The five cleavage-stage (CS) histones of the sea urchin are encoded by a maternally expressed family of replacement histone genes: functional equivalence of the CS H1 and frog H1M (B4) proteins.

The cleavage-stage (CS) histones of the sea urchin are known to be maternally expressed in the egg, have been implicated in chromatin remodeling of the male pronucleus following fertilization, and are the only histone variants present in embryonic chromatin up to the four-cell stage. With the help of partial peptide sequence information, we have isolated and identified CS H1, H2A, H2B, H3, and H4 cDNAs from egg poly(A)+ mRNA of the sea urchin Psammechinus miliaris. All five CS proteins correspond to replacement histone variants which are encoded by replication-independent genes containing introns, poly(A) addition signals, and long nontranslated sequences. Transcripts of the CS histone genes could be detected only during oogenesis and in development up to the early blastula stage. The CS proteins, with the exception of H4, are unique histones which are distantly related in sequence to the early, late, and sperm histone subtypes of the sea urchin. In contrast, the CS H1 protein displays highest sequence homology with the H1M (B4) histone of Xenopus laevis. Both H1 proteins are replacement histone variants with very similar developmental expression profiles in their respective species, thus indicating that the frog H1M (B4) gene is a vertebrate homolog of the CS H1 gene. These data furthermore suggest that the CS histones are of ancient evolutionary origin and may perform similar conserved functions during oogenesis and early development in different species.

A histone H2B variant from the embryo of the sea urchin Parenchinus angulosus.

A variant of histone H2B has been isolated from sea urchin embryo (Parenchinus angulosus). Out of the 53 amino acids positioned in the three CNBr-peptides only 26 residues are identical to those in the corresponding positions of calf thymus histone H2B. A similar degree of homology exists between the embryonic variant and the previously characterized variants from sperm cells of the same organism.

The identification by sequence homology of stage-specific sea urchin embryo histones H1.

The histone H1 fraction from gastrula of the sea urchin Parechinus angulosus consists of a multitude of polypeptides with different electrophoretic mobilities. The synthesis of these proteins is programmed. Amino acid composition, electrophoretic properties and sequence homologies identify these as isohistones H1. One of these isohistones atypically binds the non-ionic detergent Triton X-100.

The partial amino acid sequences of the two H2B histones from sperm of the sea urchin Psammechinus miliaris.

Two new histone H2B variants have been isolated from sperm cells of the sea urchin Psammechinus miliaris. They have been designated sperm histone H2B(1) Psammechinus and sperm histone H2B(2) Psammechinus. Both histones are highly homologous to the previously described sperm histones from Parechinus angulosus (Strickland et al. (1977) Eur. J. Biochem. 77, 263--275 and 277--286). The amino acid sequences of the Ps. miliaris sperm histones, though highly homologous, are not identical to the amino acid sequence derived from the codon sequence of a histone H2B gene, characterized from the same organism by Birnstiel et al. ((1977) Nature 266, 603--607).

Plant histone 2 from wheat germ, a family of histone H2a variants. Partial amino acid sequences.

1. The 0.5 M perchloric acid extract prepared from chromatin of wheat germ, Triticum aestivum, contains a group of histones formerly called plant histones. These can be resolved by gel filtration on Bio-Gel P-60 with subsequent CM-cellulose ion-exchange chromatography into five histone fractions containing families of histones H2A and H2B. 2. The partial amino acid sequences of histone H2A variants H2A(1)Triticum, H2A(2)Triticum and H2A(3)Triticum are presented. Extensive sequence homology exists between calf thymus histone H2A and wheat embryo H2A histones. Differences are largely due to conservative amino acid substitutions and in two of the variants, viz. H2A(2) and H2A(3) to N-terminal extensions of the polypeptide chains.

Histone H2B variants from the erythrocytes of an amphibian, a reptile and a bird.

Histones H2B have been isolated from the terminally differentiated diploid erythrocytes of three different classes, amphibia (Xenopus laevis), reptilia (Crocodilus niloticus) and aves (Gallus domesticus). Partial amino acid sequences revealed three regions of sequence variation, each variant involving a single amino acid substitution.

Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro.

A heat-soluble protein present in substantial quantities in Typha latifolia pollen was purified to homogeneity. The protein was subjected to cyanogen bromide cleavage, and the peptides produced were separated by HPLC chromatography and sequenced. The two sequences determined were found to be related to the putative D76 LEA protein from Brassica napus seeds and one of them to the D-7 LEA protein from upland cotton. This suggests the pollen protein to be a member of the LEA group III family of proteins. The secondary structure of the protein in solution and in the dry state was investigated using Fourier transform IR spectroscopy. Whereas the protein in solution was highly unordered, being largely in a random coil conformation, the conformation was largely alpha-helical after fast drying. Slow drying reversibly led to both alpha-helical and intermolecular extended beta-sheet structures. When dried in the presence of sucrose, the protein adopted alpha-helical conformation, irrespective of drying rate. The effect of the protein on the stability of sucrose glasses was also investigated. The dehydrated mixture of sucrose and the LEA protein had higher glass transition temperatures and average strength of hydrogen bonding than dehydrated sucrose alone. We suggest that LEA proteins may play a role together with sugars in the formation of a tight hydrogen bonding network in the dehydrating cytoplasm, thus conferring long-term stability.

Mr determination of histones from their electrophoretic mobility in acidic urea gels in the absence of detergents.

The Mr of histones can be determined from their electrophoretic mobility at pH 2.3, 8 M urea in a polyacrylamide gel by correcting for differences in their charge density and properties of the gel matrix. The applicability of this method to other proteins is considered.

Manual gas-phase isothiocyanate degradation.

We describe a manual gas-phase isothiocyanate degradation procedure for the primary structure determination of proteins and peptides. The proteins and peptides are applied to a polybrene-coated glass fiber filter wedged into a small glass column. The phenylisothiocyanate is directly pipetted onto the filter disk. The coupling and cleavage reactions are performed in small desiccators containing trimethylamine and trifluoroacetic acid vapors, respectively. The wash and extraction steps are performed by allowing the suitable solvents to percolate through the filter disk. The extracted anilinothiazolinone is then converted to the phenylthiohydantoin and identified by any one of a number of described methods. Our results show that this method is very sensitive and that the reactions proceed faster than those of the published automated procedure. No expensive equipment is required and the manual degradation can be performed by a laboratory assistant. A large number of samples can be simultaneously subjected to the degradation under identical conditions, making this an ideal method for physicochemical investigations into the isothiocyanate degradation. We also use this method to screen HPLC fractions after enzymatic protein fragmentation. Manually sequenced glass filters can be transferred to the automated instrument for more extended degradations.