Figure 5.

Phaseolus has two MIPS genes. One of two bean MIPS genes was isolated from a bacterial artificial chromosome (BAC) library, characterized, sequenced, and used to design primers for RT-PCR experiments. A, Southern-blot analysis of genomic DNA and BAC DNA digested with restriction enzymes not present in the gene sequence, EcoRI (E) and Bam Hl (B), detected two hybridizing fragments (9 and 6 kb) in genomic DNA and one hybridizing fragment (9 kb) in the BAC DNA. The deduced amino acid sequence of the clone is 89% identical to the bean root cDNA used as probe for library screening (Wang and Johnson, 1995). These results suggest that there are two MIPS genes in the bean genome. B, Bean upstream region contains ORFs interspersed with consensus RNA splice sites (lowercase bold type) and the first exon (uppercase bold type). C, RT-PCR reactions were performed using a reverse primer (5′-CCTTGGCCC TACCCATGGC-3′) made from a sequence in the third exon and four different forward primers (underlined sequence). All lanes were loaded with RT-PCR reaction products (5 μL) generated from leaf or root mRNA.