Journal: PLoS ONE
Article Title: Intron 4 Containing Novel GABAB1 Isoforms Impair GABAB Receptor Function
Figure Lengend Snippet: Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same RNA sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal ORFs of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).
Article Snippet: Cloning novel isoforms, GABAB1k, GABAB1l, GABAB1m, and GABAB1n To clone full ORFs of the novel isoforms, cDNAs were generated with oligo(dT) primer from human brain total RNA (Ambion, Austin, TX, USA) and mouse midbrain total RNA.
Techniques: Clone Assay, Sequencing, Microarray, Polymerase Chain Reaction, Cell Culture