Estimation of gene expression in heterocysts of Anabaena variabilis by using DNA-RNA hybridization

In the filamentous cyanobacterium Anabaena variabilis, specialized cells called heterocysts occur in a regular pattern along the filament and are the sites of nitrogen fixation. We used two different types of DNA-excess RNA hybridization techniques to estimate the number of genes expressed in recently differentiated, mature heterocysts. In the first, RNA and DNA were incubated in a phosphate buffer at 60 degrees C, and the hybrids were separated from the unhybridized material by hydroxylapatite chromatography. In the second, the nucleic acids were incubated at 50 degrees C in a buffer containing 50% formamide, and the fraction of DNA in duplexes was assayed by S1 nuclease digestion. Both techniques revealed that approximately 65% of the A. variabilis genome was expressed in vegetative cells and 45% of the genome was expressed in heterocysts. Two experiments were conducted to estimate the number of heterocyst-specific mRNA transcripts. In one, hybridization of heterocyst RNA to a null DNA probe (DNA not transcribed in vegetative cells) revealed that heterocyst-specific transcripts were encoded by 25% of the DNA sense strand, representing approximately 1,000 genes (assuming each to be 1,500 nucleotides in length). The second approach, in which total cell DNA was hybridized to a mixture of heterocyst and vegetative cell RNA, indicated that 14.7% of the DNA sense strand, or about 600 genes, was transcribed exclusively in the heterocyst. The remaining 900 to 1,300 transcripts present in the heterocyst appeared to be constitutively produced in both vegetative cells and heterocysts. The heterocyst-specific transcripts were present in abundant copies in the cell, while transcripts that occurred in both cell types were present at much lower frequency.