Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.     

Enhancement of pneumococcal transfection by protamine sulfate.

Protamine sulfate enhanced transfection of Streptococcus pneumoniae by DNA of omega 3 phage by factors as large as 10(5)-fold, provided it was present at the time the cells were added to the DNA. For DNA concentrations well below 1 microgram/ml, the optimum amount of protamine sulfate was near 1 microgram/ml of cells. Higher DNA concentrations required more protamine for maximum effect, and in all cases transfection fell when protamine was in excess. Transformation was not enhanced by low protamine levels and was inhibited by higher levels. A recipient strain with low but finite endonuclease activity and normal transformability showed higher transfection than did the wild type at low DNA concentrations but less than did the wild type at high DNA concentrations. Protamine sulfate enhanced its transfection at low, but not high, DNA concentrations. The behavior of this strain and the enhancement of transfection by protamine sulfate of wild-type cells were each consistent with less cutting of the donor DNA at the cell surface, which is part of the normal entry process in naturally competent gram-positive bacteria. Less cutting would lead to entry of fewer but longer strands that would be more efficient in reconstruction of the 33-megadalton phage replicon. We suggest that in this system protamine enhances transfection by inhibition of the surface nuclease action that is part of the normal entry process.     

Nucleotide sequence of a protamine component CII gene of Salmo gairdnerii.

We have isolated, using nick-translated cloned protamine cDNA's as probes, several genomic clones containing protamine gene sequences from a Charon 4A library of Eco R1 digested rainbow trout (Salmo gairdnerii) DNA. One clone was chosen for detailed study and the 2.5 kbp Bam HI-Eco R1 restriction fragment containing the gene was subcloned in the plasmid pBR322. A 920 bp Bg1 II - Bam HI restriction fragment contains a sequence coding for protamine component CII as well as regions 5' and 3' to the mRNA coding portion. Present in the region 5' to the mRNA coding sequence are the promoter associated signals "TATA" box and "CAAT" box. The 5' untranslated region of the mRNA whose length and sequence were not established from the cDNA clones (1) was determined by nuclease mapping and starts within a sequence similar to the "capping signal" found in other genes. The protamine gene for CII contains no introns, a situation common to most histone genes, but, unlike the histone genes does not occur close to other protamine genes in a "cluster".     

Protamine induces autophosphorylation of protein kinase C: stimulation of protein kinase C-mediated protamine phosphorylation by histone.

Protein kinase C (PKC), a protein phosphorylating enzyme, is characterized by its need for an acidic phospholipid and for activators such as Ca2+ and diacylglycerol. The substrate commonly used in experiments with PKC is a basic protein, histone III-S, which needs the activators mentioned. However, protamine, a natural basic substrate for PKC, does not require the presence of cofactor/activator. We report here that protamine can induce the autophosphorylation of PKC in the absence of any PKC-cofactor or activator; this may represent a possible mechanism of cofactor-independent phosphorylation of this protein. It was investigated if protamine itself can act as a PKC-activator and stimulate histone phosphorylation in the manner of Ca2+ and phospholipids. Experiments however showed that protamine is not a general effector of PKC. On the contrary, histone stimulated PKC-mediated protamine phosphorylation and protamine-induced PKC-autophosphorylation. Histone alone did not induce PKC-autophosphorylation. Kinetic studies suggest that histone increases the maximal velocity (Vmax) of protamine kinase activity of PKC without affecting the affinity (Km). Other polycationic proteins such as polyarginine serine and polyarginine tyrosine were not found to influence PKC-mediated protamine phosphorylation, indicating that the observed effects are specific to histone, and are not general for all polycationic proteins. These results suggest that histone can modulate the protamine kinase activity of PKC by stimulating protamine-induced PKC-autophosphorylation.     

Divergence of protamine gene sequences in fish

Complementary DNA to trout protamine mRNA was hybridized to excess genomic DNA from trout, salmon and yellow perch. Although there was extensive hybridization of the cDNA to trout DNA, no cross-reaction with yellow perch DNA was observed and the hybridization to salmon DNA was noticeably less than in the homologous reaction. To confirm these results, yellow perch protamine mRNA was purified and compared directly to trout protamine mRNA. Yellow perch protamine mRNA was shorter than trout protamine mRNA, when measured by agarose gel electrophoresis in the presence of methyl mercury hydroxide. The two mRNAs did not cross-react in cDNA/RNA hybridizations, although the homologous reactions went to 90% of completion. This lack of sequence homology was confirmed when the oligopyrimidine tracts from the cDNAs were compared. No sequences longer than tetranucleotides were common to both species. Trout protamine cDNA contained oligopyrimidines of composition C₇T₄, C₄T₂, C₃T₂, C₂T₄, C₂T₃, C₁T₅ and C₁T whereas yellow perch protamine cDNA contained C₆T₃ and C₄T₃.     

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.     

Red cell ghost-mediated microinjection of RNA into HeLa cells. II. Cellular translation of protamine mRNA; post-translational modifications and nuclear binding of newly-synthesized protamine

Red cell ghosts loaded with protamine messenger RNA (pmRNA) were fused to HeLa cells using polyethylene glycol, as a means of introducing the mRNA into heterologous cells. The recipient cells were capable of translating the RNA into the three protamine polypeptides, which may be resolved as three peaks (CI, CII, and CIII) by cation exchange chromatography. The synthesis of components CII and CIII was easily observed with possible traces of CI as well. The HeLa cells also phosphorylated CII after synthesis. However, this phosphorylation did not occur with CIII. In addition, CII but not CIII localized in the nucleus of the HeLa cells after synthesis. Thus, a correlation of post-translational modification with nuclear entry was observed. Localization in the nucleus, however, was not accompanied by the same tight binding of protamine to chromatin as is seen in the homologous trout testis spermatid cells. In the spermatid cells, protamine elutes from chromatin at a salt concentration of 1.2 M NaCl. In contrast, in the HeLa cells, the newly synthesized CII which had entered the nucleus, could be eluted with 0.6 M NaCl. Thus, the tight binding of protamine to chromatin in trout testis may require a series of concomitant developmental events, such as core histone hyper-acetylation (Christensen, M E & Dixon, G-H. In press) [17], which would be lacking in the HeLa cells.     

Replacement of protamine by F1 histone during reactivation of fused human sperm nuclei.

Rabbit antisera, specific for the histones F1, F2a2, F2b, F3 and for protamine were used to monitor a possible transition from protamine towards somatic-type histones during sperm nucleus reactivation, following human sperm fusion with mouse fibroblasts. Mature human sperm nuclei were shown to contain the histones F2a2, F2b, F3 and protamine, but were missing F1 histone by immuno cytochemistry using the indirect fluorescence method. However, a gradual disappearance of protamine from fused sperm nuclei, could be observed during the first 24 h of reactivation. Subsequently, F1 histone could be detected in increasing concentrations in 60% of reactivated sperm nuclei, during the next four days. The shift from protamine towards F1 histone could also be visualized cytochemically via staining with brilliant sulphaflavine, which appears to discriminate between sperm nuclei on the basis of their F1 histone content.     

Purification and properties of a protamine kinase from bovine kidney microsomes.

About an eightfold increase in protamine kinase activity was detected following extraction of highly purified microsomes from bovine kidney with 1% Triton X-100. Relative to the soluble fraction, the microsomes contained about 30% protamine kinase activity. The microsomal protamine kinase was purified to apparent homogeneity. The purified enzyme exhibited an apparent M(r) approximately 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-200. Relative to protamine, the purified kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5, 8, and less than 0.1% activity with casein, histone H2B, and histone H1, respectively. The purified kinase phosphorylated several 40 S ribosome polypeptides. One of these polypeptides was identified as ribosomal protein S6 by N-terminal sequencing. About 2.5 mol of phosphoryl groups was incorporated per mole of ribosomal protein S6 following incubation of the 40 S ribosomes with the purified kinase. Following incubation with protein phosphatase 2A2, purified preparations of the protamine kinase were inactivated. These properties were identical to those of purified preparations of a protamine kinase from extracts of bovine kidney cytosol (Z. Damuni, G.D. Amick, and T.R. Sneed, 1989, J. Biol. Chem. 264, 6412-6418). Near identical peptide patterns were obtained following incubation of purified preparations of the microsomal and cytosolic protamine kinases with Staphylococcus aureus V8 proteinase. The results indicate that a form of the cytosolic protamine kinase is present in microsomes.     

Evolution of protamine P1 genes in primates

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine Pls indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10–13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24–29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39–44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest neighbor matrices and trees based on maximum parsimony indicates that PI genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas. are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine Pls are expressed in germ line rather than somatic cells. In contrast to the variability of the exon regions of the protamine P1 genes, the sequence of the single intron is highly conserved.     

A study of the interaction of histones with DNA using isopycnic centrifugation in metrizamide gradients.

Isopycnic sedimentation in metrizamide gradients has shown that mouse-liver histones bind co-operatively to both homologous and bacterial DNA's. However, at low input ratios of histone to DNA, two types of stable complex are formed, depending on the histone concentration. One complex contains half as much histone as DNA while the other contains approximately equal amounts of histone and DNA. At high input ratios of histone to DNA extra histone is bound giving complexes containing up to twice as much histone as DNA. Poly-L-lysine and protamine were also found to bind co-operatively to DNA.     

DNA supercoiling by core histone fractions and protamine

The nuclear extract isolated from late Drosophila melanogaster embryos has Mg2+-dependent DNA topoisomerase 1 and nuclease activities. The extract facilitates the closed circular duplex DNA supercoiling in the presence of calf histone fractions H2A, H2B, H3 and H4, and fish protamine but not HI histone.     

Improved gene expression using low molecular weight peptides produced from protamine sulfate

DNA condensation plays a key role in non-viral gene delivery by affecting gene transfection, nuclear targeting, and eventual gene expression efficiency. Theoretically, a DNA condenser with the appropriate DNA condensation ability but without affecting DNA dissociation from DNA condensates inside the cytoplasm should be a perfect carrier for gene delivery. Protamine is a natural DNA condensation agent and has been widely used in gene delivery. In this work, protamine was selectively digested enzymatically to produce low molecular weight protamine fragments (LMWPs) of various lengths and amino acid compositions. The DNA condensation ability and gene transfection efficiency of these LMWP peptides were tested. Compared to protamine, all the LMWP peptides showed lower DNA binding strength. However, some LMWP peptides demonstrated excellent DNA condensation ability and could form very compact DNA condensates with small particle size (∼100 nm). More interestingly, LMWP peptide-mediated in vitro gene delivery showed prolonged (up to 12 days) gene expression. Results from this study suggest that designing DNA condensers with appropriate and tunable DNA binding strengths and condensation abilities would be an effective means to improve gene expression and thus gene therapy efficiency. Since LMWP peptides have low immunogenicity, they would be safer than protamine for use in gene therapies. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 10, pp 1447–1455.     

Replacement of protamine by F1 histone during reactivation of fused human sperm nuclei

Summary Rabbit antisera, specific for the histones F1, F2a2, F2b, F3 and for protamine were used to monitor a possible transition from protamine towards somatic-type histones during sperm nucleus reactivation, following human sperm fusion with mouse fibroblasts.Mature human sperm nuclei were shown to contain the histones F2a2, F2b, F3 and protamine, but were missing F1 histone by immuno cytochemistry using the indirect fluorescence method. However, a gradual disappearance of protamine from fused sperm nuclei, could be observed during the first 24 h of reactivation. Subsequently, F1 histone could be detected in increasing concentrations in 60% of reactivated sperm nuclei, during the next four days.The shift from protamine towards F1 histone could also be visualized cytochemically via staining with brilliant sulphaflavine, which appears to discriminate between sperm nuclei on the basis of their F1 histone content.     

A novel label-free upconversion fluorescence resonance energy transfer-nanosensor for ultrasensitive detection of protamine and heparin

A novel label-free fluorescence nanosensor was developed for ultrasensitive detection of protamine and heparin based on fluorescence resonance energy transfer (FRET) between NaYF₄:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The FRET system was formed by the electrostatic adsorption of AuNPs on UCNPs, and the fluorescence of UCNPs was significantly quenched. When protamine was added to the mixture of UCNPs–AuNPs, the AuNPs interacted with protamine and then desorbed from the surface of UCNPs and aggregated, resulting in the recovery of the fluorescence of UCNPs. On the addition of both protamine and heparin, the FRET system formed owing to the stronger interaction between heparin and protamine than that with AuNPs, leading to a marked fluorescence quenching of UCNPs. The concentrations of protamine and heparin were proportional to the changes of the fluorescence of UCNPs. The linear response range was obtained over the concentration ranges of 0.02 to 1.2 μg/ml and 0.002 to 2.0 μg/ml with low detection limits of 6.7 and 0.7 ng/ml for protamine and heparin, respectively. Simultaneous measurement of protamine and heparin in human serum can be achieved, suggesting that the nanosensor can be used in a complex biological sample matrix.     

Vertebrate protamine gene evolution I. Sequence alignments and gene structure

Summary The availability of the amino acid sequence for nine different mammalian P1 family protamines and the revised amino acid sequence of the chicken protamine galline (Oliva and Dixon 1989) reveals a much close relationship between mammalian and avian protamines than was previously thought (Nakano et al. 1976). Dot matrix analysis of all protamine genes for which genomic DNA or cDNA sequence is available reveals both marked sequence similarities in the mammalian protamine gene family and internal repeated sequences in the chicken protamine gene. The detailed alignments of the cis-acting regulatory DNA sequences shows several consensus sequence patterns, particularly the conservation of a cAMP response element (CRE) in all the protamine genes and of the regions flanking the TATA box, CAP site, N-terminal coding region, and polyadenylation signal. In addition we have found a high frequency of the CA dinucleotide immediately adjacent to the CRE element of both the protamine genes and the testis transition proteins, a feature not present in other genes, which suggests the existence of an extended CRE motif involved in the coordinate expression of protamine and transition protein genes during spermatogenesis. Overall these findings suggest the existence of an avian-mammalian P1 protamine gene line and are discussed in the context of different hypotheses for protamine gene evolution and regulation.     

In situ protamine release: A versatile sample preparation method for the electrophoretic analysis of nuclear proteins on acid/urea-based gels

Protamine sulfate is used to release histones and other basic proteins from the DNA of chromatin. This phenomenon becomes the basis of a versatile method for analysis of the nucleic acid and protein composition of nucleoprotein samples, which is termed here in situ protamine release. When protamine is added to a nucleoprotein sample in 5% acetic acid and 8 m urea, at a concentration of 1.0%, ≥94% of the histones are released from the DNA of chromatin, comparable to the release of histones using sodium dodecyl sulfate. This makes in situ protamine release the method of choice for the analysis of acid-soluble proteins on acid/urea-based gels, where the DNA must be removed from the protein prior to electrophoresis. Compared to DNase I release or acid extraction, protamine release is found to be the simplest, most reliable, and most effective method for removing the acid-soluble proteins from DNA. Protamine is either added to the sample (very much like the detergent, sodium dodecyl sulfate), or electrophoresed through a gel containing nucleoprotein, thus displacing proteins in its path. A serendipitous advantage of protamine is that it can also serve as a carrier for the precipitation of dilute nucleoprotein samples with ethanol, and 3 mm Mg²⁺, to concentrate the nucleoprotein in preparation for analysis. A unique feature of the in situ protamine-release method is that the DNA is not lost or destroyed and can therefore be used for subsequent analysis.     

Identification of bull protamine disulfides

We have identified the disulfide cross-links in bull protamine by titrating intact bull sperm with dithiothreitol (DTT) and following the modification of each cysteine residue with tritiated iodoacetate. The derivatization of each cysteine was monitored by a combination of HPLC, peptide mapping, and protein sequencing. Analyses of total free sulfhydryls show that all seven of the bull protamine cysteines are cross-linked as disulfides in mature sperm. The first disulfide is reduced at a DTT:protamine cysteine (DTT:Cys) ratio of 0.3 and the last at a ratio of 2.0. Intra- and intermolecular disulfides were identified by correlating the reduction of specific disulfides with the dissociation of protamine from DNA in partially reduced sperm and sperm treated with N,N'-ethylenedimaleimide, a bifunctional disulfide cross-linking agent. Three intermolecular and two intramolecular disulfides were identified. The results of these experiments demonstrate that the amino- and carboxy-terminal ends of the bull protamine molecule are folded inward toward the center of the molecule and are locked in place, each by a single intramolecular disulfide bridge. Three intermolecular disulfides cross-link neighboring protamine molecules around the DNA helix in such a manner that the protamines cannot be dissociated from DNA without first reducing the interprotamine disulfides.