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Product Focus: SV Total RNA Isolation System---Access RT-PCR System

HMG-1 as a late mediator of endotoxin lethality in mice.

Endotoxin (lipopolysaccharide or LPS), a cell-wall constituent of Gram-negative bacteria, stimulates the release of cytokines such as TNF and IL-1beta from macrophages. Bacteria do not directly cause tissue injury and lethality, but instead these host cytokines mediate the damage. Antagonists to TNF and IL-1 have been shown limited efficacy in clinical trials, possibly because these cytokines are early mediators in pathogenesis. Studies by this group have identified a potential late mediator of endotoxin lethality as high mobility group-1 (HMG-1) protein, which was found to be released by cultured macrophages more than 8 hours following stimulation with endotoxin, TNF, or IL-1. Total RNA was isolated from the endotoxin-treated macrophages using the SV Total RNA Isolation System^(a) (Cat.# Z3100), and subsequent analysis using the Access RT-PCR System^(b) (Cat.# A1250) demonstrated that HMG-1 mRNA levels were unaffected by LPS treatment, indicating that HMG-1 release is unlikely to be linked to increased gene transcription. Mice treated with endotoxin showed increased serum levels of HMG-1 from 8 to 32 hours after exposure. Administration of antibodies to HMG-1 attenuated the endotoxin lethality in mice, and administration of HMG-1 itself was also lethal. Thus the HMG-1 protein may warrant further investigation as a therapeutic target for endotoxemia.

Wang, H.^1,3, Bloom, O.³, Zhang, M.3, Vishnubhakat, J.M.³, Ombrellino, M.^2,3, Che, J.³, Frazier, A.^2,3, Yang, H. ³, Ivanova, S.³, Borovikova, L.³, Manogue, K.R.³, Faist, E.⁴, Abraham, E.⁵, Andersson, J.⁶, Andersson, U.⁷, Molina, P.E.², Abumrad N.N.², Sama, A.¹, and Tracey, K.J.^2,3 (1999) Science 285, 248.

¹Department of Emergency Medicine and ²Department of Surgery, North Shore University Hospital – New York University School of Medicine, Manhasset, NY 11030 USA. ³The Picower Institute for Medical Research, Manhasset, NY   11030 USA. ⁴Department of Surgery, Klinicum Grosshadern, Ludwig-Maximilians University, Munich, Germany. ⁵Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Science Center, Denver, CO   80262 USA. ⁶Department of Infectious Disease, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden. ⁷Department of Rheumatology, Astrid Lindgren’s Children’s Hospital, Karlinska Institute, Stockholm, Sweden.

Product Focus: Dual-Luciferase^® Reporter Assay System---Beta-Galactosidase Enzyme Assay System---pRL-null Vector

Light-dependent sequestration of TIMELESS by CRYPTOCHROME.

Most organisms display an endogenous timekeeping mechanism, or circadian clock, which consists of negative feedback loops of gene regulation that facilitate adaptation to cycles of light and darkness. In Drosophila, as well as other organisms, several of the molecules involved in sustaining this circadian clock have been identified. A gene product required for circadian photoreception has recently been identified in Drosophila, and termed crytochrome (CRY). These researchers investigated whether CRY could interact directly with the core clock proteins PERIOD (per) and TIMELESS (tim). A Drosophila cell line was transiently transfected with a firefly luciferase reporter construct under control of the tim promoter, in conjunction with different combinations of constructs expressing clk, per, tim, and cry. Data was normalized to a cotransfected reporter plasmid, either the pRL-null Vector^(c,d) (Cat.# E2271), a Renilla luciferase vector, or a beta-galactosidase expression vector, and the resulting activities measured using either the Dual-Luciferase^® Reporter Assay System^(e,f) (Cat.# E1910) or the Beta-Galactosidase Enzyme Assay System^(c) (Cat.# E2000). These transfection studies, along with coimmuneprecipitation assays, a yeast two-hybrid assay, and immunolocalization studies, show that CRY can block the function of PER/TIM heterodimeric complexes in a light-dependent fashion. In addition, PER/TIM and CRY influence the subcellular distribution of these protein complexes. Thus, CRY acts as a circadian photoreceptor by directly interacting with the core protein components of the circadian clock.

Ceriani, M.F.¹, Darlington, T.K.¹, Staknis, D.², Mas, P.¹, Petti, A.A.², Weitz, C.J.², and Kay, S.A.¹ (1999) Science 285, 553.

¹Department of Cell Biology and NSF Center for Biological Timing, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 USA. ²Department of Neurobiology, Harvard Medical School, Boston, MA  02115 USA.

Product Focus: TNT^® Coupled Reticulocyte Lysate System

A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA-editing enzyme.

The apolipoprotein B (apoB) mRNA is edited by deamination of a specific cytidine to form uridine. This deamination changes a glutamine codon (CAA) to a translation termination codon (UAA) and results in the formation of a truncated apoB protein (apoB48). Expression of the apolipoprotein B mRNA-editing enzyme (APOBEC-1) results in dysplasia and carcinoma in mouse and rabbit liver. Using a modified differential display technique, these researchers have identified a novel mRNA target (NAT1) that is edited at multiple sites in these livers by the APOBEC-1 enzyme. The NAT1 cDNA was expressed in vitro using the TNT^® Coupled Reticulocyte Lysate System^(e,g,h) (Cat.# L4600), where a protein larger than anticipated was synthesized, suggesting that an upstream non-ATG start codon was utilized for translation initiation. Site-directed mutagenesis confirmed that a GTG codon was utilized as the start codon for translation, demonstrating that non-ATG start codons may be utilized in this in vitro expression system. NAT1 was found to be ubiquitously expressed and highly conserved among species, with homology to the carboxy-terminal portion of the eukaryotic translation initiation factor (eIF) 4G, which binds to eIF4A and eIF4E to from eIF4F. NAT1 binds to eIF4A, but not eIF4E, and was found to inhibit both cap-dependent and cap-independent translation.

Yamanaka, S.¹, Poksay, K.S.², Arnold, K.S^.2, and Innerarity, T.L.^2-4 (1997) Genes Dev. 11, 321.

¹Department of Pharmacology, Osaka City University Medical School, Osaka 545 Japan. ²Gladstone Institute of Cardiovascular Disease, ³Cardiovascular Research Institute, and ⁴Department of Pathology, University of California, San Fransisco, CA  94141-9100 USA.

Product Focus: CheckMate™ Mammalian Two-Hybrid System

CREB binding protein coordinates the function of multiple transcription factors including nuclear factor I to regulate phosphoenolpyruvate carboxykinase (GTP) gene transcription.

Nuclear factor 1 (NF1) binds to a region of the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter and inhibits the induction of transcription from the promoter caused by protein kinase A. The effects of NF1 on the PEPCK promoter were observed even in the absence of the NF1 binding site, suggesting the possibility of other binding sites on the promoter or an interaction of NF1 with a transcriptional co-activator. The authors utilized the CheckMate™ Mammalian Two-Hybrid System^(c,d,h,i) (Cat.# E2440) to demonstrate the direct interaction between the transactivation domain of NF1-C and the CREB binding domain of the CREB-binding protein (CBP). The inhibitory effect of NF1 on transcription of the PEPCK gene induced by PKA appears to be the result of an interaction between NF1 and the CREB-binding protein in which NF1 competes with CREB for binding to the CREB-binding site on CBP. These results also suggest that CBP acts to coordinate the action of multiple transcription factors known to regulate transcription of the PEPCK gene.

Leahy, P.¹, Crawford, D.R.¹, Grossman, G.¹, Gronostajski, R.M.², and Hanson, R.W.¹ (1999) J. Biol. Chem. 274, 8813.

¹Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH  44106 USA, and ²Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH  44106 USA.

Product Focus: Tfx™-10 Reagent

Antisense oligonucleotides directed against the viral RNA polymerase gene enhance survival of mice infected with influenza A.

Influenza A viruses can cause acute respiratory disease. The influenza A viral RNA polymerase is encoded by three genes (PB1, PB2, and PA), two of which (PB1 and PB2) function cooperatively in cap-dependent mRNA transcription. The authors tested the ability of liposome-mediated delivery of antisense phosphorothioate oligonucleotides directed against the translation initiation codons of the viral PB2 or PA genes to enhance the survival of mice infected with the influenza A virus. Intravenous administration of the PB2 antisense oligonucleotides in a complex with the cationic liposome, Tfx™-10 Reagent^(j) (Cat.# E2381), significantly prolonged the survival time and rate of the influenze A-infected mice. The Tfx™-10-encapsulated PB2 antisense oligonucleotides also inhibited viral growth in lung tissue and reduced pulmonary consolidations. The size distribution of the lipid complex was determined by quasi-elastic light scattering and the mean diameter of the liposome was <50nm for Tfx™-10. The antiviral effect was found to correlate with a decrease in PB2 mRNA levels, a reduction in virus titer in the lung tissues, and a reduction in pulmonary damage.

Mizuta, T.^1,3, Fujiwara, M.^1,3, Hatta, T.², Abe, T.², Miyano-Kurosaki, N.², Shigeta, S.³, Yokota, T.^1,3, and Takaku, H.² (1999) Nat. Biotech. 17, 583.

¹Rational Drug Design Laboratories, 4-1-1 Misato, Matsukawa-Machi, Fukushima 960-1242 Japan. ²Department of Industrial Chemistry, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016 Japan. ³Department of Microbiology, Fukushima University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295 Japan.

Product Focus: Pfu DNA Polymerase

Functional effect of deletion and mutation of the Escherichia coli ribosomal RNA and tRNA pseudouridine synthase RluA.

The Escherichia coli gene rluA encodes for the pseudouridine synthase enzyme RluA that forms pseudouridine 746 in 23S rRNA and pseudouridine 32 in tRNA. Plasmid rescue of rluA- bacterial strains using an rluA gene containing asparagine or threonine replacements for the highly conserved aspartic acid 64 demonstrated that neither mutant could form 23S RNA pseudouridine 746 or tRNA pseudouridine 32 in vivo, illustrating the importance of this conserved aspartate in the enzyme-catalyzed formation of both pseudouridines. No difference was observed in the exponential growth rate between the wildtype and mutants, either in rich medium or minimal medium at various temperatures, but when both strains were grown together, the wildtype strain exhibited a strong selective advantage. The authors used Pfu DNA Polymerase^(k) (Cat.# M7741) in a megaprimer PCR mutagenesis procedure to generate the asparagine and threonine point mutations. DNA sequencing of the isolated plasmids verified that the expected mutations had been produced at the desired site.

Raychaudhuri, S.¹, Niu, L.², Conrad, J.¹, Lane, B.G.³, and Ofengand, J.¹ (1999) J. Biol. Chem. 274, 18880.

¹Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33101 USA. ²Program in Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 USA. ³Department of Biochemisty, University of Toronto, Toronto, Ontario,   M5S 1A8.

^(a)Patent Pending.

^(b)The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.

^(c)Certain applications of this product may require licenses from others.

^(d)The cDNA encoding luciferase from Renilla reniformis is covered by U.S. Pat. No. 5,292,658 assigned to the University of Georgia Research Foundation, Inc., and sublicensed from SeaLite Sciences, Inc., Norcross, GA. The pRL family of Renilla luciferase cDNA vectors is for research use only. Commercial manufacture would require a license from SeaLite Sciences, Inc.

^(e)U.S. Pat. Nos. 5,283,179, 5,641,641, 5,650,289, Australian Pat. No. 649289, and European Pat. No. 0 553 234 have been issued to Promega Corporation for a firefly luciferase assay method, which affords greater light output with improved kinetics as compared to the conventional assay. Certain applications of this product may require licenses from others.

^(f)U.S. Pat. No. 5,744,320 has been issued to Promega Corporation for quenching reagents and assays for enzyme-mediated luminescence.

^(g)U.S. Pat. Nos. 5,324,637, 5,492,817 and 5,665,563, European Pat. No. 0 566 714 B1, Australian Pat. No. 660329 and Japanese Pat. No. 2904583 have been issued to Promega Corporation for coupled transcription/translation systems that use RNA polymerases and eukaryotic lysates.

^(h)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673.

⁽ⁱ⁾The CMV promoter and its use are covered under U.S. Pat. Nos. 5,168,062 and 5,385,839 owned by the University of Iowa Research Foundation, Iowa City, Iowa, and licensed FOR RESEARCH USE ONLY. Commercial users must obtain a license to these patents directly from the University of Iowa Research Foundation.

^(j)The cationic lipid component of the Tfx^TM Reagents is covered by U.S. Pat. Nos. 5,527,928, 5,744,625 and pending foreign patents.