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A cDNA library prepared from mRNA extracted from immature male gonads of the bivalve mollusc Ensis minor (razor shell) was probed with a 133-bp reverse-transcriptase PCR product corresponding to a segment of the sperm protein EM6 [Giancotti, V., Russo, E., Gasparini, M., Serrano, D., Del Piero, D., Thorne, A. W., Cary, P.D. & Crane-Robinson, C. (1993) Eur. J. Biochem. 136, 509-516]. A single 1.5-kb clone was found to encode both sperm proteins EM1 and EM6. Mass spectrometry was used to define the C-terminus of EM1, and since the N-terminus of EM6 is known from Edman degradation, this showed that the pentapeptide NTNNS must be lost on proteolytic processing. Both EM1 and EM6 contain highly repeated amino acid sequences, suggestive of extended structures. EM1 contains seven tandem repeats of the dipeptide S(K/R), followed by six potential cdc2 phosphorylation sites and seven repeats of the octapeptide KRSASKKR, with occasional K/R substitutions. EM6 contains a globular domain preceded by 17 almost identical uninterupted tandem repeats of the motif KKRSXSRKRSAS, where X is charged. Its C-terminus contains 15 short basic clusters. Assignment of EM1 and EM6 to the established categories of molluscan sperm proteins [PLI, PLII, PLIII, PLIV; Ausio, J. (1992) Mol. Cell. Biochem. 115, 163-172] is discussed.

Exhaustive extraction of mouse tissues with perchloric acid has been used together with reverse-phase HPLC and electrophoresis to quantify the amounts of chromosomal proteins HMG17, HMG14 and HMGI, relative to histone H1. Normal lung and thymus contain approximately 3% HMG17/HMG14 but only approximately 2% HMGI. In tumor tissues (Lewis lung carcinoma and lymphoma NQ35), the amount of HMG17/HMG14 is not greatly altered but HMGI levels rise considerably, reaching 10% in Lewis lung carcinoma. HMGI synthesis does not replace HMG17/HMG14 proteins, suggesting that HMGI proteins contribute to the structure of chromatin regions in a manner distinct from those of HMG17/HMG14. Ion-spray mass spectrometry has been used to determine the molecular masses of H1 subtypes from the same four mouse tissues. In addition to the six known species H1 zero, H1a, H1b, H1c, H1d and H1e, a newly defined subtype of mass 21,756 Da from Lewis lung carcinoma, named H1L was identified. Several phosphorylated H1 subtypes have also been defined by mass spectrometry. The combined use of reverse-phase HPLC and electrophoresis permitted quantification of these seven histone H1 subtypes in the four mouse tissues. Increased phosphorylation of H1 subtypes in tumors parallels the phosphorylation of HMGI proteins which are present in great amounts, showing that both are involved as post-translational-modified forms in the structure of the chromatin of neoplastic systems.

The class I of the high mobility group (HMG) proteins is formed by phosphoproteins which are associated with AT-rich DNA sequences in the nucleus. Three HMGI proteins have previously been described in proliferating rodent cells (HMG Y, HMG I and HMGI-C). All three proteins exhibit microheterogeneity. The microheterogeneity of mouse HMG Y has been investigated in detail and shown to be due to phosphorylation of the protein which is sensitive to alkaline-phosphatase treatment. HMG I is similarly modified. Human cells have up to now only been found to contain HMG Y and HMG I. A search for the third protein, HMGI-C, in human cells was carried out and the protein was found in a hepatoma cell line, but not in normal or transformed T-cells. This HMGI-C protein was found to be modified by phosphorylation, part of which was found to be phosphatase insensitive. An unexpected additional finding in this study was that human cells contain two HMG17 proteins which differ in their N-terminal primary sequences.

A test has been made of the postulate that interaction between histones H2A and H2B occurs only in the C-terminal domain of H2B and is independent of the N-terminal domain in which the sites of chemical modification occur. Sea urchin sperm H2B's have much extended N-terminal domains when compared to other studied H2B's and the interactions of Sphaerechinus granularis H2B1 with homologous H2A and with calf thymus H2A have been studied. Continuous variation analysis of circular dichroism results indicates that both homologous and heterologous H2A/H2B pairs can cooperatively form complexes, which in all cases involve the same number of helical residues (approximately equal to 80). It is concluded that although the sequence of the N-terminal domain of H2B can indirectly influence the free solution association constant with histone H2A, it does not take part in the interaction.

The nuclear magnetic resonance (NMR) spectrum of chromatin at ionic strengths below about 0.5 M may be attributed solely to its histone H1 component. The effect of various ions and urea on the complex has been investigated using NMR and confirm that the contraction of the complex on increase of ionic strength is largely due to electrostatic interactions. A detailed study of the H1 - DNA complex has also been undertaken. The behaviour of H1 in the two cases is virtually identical, implying that in chromatin the H1 is complexed with the DNA rather than with the other histones. Microcalorimetric measurements reveal that the binding of H1 to DNA is athermic or involves a heat of reaction which is very small indeed.

Analysis of the total protein of the mature sperm of the bivalve mollusc Ensis minor (razor shell) using gel electrophoresis, amino acid analysis, nuclear magnetic resonance, circular dichroism and trypsin digestion, show it to contain all five histones plus a protamine-like protein. The histones H3, H4 and probably H2A are similar to those from calf thymus or sea urchin sperm, but the putative H2B appears to have a very high molecular mass (about 20 kDa). The histone H1 molecule is unusual, having little or no proline and 8-10 residues of histidine. The protamine-like species is rich in both lysine as well as arginine and is of much higher molecular mass than fish sperm protamines. Nucleosomes containing the four core histones have been prepared and the nucleosomal repeat shown to be 200 +/- 5 base pairs. Checks for the absence of contaminating cells reinforce the conclusion that a histone-containing nucleosomal structure co-exists with a protamine-like protein in this sperm chromatin.