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R Balhorn

Publications and source records attributed to R Balhorn.

At least 55 records · Page 3Linked to original sources
PubMed1990

Expression of the rat protamine 2 gene is suppressed at the level of transcription and translation.

We have compared the rat protamine 2 gene sequence (rP2) to that of the mouse protamine 2 (mP2) gene. The sequence encompasses 435 nucleotides of the coding region which includes an intron of 120 nucleotides, 461 nucleotides 5' to the coding sequence and 181 bases 3' to it. In the mouse the protamine 2 gene is abundantly transcribed and translated. The mP2 protein is initially synthesized as a precursor and then proteolytically processed to yield the mature protein. In contrast, in the rat, protamine 2 transcripts are present at 2-5% that found in the mouse and the mature protein has never been detected in spermatozoa. Although there is 92% nucleotide similarity between rat and mouse genes and 91% similarity of the predicted amino acid sequences, in vitro runoff transcription assays performed in either rat or mouse testis-derived transcription systems reveal that the rP2 promoter is only 30% as efficient a promoter as the mP2 promoter. Analyses of total sperm basic nuclear proteins extracted from epididymal sperm using a monoclonal antibody specific for protamine 2 suggest that the rat P2 mRNA is translated in vivo but is not properly processed. These results suggest that the lowered transcription rate and altered processing sites of the rat protamine 2 gene are likely to contribute to the lack of protamine 2 in rat spermatozoa.

Amino Acid Sequence↗
PubMed1989

Direct observation of native DNA structures with the scanning tunneling microscope.

Uncoated double-stranded DNA dissolved in a salt solution was deposited on graphite and imaged in air with the scanning tunneling microscope (STM). The resolution was such that the major and minor grooves could be distinguished. The pitch of the helix varied between 27 and 63 angstroms in the images obtained. Thus the STM can be useful for structural studies of a variety of uncoated and isolated biomolecules.

Animals↗
PubMed1988

Aberrant protamine 1/protamine 2 ratios in sperm of infertile human males.

Protamines were extracted from the sperm of fertile and infertile human males and the relative proportion of protamines 1, 2, and 3 were determined by scanning microdensitometry following electrophoresis of total protamine in polyacrylamide gels. The proportion of the three protamines was found to be similar in sperm obtained from different normal males. The distribution of protamines in sperm obtained from a select group of infertile males producing an elevated level of large sperm heads, in contrast, was different from that of the fertile males.

Densitometry↗
PubMed1987

Sequence-specific packaging of DNA in human sperm chromatin.

The DNA in human sperm chromatin is packaged into nucleoprotamine (approximately 85%) and nucleohistone (approximately 15%). Whether these two chromatin fractions are sequence-specific subsets of the spermatozoon genome is the question addressed in this report. Sequence-specific packaging would suggest distinct structural and functional roles for the nucleohistone and nucleoprotamine in late spermatogenesis or early development or both. After removal of histones with 0.65M NaCl, exposed DNA was cleaved with Bam HI restriction endonuclease and separated by centrifugation from insoluble nucleoprotamine. The DNA sequence distribution of nucleohistone DNA in the supernatant and nucleoprotamine DNA in the pellet was compared by cloning size-selected single-copy sequences and by using the derived clones as probes of nucleohistone DNA and nucleoprotamine DNA. Two clones derived from nucleohistone DNA preferentially hybridized to nucleohistone DNA, and two clones derived from nucleoprotamine DNA preferentially hybridized to nucleoprotamine DNA, which demonstrated the existence of sequence-specific nucleohistone and nucleoprotamine components within the human spermatozoon.

Chromatin↗
PubMed1987

Monoclonal antibodies to human protamines.

Nine monoclonal antibodies to human protamine, hup1a, 1b, 1c, 1d, 1e, 2a, 2b, A, and B, have been isolated and partially characterized. Enzyme-linked immunoabsorption assay analyses with HPLC-separated human protamine 1 and protamine 2+3 mixture identified five of these antibodies as specific for human protamine 1, two antibodies specific for protamine 2+3 mixture and two monoclonal antibodies reactive with all three human protamines. These findings were confirmed by immunoblotting. None of the antibodies reacted with poly-arginine or somatic histone proteins. Additional analyses with bull, boar, and ram protamines indicated that all of the monoclonal antibodies except hupA are specific for human protamine. HupA reacted with protamines from all of the species tested. These studies suggest that each of the antibodies recognizes one of at least four distinct epitopes on protamine.

Animals↗
PubMed1987

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not.

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.

Amino Acid Sequence↗
PubMed1987

High-performance liquid chromatographic separation and partial characterization of human protamines 1, 2, and 3.

A method for separating the three human protamines by HPLC of underivatized, total protamine extracts on a Nucleosil RP-C18 column is described. The identities of the three proteins have been confirmed by a combination of disc gel electrophoresis, amino acid composition, and primary sequence analysis. The results show that human protamine 3 elutes first, closely followed by protamine 2. Protamine 1 elutes later. The amino acid compositions and partial amino terminal sequences of human protamines 2 and 3 indicate that these two proteins are very closely related and suggest that they differ only by three amino-terminal amino acids.

Amino Acids↗
PubMed1986

A corrected primary sequence for bull protamine.

We have redetermined the primary sequence for bull protamine using HPLC peptide mapping and automated amino-acid sequencing techniques and report, on the basis of these findings, that the previously published amino-acid sequence for this protein is incorrect. The correct protamine sequence is 50 amino acids in length and differs from the original published sequence by the tripeptide -Cys-39-Arg-40-Arg-41-. Analyses of protamine tryptic peptides derived from nine diverse breeds of Bos tarus and Bos indicus indicate that this sequence is present in the protamine of each breed and that it does not represent a variant or mutation.

Amino Acid Sequence↗
PubMed1985

Application of HPLC to the isolation of molecular targets in dosimetry studies.

High-performance liquid chromatography (HPLC) methods are described which permit the rapid isolation of multiple target macromolecules from the tissues of animals exposed to chemical mutagens. DNA and selected chromosomal proteins are isolated in a simple two step separation scheme. Isolated nuclei are dissolved in 3 M guanidine hydrochloride and the DNA and chromosomal proteins separated on a TSK 3000 SW column. The DNA peak is retained for analysis and the chromatin proteins are dialyzed, lyophylized, and rechromatographed on a PRP-1 column to separate individual histones. Hemoglobin and albumin, two proteins that may prove useful for monitoring mutagen exposure, are isolated from 100 microL of blood by HPLC on a Poly Cat A cation exchange column. Using this approach, we have monitored the kinetics and dose response of adduct formation (and repair) to DNA, histone, hemoglobin and albumin in mice exposed to 7-bromomethylbenzanthracene and benzo[a]pyrene. The results of these studies are described and briefly discussed. Experiments with other mutagens demonstrate the limits to which DNA adduct quantification may be pushed using radioisotopes. Exposures to very high specific activity (200 Ci/mmole) benzo(a)pyrene have allowed DNA adduct quantification down to a few adducts per cell.

Animals↗
PubMed1984

DNA packaging in mouse spermatids. Synthesis of protamine variants and four transition proteins.

A comparison of the protein compositions of mouse late-step spermatids and cauda epididymal sperm has revealed that the relative distribution of the two amino acid sequence variants of mouse protamine differ markedly in spermatids and sperm. Sonication-resistant spermatids contain the two variants in a ratio of 1:1, while the ratio of these two proteins in cauda epididymal sperm is approx. 2:1. Labeling studies in vivo have shown that this difference is due, in part, to an asynchrony in the time of synthesis of the two protamine variants. Both proteins are synthesized in late-step spermatids, but synthesis of the tyrosine variant in sperm chromatin begins approximately one day before synthesis of the more predominant histidine variant. Analyses of the time of synthesis of protamine and the four transition proteins in late-step spermatids allowed us to estimate the spermatid stage in which these proteins are deposited on DNA and relate these events to the onset of sonication resistance in maturing spermatids. These results indicate that: (1) synthesis and deposition of protamine begins coincident with the onset of sonication resistance in early step 12 spermatids; (2) protamine deposition is complete by mid-step 15; and (3) synthesis of the transition proteins occurs coincident with protamine synthesis.

Animals↗
PubMed1982

A model for the structure of chromatin in mammalian sperm.

DNA in mammalian, and most vertebrate sperm, is packaged by protamines into a highly condensed, biochemically inert form of chromatin. A model is proposed for the structure of this DNA-protamine complex which describes the site and mode of protamine binding to DNA and postulates, for the first time, specific inter- and intraprotamine interactions essential for the organization of this highly specialized chromatin. In this model, the central polyarginine segment of protamine binds in the minor groove of DNA, crosslinking and neutralizing the phosphodiester backbone of DNA while the COOH- and NH2-terminal ends of protamine participate in the formation of inter- and intraprotamine hydrogen, hydrophobic, and disulfide bonds. Each protamine segment is of sufficient length to fill one turn of DNA, and adjacent protamines are locked in place around DNA by multiple disulfide bridges. Such an arrangement generates a neutral, insoluble chromatin complex, uses all protamine sulfhydryl groups for cross linking, conserves volume, and effectively renders the chromatin invulnerable to most external influences.

Amino Acid Sequence↗
PubMed1979

Separation of satellite DNA chromatin and main band DNA chromatin from mouse brain.

Using restriction endonucleases which preferentially digest mouse main band DNA and leave satellite DNA intact, we have isolated highly purified chromatin fractions containing only mouse satellite or main band DNA. Following the digestion of mouse brain nuclei with EndoR Alu I, main band DNA chromatin is selectively extracted with 10mM Tris, 10mM EDTA. Satellite DNA chromatin is subsequently extracted from the nuclear pellet with Tris-3M urea and further purified on sucrose gradients. Chromatin extracted from digested nuclei with Tris-EDTA contains only main band DNA and has a molecular weight lower than 2 x 10(6). Chromatin fractions obtained from the lower regions of sucrose gradients of the Tris-Urea extracts contain 40--95% satellite DNA and have a molecular weight of 6 to 8 x 10(6). Both the satellite DNA and main band DNA chromatins contain all five histones and have a protein to DNA ratio of 1.3 to 1.

Animals↗
PubMed1977

Mouse sperm chromatin proteins: quantitative isolation and partial characterization.

Conditions are described that permit the quantitative extraction of chromatin proteins from the epididymal sperm of the mouse. These proteins have been isolated free of contaminating tail proteins following removal of the tails with cetyltrimethylammonium bromide (CTAB). Without this treatment, numerous acid-soluble tail proteins coextract with the nuclear proteins isolated from partially purified heads. The proteins isolated in this manner do not require prior modification with iodoacetamide and show no evidence of proteolytic degradation. In acid-urea polyacrylamide gels, 99% of the sperm protein migrates as one electrophoretic band. Evidence is presented that suggests that this single band contains two protamine-like proteins.

Amino Acid Sequence↗
PubMed1975

Phosphorylation of the lysine-rich histones throughout the cell cycle.

The phosphorylating of the lysine-rich histone at various stages in the cell cycle has been studied. In rapidly dividing cell populations the lysine-rich histone is phosphorylated rapidly after synthesis and more slowly once bound to the chromosome. The half-life of hydrolysis of such interphase phosphorylation in 5 hr except during mitosis when the phosphata hydrolysis increases almost three-fold. During mitosis there is extensive phosphorylation at sites different from those phosphorylated during interphase and a smaller measure of sites common to both mitotic and interphase cells. The sites of mitotic phosphorylation are most critically distinguished from those phosphorylated in interphase by the rapidly hydrolysis of M-phase phosphohistone when the cells divide and enter the G1 phase of the cell cycle.

Autoradiography↗
PubMed1975

The phosphorylation region of lysine-rich histone in dividing cells.

N-Bromosuccinimide cleavage of in vivo 32P-labelled lysine-rich histone isolated from rapidly dividing cells has been studied. N-Bromosuccinimide cleaves F1-histone into two fragments, a small N-terminal piece and a larger C-terminal portion. The phosphate-induced microheterogeneity and associated radioactivity which has been linked to cell replication, is found in the carboxyterminal fragment, No phosphorous is found associated with the amino-terminal fragment when histone phosphorylation is associated with cell division. The specific tryptic phosphopeptides obtained from in vivo labelled F1 are clearly different from those obtained from in vitro incubations of free F1-histones and cytoplasmic protein kinase.

Animals↗