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PubMed2006

Chromosome healing by de novo telomere addition in Saccharomyces cerevisiae.

The repair of spontaneous or induced DNA damage by homologous recombination (HR) in Saccharomyces cerevisiae will suppress chromosome rearrangements. Alternative chromosome healing pathways can result in chromosomal instability. One of these pathways is de novo telomere addition where the end of a broken chromosome is stabilized by telomerase-dependent addition of telomeres at non-telomeric sites. De novo telomere addition requires the recruitment of telomerase to chromosomal targets. Subsequently, annealing of the telomerase reverse transcriptase RNA-template (guide RNA) at short regions of homology is followed by extension of the nascent 3'-end of the broken chromosome to copy a short region of the telomerase guide RNA; multiple cycles of this process yield the new telomere. Proteins including Pif1 helicase, the single-stranded DNA-binding protein Cdc13 and the Ku heterocomplex are known to participate in native telomere functions and also regulate the de novo telomere addition reaction. Studies of the sequences added at de novo telomeres have lead to a detailed description of the annealing-extension-dissociation cycles that copy the telomerase guide RNA, which can explain the heterogeneity of telomeric repeats at de novo and native telomeres in S. cerevisiae.

Chromosome Breakage↗
PubMed1998

Yeast nuclear PET127 gene can suppress deletions of the SUV3 or DSS1 genes: an indication of a functional interaction between 3' and 5' ends of mitochondrial mRNAs.

Saccharomyces cerevisiae nuclear genes SUV3 and DSS1 encode putative RNA helicase and RNase II, respectively, which are subunits of the mitochondrial degradosome (mtEXO): a three-protein complex which has a 3' to 5' exoribonuclease activity and plays a major role in regulating stability of mitochondrial RNA. Lack of either of the two gene products results in a respiratory negative phenotype, while on the molecular level it causes a total block of mitochondrial translation, loss of the in vitro exoribonuclease activity and changes in stability and processing of many mtRNAs. We have found that the yeast nuclear gene PET127 present on a low or high copy number vector can effectively suppress the effects of the SUV3 or DSS1 gene disruptions. Since the product of the PET127 gene is involved in processing of the 5' ends of mitochondrial mRNAs, we suggest that there is a functional coupling between the 5' and 3' ends of mitochondrial mRNAs.

Blotting, Northern↗
PubMed1998

Soft-copy sonography: cost reduction sensitivity analysis in a pediatric hospital.

OBJECTIVE: Our objective was to determine whether interpreting sonograms of pediatric patients using soft-copy (computer workstation) instead of laser-printed film could reduce costs for a pediatric radiology department. We used theoretic models of growth to analyze costs. MATERIALS AND METHODS: The costs of a sonographic picture archiving and communication system (three interface devices, two workstations, a network server, maintenance expenses, and storage media costs) were compared with the potential savings of eliminating film and increasing technologist efficiency or reducing the number of technologists. The model was based on historic trends and future capitation estimates that will reduce fee-for-service reimbursement. The effects of varying the study volume and reducing technologists' work hours were analyzed. RESULTS: By converting to soft-copy interpretation, we saved 6 min 32 sec per examination by eliminating film processing waiting time, thus reducing examination time from 30 min to 24 min. During an average day of 27 examinations, 176 min were saved. However, 33 min a day were spent retrieving prior studies from long-term storage; thus, 143 extra minutes a day were available for scanning. This improved efficiency could result in five more sonograms a day obtained by converting to soft-copy interpretation, using existing staff and equipment. Alternatively, five examinations a day would equate to one half of a full-time equivalent technologists position. Our analysis of costs considered that the hospital's anticipated growth of sonography and the depreciation of equipment during 5 years resulted in a savings of more than $606,000. Increasing the examinations by just 200 sonograms in the first year and no further growth resulted in a savings of more than $96,000. If the number of sonograms stayed constant, elimination of film printing alone resulted in a loss of approximately $157,000; reduction of one half of a full-time equivalent technologist's position would recuperate approximately $134,000 of that loss. CONCLUSION: Soft-copy sonography can save money through improved technologist efficiency, thereby increasing the number of sonograms obtained and revenue generated. If the number of sonograms does not increase, elimination of printing costs and reduction of staff technologists will not result in a savings.

Cost Savings↗
PubMed2026

Structural insights into RNA phosphorylation by the RNase PNK module of the human rixosome complex.

The mammalian rixosome complex is a large multi-subunit complex that plays essential roles in ribosome assembly and heterochromatin maintenance. Three structural proteins form the stable core of the rixosome to which three enzymatic modules are flexibly tethered including an RNA processing module, AAA-ATPase, and SUMO protease. The RNA processing module is formed by RNase PNK, a tetrameric assembly comprising two copies each of the LAS1L endoribonuclease (RNase) and the NOL9 polynucleotide kinase (PNK). Using single particle cryo-EM, we determined ATPγS and AMP-PNP/RNA-bound structures of human RNase PNK. The structures revealed the overall butterfly-like architecture of the complex and provide new insights into the mechanism of RNA accommodation and 5' hydroxyl phosphorylation within the NOL9 active site. Through reconstitution studies and molecular modeling, we establish how RNase PNK is incorporated into the larger rixosome complex by a distinct domain of LAS1L. Finally, we show that the human 5'-3' exoribonuclease XRN2 directly associates with RNase PNK and selectively degrades NOL9-phosphorylated RNA in vitro, thereby linking ITS2 processing by the rixosome to processive exonucleolytic decay. Collectively this work establishes an updated model for how the rixosome integrates its diverse enzymatic activities to regulate ITS2 processing.

Humans↗
PubMed2007

Unit-length, single-stranded circular DNAs of both polarity of begomoviruses are generated in Escherichia coli harboring phage M13-cloned begomovirus genome with single copy of replication origin.

Replication of genomic DNAs of plant-pathogenic begomoviruses has been demonstrated in prokaryotes, which supported the possibility of analyzing DNA replication process of begomoviruses in bacteria. However, previous studies indicated that the replication of begomovirus DNAs in prokaryotes requires tandem constructs of viral genomes with at least two copies of the origin of replication (ori). In this study, phage M13 vector harboring the unit-length genome with only a single copy of ori of a mono-partite begomovirus, Ageratum yellow vein virus PD isolate (AYVV-[PD]), was constructed and used to investigate the replication of AYVV-[PD] DNAs in Escherichia coli. The generation of single-stranded, circular DNAs (sscDNAs) corresponding to the unit-length AYVV-[PD] genome of both polarity was observed and verified. Replication-associated (Rep) protein of AYVV-[PD] was detected only in bacteria generating the corresponding sscDNAs, whereas disruption of the Rep gene abolished the phenomenon. The results suggested that a single copy of ori is sufficient for the prokaryotes to support the generation of unit-length, genomic sscDNAs of begomoviruses, which requires the presence of functional Rep protein.

Bacteriophage M13↗
PubMed1990

Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit.

The entire vir regulon of Agrobacterium tumefaciens was subcloned and the complete 28.6-kbp nucleotide sequence was determined. The regulon was cloned as a single unit into two replicons, one of which replicates at a high copy number in this bacterium, and a second which has broad-host-range features to replicate in other Gram-negative bacteria. These vir region plasmids are able to confer in trans the processing and transfer activities on a second plasmid containing the T-DNA. In the high copy number vir region plasmid pUCD2614, a moderate increase in basal vir gene expression was observed as judged by virE::cat fusion expression assays relative to the wild-type control plasmid. Furthermore, higher efficiencies of tobacco leaf disk transformation were observed than with the widely used vir helper plasmid pAL4404. The nucleotide sequence studies showed that the vir region consists of 28,631 bp comprising 24 open reading frames which encode proteins involved in tumorigenicity. Two open reading frames not previously characterized, virH and ORF5, were uncovered within the virD/virE intervening spacer region. Together these studies more completely characterize the structure and function of the vir regulon.

Base Sequence↗
PubMed1991

Characterization of two novel human retropseudogenes related to the calmodulin-encoding gene, CaMII.

Two human genomic clones (lambda hg22 and lambda hg29), containing two novel calmodulin (CaM) retropseudogenes, were isolated and characterized. The two pseudogenes show high similarity with the human CaMII cDNA, hCE1 [SenGupta et al., J. Biol. Chem. 262 (1987) 16663-16670] and the CaMII-type retropseudogene, hCE2 (CaMII-psi 1) [SenGupta et al., Nucleic Acids Res. 17 (1989) 2868]. One of them, in clone lambda hg22 (CaMII-psi 2), shows all the characteristics of a processed pseudogene. In clone lambda hg29 (CaMII-psi 3), however, an Alu repetitive sequence was detected immediately upstream from the ancestral 5'-untranslated region. Downstream from the truncated 3'-untranslated region, three additional copies of Alu repetitive sequences flanking about 750 nucleotides of unknown origin were found. Such a processed retropseudogene flanked by multiple Alu repeats may be a target for further recombination events. The three retropseudogenes CaMII-psi 1, CaMII-psi 2 and CaMII-psi 3 are estimated to be about 49, 21 and 25 million years old, respectively.

Animals↗
PubMed1989

Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus.

EBNA1 activates the EBV plasmid maintenance sequence oriP by binding to its two essential regions. One region is a family of 30-base-pair (bp) repeats and is activated by EBNA1 to act as a transcriptional enhancer. The other region contains a 65-bp dyad symmetry and lacks enhancer function. To explore the functional differences between the two regions, we determined oriP activities as functions of the number of 30-bp repeats and compared them with activities determined when tandem copies of the dyad symmetry region were used to replace the 30-bp repeats. Three conclusions have been drawn. (i) Activation of the 30-bp repeats by EBNA1 to enhance transcription or to permit plasmid maintenance is a highly cooperative process involving at least six or seven 30-bp repeats for full activity. (ii) Tandem copies of the dyad symmetry region cooperatively enhance transcription but are less effective than 30-bp repeats providing a similar number of EBNA1-binding sites. (iii) Tandem copies of the dyad symmetry region alone cooperatively activate replication, suggesting that the region contains the actual origin of replication. We also report that while rodent-derived cell lines do not support replication of EBV-derived plasmids they do permit EBNA1-dependent enhancer activity. EBV plasmid replication thus requires the interaction of EBNA1 or oriP with a host factor that is not required for enhancement of transcription.

Antigens, Viral↗
PubMed2000

Site-specific photocrosslinking to probe interactions of Arf1 with proteins involved in budding of COPI vesicles.

ADP-ribosylation factor 1 (Arf1) plays an important role in early and intra-Golgi protein trafficking. During this process, Arf1 interacts with many different proteins and other molecules that regulate its state of activation or are involved in its intracellular function. To determine which of these proteins interact directly with Arf1 during coat protein type I (COPI) vesicle biogenesis, we probed the molecular environment of Arf1 by use of site-specific photocrosslinking. This method was first used successfully in the field of protein trafficking to study the mechanisms involved in protein translocation across the endoplasmic reticulum during protein synthesis. In such a hydrophobic environment, crosslink yields of up to 30% have been observed. We have now applied this method to study the mechanism of vesicle budding from the cytosolic face of the Golgi apparatus, an aqueous environment. Although the crosslink yield is significantly lower under these conditions, due to predominant reaction of the photolabile probes with water, a specific interaction of Arf1 with subunits of coatomer, the major coat protein of COPI vesicles, could readily be identified.

ADP-Ribosylation Factor 1↗
PubMed1994

[Disorganized eye movements and visuospatial dysfunctions in an early stage of the patients with Alzheimer's disease--the effects of language and visual information processing on constructional performances].

In Alzheimer's disease higher brain function disorders such as geographical disorientation, visual agnosia and constructional disability are frequently observed in addition to the progressive memory loss. Visual information processing was studied through newly developed technology, vision analyzer, since visuospatial functions play an important role in the pathophysiology underlying these neuropsychiatric symptoms and dementia itself. We found characteristic findings as to eye movements of focus indicating visual cognitive disorders. Language or language functions, however, are considered to be essentially involved in visuospatial functions. We examined eye movements of patients suffering from Alzheimer's disease (15 cases), MID (5 cases), Parkinson's disease (5 cases) and 10 control subjects during constructional behavior under various conditions in terms of visual information processing. 1. Alzheimer's disease (n = 15): All patients showed constructional disabilities when copying geometrical figures. 1) Three dimensional drawing was possible following verbal instructions in 7 out of 15 patients. 2) Three dimensional drawing was successful when the model figure to be copied was covered in 4 out of these 7 patients. 3) Three dimensional drawing was possible following verbal instruction in a dark room, although they were not so accurate but better than those when copied in 4 out of 7 patients. 4) The analysis of eye movements revealed that typical eye movements were confirmed during copying geometric figures. The distribution of the gazing points of focus, and the average eye movement velocity distribution were also distinctive. But these disorganized eye movements in description of graphic figures following verbal instruction became more similar to those of healthy controls when compared to those in the copying graphic figures. 2. MID (n = 5), Parkinson disease (n = 5), healthy controls (n = 10): 1) No subject showed constructional disability when copying a graphic figure. They were also capable to describe a graphic figure both in the dark and following verbal instruction. 2) No remarkable change was observed in the distribution of the eye movement, or in the average eye movement velocity on these subjects. 3. These results indicate that in an early stage of the patients with Alzheimer's disease, primary or fundamental disorders seem to lay in the verification process of visual information, which results in the difficulties in categorization process of the visual objects. The categorization process is involved with language function, which was preserved in a certain period at an early stage of the disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Alzheimer Disease↗
PubMed2002

Classical no-cloning theorem.

A classical version of the no-cloning theorem is discussed. We show that an arbitrary probability distribution associated with a (source) system cannot be copied onto another (target) system while leaving the original distribution of the source system unperturbed. For classical dynamical systems such a perfect cloning process is not permitted by the Liouvillian (ensemble) evolution associated with the joint probability distribution of the composite source-target-copying machine system.

Journal Article↗
PubMed2001

The effect of changes in laboratory practices on the rate of false-positive cultures for Mycobacterium tuberculosis.

CONTEXT: False-positive cultures for Mycobacterium tuberculosis have been found in nearly all DNA fingerprinting studies, but the effectiveness of interventions to reduce cross-contamination has not been evaluated. OBJECTIVE: To evaluate whether changes in laboratory policies and procedures reduced the rate of false-positive cultures. DESIGN: Retrospective study of isolates with matching DNA fingerprints. SETTING: A mycobacteriology laboratory serving an urban tuberculosis control program and public hospital system. PATIENTS: All M tuberculosis isolates processed from July 1994 to December 1999. METHODS: Isolates were fingerprinted using IS6110; pTBN12 was used to fingerprint isolates having fewer than 6 copies of IS6110. We further evaluated all patients having only one positive culture whose DNA fingerprint matched that of another isolate processed in the laboratory within 42 days. INTERVENTIONS: We changed laboratory policy to reduce the number of smear-positive specimens processed and changed laboratory procedures to minimize the risk of cross-contamination during batch processing. MAIN OUTCOME MEASURE: The rate of false-positive cultures. RESULTS: Of 13 940 specimens processed during the study period, 630 (4.5%) from 184 patients and 48 laboratory proficiency specimens grew M tuberculosis. There were no cases (0/184) of probable or definite cross-contamination, compared with the 4% rate (8/199) identified in our previous study (P =.008). We also fingerprinted a convenience sample of isolates from other laboratories in Denver; 13.6% (3/22) of these were false-positive, a rate similar to the 11.9% rate (5/42) identified for other laboratories in our previous study (P =.84). CONCLUSIONS: Laboratory cross-contamination decreased significantly after relatively simple, inexpensive changes in laboratory policies and practices. Cross-contamination continued to occur in other laboratories in Denver.

Colorado↗
PubMed1986

Characterization of Fischer rat embryo (CREF) cells transformed by bovine papillomavirus type 1.

Transformation of an established Fischer rat embryo (CREF) cell line by bovine papillomavirus type 1 (BPV-1), in contrast to transformation by type 5 adenovirus or the T24 (Ha-ras) oncogene, resulted in transformants which did not exhibit a major increase in saturation density or a decrease in 125I-epidermal growth factor binding. BPV-1-transformed CREF clones did, however, grow in agar suspension culture and were tumorigenic in both nude mice and Fischer rats. The majority of transformed clones contained multiple extrachromosomal copies of BPV DNA. One transformed CREF clone contained integrated BPV-1 DNA which underwent sequence rearrangements following tumor formation in a Fischer rat and reestablishment in cell culture. BPV copy number varied in subclones of transformants isolated from the same monolayer focus, in agar-derived subclones of the same transformed focus, in tumors and in tumor-derived BPV-transformed CREF subclones. The degree of expression of specific transformation-related phenotypes, i.e., saturation density, growth in agar, and tumorigenicity, was not correlated with BPV copy number in transformed clones. Analysis of the biological properties of tumor-derived BPV-1-transformed CREF subclones indicated that certain transformants developed a stable increase in expression of transformation-related properties, a process termed "progression." TPA did not enhance the frequency of BPV-1 transformation or BPV DNA copy number in transformed CREF cells. The present study demonstrates that the CREF- transformation system can be utilized to study the molecular basis of BPV-1 transformation and should prove useful in studying the role of specific BPV-1 transforming proteins in regulating expression of the transformed phenotype and the mechanism by which transformed cells undergo progression of the transformed state.

Adenoviruses, Human↗
PubMed1998

Enhanced secretion of human granulocyte colony-stimulating factor directed by a novel hybrid fusion peptide from recombinant Saccharomyces cerevisiae at high cell concentration.

The synthesis and secretion of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are investigated in fed-batch cultures at high cell concentration of recombinant Saccharomyces cerevisiae, and some important characteristics of the secreted rhG-CSF are demonstrated. Transcription of the recombinant gene is regulated by a GAL1-10 upstream activating sequence (UASG), and the rhG-CSF is expressed in a hybrid fusion protein consisting of signal sequence of Kluyveromyces lactis killer toxin and N-terminal 24 amino acids of human interleukin 1beta. The intracellular KEX2 cleavage leads to excretion of mature rhG-CSF into extracellular culture broth, and the cleavage process seems to be highly efficient. In spite of relatively low copy number the plasmid propagation is stably maintained even at nonselective culture conditions. The rhG-CSF synthesis does not depend on galactose level, whereas the production of extracellular rhG-CSF was significantly enhanced by increasing the inducer concentration above a certain level and also by supplementing the nonionic surfactant to the culture medium, which is notably due to the enhanced secretion efficiency. Various immunoblotting analyses demonstrate that none of the rhG-CSF is accumulated in the cell wall fraction and that a significant amount of intracellular rhG-CSF antibody-specific immunoreactive proteins is located in the ER. A core N-glycosylation at fused IL-1beta fragment is likely to play a critical role in directing the high-level secretion of rhG-CSF, and the O-glycosylation of secreted rhG-CSF seems nearly negligible. Also the extracellular rhG-CSF is observed to exist as various multimers, and the nature of molecular interaction is evidently not the covalent disulfide bridges. The CD spectra of purified rhG-CSF and Escherichia coli-derived standard show that the conformations of both are similar and are almost identical to that reported for natural hG-CSF.

Biotechnology↗
PubMed2001

Granulocytic differentiation of HL-60 cells, both spontaneous and drug-induced, might require loss of extrachromosomal DNA encoding a gene(s) not c-MYC.

Treatment of HL-60 cells with drugs induces granulocytic differentiation and c-MYC down-regulation that is irreversible and associated with loss of DNase I-hypersensitive sites in c-MYC promoter. The expression of these phenotypes requires a slow process that appears to accompany a loss of c-MYC copies in double minutes via micronuclei. However, the drug treatment induced c-MYC down-regulation very early, though only reversibly. Here we show that we can resolve this paradox by assuming a gene(s) in other extrachromosomal, acentromeric DNA. Treatment with drugs might induce no down-regulation of this gene, but its complete elimination via micronuclei might be necessary and sufficient for the expression of the above phenotypes. Loss of c-MYC copies is unavoidable because the exclusion of extrachromosomal DNAs via micronuclei is at random. This conclusion is based on the observation of a substantial number of c-MYC copies in certain differentiated cells, irrespective of whether the differentiation was induced with drugs or without.

Cell Differentiation↗
PubMed1998

Mouse models dissect the role of p53 in cancer and development.

Mice lacking one or two copies of the p53 gene have provided invaluable insight into the process of tumorigenesis. The importance of apoptosis in suppression of tumorigenesis in vivo became evident from analysis of these mice. Moreover, the timing and kinds of tumors that develop in these mice are altered by the presence of additional inherited mutations, by strain differences, and by food intake. Developmental abnormalities are also visible in mice with loss of p53 and with overexpression of p53 suggesting that p53 levels are critical for normal cellular processes. While mice do not necessarily recapitulate all the tumor types found in inherited cancers, they offer the unique opportunity to decipher the critical pathways in tumorigenesis. These findings can then be applied to humans.

Animals↗
PubMed1994

DNA amplification: new insights into its mechanism.

DNA amplification is a process whereby a limited part of the genome is increased in copy number with various consequences for the cell. It is frequently observed in cancer cells and it can be induced in mammalian cells grown in culture as well as in tumor cells when these are subjected to growth inhibiting drugs. In recent years new insights into the mechanisms involved in DNA amplification have been obtained; discussion of these will form the major subject of this short review.

Animals↗