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J. Immunol. 162, 6562-6571. Molecular cloning and characterization of a novel CD1 gene from the pig. 1999

Chun, T., Wang, K., Zuckermann, F.A., Gaskins, H.R.

Notes: The complete cDNA for the CD1.1 gene was amplified and subcloned into the pTARGET™ Mammalian Expression Vector. The 1020bp, 339 amino acid protein was stably expressed in CHO cells following selection with G-418 sulfate. Expression was confirmed by Northern blot and FACS analysis with an mAb to the CD1.1 protein. The pGEM®-T Vector System was used for routine cloning of both PCR and RT-PCR products. The Prime-a-gene® Labeling System was used create probes for cosmid library screening. (1303)

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J. Biol. Chem. 274, 24865-24872. Nuclear localization of protein kinase U-alpha is regulated by 14-3-3. 1999

Zhang, S., Xing, H., Muslin, A.J.

Notes: The protein named KIAA (identified by yeast two-hybrid screen) and the 14-3-3zeta protein were each amplified to add epitope tags. The resulting products were subcloned into the pTARGET™ Mammalian Expression Vector and stably expressed in NIH/3T3 cells. (0096)

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J. Biol. Chem. 274, 17789-17793. The mechanism of adenosine formation in cells: Cloning of cytosolic 5'-nucleotidase I 1999

Sala-Newby, G.B., Skladanowski, A.C., Newby, A.C.

Notes: A clone of the cytosolic 5'-nucleotidase I cDNA into the available BamHI-KpnI site of the pTargeT™ Mammalian Expression Vector System. The expression construct was cotransfected into COS-7 cells with the pSV-betaGalactosidase Vector to control for transfection efficiency. (0432)

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Biochemistry 37, 15050-15056. A novel Arg362Ser mutation in the sterol 27-hydroxylase gene (CYP27): Its effects on pre-mRNA splicing and enzyme activity. 1998

Chen, W., Kubota, S., Ujike, H., Ishihara, T., Seyama, Y.

Notes: A minigene was constructed with five exons and four introns from exons 5-9 of the CYP27 gene. The 2111bp product was generated with primers containing a start codon and a stop codon. The product was directly cloned into the pTARGET™ Mammalian expression vector and transfected into COS-1cells. RNA was isolated from these cells and RT-PCR was performed to amplify the spliced product. This assay could distinguish between normal splicing of the third intron and aberrant splicing of due to the Arg362Ser mutation. The proteins produced by the normal and mutant minigenes were also assessed by immunoblotting. (1333)

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J. Lipid Res. 39, 509-517. Alternative pre-mRNA splicing of the sterol 27-hydroxylase gene (CYP27) caused by a G to A mutation at the last nucleotide of exon 6 in a patient with cerebrotendinous xanthomatosis 1998

Chen, W., Kubota, S., Seyama, Y.

Notes: To confirm the G to A mutation caused alternative splicing, a minigene containing exon 6 with or without the G to A mutation was amplified. The 2111bp minigene was T/A cloned into the pTargeT™ Mammalian Expression Vector and transfected into COS cells. The RNA was isolated from the transfected cells and analyzed by RT-PCR. The G to A mutation resulted in a truncated RT-PCR product. To confirm the mutation would cause a truncated, inactive protein. A cDNA was constructed that would be the result of the alternative splicing and subcloned into the pTargeT™ Vector. COS cells expressing the mutant had no detectable sterol 27 hydroxylase activity. (1330)

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Proc. Natl. Acad. Sci. USA 95, 9654-9659. Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzymes. 1998

Ohno, K., Brengman, J., Tsujino, A., Engel, A.G.

Notes: The entire coding region of the T isoform of the catalytic subunit of the acetycholinesterase (ACHET)was amplified and cloned into the pTargeT™ Vector and sequenced. The ColQ cDNA was cloned into the pTargeT™ Vector as well and mutated with a Pfu DNA polymerase-based mutagenesis system. The ACHET and ColQ mutants were coexpressed in COS cells and interactions examined with sedimentation analysis through sucrose gradients. (0595)

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Biochemistry 37, 4420-4428.. Silent nucleotide substitution in the sterol 27-hydrosylase gene (CYP 27) leads to alternative pre-mRNA splicing by activating a crytic 5' splice site at the mutant codon in cerebrotendinous xanthomatosis patients. 1998

Chen, W., Kubota, S., Teramoto, T., Nishimura, Y., Yonemoto, K., Seyama, Y.

Notes: Genomic analysis noted a single base change between wild type and mutant. To see if this base change was enough for alternative mRNA splicing, minigenes were constructed and subcloned into the pTargeT™ Vector. The mutant and wild-type minigenes were transfected into COS-1 cells. Forty-eight hours post-transfection total RNA was isolated and analyzed by RT-PCR. Transfectants with the mutant sequence did produce a different product due to alternative splicing. (1331)

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Am. J. Physiol. 273, C1937-C1946. Functional and molecular characterization of NHE3 expression during ontogeny in rat jejunal epithelium. 1997

Collins, J.F., Xu, H., Kiela, P.R., Zeng, J., Ghishan, F.K.

Notes: The pTargeT™ Vector was used to subclone the 2.5kb Na+/H+ Exchanger 2 and the 2.7kb Na+/H+ Exchanger 3 directly from PCR reactions. The constructs were stably transfected into PS120 Chinese hamster lung fibroblasts and selected for resistance to the neomycin analog, G-418. After 7-10 days of selection, the cells were further tested for expression by RT-PCR and immunoblotting. (1318)

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