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PubMed1976

Structural organization and processing of the genetic transcript in the cellular slime mold Dictyostelium discoideum.

The organization of the genome and the synthesis and processing of heterogeneous nuclear RNA (HNRNA) in the cellular slime mold Dictysotelium discoideum have been analyzed. Approximately 60-70% of the genome of Dictyostelium consists of interspersed reiterated and single-copy sequences. The interspersed reiterated sequences have an average length of 250-400 nucleotides. Approximately 50% of the reiterated DNA sequences consist of long noninterspersed sequences. The results of analyses of ynRNA synthesis and processing have been incorporated into a model. According to the model the primary genetic transcript of Dictyostelium is synthesized as a molecule that is 25% larger than mRNA. The bulk of the hnRNA is synthesized from a unit consisting of a short reiterated DNA sequence transcript at the 5' end of the molecule and a single-copy sequence of approximately 1,200 nucleotides in length. In the processing of the mRNA precursor, there appears to be a loss of the majority of the repetitive sequence at the 5' end. The genome contains interspersed sequences of poly (dT)25. These sequences, which appear to be at the 3' terminus of the transcription unit, are transcribed directly into the heterogenous nuclear RNA and are contained within the messenger RNA. During the processing of the heterogeneous nuclear RNA, a poly (A) sequence of approximately 125 nucleotides in length is added posttranscriptionally to the 3' end of the molecule.

Base Sequence↗
PubMed2006

Strategies to determine the biological function of microRNAs.

MicroRNAs (miRNAs) are regulators of gene expression that control many biological processes in development, differentiation, growth and metabolism. Their expression levels, small size, abundance of repetitive copies in the genome and mode of action pose unique challenges in studies elucidating the function of miRNAs. New technologies for identification, expression profiling and target gene validation, as well as manipulation of miRNA expression in vivo, will facilitate the study of their contribution to biological processes and disease. Such information will be crucial to exploit the emerging knowledge of miRNAs for the development of new human therapeutic applications.

Animals↗
PubMed2004

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes.

Oxygenic photosynthesis produces various radicals and active oxygen species with harmful effects on photosystem II (PSII). Such photodamage occurs at all light intensities. Damaged PSII centres, however, do not usually accumulate in the thylakoid membrane due to a rapid and efficient repair mechanism. The excellent design of PSII gives protection to most of the protein components and the damage is most often targeted only to the reaction centre D1 protein. Repair of PSII via turnover of the damaged protein subunits is a complex process involving (i) highly regulated reversible phosphorylation of several PSII core subunits, (ii) monomerization and migration of the PSII core from the grana to the stroma lamellae, (iii) partial disassembly of the PSII core monomer, (iv) highly specific proteolysis of the damaged proteins, and finally (v) a multi-step replacement of the damaged proteins with de novo synthesized copies followed by (vi) the reassembly, dimerization, and photoactivation of the PSII complexes. These processes will shortly be reviewed paying particular attention to the damage, turnover, and assembly of the PSII complex in grana and stroma thylakoids during the photoinhibition-repair cycle of PSII. Moreover, a two-dimensional Blue-native gel map of thylakoid membrane protein complexes, and their modification in the grana and stroma lamellae during a high-light treatment, is presented.

Light↗
PubMed2004

Processive utilization of the human telomerase template: lack of a requirement for template switching.

The ribonucleoprotein telomerase is a specialized reverse transcriptase minimally composed of an RNA, TER, and a protein catalytic subunit, TERT. The TER and TERT subunits of telomerase associate to form a dimeric enzyme in several organisms, including human. A small portion of TER, the template domain, is used by telomerase for the synthesis of tandem repeats of telomeric DNA. We studied some of the requirements for processive template usage by human telomerase. A blunt-ended duplex DNA primer was not utilized by telomerase. With a duplex telomeric DNA primer, a single-stranded 3' overhang with a minimum length of approximately 6 bases was required for efficient priming activity. Large substitutions in the human TER templating domain did not abolish enzymatic activity, although insertion of two residues into this sequence reduced processivity, as did a template mutation that results in a mismatch between the template region used for copying DNA and the region used for alignment of the substrate primer. Finally, by using a complementary pair of catalytically inactive telomerase RNA pseudoknot mutants in combination with a marked template, we demonstrated that processive synthesis by an obligatory dimer of human telomerase does not require template switching. These results indicate that processive template usage by human telomerase, like that of Tetrahymena telomerase, is strongly dependent on the base identities in the template domain and that a dimeric human telomerase can processively utilize a single template.

Base Pair Mismatch↗
PubMed1993

An efficient disk based data structure for rapid searching of quantitative two-dimensional gel databases.

Fast access of two-dimensional (2-D) gel quantitative databases is important for rapid searching for protein differences between sets of 2-D gels from an experiment. The GELLAB-II system organizes corresponding spots from the gels in the database into reference or "Rspot" sets. These Rspot numeric names index fixed regions in the paged composite gel database file. This is adequate for an existing database, but has several problems. (i) Building the initial database requires guessing how much disk space to pre-allocate for each corresponding spot (i.e. spots from different gels). If it ever runs out of pre-allocated space during this process, it must expand the size of each corresponding set of spots copying the old database data into the new in-place on the disk. (ii) When adding new gels or editing the database, if a new spot is created, the system may also go into this expansion mode. The time spent and wasted disk space can be appreciable--depending on the size of the database (order of 100 gel database). (iii) Because each set of corresponding spots is the same size, we waste space in most spot sets since they do not require the additional space a few spot sets require which contain additional fragmented spots. We present a new low-level disk object-based structure and algorithm, paged indexed buckets (PIB), which optimizes disk space usage while having similar retrieval speed to the original method.

Algorithms↗
PubMedDate not supplied

Storage options for the healthcare enterprise.

The storage objectives for the healthcare enterprise (HE) are to ensure that information (images and data) are readily available anywhere and at anytime, images and data are secure, and the storage fulfills legal requirements and the Health Insurance Portability and Accountability Act (HIPAA). These objectives must be satisfied at a minimum economic cost with respect to personnel, hardware, software, space and telecommunications. Many approaches and storage configurations meet these objectives. Which approach is chosen will depend on the size of the institution, patient population, geographic distribution of the institutions (if more than one), type of facility (such as a hospital, outpatient clinic or private imaging center), and financial investment objectives. The quantity of storage required depends on the characteristics of the modalities, the number of imaging devices and databases, the number and location of imaging sites that make up the HE, the size of the data and image, and the projected procedure volume growth. The only certainty with respect to storage requirements is that they will increase significantly with time. The types of storage required in the HE can be described by their functions: Active storage includes both online and long-term storage. Backup images are temporarily backed up on the limited storage capacity of the modality for several days or longer. Additional copies of the study are made on different media (e.g., disk, DVD or tape), in different locations. The process of backing up data and images must be automated. Effective April 21, 2005, HIPAA requires that all healthcare entities have a disaster recovery plan in effect. This requires that a copy of all medical data be secure, retrievable and maintained in a second location, such that if the primary copy of the data is destroyed or made unavailable, the disaster recovery copy would be available. Planning for the HE archive is critical if the HE is to work productively in an integrated digital environment. The information technology department must be an integral part of planning for the HE archive, which must be located in a secure data center and not under the management of any single clinical department. After the technologies currently available are evaluated, it is imperative that the chosen solution is cost-effective and scalable, and that it will allow the HE to take advantage of future storage and storage management technology.

Archives↗
PubMed1991

A new allele of the duplicated 27kD zein locus of maize generated by homologous recombination.

The 27kD zein storage protein locus in many inbred lines of maize consists of a tandem duplication of 12kb, with an expressed gene in each repeat, termed A and B. A single-copy allele with only the A gene can be generated from this duplication in particular stocks of the maize inbred line A188 by a mitotic process that includes a crossover at the 3' regions of the two genes (1). Here, we characterize a second single-copy allele with only the B gene, found in different stocks of A188. This allele arises from a homologous recombination at the highly conserved 5' regions of the two repeats, and cloning and sequencing of this allele define the crossover region. The A and B genes in the duplicated allele were previously shown to be expressed at different levels; this difference remains unchanged in either recombinant allele. Therefore, the crossover points of these two recombinant alleles define the borders of cis-acting sequences responsible for the differential expression of the two genes.

Alleles↗
PubMed1991

Recurrence of repeat-induced point mutation (RIP) in Neurospora crassa.

Duplicate DNA sequences in the genome of Neurospora crassa can be detected and mutated in the sexual phase of the life cycle by a process termed RIP (repeat-induced point mutation). RIP occurs in the haploid nuclei of fertilized, premeiotic cells before fusion of the parental nuclei. Both copies of duplications of gene-sized sequences are affected in the first generation at frequencies of approximately 50-100%. We investigated the extent to which sequences altered by RIP remain susceptible to this process in subsequent generations. Duplications continued to be sensitive to RIP, even after six generations. The fraction of progeny showing evidence of RIP decreased rapidly, however, apparently as a function of the extent of divergence of the duplicated sequences. Analysis of the stability of heteroduplexes of DNA altered by RIP and their native counterpart indicated that linked duplications diverged further than did unlinked duplications. DNA methylation, a common feature of sequences altered by RIP, did not seem to inhibit the process. A sequence that had become resistant to RIP was cloned and reintroduced into Neurospora in one or more copies to investigate the basis of the resistance. The altered sequence regained its methylation in vegetative cells, indicating that the methylation of sequences altered by RIP observed in vegetative cells is a consequence of the mutations. Duplication of the sequence restored its sensitivity to RIP suggesting that resistance to the process was due to loss of similarity between the duplicated sequences. Consistent with this, we found that the resistant sequence did not trigger RIP of the native homologous sequences of the host, even when no other partner was available. High frequency intrachromatid recombination, which is temporally associated with RIP, was more sensitive than RIP to alterations in the interacting sequences.

Biological Evolution↗
PubMed1984

A deletion that includes the signal peptidase cleavage site impairs processing, glycosylation, and secretion of cell surface yeast acid phosphatase.

We transformed Saccharomyces cerevisiae with a high-copy-number plasmid carrying either the wild-type gene coding for a repressible cell surface acid phosphatase or two modified genes whose products lack a 13- or 14-amino-acid segment spanning or immediately adjacent to the signal peptidase cleavage site. The wild-type gene product underwent proteolytic cleavage of the signal peptide, core glycosylation, and outer chain glycosylation. The deletion spanning the signal peptidase cleavage site led to an unprocessed protein. This modified protein exhibited core glycosylation, whereas its outer chain glycosylation was severely inhibited. Secretion of the deleted protein was impaired, and active enzyme accumulated within the cell. The deletion immediately adjacent to the signal peptidase cleavage site exhibited only a small decrease in the efficiency of processing and had no effect on the efficiency of secretion.

Acid Phosphatase↗
PubMed2002

Identification of a protein essential for a major pathway used by human cells to avoid UV- induced DNA damage.

When DNA replication stalls at a fork-blocking lesion, cells use damage tolerance pathways to continue replication. One pathway, "translesion synthesis," involves specialized DNA polymerases that can use damaged DNA as a template. Translesion synthesis can result in mutations (i.e., can be error-prone), but it can also be error-free. An alternative pathway has been hypothesized (sometimes called "damage avoidance"), by which cells make temporary use of an undamaged copy of the blocked sequence as a template, i.e., the newly synthesized daughter strand of the sister duplex or the allelic copy. This pathway is error-free. Evidence of the use of the daughter strand of the sister duplex as a template in intact mammalian cells has not been available heretofore. To determine whether hMms2, a ubiquitin-conjugating enzyme-like protein, plays a critical role in such damage avoidance, a human fibroblast cell strain in which both error-prone translesion synthesis and error-free damage avoidance can be detected and quantified simultaneously, and several derivative strains in which expression of hMms2 protein had been eliminated or greatly decreased, were compared for their ability to avoid translesion synthesis past UV(254nm)-induced DNA photoproducts. Loss of hMms2 protein eliminated the ability of the latter strains to use an allelic copy of a target gene for damage avoidance, i.e., to produce a wild-type gene from two nonfunctional allelic copies of that gene. Molecular analysis of the wild-type gene showed that this process involves gene conversion unassociated with crossing-over. That the loss of hMms2 also eliminated use of the daughter strand of the sister duplex as a template for damage avoidance could be inferred from the fact that the frequency of mutations induced by UV in the single copy HPRT gene of the derivative strains was significantly higher than that observed in the parental strain. These data indicate that hMMS2 is essential for human cells to carry out damage avoidance by using either type of homolog, and that damage avoidance and translesion synthesis are alternative pathways for tolerating fork-blocking photoproducts.

DNA↗
PubMed2005

Generating single-copy nuclear gene data for a recent adaptive radiation.

Recent adaptive radiations provide an exceptional opportunity to understand the processes of speciation and adaptation. However, reconstructing the phylogenetic history of recent and rapidly evolving clades often requires the use of multiple, independent gene genealogies. Nuclear introns are an obvious source of the necessary data but their use is often limited because degenerate primers can amplify paralogous loci. To identify PCR primers for a large number of loci in an especially rapid adaptive radiation, that of the flowering plant genus Aquilegia (Ranunculaceae), we developed an efficient method for amplifying multiple single-copy nuclear loci by sequencing a modest number of clones from a cDNA library and designing PCR primers; with one primer anchored in the 3' untranslated region (3'-UTR) and one primer in the coding region of each gene. Variation between paralogous loci evolves more quickly in 3'-UTR regions compared to adjacent exons, and therefore we achieved high specificity for isolating orthologous loci. Furthermore, we were able to identify genes containing large introns by amplifying genes from genomic DNA and comparing the PCR product size to that predicted from their cDNA sequence. In Aquilegia eight out of eleven loci were isolated with this method and six of these loci had introns. Among four genes sequenced for samples spanning the phylogenetic breadth of the genus, we found sequence variation at levels similar to that observed in ITS, further supporting the recent and rapid radiation in Aquilegia. We assessed the orthology of amplification products by phylogenetic congruence among loci, the presence of two well established phylogenetic relationships, and similarity among loci for levels of sequence variation. Higher levels of variation among samples for one locus suggest possible paralogy. Overall, this method provides an efficient means of isolating predominantly single-copy loci from both low and high-copy gene families, providing ample nuclear variation for reconstructing species-level phylogenies in non-model taxa.

3' Untranslated Regions↗
PubMed1992

The evolution of the amplified adenylate deaminase 2 domains in Chinese hamster cells suggests the sequential operation of different mechanisms of DNA amplification.

Fluorescent in situ hybridization was used to localize the adenylate deaminase 2 (AMPD2) genes and flanking sequences on the chromosomes of the Chinese hamster line GMA32 and to study the distribution of additional copies of these genetic sequences in amplified mutants selected at several early stages of the amplification process. The synteny of AMPD2 genes and MDR1 genes, located on chromosomes 1, was demonstrated; in GMA32 the existence of a rearrangement positioning the two AMPD2 genes at different distances from the telomeres was disclosed. Using this structural marker, we showed that the amplified copies distribute along only one of the chromosomes 1. Their organization in different cells of clonal mutant populations at a very early stage of amplification was extremely heterogeneous; classes of organization could be recognized however. Their quantitative distribution at this stage and in cells which went through 10 more division cycles suggests an evolution pathway common to the mutant clones under study: as a rule, tandems of few units of identical and very large size (47 Mb) appear to be the first detected product of amplification; this organization is progressively overtaken by structures with more units of reduced and irregular size, while, in a growing number of cells, clusters of much shorter units can be observed. The nature of segregative amplification mechanisms operating in these processes and the possible involvement of replicative ones are discussed.

AMP Deaminase↗
PubMed1985

Iperalimentazione. A program to calculate hyperalimentation needs.

Parenteral hyperalimentation is an important means of nutritional support for critical care patients. Calculation of the amount of calories, components and additives necessary for the appropriate compounding of hyperalimentation solutions is a lengthy, tedious process with many potential opportunities for computation error. A new computer program written in Pascal has been created in order to reduce calculation time and error. Menus and single-keystroke entry are implemented wherever practicable. A review screen is employed to reduce user input errors. A hard-copy printout is generated on command. Program testing has resulted in a 90% reduction in processing time.

Computers↗
PubMed1999

Taming of transposable elements by homology-dependent gene silencing.

Transposable elements can invade virgin genomes within a few generations, after which the elements are 'tamed' and retain only limited transpositional activity. Introduction of the I element, a transposon similar to mammalian LINE elements, into Drosophila melanogaster genomes devoid of such elements initially results in high-frequency transposition of the incoming transposon, high mutation rate, chromosomal nondisjunction and female sterility, a syndrome referred to as hybrid dysgenesis (for review, see refs 2-4); a related syndrome has also been described in mammals. High-frequency transposition is transient, as the number of I elements reaches a finite value and transposition ceases after approximately ten generations. It has been proposed that the I elements encode a factor that negatively regulates their own transcription, but evidence for such a mechanism is lacking. Using the hybrid dysgenesis syndrome in Drosophila as a model, we show here that transpositional activity of the I element can be repressed by prior introduction of transgenes expressing a small internal region of the I element. This autoregulation presents features characteristic of homology-dependent gene silencing, a process known as cosuppression. Repression does not require any translatable sequence, its severity correlates with transgene copy number and it develops in a generation-dependent manner via germline transmission of a silencing effector in females only. These results demonstrate that transposable elements are prone to and can be tamed by homology-dependent gene silencing, a process that may have emerged during the course of evolution as a specific defense mechanism against these elements.

Animals↗
PubMed1997

Isolation of a Schizosaccharomyces pombe gene which in high copy confers resistance to the nucleoside analogue 5-azacytidine.

Treatment of Schizosaccharomyces pombe with the C5 DNA methyltransferase (C5Mtase) inhibitor 5-azacytidine (5-azaC) has previously been shown to induce G2 checkpoint-dependent cell cycle arrest. S. pombe strains defective in both the checkpoint control pathways and in DNA repair processes are sensitive to 5-azaC. Here we describe the isolation of azr1+, as a multi-copy suppressor of the 5-azaC sensitivity of G2 checkpoint and DNA repair-deficient strains. azr1+ encodes a putative 25 kDa protein with limited homology to a Saccharomyces cerevisiae open reading frame of unknown function. The azr1+ gene is not essential and the null mutant shows no alteration in either DNA repair or checkpoint properties. We also report the sequence of the putative fission yeast cytidine deaminase gene, designated pcd1+, which lies immediately adjacent to azr1+ but which plays only a moderate role in suppression of 5-azaC sensitivity.

Amino Acid Sequence↗
PubMed1998

Molecular instability in the COII-tRNA(Lys) intergenic region of the human mitochondrial genome: multiple origins of the 9-bp deletion and heteroplasmy for expanded repeats.

We have identified two individuals from Glasgow in Scotland who have a deletion of one of two copies of the intergenic 9-bp sequence motif CCCCCTCTA, located between the cytochrome oxidase II (COII) and lysine tRNA (tRNA(Lys)) genes of the human mitochondrial genome. Although this polymorphism is common in Africa and Asia, it has not been reported in Northern Europe. Analysis of the mitochondrial DNA control region sequences of these two individuals suggests that they belong to a lineage that originated independently of the previously characterized African and Asian 9-bp deleted lineages. Among the Scottish population we have also identified a maternal lineage of three generations exhibiting heteroplasmy for two, three and four copies of the CCCCCTCTA motif. Polymerase chain reaction amplification across the COII-tRNA(Lys) intergenic region of these individuals gives different ratios of the three product lengths that are dependent on the concentration of the DNA-binding dye crystal violet. To investigate whether changes in repeat number were generated de novo, we constructed clones containing known numbers of the CCCCCTCTA motif. In the presence of high concentrations of crystal violet we obtained two, three and four copies of this motif when the amplification template contained only four copies. Various DNA-binding drugs are known to stabilize bulged structures in DNA and contribute to the process of slipped-strand mispairing during DNA replication. These results suggest that the COII-tRNA(Lys) intergenic region is unstable owing to slipped-strand mispairing. Although sequences containing four copies of the CCCCCTCTA motif are less stable in vitro, we observed an increase in the proportion of mitochondrial genomes with four repeats between-a mother and a daughter in the heteroplasmic lineage. From this we conclude that drift in the germ-line lineage is a main factor in the maintenance or loss of heteroplasmy.

Africa↗
PubMed1991

A copy of exon 3-intron 3 from the barley aleurain gene is present on chromosome 2.

A genomic clone from Hordeum vulgare L. cv. Himalaya contains 700 bp of DNA that is homologous with a high degree of nucleotide sequence similarity to exon 3-intron 3 from the gene for the thiol protease, aleurain. Genomic Southern blot mapping data indicate that this clone in phage lambda had not undergone rearrangement, and no other sequences homologous to aleurain are present on it. Although exon 3 in aleurain encodes the polypeptide region cleaved during proteolytic processing of the proenzyme to its mature form, we do not know if the copy is expressed in some other protein. We have mapped the aleurain gene to chromosome 1, and this copy of exon 3-intron 3 to chromosome 2.

Base Sequence↗
PubMed2003

ExpressYourself: A modular platform for processing and visualizing microarray data.

DNA microarrays are widely used in biological research; by analyzing differential hybridization on a single microarray slide, one can detect changes in mRNA expression levels, increases in DNA copy numbers and the location of transcription factor binding sites on a genomic scale. Having performed the experiments, the major challenge is to process large, noisy datasets in order to identify the specific array elements that are significantly differentially hybridized. This normally requires aggregating different, often incompatible programs into a multi-step pipeline. Here we present ExpressYourself, a fully integrated platform for processing microarray data. In completely automated fashion, it will correct the background array signal, normalize the Cy5 and Cy3 signals, score levels of differential hybridization, combine the results of replicate experiments, filter problematic regions of the array and assess the quality of individual and replicate experiments. ExpressYourself is designed with a highly modular architecture so various types of microarray analysis algorithms can readily be incorporated as they are developed; for example, the system currently implements several normalization methods, including those that simultaneously consider signal intensity and slide location. The processed data are presented using a web-based graphical interface to facilitate comparison with the original images of the array slides. In particular, Express Yourself is able to regenerate images of the original microarray after applying various steps of processing, which greatly facilities identification of position-specific artifacts. The program is freely available for use at http://bioinfo.mbb.yale.edu/expressyourself.

Algorithms↗