Michele Corzett

Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 ± 172 base pairs per second (0.30 µm s-1), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

DETECTION OF P2 PRECURSORS IN THE SPERM CELLS OF INFERTILE PATIENTS WHO HAVE REDUCED PROTAMINE P2 LEVELS

OBJECTIVE To determine whether the reduction in the protamine P2 content (increased P1/P2 ratio) reported in some infertile patients could result from incomplete processing of protamine P2 precursors. DESIGN Analysis of samples with a marked reduction in the protamine P2 content using polyacrylamide gel electrophoresis and subsequent detection of protamine P2 precursors through Western blot analysis. SETTING University departments and laboratories. PATIENT(S) One hundred eighty-four men undergoing an evaluation for infertility. MAIN OUTCOME MEASURE(S) Comparative Western blot analysis of nuclear sperm proteins using specific antibodies to protamine P1 and protamine P2. RESULT(S) After selection of the samples with a marked reduction of the protamine P2 content and subsequent analysis by Western blot, a small proportion of putative P2 precursors was detected in most samples, whereas a significant increase was detected in two of them. CONCLUSION(S) In some infertile men, a reduction in the protamine P2 content relative to protamine P1 (increased P1/P2 ratio) is detected concomitant with an increase in the amount of putative P2 precursors. This could represent the first report of incomplete processing of a nuclear sperm protein in humans.

DNA AND TOTAL PROTAMINE MASSES IN INDIVIDUAL SPERM FROM FERTILE MAMMALIAN SUBJECTS

The total amount of phosphorus and sulfur inside the nuclei of individual bull, stallion, hamster, human, and mouse sperm from fertile subjects has been measured using Particle Induced X-ray Emission (PIXE). Using the sulfur masses, we determined the total protamine (protamine 1 plus protamine 2) mass within the sperm nuclei of each species. Using the phosphorus masses, we determined the DNA mass present within the sperm nuclei of each species. The results reveal that although the relative proportion of protamine 1 to protamine 2 varies among the species examined, the total protamine mass to DNA mass ratio is similar in bull, stallion, hamster, and mouse sperm nuclei. In contrast, mature human sperm nuclei were found to contain significantly less protamine. This observation is consistent with other studies, which suggest that as much as 15% of the DNA in human sperm remain packaged by histones. Using the data obtained for bull sperm, the length of DNA that could be covered by each protamine 1 molecule in bull sperm has been estimated. Making the assumption that the size of the protamine 1 binding site on DNA is similar in the sperm of these species, the length of DNA covered by a single protamine 2 molecule also has been estimated.

DNA and protein content of mouse sperm. Implications regarding sperm chromatin structure.

Abstract A variety of biochemical and histochemical techniques have been used to compare the composition of chromatin in sperm nuclei isolated from the epididymides of five mouse strains. The DNA content was determined by phosphorus analysis, deoxyribose analysis, absorption spectroscopy at 260 nm, and cytomorphometry following gallocyanine chrome alum staining. All four methods indicate that the mouse sperm nucleus contains approx. 3.3 pg DNA and that the DNA content does not vary significantly among the strains tested. Three different techniques, quantitative amino acid analysis, absorption spectroscopy at 230 nm, and sperm head density analysis in cesium chloride, were used to determine the protein content. Sperm nuclei from each strain of mouse were found to have a protein to DNA ratio of 0.9 and a chromatin protein content of 3 pg/nucleus. Comparisons of the basic proteins by disc gel electrophoresis demonstrate that the sperm nuclei contain only protamine and lack significant levels of somatic histones or transition proteins. The sperm from each strain contained both mouse protamine variants and the relative distribution of the two proteins did not appear to differ among strains. Using this information, we have been able to draw certain conclusions regarding DNA-protamine interactions and the mode of DNA packaging in the sperm nucleus. The most important of these is that the DNA in the mouse sperm nucleus cannot be packaged in nucleosomes. The protamines in sperm chromatin do not function as structural proteins, providing a subunit core around which the DNA is wrapped, but appear to completely neutralize the phosphodiester backbone of the DNA molecule, thereby minimizing the repulsion between neighboring segments of DNA and allowing it to be condensed into a biochemically inactive particle of genetic information.

Raman Spectroscopy of DNA Packaging in Individual Human Sperm Cells distinguishes Normal from Abnormal Cells

Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA-packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in-vitro fertilization.

DNA condensation by protamine and arginine-rich peptides: Analysis of toroid stability using single DNA molecules

Both somatic cells and sperm have been shown to take up exogenous DNA, but the frequency of its integration is usually low. Scanning probe microscopy studies of sperm chromatin and synthetic DNA‐protamine complexes indicate that the coiling of DNA into toroidal subunits, a process initiated in the maturing spermatid to prepare its genome for delivery into the egg, can be mimicked by simply adding protamine to DNA in vitro. The increased resistance of DNA‐protamine complexes to nuclease digestion and their structural similarity to native sperm chromatin suggest that the packaging of DNA by protamine might offer a new approach for improving the efficiency of DNA uptake by sperm. Decondensation experiments performed with individual DNA molecules have provided a direct measure of the stability of toroids produced using salmon protamine and smaller arginine‐rich peptides. These experiments show that the arginine content of protamine‐related sequences can have a dramatic effect on their rate of dissociation from DNA. This technique and the information it provides can be used to identify protamine analogs that can be bound to DNA to increase the efficiency of its uptake by sperm and other cells. Mol. Reprod. Dev. 56:230–234, 2000. Published 2000 Wiley‐Liss, Inc.

Monitoring Dynamic Protein Expression in Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label‐free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein [MOG(1‐120)].

Protein and DNA contents in sperm from an infertile human male possessing protamine defects that vary over time.

Sperm from 2 semen samples collected 6 months apart from an infertile male and 3 semen samples collected over an 18‐month period from a fertile human male volunteer have been analyzed for their protamine and DNA content. Hup1M and Hup2b antibodies were used to detect the presence of protamines and protamine precursors in western blots of nuclear proteins isolated from pools of sperm. Phosphorus and sulfur contents, which can be used to estimate the nuclear DNA and protamine contents of sperm from fertile males, were measured within individual sperm heads from each semen sample by particle induced x‐ray emission (PIXE). The single‐cell data reveal no significant differences in the phosphorus and sulfur contents of sperm heads in the three semen samples obtained from the fertile male. For the initial semen sample produced by the infertile male, Western blot data show a normal complement of protamine 1, small amounts of mature protamine 2, and reveal large amounts of anti‐protamine 2 reactive proteins with electrophoretic mobilities similar to protamine 2 precursors. Data from PIXE show elevated levels of sulfur within sperm heads compared with sperm from the fertile male. Western blot data exhibit no evidence of protamines or protamine 2 precursors in the second semen sample produced by the infertile male. Data from PIXE suggest that these sperm are highly deficient in sulfur and protamines. These results show that the degree of maturation of sperm cells present in the semen of some infertile males can vary with time. Mol. Reprod. Dev. 50:345–353, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

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.

Cadmium concentrations in the testes, sperm, and spermatids of mice subjected to long‐term cadmium chloride exposure

BACKGROUND Exposures to cadmium have been reported to reduce male fertility and there are several hypotheses that suggest how reduced male fertility may result from incorporation of cadmium into sperm chromatin. The purpose of this study was to determine whether mice subjected to long-term intraperitoneal cadmium exposure incorporated cadmium into their sperm chromatin. METHODS Male mice were exposed to 0.1 mg/kg body weight cadmium in the form of CdCl2 via intraperitoneal injection once per week for 4, 10, 26, and 52 weeks and then sacrificed. The cadmium contents of the liver, testes, pooled sperm, and pooled spermatids from dosed and control animals were determined by atomic absorption spectroscopy. Cadmium and zinc contents in individual sperm and spermatid heads were determined by particle-induced x-ray emission. RESULTS Atomic absorption spectroscopy revealed that although cadmium accumulated in the liver and testes, cadmium was not detected in pooled sperm or spermatid samples down to minimum detectable limits of 0.02 microg/g dry weight. Particle-induced x-ray emission analyses did not show the presence of cadmium in any sperm or spermatid head down to minimum detectable limits of 15 microg/g dry weight. Particle-induced x-ray emission analyses also demonstrated that phosphorus, sulfur, and zinc concentrations in individual sperm and spermatid heads were not altered by exposure to CdCl2. CONCLUSIONS Because cadmium was not incorporated into sperm chromatin at levels above 0.02 microg/g dry weight, the data cast doubt on hypotheses that suggest that reduced male fertility may result from incorporation of cadmium into sperm chromatin.