Figure 3

The structure of histone mRNA 3' ends: RNase protection assays. Top: schematic representation of RNase protection assays showing protected fragments produced when histone mRNAs end after the hairpin structure or after the AAUAAA polyadenylation signals. his-62: internally ³²P labelled his-62 probe was hybridised with either total RNA from N2 animals or yeast RNA and subjected to treatment with RNase A/T1 as described, where indicated. Probe marks radioabelled his-62 probe. Size standards were ³²P labelled pBR322 Msp I marker (M) and a KOH single nucleotide ladder (KOH) produced from 5' ³²P end labelled probe. Note that this combination of RNA and DNA marker does not allow for a precise determination of fragment length. We based our estimate on DNA marker fragments and used the nucleotide ladder to determine the fragment length between the 34 and 67 nucleotide DNA marker fragments. The ~50 nucleotide product resulting from hybridisation to mRNA ending after the hairpin is indicated by the arrow. Analysis was by denaturing PAGE followed by autoradiography as described. his-9: the internally³²P labelled his-9 probe was hybridised with total RNA from N2 animals or yeast RNA, and subjected to treatment with RNAse A/T1. Size standard (M) was ³²P labelled pBR322 Msp I DNA. Probe marks radioabelled his-9 probe. The major ~80 nucleotide product is indicated by the arrow. Analysis was as for his-62 RNase protection assay.