Radiolabeled RNA probes transcribed from various areas of tropoelastin cDNA in both the sense or antisense orientation have been incubated with nuclear and cytoplasmic extracts and were then handled with T1 RNase to digest unprotected RNA. Heparin was extra to disrupt nonspecic binding and also to inhibit endog enous RNases. The reaction merchandise, which consisted from the radiolabeled RNA element more helpful hints and bound extract issue, had been resolved beneath nondenaturing situations, and protected prod ucts had been detected by autoradiography. For these initial map ping research, we made use of ALF extracts, due to the fact we believed that tropoelastin mRNA binding component or exercise could be extra abundant throughout periods of accelerated transcript decay.
A protected band was detected only with RNA fragments containing sequences coded by exon 30 incubated Chelerythrine with cyto plasmic extract from ALFs, No binding activity was detected with RNA probes covering exons one to 18 or even the 3 UTR, In contrast, a prominent band was seen with an RNA probe transcribed from exons 17 to 36, In agreement with the selective, accelerated degra dation of thoroughly processed tropoelastin mRNA, bind ing action was only seen with RNA probes incubated with cytosolic extract, A weak protected band with the very same mobility as that produced with cytoplasmic extract was detected with RNA from exons 17 to 36 incubated with nuclear extracts, but this binding activity was probably because of some carryover of cytoplasmic parts during nuclear iso lation. Incubation of progressively smaller RNA probes indi cated that binding activity was conferred by sequences coded by exon 30, No binding action was detected with radiolabeled antisense RNA transcribed from exons 17 to 36, The specicity of binding to exon thirty was demonstrated by competitors with unlabeled RNA.
Binding action to radiola beled RNA from exons 17 to 36 was properly inhibited by a twenty fold or 60 fold molar extra of cold exon 30 RNA but was only minimally reduced by a a hundred fold excess of cold plasmid RNA, Also, no protected bands had been noticed with RNA probes transcribed in both course from linearized parental plasmid, Occasionally, the protected

band appeared being a doublet, which might repre sent incomplete digestion from the RNA target. These observa tions have been fully reproducible amid a lot of experiments with extracts from at least seven diverse ALF cell strains. Together, these data demonstrate the specicity on the binding interaction with sequences in exon thirty. We then employed very similar techniques to map the binding area within the mRNA sequences coded by exon thirty. Inside the rat tropoelastin gene, exon 30 consists of a 72 bp inser tion not identified in greater mammals, The bases anking this insert, even so, are conserved between species, Utilizing distinct restriction enzymes, we had been capable of transcribe progressively smaller sized RNAs of exon thirty.