Subcloning of the Histone DNA Sequences

The purification and characterization of histone mRNA is a relatively straightforward procedure if conducted with the RNA from early cleavage stage sea urchin embryos [1 -6 ] where histone mRNA is the most abundant mRNA component found in small polysomes [1, 2]. Consequently this has led to an extensive characterization of the different subtypes of histone mRNA [4 -9 ] as well as to their use as hybridization probes to detect and characterize cloned histone DNA sequences from various sea urchin species [10-13]. Obtaining pure histone mRNA from other sources is, however, a difficult and tedious task. Therefore, the characterization of the histone genes in these sys­ tems either by in situ hybridization [14-17] or by cloning of genomic DNA fragments has been based mainly on the use of the heterologous sea urchin histone mRNA or cloned sea urchin histone DNA as hybridization probes [18]. Sea urchin histone DNA is basically organized in regular tandemlike clusters containing all five his­ tone genes. W ithin this basic repeat is contained an excess of spacer DNA [9]. Therefore, the original cloning experiments have led to cloned clusters instead of to individual histone genes. However, when analyzing differential histone mRNA expres­ sion in a given organism it is desirable to have hybridization probes which are specific for each gene. This is also true when analyzing whether a cloned histone specific genomic DNA sequence con­ tains more than one of the five histone genes. To cleave the cloned sea urchin histone DNA clusters