Insulin transcription in the islets. (a) Total RNA from islets was reverse transcribed to cDNA using oligo-(dT)₁₇ primer. β-actin and insulin cDNAs were then amplified by PCR. The cycle numbers used for the PCR were 18 (lanes 1 and 2), 21 (lanes 3 and 4), 24 (lanes 5 and 6), 27 (lanes 7 and 8), and 30 (lanes 9 and 10), respectively. The amounts of cDNA from control C57BL/6J (odd lanes) and Mody mice (even lanes) were adjusted by the levels of amplified β-actin (upper panels). Note that both Ins1 and Ins2 transcripts should be amplified with equal efficiencies because primers were derived from the common sequences between them. The PCR products of Ins2 (263 bp) and Ins1 (257 bp) were not resolved in this gel system. The total insulin levels in the islets of Mody mice were approximately 85%–90% of those of the control mice (lower panels). (b) The insulin transcripts amplified from islet RNA of either C57BL/6J (lanes 1, 3, and 5) or Mody mice (lanes 2, 4, and 6) were run without digestion (lanes 1 and 2). They were then digested with Bst EII for discrimination between Ins1 (257 bp) and Ins2 (111 bp) transcripts (lanes 3 and 4). Similarly, they were digested with Fnu 4HI to separate Ins1 (167 bp), wild-typeIns2 (174 bp), and mutant Ins2 (263 bp) transcripts (lanes 5 and 6). The left lane shows radiolabeled φX174/Hinf I digested DNA markers. Because the PCR products were labeled with an end-labeled 5′ primer, the radioactivity of each band corresponds to the expression level, irrespective of its size. The measurement of the radioactivity of each band revealed that 27% and 73% of the total insulin transcripts in C57BL/6J mice are derived from Ins1 and Ins2, respectively. Similar values, 24% for Ins1 and 76% for Ins2, were obtained from Mody mice. Furthermore, 39% of the total insulin transcripts, which is approximately half the value of total Ins2, were derived from the mutant Ins2 allele in Mody mice, suggesting that both normal and mutant Ins2 alleles are transcribed similarly.