Abstract
In situ hybridization of complementary DNA (cDNA) synthesized from total cytoplasmic polyadenylated RNA isolated from Chinese hamster cells was employed to investigate the distribution of messenger specifying sequences on mammalian chromosomes. The kinetics of cDNA-nuclear DNA annealing indicate that about 85% of the cDNA represents sequences which are transcribed from non-repetitive DNA sequences. When cDNA is hybridized back to its template RNA, the reaction kinetics show that more than 60% of the poly(A) RNA is at least 104 times more complex than rabbit globin mRNA. In situ hybridization of cDNA to Chinese hamster cells fixed on slides shows no significant clustering of silver grains on interphase nuclei. On metaphase chromosomes the majority of silver grains are localized in euchromatic areas. It appears that all euchromatic segments have similar grain densities. Chromosomes 1 and 2, which have relatively little heterochromatin, do not have a higher grain density than the other chromosomes. However, the Y chromosome, which is entirely heterochromatic, contains only about 1/3 the grain density of the chromosomes 1 or 2. — When the cDNA, which anneals only to the high abundancy class of poly(A) RNA was fractionated and hybridized in situ to Chinese hamster chromosomes, the distribution of silver grains is localized in the euchromatic areas. The Y chromosome and the heterochromatic arm of the X chromosome contain less grains; telomeres of some autosomes have higher grain densities. The oligo-(dT) primer in cDNA did not affect the results of this study since no grains are found when 3H-poly(dT) was used as probe for in situ hybridization. The majority (>90%) of the grains could be blocked by competition with excess repetitive DNA in the hybridization reaction, indicating that the in situ hybridization involved predominantly repetitive sequences.
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Kuo, M.T., Saunders, G.F. Location of messenger specifying sequences in mammalian chromosomes. Chromosoma 63, 241–252 (1977). https://doi.org/10.1007/BF00327452
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DOI: https://doi.org/10.1007/BF00327452