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| 1. |
Rahmani, F., Hummel, M., Schuurmans, J., Wiese-Klinkenberg, A., Smeekens, S. and Hanson, J.
(2009)
Sucrose control of translation mediated by an upstream open reading frame-encoded peptide.
Plant Physiol.
150
,
1356–1367
.
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Notes:
The authors were wanted to study the upstream open reading frame 2 (uORF2) of the 5’ leader of bZIP11 mRNA, which has a role in sucrose regulation. The whole 5’ leader fragment of bZIP11 was subcloned into the pALTER® Vector and amino acid substitutions were introduced using the Altered Sites® II in vitro Mutagenesis System. The pGEM®-T Easy Vector was used to clone two PCR fragments that were then subcloned using restriction enzymes to create a fusion of uORF2 to a different 5’ leader. Arabidopsis seedlings were transformed via particle bombardment. 20mg of plant tissue was ground in Passive Lysis Buffer, centrifuged, and 20µl of the supernatant was assessed for reporter gene expression using the Dual-Luciferase® Reporter Assay System.
(0004023) |
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Products: Altered Sites® II in vitro Mutagenesis System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pALTER®-1 Vector | pGEM®-T Vector | pGEM®-T Vector System I | pGEM®-T Vector System II |
| 2. |
Cameron, A.D., Volar, M., Bannister, L.A. and Redfield, R.J.
(2008)
RNA secondary structure regulates the translation of sxy and competence development in Haemophilus influenzae.
Nucleic Acids Res.
36
,
10–20
.
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Notes:
The authors examined the role of sxy expression in hypercompetence of Haemophilus influenza. The 1.8kb sxy gene was subcloned into the pALTER®-1 Vector and point mutations made using the Altered Sites® II in vitro Mutagenesis System. The mutated gene was sequenced and subcloned back into the original plasmid. The wildtype and mutant sxy genes were transcribed, dephosphorylated and labeled with 32P ATP. The end-labeled RNAs were then subjected to S1 nuclease mapping. The E. coli S30 Extract System for Linear Templates was used with the wildtype and mutant sxy constructs and the expression levels of the expressed protein measured by spot blotting.
(0003995) |
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Products: E. coli S30 Extract System for Linear Templates | Altered Sites® II in vitro Mutagenesis System |
| 3. |
Rabadan-Diehl, C., Martínez, A., Volpi, S., Subburaju, S. and Aguilera, G.
(2007)
Inhibition of vasopressin V1b receptor translation by upstream open reading frames in the 5'-untranslated region.
J. Neuroendocrinol.
19
,
309-319
.
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Notes:
The authors studied the rat VP V1b receptor (V1bR) gene including the 826 base 5´ UTR, which has five ORFs upstream (uORF) of the V1bR protein start codon, to examine the effect of the upstream peptides on V1bR translation. The V1bR gene was cloned into the pALTER®-MAX Vector, and substitution mutations were created in the uORF translation initiation codons with the Altered Sites® II in vitro Mutagenesis System. One microgram of the linearized pALTER®- V1bR construct was transcribed using the Riboprobe® System and translated using Wheat Germ Extract with or without 35S-methionine. The unlabeled protein was analyzed by Western blot. The radiolabeled protein was spun through a 3% sucrose cushion, precipitated and separated by SDS-PAGE. For a possible membrane-targeted protein, the uORF1 was transcribed in vitro and then translated using Rabbit Reticulocyte Lysate, Canine Pancreatic Microsomal Membanes and 35S-methionine. The proteins were spun through a 3% sucrose cushion and then run on a 15% SDS-PAGE gel to determine membrane presence.
(0003749) |
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Products: Altered Sites® II Mammalian in vitro Mutagenesis System | Canine Pancreatic Microsomal Membranes | pALTER®-MAX Vector | Rabbit Reticulocyte Lysate System, Nuclease Treated | Riboprobe® System—SP6 | Riboprobe® System—T3 | Riboprobe® System—T7 | Wheat Germ Extract |
| 4. |
Ellison, T.I., Eckert, R.L. and MacDonald, P.N.
(2007)
Evidence for 1,25-dihydroxyvitamin D3-independent transactivation by the vitamin D receptor: uncoupling the receptor and ligand in keratinocytes.
J. Biol. Chem.
282
,
10953–10962
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Notes:
While the absence of the Vitamin D receptor (VDR) has profound effects in skin cells, mutation of 25-hydroxyvitamin D 1α-hydroxylase (24OHase), the enzyme required for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone biosynthesis, has little effect on the skin. To determine how VDR may transactivate independent of the 1,25(OH)2D3 ligand, the human 24-hydroxylase promoter was amplified from MCF-7 genomic DNA, digested with XhoI and HindIII and inserted into the pGL3-Basic Vector. Mutations in the proximal and distal vitamin D response elements in the human 24-hydroxylase promoter were introduced using the GeneEditor™ Site-Directed Mutagenesis System. HaCaT cells, primary human fibroblasts or primary human keratinocytes were seeded at a density of 3.2 × 104 cells/well in 12-well plates and transiently transfected with reporter constructs. After 18 hours, the cells were exposed to 1,25(OH)2D3, 9-cis-retinoic acid, ethanol vehicle, or no additive and harvested 24 hours later. The luciferase activity of the cell lysates was measured using the Dual-Luciferase® Reporter Assay System. Five micrograms of RNA purified from mouse keratinocyte and fibroblast cultures was reverse transcribed and amplified for the 24OHase transcripts using the PCR Master Mix. The products were analyzed on ethidium bromide-stained 2% agarose gels.
(0003695) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | GeneEditor™ in vitro Site-Directed Mutagenesis System | PCR Master Mix | pGL3-Basic Vector |
| 5. |
Tseng, G.N., Sonawane, K.D., Zhang, M., Liu, J. and Guy, H.R.
(2007)
Probing the outer mouth structure of the hERG channel with peptide toxin footprinting and molecular modeling.
Biophys. J.
92
,
3524–3540
.
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Notes:
The Altered Sites® II Mammalian in vitro Mutagenesis System was used for cysteine scanning mutagenesis of the residues lining the outer vestibule of the wildtype human ether a-go-go related gene (hERG) channel. A total of 51 mutants were created and tested for hERG channel activity with 19 perturbed channel function, and 32 resulted in normal channel function.
(0003577) |
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Products: Altered Sites® II Mammalian in vitro Mutagenesis System |
| 6. |
Qi, J. and Forgac, M.
(2007)
Cellular environment is important in controlling V-ATPase dissociation and its dependence on activity.
J. Biol. Chem.
282
,
24743–24751
.
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Notes:
The role of V0 subunit isoform and its cellular environment in the disassociation of vacuolar (H+)-ATPases (V-ATPases) in yeast was explored in this paper. An EcoRI-BamHI fragment of HA-Stv1p, a hemagglutanin-tagged Golgi-targeting subunit of V-ATPase, had two different mutations introduced (R795K and R795Q) using the Altered Sites® II in vitro Mutagenesis System. The mutant vectors were then transformed into yeast strain MM112 and used in studies with vacuolar protein sorting (vps) mutants.
(0003693) |
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Products: Altered Sites® II in vitro Mutagenesis System |
| 7. |
Ruan, X., Bhattacharjee, H. and Rosen, B.P.
(2006)
Cys-113 and Cys-422 form a high affinity metalloid binding site in the ArsA ATPase.
J. Biol. Chem.
281
,
9925–9934
.
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Notes:
To examine which amino acids of ArsA may be important for metalloid binding and transport, substitution mutations were introduced in the arsA gene using the Altered Sites® II in vitro Mutagenesis System. Plasmids were purified using the Wizard® Plus Minipreps DNA Purification System and subjected to restriction enzyme digestion.
(0003516) |
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Products: Altered Sites® II in vitro Mutagenesis System | Wizard® Plus Minipreps DNA Purification System |
| 8. |
Nasu, M., Ito, M., Ishida, Y., Numa, N., Komaru, K., Nomura, S. and Oda, K.
(2006)
Aberrant interchain disulfide bridge of tissue-nonspecific alkaline phosphatase with an Arg433-->Cys substitution associated with severe hypophosphatasia.
FEBS J.
273
,
5612-5624
.
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Notes:
The wildtype tissue-nonspecific alkaline phosphatase (TNSALP) gene was cloned into the pALTER®-MAX Vector, and codons for specific arginines were mutated using the Altered Sites® II Mammalian in vitro Mutagenesis System. The mutant constructs were subcloned and used for either transient or stable transfection experiments.
(0003747) |
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Products: Altered Sites® II Mammalian in vitro Mutagenesis System | pALTER®-MAX Vector |
| 9. |
Zhang, X.Q., Ahlers, B.A., Tucker, A.L., Song, J., Wang, J., Moorman, J.R., Mounsey, J.P., Carl, L.L., Rothblum, L.I. and Cheung, J.Y.
(2006)
Phospholemman inhibition of the cardiac Na /Ca2+ exchanger. Role of phosphorylation.
J. Biol. Chem.
281
,
7784–7792
.
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Notes:
Phospholemman (PLM), a membrane phosphoprotein, was shown to inhibit the cardiac
Na+/Ca2+ exchanger (NCX1) and is a substrate for PKA and PKC. The PLM coding region was cloned into the pALTER®-1 Vector and serine substitution mutations generated using the Altered Sites® II in vitro Mutagenesis System. The substitutions were verified by DNA sequencing and subcloned into a mammalian expression vector. These mutant constructs were transfected and tested for changes in current by patch clamp testing.
(0003517) |
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Products: Altered Sites® II in vitro Mutagenesis System |
| 10. |
Dai, E., Viswanathan, K., Sun, Y.M., Li, X., Liu, L.Y., Togonu-Bickersteth, B., Richardson, J., Macaulay, C., Nash, P., Turner, P., Nazarian, S.H., Moyer, R., McFadden, G. and Lucas, A.R.
(2006)
Identification of myxomaviral serpin reactive site loop sequences that regulate innate immune responses.
J. Biol. Chem.
281
,
8041–8050
.
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Notes:
To determine which amino acids in Serp-1, a myxomaviral serine proteinase inhibitor, were important for its anti-inflammatory activity, the complete sequence of Serp-1 was subjected to mutation by several techniques including the Altered Sites® II in vitro Mutagenesis System. The products were used to construct vaccinia virus mutants and reactive center loop (RCL) chimeras for infection in mice.
(0003518) |
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Products: Altered Sites® II in vitro Mutagenesis System |
| 11. |
Komaru, K., Ishida, Y., Amaya, Y., Goseki-Sone, M., Orimo, H. and Oda, K.
(2005)
Novel aggregate formation of a frame-shift mutant protein of tissue-nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C-terminal extension. Retarded secretion and proteasomal degradation.
FEBS Lett.
272
,
1704–1717
.
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Notes:
To examine further the phenotype of a known tissue-nonspecific alkaline phosphatase (TNSALP) frameshift mutant, the cDNAs for wildtype TNSALP and TNSALP (1559delT) were subcloned into pALTER®-MAX Vector. Three cysteine residues were replaced with serines using the Altered Sites® II Mammalian Mutagenesis System. The substitution mutations were confirmed by DNA sequencing and the plasmids transfected into CHO cells. The size of the TNSALP (1559delT) product was compared to wildtype TNSALP using the TNT® T7 Coupled Reticulocyte Lysate System and [35S]methionine ⁄ cysteine with or without Canine Pancreatic Microsomal Membranes. The proteins were analyzed using SDS-PAGE and fluorography.
(0003519) |
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Products: Altered Sites® II Mammalian in vitro Mutagenesis System | Canine Pancreatic Microsomal Membranes | pALTER®-MAX Vector | TNT® T7 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System, Trial Size |
| 12. |
Baba, T., Shimizy, T., Suzuki, Y-i., Ogawara, M., Isono, K-i., Kosek, H., Kurosawa, H. and Shirasawa, T.
(2005)
Estrogen, insulin, and dietary signals cooperatively regulate longevity signals to enhance resistance to oxidative stress in mice
J. Biol. Chem.
280
,
16417-16426
.
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Notes:
The authors of this study created a mouse model of a C. elegans insulin receptor mutation. The Altered Sites® II in vitro Mutagenesis System was used to create a transgenic mouse containing a Pro1195Leu substitution in the insulin receptor. Mice homozygous for this mutation died as neonates. Heterozygous mice grew normally, but lifespan was increased by 33% in females and 18% in males. Manganese superoxide dismutase was upregulated in both male and female heterozygotes. Estrogen further increased longevity of the mutant mice as did caloric restriction.
(0003669) |
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Products: Altered Sites® II in vitro Mutagenesis System |
| 13. |
Tiainen, M., Vaahtomeri, K., Ylikorkala, A., and Makela, T.P.
(2002)
Growth arrest by LKB tumor suppressor: induction of p21^WAF1/CIP1.
Hum. Mol. Genet.
11
,
1497-1504
.
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Notes:
In this paper, Promega's GeneEditor™ in vitro Site-Directed Mutagenesis System was used to change amino acids important for the nuclear localization signal and the kinase domain of the LBK1 tumor suppressor. Mutants were tested for their ability to arrest the cell cycle as well as for cellular localization. The pCI-neo Mammalian Expression Vector and the Dual Luciferase® Reporter Assay System were also used during the course of this study.
(0002494) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System |
| 14. |
Zou, Z., DiRusso, C.C., Ctrnacta, V., and Black, P.N.
(2002)
Fatty acid transport in Saccharomyces cerevisiae.
J. Biol. Chem.
277
,
31062-31071
.
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Notes:
The authors used Promega's Altered Sites® in vitro Mutagenesis System with the vector pALTER-1 (pSELECT) to perform single amino acid substitutions in the FAT1 gene of Saccharomyces cerevisiae. This gene is involved in fatty acid transport as well as intrinsic fatty acyl-CoA synthetase activity. The mutations were targeted in three regions: the ATP/AMP signature motif, the FATP/VLACS signature motif and the C-terminal amino acid. The signature regions were conserved in both mammalian and non-mammalian systems. The effect of amino acid changes in the highly conserved regions on growth rate, fatty acid import and CoA synthetase activity was evaluated.
(0002561) |
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Products: Altered Sites® II in vitro Mutagenesis System |
| 15. |
Kraichely, D.M., Collins, J.J. III, DeLisle, R.K. , MacDonald, P.N.
(1999)
The autonomous transactivation domain in helix H3 of the vitamin D receptor is required for transactivation and coactivator interaction.
J. Biol. Chem.
274
,
14352-14358
.
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Notes:
The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce a variety of different point mutations into the vitamin D receptor. The mutant proteins were also expressed in vitro with the TNT® Coupled Reticulocyte Lysate System and used for in vitro protein interaction assays.
(0000889) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System | TNT® SP6 Coupled Reticulocyte Lysate System | TNT® SP6 Coupled Reticulocyte Lysate System, Trial Size | TNT® T3 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System, Trial Size | TNT® T7/SP6 Coupled Reticulocyte Lysate System | TNT® T7/T3 Coupled Reticulocyte Lysate System |
| 16. |
Guo, S., Rena, G., Cichy, S., He, X., Cohen, P., Unterman, T.
(1999)
Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence.
J. Biol. Chem.
274
,
17184-17192
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Notes:
The pALTER®-MAX Vector was used to create mutations causing three separate amino acid substitutions on the FKHR cDNA. The Reverse Transcriptase System was used for RT-PCR.
(0001088) |
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Products: Altered Sites® II Mammalian in vitro Mutagenesis System | pALTER®-MAX Vector | Reverse Transcription System |
| 17. |
Calvo, P.A., Frank, D.W., Bieler, B.M., Berson, J.F., Marks, M.S.
(1999)
A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery
J. Biol. Chem.
274
,
12780-12789
.
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Notes:
The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce as many as three point mutations into the same cDNA. Two deletion mutants were also introduced into the tyrosinase cDNA by the insertion of premature stop codons. The mutations were either performed in the pCI Mammalian Expression Vector or the vector pCDM8.1.
(0001382) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System | pCI-neo Mammalian Expression Vector |
| 18. |
Guidotti, L.G., Rochford, R., Chung, J., Shapiro, M., Purcell, R., Chisari, F.V.
(1999)
Viral clearance without destruction of infected cells during acute HBV infection.
Science
284
,
825-829
.
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Notes:
The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce a novel EcoR I site into an HBV envelope protein cDNA. No indication of which plasmid was used for the mutagenesis.
(0001084) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System |
| 19. |
Chen, D., Man, H., Hong, H., Koh, S.S., Huang, S.-M., Schurter, B.T., Aswad, D.W., Stallcup, M.R.
(1999)
Regulation of transciptionby a protein methyltransferase
Science
284
,
2174-2177
.
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Notes:
The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to make a three amino acid change: VLD to AAA.
(0001367) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System |
| 20. |
Ma, T., Zou, N., Lin, B.Y., Chow, L.T., Harper, J.W.
(1999)
Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication.
Proc. Natl. Acad. Sci. U S A
96
,
382-387
.
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Notes:
The GeneEditor™ Site-Directed Mutagenesis System was used to produce a deletion of the RXL cyclin E association site in the E1 protein. The mutations were performed in a mammalian expression vector.
(0000761) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System |
| 21. |
Procopio, W.N., Pelavin, P.I., Lee, W.M.F., Yeilding, N.M.
(1999)
Angiopoietin-1 and -2 coiled coil domains mediate distinct homo-oligomerization patterns, but fibrinogen-like domains mediate ligand activity.
J. Biol. Chem.
274
,
30196-30201
.
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Notes:
The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce an Xho I site into one cDNA so that a chimeric protein could be created.
(0000008) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System |
| 22. |
Zhou, Z., Gong, Q., January, C.T.
(1999)
Correction of defective protein trafficking of a mutant HERG potassium channel in human long QT syndrome: Pharmacological and temperature effects.
J. Biol. Chem.
274
,
31123-31126
.
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Notes:
The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to produce the N470D mutation in the LQT2 cDNA. The mutation was produced in an undefined mammalian expression vector.
(0000062) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System |
| 23. |
Tokymitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., Muramatsu, M.
(1999)
Substrate recognition by Ca2+/calmodulin-dependent protein kinase kinase: Role of the Arg-Pro-rich insert domain.
J. Biol. Chem.
274
,
15803-15810
.
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Notes:
The GeneEditor™ Site-Directed Mutagenesis System was used to create point mutations in the CaM KK protein within the mammalian expression vector pME18s.
(0000271) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System |
| 24. |
Sutor, S. L., Vroman, B. T., Armstrong, E. A., Abraham, R. T., Karnitz, L. M.
(1999)
A phosphatidylinositol 3-kinase-dependent pathway that differentially regulates c-Raf and A-Raf
J. Biol. Chem.
274
,
7002-7010
.
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Notes:
The authors used the GeneEditor™ in vitro Site Directed Mutagenesis Kit to introduce a lysine to tryptophan mutation in A-raf that inactivates the kinase activity of this protein. The mutant A-raf was expressed in FDC-P1 cells.
(0000325) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System |
| 25. |
Strassheim, D., May, L.G., Varker, K.A., Puhl, H.L., Phelps, S.H., Porter, R.A., Aronstam, R.S., Williams, C.L.
(1999)
M3 muscarinic acetylcholine receptors regulate cytoplasmic myosin by a process involving RhoA and requiring conventional protein kinase C isoforms
J. Biol. Chem.
274
,
18675-18685
.
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Notes:
The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to create an amino acid substitution of asparagine for threonine. The mutation was accomplished in a mammalian expression vector.
(0000342) |
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Products: GeneEditor™ in vitro Site-Directed Mutagenesis System |