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PubMed2003

Function and regulation of Aurora/Ipl1p kinase family in cell division.

During mitosis, the parent cell distributes its genetic materials equally into two daughter cells through chromosome segregation, a complex movements orchestrated by mitotic kinases and its effector proteins. Faithful chromosome segregation and cytokinesis ensure that each daughter cell receives a full copy of genetic materials of parent cell. Defects in these processes can lead to aneuploidy or polyploidy. Aurora/Ipl1p family, a class of conserved serine/threonine kinases, plays key roles in chromosome segregation and cytokinesis. This article highlights the function and regulation of Aurora/Ipl1p family in mitosis and provides potential links between aberrant regulation of Aurora/Ipl1p kinases and pathogenesis of human cancer.

Aneuploidy↗
PubMed1974

The three-dimensional organization of the amplified ribosomal DNA complex in Acheta.

The major chromomere of pachytene chromosome 6 in the oocytes of Acheta (Orthoptera) is a region of amplification for ribosomal RNA cistrons (28 S and 18 S). This chromomere was isolated and studied with the scanning electron microscope. It has an outer shell that is smooth at early stages but divides into polygonal plates as indentations appear on its surface. These indentations increase in number as the DNA copies separate from each other and as the ribosomal RNA synthesis progresses. The vertices of the polygonal plates coincide with the positions at which the DNA copies reach the surface of the chromomere during their "puffing" process. The combined information gained from scanning and transmission electron microscopy, DNA measurements, DNA.RNA hybridization, and cytochemistry, leads to a three-dimensional picture of the organization of the amplified ribosomal DNA in its association with ribosomal RNA and protein.

Animals↗
PubMed1993

Molecular cloning of the gene for the allatostatin family of neuropeptides from the cockroach Diploptera punctata.

Allatostatins (ASTs) are insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have isolated a cDNA from the cockroach Diploptera punctata that encodes a 41.5-kDa precursor polypeptide containing the AST family of peptides. Translation of the cDNA revealed a 370-amino acid pre-pro-peptide consisting of 13 AST-type peptides and appropriate processing sites for endoproteolytic cleavage and amidation. The 13 potential AST sequences are characterized by the C-terminal AST corestructure Phe-Gly-Leu-NH2, with only one exception. Separating the clustered ASTs in the precursor, three acidic spacer regions are found. Contained within the largest of these are two potentially related peptides that may also be processed. Southern blot analysis revealed the presence of a single copy of the AST gene per haploid genome, as well as the probability that the gene may be present in at least two allelic forms. In situ hybridization indicated the AST-encoding gene is expressed in neurosecretory cells of D. punctata brain.

Amino Acid Sequence↗
PubMed2002

PMP22 overexpression causes dysmyelination in mice.

Charcot-Marie-Tooth (CMT) disease is the most frequent hereditary peripheral neuropathy in humans. Its prevalence is about one in 2500. A subform, CMT1A, is transmitted as an autosomal dominant trait. An estimated 75% of patients are affected. This disorder has been shown to be associated with the duplication of a 1.5 Mb region of the short arm of chromosome 17, in which the PMP22 gene has been mapped. We have constructed a murine model of CMT1A by inserting into the murine genome a human YAC containing peripheral myelin protein 22 (PMP22) and its flanking controlling elements. We describe the behaviour of the C22 line (seven copies of YAC, 2.1 times PMP22 overexpression) during the myelination process. Electron microscopy, morphometry, electrophysiology, nerve conduction and expression of specific markers (e.g. Krox20) in normal and pathological Schwann cells demonstrated that PMP22 overexpression leads to a defect in the myelination of axons. The largest axons are the most affected. Only a few demyelination/remyelination processes were observed. Moreover, PMP22 overexpression probably enhances collagen synthesis by fibroblasts, before myelination, demonstrating that structures other than Schwann cells are affected by PMP22 overexpression. Classically, CMT1A was thought to be induced by a demyelination process following a phase of normal myelination, yet our data suggest that dysmyelination should be considered as a major factor for the disease.

Animals↗
PubMed2004

FtsH-mediated repair of the photosystem II complex in response to light stress.

A common feature of light stress in plants, algae, and cyanobacteria is the light-induced damage to the photosystem II complex (PSII), which catalyses the photosynthetic oxidation of water to molecular oxygen. A repair cycle operates to replace damaged subunits within PSII, in particular, the D1 reaction centre polypeptide, by newly synthesized copies. As yet the molecular details of this physiologically important process remain obscure. A key aspect of the process that has attracted much attention is the identity of the protease or proteases involved in D1 degradation. The results are summarized here of recent mutagenesis experiments that were designed to assess the functional importance of the DegP/HtrA and FtsH protease families in the cyanobacterium Synechocystis sp. PCC 6803. Based on these results and the analysis of Arabidopsis mutants, a general model for PSII repair is suggested in which FtsH complexes alone are able to degrade damaged D1.

ATP-Dependent Proteases↗
PubMed2025

Unequally Abundant Chromosomes and Unusual Collections of Transferred Sequences Characterize Mitochondrial Genomes of Gastrodia (Orchidaceae), One of the Largest Mycoheterotrophic Plant Genera.

The mystery of genomic alternations in heterotrophic plants is among the most intriguing in evolutionary biology. Compared to plastid genomes (plastomes) with parallel size reduction and gene loss, mitochondrial genome (mitogenome) variation in heterotrophic plants remains underexplored in many aspects. To further unravel the evolutionary outcomes of heterotrophy, we present a comparative mitogenomic study with 13 de novo assemblies of Gastrodia (Orchidaceae), one of the largest fully mycoheterotrophic plant genera, and its relatives. Analyzed Gastrodia mitogenomes range from 0.56 to 2.1 Mb, each consisting of numerous, unequally abundant chromosomes or contigs. Size variation might have evolved through chromosome rearrangements followed by stochastic loss of "dispensable" chromosomes, with deletion-biased mutations. The discovery of a hyper-abundant (∼15 times intragenomic average) chromosome in two assemblies represents the hitherto most extreme copy number variation in any mitogenomes, with similar architectures discovered in two metazoan lineages. Transferred sequence contents highlight asymmetric evolutionary consequences of heterotrophy: despite drastically reduced intracellular plastome transfers convergent across heterotrophic plants, their rarity of horizontally acquired sequences sharply contrasts parasitic plants, where massive transfers from their hosts prevail. Rates of sequence evolution are markedly elevated but not explained by copy number variation, extending prior findings of accelerated molecular evolution from parasitic to heterotrophic plants. Putative evolutionary scenarios for these mitogenomic convergence and divergence fit well with the common (e.g. plastome contraction) and specific (e.g. host identity) aspects of the two heterotrophic types. These idiosyncratic mycoheterotrophs expand known architectural variability of plant mitogenomes and provide mechanistic insights into their content and size variation.

Genome, Mitochondrial↗
PubMed2004

Human telomerase catalyzes nucleolytic primer cleavage.

Telomerase is a reverse transcriptase that uses an integral RNA molecule to add de novo G-rich repeats onto telomeric DNA, or onto nontelomeric DNA generated during chromosome fragmentation and breakage events. A telomerase-mediated DNA substrate cleavage activity has been reported in ciliates and yeasts. Nucleolytic cleavage may serve a proofreading function, enhance processivity or ensure that nontemplate telomerase RNA sequences are not copied into DNA. We identified and characterized a human telomerase-mediated nucleolytic cleavage activity using enzyme reconstituted in a rabbit reticulocyte lysate in vitro transcription/translation system and native enzyme extracted from cells. We found that telomerase catalyzed the removal of nucleotides from DNA substrates including those that can form a mismatch with the RNA template or that contain nontelomeric sequences located 3' to a telomeric sequence. Unlike Tetrahymena telomerase, human telomerase catalyzed the removal of more than one nucleotide (up to 13) from telomeric primers. DNA substrates predicted to align at the 3'-end of the RNA template were not cleaved, consistent with cleavage being dictated by the template 5'-end. We also found some differences in the nuclease activity between RRL-reconstituted human telomerase and native enzyme.

Animals↗
PubMed2004

Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis.

We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets. This multiplexing approach was validated by examining a panel of inflammation responsive genes in human umbilical vein endothelial cells stimulated with inflammatory cytokine interleukin 1beta. The laser-induced fluorescence detection and electrokinetic sample injection process in capillary electrophoresis allows sensitive quantification of thousands of copies of mRNA molecules in a reaction. The assay is precise, as evaluated by measuring qualified Z' factor, a dimensionless and simple characteristic for applications in high-throughput screening using mRNA assays. Our data demonstrate the synergy between the multiplexing capability of eTag molecules by sensitive capillary electrophoresis detection and the isothermal linear amplification characteristics of the Invader assay. eTag multiplex mRNA assay presents a unique platform for sensitive, high sample throughput and multiplex gene expression analysis.

Cells, Cultured↗
PubMedDate not supplied

Natural instability of Agrobacterium vitis Ti plasmid due to unusual duplication of a 2.3-kb DNA fragment.

The octopine/cucumopine (o/c) Ti plasmids of Agrobacterium vitis carry two T regions, TA and TB. The TA region resembles the octopine TL region. The TB region contains the auxin synthesis genes TB-iaaM and TB-iaaH and the cucumopine synthesis gene cus. Within the group of o/c isolates, strains 2608 and 2641 are closely related. However, 2641 lacks the TB region. The restriction maps of pTi2608 and pTi2641 were established and showed that the TB deletion resulted from intramolecular recombination between two directly repeated sequences separated by 66 kb in pTi2608. The 2,294-bp repeated sequence lacks inverted repeats and does not duplicate its target site, indicating that it is not a classical bacterial insertion sequence (IS element). It was therefore called an RSAv element (repeated sequence of A. vitis). The RSAv element carries two open reading frames: ORF234 is homologous to the traR gene of the A. tumefaciens nopaline Ti plasmid pTiC58; ORF488 is homologous to the sucrose phosphorylase gene of Leuconostoc mesenteroides and the glucosyl transferase A gene of Streptococcus mutans. The RSAv repeat starts precisely at the start codon of ORF488 and ends two base pairs 3' of the stop codon of ORF234. The structural organization of the RSAv element suggests that the amplification event did not result from a random amplification process. A study of the distribution of the two RSAv copies (RSAv-1 and RSAv-2) in pTi2608 and other o/c isolates indicates that the ancestor o/c Ti plasmid contained only RSAv-1 and that this sequence was duplicated at one point during the divergent evolution of the o/c Ti plasmids.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Evaluation of an enhanced digital film-duplication system by receiver operating characteristic analysis.

RATIONALE AND OBJECTIVES: Over- or underexposure often results in poor quality radiographs. Using receiver operating characteristic analysis, the authors investigated the potential utility of a system that corrects densities in such images to improve the accuracy of lung nodule detection. METHODS: Chest radiographs were digitized by a laser film digitizer. Simulated nodules were superimposed on 36 improperly exposed chest radiographs. All images, with and without nodules, were then processed by a nonlinear density-correction technique. Hard-copy images were produced by a laser film printer. Eight radiologists evaluated the corrected and uncorrected images for the presence or absence of lung nodules using a continuous (0-100) confidence-rating scale. RESULT: The detectability of lung nodules improved significantly in the retrocardiac/retrodiaphragmatic regions of underexposed images and in the peripheral lung region of overexposed images. CONCLUSIONS: This system may increase diagnostic accuracy in detection of lung nodules in improperly exposed images.

Confidence Intervals↗
PubMed2006

Asymptotic violation of Bell inequalities and distillability.

A multipartite quantum state violates a Bell inequality asymptotically if, after jointly processing by general local operations an arbitrarily large number of copies of it, the result violates the inequality. In the bipartite case we show that asymptotic violation of the Clauser-Horne-Shimony-Holt inequality is equivalent to distillability. Hence, bound entangled states do not violate it. In the multipartite case we consider the complete set of full-correlation Bell inequalities with two dichotomic observables per site. We show that asymptotic violation of any of these inequalities by a multipartite state implies that pure-state entanglement can be distilled from it, although the corresponding distillation protocol may require that some of the parties join into several groups. We also obtain the extreme points of the set of distributions generated by measuring N quantum systems with two dichotomic observables per site.

Journal Article↗
PubMed2004

Sequence and comparative analysis of the maize NB mitochondrial genome.

The NB mitochondrial genome found in most fertile varieties of commercial maize (Zea mays subsp. mays) was sequenced. The 569,630-bp genome maps as a circle containing 58 identified genes encoding 33 known proteins, 3 ribosomal RNAs, and 21 tRNAs that recognize 14 amino acids. Among the 22 group II introns identified, 7 are trans-spliced. There are 121 open reading frames (ORFs) of at least 300 bp, only 3 of which exist in the mitochondrial genome of rice (Oryza sativa). In total, the identified mitochondrial genes, pseudogenes, ORFs, and cis-spliced introns extend over 127,555 bp (22.39%) of the genome. Integrated plastid DNA accounts for an additional 25,281 bp (4.44%) of the mitochondrial DNA, and phylogenetic analyses raise the possibility that copy correction with DNA from the plastid is an ongoing process. Although the genome contains six pairs of large repeats that cover 17.35% of the genome, small repeats (20-500 bp) account for only 5.59%, and transposable element sequences are extremely rare. MultiPip alignments show that maize mitochondrial DNA has little sequence similarity with other plant mitochondrial genomes, including that of rice, outside of the known functional genes. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly three-fourths of the maize NB mitochondrial genome is still of unknown origin and function.

Base Sequence↗
PubMed2004

Simulating evolution by gene duplication of protein features that require multiple amino acid residues.

Gene duplication is thought to be a major source of evolutionary innovation because it allows one copy of a gene to mutate and explore genetic space while the other copy continues to fulfill the original function. Models of the process often implicitly assume that a single mutation to the duplicated gene can confer a new selectable property. Yet some protein features, such as disulfide bonds or ligand binding sites, require the participation of two or more amino acid residues, which could require several mutations. Here we model the evolution of such protein features by what we consider to be the conceptually simplest route-point mutation in duplicated genes. We show that for very large population sizes N, where at steady state in the absence of selection the population would be expected to contain one or more duplicated alleles coding for the feature, the time to fixation in the population hovers near the inverse of the point mutation rate, and varies sluggishly with the lambda(th) root of 1/N, where lambda is the number of nucleotide positions that must be mutated to produce the feature. At smaller population sizes, the time to fixation varies linearly with 1/N and exceeds the inverse of the point mutation rate. We conclude that, in general, to be fixed in 10(8) generations, the production of novel protein features that require the participation of two or more amino acid residues simply by multiple point mutations in duplicated genes would entail population sizes of no less than 10(9).

Amino Acids↗
PubMed2003

Insertion sequence ISEcp1B is involved in expression and mobilization of a bla(CTX-M) beta-lactamase gene.

The genetic structures (ca. 10-kb DNA fragment) surrounding the plasmid-borne extended-spectrum beta-lactamase bla(CTX-M-19) gene in a Klebsiella pneumoniae clinical isolate were determined. This beta-lactamase gene was part of a 4,797-bp transposon inserted inside orf1 of Tn1721. Inside this transposon, bla(CTX-M-19) was bracketed upstream and downstream by insertion sequences ISE cp1B and IS903D, respectively, and further downstream by a truncated gene encoding an outer membrane protein for iron transport. The single-copy ISEcp1B element was probably involved alone in the mobilization process that led to a 5-bp duplication at the target site of the transposed fragment. This mobilization event probably involved one inverted repeat of ISE cp1B and a second sequence farther away, resembling its second inverted repeat. Additionally, ISEcp1B provided -35 and -10 promoter sequences, contributing to the high-level expression of the bla(CTX-M-19) gene. Southern blot analysis failed to identify a reservoir of ISEcp1-like sequences among a series of gram-negative and gram-positive bacterial species usually found in the skin and intestinal human floras. The ability of ISEcp1-like elements to mobilize and to promote the expression of beta-lactamase genes may explain, in part, the current spread of CTX-M-type enzymes worldwide.

Anti-Bacterial Agents↗
PubMed2000

Knockout of the p-coumarate decarboxylase gene from Lactobacillus plantarum reveals the existence of two other inducible enzymatic activities involved in phenolic acid metabolism.

Lactobacillus plantarum NC8 contains a pdc gene coding for p-coumaric acid decarboxylase activity (PDC). A food grade mutant, designated LPD1, in which the chromosomal pdc gene was replaced with the deleted pdc gene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector. The LPD1 mutant strain remained able to weakly metabolize p-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids. We have shown that L. plantarum has a second acid phenol decarboxylase enzyme, better induced with ferulic acid than with p-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when glucose is added. Those two enzymatic activities are in competition for p-coumaric and ferulic acid degradation, and the ratio of the corresponding derivatives depends on induction conditions. Moreover, PDC appeared to decarboxylate ferulic acid in vitro with a specific activity of about 10 nmol. min(-1). mg(-1) in the presence of ammonium sulfate. Finally, PDC activity was shown to confer a selective advantage on LPNC8 grown in acidic media supplemented with p-coumaric acid, compared to the LPD1 mutant devoid of PDC activity.

Carboxy-Lyases↗
PubMed1991

Defective retroviruses can disperse in the human genome by intracellular transposition.

Using an assay for retrotransposition detection (T. Heidmann, O. Heidmann, and J. F. Nicolas, Proc. Natl. Acad. Sci. USA 85:2219-2223, 1988), we demonstrated that a defective retrovirus deleted for the gag, pol, and env open reading frames can disperse in the genome of human HeLa cells by intracellular transposition, at a frequency close to 10(-6) events per cell per generation. Transposition requires cooperation in trans for the gag and pol gene products and may be associated with the release of low amounts of noninfectious retroviruslike particles which are the hallmarks but not the intermediates of this transposition process. Similar events could account for the dispersion at high copy number of some of the human endogenous sequences related to retroviruses and for the occurrence of noninfectious retroviruslike particles in human placenta and several tumor cell lines (reviewed by E. Larsson, N. Kato, and M. Cohen, Curr. Top. Microbiol, Immunol, 148:115-132, 1989).

Defective Viruses↗
PubMed1988

Genetic analysis of small nuclear RNAs in Saccharomyces cerevisiae: viable sextuple mutant.

Saccharomyces cerevisiae contains at least 24 distinct small nuclear RNAs (snRNAs), several of which are known to be essential for viability and to participate in the splicing of pre-mRNAs; the RNAs in this subset contain binding sites for the Sm antigen, a hallmark of metazoan snRNAs involved in mRNA processing. In contrast, we showed previously that the single-copy genes for three other snRNAs (snR3, snR4, and snR10) are not required for viability, although cells lacking snR10 are growth impaired at low temperature. None of these RNAs associates with the Sm antigen. To assess this apparent correlation, we cloned and sequenced the genes encoding three additional non-Sm snRNAs. Comparison of these genes with nine additional yeast snRNA genes revealed a highly conserved TATA box located 92 +/- 8 nucleotides 5' of the transcriptional start site. By using the technique of gene replacement with null alleles, each of these three single copy genes was shown to be completely dispensable. We constructed multiple mutants to test the hypothesis that, individually, each of these snRNAs is nonessential because the snRNAs play functionally overlapping roles. A mutant lacking five snRNAs (snR3, snR4, snR5, snR8, snR9) was indistinguishable from the wild type, and growth of the sextuple mutant was no more impaired than that in strains lacking only snR10. This widespread dispensability of snRNAs was completely unexpected and forces us to reconsider the possible roles of these ubiquitous RNAs.

Base Sequence↗
PubMed1994

Extraocular muscle proprioception functions in the control of ocular alignment and eye movement conjugacy.

1. The function of extraocular muscle proprioception in the control of eye movements is uncertain. We tested the hypothesis that proprioception contributes to the long-term regulation of ocular alignment and eye movement conjugacy. 2. Eye movements were recorded in monkeys with unilateral extraocular muscle palsies, before and after proprioceptive deafferentation of the paretic eye. Following deafferentation, ocular alignment and saccade conjugacy gradually worsened over several weeks. In contrast, disconjugate adaptation induced by habitual binocular viewing with a prism (disparity-mediated adaptation) occurred normally after deafferentation. 3. These results provide the first evidence that proprioception functions in the control of eye movements in primates, and indicate that proprioception contributes to the long-term adaptive mechanisms that regulate ocular alignment during fixation and saccades. The error signal used in this process may be derived from a mismatch between the efference copy and proprioceptive afference.

Afferent Pathways↗