A rough guide to molecular biology.
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DNA↗
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[Infections by Marburg and Ebola viruses: guide for their diagnosis, treatment and control].
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Animals↗
Role of short RNAs in gene silencing.
Recent research has revealed the existence of an elegant defence mechanism in plants and lower eukaryotes. The mechanism, known in plants as post-transcriptional gene silencing, works through sequence-specific degradation of RNA. It appears to be directed by double-stranded RNA, associated with the production of short 21-25 nt RNAs, and spread through the plant by a diffusible signal. The short RNAs are implicated as the guides for both a nuclease complex that degrades the mRNA and a methyltransferase complex that methylates the DNA of silenced genes. It has also been suggested that these short RNAs might be the mobile silencing signal, a suggestion that has been challenged recently.
Adaptation, Physiological↗
P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction.
The molecular basis for virus-induced disease in plants has been a long-standing mystery. Infection of Arabidopsis by Turnip mosaic virus (TuMV) induces a number of developmental defects in vegetative and reproductive organs. We found that these defects, many of which resemble those in miRNA-deficient dicer-like1 (dcl1) mutants, were due to the TuMV-encoded RNA-silencing suppressor, P1/HC-Pro. Suppression of RNA silencing is a counterdefensive mechanism that enables systemic infection by TuMV. The suppressor interfered with the activity of miR171 (also known as miRNA39), which directs cleavage of several mRNAs coding for Scarecrow-like transcription factors, by inhibiting miR171-guided nucleolytic function. Out of ten other mRNAs that were validated as miRNA-guided cleavage targets, eight accumulated to elevated levels in the presence of P1/HC-Pro. The basis for TuMV- and other virus-induced disease in plants may be explained, at least partly, by interference with miRNA-controlled developmental pathways that share components with the antiviral RNA-silencing pathway.
Arabidopsis↗
Analysis of connective tissues by laser capture microdissection and reverse transcriptase-polymerase chain reaction.
Studies of gene expression from bone, cartilage, and other tissues are complicated by the fact that their RNA, collected and pooled for analysis, often represents a wide variety of composite cells distinct in individual phenotype, age, and state of maturation. Laser capture microdissection (LCM) is a technique that allows specific cells to be isolated according to their phenotype, condition, or other marker from within such heterogeneity. As a result, this approach can yield RNA that is particular to a subset of cells comprising the total cell population of the tissue. This study reports the application of LCM to the gene expression analysis of the cartilaginous epiphyseal growth plate of normal newborn mice. The methodology utilized for this purpose has been coupled with real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) to quantitate the expression of certain genes involved in growth plate development and calcification. In this paper, the approaches used for isolating and purifying RNA from phenotypically specific chondrocyte populations of the murine growth plate are detailed and illustrate and compare both qualitative and quantitative RT-PCR results. The technique will hopefully serve as a guide for the further analysis of this and other connective tissues by LCM and RT-PCR.
Animals↗
Computer simulation of chaperone effects of Archaeal C/D box sRNA binding on rRNA folding.
Archaeal C/D box small RNAs (sRNAs) are homologues of eukaryotic C/D box small nucleolar RNAs (snoRNAs). Their main function is guiding 2'-O-ribose methylation of nucleotides in rRNAs. The methylation requires the pairing of an sRNA antisense element to an rRNA target site with formation of an RNA-RNA duplex. The temporary formation of such a duplex during rRNA maturation is expected to influence rRNA folding in a chaperone-like way, in particular in thermophilic Archaea, where multiple sRNAs with two binding sites are found. Here we investigate possible mechanisms of chaperone function of Archaeoglobus fulgidus and Pyrococcus abyssi C/D box sRNAs using computer simulations of rRNA secondary structure formation by genetic algorithm. The effects of sRNA binding on rRNA structure are introduced as temporary structural constraints during co-transcriptional folding. Comparisons of the final predictions with simulations without sRNA binding and with phylogenetic structures show that sRNAs with two antisense elements may significantly facilitate the correct formation of long-range interactions in rRNAs, in particular at elevated temperatures. The simulations suggest that the main mechanism of this effect is a transient restriction of folding in rRNA domains where the termini are brought together by binding to double-guide sRNAs.
Archaeoglobus fulgidus↗
Distinct functions of two RNA ligases in active Trypanosoma brucei RNA editing complexes.
Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.
Adenosine Triphosphate↗
The human immunodeficiency virus type 1 Rev protein shuttles between the cytoplasm and nuclear compartments.
A retroviral regulatory protein, Rev (regulator of virion protein expression), is made in cells infected by human immunodeficiency virus (HIV). Rev is essential for the completion of the retroviral life cycle and interacts with the host cell at some posttranscriptional step in order to express the incompletely spliced HIV mRNAs from which HIV structural proteins are translated. Neither the host cell components nor the mechanisms responsible for this important regulation have been defined. We now report that Rev is a nucleocytoplasmic shuttle protein which is continuously transported between the cytoplasm, the nucleoli, and nucleoplasmic speckles enriched in RNA splicing and processing factors. The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation.
Amino Acid Sequence↗
Efficient inhibition of both syncytium-inducing and non-syncytium-inducing wild-type HIV-1 by lamivudine in vivo.
OBJECTIVE: To determine the effect of lamivudine (3TC) on syncytium-inducing (SI) and non-SI (NSI) HIV-1 populations in vivo. DESIGN: Responses in virus load and 3TC resistance in 40 3TC-treated subjects were analysed in relation to the presence or absence of SI HIV-1 variants. METHODS: Peripheral blood samples were collected at regular intervals from 40 HIV-1-infected subjects during 3TC treatment. Virus isolates obtained at the start of treatment were typed for SI-capacity by coculture with MT-2 cells. Changes in levels of viral RNA in plasma were determined by quantitative reverse transcriptase PCR. The relative amount of wild-type and mutant virus at codon 184, associated with HIV resistance to 3TC, was determined using a primer-guided nucleotide incorporation assay after amplification of part of the reverse transcriptase gene. In five subjects the frequency of productively infected CD4+ cells with SI or NSI variants was determined in relation to codon 184 genotype. RESULTS: Twenty-six subjects harboured only NSI variants at baseline, whereas 14 subjects also harboured SI variants. Although baseline plasma viral RNA load and CD4 cell counts were different between the two groups, no differences in the response to 3TC therapy were observed for these parameters. In-depth analysis of five subjects showed that the kinetics of virus load changes and emergence of 3TC resistance mutations were similar in plasma and cells, and comparable for the SI and NSI populations present in one individual. CONCLUSIONS: These data show that, in contrast to didanosine and zidovudine, the pressure exerted by 3TC is similar for SI and NSI M184 populations.
Anti-HIV Agents↗
Silence of the strands: RNA interference in eukaryotic pathogens.
Double-stranded (ds) RNA interference (RNAi) is a recent technological advance that enables researchers to reduce gene expression at the post-transcriptional level. This form of RNA silencing is initiated by dsRNA, expressed in or introduced into a cell of interest, which triggers homology-dependent degradation of the corresponding mRNA. This versatile technique has remarkable promise as a tool for the study of eukaryotic pathogens. Protozoan parasites and pathogenic fungi often resist manipulation using standard molecular genetic approaches. Researchers studying these organisms need flexible molecular tools, particularly to exploit newly sequenced genomes; this review offers a practical guide to establishing RNAi in pathogenic eukaryotes.
Animals↗
Investigation of infection control practices and knowledge of hepatitis C among body-piercing practitioners.
BACKGROUND: Body piercing has become increasingly popular, leading to concerns about the associated risk of hepatitis C virus (HCV) transmission during piercing. Many body-piercing practitioners (BPPs) have recently entered the industry but little is known about their training and understanding of HCV transmission. This study measured BPP knowledge about HCV and infection control procedures. It also tested for HCV contamination within body-piercing establishments. METHODS: BPPs completed a questionnaire about the number and type of piercings performed, their methods for disposing of and reprocessing piercing equipment, and their training and knowledge of HCV. Environmental swabs were collected and tested for HCV RNA. RESULTS: BPPs at 35 establishments were recruited. A total of 31 BPPs had training as a BPP, ranging from 1 hour to 6 years (median: 15 days). Reprocessing of equipment was variable; 8 establishments inadequately reprocessed piercing guns and 4 inadequately reprocessed forceps or guiding equipment. All BPPs were aware of HCV but many did not know how the virus was transmitted. A total of 19 BPPs performed extra cleaning after piercing a customer known to be HCV positive. No environmental swabs tested were positive for HCV RNA. CONCLUSIONS: This study showed that many BPPs had inadequate training, and lacked knowledge and understanding of HCV transmission, infection control, and universal precautions. To reduce the risk of HCV transmission, BPPs should be required to undergo formal training in infection control before being registered as BPPs.
Chi-Square Distribution↗
The many facets of H/ACA ribonucleoproteins.
The H/ACA ribonucleoproteins (RNPs) are known as one of the two major classes of small nucleolar RNPs. They predominantly guide the site-directed pseudouridylation of target RNAs, such as ribosomal and spliceosomal small nuclear RNAs. In addition, they process ribosomal RNA and stabilize vertebrate telomerase RNA. Taken together, the function of H/ACA RNPs is essential for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. Every cell contains 100-200 different species of H/ACA RNPs, each consisting of the same four core proteins and one function-specifying H/ACA RNA. Most of these RNPs reside in nucleoli and Cajal bodies and mediate the isomerization of specific uridines to pseudouridines. Catalysis of the reaction is mediated by the putative pseudouridylase NAP57 (dyskerin, Cbf5p). Unexpectedly, mutations in this housekeeping enzyme are the major determinants of the inherited bone marrow failure syndrome dyskeratosis congenita. This review details the many diverse functions of H/ACA RNPs, some yet to be uncovered, with an emphasis on the role of the RNP proteins. The multiple functions of H/ACA RNPs appear to be reflected in the complex phenotype of dyskeratosis congenita.
Animals↗
Efficient trans cleavage and a common structural motif for the ribozymes of the human hepatitis delta agent.
Cis-active ribozymes are potential therapeutic agents; however, to be used in this capacity, they must first be converted to trans-active ribozymes, a process facilitated by analysis of their structures. We present evidence that the genomic and antigenomic ribozymes of the human delta hepatitis agent share a structural ("axehead") motif that has conserved sequence elements and a stable hairpin. Guided by the features of the axehead, we divided each of the delta ribozymes into two subdomains, which we synthesized as separate RNA transcripts to give an enzyme and substrate for each ribozyme. Incubation of a substrate subdomain with its matching enzyme resulted in efficient and accurate trans cleavage. This work forms the basis for kinetic studies and for adapting the delta ribozymes for cleavage of selected target RNAs.
Base Sequence↗
Predicting the therapeutic response in patients with chronic hepatitis C: the role of viral kinetic studies.
A substantial proportion of patients infected with hepatitis C virus (HCV) genotype 1 still does not respond to pegylated interferon-alfa/ribavirin (IFN/RBV) therapy. Factors which identify potential non-responders are needed to limit exposure to drugs in patients unlikely to benefit from treatment and to save health care resources. Host predictive factors have a low negative predictive value. In contrast, viral factors have a high precision in predicting outcome of therapy. Viral kinetics are the basis for the study of response of therapy. The decrease in viral load within 24 h after administration of a single test dose of conventional IFN reflects the IFN-sensitivity of the virus strain and predicts the outcome of conventional IFN/RBV therapy even before treatment with a specificity of 100% and a sensitivity of 83%. In contrast to conventional IFN, the two available PEG-IFN preparations differ considerably in how they suppress viral replication, and cut-off values have to be prospectively established separately for each drug. Patients without an early virological response (HCV-RNA either undetectable or decrease by >or=2 log10 after 12 weeks) (EVR), do not achieve a sustained virological response (SVR; negative predictive value: 97-98%). Thus, in the absence of an EVR, treatment should be stopped. The outcome of PEG-IFN alfa-2a/RBV combination therapy is dependent on the rapidity of the virological response. Patients who become HCV-RNA negative after 4 weeks have the best chance of achieving an SVR. The rapidity of viral elimination may be a useful guide to tailoring the length of treatment in patients with an EVR.
Antiviral Agents↗
Gene therapy: a primer for radiologists.
Gene therapy is one of the most rapidly evolving areas in medicine. Radiologists should have an understanding of basic techniques used to identify and clone a gene and insert it into a vector capable of directing expression in mammalian tissues. DNA delivery systems include retroviral vectors (RNA viruses), adenoviral vectors (DNA viruses), and cationic liposomes, along with strategies that involve ultrasound-directed gene transfer, computed tomography-guided gene transfer, and transcatheter gene delivery, in particular via the hepatic artery. Genes being evaluated in preclinical and clinical trials include oncogenes, antioncogenes (tumor suppressor genes), suicide genes, conventional antimetabolites, antiangiogenesis factors, secreted immunostimulatory cytokines such as interleukins and interferons, and immunomodulatory cell surface proteins, including foreign HLA proteins and costimulatory molecules. A foundation in molecular biology is needed for the practicing radiologist interested in but unfamiliar with current gene therapy terminology and experimental strategies. Such a foundation will encourage the dissemination of basic biologic, diagnostic imaging, and interventional oncoradiologic developments and should facilitate integration of the radiologist into the gene therapy team.
Cystic Fibrosis↗
[Primary non-Hodgkin's lymphomas of the liver in human immunodeficiency virus infected patients. Three cases].
The occurrence of primary non Hodgkin's lymphoma of the liver has rarely been reported in patients with the human immunodeficiency virus infection. We report 3 cases in these patients, presenting with a single liver tumor in 2 cases and multiple tumors in one case. Diagnosis was made by guided-biopsy. Histologically, there was B diffuse large cell lymphoma in 2 cases, and immunoblastic lymphoma in one case. In one case, RNA of Epstein-Barr virus was found in the nucleus of tumor cells by in situ hybridization. No extrahepatic localization was found in any of the 3 cases. One patient died one week after diagnosis, but the 2 other patients were treated by chemotherapy and were still alive 6 and 30 months later. In patients infected by human immunodeficiency virus, primary non Hodgkin's lymphoma should be investigated in case of liver tumor in order to provide early treatment.
AIDS-Related Opportunistic Infections↗
RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway.
RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.
Arabidopsis↗
RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.
Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage.
Adenosine Triphosphate↗