Abstract
The kinetics of the cytological or in situ DNA-RNA hybridization reaction between 125I HeLa cell 18S and 28S rRNA and the interphase nuclei of Chinese hamster cells were studied. The reaction is consistent with the expected first order kinetics, and the value of the rate constant was very similar to the value obtained from analogous filter disc DNA-RNA hybridization experiments. This similarity in the rate constants and the known relationship between the rate constant and the complexity of the RNA hybridized, define conditions to optimize the in situ hybridization reaction.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 647-654 |
| Number of pages | 8 |
| Journal | Nucleic acids research |
| Volume | 2 |
| Issue number | 5 |
| DOIs | |
| State | Published - May 1975 |
Funding
initiate equally well. More importantly, the rate constant for an JLn situ experiment has a very similar value as the rate constant for a filter disc experiment. This result implies that the initiation events are taking place on nure DNA and that the uneven distribution of the DNA on the solid support is not affecting the hybridization reaction. In filter disc DNA-RNA hybridization experiments, the value of the rate constant has been found to be inversely proportional to the complexity of the RNA as would be expected for a reaction determined by the initiation event (10) . Since no long stretches of repeated sequence are present in rRNA, k can be related directly to the molecular weight of the RNA and the value of k is expected to be similar for all rRNA hybridization experiments. It is of considerable interest, therefore, to determine whether the same relationship is true for _iri situ hybridization experiments as would be suggested by the similarity of the rate constants for the two systems. We have started to study the kinetics of Drosonhila melanogaster 28S rRNA binding to the nucleolus and 5S rRNA binding to chromomere 56F on the polytene chromosome (2) in order to determine this point. Preliminary data give approximately the expected value for the rate constant of 28S rRNA but' give an unexpectedly slow rate for the 5S rRNA. However, other variables that may affect the rate are the length of the RNA molecules and the possibility of diffusion limiting the reaction rate as can also occur in filter disc hvbridization (10,13). It is clear that when in_ situ hybridization is to used to localize DNA sequences in chromosomes, the concentration of RNA and the time of hybridization must be carefully controlled. Since the reaction is essentially complete (97%) when run at 5 times the Crtj/2 value, longer incubation times should be avoided to prevent the detection of small amounts of impurity present in an RNA preparation. This precaution is especially important if the impurity hybridizes to a much higher saturation value than the RNA being tested. Indeed, it is probably best to perform an in situ localization experiment at a variety of conditions to ascertain that the kinetics give the behavior expected for the RNA being used. ACKNOWLEDGEMENTS The authors than Prof. D. Steffensen for encouragement and the use of his facilities. Paul Szabo is a Damon Runyon Postdoctoral Fellow, Robert Elder is a NSF Predoctoral Fellow and 0. C. Uhlenbeck is a NIH Career Development Awardee. The research was aided by Grant DRG-28-F from the Damon Runyon Memorial Fund for Cancer Research.
ASJC Scopus subject areas
- Genetics