The Use of Iodinated RNA for Gene Localization

Abstract

Carrier-free iodination of 5S RNA with 1261 yields a probe suitable for use in cytological RNA-DNA hybridization studies. The genes coding for 5S ribosomal RNA in Drosophila could be localized by autoradiography after a 2-day exposure, whereas a 2-month exposure is needed when the best available ['H]RNA is used. The procedure introduces one covalently bound 12"I atom per 100 nucleotides, resulting in a specific activity of over 108 dpm/,ug of RNA. The method may be readily applied to other problems involving molecular hybridization techniques.Molecular hybridization of radioactive RNA or DNA to chromosomes offers the opportunity for localizing genes or specific DNA sequences by autoradiography. One needs to isolate in high purity a specific gene product that is either labeled or can be used as a template to transcribe a labeled probe; the probe must have specific activity high enough to yield a detectable number of disintegrations at the site where it hybridizes; there must be a sufficient number of complementary nucleotides in one region of the chromosome to bind detectable amounts of the probe. The higher the specific activity of the probe, the smaller is the template that may be detected.Molecular hybridization in situ has been successfully applied to diploid cells that contain repeated large DNA sequences (1-5) or highly amplified specific genes during specific times of cell development (6). Localization of smaller sequences that are not highly redundant has been successful only in giant chromosome preparation of some dipterans (7, 8); identification of hybridized regions required months of autoradiographic exposure to obtain convincing numbers of silver grains.All successful gene localizations have been obtained with 'H-labeled RNA or DNA. The main requirement for direct chemical labeling of isolated RNA is that sufficient label be introduced into the RNA without loss of its ability to hybridize specifically with its template. The labeling isotope should have a reasonably short half-life since the maximal specific activity of an isotope is inversely related to its halflife. We have chosen to work with 125I, which has a 60-day half-life, 1/70th that of 3H. Commerford (9) has developed mild conditions for iodinating nucleic acids. He showed that the iodinated nucleic-acid product contained iodine almost exclusively at the C5 position on cytosine, and that DNA containing 10% substitution of iodine in cytosine preserved Abbreviation: SSC, 0.15 M NaCl-0.015 M Na citrate.

Application of Fingerprinting Techniques to Iodinated Nucleic Acids

“…Several reports have appeared describing methods for iodination of nucleic acids with 125I (12,13). Prensky et al (13) have described a modification of Commerford's iodination procedure (12) by which they obtain RNA of very high specific radioactivity (106-101 dpm/,g).…”

“…Prensky et al (13) have described a modification of Commerford's iodination procedure (12) by which they obtain RNA of very high specific radioactivity (106-101 dpm/,g). These authors showed the specific in situ hybridization of Drosophila 5S RNA with band 56F of Drosophila polytene chromosomes.…”

Molecular hybridization of iodinated 4S, 5S, and 18S + 28S RNA to salamander chromosomes.

“…RNA iodinated in vitro with carrier-free 125I produces a hybrid that can be rapidly detected with a resolution comparable to that obtained with tritiated RNAs (33,42,43). Ribosomal and transfer RNAs can be easily purified, and their complementary sequences are repetitious in the genome of eukaryotes (4,8,35).…”

Hybridization of prokaryotic and eukaryotic 5S rRNA to Euglena gracilis chloroplast DNA

“…Each 5S rRNA was labelled in vitro with 250/ICi of 12sI (Radiochemical Centre) by thallium trichloride oxidation at a carrier iodide concentration of 10 -s M. Conditions of reaction and removal of unstable intermediates were essentially as described [21].…”