNase MRP in S. pombe, mrp-1, by homology to vertebrate RNase MRP RNAs. The mrp-1 gene encodes an RNA of maximum mature length 400 nucleotides that shares a high degree of identity, in evolutionarily conserved regions, to both vertebrate RNase MRP RNAs and S. pombe RNase P RNA. Disruption of mrp-1 in the diploid strain SP826 and sporulation of tetrads resulted in a 2 dead:2 viable segregation, consistent with the gene being essential. Lethality is rescued by a plasmid-borne copy of mrp-1. Partially purified ribonucleoprotein RNase MRP activity correctly and efficiently processed all previously characterized heterologous mitochondrial RNA substrates. The compact mitochondrial genome of S. pombe contains sequence elements with > 50% identity to mammalian D-loop CSBI and CSBII elements. The identification of mrp-1 in S. pombe should facilitate not only comparisons between the related ribonucleoproteins RNase MRP and RNase P, but should also provide an opportunity for genetic elucidation of RNase MRP function in a situation reflective of the animal kingdom.

Animals↗
PubMed1995

The phosphodiesterase secreted by prestalk cells is necessary for Dictyostelium morphogenesis.

Dictyostelium discoideum secretes a cyclic nucleotide phosphodiesterase to control cAMP levels during development. Three promoters control expression of the gene--one during vegetative growth, one during aggregation, and one which constrains phosphodiesterase synthesis to prestalk cells. In this report we show that the expression of phosphodiesterase (PDE) in prestalk cells is necessary for morphogenesis. A gene that codes for a specific glycoprotein inhibitor of the phosphodiesterase (Kd = 0.1 nM) was fused to the prestalk-specific promoter of the PDE gene. Transformants carrying multiple copies of this construct secreted inhibitor in 100-fold excess after the aggregation process had occurred. The first effect seen was an elongated tip, followed by a block in slug formation and an inability to culminate. Stalk and spores cells are produced but morphogenesis is uncoupled from cellular differentiation. Overproduction of inhibitor during earlier stages delayed aggregation, but did not affect fruiting body formation. A phosphodiesterase mutant was transformed with a plasmid that expresses PDE only during aggregation and not in prestalk cells. The defect in aggregation was rescued, but the defect in later development was not. The combined results indicate that PDE expression in prestalk cells is critical to morphogenesis. To ask whether the inhibitor gene under its normal regulation had a role in aggregation or later morphogenesis, it was destroyed by homologous recombination. The loss of the gene did not prevent development under the conditions used.

Amino Acid Sequence↗
PubMed1997

Genetic relationships between the mutations spade and Sternopleural and the wingless gene in Drosophila development.

In Drosophila melanogaster, there are cases in which gene products contributing to the same developmental event may derive from closely adjacent transcription units and may even share cis-regulatory sequences. Correct recognition of such genomic organization is central to an understanding of developmental mechanisms. The adult phenotypes of combinations between the mutations spade, Sternopleural, and wingless suggest that they are lesions in functionally related genes within the same chromosomal region. wingless mutations fail to complement the recessive mutation spade. The spade mutation, as previously shown, behaves as a lesion in a regulatory site of wingless, sited 5' to the transcription unit, and is concerned with particular postembryonic functions of wingless. While showing wingless-like phenotypes in combination with Sternopleural, even lethal alleles of wingless complement the recessive lethality of Sternopleural alleles. Mutations in Sternopleural increase the severity of wingless phenotypes in many wingless-dependent processes during postembryonic development, and this interaction can occur when the only functional copies of Sp or wg are located in either opposing chromosomes or the same chromosome. This is inconsistent with previous attempts to define Sp as a regulatory allele of wg and explain the phenotypes that result from combinations of Sp and wg by means of transvection. We have analyzed a new EMS-induced allele of Sternopleural that is more severe than the original allele, which also argues for Sp being a separate, mutable genetic locus rather than a regulatory allele of wg. Finally, we have a revertant of Sternopleural (Sp[Rv1]) that behaves as a genetic null allele of wg, but causes ventral-to-dorsal transformations in combination with wg(P), which is not observed in combinations of wg null alleles with wg(P). Because wg(P) is the result of an inversion and because inversions inhibit transvection, the increased severity observed in Sp(Rv1)/wg(P) in comparison to wg(null)/Sp(Rv1) animals cannot be explained by an absence of transvection. Therefore, the two Sternopleural mutations most reasonably define an independent gene located 3' to the wingless gene and having strong functional synergism with it.

Animals↗
PubMed1985

Construction of Co1E1 RNA1 mutants and analysis of their function in vivo.

We have carried out experiments designed to investigate the relationship between structure and function for the Co1E1 RNA1 species. RNA1 is a small RNA (108 nucleotides) that has been implicated in copy number control of the multicopy plasmid Co1E1. In vitro, RNA1 inhibits the processing of the primer precursor required for initiation of DNA replication. The RNA1 gene is entirely complementary to the 5'-terminal region of the primer. We have functionally separated these 2 RNA species by cloning the RNA1 gene downstream from the S. marcescens trp promoter. When cloned in a Co1E1-compatible plasmid, a trp-RNA1 fusion has been shown to mediate Co1E1-type incompatibility in vivo. The construction scheme described here also generates mutant RNA1 species with altered sequences at the 5' terminus of RNA1 which have been assayed for function in vivo. These experiments have indicated that sequences at the 5' terminus play a critical role in RNA1 function.

Bacteriocin Plasmids↗
PubMed1994

The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment.

The hns gene is a member of the cold-shock regulon, indicating that the nucleoid-associated, DNA-binding protein H-NS plays an important role in the adaptation of Escherichia coli to low temperatures. We show here that the ability to cope efficiently with a cold environment (12 degrees C and 25 degrees C) is strongly impaired in E. coli strains carrying hns mutations. Growth inhibition is much more pronounced in strains carrying the hns-206 allele (an ampicillin resistance cassette inserted after codon 37) than in those carrying the hns-205 mutation (a Tn10 insertion located in codon 93). A protein fragment (H-NS*) is synthesized in strains carrying the hns-205::Tn10 mutation, suggesting that this truncated polypeptide is partially functional in the cold adaptation process. Analysis of the growth properties of strains harbouring four different low-copy-number plasmid-encoded hns' genes that result in the production of C-terminally truncated H-NS proteins supports this proposal. H-NS* proteins composed of 133, 117 or 94 amino-terminal amino acids partially complemented the severe cold-sensitive growth phenotype of the hns-206 mutant. In contrast, synthesis of a truncated H-NS protein with only 75 amino-terminal amino acids was insufficient to restore growth at low temperature.

Adaptation, Physiological↗
PubMed1999

Dynamic diversification from a putative common ancestor of scorpion toxins affecting sodium, potassium, and chloride channels.

Scorpions have survived successfully over millions of years without detectable changes in their morphology. Instead, they have developed an efficient alomonal machinery and a stinging device supporting their needs for prey and defense. They produce a large variety of polypeptidic toxins that bind and modulate ion channel conductance in excitable tissues. The binding site, mode of action, and chemical properties of many toxins have been studied extensively, but little is known about their genomic organization and diversity. Genes representing each of the major classes of Buthidae scorpion toxins, namely, "long" toxins, affecting sodium channels (alpha, depressant, and excitatory), and "short" toxins, affecting potassium and chloride channels, were isolated from a single scorpion segment and analyzed. Each toxin type was found to be encoded by a gene family. Regardless of toxin length, 3-D structure, and site of action, all genes contain A+T-rich introns that split, at a conserved location, an amino acid codon of the signal sequence. The introns vary in length and sequence but display identical boundaries, agree with the GT/AG splice junctions, and contain T-runs downstream of a putative branch point, 5'-TAAT-3'. Despite little sequence similarity among all toxin classes, the conserved gene organization, intron features, and common cysteine-stabilized alpha-helical (CSH) core connecting an alpha-helix to a three-stranded beta-sheet suggest, that they all evolved from an ancestral common progenitor. Furthermore, the vast diversity found among genomic copies, cDNAs, and their protein products for each toxin suggests an extensive evolutionary process of the scorpion "pharmaceutical factory," whose success is due, most likely, to the inherent permissiveness of the toxin exterior to structural alterations.

Amino Acid Sequence↗
PubMed2000

Copper-dependence of mitochondrial DNA rearrangements in Podospora anserina.

Rearrangements of the mitochondrial DNA (mtDNA) are a hallmark of senescence in wild-type strains of the ascomycete P. anserina. These rearrangements include the systematic amplification of the first intron (p1-intron) of the cytochrome oxidase subunit-I gene (CoI) as a circular DNA molecule (p1DNA). In addition, deletions and amplifications of other regions of the mtDNA occur. The molecular basis of the underlying processes is not understood in detail. A comparative analysis of the wild-type strain and of the long-lived mutant grisea, affected in the uptake of copper, revealed that mtDNA instabilities are dependent on the availability of cellular copper. In the mutant, the first steps in the corresponding pathway, including the transcription of the CoI gene, the splicing of the p1-intron and the transposition of this mobile element, are not impaired. In contrast, recombination processes between short direct repeats, as well as rearrangements between two tandem intron copies leading to the formation of p1DNA, appear to be affected. Additional copper in the growth medium rescues this molecular phenotype. We suggest that copper is a cofactor of a component of the molecular machinery leading to the characteristic age-related mtDNA rearrangements.

Ascomycota↗
PubMed2003

Behavioural development in a matching-to-sample task and token use by an infant chimpanzee reared by his mother.

We investigated the behavioural and cognitive development of a captive male infant chimpanzee, Ayumu, raised by his mother, Ai. Here we report Ayumu's achievements up to the age of 2 years and 3 months, in the context of complex computer-controlled tasks. From soon after birth, Ayumu had been present during an experiment performed by his mother. The task consisted of two phases, a matching-to-sample task in which she received token rewards, and the insertion of these tokens into a vending machine to obtain food rewards. Ayumu himself received no reward or encouragement from humans for any of the actions he exhibited during the experiment. At the age of 9 months and 3 weeks, Ayumu performed his first matching-to-sample trial. At around 1 year and 3 months, he began to perform them consistently. Also during this period, he frequently stole food rewards from his mother. At 2 years and 3 months, Ayumu succeeded for the first time in inserting a token into the vending machine. Once he had succeeded in using a token, he performed both phases of the task in sequence 20 times consecutively. The infant's behaviour was not shaped by food rewards but by a strong motivation to copy his mother's behaviour. Our observations of Ayumu thus mirror the learning processes shown by wild chimpanzees.

Animals↗
PubMed1991

Characterization of the aldose reductase-encoding gene family in rat.

Although the enzyme aldose reductase (AR) is implicated in the development of tissue pathology in diabetes, the exact mechanism of this involvement remains unclear. To better understand the role that expression of the aldose reductase-encoding gene (ALR) may play in diabetic complications, we have begun to analyze the gene and its regulatory regions, and we present here the sequence of four ALR genes in the rat. The putative functional gene is 14.1 kb long, has ten exons which show perfect sequence identity to the rat lens AR RNA sequence, and nine introns with classical splice-site consensus sequences. Potential regulatory elements in the 5'-flanking region of this gene include a TATA box and two CCAAT boxes. Probing rat genomic Southern blots with a fragment from the first intron indicates that there is probably only one copy of this gene in the rat genome. The other three genes are processed pseudogenes which show approx. 90% identity to the rat lens AR RNA sequence, contain no introns, and have poly(A) regions at their 3' ends. Chromosomal localization studies show the presence of ALR genes on chromosomes 3, 4 and 6 in the rat with the putative functional gene mapped on chromosome 4.

Aldehyde Reductase↗
PubMed1993

Characterization of rat pseudogenes for enhancer factor I subunit A: ripping provides clues to the evolution of the EFIA/dbpB/YB-1 multigene family.

Genomic Southern blot analysis of rat EFIA (gene encoding enhancer factor I subunit A) reveals a complex band pattern when cDNA subfragment probes are used. Screening a rat genomic library with a rat EFIA cDNA probe yields two different processed EFIA pseudogenes, designated rat psi EFIA#(2/3) and #(4/7), in addition to two other different, but less extensively characterized clones. psi EFIA#(4/7) has no open reading frame (ORF) sequences. psi EFIA#(2/3) contains two ORFs (83 and 178 codons), the products of which (if expressed) might be negative-acting EFIA transcription factors. Located nearly 0.6 kb upstream from psi EFIA#(2/3) is a perfect 69-bp dinucleotide (CT) tandem repeat, a sequence element associated with other isolated pseudogenes. Additionally, the 3' end of this processed gene is interrupted by an unusual retroposon, an inverted dimeric B1-like short interspersed repetitive element (SINE). The isolation of several independent clones of the same EFIA processed pseudogenes indicates that they comprise a significant component of the rat EFIA copy multiplicity. The phenomenon of repeat induced point mutagenesis (ripping) at rat EFIA pseudogene CpG doublets occurs at a frequency at least 6.5 times higher than predicted from random mutagenesis. This is consonant with the proposal that ripping may be the mechanism which inactivates the ectopic recombination potential of the rat EFIA pseudogenes.

Amino Acid Sequence↗
PubMed2004

Molecular biology of sporadic gastric cancer: prognostic indicators and novel therapeutic approaches.

Both the availability of multiple treatment modalities and novel therapeutic targets make the correct prognostic stratification and the identification of truly predictive factors an issue of major debate in gastric cancer. Along with "classic" prognostic factors such as those related to the diffusion of the tumour at diagnosis (i.e., depth of gastric wall infiltration, locoregional lymph nodes or distant metastases) or those concerning the pathologic characteristics of the tumour, other, innovative, factors should be considered if a better definition of the characteristics of the tumour is to be given. These biological factors are often derived from the genetic process, which is thought to represent a crucial step to gastric cancer (DNA copy number changes, microsatellite instability, thymidilate synthase, E-cadherin, beta-catenin, mucin antigen, p53, c-erb B-2, COX-2, matrix metalloproteinases, VEGFR and EGFR). Some of those putative prognostic indicators can also be considered predictive of response to therapy as they are a molecular target either to chemotherapeutics (i.e., thymidilate synthase that is targeted by 5FU) or to a new class of antineoplastic molecules (i.e., c-erb B-2 targeted by trastuzumab, COX-2 by NSAIDs, matrix metalloproteinases, EGFR and VEGFR by specific inhibitors).

Apoptosis↗
PubMed2003

Production of supercoiled multimeric plasmid DNA for biopharmaceutical application.

Production of nucleic acids as an active pharmaceutical ingredient (API) in gene therapy and genetic vaccination is gaining more and more importance. Non-viral vectors like plasmid DNA are currently investigated in various clinical trials. Supercoiled multimeric plasmids are of particular interest for pharmaceutical purpose because they contain multiple copies of a therapeutic gene and can therefore be more efficient vectors. A process for the preparation of Escherichia coli strains replicating dimers, trimers, and tetramers of a 4.6 kb plasmid is presented. Cultivation of these clones on semi-defined glycerol medium in a 7 l bioreactor shows structural stability of dimers and trimers during the whole cultivation process. Plasmid concentrations and selectivities are compared to the corresponding cultivation with the plasmid monomer. Cultivation of the tetramer replicating strain shows a disintegration of the plasmid multimer and reconstitution of the monomer and smaller multimers.

Biopharmaceutics↗
PubMed2002

When writing 0 (zero) is easier than writing O (o): a neuropsychological case study of agraphia.

Though a few case studies reported a dissociation between intact writing of Arabic and impaired writing of alphabetical script, a detailed experimental analysis of such a dissociation is still lacking. We report a follow-up study of a patient with a parieto-occipital lesion who is affected by severe peripheral agraphia for letters, but not for Arabic digits. While letters in writing to dictation are frequently illegible, distorted, or consist in meaningless strokes, Arabic digits are well-formed and fluently produced. In a series of tasks, including copying of letters with tachistoscopic presentation and handwriting on a digitizing tablet, several processing levels are assessed in order to localize JS' functional writing impairment and to determine different processing routes for letters and for numbers. Overall, the results of the experimental investigation suggest a notation specific deficit in the activation of graphomotor patterns for letters, but not for digits. The study thus adds evidence to the so far reported dissociations between Arabic and alphabetical scripts.

Aged↗
PubMed2005

Analytical methods for studying the evolution of paralogs using duplicate gene datasets.

Gene duplication is widely viewed as an important source of raw material for functional innovation in proteins because at least some duplicate copies will evolve new or slightly modified functions. The study of the molecular processes by which functional innovation occurs interests both evolutionary biologists and protein chemists, and the development of methods to investigate these processes has led to a productive meeting of disciplines and an availability of complementary approaches for exploring datasets. This has resulted in insights into past events, prediction of current function, and prediction of future change. The methods fall broadly into two categories: those that rely on detection of shifts in selective constraints and those that rely on detection of correlations between molecular changes and functional shifts. Strengths and limitations of the methods are evaluated here in the context of the question being addressed, the input required, and the specific metric that is evaluated in each test.

Databases, Genetic↗
PubMed1997

Structure and transcription of the gene for translation elongation factor 1 subunit alpha of zebrafish (Danio rerio).

The zebrafish gene for translation elongation factor 1 alpha (EF1 alpha) was isolated from a phage Lambda genomic library and sequence and structure determined. One gene copy of EF1 alpha per haploid set of chromosomes was found and no processed pseudogenes. A highly active promoter region was localized to a 277 bp PstI/PvuII fragment beginning 240 bp upstream from the tsp, but no transcription enhancing, or silencing activity was observed within 1 kbp upstream, or downstream from the promoter. Expression of EF1 alpha appears to be developmentally regulated.

Animals↗
PubMed2000

It takes two transposons to tango: transposable-element-mediated chromosomal rearrangements.

Transposable elements (TEs) promote various chromosomal rearrangements more efficiently, and often more specifically, than other cellular processes(1-3). One explanation of such events is homologous recombination between multiple copies of a TE present in a genome. Although this does occur, strong evidence from a number of TE systems in bacteria, plants and animals suggests that another mechanism - alternative transposition - induces a large proportion of TE-associated chromosomal rearrangements. This paper reviews evidence for alternative transposition from a number of unrelated but structurally similar TEs. The similarities between alternative transposition and V(D)J recombination are also discussed, as is the use of alternative transposition as a genetic tool.

Animals↗
PubMed1998

Direct selection cloning vectors adapted to the genetic analysis of gram-negative bacteria and their plasmids.

A range of specific and unusual biological pathways are found in Gram-negative bacteria. It is possible to express the genes involved in these processes in Escherichia coli, however, some genes prove lethal when cloned into high copy number vectors in common usage. Conversely, various genetic functions remain silent in E. coli and require to be transferred into their original host for expression and subsequent analysis. To facilitate the cloning and the characterisation of bacterial genes, we have constructed CcdB 'positive-selection' vectors that possess one or more of the following properties: (i) low or medium copy number; (ii) narrow or broad replication host range; (iii) conjugational mobilisation. In this communication, we illustrate the use of these new cloning tools and analyse the CcdB toxicity in different bacterial species.

Bacterial Proteins↗
PubMed2006

Real-time polymerase chain reaction (PCR) quantitative detection of Brassica napus using a locked nucleic acid TaqMan probe.

Several countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms. Real-time quantitative Polymerase Chain Reaction (PCR) has quickly become the method of choice in support of these regulations and requires the development of separate PCR assays targeting the transgenic sequence as well as a specific endogenous gene sequence. To develop a Brassica napus-specific PCR assay, partial sequences of the acetyl-CoA carboxylase BnACCg8 gene from B. napus and the closely related Brassica rapa were determined and compared, and a region of unique nucleotide sequence was identified. Universal amplification primers were designed to either side of this region, and a locked nucleic acid TaqMan probe was designed to the B. napus-specific sequence. Evaluation of this primer/probe combination indicated a high level of specificity to B. napus: no amplification signal was observed with any other species tested, including five closely related Brassica species. The method was assayed with 14 different B. napus cultivars, and comparable amplification curves were consistently obtained for all. The assay was highly sensitive, with a limit of detection between 1 and 10 haploid copies. Practically, the method was demonstrated to be effective for the detection of processed food samples and for the quantification of Roundup Ready canola content in mixed samples.

Acetyl-CoA Carboxylase↗