Pierre Sautiere

Purification and characterization of nuclear basic proteins of human sperm

Highly purified nuclei were obtained from human sperm without protein loss through the use of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), a newly available detergent. The basic protein complement of these nuclei is highly heterogeneous and comprises histones (some of which are testis-specific), protamines and proteins of intermediate basicity and molecular size. The protamines belong to two different classes of protein. Microheterogeneity observed in some of these protamines originates from slight variations in their amino acid composition as well as from post-synthetic modifications. Two of these protamines previously considered as two different proteins are in fact the same protein with different degrees of phosphorylation. All these protamines and intermediate basic proteins are characterized by high amounts of arginine and cysteine. Three of the protamines and all five intermediate basic proteins are also histidine-rich.

The structure of ubiquitinated histone H2B.

Ubiquitinated histone H2B (uH2B) has been purified from both calf and pig thymus by exclusion chromatography in 7 M urea. Digestion of uH2B with Staphylococcus aureus V8 protease yielded the peptide 114‐125 containing the ubiquitin moiety. Further digestion of this peptide with trypsin removed the ubiquitin and three H2B residues from the N‐terminus. Edman degradations of both peptides established that ubiquitin is attached to the epsilon‐amino group of lysine 120 in both calf and pig uH2B by an iso‐peptide bond to the C‐terminal glycine 76 of ubiquitin.

P2 protamines from human sperm are zinc -finger proteins with one Cys2His2 motif

P1 (HP1) and P2 (HP2, HP3, HP4) protamines were isolated from human sperm nuclei in the reduced form and their interaction with zinc and cobalt was studied. One zinc atom per molecule of P2 protamines but not of P1 protamine was found. Absorption spectra of P2 protamines with cobalt were characteristic of a tetrahedral complex involving two histidine and two cysteine residues and with one cobalt per molecule. A tetrahedral complex was found neither in P1 protamines nor in P2 protamines alkylated at cysteine or at histidine residues. The zinc finger motif Cys2/His2 of P2 protamines may play a role in stabilization of human sperm chromatin and in inhibition of transcription.

Isolation and characterization of two protamines St1 and St2 from stallion spermatozoa, and amino-acid sequence of the major protamine St1.

Two protamines, St1 and St2, were isolated from stallion sperm nuclei, where they represent about 75 and 25%, respectively, of the total basic protein complement. The primary structure of protamine St1 (49 residues; Mr approximately equal to 6600) has been determined. The structure of this protamine is compared to the amino-acid sequence of other mammalian protamines already known.

Antigenic structure of histone H2B

Antigenic determinants of histone H2B were localized using a series of 23 overlapping fragments of H2B obtained either by chemical and enzymatic cleavage of the histone or by solid-phase peptide synthesis. The ability of peptides to bind H2B antibodies was measured in an enzyme-linked immunosorbent assay, using antisera directed against calf thymus and chicken erythrocyte H2B as well as four anti H2B monoclonal antibodies obtained from autoimmune mice. Seven antigenic determinants were localized in the H2B molecule in the vicinity of residues 1-11, 6-18, 15-25, 26-35, 50-65, 94-113 and 114-125. Two of these determinants (residues 6-18 and 26-35) were revealed only through the binding properties of antibodies isolated from autoimmune mice. The usual correlation between hydrophilicity and antigenicity was found to hold for four of the epitopes, and the N- and C-termini of H2B were both antigenically active.

Immunochemical detection of changes in chromatin subunits induced by histone H4 acetylation.

Native, reassociated, and reconstituted core particles from chicken erythrocytes were compared by both biophysical and immunochemical methods. No significant difference between the three types of core particles could be demonstrated by electron microscopy, circular dichroism, or immunochemical analysis with antisera to histone H2B, H2A, and H3. Core particles were also reconstituted with calf thymus non‐acetylated H3, H2A, and H2B with either mono‐, di‐, or tri‐acetylated H4 isolated from cuttle ‐fish testes. The hyperacetylation of H4 did not significantly alter the biophysical characteristics of core particles but it induced several changes in their immunochemical reactivity. Binding to core particles of antibodies specific for H2A, H3, and for the IRGERA (synthetic C‐terminal) peptide of H3 was considerably decreased when di‐ or tri‐acetylated H4 was used for reconstitution, whereas binding of H2B antibodies remained the same. Our results suggest that the presence of hyperacetylated H4 within core particles leads to conformational changes that alter the antigenic determinants of several of the histones present at the surface of chromatin subunits. Since histone acetylation is correlated with the open structure of active chromatin, it may become possible to monitor the activity of chromatin by immunochemical methods.

Isolation and characterization of the ram spermatidal nuclear proteins P1, 3 and T

Ram spermatidal proteins P1, 3 and T were isolated from non-round spermatid nuclei and characterized by amino acid analysis. Spermatidal proteins are small arginine- and cysteine-rich basic proteins. Proteins P1 and T are unusually rich in serine and the histidine content of P1 is particularly high. The NaCl molarities required to dissociate these proteins from the spermatid nuclei were determined. These proteins are present only during the reorganization of the spermatid chromatin.

Characterization of the chromosomal protein MC1 from the thermophilic archaebacterium Methanosarcina sp. CHTI 55 and its effect on the thermal stability of DNA.

In the deoxyribonucleoprotein complex of Methanosarcina sp. CHTI 55, DNA is associated with two proteins, named MC1 (methanogen chromosomal protein 1) (Mr 10,760) and MC2 (Mr 17,000). Protein MC1, the most abundant of these proteins, is closely related to the Methanosarcina barkeri MS protein MC1. The effect of Methanosarcina sp. CHTI 55 protein MC1 on the thermal stability of DNA has been studied in native deoxyribonucleoprotein complex, as well as in reconstituted complexes, and it has been compared to the effect of E. coli DNA-binding protein II. Both proteins are able to protect DNA against thermal denaturation, but the differences observed in the melting profiles suggest that they interact by different mechanisms. Moreover, our studies indicate that one molecule of protein MC1 protects eight base pairs of DNA.

Characterization of the chromosomal protein HMb isolated from Methanosarcina barkeri

Archaebacteria Methanogen Chromosomal protein

Structure primaire de l'histone riche en glycine et en arginine isolée de la tumeur de chloroleucémie du Rat

Summary The primary structure of the glycine-rich, arginine-rich histone isolated from the chloroleukaemic tumor of the rat has been established. The amino-acid sequence deduced from the study of the tryptic peptides and of the COOH-terminal fragment resulting from cleavage with cyanogen bromide, is identical to that of homologous histones isolated from calf thymus and hog thymus.