Abstract

The trout testis contains two major high mobility group (HMG) proteins HMG-T and H6 which, although related to the four mammalian HMGs, exhibit distinct variation as evidenced by differences in electrophoretic mobility and amino acid sequence. Previous work using various endonucleases as probes has shown that HMG-T and H6 are located at specific sites in the testis chromatin. The differentiation of testis cells during spermatogenesis is characterized by a unique transition from a histone-packaged genome to one bound by a class of small molecular weight, highly basic proteins, the protamines. Questions arise as to whether any of the HMG variability may be unique to the process of spermatogenesis and whether the histone-protamine transition occurring in most testis cells affects the HMG protein distribution and/or the specificity of the probe. In an attempt to answer these questions, the distribution of the HMG proteins in the chromatin of trout liver, a tissue lacking protamine, has been studied and comparisons made with testis. Liver HMGs exhibit the same electrophoretic characteristics as the testis HMGs indicating that the variability when compared to mammalian HMGs is primarily phylogenetic in origin rather than tissue-specific. Furthermore, micrococcal nuclease digestion of liver nuclei and its effect on the subsequent HMG protein distribution during chromatin fractionation yields a pattern very similar to that for testis, suggesting that the interaction of the HMGs with the remaining testis nucleohistone is not significantly altered by the ongoing transition to nucleoprotamine. Finally, the HMGs represent an unusually high proportion of the total testis non-histone protein population; the implications of this are discussed.

Highlights

  • The high mobility group (HMG)' proteins in mammalian cells consist of four major species, HMGs 1, 2, 14, and 17 [1, 2], which, based on biochemical and sequence data [3, 4], appear to fall into two classes; one comprising HMGs 1and 2, the other comprising HMGs 14 and 17

  • Since the nuclease digestion studies described abovewere conducted with testes at the protamine stage, it was not clear what effect the ongoing replacement of histones with protamine had on the nuclease digestion properties of the nuclei or on the association of the HMG proteins with specific sites in the remaining fraction of nucleohistone

  • Micrococcal nuclease al. [14] wasused to extract and isolate the HMG protein (Worthington) is added to a final concentration of either 100 units/ ml or 1000 units/ml andthe mixture is incubated for the periods of time stated in the text

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Summary

Introduction

The HMG' proteins in mammalian cells consist of four major species, HMGs 1, 2, 14, and 17 [1, 2], which, based on biochemical and sequence data [3, 4], appear to fall into two classes; one comprising HMGs 1and 2, the other comprising HMGs 14 and 17. Since the nuclease digestion studies described abovewere conducted with testes at the protamine stage, it was not clear what effect the ongoing replacement of histones with protamine had on the nuclease digestion properties of the nuclei or on the association of the HMG proteins with specific sites in the remaining fraction of nucleohistone. To investigate this issue,we have studied the HMG proteins and their relationship to chromatin in a trout tissue, liver, which does not contain protamine. Direct comparison of the results obtained with testis and liver indicate that thepresence of protamine in the testis hasno apparent effect onthe HMG proteins or their distribution in that fraction of chromatin not yet converted to nucleoprotamine (from here on referred to as theresidual nucleohistone)

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