Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 2. Expression of mouse CST gene in various tissues. (A) Northern blot of CST mRNAs. First, 20 mg total RNA from indicated organs was electrophoresed, blotted, and hybridized with a digoxigenin-labeled (±) strand RNA probe made from the full-length mouse CST cDNA. Lane 1, kidney; lane 2, brain; lane 3, testis; lane 4, heart; lane 5, skeletal muscle; lane 6, small intestine; lane 7, stomach; lane 8, liver; lane 9, lung; lane 10, spleen. (B) RT-PCR analysis of CST and actin mRNAs. Next, 1 mg total RNA from indicated organs was reverse-transcribed with a random primer. The resulting cDNAs were separately amplified by PCR using a set of CST primers, 50-GGGTTTCCTGAGATGAC-30 (nucleotides 1±17 in Fig. 1) and 50-TAGTGCGCGTTGTAGCT-30 (nucleotides 634±650) and actin primers, 50-TTACCAACTGGGACGACATG-30 (nucleotides 149±168 in [16]) and 50-AGGAGCCAGAGCAGTAATCT-30 (nucleotides 869±888). The PCR products were separately electrophoresed, blotted, and hybridized with CST and actin probes. The observed PCR products showed the predicted sizes of 650 (CST) and 740 bases (actin). Lanes: 1, brain; 2, heart; 3, skeletal muscle; 4, kidney; 5, stomach; 6, small intestine; 7, lung; 8, liver; 9, spleen; 10, testis. (C) CST activity. Whole homogenates from indicated organs were assayed for CST activity and protein concentration as described in Materials and methods. 1, kidney; 2, brain; 3, testis; 4, heart; 5, skeletal muscle; 6, small intestine; 7, stomach; 8, liver; 9, lung; 10, spleen.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Expressing, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Amplification, Activity Assay, Protein Concentration

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 3. Multiple forms of mouse CST cDNA with 50-UTR unique sequences. Seven types of unique DNA sequences on the 50 end of mouse CST cDNAs are represented by boldface letters. The 50-terminus of the homologous sequence is indicated by arrows. The translation-initiation codon ATG is boxed and indicated by arrowheads. In forms A, B2, and C, type a, b, and c sequences are underlined with continuous lines and type d sequence is underlined with broken lines.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Sequencing

Journal: European journal of biochemistry

Article Title: cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

doi: 10.1046/j.1432-1327.2000.01139.x

Figure Lengend Snippet: Fig. 5. RT-PCR analysis of tissue-specific use of alternative exon 1. (A) Position of each primer and probe. Boxes represent exons. Symbols for exons are the same as in Fig. 4. Black boxes are coding exons. Total RNAs from various tissues were reverse transcribed using GSP1 primer. After isolation of the first cDNA strand, PCR was performed using a combination of a common antisense-primer 2A, corresponding to exon 2, and seven sense-primers relating to the seven exons 1, aS, bS, cS, dS, eS, fS, or gS. The PCR products were subjected to Southern hybridization with a probe EX2, corresponding to exon 2. (B) RNA from indicated organs was examined. PCR was performed using sense-primer aS (lane a), bS (lane b), cS (lane c), dS (lane d), eS (lane e), fS (lane f ), or gS (lane g). Minor bands in lanes a and b are represented by asterisks in small intestine.

Article Snippet: According to the manufacturer's manual, 1 mg total RNA, isolated from mouse brain, kidney, testis, stomach and small intestine, was reverse transcribed with a CST-genespecific antisense primer GSP1, 5 0-AAGTACGACGGGTAGTG-3 0, corresponding to mouse CST cDNA nucleotides 358±374 (Fig. 1).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Isolation, Hybridization