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Growth failure is a common problem in HIV-infected children. The extent to which this growth failure could be reversed after the children receive antiretroviral therapy (ART) is not known. This study assessed the incidence of growth failure in HIV-infected Thai children, impact of ART on growth, and the predictors of growth reversal after initiating ART. Growth parameters and other characteristics were extracted from the database of a prospective cohort of HIV-infected children (age [less than or equal to]15 years) who were enrolled to initiate non-nucleoside reverse transcriptase inhibitor-based ART between August 2002 and May 2007. Body weight and height measurements, CD4 cell counts, plasma HIV RNA levels were collected at baseline and 24-week intervals. A total of 225 HIV-infected children were included, 116 (51%) were males. The median age at baseline was 7.4 years (interquartile range [IQR] 5.2-9.8). Fifty-three percent were in Centers for Disease Control and Prevention (CDC) category C and 54% had CD4 percentage 5 or less. The mean (standard deviation [SD]) of baseline weight-for-age (WAZ) and height-for-age (HAZ) z-scores were -2.02 (1.17) and -2.22 (1.51). The median follow-up time was 216 weeks (IQR 120-240). The cumulative probability of growth reversal among the 179 subjects with growth failure at entry was 58% (95% confidence interval [CI] 49-67) at week 240. In a multivariate Cox regression model, higher entry WAZ (p < 0.001) and HAZ (p < 0.001), use of a nevirapine-based regimen (compared to efavirenz, p = 0.027) and larger CD4% gains to week 48 (p < 0.001) were significant predictors of growth reversal after initiating ART. NNRTI-based ART leads to a substantial improvement in growth of HIV-infected children. Initiation of ART before the children developed growth failure should be encouraged. DOI: 10.1089/apc.2009.0093

The study aims to investigate the clinical characteristics of patients hospitalized with 2009 H1N1 influenza in the United States during the period of April to June, 2009. The results indicate that nearly 3/4th of the patients had some underlying medical conditions and also seemed to benefit from antiviral therapy.

Constitutive expression of telomerase in human cells prevents the onset of senescence and crisis by maintaining telomere homeostasis. However, accumulating evidence suggests that the human telomerase reverse transcriptase catalytic subunit (TERT) contributes to cell physiology independently of its ability to elongate telomeres. Here we show that TERT interacts with the RNA component of mitochondrial RNA processing endoribonuclease (RMRP), a gene that is mutated in the inherited pleiotropic syndrome cartilage-hair hypoplasia. Human TERT and RMRP form a distinct ribonucleoprotein complex that has RNA-dependent RNA polymerase (RdRP) activity and produces double-stranded RNAs that can be processed into small interfering RNA in a Dicer (also known as DICER1)-dependent manner. These observations identify a mammalian RdRP composed of TERT in complex with RMRP., Telomerase is a ribonucleoprotein complex that elongates telomeres. Although several proteins interact with telomerase (1-4), the minimal components of active telomerase include the catalytic telomerase reverse transcriptase (TERT) and a [...]

The collection of gene expression data from human heart biopsies is important for understanding the cellular mechanisms of arrhythmias and diseases such as cardiac hypertrophy and heart failure. Many clinical and basic research laboratories conduct gene expression analysis using RNA from whole cardiac biopsies. This allows for the analysis of global changes in gene expression in areas of the heart, while eliminating the need for more complex and technically difficult single-cell isolation procedures (such as flow cytometry, laser capture microdissection, etc.) that require expensive equipment and specialized training. The abundance of fibroblasts and other cell types in whole biopsies, however, can complicate gene expression analysis and the interpretation of results. Therefore, we have designed a technique to quickly and easily purify cardiac myocytes from whole cardiac biopsies for RNA extraction. Human heart tissue samples were collected, and our purification method was compared with the standard nonpurification method. Cell imaging using acridine orange staining of the purified sample demonstrated that >98% of total RNA was contained within identifiable cardiac myocytes. Real-time RT-PCR was performed comparing nonpurified and purified samples for the expression of troponin T (myocyte marker), vimentin (fibroblast marker), and a-smooth muscle actin (smooth muscle marker). Troponin T expression was significantly increased, and vimentin and n-smooth muscle actin were significantly decreased in the purified sample (n = 8; P < 0.05). Extracted RNA was analyzed during each step of the purification, and no significant degradation occurred. These results demonstrate that this isolation method yields a more purified cardiac myocyte RNA sample suitable for downstream applications, such as real-time RTPCR, and allows for more accurate gene expression changes in cardiac myocytes from heart biopsies. real-time reverse transcriptase-polymerase chain reaction; vimentin; troponin T, smooth muscle actin; cardiomyocytes

Molecular dynamics simulations are used for examining the complexes of HIV-1 reverse transcriptase (RT) with the substrate and the antiretrovirals AZT and d4t. Results suggest that the finger residues K65 and R72 form hydrogen bonds with the substituted nucleotides.

The effects of copper (Cu) on trace metal and antibiotic resistance of Pseudomonas aeruginosa have been investigated. Cu treatments induced resistance not only to this metal but also, surprisingly, to zinc (Zn). Quantitative reverse transcription-PCR (qRT-PCR) revealed that after Cu treatment the transcription of the czcRS two-component system (TCS) operon was enhanced as well as that of the czcCBA operon encoding an efflux pump specific for zinc, cadmium, and cobalt. Cu treatments at the same time caused a decrease in the production of OprD porin, resulting in resistance to the carbapenem antibiotic imipenem. The CzcR regulator was known to repress oprD. However, Cu was still able to decrease the production of OprD and induce imipenem resistance in a czcRS knockout mutant. This strongly suggested that another Cu-dependent regulatory system was acting negatively on oprD expression. TCS regulator genes copR-copS have been shown to be involved in Cu tolerance in P. aeruginosa, qRT-PCR showed that overproduction of the CopR or of the CzcR regulator resulted in increased transcription of the czcC gene as well as in a decrease in oprD gene transcription, either in the wild-type strain or in the czcRS knockout mutant. Overproduction experiments suggest that a metal-dependent mechanism operates at the posttranscriptional level to control the production of the CzcCBA efflux pump. This study shows that CopR is a new negative regulator of OprD porin and that it links Zn, Cu, and imipenem resistances by interacting with the CzcRS TCS.

The evolutionary origin of telomerases, enzymes that maintain the ends of linear chromosomes in most eukaryotes, is a subject of debate. Penelope-like elements (PLEs) are a recently described class of eukaryotic retroelements characterized by a GIY-YIG endonuclease domain and by a reverse transcriptase domain with similarity to telomerases and group II introns. Here we report that a subset of PLEs found in bdelloid rotifers, basidiomycete fungi, stramenopiles, and plants, representing four different eukaryotic kingdoms, lack the endonuclease domain and are located at telomeres. The 5' truncated ends of these elements are telomereoriented and typically capped by species-specific telomeric repeats. Most of them also carry several shorter stretches of telomeric repeats at or near their 3' ends, which could facilitate utilization of the telomeric G-rich 3' overhangs to prime reverse transcription. Many of these telomere-associated PLEs occupy a basal phylogenetic position close to the point of divergence from the telomerase-PLE common ancestor and may descend from the missing link between early eukaryotic retroelements and present-day telomerases. reverse transcriptase | telomerase | transposable elements

Bacillus subtilis has multiple stress response systems whose integrated action promotes growth and survival under unfavorable conditions. Here we address the function and transcriptional organization of a five-gene cluster containing ssrA, previously known to be important for growth at high temperature because of the role of its tmRNA product in rescuing stalled ribosomes. Reverse transcription-PCR experiments detected a single message for the secG-yvaK-rnr-smpB-ssrA cluster, suggesting that it constitutes an operon. However, rapid amplification of cDNA ends-PCR and lacZ fusion experiments indicated that operon transcription is complex, with at least five promoters controlling different segments of the cluster. One [[sigma].sup.A]-like promoter preceded secG ([P.sub.1]), and internal [[sigma].sup.A]-like promoters were found in both the rnr.smpB ([P.sub.2]) and smpB-ssrA intervals ([P.sub.3] and [P.sub.HS]). Another internal promoter lay in the secG-yvaK intercistronic region, and this activity ([P.sub.B]) was dependent on the general stress factor [[sigma].sup.B]. Null mutations in the four genes downstream from [P.sub.B] were tested for their effects on growth. Loss of yvaK (carboxylesterase E) or rnr (RNase R) caused no obvious phenotype. By contrast, smpB was required for growth at high temperature (52[degrees]C), as anticipated if its product (a small ribosomal binding protein) is essential for tmRNA (ssrA) function. Notably, smpB and ssrA were also required for growth at low temperature (16[degrees]C), a phenotype not previously associated with tmRNA activity. These results extend the known high-temperature role of ssrA and indicate that the ribosome rescue system is important at both extremes of the B. subtilis temperature range. doi:10.1128/JB.00062-.07

Mobile DNAs use many mechanisms to minimize damage to their hosts. Here we show that a subclass of group II introns avoids host damage by inserting directly after transcriptional terminator motifs in bacterial genomes (stem-loops followed by Ts). This property contrasts with the site-specific behavior of most group II introns, which insert into homing site sequences. Reconstituted ribonucleoprotein particles of the Bacillus halodurans intron B.h.I1 are shown to reverse-splice into DNA targets in vitro but require the DNA to be single-stranded and fold into a stem-loop analogous to the RNA structure that forms during transcription termination. Recognition of this DNA stem-loop motif accounts for in vivo target specificity. Insertion after terminators is a previously unrecognized strategy for a selfish DNA because it prevents interruption of coding sequences and restricts expression of the mobile DNA after integration. reverse transcriptase | ribozyme | mobile DNA | bacteria

Staphylococcus epidermidis is both a human skin commensal and an opportunistic pathogen, causing infections linked to implanted medical devices. This paper describes localized tufts of fibrillar appendages on a subpopulation (25%) of wild-type (WT) S. epidermidis NCTC 11047 cells. The fibrils (122.2 [+ or -] 10.8 nm long) are usually in a lateral position on the cells. Fibriilar ([Fib.sup.+]) and nonfibrillar ([Fib.sup.-]) subpopulations were separated (enriched) by 34 sequential partitions of WT cells between a buffer phase and a hexadecane phase. Following enrichment, hydrophobic cells from the hexadecane phase comprised 70% [Fib.sup.-] cells and the less hydrophobic cells from the buffer phase entirely comprised [Fib.sup.-] cells. The [Fib.sup.+] and [Fib.sup.-] subpopulations did not revert on subculture (34 times) on solid medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell surface proteins from WT, [Fib.sup.+], and [Fib.sup.-] cells revealed two high-molecular-mass proteins (280 kDa and 230 kDa) on the WT and [Fib.sup.+] cells that were absent from the [Fib.sup.-] cells. Amino acid sequencing revealed that fragments of both the 280- and 230-kDa proteins had 100% identity to the accumulation-associated protein (Aap). Aap is known to cause biofilm formation if it is truncated by loss of the terminal A domain. Immunogoid staining with anti-Aap antibodies labeled tuft fibrils of the WT and [Fib.sup.+] cells but not the cell surface of Fibcells. The tufts were labeled with N-terminally directed antibodies (anti-A domain), showing that the fibrillar Aap was not truncated on the cell surface. Thus, the presence of full-length Aap correlated with the low biofilm-forming abilities of both WT and [Fib.sup.+] S. epidermidis NCTC 11047 populations. Reverse transcriptionPCR showed that aap was transcribed in both [Fib.sup.+] and [Fib.sup.-] cells. We therefore propose that full-length Aap is expressed on cells of S. epidermidis NCTC 11047 as tufts of short fibrils and that fibril expression is regulated at a posttranscriptional level.

HIV-1 reverse transcription requires several nucleic acid rearrangement steps that are 'chaperoned' by the nucleocapsid protein (NC), including minus-strand transfer, in which the DNA transactivation response element (TAR) is annealed to the complementary TAR RNA region of the viral genome. These various rearrangement processes occur in NC bound complexes of specific RNA and DNA structures. A major barrier to the investigation of these processes in vitro has been the diversity and heterogeneity of the observed nucleic acid/protein assemblies, ranging from small complexes of only one or two nucleic acid molecules all the way up to large-scale aggregates comprised of thousands of NC and nucleic acid molecules. Herein, we use a flow chamber approach involving rapid NC/nucleic acid mixing to substantially control aggregation for the NC chaperoned irreversible annealing kinetics of a model TAR DNA hairpin sequence to the complementary TAR RNA hairpin, i.e., to form an extended duplex. By combining the flow chamber approach with a broad array of fluorescence single-molecule spectroscopy (SMS) tools (FRET, molecule counting, and correlation spectroscopy), we have unraveled the complex, heterogeneous kinetics that occur during the course of annealing. The SMS results demonstrate that the TAR hairpin reactant is predominantly a single hairpin coated by multiple NCs with a dynamic secondary structure, involving equilibrium between a 'Y' shaped conformation and a closed one. The data further indicate that the nucleation of annealing occurs in an encounter complex that is formed by two hairpins with one or both of the hairpins in the 'Y' conformation. HIV-1 nucleocapsid protein | nucleic acid aggregation | transactivation response element DNA/RNA annealing | wild-type (WT)

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction. endonuclease | retrotransposition | reverse transcription | RNA protein interactions

Human pregnane X receptor (hPXR) gene polymorphisms (spanning exon 2 to exon 5) and alternative mRNA splicing were investigated as possible contributors to individual variability in CYP3A metabolic activity measured [...]

Drug exporters contribute to the intrinsic drug resistance in many organisms. Although there are at least 20 exporter genes in Escherichia coli, most of them apparently do not confer drug resistance in complex laboratory media except for the AcrAB, EmrE, and MdfA efflux systems. In this study, we comprehensively investigated the growth phase-dependent expression of drug exporter genes. The expression of acrAB, emrAB, emrD, emrE, emrKY, mdfA, and ydgFE is stable at moderate levels during any growth phase, whereas mdtEF promoter activity greatly increased with cell growth and reached the maximum level at the late stationary phase. The growth phase-dependent increase in mdtEF expression was also observed on quantitative reverse transcription-PCR analysis. As expected from the transporter expression, the stationary-phase cells actually showed MdtEF-dependent tolerance to drugs and toxic dyes. Growth phase-dependent elevation of mdtEF expression was found to be mediated by the stationary-phase [sigma] factor rpoS and the RpoS-dependent signaling pathway, Hfq, GadY, and GadX. The induction level was decreased by tnaAB deletion, suggesting that indole sensing stimulates this process.

The potential relationship between helicase structural organization and unwinding mechanism is examined by performing in vitro Rho helicase experiments with model substrates containing an RNA-DNA helix downstream from a Rho loading site. The results have outlined the differences as well as the similarities in the mechanism of unwinding between Rho and other hexameric helicases, which are discussed in relation with the biological function of the Rho helicase.

The genetic mechanisms mediating the adaptation of Mycobacterium tuberculosis within the host are poorly understood. The best-characterized regulatory systems in this organism include sigma factors and two-component signal transduction systems, mprAB is a two-component system required by M. tuberculosis for growth in vivo during the persistent stage of infection. In this report, we demonstrate that MprAB is stress responsive and regulates the expression of numerous stress-responsive genes in M. tuberculosis. With DNA microarrays and quantitative real-time reverse transcription-PCR, genes regulated by MprA in M. tuberculosis that included two stress-responsive sigma factors were identified. Response regulator MprA bound to conserved motifs in the upstream regions of both sigB and sigE in vitro and regulated the in vivo expression of sigB and sigE in M. tuberculosis. In addition, mprA itself was induced following exposure to stress, establishing a direct role for this regulatory system in stress response pathways of M. tuberculosis. Induction of mprA and sigE by MprA in response to stress was mediated through the cognate sensor kinase MprB and required expression of the extracytoplasmic loop domain. These results provide the first evidence that recognition of and adaptation to specific stress in M. tuberculosis are mediated through activation of a two-component signal transduction system that directly regulates the expression of stress-responsive determinants.

The P2-1ike coliphages are highly similar; the structural genes show at least 96% identity. However, at two loci they have genes believed to be horizontally transferred. We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames. The product of one of them exhibits reverse transcriptase activity and blocks infection of phage T5.

The Yersinia enterocolitica LuxI homologue YenI directs the synthesis of N-3-(oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL). In a Y. enterocolitica yenI mutant, swimming motility is temporally delayed while swarming motility is abolished. Since both swimming and swarming are flagellum dependent, we purified the flagellin protein from the parent and yenI mutant. Electrophoresis revealed that in contrast to the parent strain, the yenI mutant grown for 17 h at 26[degrees]C lacked the 45-kDa flageilin protein FleB. Reverse transcription-PCR indicated that while mutation of yenI had no effect on yenR, flhDC (the motility master regulator) orfliA (the flagellar sigma factor) expression, fleB (the flagellin structural gene) was down-regulated. Since 3-oxo-C6-HSL and C6-HSL did not restore swimming or swarming in the yenI mutant, we reexamined the N-acylhomoserine lactone (AHL) profile of Y. enterocolitica. Using AHL biosensors and mass spectrometry, we identified three additional AHLs synthesized via YenI: N-(3-oxodecanoyl)homoserine lactone, N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL), and N-(3-oxotetradecanoyl)homoserine lactone. However, none of the long-chain AHLs either alone or in combination with the short-chain AHLs restored swarming or swimming in the yenI mutant. By investigating the transport of radiolabeled 3-oxo-C12-HSL and by introducing an AHL biosensor into the yenI mutant we demonstrate that the inability of exogenous AHLs to restore motility to the yenI mutant is not related to a lack of AHL uptake. However, both AHL synthesis and motility were restored by complementation of the yenI mutant with a plasmid-borne copy of yenI.

A new double-ARMS (amplification refractory mutation system) real-time polymerase chain reaction assay was developed to detect and quantify 4 populations in the CD45RO(super +) T-cell compartment. Twenty-one patients, 18 lamivudine and efavirenz/nevirapine experienced, were enrolled in a cross-sectional study.

In the murine model of infection, a Mycobacterium tuberculosis mce1 operon mutant elicits an aberrant granulomatous response, resulting in uncontrolled replication and failure to enter a persistent state. In this study, we demonstrate that the mce1 genes can be transcribed as a 13-gene polycistronic message encompassing Rv0166 to Rv0178. Quantitative reverse transcriptase PCR and immunoblot analyses revealed that the mce1 genes and proteins are expressed during in vitro growth but are significantly down-regulated in intracellular bacilli isolated from murine macrophages. A homologue of the FadR subfamily of GntR transcriptional regulators, Rv0165c (designated Mce1R), lies upstream and is divergently transcribed from the operon. To investigate whether this gene plays a role in regulation of mce1 expression, we created an M. tuberculosis mce1R deletion mutant. There was no difference in expression of mce1 operon genes in [DELTA]mce1R compared to expression in the wild type during logarithmic growth in vitro. However, in bacilli isolated from murine macrophages, expression of mce1 genes was significantly higher in [DELTA]mce1R. In addition, overexpression of mce1R resulted in repression of the mce1 genes. These data demonstrate that Mce1R is a negative regulator that acts intracellularly to repress expression of the mce1 operon. We propose that Mce1R facilitates balanced temporal expression of the mce1 products required for organized granuloma formation, which is both protective to the host and necessary for the persistence of M. tuberculosis.

In vivo expression of human telomerase is significantly different from that of mouse telomerase. To assess the basis for this difference, a bacterial artificial chromosome clone containing the entire hTERT (human telomerase reverse transcriptase) gene was introduced in mice. In these transgenic mice, expression of the hTERT transgene was similar to that of endogenous hTERT in humans, rather than endogenous mTERT (mouse telomerase reverse transcriptase). In tissues and cells showing a striking difference in expression levels between hTERT in humans and mTERT in mice (i.e., liver, kidney, lung, uterus, and fibroblasts), expression of the hTERT transgene in transgenic mice was repressed, mimicking hTERT in humans. The transcriptional activity of the hTERT promoter was much lower than that of the mTERT promoter in mouse embryonic fibroblasts or human fibroblasts. Mutational analysis of the hTERT and mTERT promoters revealed that a nonconserved GC-box within the hTERT promoter was responsible for the human-specific repression. These results reveal that a difference in cisregulation of transcription, rather than transacting transcription factors, is critical to species differences in tissue-specific TERT expression. Our data also suggest that the GC-box-mediated, human-specific mechanism for TERT repression is impaired in human cancers. This study represents a detailed characterization of the functional difference in a gene promoter of mice versus humans and provides not only important insight into species-specific regulation of telomerase and telomeres but also an experimental basis for generating mice humanized for telomerase enzyme and its pattern of expression.

Site-specific fluorescent labeling of RNA molecules is achieved by specific transcription using an unnatural base pair system. This method is useful for analyzing conformational changes of RNA molecules and for detecting interactions between RNA and its binding species.

The distribution of the metalloprotease gene zmpA was determined among strains of the Burkholderia cepacia complex (Bcc). The zmpA gene was present in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria and B. pyrrocinia but absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The presence of zmpA generally correlated with extracellular proteolytic activity with the exception of five strains, which had zmpA but had no detectable proteolytic activity when skim milk agar was used as a substrate (zmpA protease deficient). Western immunoblot experiments with anti-ZmpA antibodies suggest that the zmpA protease-deficient strains do not secrete or accumulate detectable ZmpA. Transcriptional zmpA::lacZ fusions were introduced in selected strains of the Bcc. zmpA::lacZ was expressed in all strains, but expression was generally lower in the zmpA protease-deficient strains than in the zmpA protease-proficient strains. Quantitative reverse transcriptase real-time PCR demonstrated that zmpA protease-deficient strains did express zmpA mRNA, although at various levels. ZmpA has previously been shown to be positively regulated by the CepIR quorum-sensing system. Addition of exogenous AHLs did not restore extracellular protease production to any of the zmpA protease-deficient strains; however, introduction of cepR in trans complemented protease activity in two of five strains. Extra-cellular proteolytic activity was restored by the presence of zmpA in trans in two of the five strains. These studies suggest that although some strains of the Bcc contain the zmpA gene, multiple factors may influence its expression.

Results from a new kind of single-molecule kinetic measurement on the human immunodeficiency virus (HIV) reverse transcriptase are reported. The intrinsic processive DNA-dependent polymerization of HIV reverse transcriptase (RT) is approximately Poissionian, wherein each nucleotide is added sequentially, with a rate of about 100 bases per second at 21 degree Celsius.

Telomerase deficiency leads to a progressive loss of telomeric DNA that eventually triggers cell apoptosis in human primary cells during prolonged growth in culture. Rare survivors can maintain telomere length through either activation of telomerase or recombination-based telomere lengthening, and thus proliferate indefinitely. We have explored the possibility that telomeres may be maintained through telomere sister chromatid exchange (T-SCE) in murine telomere reverse transcriptase-deficient ([mTert.sup.-/-]) splenocytes and ES cells. Because telomerase deficiency leads to gradual loss of telomeric DNA in [mTert.sup.-/-] splenocytes and ES cells and eventually to chromosomes with telomere signal-free ends (SFEs), we examined these cell types for evidence of sister chromatid exchange at telomeres, and observed an increase in T-SCEs only in a subset of [mTert.sup.-/-] splenocytes or ES cells that possessed multiple SFEs. Furthermore, T-SCEs were more often detected in ES cells than in splenocytes that harbored a similar frequency of SFEs. In mTert heterozygous ([mTert.sup.+/-]) ES cells or splenocytes, which are known to exhibit a decrease in average telomere length but no SFEs, no increase in T-SCE was observed. In addition to T-SCE, other genomic rearrangements (i.e., SCE) were also significantly increased in [mTert.sup.-/-] ES cells possessing critically short telomeres, but not in splenocytes. Our results suggest that animals and cell culture differ in their ability to carry out genomic rearrangements as a means of maintaining telomere integrity when telomeres become critically shortened. murine telomerase reverse transcriptase | telomere signal-free end

Telomerase is a special reverse transcriptase that extends one strand of the telomere repeat by using a template embedded in an RNA subunit. Like other polymerases, telomerase is believed to use a pair of divalent metal ions (coordinated by a triad of aspartic acid residues) for catalyzing nucleotide addition. Here we show that, in the presence of manganese, both yeast and human telomerase can switch to a template- and RNA-independent mode of DNA synthesis, acting in effect as a terminal transferase. Even as a terminal transferase, yeast telomerase retains a species-dependent preference for GT-rich, telomere-like DNA on the 5' end of the substrate. The terminal transferase activity of telomerase may account for some of the hitherto unexplained effects of telomerase overexpression on cell physiology. reverse transcriptase | telomere | divalent metal ion

The steady-state kinetic measurements are extended to two replicative DNA polymerases, exonuclease-deficient T7 DNA polymerase T7(super -) and HIV reverse transcriptive (HIV-RT). The influences of G[8-5]C on the thermal stability and base pairing properties of DNA duplexes are examined.

Bacterial gene expression was studied during in vitro exponential and early stationary growth in medium with and without sufficient iron, respectively. The aims of the investigation were to develop and test a sensitive and reproducible method for the study of gene expression in Actinobacillus pleuropneumoniae by real-time quantitative reverse transcription (RT)-PCR and to evaluate a number of suitable internal controls.

The study developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII noroviruses (NoV) in fecal samples, as well as shellfish samples. These assays provide rapid, sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical ad shellfish samples.

Susceptibility results of HIV-1 isolates lacking drug resistance mutations and containing matching patterns of drug resistance mutations are examined in order to assess the precision of the Antivirogram and PhenoSense assays. The PhenoSense assay is more precise than the Antivirogram assay and superior at detecting resistance to abacavir, didanosine, and stavudine.

Understanding the mechanisms of HIV-1 drug resistance is critical for developing more effective antiretroviral agents and therapies. Based on our previously described dynamic copy-choice mechanism for retroviral recombination and our observations that nucleoside reverse transcriptase inhibitors (NRTIs) increase the frequency of reverse transcriptase template switching, we propose that an equilibrium exists between (i) NRTI incorporation, NRTI excision, and resumption of DNA synthesis and (ii) degradation of the RNA template by RNase H activity, leading to dissociation of the template-primer and abrogation of HIV-1 replication. As predicted by this model, mutations in the RNase H domain that reduced the rate of RNA degradation conferred high-level resistance to 3'azido-3'-deoxythymidine and 2,3-didehydro-2,3-dideoxythymidine by as much as 180- and 10-fold, respectively, by increasing the time available for excision of incorporated NRTIs from terminated primers. These results provide insights into the mechanism by which NRTIs inhibit HIV-1 replication and imply that mutations in RNase H could significantly contribute to drug resistance either alone or in combination with NRTI-resistance mutations in reverse transcriptase. drug resistance | recombination | NRTI | reverse transcriptase | dynamic copy choice

Penelope-like elements are a class of retroelement that have now been identified in >50 species belonging to at least 10 animal phyla. The Penelope element isolated from Drosophila virilis is the only transpositionally active representative of this class isolated so far. The single ORF of Penelope and its relatives contains regions homologous to a reverse transcriptase of atypical structure and to the GIY-YIG, or Uri, an endonuclease (EN) domain not previously found in retroelements. We have expressed the single ORF of Penelope in a baculovirus expression system and have shown that it encodes a polyprotein with reverse transcriptase activity that requires divalent cations ([Mn.sup.2+] and [Mg.sup.2+]). We have also expressed and purified the EN domain in Escherichia coli and have demonstrated that it has EN activity in vitro. Mutations in the conserved residues of the EN catalytic module abolish its nicking activity, whereas the DNA-binding properties of the mutant proteins remain unaffected. Only one strand of the target sequence is cleaved, and there is a certain degree of cleavage specificity. We propose that the Penelope EN cleaves the target DNA during transposition, generating a primer for reverse transcription. Our results show that an active Uri EN has been adopted by a retrotransposon. GIY-YIG endonuclease | retrotransposons | Drosophila virilis | Uri domain

Activation of telomerase is crucial for cells to gain immortality. Most normal human somatic cells have a limited proliferative life span, and expression of the rate-limiting telomerase catalytic subunit, known as human telomerase reverse transcriptase (hTERT), has been believed to be tightly repressed. This model of hTERT regulation is challenged by the recent identification of the induction of hTERT in normal cycling human fibroblasts during their transit through S phase. Here we show the small-molecule-based identification of the assembly and disassembly of E2F-pocket protein-histone deacetylase (HDAC) complex as a key mechanistic basis for the repression and activation of hTERT in normal human cells. A cell-based chemical screen was used to identify a small molecule, CGK1026, that derepresses hTERT expression. CGK1026 inhibits the recruitment of HDAC into E2F-pocket protein complexes assembled on the hTERT promoter. Chromatin immunoprecipitation analysis reveals dynamic alterations in hTERT promoter occupancy by E2F and pocket proteins according to the cell cycle-dependent regulation of hTERT. Dominant-negative or protein-knockout strategies to disrupt the assembly of E2F-pocket protein-HDAC complex derepress hTERT and telomerase activity. Taken together with the results on the regulatory function of these complexes in cellular senescence and tumorigenesis, our findings suggest that dynamic assembly of E2F-pocket protein-HDAC complex plays a central role in the regulation of hTERT in a variety of proliferative conditions (e.g., normal cycling, senescent, and tumor cells).

An approach for inhibiting the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-I) reverse transcriptase (RT) is demonstrated by targeting RNase H RNA.DNA hybrid substrates. In this approach the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin and ribostamycin, to two different chimeric RNA-DNA duplexes inhibits specific RT-mediated RNase cleavage in competitive manner.

The prevalence of mutations associated with resistance to antiretroviral drugs and their interpretation in patients infected with non-B HIV-1 variants is compared with HIV-1 subtype B-infected patients with similar treatment regimens. The results suggested that genetic diversity of HIV-1 does not play major role in the development of resistance to antiretroviral drugs.

Byline: A. B. Chetverin (1), H. V. Chetverina (1) Keywords: molecular diagnostics; nucleic acids; exponential amplification; polymerase chain reaction; reverse transcription; isothermal DNA amplification systems; Q[beta] replicase Abstract: The accuracy and reliability of diagnostic tests for infections and cancer can be substantially improved by using a gel, rather than a liquid medium, to amplify nucleic acids and thereby to obtain molecular colonies. Author Affiliation: (1) Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia Article History: Registration Date: 09/10/2004

Byline: O. I. Andreeva (1), E. V. Efimtseva (1), N. Sh. Padyukova (1), S. N. Kochetkov (1), S. N. Mikhailov (1), H. B. F. Dixon (2), M. Ya. Karpeisky (1) Keywords: T7 RNA polymerase; HIV-1 reverse transcriptase; methylenebisphosphonates; competitive inhibition Abstract: We have examined the interaction of human immunodeficiency virus reverse transcriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleoside triphosphates and inorganic pyrophosphate (PP.sub.i) analogs containing nonhydrolyzable bisphosphonate groups. We have synthesized a number of derivatives of bisphosphonic acid having different aromatic and nonaromatic side substituents, as well as the NTP derivatives whose incorporation into the growing nucleotide chain during the polymerization reaction results in formation of bisphosphonates as leaving groups. The competitive character of inhibition of both enzymes has been revealed for all the compounds under study, and the inhibition constants have been estimated. One of P[P.sub.i]analogs containing a bulky aromatic substituent is characterized by similar inhibition constants for both T7 RNAP and RT. The universal character of this inhibitor can serve as evidence for a similar structure of the NPT-binding sites in the two polymerases. It has been shown that nonsubstituted methylenebisphosphate is a better leaving group than that containing additional methyl and hydroxyl groups. The NTP analogs are very weak inhibitors of T7 RNAP, whereas HIV RT is more sensitive to this type of compounds. On the basis of the X-ray crystallographic data on the T7 RNAP complex with a template and NTP, we have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme. The peculiarities observed in the model correlate well with the experimental data on inhibition. Author Affiliation: (1) Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia (2) Department of Biology, King's College, Cambridge, UK Article History: Registration Date: 08/10/2004

The polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed tumor rejection antigen because telomerase is silent in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces immunity against tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL), which lysed melanoma and thymoma tumor cells and inhibited the growth of three unrelated tumors in mice of distinct genetic backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used as surrogate targets in the CTL assays, obviating the difficulties in obtaining tumor cells from cancer patients. In one instance, where a tumor cell line was successfully established in culture from a patient with renal cancer, the patient&apos;s tumor cells were efficiently lysed by the CTL. Immunization with tumor RNA was generally more effective than immunization with TERT RNA, suggesting that an optimal immunization protocol may have to include TERT as well as additional tumor antigens., Author(s): Smita K. Nair [1]; Axel Heiser [2]; David Boczkowski [1]; Anish Majumdar [3]; Michio Naoe [2]; Jane S. Lebkowski [3]; Johannes Vieweg [2]; Eli Gilboa (corresponding author) [1] Cytotoxic [...]