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We describe a method that can be used to obtain and sequence 3' and 5' ends of cDNA transcripts directly from PCR products. The method employs a modified oligo(dT) primer that enables it to "lock-dock" at the junction of gene-specific cDNA sequence and a natural (3') or appended (5') poly(A) tail. As a result, discrete, first-round PCR products are obtained(More)
To elucidate glucose transport mechanisms in brain and to demonstrate the cellular expression of the brain-type glucose transporter (GLUT3), antisera to a synthetic peptide corresponding to the C terminus were prepared and used as probes for this isoform of the facilitative glucose transporter family. Immunocytochemistry of frozen sections of dog and rat(More)
GLUT1 and GLUT3 mRNAs in normal and post-ischemic gerbil brains were examined qualitatively and semi-quantitatively using in situ hybridization in conjunction with image analysis. Coronal brain sections at the level of the anterior hippocampus were prepared three hours, one day, and three days after animals were subjected to six min of ischemia. The(More)
Glucose transporter (GLUT) expression and regulation were studied in rat brain endothelial cells in primary culture (RBEC) and in immortalised RBE4 cells. Immunoblotting analysis showed a low expression of the endothelium-specific GLUT1 in RBEC and RBE4 cells compared to isolated brain capillaries. RBEC and RBE4 cells also expressed the GLUT3 isoform,(More)
There has been a sparsity of various mammalian neuronal glucose transporter 3-encoding sequences(Glut3) available for the purposes of alignment studies. We report here a 2355-bp sequence of canine Glut3 that encodes a deduced protein of 496 amino acids (aa). The full-length canine aa sequence was compared to those of the human, mouse and rat glucose(More)
We employ here a modified oligo(dT) primer called a "lock-docking" primer which enables the production of a cDNA template that can subsequently be amplified in the 3'-RACE PCR procedure to produce discrete, first-round products for 3'-cDNA ends of any reverse-transcribed poly(A) RNAs. An upstream consensus primer targeted to a highly conserved region (amino(More)
The carboxyl-terminal amino acid sequences of the canine and gerbil glucose transporter GLUT3 were determined and compared to the published rat sequence. Eleven of 16 amino acids comprising the carboxyl terminus of GLUT3 were found to be identical in rat and dog. However, the canine sequence "ATV" substitutes for the rat sequence "PGNA" at the end of the(More)
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