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Biomedical subjects

L Kuo

Publications and source records attributed to L Kuo.

At least 19 recordsLinked to original sources
PubMed1999

Intravascular flow decreases erythrocyte consumption of nitric oxide.

Nitric oxide (NO) produced by the endothelium diffuses both into the lumen and to the smooth muscle cells according to the concentration gradient in each direction. The extremely high reaction rate between NO and hemoglobin (Hb), k(Hb)= 3-5 x 10(7) M(-1).s(-1), suggests that most of the NO produced would be consumed by Hb in the red blood cells (RBCs), which then would block the biological effect of NO. Therefore, specific mechanisms must exist under physiological conditions to reduce the NO consumption by RBCs, in which the Hb concentration is very high (24 mM heme). By using isolated microvessels as a bioassay, here we show that physiological concentrations of RBCs in the presence of intravascular flow does not inhibit NO-mediated vessel dilation, suggesting that RBCs under this condition are not an NO scavenger. On the other hand, RBCs (50% hematocrit) without intravascular flow reduce NO-mediated dilation to serotonin by 30%. In contrast, free Hb (10 microM) completely inhibits NO-mediated dilation with or without intravascular flow. The effect of flow on NO consumption by RBCs may be attributed to the formation of an RBC-free zone near the vessel wall, which is caused by hydrodynamic forces on particles. Intravascular flow does not affect the reaction rate between NO and free Hb in the lumen, because the latter forms a homogeneous solution and is not subject to the hydrodynamic separation. However, intravascular flow only partially contributes to the reduced consumption of NO by RBCs, because without the flow, the NO consumption by RBCs is already about 3 orders of magnitude slower than free Hb.

Animals↗
PubMed1999

A concise promoter region of the heart fatty acid-binding protein gene dictates tissue-appropriate expression.

The heart fatty acid-binding protein (HFABP) is a member of a family of binding proteins with distinct tissue distributions and diverse roles in fatty acid metabolism, trafficking, and signaling. Other members of this family have been shown to possess concise promoter regions that direct appropriate tissue-specific expression. The basis for the specific expression of the HFABP has not been previously evaluated, and the mechanisms governing expression of metabolic genes in the heart are not completely understood. We used transient and permanent transfections in ventricular myocytes, skeletal myocytes, and nonmyocytic cells to map regulatory elements in the HFABP promoter, and audited results in transgenic mice. Appropriate tissue-specific expression in cell culture and in transgenic mice was dictated by 1.2 kb of the 5'-flanking sequence of FABP3, the HFABP gene. Comparison of orthologous murine and human genomic sequences demonstrated multiple regions of near-identity within this promoter region, including a CArG-like element close to the TATA box. Binding and transactivation studies demonstrated that this element can function as an atypical myocyte enhancer-binding factor 2 site. Interactions with adjacent sites are likely to be necessary for fully appropriate, tissue-specific, developmental and metabolic regulation.

Animals↗
PubMed1999

Coronary arteriolar dilation to acidosis: role of ATP-sensitive potassium channels and pertussis toxin-sensitive G proteins.

BACKGROUND: We previously demonstrated that coronary arteriolar dilation in response to acidosis is mediated by the opening of ATP-sensitive potassium (KATP) channels. However, the signal transduction involved in the KATP-channel activation during acidosis has not been elucidated. A recent study in cardiac myocytes implied that pertussis toxin (PTX)-sensitive G proteins may be involved in the signal transduction for KATP-channel activation. However, it remains unclear whether this transduction process also occurs in the vascular tissue and, in particular, whether it exerts functional dilation in response to acidosis. METHODS AND RESULTS: To examine the signaling pathway for acidosis-induced dilation, porcine coronary arterioles were isolated, cannulated, and pressurized for in vitro study. The GTPase activity in reconstituted G proteins was examined at different levels of pH. Extravascular acidosis (pH 7.3 to 7.0) produced a graded dilation of coronary arterioles. This dilation was not affected by removal of endothelium but was significantly attenuated after inhibition of KATP channels and G proteins by glibenclamide and PTX, respectively. Glibenclamide and PTX attenuated the acidosis-induced arteriolar dilation to the same extent, and combined administration of both inhibitors did not further inhibit the vasodilation. These results indicated that both inhibitors act on the same vasodilatory pathway. Furthermore, vasodilation of coronary arterioles to the KATP-channel opener pinacidil and to the endothelium-independent vasodilator sodium nitroprusside was not affected by PTX. Because PTX inhibited acidosis-induced vasodilation without inhibiting KATP-channel function, it is suggested that PTX inhibits the vasodilatory pathway upstream from KATP channels. GTPase activity in reconstituted G proteins was significantly enhanced by a reduction in pH, indicating that G proteins were directly activated by acidosis. CONCLUSIONS: On the basis of these findings, we conclude that acidosis-induced coronary arteriolar dilation is mediated by the opening of smooth muscle KATP channels through the activation of PTX-sensitive G proteins.

Acidosis↗
PubMed1999

Changes in coronary endothelial cell Ca2+ concentration during shear stress- and agonist-induced vasodilation.

Increases in intraluminal shear stress are thought to cause vasodilation of coronary arterioles by activation of Ca2+-dependent endothelial nitric oxide synthase followed by release of nitric oxide. We tested the hypothesis that endothelium-dependent vasodilation of isolated coronary arterioles to shear stress and agonists is necessarily preceded by an increase in endothelial cell Ca2+ concentration ([Ca2+]i). After selective loading of endothelium in isolated rabbit coronary arterioles with fura 2, simultaneous changes in diameter and [Ca2+]i were recorded. Vasodilations recorded in response to ACh, substance P, or shear stress were accompanied by significant increases in endothelial cell [Ca2+]i. Vasodilations to shear stress were accompanied by smaller changes in endothelial cell [Ca2+]i than equivalent dilations evoked by substance P or ACh. To test the role for Ca2+ as an activator of endothelial nitric oxide synthase, the endothelium was treated with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid. 1,2-Bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid eliminated significant changes in endothelial cell [Ca2+]i and inhibited dilations to ACh and substance P but did not significantly affect shear stress-induced vasodilation. The data indicate that endothelium-dependent vasodilation of coronary arterioles in response to agonists and shear stress is mediated in part through a rise in endothelial cell [Ca2+]i but that a substantial component of the shear stress-induced response occurs through a Ca2+-insensitive pathway.

Acetylcholine↗
PubMed1998

LDLs impair vasomotor function of the coronary microcirculation: role of superoxide anions.

Oxidized LDLs (Ox-LDLs) inhibit endothelium-dependent dilation of isolated conduit arteries in a manner comparable to the impairment demonstrated in atherosclerotic vessels. However, it is not known whether the microvessels, which do not develop atherosclerotic lesions, are susceptible to Ox-LDL. Since endothelial release of NO plays an important role in vasodilation and since its dysfunction associated with atherosclerosis has been shown to extend into the coronary microcirculation, we hypothesized that Ox-LDLs impair endothelium-dependent vasodilation of coronary arterioles by reducing the synthesis and/or release of NO. To test this hypothesis, porcine subepicardial vessels (50 to 100 microm) were isolated, cannulated, and pressurized to 60 cm H2O without flow for in vitro study. Isolated vessels developed basal tone and dilated in a dose-dependent manner to the endothelium-dependent vasodilators serotonin, ATP, and ionomycin. These vasodilatory responses were inhibited by the NO synthase inhibitor NG-monomethyl-L-arginine and were subsequently reversed by extraluminal administration of the NO precursor L-arginine (3 mmol/L), suggesting the involvement of NO in these vasomotor responses. Intraluminal incubation of the vessels with native LDL (N-LDL) or Ox-LDL (1 mg protein/mL) significantly attenuated dilations to serotonin, ATP, and ionomycin. Ox-LDL produced more severe inhibition than did N-LDL, and the inhibitory effect was comparable to that of NG-monomethyl-L-arginine. The inhibitory effects of N-LDL and Ox-LDL were reversed by exogenous L-arginine (3 mmol/L) and were prevented by sodium dihydroxybenzene disulfonate (Tiron), a cell-permeable superoxide scavenger. In contrast, administration of the cell-impermeable superoxide scavenger superoxide dismutase prevented the inhibitory effect of N-LDL but not of Ox-LDL. In addition, the inhibitory effects of LDL were not restored by D-arginine or by removal of intraluminal LDL. Neither N-LDL nor Ox-LDL altered endothelium-independent vasodilation to sodium nitroprusside. These results indicate that coronary arterioles are susceptible to LDLs that specifically impair endothelium-dependent vasodilation by reducing NO synthesis. It is suggested that the initiation of superoxide anion production and the subsequent L-arginine deficiency may be responsible for the detrimental effect of LDL.

Animals↗
PubMed1998

Lipopolysaccharide activates endothelial nitric oxide synthase through protein tyrosine kinase.

Vascular endothelial cell injury or activation by lipopolysaccharide (LPS) plays an important role in the pathogenesis of endotoxin shock. However, the effect of LPS on NO production from vascular endothelial cells (ECs) is incompletely understood. In this study, bovine coronary venular ECs were treated with LPS and the release of NO and expression of the endothelial NO synthase (ecNOS) were examined. We found that the ecNOS activity is transiently enhanced by LPS within the time scale of about 10 h due to the interplay between two LPS-induced mechanisms. Within the first 10 h of LPS treatment, the specific activity of ecNOS is increased by a post-translational modification mediated through a protein tyrosine kinase cascade. After about 10 h of treatment, however, LPS destabilizes the transcript of ecNOS and thus decreased the expression level and total activity.

Animals↗
PubMed1998

Targeted recombination between MHV-2 and MHV-A59 to study neurotropic determinants of MHV.

MHV-A59 produces acute encephalitis, acute hepatitis and chronic demyelination in infected mice. MHV-2 produces only hepatitis and mild meningitis but without encephalitis or demyelination. We have previously studied a set of recombinant viruses between these two strains. The common denominator of viruses that produced encephalitis was a membrane (M) gene derived from MHV-A59. Thus to study the potential contribution of the M gene to acute encephalitis, chimeric viruses were produced in which the M gene of MHV-A59 was substituted with the M gene of MHV-2 by targeted recombination. A control virus was produced in which the M gene of A59 was recombined back into an A59 background. Viruses were then analyzed for their biologic properties and compared with the phenotypes of MHV-A59 and MHV-2 by histopathology and plaque assays for viral titers in organs following intracerebral (IC) inoculation. All three chimeric viruses had a phenotype similar to MHV-A59. Thus, the replacement of the M gene of MHV-A59 with that of MHV-2 is insufficient to produce a phenotype that lacks encephalitis similar to MHV-2.

Animals↗
PubMed1998

Coronavirus particle assembly: primary structure requirements of the membrane protein.

Coronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly.

Amino Acid Sequence↗
PubMed1998

Arginase modulates nitric oxide production in activated macrophages.

In macrophages and many other cell types, L-arginine is used as a substrate by both nitric oxide synthase (NOS) and arginase to produce nitric oxide (NO) and urea, respectively. Because the availability of L-arginine is a major determinant for NO synthesis in the activated macrophage, we hypothesized that NO production may be reduced by arginase via depleting the common substrate in this cell type. To test this hypothesis, we investigated the effect of an arginase inhibitor, L-norvaline, on NO production in J774A.1 mouse macrophages activated by lipopolysaccharide (LPS, 1.0 microgram/ml) for 22 h. In the absence of LPS, macrophages produced a low level of NO. In contrast, NO production from these cells was significantly increased in the presence of LPS. Increasing extracellular levels of L-arginine (0.01-0.8 mM) produced a concomitant increase in NO production of activated macrophages. L-Norvaline (10 mM), which specifically inhibits arginase activity (i.e., reducing urea production by 50%) without altering NOS activity, enhanced NO production (by 55%) from activated macrophages. The enhancement of NO production by L-norvaline was inversely related to the extracellular level of L-arginine. A more pronounced increase in NO production was observed at the lower level of extracellular L-arginine, i.e., a 55 vs. 28% increase for 0.05 and 0.1 mM extracellular L-arginine, respectively. When the L-arginine concentration exceeded 0.5 mM, the L-norvaline effect was abolished. These results indicate that arginase can compete with NOS for their common substrate and thus inhibit NO production. This regulatory mechanism may be particularly important when the extracellular supply of L-arginine is limited.

Animals↗
PubMed1998

Effective diffusion distance of nitric oxide in the microcirculation.

Despite its well-documented importance, the mechanism for nitric oxide (NO) transport in vivo is still unclear. In particular, the effect of hemoglobin-NO interaction and the range of NO action have not been characterized in the microcirculation, where blood flow is optimally regulated. Using a mathematical model and experimental data on NO production and degradation rates, we investigated factors that determine the effective diffusion distance of NO in the microcirculation. This distance is defined as the distance within which NO concentration is greater than the equilibrium dissociation constant (0.25 microM) of soluble guanylyl cyclase, the target enzyme for NO action. We found that the size of the vessel is an important factor in determining the effective diffusion distance of NO. In approximately 30- to 100-micron-ID microvessels the luminal NO concentrations and the abluminal effective diffusion distance are maximal. Furthermore, the model suggests that if the NO-erythrocyte reaction rate is as fast as the rate reported for the in vitro NO-hemoglobin reaction, the NO concentration in the vascular smooth muscle will be insufficient to stimulate smooth muscle guanylyl cyclase effectively. In addition, the existence of an erythrocyte-free layer near the vascular wall is important in determining the effective NO diffusion distance. These results suggest that 1) the range of NO action may exhibit significant spatial heterogeneity in vivo, depending on the size of the vessel and the local chemistry of NO degradation, 2) the NO binding/ reaction constant with hemoglobin in the red blood cell may be much smaller than that with free hemoglobin, and 3) the microcirculation is the optimal site for NO to exert its regulatory function. Because NO exhibits vasodilatory function and antiatherogenic activity, the high NO concentration and its long effective range in the microcirculation may serve as intrinsic factors to prevent the development of systemic hypertension and atherosclerotic pathology in microvessels.

Animals↗
PubMed1998

Estimation of nitric oxide production and reaction rates in tissue by use of a mathematical model.

Nitric oxide (NO) produced by the vascular endothelium is an important biologic messenger that regulates vessel tone and permeability and inhibits platelet adhesion and aggregation. NO exerts its control of vessel tone by interacting with guanylyl cyclase in the vascular smooth muscle to initiate a series of reactions that lead to vessel dilation. Previous efforts to investigate this interaction by mathematical modeling of NO diffusion and reaction have been hampered by the lack of information on the production and degradation rate of NO. We use a mathematical model and previously published experimental data to estimate the rate of NO production, 6.8 x 10(-14) micromol . micron-2 . s-1; the NO diffusion coefficient, 3,300 micron2 s-1; and the NO consumption rate coefficient in the vascular smooth muscle, 0.01 s-1 (1st-order rate expression) or 0.05 microM-1 . s-1 (2nd-order rate expression). The modeling approach is discussed in detail. It provides a general framework for modeling the NO produced from the endothelium and for estimating relevant physical parameters.

Endothelium, Vascular↗
PubMed1997

Analysis of the gene start and gene end signals of human respiratory syncytial virus: quasi-templated initiation at position 1 of the encoded mRNA.

The gene start (GS) and gene end (GE) transcription signals of human respiratory syncytial virus (RSV) strain A2 were analyzed in helper-dependent monocistronic and dicistronic minireplicons which were complemented by a standard RSV strain. The GS signal, which is the start site for mRNA synthesis, is highly conserved for the first nine genes: 3'-CCCCGUUUA(U/C) (negative sense). This conserved version of the signal was analyzed by "saturation" mutagenesis, in which all 10 positions, as well as one downstream and one upstream position, were changed one at a time into each of the other three nucleotides. Most of the positions appear to contribute to the signal: positions 1, 3, 6, 7, and, in particular, 9 were the most sensitive, whereas position 5 was relatively insensitive. The effect of nucleotide substitution in the first position of the signal was examined further by cDNA cloning and sequence analysis of the residual mRNA which was produced. For the two mutants examined (1C to U, and 1C to A), the site of initiation was unchanged. However, the mRNAs were dimorphic with regard to the assignment of the 5'-terminal nucleotide: two-thirds contained the predicted mutant substitution, and one-third contained the parental assignment. Intracellular minigenome contained only the mutant assignment, indicating that the heterogeneity was at the level of transcription by the RSV polymerase. This suggests that the templated mutant assignment at position 1 can sometimes be overridden by an innate preference for the parental assignment, a phenomenon which we dubbed quasi-templated initiation. The GS signal of the L gene, encoding the 10th RSV mRNA, contains three differences (3'-CCCUGUUUUA) compared to the conserved version. It was shown to be equal in efficiency to the conserved version. This was unexpected, since the saturation mutagenesis described above indicated that U in place of A at position 9 should be highly inhibitory. Instead, the A at position 10 of the L GS signal was found to be critical for activity, indicating that an essential A residue indeed was present in both versions of the GS signal but that its spacing differed. The GE signal, which directs termination and polyadenylation, has more sequence diversity in nature than does the GS signal. The naturally occurring GE signals of strain A2 were compared by their individual incorporation into a dicistronic minigenome. They were similar in the ability to produce translatable mRNA except in the cases of NS1 and NS2, which were approximately 60% as efficient.

Blotting, Northern↗
PubMed1997

Interaction between adenosine and flow-induced dilation in coronary microvascular network.

Previous studies have demonstrated that coronary microvessels are regulated by at least three possible means: metabolite-induced, shear-induced, and pressure-induced (myogenic) mechanisms. Adenosine, a putative metabolic vasodilator, preferentially dilates downstream coronary microvessels, whereas the shear-sensitive mechanism is detected predominantly in upstream larger microvessels. However, the interaction of these mechanisms and the significance of the heterogeneous vascular responsiveness in flow regulation have not been evaluated. These tasks cannot be performed experimentally because of several confounding factors that cannot be separated. Therefore, the present study employed a data-based modeling approach to investigate the role of response heterogeneity in a coronary vascular network and to test the hypothesis that shear-sensitive mechanism or the myogenic mechanisms will enhance the vascular sensitivity to adenosine due to the heterogeneity of the vascular responsiveness. We obtained necessary data and developed empirical models for the responsiveness of single vessels to pressure, shear stress, and adenosine. With the single-vessel models, a network model was established based on the branching pattern of coronary microvessels, mass balance, and fluid mechanics laws. Model simulation predicted an enhanced vascular response to adenosine in the network. Such an enhancement is caused by the heterogeneous vascular response to adenosine and the predominant flow-induced dilation in the large arterioles. Preferential dilation of the downstream small arterioles to adenosine initiates an increase in flow and a decrease in pressure at upstream vessels. The increased flow activates the shear-sensitive mechanism of the upstream large arterioles and further enhances the flow. This hemodynamic interaction contributes up to approximately 20% of the adenosine-induced flow increase and also reduces the adenosine-induced pressure drop. In contrast to the shear-sensitive mechanism, myogenic response contributes relatively little to the vascular response to adenosine. These results suggest that various vascular regulation mechanisms and the response heterogeneity of vessels of different sizes may act in an integrative fashion for the optimal control of microvascular perfusion.

Adenosine↗
PubMed1997

Endothelial ATP-sensitive potassium channels mediate coronary microvascular dilation to hyperosmolarity.

Coronary arterial occlusion has been shown to increase osmolarity in the myocardial interstitium. Intracoronary injection of hyperosmolar solutions reduces coronary vascular resistance. However, the response of coronary microvessels to an abluminal increase in osmolarity is unclear, and the underlying mechanism for its vasomotor regulation has not been elucidated. In this regard, porcine coronary arterioles (81 +/- 2 microns) were isolated, cannulated, and pressurized for in vitro study. Hyperosmolarity (300-345 mosM) was produced by adding D-glucose or D-sucrose to the extravascular solution. After the arterioles developed a stable vascular tone, a graded vasodilation was observed when glucose or sucrose was incrementally administered. This hyperosmotic vasodilation was abolished after endothelial removal. Intraluminal administration of KCl (80 mM) or the ATP-sensitive potassium (KATP)-channel inhibitor glibenclamide (1 microM) to the intact vessels significantly attenuated the hyperosmotic vasodilation. Inhibition of inward rectifier potassium channels by a low concentration of BaCl2 (10 microM) did not affect vasodilation. However, a high concentration of BaCl2 (100 microM), which has been reported to inhibit KATP channels, attenuated the hyperosmotic vasodilation. Iberiotoxin (100 nM), a calcium-activated potassium (KCa)-channel inhibitor had no effect on hyperosmolarity-induced vasodilation. Inhibition of the synthesis of endothelial nitric oxide, prostaglandins, and arachidonic acid metabolites from cytochrome P-450 had no effect on hyperosmotic vasodilation. Furthermore, inhibition of vascular smooth muscle KATP channels and the large- and small-conductance KCa channels by extraluminal administration of glibenclamide, iberiotoxin, and apamin, respectively, did not alter vasodilation in response to hyperosmolarity. These results indicate that dilation of coronary arterioles in response to hyperosmotic stimulation requires an intact endothelium. However, the response is independent of the release of nitric oxide, prostaglandins, or cytochrome P-450-related endothelium-derived hyperpolarizing factor and is not a result of activation of KATP and KCa channels in vascular smooth muscle. It is suggested that the opening of KATP channels in vascular endothelium and subsequent hyperpolarization of that cell type mediate coronary microvascular dilation in response to hyperosmolarity.

Animals↗
PubMed1996

Downregulation of endothelial constitutive nitric oxide synthase expression by lipopolysaccharide.

Lipopolysaccharide (LPS), a causal agent of sepsis, has been shown to induce systemic nitric oxide (NO) synthesis through complex mechanisms. However, the effect of LPS on endothelial cells is incompletely understood. To investigate the mechanism by which LPS influences the release of NO from endothelial cells, the effect of this compound on endothelial constitutive nitric oxide synthase (ecNOS) was studied in cultured bovine coronary venular endothelial cells. Western and Northern analyses showed that LPS decreased ecNOS expression at the protein and mRNA levels in a time-dependent and dose-responsive manner. Concurrent treatment of the endothelial cells with LPS and a transcription inhibitor, actinomycin D, resulted in decreased ecNOS mRNA within 8 hours. In contrast, treatment with actinomycin D had only a relatively insignificant effect on the ecNOS transcript level. This result suggests that the reduction of ecNOS by LPS resulted from an increased degradation rate of its transcript.

Animals↗
PubMed1996

Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus.

Preceding and following each gene of respiratory syncytial virus (RSV) are two conserved sequences, the gene-start (GS) and gene-end (GE) motifs, respectively, which are thought to be transcription signals. The functions and boundaries of these signals and the process of sequential transcription were analyzed with cDNA-encoded RNA analogs (minigenomes) of nonsegmented negative-sense RSV genomic RNA. Two minigenomes were used. The monocistronic RSV-CAT minigenome consists of the chloramphenicol acetyltransferase (CAT) translational open reading frame (ORF) bordered by the GS and GE motifs and flanked by the 3' leader and 5' trailer extragenic regions of genomic RNA. The dicistronic RSV-CAT-LUC minigenome is a derivative of RSV-CAT into which the ORF for luciferase (LUC), bordered by GS and GE motifs, was inserted downstream of the CAT gene with an intergenic region positioned between the two genes. Each minigenome was synthesized in vitro and transfected into RSV-infected cells, where it was replicated and transcribed to yield the predicted polyadenylated subgenomic mRNA(s). The only RSV sequences required for efficient transcription and RNA replication were the 44-nucleotide 3' leader region, the last 40 nucleotides of the 5' trailer region, and the 9- to 10-nucleotide GS and 12- to 13-nucleotide GE motifs. The GS and GE motifs functioned as self-contained, transportable transcription signals which could be attached to foreign sequences to direct their transcription into subgenomic mRNAs. Removal of the GS motif greatly reduced transcription of its gene, and the requirement for this element was particularly strict for the gene in the downstream position. Ablation of the promoter-proximal GS signal was not associated with increased antigenome synthesis. Consistent with its proposed role in termination and polyadenylation, removal of the CAT GE signal in RSV-CAT resulted in the synthesis of a nonpolyadenylated CAT mRNA, and in RSV-CAT-LUC the same mutation resulted in readthrough transcription to yield a dicistronic CAT-LUC mRNA. The latter result showed that a downstream GS signal is not recognized for reinitiation by the polymerase if it is already engaged in mRNA synthesis; instead, it is recognized only if the polymerase first terminates transcription at an upstream termination signal. This result also showed that ongoing transcription did not open the downstream LUC gene for internal polymerase entry. Removal of both the GS and GE signals of the upstream CAT gene in RSV-CAT-LUC silenced expression of both genes, confirming that independent polymerase entry at an internal gene is insignificant. Remarkably, whereas both genes were silent when the CAT GS and GE signals were both absent, restoration of the CAT GE signal alone restored a significant level (approximately 10 to 12% of the wild-type level) of synthesis of both subgenomic mRNAs. This analysis identified a component of sequential transcription that was independent of the promoter-proximal GS signal and appeared to involve readthrough from the leader region.

Animals↗
PubMed1996

The structurally diverse intergenic regions of respiratory syncytial virus do not modulate sequential transcription by a dicistronic minigenome.

The first nine genes of respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus, are separated by intergenic regions which range in size from 1 to 52 nucleotides for strain A2 and lack obvious consensus elements except that each ends in an A (genome sense). Their significance for gene expression was investigated by using RSV-CAT-LUC RNA, a helper-dependent cDNA-encoded dicistronic analog of RSV genomic RNA in which the viral genes were replaced by a negative-sense copy of the translational open reading frame (ORF) encoding chloramphenicol acetyltransferase (CAT) as the upstream, leader-proximal gene and that encoding luciferase (LUC) as the downstream gene. These foreign ORFs were flanked by the RSV gene-start (GS) and gene-end (GE) transcription signals and separated by the naturally occurring G/F intergenic region. The RSV-CAT-LUC minigenome was synthesized in vitro and transfected into RSV-infected cells, and synthesis of the CAT and LUC mRNAs was monitored by enzyme assay and Northern (RNA) blot hybridization. Surprisingly, substitution of each of the other naturally occurring RSV intergenic regions in turn did not significantly alter the absolute or relative amounts of the two mRNAs. Substitution of a nonnatural 10-nucleotide intergenic region, or elimination of the intergenic region altogether, also had little effect on the level of expression of the two genes. Four of the minigenome variants containing naturally occurring intergenic regions were modified further by replacing part of the LUC ORF with a second copy of the CAT ORF, so that each of the two mRNAs would hybridize equally with a CAT-specific probe and their relative molar amounts could be determined. The level of expression of the downstream gene was 0.30 to 0.36 that of the upstream one. This determined the magnitude of RSV transcriptional polarity across a gene pair and confirmed that this value was very similar among the various intergenic regions. Minigenome transcription also yielded a CAT-LUC readthrough mRNA at a level 0.10 to 0.13 that of the LUC mRNA. In summary, the structurally diverse RSV intergenic regions do not appear to play a role in modulating RSV gene expression.

Base Sequence↗