A rat H1t‐GFP transgene recapitulates endogenous H1t expression pattern in mouse

H1 histones bind to linker DNA. H1t (H1f6), a testis‐specific linker histone variant, is present in pachytene spermatocytes and spermatids. The expression of H1t histone coincides with the acquisition of metaphase I competence in pachytene spermatocytes. Here we report the generation of H1t‐GFP transgenic mice. The H1t‐GFP (H1 histone testis‐green fluorescence protein) fusion protein expression recapitulates the endogenous H1t expression pattern. This protein appears first in mid pachytene spermatocytes in stage V seminiferous tubules, persists in round spermatids and elongating spermatids, but is absent in elongated spermatids. The strong green fluorescence signal, due to the high abundance of H1t‐GFP, is maintained in spermatocytes after induction towards metaphase I through treatment with okadaic acid. Therefore, H1t‐GFP can be used as a visual marker for monitoring the progression of meiosis in vitro and in vivo, as well as fluorescence‐activated cell sorting (FACS) sorting of germ cells.     

The effect of manganese(II) on the structure of DNA/HMGB1/H1 complexes: electronic and vibrational circular dichroism studies

The interactions were studied of DNA with the nonhistone chromatin protein HMGB1 and histone H1 in the presence of manganese(II) ions at different protein to DNA and manganese to DNA phosphate ratios by using absorption and optical activity spectroscopy in the electronic [ultraviolet (UV) and electronic circular dichroism ECD)] and vibrational [infrared (IR) and vibrational circular dichroism (VCD)] regions. In the presence of Mn2+, the protein-DNA interactions differ from those without the ions and cause prominent DNA compaction and formation of large intermolecular complexes. At the same time, the presence of HMGB1 and H1 also changed the mode of interaction of Mn2+ with DNA, which now takes place mostly in the major groove of DNA involving N7(G), whereas interactions between Mn2+ and DNA phosphate groups are weakened by histone molecules. Considerable interactions were also detected of Mn2+ ions with aspartic and glutamic amino acid residues of the proteins.     

Purification and characterization of monomeric isocitrate dehydrogenase with NADP(+)-specificity from Vibrio parahaemolyticus Y-4.

NADP(+)-dependent isocitrate dehydrogenase [IDH: EC 1.1.1.42] was purified to electrophoretic homogeneity from Vibrio parahaemolyticus Y-4, and shown to be a monomeric protein of molecular weight 80,000 with a pI of 5.0. The amino acid composition and partial sequence at the N-terminus resembled those reported for other bacterial monomeric IDHs. Immunotitration with antisera to the monomeric and dimeric enzymes (antisera to IDH-II and -I of Vibrio ABE-1) showed an immunochemical distinction between the monomeric and dimeric IDHs, but there is similarity within the IDHs of each group. The circular dichroism spectra of the native and heat-denatured enzyme are also similar to those of monomeric IDH (IDH-II of Vibrio ABE-1). These monomeric IDHs are proteins comprising 17-22% helix and 25-35% beta-pleated sheet in the native state.     

Condensation of DNA by the C-terminal domain of histone H1. A circular dichroism study

The condensation of DNA by the C-terminal domain of histone H1 has been studied by circular dichroism in physiological salt concentration (0.14 M NaF). As the intact H1 molecule, its C-terminal domain induces the so-called psi state of DNA that is characterized by a nonconservative circular dichroism spectrum which is currently attributed to ordered aggregation of the DNA molecules. On a molar basis, intact H1 and its C-terminal domain give spectra of similar intensity. Neither the globular domain of H1 nor an N-terminal fragment, that includes both the globular and N-terminal domains, has any effect on the conservative circular dichroism of DNA. From these results it is concluded that the condensation of DNA mediated by histone H1 is mainly due to its C-terminal domain. The effect of the salt concentration and the size of DNA molecules on the circular dichroism of the complexes are also examined.     

Structure of DNA complexes with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions: 2. Vibrational circular dichroism spectroscopy

DNA complexes with nonhistone HMGB1 chromatin protein and histone H1 in the presence of manganese ions were studied using methods of absorption and circular dichroism spectroscopy in the infrared region. It was demonstrated that the method provides good results, even for solutions that contain large particles, which cause scattering in UV region. It was determined that manganese ions in the complex are able to coordinate not only to different chemical groups in DNA, but also to dicarboxylic acid residues of the HMGB1 protein, which stimulates DNA condensation and slightly weakens DNA-protein interactions in the complex.     

Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently

We analyzed the DNA-binding and DNA-bending properties of recombinant HMGB1 proteins based on a rice HMGB1 cDNA. Electrophoretic mobility shift assay demonstrated that rice HMGB1 can bind synthetic four-way junction (4H) DNA and DNA minicircles efficiently but the binding to 4H can be completed out by HMGA and histone H1. Conformational changes were detected by circular dichroism analysis with 4H DNA bound to various concentrations of HMGB1 or its truncated forms. T4 ligase-mediated circularization assays with short DNA fragments of 123 bp showed that the protein is capable of increasing DNA flexibility. The 123-bp DNA formed closed circular monomers efficiently in its presence, similar to that in an earlier study on maize HMG. Additionally, our results show for the first time that the basic N-terminal domain enhances the affinity of the plant HMGB1 protein for 4H DNA, while the acidic C-terminal domain has the converse effects.     

In vitro poly(ADP‐ribosyl)ated histones H1a and H1t modulate rat testis chromatin condensation differently

Rat testis H1 proteins were poly(ADP‐ribosyl)ated in vitro. The modifying product, poly(ADP‐ribose), was found covalently bound to each histone variant at various extents and exhibited distinct structural features (linear and short, rather than branched and long chains). Interest was focused on the somatic H1a, particularly abundant in the testis, as compared with other tissues, and the testis‐specific H1t, which appears only at the pachytene spermatocyte stage of germ cell development. These H1s were modified with poly(ADP‐ribose) by means of two in vitro experimental approaches. In the first system, each variant was incubated with purified rat testis poly(ADP‐ribose)polymerase in the presence of [³²P] NAD. In parallel, poly(ADP‐ribosyl)ated H1s were also prepared following incubation of intact rat testis nuclei with [³²P] NAD. In both experiments, the poly(ADP‐ribosyl)ated proteins were purified from the native forms by means of phenyl boronic agarose chromatography. The results from both analyses were in agreement and showed qualitative differences with regard to the poly(ADP‐ribose) covalently associated with H1a and H1t. Comparison of the bound polymers clearly indicated that the oligomers associated with H1a were within 10–12 units long, whereas longer chains (≤20 ADP‐R units) were linked to H1t. Individual poly(ADP‐ribosyl)ated H1s were complexed with homologous H1‐depleted oligonucleosomes (0.5–2.5 kbp) in order to measure their ability to condensate chromatin, in comparison with the native ones. Circular dichroism showed that the negative charges of the oligomeric polyanion, although present in limited numbers, highly influenced the DNA‐binding properties of the analyzed H1s. In particular, the poly(ADP‐ribosyl)ated H1a and H1t had opposite effects on the condensation of H1‐depleted oligonucleosomes. J. Cell. Biochem. 76:20–29, 1999. © 1999 Wiley‐Liss, Inc.     

Intrinsic structural disorder and sequence features of the cell cycle inhibitor p57Kip2.

The cell cycle inhibitor p57Kip2 induces cell cycle arrest by inhibiting the activity of cyclin-dependent kinases. p57, although active as a cyclin A-CDK2 inhibitor, is largely unfolded or intrinsically disordered as shown by circular dichroism and fluorescence spectra characteristic of an unfolded protein and a hydrodynamic radius consistent with an unfolded structure. In addition, the N-terminal domain of p57 is both functionally independent as a cyclin A-CDK2 inhibitor and unstructured, as demonstrated by circular dichroism and fluorescence spectra indicative of unfolded proteins, a lack of 1H chemical shift dispersion and a hydrodynamic radius consistent with a highly unfolded structure. The amino acid compositions of full-length p57 and the excised QT domain of p57 exhibit significant deviations from the average composition of globular proteins that are consistent with the observed intrinsic disorder. However, the amino acid composition of the CDK inhibition domain of p57 does not exhibit such a striking deviation from the average values observed for proteins, implying that a general low level of hydrophobicity, rather than depletion or enrichment in specific amino acids, contributes to the intrinsic disorder of the excised p57 CDK inhibition domain.     

A novel structure-specific endonuclease activity associated with polypyrimidine-tract binding (PTB) related protein from rat testis

Nucleases are involved in the processing of various intermediates generated during crucial DNA metabolic processes such as replication, repair, and recombination and also during maturation of RNA precursors. An endonuclease, degrading specifically single-stranded circular DNA, was identified earlier in rat testis nuclear extract while purifying a strand-transfer activity. We are now reporting the purification of this endonuclease, which is a monomeric 42 kDa protein, from rat testis to near-homogeneity. In addition to degrading single-stranded circular DNA, it nicks supercoiled plasmid DNA to generate relaxed DNA and does not act on linear single-stranded or double-stranded DNA. It also makes specific incisions at the single-strand/duplex junction of pseudo-Y, 3'- and 5'-overhangs and 3'- and 5'-flap structures. Other structures such as mismatch, insertion loop, and Holliday junction are not substrates for the testis endonuclease. In contrast to FEN1, the testis endonuclease makes asymmetric incisions on both strands of the branched structures, and free single-stranded ends are not necessary for the structure-specific incisions. Neither 5'-3' nor 3'-5' exonuclease activity is associated with the testis endonuclease. The amino acid sequences of tryptic peptides of the 42 kDa endonuclease show near-identity to polypyrimidine-tract binding protein (PTB) that is involved in the regulation of splicing of eukaryotic mRNA. The significance of the results on the association of structure-specific endonucleae activities with PTB-related protein is discussed.     

Acid and alkaline phosphatase activities in lithium treated testis, prostate and seminal vesicles of adult albino rats: evidence of duration and dose dependent response

Changes in the activities of acid and alkaline phosphatase were observed in the testis, prostate and seminal vesicle after the injection of lithium chloride at the doses of 100, 200 and 400 micrograms/100 g body weight/day for 7, 14 and 21 days. The studies indicate that 200 and 400 micrograms/100 g body weight for 14 days and 21 days showed a significant inhibition in the activity of acid phosphatase in all the above reproductive organs. There is a significant stimulation of alkaline phosphatase activity at the doses of 200 and 400 micrograms of lithium after 21 days of treatment in testis, prostate and seminal vesicle along with significant decrease in accessory sex organs weight in comparison to control animal. Therefore, it is evident that the effect of lithium on male reproductive organs mainly depends on the amount of the drug being injected and the duration of treatment to it.     

The histone H1 globular region. A possible supersecondary structure from spectroscopic and statistical studies

The central region of the basic nuclear protein, histone H1, has a highly conserved amino acid sequence and a globular structure which is still not known at atomic resolution. A possible secondary and supersecondary structure was predicted by combining experimental measurements of circular dichroism and NMR spectroscopy with a statistical method based on the amino acid sequence. Our results showed the protein fragment as being highly structured and having a total alpha-helix content of about 40%.     

Conformational peculiarities of nuclear protein HMGB1 and specificity of its interaction with DNA

Changes in the secondary structure of DNA and non-histone chromosomal protein HMGB1 were studied by circular dichroism and UV spectroscopy. We have demonstrated that the HMGBI protein is able to change its secondary structure upon binding to DNA. We estimated the proportion of bound protein on the assumption that there were two spectrally distinguishable forms of the HMGB1 in solution. The bound protein fraction decreases with increasing protein to DNA ratios (r) from 0.48 at r = 0.13 to 0.06 at r = 2.43. It has been shown that HMGB1 is able to induce considerable changes in DNA structure even when the amount of the protein directly associated with DNA is low.     

Physical properties of inner histone-DNA complexes.

Chicken-erythrocyte inner histone tetramer has been complexed with several natural and synthetic DNA duplexes by salt-gradient dialysis at various protein/DNA ratios. The resulting complexes, in low-ionic-strength buffer, have been examined by electron microscopy, circular dichroism, and thermal denaturation. Electron microscopy reveals nucleosomes (nu bodies) randomly arranged along DNA fibers, including poly(dA-dT)-poly(dA-dT), poly(dI-dC)-poly(dI-dC), but not poly(dA)-poly(dT). Circular dichroism studies showed prominent histone alpha-helix and "suppression" of nucleic acid ellipticity (lambda less than 240 nm). Thermal denaturation experiments revealed Tm behavior comparable to that of H1- (or H5-) depleted chromatin. Tm III and Tm IV increased linearly with G + C%(natural DNAs), but were virtually independent of the histone/DNA ratio; therefore, the melting of nucleosomes along a DNA chain is insensitive to adjacent "spacer" DNA lengths. This suggests that Tm III and Tm IV arise from the melting of different domains of DNA associated with the core nu body.     

Conformational effects of histones H1 on DNA structure. Comparative study between H1-1, H1(0), H5 and sperm holothuria phi 0

Interactions of mammalian histones, H1-1 and H1(0), phi 0 from holothuria sperm and H5 with poly(dA-dT), poly(dG-dC) and poly(dG-me5dC) were measured by a nitrocellulose filter binding assay and circular dichroism. All of the proteins bound to every one of the polymers, but differed in the extent of binding, which depended on the polynucleotide/protein ratios and ionic strength. The order of retention of all polymers was phi 0 greater than H1-1 greater than H1(0). The binding of H1(0) to poly(dG-me5dC) was remarkably sensitive to ionic strength. The proteins caused changes in the spectral features of the polynucleotides, but differed in the type and extent of the change. Complexes prepared with H1-1 and H1(0) with all polymers showed a strongly negative psi spectrum. Complexes of poly(dA-dT) and phi 0, at a protein/polynucleotide ratio of 0.4, displayed a distinctive spectrum, giving the appearance of a Z-like DNA spectrum, at low ionic strength. At higher ionic strength the complexes showed a psi spectrum. Complexes of poly(dG-me5dC) in the Z or B conformation with phi 0 showed spectral features characteristic of a mixture of a Z-like and a psi spectrum. In contrast, H5 reduced the Z-DNA spectral features in the presence of Mg, and produced an inversion of the B spectrum up to a polynucleotide/protein ratio of 0.24. These findings demonstrate the ability of different proteins to produce changes in the conformation of DNA. This may reflect the ability of chromatin to undergo differential condensation, depending on both the base composition of DNA and the type of H1 histone bound to it.     

Conformation of allantoicase in aqueous solution

1. The separation of 0.9-S and 10.8-S allantoicase with the aid of a 2H2O-H2O gradient was described. The resulting preparations were subjected to sedimentation equilibrium, optical rotatory dispersion (ORD), circular dichroism (CD) and infrared studies. 2. The molecular weight of 0.9-S allantoicase was determined to be about 1.1 x 10(4) g/mole in studies on the sedimentation behavior, the metal content and amino acid composition. The molecular weight of 10.8-S allantoicase was about 15.4 x 10(4) g/mole. 3. Optical rotatory dispersion, circular dichroism and infrared studies indicated that both molecules contain alpha-helix, beta conformation and random coil. A Cotton effect at 418 nm was ascribed to the asymmetric binding of Mn2+ to the enzyme. Competitive inhibitors decreased the absorption and circular dichroism bands at about 280 nm and 418 nm. These phenomena suggested that the aromatic groups may play an essential role in the binding of substrates and inhibitors by the Mn(2+)-enzyme complex. 4. Comparison of alpha-helical contents of metalloallantoicases showed that the enzymes with low helical contents exhibited high enzymic activities. 5. The nearly identical physicochemical behavior and specific enzymic activity of 0.9-S and 10.8-S allantoicase indicated that they are very similar in structure and conformation.     

Linear dichroism of the complex between the gene 32 protein of bacteriophage T4 and poly(1,N6-ethenoadenylic acid)

We performed linear dichroism measurements in compressed polyacrylamide gels on the complex between the helix-destabilizing protein of bacteriophage T4, GP32 and poly(1,N6-ethenoadenylic acid), which is used as a model system for single-stranded DNA. A strong hyperchromism for poly(1,N6-ethenoadenylic acid) in the complex indicates a strongly altered conformation. The positive linear dichroism in the wavelength region where the bases absorb must be explained by a strong tilting of the bases in the complex. This finding is in accordance with results from earlier studies, using electric birefringence and circular dichroism measurements. Our measurements show that the angle between the bases and the local helix axis is 42(+/- 6)degrees. In addition, a pronounced contribution from the tryptophan residues of GP32 can be recognized, indicating that several of these residues have a specific orientation in the complex. The sign of the dichroism due to the tryptophan residues is the same as that due to the DNA bases. However, it is not sufficient to assume that all the observed dichroism is due to one or more intercalated tryptophan residues and there must be one or more additional tryptophan residues that make an angle of less than 40 degrees with the local helix axis. Some possible structures of the DNA-protein complex are discussed.