Denaturation of mouse satellite DNA upon melting of chromatin in solution

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.     

Intrastrand self-complementary sequences in Bacillus subtilis DNA.

Intrastrand self-complementary sequences have been isolated from the DNA of Bacillus subtilis by hydroxyapatite (HA) chromatography following thermal renaturation of strands separated by chromatography on methylated albumin kieselguhr (MAK). The instrastrand structures derived from the MAK H strand (HA HII) were biologically active showing transforming activity for a wide variety of markers, as well as hybridization to both pulse-labelled and ribosomal RNA. Removal of regions of single-strand DNA with S1 nuclease did not significantly alter the biological activity of the self-annealed molecules. The overall efficiency of transformation and hybridization of the intrastrand self-annealing DNA was low suggesting that many sequences in the population are neither active in transformation to prototrophy nor transcribed into RNA.     

Amplification of alpha-fetoprotein complementary DNA by insertion into a bacterial plasmid.

A fragment of the α-fetoprotein (AFP) structural gene was purified and amplified by bacterial cloning techniques. Double-stranded DNA_AFP was constructed from a cDNA copy of greater than 95% pure mRNA_AFP and inserted into E. coli plasmid pBR322 by poly(dA-dT)-linkers. Chimeric plasmid DNA isolated from transformants of E. coli strain χ1776 have been shown to contain α-fetoprotein sequences by hybridization to labeled mRNA_AFP. One clone, designated pA5 (chimeric plasmid pBR322 containing a cDNA_AFP sequence isolated from clone 5), has been studied in more detail. The inserted sequence of approximately 950 nucleotide pairs was positively identified by a hybridization-translation procedure. Hybridization of [³H]uridine-labeled poly(A)-containing RNA from an AFP-secreting cell line to excess pA5 DNA immobilized on nitrocellulose filters was used to show the selectivity of this probe for detecting expression of the AFP gene.