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PubMed2006

RNA editing site recognition in heterologous plant mitochondria.

RNA editing is a process that modifies the information content of mitochondrial messenger RNAs in flowering plants changing specific cytosine residues into uridine. To gain insight into editing site recognition, we used electroporation to introduce engineered wheat (Triticum aestivum) or potato (Solanum tuberosum) mitochondrial cox2 genes, and an atp9-containing chimeric gene, into non-cognate mitochondria, and observed the efficiency of editing in these contexts. Both wheat and potato mitochondria were able to express "foreign" constructs, and their products were properly spliced. Seventeen and twelve editing sites are present in the coding regions of wheat and potato cox2 transcripts, respectively. Eight are common to both plants, whereas nine are specific to wheat, and four to potato. An analogous situation is found for the atp9 mRNA coding regions from these species. We found that both mitochondria were able to recognize sites that are already present as T at the genomic level, making RNA editing unnecessary for that specific residue in the cognate organelle. Our results demonstrate that non-cognate mitochondria are able to edit residues that are not edited in their own transcripts, and support the hypothesis that the same trans-acting factor may recognize several editing sites.

Arabidopsis Proteins↗
PubMedDate not supplied

Health plan member experience with point-of-service prescription step therapy.

OBJECTIVE: To better understand health plan member experience with point-of-service prescription step-therapy edits and outcomes in terms of drug received. METHODS: Self-administered mailed surveys were sent to 1,000 members who experienced a step-therapy edit from September 1, 2002, through January 31, 2003, for proton pump inhibitors or nonsteroidal anti-inflammatory drugs. Based upon these findings, a second survey was conducted by telephone among 617 members who experienced a step-therapy edit from January through April 25, 2003, and who had no subsequent prescription claim for the drug therapy class associated with the edit. RESULTS: The mailed survey generated a 23% response rate, and the telephone survey generated a 33% response rate. Just over 66% of the mail survey respondents indicated that they contacted their physician directly to try to remedy the situation, while 40% indicated that their pharmacist contacted their physician. Forty-four (44%) percent of members indicated that they received a different medication than was originally prescribed, 15% obtained prior authorization for the brand medication, 11% received no medication, 11% paid full price for the branded medication, 8% got an over-the-counter medication, and 4% received samples from their physician. Approximately 7% sought other means of obtaining coverage (i.e., they used spouse.s insurance) or did not remember the outcome. Member and pharmacy contact with the physician significantly influenced whether the member obtained a medication covered by their health plan (odds ratio [OR] = 6.5; 95% confidence interval [95% CI], 2.76-15.12 and OR = 4.6; 95% CI, 1.96-10.60, respectively). In a separate survey conducted by telephone among a different group of members, insight into reasons why members did not obtain any medication was obtained. Using a closed-ended question, 12% (n = 25) of members indicated receiving no medication. Upon further questioning, however, 32% of those who indicated that they had not received a medication said that they had in fact received a medication to treat their condition some time after the step edit. The second most common reason for not receiving a medication included issues related to cost (i.e., willingness to pay) or affordability (16% and 28%, respectively). CONCLUSIONS: The results of this study suggest that a majority of members receive a medication covered by their health plan subsequent to rejection of a claim for a prescribed drug that is the target of a step-therapy edit. However, there are opportunities for better member and provider communication designed to increase the use of first-line drugs and reduce the number of members paying out-of-pocket or receiving no medication.

Adolescent↗
PubMed1990

A microcomputer data base system for an in vitro fertilization clinic programmed in BASIC.

A computer program was developed for the IBM personal computer to be used for in vitro fertilization and gamete intrafallopian transfer clinics. This program, written in BASIC, allows input, editing, updating, sorting, and printing of patient data. Statistical functions permit summation of patient data based on various combinations of user-defined treatment cycles, diagnoses, and protocols, thus making possible comparison of pregnancy and other patient data between and among various treatment groups and diagnoses. The statistical information can be continually updated and revised when new data become available on patients (such as confirmation of pregnancy by ultrasound or live births) and at the end of each cycle. The formats used are useful in assimilating individual clinic data for various surveys and other reporting requirements. The program can be easily modified by anyone with minimal training in the BASIC programming language.

Ambulatory Care Facilities↗
PubMed1997

Involvement of 5' flanking sequence for specifying RNA editing sites in plant mitochondria.

Unsuccessful insertion of foreign DNA into plant mitochondrial genomes has hindered scientific evaluation of cis-elements needed for RNA editing. Both a normal atp6 gene and a chimeric atp6 sequence are present in rice mitochondria. The chimeric atp6 contains one-half of the normal atp6 sequence in its 5' portion and an unknown sequence in its downstream portion. The C-nucleotide at position 511, located just upstream of the unknown sequence recombined in the chimeric atp6 sequence, is edited, as are other possible editing sites upstream from position 511. We report here that the 5' sequence adjacent to the editing site of atp6 contains cis-information required for RNA editing and that the 3' sequence flanking the editing site provides little contribution to editing-site recognition.

Amino Acid Sequence↗
PubMed1995

Minicircle-encoded guide RNAs from Crithidia fasciculata.

Although the mitochondrial uridine insertion/deletion, guide RNA (gRNA)-mediated type of RNA editing has been described in Crithidia fasciculata, no evidence for the encoding of gRNAs in the kinetoplast minicircle DNA has been presented. There has also been a question as to the capacity of the minicircle DNA in this species to encode the required variety of gRNAs, because the kinetoplast DNA from the C1 strain has been reported as essentially containing a single minicircle sequence class. To address this problem, the genomic and mature edited sequences of the MURF4 and RPS12 cryptogenes were determined and a gRNA library was constructed from mitochondrial RNA. Five specific gRNAs were identified, two of which edit blocks within the MURF4 mRNA, and three of which edit blocks within the RPS12 mRNA. The genes for these gRNAs are all localized with identical polarity within one of the two variable regions of specific minicircle molecules, approximately 60 bp from the "bend" region. These minicircles were found to represent minor sequence classes representing approximately 2% of the minicircle DNA population in the network. The major minicircle sequence class also encodes a gRNA at the same relative genomic location, but the editing role of this gRNA was not determined. These results confirm that kinetoplast minicircle DNA molecules in this species encode gRNAs, as is the case in other trypanosomatids, and suggest that the copy number of specific minicircle sequence classes can vary dramatically without an overall effect on the RNA editing system.

Amino Acid Sequence↗
PubMed2000

Transcription and RNA editing in a soluble in vitro system from Physarum mitochondria.

The dissection of RNA editing mechanisms in PHYSARUM: mitochondria has been hindered by the absence of a soluble in vitro system. Based on our studies in isolated mitochondria, insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs is closely linked to transcription. Here we have fractionated mitochondrial lysates, enriching for run-on RNA synthesis, and find that editing activity co-fractionates with pre-formed transcription elongation complexes. The establishment of this soluble transcription-editing system allows access to the components of the editing machinery and permits manipulation of transcription and editing substrates. Thus, the availability of this system provides, for the first time, a means of investigating roles for cis-acting elements, trans-acting factors and nucleotide requirements for the insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs. This methodology should also be broadly applicable to the study of RNA processing and editing mechanisms in a wide range of mitochondrial systems.

Adenosine Triphosphate↗
PubMedDate not supplied

[Cochrane EPOC group: closing the gap between quality assurance and organization of care research and front line professionals].

Keeping physicians informed on an ongoing basis is a new challenge for continuing medical education and quality assurance. In Italy over the last 5 years interest in evidence based literature is growing. This is demonstrated by the launch of an Italian edition of Clinical Evidence and by the growing number of guidelines and systematic reviews produced by Italian authors and institutions. However, there is some uncertainty concerning the familiarity of Italian policy makers and public health physicians with the evidence-based resources, including also how to access them. This article attempts to close this gap, by describing the activities of the Cochrane Collaboration and, within it, of the Cochrane Effective Practice and Organisation of Care Group (EPOC), both aim to prepare and maintaining SR of health care interventions. Specifically, the EPOC group develops systematic reviews of professional, financial, organisational and regulatory interventions that are designed to improve professional practice and the delivery of effective health services. EPOC has 31 reviews and 24 protocols published in Issue 4, 2004 of the Cochrane Library and has developed standard methods to assist people, such as quality criteria for study design specific to health services research. The EPOC specialized register contains details of over 2200 studies that fall within the group's scope. Systematic reviews provide a valuable and efficient source of information for policy makers and health care professionals aimed at implementing effective and efficient strategies to encourage medical behavioural change and deliver of high quality services.

Biomedical Research↗
PubMed1998

Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2.

We have identified an RNA-specific adenosine deaminase (termed Tad1p/scADAT1) from Saccharomyces cerevisiae that selectively converts adenosine at position 37 of eukaryotic tRNAAla to inosine. The activity of purified recombinant Tad1p depends on the conformation of its tRNA substrate and the enzyme was found to be inactive on all other types of RNA tested. Mutant strains in which the TAD1 gene is disrupted are viable but lack Tad1p enzyme activity and their tRNAAla is not modified at position A37. Transformation of the mutant cells with the TAD1 gene restored enzyme activity. Tad1p has significant sequence similarity with the mammalian editing enzymes which act on specific precursor-mRNAs and on long double-stranded RNA. These findings suggest an evolutionary link between pre-mRNA editing and tRNA modification.

Adenosine Deaminase↗
PubMed1993

RNA editing in kinetoplastid mitochondria.

RNA editing in the mitochondrion of kinetoplastid protozoa results in the posttranscriptional addition and deletion of uridine residues in mRNAs. Editing of mRNAs can lead to the formation of initiation codons for mitochondrial translation, the correction of frame-shifted genes at the RNA level, and in extensively edited mRNAs, the formation of complete reading frames. Kinetoplastid RNA editing requires that genetic information from two or more separately transcribed genes be brought together to form the mature, edited mRNA. The information necessary for the proper insertion or deletion of uridines in the mRNA is present in small mitochondrial transcripts termed guide RNAs (gRNAs). Editing of mRNAs appears to be associated with a high molecular weight complex, called the editosome, containing specific gRNAs, unedited mRNAs, and proteins. Editing is likely a two-step process involving first the breakage of a phosphodiester bond at the editing site and formation of a chimeric molecule with a gRNA covalently joined to the 5' end of the 3' portion of an mRNA. The chimera is resolved by the rejoining of the 5' end of the mRNA to the 3' portion of the mRNA with the addition or deletion of a uridine at the junction point. Two models are proposed for the biochemical mechanism of RNA editing. The first is an enzymatic cascade of cleavage and ligation while the other supports successive rounds of transesterification. The obvious functional necessity for editing in kinetoplastid mitochondria is the formation of translatable mRNAs. Far less clear is the evolutionary origin of editing and the role editing plays in regulating mitochondrial gene expression.

Animals↗
PubMed2006

RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction.

RBP16 is an abundant RNA binding protein from Trypanosoma brucei mitochondria that affects both RNA editing and stability. We report here experiments aimed at elucidating the mechanism of RBP16 function in RNA editing. In in vitro RNA editing assays, recombinant RBP16 is able to significantly stimulate insertion editing of both CYb and A6 pre-mRNAs. Enhancement of in vitro editing activity occurs at, or prior to, the step of pre-mRNA cleavage, as evidenced by increased accumulation of pre-mRNA 3' cleavage products in the presence of RBP16. Mutated RBP16 that is severely compromised in cold shock domain (CSD)-mediated RNA binding was able to enhance editing to levels comparable to the wild-type protein in some assays at the highest RBP16 levels tested. However, at low RBP16 concentrations or in assays with native, oligo(U)-tail-bearing gRNAs, editing stimulation by mutant RBP16 was somewhat compromised. Together, these results indicate that both the N-terminal CSD and C-terminal RGG RNA binding domains of RBP16 are required for maximal editing stimulation. Finally, the relaxed specificity of RBP16 for stimulation of both CYb and A6 editing in vitro implicates additional specificity factors that account for the strict CYb specificity of RBP16 action in editing in vivo. Our results constitute the first report of any putative RNA editing accessory factor eliciting an effect on editing in vitro. Overall, these results support a novel accessory role for RBP16 in U insertion editing.

Amino Acid Sequence↗
PubMed2001

Involvement of a site-specific trans-acting factor and a common RNA-binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system.

RNA editing in higher plant chloroplasts involves C-->U conversion at approximately 30 specific sites. An in vitro system supporting accurate editing has been developed from tobacco chloroplasts. Mutational analysis of substrate mRNAs derived from tobacco chloroplast psbL and ndhB mRNAs confirmed the participation of cis-acting elements that had previously been identified in vivo. Competition analysis revealed the existence of site-specific trans-acting factors interacting with the corresponding upstream cis-elements. A chloroplast protein of 25 kDa was found to be specifically associated with the cis-element involved in psbL mRNA editing. Immunological analyses revealed that an additional factor, the chloroplast RNA-binding protein cp31, is also required for RNA editing at multiple sites. This combination of site-specific and common RNA-binding proteins recognizes editing sites in chloroplasts.

Base Sequence↗
PubMed1995

Toward the therapeutic editing of mutated RNA sequences.

If RNA editing could be rationally directed to mutated RNA sequences, genetic diseases caused by certain base substitutions could be treated. Here we use a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA. The complementary RNA oligonucleotide was hybridized to a premature stop codon and the hybrid was treated with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. When the treated RNAs were translated in vitro, a dramatic increase in expression of a downstream luciferase coding region was observed. The cDNA sequence data are consistent with deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. Injection of oligonucleotide-mRNA hybrids into Xenopus embryos also resulted in an increase in luciferase expression. These experiments demonstrate the principle of therapeutic RNA editing.

Adenosine Deaminase↗
PubMed1997

No editing of mitochondrial plasmid transcripts in mitochondria of Physarum that have an RNA-editing system.

Two types of RNA editing have been reported in the mitochondria of Physarum; extensive insertions of nucleotides and single-base substitutions. In the Ng strain of P. polycephalum and its derivatives, mitochondria have a specific plasmid (mF) that promotes fusion of mitochondria. We examined the editing of transcripts derived from the mF plasmid. For analysis, we selected three regions of the plasmid, including a DNA fragment that corresponded to missing conserved domains of the RNA polymerase. In contrast to the mitochondrial DNA (mtDNA), no RNA editing of the transcripts of the mF plasmid was detected. Our results suggest that the mechanism of transcription of the mitochondrial plasmid is independent of that of mtDNA, indicating that the plasmid has a different evolutionary origin from the mtDNA.

Animals↗
PubMed2004

Reinstatement of Rhodotorula colostri (Castelli) Lodder and Rhodotorula crocea Shifrine & Phaff, former synonyms of Rhodotorula aurantiaca (Saito) Lodder.

Rhodotorula aurantiaca (Saito) Lodder is an anamorphic basidiomycetous yeast species that belongs to the so-called "Erythrobasidium lineage" of the Urediniomycetes, according to molecular phylogenetic studies based on nucleotide sequence analyses of different ribosomal DNA regions. In the most recent editions of the yeast taxonomy treatises the species Rhodotorula colostri (Castelli) Lodder and Rhodotorula crocea Shifrine & Phaff were listed as synonyms of R. aurantiaca. Taxonomic heterogeneity within R. aurantiaca was demonstrated in a study based on whole-cell protein profiles and is also hinted at by the observed differences in physiological and biochemical characteristics among the different strains under that species name. We determined partial nucleotide sequences of the 26S rRNA gene (D1/D2 domains) of strains maintained in the CBS culture collection under R. aurantiaca, including the type strains of its synonyms. The results showed that R. colostri and R. crocea are clearly distinct from R. aurantiaca and from any other currently recognised basidiomycetous yeast species. Furthermore, phylogenetic analysis of the sequence data placed the former two species in separate lineages of the Microbotryomycetidae: R. colostri in the "ruineniae clade" (Sporidiobolus lineage or Sporidiobolales) and R. crocea loosely linked to Rhodotorula javanica (Microbotryum lineage).

DNA, Fungal↗
PubMed1996

Content validity of the Harris Infant Neuromotor Test.

BACKGROUND AND PURPOSE: The purpose of this study was to assess the content validity of the Harris Infant Neuromotor Test (HINT), a new screening tool designed to detect early signs of cognitive and neuromotor delays in infants with known risk factors. SUBJECTS: A multidisciplinary panel of 26 international experts, including researchers and clinicians in physical therapy, occupational therapy, developmental pediatrics, child neurology, and psychology, was selected to assess, review, and suggest modifications to the HINT. METHODS: A validity questionnaire that was keyed to the three parts of the HINT (background information, parent questionnaire, and infant assessment) and to individual items within each part was mailed to the selected content experts. Data were analyzed using descriptive statistics. RESULTS: Twenty experts completed the validity questionnaires, and the remaining 6 experts provided written comments. The experts generally agreed that the items on the initial development edition of the HINT were clearly worded and free of cultural bias. Based on written feedback, additional background information items were added, a fifth question was added to the parent questionnaire portion, and 20 of the 22 infant assessment items were modified. CONCLUSION AND DISCUSSION: After receiving questionnaire responses and written comments from a panel of 26 experts, the original version of the HINT was modified and printed as development edition 2 in November 1993. This revised edition is currently being used to examine the reliability and validity of the HINT in assessing infants at risk.

Adult↗
PubMed1994

Trypanosoma brucei mitochondria contain RNA helicase activity.

Mitochondrial gene expression in kinetoplastid organisms such as Trypanosoma, Leishmania and Crithidia requires a posttranscriptional RNA processing event known as kRNA editing. During editing, uridine nucleotides get inserted and deleted into pre-mRNAs directed by small, metabolically stable RNAs, termed guide RNAs. Although the precise mechanism of the reaction is not understood, the accepted working model describes the formation of extended anti-parallel RNA helices between gRNA molecules with pre- and partially edited mRNAs as intermediates. These duplex structures must be separated to ensure the sequential action of multiple gRNAs in a 3' to 5' polarity on the mRNA molecule. In spite of this fact, no unwinding activity has heretofore been identified in kinetoplastid mitochondria. We report the characterisation of a RNA helicase activity within Trypanosoma brucei mitochondrial extracts. The activity unwinds 25- and 48 bp, tailed RNA duplex structures but fails to separate DNA strands. It can be destroyed by heat denaturation as well as by proteinase K treatment. The activity requires magnesium cations and acts in a NTP/dNTP dependent manner. Hydrolysis of a nucleoside triphosphate is required rather than mere NTP binding as deduced from a comparison of unwinding in the presence of ATP and AMP-PCP. RNA duplexes mimicking presumed kRNA editing intermediates are substrates of the unwinding activity and therefore, we address the possible involvement of a RNA helicase activity during kRNA editing.

Adenosine Triphosphate↗