Non-histone nuclear proteins as gene regulators?

ALEXANDER J. MAcGILLIVRAY* Beatson Institute for Cancer Research, Wolfson Laboratory for Molecular Pathology, Garscube Estate, Switchback Road, Bearsden, Clasgow G61 1 BD, Scotland, U.K. One of the most intriguing problems in molecular biology today concerns the mechanisms whereby control is exerted over the expression of specific genes. The situation in prokaryotes has been well elucidated, with both positive and negative forms of control having been characterized, e.g. the Catabolic Gene Activator Protein and repressor proteins of the lac gene system in Escherichia coli (see Dickson et al., 1975). The situation in eukaryotes, by contrast, is only beginning to be understood and, indeed, many problems exist in determining the operation of genes in higher organisms. For example, many such organisms possess large amounts of DNA, much of which does not code for protein; the DNA is complexed with large amounts of protein and is separated from the rest of the cell by the nuclear membrane; in multicellular organisms the processes of differentiation and development have led to the DNA of the different tissues being genetically similar, but only a small portion of the sequences are actually expressed in the cells of each tissue. Hence the study of the control of geneexpression in eukaryotes depends upon a number of factors. (1) Our ability to isolate the genetic material in as native a state as possible. In this respect, DNA together with its associated proteins can be isolated by various procedures as a nucleoprotein complex referred to as ‘chromatin’, a product usually accepted as representing a preparation of interphase chromosomes. (2) The ability of genes in isolated chromatin to be expressed in vitro and the availability of techniques to assay their transcription products. Much interest in studyinggene control was aroused when the expression of specific genes, e.g. those coding for globin (Wilson et al., 1975), histone (Stein et al., 1975) and ovalbumin (Harris et al., 1976), was demonstrated by using radioactively labelled complementary DNA species as probes after transcription of the chromatin with bacterial RNA polymerase. (3) The need for chromatin to be fractionated into its primary constituents. Apart from DNA, chromatin consists of a small amount of RNA and two types of protein, namely the five basic histones, which together are present in an amount equal to that of DNA and a heterogeneous group referred to as the non-histone proteins, whose quantity varies from tissue to tissue. There are numerous procedures available for the preparation of those proteins (see MacGillivray, 1976), perhaps the most successful being those based on dissociation of chromatin in solutions containing high concentrations of salt and urea. (4) The requirement that chromatin can be dissociated and then reassembled without loss of its specific template properties. Although chromatin can be easily dissociated in salt/urea solutions, little is known concerning its reassembly, the only procedure available being reconstitution by removal of the salt etc. through gradient dialysis. When such chromatin is transcribed by bacterial RNA polymerase and the RNA product analysed by means of complementary DNA prepared from poly(A)-containing nuclear RNA, then it appears that at least 50 % of the DNA sequences expressed in vivo are not transcribed from the reconstituted chromatin (A. Alonso, unpublished work). Nevertheless, by using similar conditions other authors have demonstrated the expression of specific genes from reconstituted chromatins, e.g. genes for globin (Gilmour & Paul, 1975; Barrett et al., 1974) and histone (Stein et al., 1975) and this has formed the basis of further investigations. In the main the above criteria would appear to have been fulfilled and this has allowed a number of studies to be initiated, particularly of the globinand histone-gene systems. * Present address: Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton BNl 9QG, U.K.