Inosine incorporation in GC rich RNA probes increases hybridization sequence specificity

Abstract

Human metallothioneins (MT) are represented by a multigene family. Many of these genes are expressed differentially and in a cell type/tissute specific manner in response to a variety of inducers including heavy metals (eg. Cd, Zn, Cu) and dexamethasone (Dex) (1). One of the MT genes, MT-IC (2) is expressed in a hepatoblastoma cell line (Hep-G2) in response to heavy metals but not in a lymphoblastoid cell line (Wi-L2). This is shown in Fig.l (panel A) where a Northern blot of total RNA (5Ag) from these cell lines was probed with a nick translated MT-I0 specific DNA probe derived from its 3'-terminus. When a similar blot was hybridized to an RNA probe derived from the same region of the DNA, hybridization to the Wi-L2 RNA was also seen (Fig.l, panel B). This was demonstrated to be due to nonspecific hybridization of the MT-I RNA probe to the MT-IIA mRNA because of a region of 34 nucleotides (&70% GC) which is partially homologous within these genes (3). Since RNA/RNA hybrids of high GC contents are very stable it was difficult to melt this region even under stringent conditions of hybridization (3xSSC; 650C; 50% formamide) and washings (up to O.lxSSC; 650C). To eliminate this nonspecific hybridization due to high GC contents, the RNA probe was sythesized (4) in the presence of inosine 5'-triphosphate (ITP replacing GTP). When this probe was used, nonspecific hybridization was completely eliminated (Fig.l, panel C) even under less stringent conditions of hybridization (3xSSC; 65'C) and washings (up to lxSSC; 65°C). Figure 2 shows that even though the size of the RNA synthesized in the ITP containing reaction (I) is the same as that from the reaction containing GTP (G) (suggesting specific transcription initiation), the extent of its synthesis is reduced. It can , however, be increased by incubating the ITP containing reaction for up to 3 h with additional supplements of ITP and RNA3 olymerase. Under these conditions the extent of radioactive incorporation6(a P-UTP) 6 was increased up to -30% of the GTP containing reaction (12x10 versus 37x10 cpm from reactigns containing 0.5 Ag of the linearized DNA template). Since only about lxlO cpm/filter were used in hybridization, the reduced extent of RNA synthesis is not a limitation.