猕猴桃叶DNA的AFLP分析

以猕猴桃幼叶为材料提取基因组DNA并进行AFLP扩增。对主要实验步聚包括DNA纯化、DNA的酶切、酶切片段与接头的连接、PCR扩增、电泳、以及银染等方面的反应参数进行比较和优选,初步摸索出适合于猕猴桃叶为材料的AFLP程序,并得到较为清晰可辩的AFLP指纹图谱,为开展猕猴桃遗传多态性研究和在分子水平上开展物种生物学分析提供一个实用的手段。     

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa

The aim of the present study was to evaluate the effect of selecting a sperm subpopulation by means of a discontinuous density gradient centrifugation (DGC) on the quality of ram thawed semen, and the relationships between sperm parameters assessed in unselected and in selected sperm samples with in vivo fertility after intrauterine artificial insemination (IUI) using unselected sperm samples. Semen samples from twenty males were collected by artificial vagina and cryopreserved following a standard protocol. After thawing, unselected sperm samples were used in an in vivo fertility trial and sperm motility (subjective and objective, assessed by means of CASA) and membrane and acrosomal integrities (microscopy) were evaluated on unselected and selected sperm samples. In addition, plasmalemma integrity (YO-PRO-1/PI), membrane fluidity (Merocyanine 540/YO-PRO-1), mitochondrial activity (Mitotracker Deep Red/YO-PRO-1), and DNA fragmentation index (%DFI) assessed by Sperm Chromatin Structure Assay (SCSA^®) were evaluated by flow cytometry before and after sperm processing using DGC. Results showed that DGC improved all sperm parameters significantly, except the %DFI, which increased after the selection procedure. No relationships were found between sperm parameters evaluated in unselected sperm samples and in vivo fertility. However, we found a positive correlation between spermatozoa with high membrane fluidity within the viable sperm population (VIABMerocyanine+) evaluated in selected sperm samples and in vivo fertility (r = 0.370, P = 0.019). In conclusion, our results suggest that selected spermatozoa represent a sperm subpopulation different to the unselected one that could be related with the in vivo fertility.     

Cytometry of breast carcinoma: significance of plo?dy balance and proliferation index

Image cytometry of DNA distribution in fine needle biopsies of breast carcinomas at first diagnosis was performed to see if there were significant differences in DNA histograms between patients having very different outcome but same tumor histological typing and similar therapy. Two groups of patients were considered retrospectively: the first (20 patients) with survival time shorter than 5 years and the second (20 patients) with survival time longer than 10 years. Seven benign tumors were used as controls. Ten plo?dy classes were defined. The frequencies of cells in those classes were used as independent features in a supervised multivariate analysis. The advantages of this approach was pointed out with respect to the four-type classification of Auer. The scattering of DNA histograms within the feature space showed that a subgroup of patients with poor prognosis was clearly separated from a subgroup of patients with good prognosis but both long survival patients and short survival patients were scattered in between. In order to replace the multivariate classification of histograms by a simpler approach, two parameters were computed which explained most of the scattering in the feature space: the plo?dy balance (difference between the percentages of euploid and aneuplo?d cells) and the proliferation index (percentage of cells between peaks). The scattergram of patients according to these parameters showed again that some DNA distributions were specific for either good or bad prognosis. But the separation was uncertain for seven short-survival patients and six long-survival patients. For six patients, the DNA distributions were very similar between long and short survival times. Those patients thus could not be separated even by means of discriminant analysis. The main conclusion of this study was that, for a significant number of patients, the objective multivariate classification of tumors DNA profiles is of little assistance to the pathologist who has to give a prognosis for the one patient under consideration.     

Antioxidative activity and protective effect of probiotics against high-fat diet-induced sperm damage in rats

In this study, antioxidant capability and protective effect of probiotics on reproductive damage induced by diet oxidative stress were investigated. Thirty male Sprague-Dawley rats were randomly divided into three groups with 10 rats in each group. The control group consumed a normal standard diet (5% fat, w/w). The other two treatment groups were fed with a high-fat diet (20% fat, w/w), and a high-fat diet supplemented with 2% probiotics (w/w), respectively. At the end of the experimental period, that is, after 6 weeks, rats were killed. Activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), contents of nitric oxide (NO) free radical and malondialdehyde (MDA) in serum and sperm suspension were examined. Sperm parameters including sperm concentration, viability, motility and DNA integrity were analyzed. The results showed that high-fat diet could induce oxidative stress, shown as significant increases in lipid peroxidation, NO free radical, significant decrease in activities of SOD, GSH-Px, significant reduction in sperm concentration, viability and motility, and damage in sperm DNA (P < 0.05), compared with the control group. These alterations were significantly reversed in the probiotics-supplemented group and had no significant difference in antioxidant capability, lipid peroxidation and sperm parameters compared with the control group. The percentage of sperm with DNA damage was significantly lower than the high-fat diet group and still higher than the control group, which means that probiotics could attenuate sperm damage to some extent. The present results indicated that dietary probiotics had antioxidant activity and the protective effect against sperm damage induced by high-fat diet to some extent.     

Morphometric differences between cytologically benign and malignant serous effusions

The morphometric differences between benign and malignant serous effusions, as diagnosed by standard cytologic criteria in 95 unselected cases (50 benign and 45 malignant), were studied using the IBAS semi-automated image analysis system, which calculates various parameters from tracings of cellular and nuclear outlines. Fourteen cases were also stained for cytokeratin proteins (with the CAM 5.2 antibody) by the immunoperoxidase technique and reanalyzed for positive cells. Significant differences were found for mean values between cytologically benign and malignant cases for cellular and nuclear areas, perimeters and maximum diameters, but not for two form factors. Some differences were enhanced in the CAM 5.2-stained cases. Real morphometric differences in samples of cells from benign and malignant cases are the basis of cytologic diagnosis. Fully automated diagnostic systems could operate on arbitrary threshold values, but there is considerable overlap in specimen means for all parameters between benign and malignant cases.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

Spin-on end-functional diblock copolymers for quantitative DNA immobilization

We demonstrate a simple means to covalently bond DNA to both hard (i.e., glass and silicon wafers) and soft (i.e., polymeric) substrates that provides quantitative and precise control of the DNA areal density. The approach is based on spin coating an alkyne-end-functional diblock copolymer, alpha-alkyne-omega-Br-poly( tBA- b-MMA), that self-assembles on both types of substrates as an ordered monolayer and thereby directs alkyne groups to the surface. Azido-functionalized DNA is covalently linked to the alkyne functionalized substrates by means of a "click" reaction between azide and alkyne groups. The density of immobilized DNA can be quantitatively controlled by varying the parameters used for spin-coating the copolymer film, that is, solution concentration and rotational speed, or by varying the copolymer molecular weight. We find the yield of the DNA coupling reaction to be dependent on the nature of the polymer underlying the reactive alkyne functional groups, being higher for more hydrophilic polymers.     

DNA interaction and nucleotide sequence cleavage of copper-streptonigrin

The copper-accelerated DNA binding and cleavage of streptonigrin have been investigated by 1H-NMR, ESR spectrometry and nucleotide sequence analysis. In the DNA breakage by the streptonigrin-Cu(II)-NADPH system, the somewhat preferred cleavage sites were several cytosine bases adjacent to purine bases such as GCGG(5'----3'), ACGC(5'----3') and GGCG(5'----3') sequences. The proton chemical shifts for the streptonigrin-Cu(I)-poly(dA-dT) complex demonstrated the interaction between the pyridine ring of the drug and the purine bases of the nucleic acid. Indeed, the temperature profile of adenine H-2 proton clearly showed the Tm to shift from 70 degrees C in the binary streptonigrin-poly(dA-dT) system to 75 degrees C in the ternary streptonigrin-Cu(I)-poly(dA-dT) system. The interaction of the streptonigrin-Cu(II) complex with DNA also induced the apparent change of ESR parameters. The tricyclic phenanthidium ring system including the copper chelate ring appears to significantly contribute to the present DNA interaction and cleavage of copper-streptonigrin.     

Single-stranded DNA binding and methylation by EcoP1I DNA methyltransferase

EcoP1I methyltransferase (M.EcoP1I) belongs to the type III restriction-modification system encoded by prophage P1 that infects Escherichia coli. Binding of M.EcoP1I to double-stranded DNA and single-stranded DNA has been characterized. Binding to both single- and double-stranded DNA could be competed out by unlabeled single-stranded DNA. Metal ions did not influence DNA binding. Interestingly, M.EcoP1I was able to methylate single-stranded DNA. Kinetic parameters were determined for single- and double-stranded DNA methylation. This feature of the enzyme probably functions in protecting the phage genome from restriction by type III restriction enzymes and thus could be considered as an anti-restriction system. This study describing in vitro methylation of single-stranded DNA by the type III methyltransferase EcoP1I allows understanding of the mechanism of action of these enzymes and also their role in the biology of single-stranded phages.     

Investigation of the interaction between ssDNA and 2-aminophenoxazine-3-one and development of an electrochemical DNA biosensor

An electrochemical method was used to probe the interaction between 2-aminophenoxazine-3-one (AP) and the short DNA sequence related to the hepatitis B virus (HBV), and an electrochemical DNA biosensor was developed. The voltammetric signals of AP have been investigated at bare glassy carbon electrode (bare GCE), hybrid double-stranded DNA-modified GCE (dsDNA/GCE), and single-stranded DNA-modified GCE (ssDNA/GCE) by means of differential pulse voltammetry (DPV), and the peak currents increased with respect to the order of electrodes. The extent of hybridization was evaluated on the basis of the difference between signals of AP with a probe before and after hybridization with the complementary sequence. Control experiments with noncomplementary were performed to test the selectivity of the biosensor. With this approach, a sequence of the HBV could be quantified over the range from 3.53 x 10(-7) to 1.08 x 10(-6) M, with a linear correlation of r = 0.9963 and a detection limit of 1.00 x 10(-7) M.     

An effective method of DNA isolation from the mature leaves of Gossypium species that contain large amounts of phenolic terpenoids and tannins

Purified and unstained nuclei were isolated from the leaves of several Gossypium species (diploid and tetraploid) by means of a citrate buffer (pH 5.0), Triton X-100 (5%), and a reducing sugar (1M glucose). DNA, previously unobtainable, was then extracted from the nuclei by conventional means. Comparisons of final DNA yield were made between three methods of purification: namely, the standardized ribonuclease procedure, hydroxyapatite chromatography and equilibrium density centrifugation in cesium chloride. The latter method produce the lowest, yet purest, yield of DNA for renaturation studies in Gossypium.     

Changes in DNA supercoiling during repair of gamma-radiation-induced damage

Enriched human B-lymphocyte and T-lymphocyte subpopulations were isolated by means of two Percoll density gradient centrifugation steps. Strand-break repair kinetics of 60Co gamma-irradiation-damaged DNA were obtained by applying a modified nucleoid sedimentation technique. Although no variations in the strand-break repair patterns of B-cells and T-cells within an individual could be detected, variations between individuals were evident. Changes in DNA supercoiling during the repair process were studied by means of ethidium bromide intercalation. The supercoiled conformation of DNA in peripheral lymphocyte subpopulations displayed similar undulating patterns within an individual, variations occurring between individuals. The results of this study indicate that alterations to the higher-order structure (supercoiling) of DNA are integral to the repair process.     

DNA and protein synthesis in cultured lens epithelial cells. II. Activity of synthetic effectors]

Cultivated bovine lens epithelium cells are highly susceptible to inhibitors of DNA-, RNA- and protein synthesis. The strict correlation between inhibition by puromycin of protein and DNA synthesis suggests that, in the cell system investigated, protein synthesis is essential for DNA synthesis to occur. Studies with actinomycin D have shown that in cultivated lens epithelium cells, part of protein synthesis is accomplished through a relatively long-lived mRNA. In long-term cultivation experiments, no further stabilization of mRNA, which is typical of lens fibre cells, could be demonstrated. There are indications that high doses of actinomycin D produce direct inhibition of DNA synthesis. By means of cytosine arabinoside a linear relationship was established between concentration of the effector and inhibition of DNA synthesis. Protein synthesis remains virtually unaffected even after high doses. The strong inhibition of DNA synthesis with protein synthesis continuing ("unbalanced growth") could not be utilized for the synchronization of lens epithelium cells, because it was only partly reversible after changing the medium and applying deoxycytidine.     

Diversification of exogenous genes in vivo in Neurospora

We have adapted the meiotic recombination hotspot cog of Neurospora crassa for shuffling exogenous DNA, providing a means of generating novel genes in situ from sequences introduced into chromosomes. Genes to be diversified are inserted between the his-3 locus and cog. Diversification crosses are heterozygous both for alleles of the exogenous DNA and for auxotrophic alleles of his-3. Progeny selected for ability to grow without histidine supplementation are enriched for exchange events within the exogenous DNA. Exchange events initiated by cog can propagate past DNA sequences mismatched for more than 370 bp and complete exchanges in patches of matched sequence as short as 24 bp, parameters that make the system suited for use in the directed evolution of genes for protein engineering. Here we demonstrate the system by shuffling human immunoglobulin kappa chain genes and also endoglucanase genes derived from different species of fungi.     

Duration time of a one-dimensional random walk as a function of the energies of the intermediate states: application for dissociation and relaxation processes in DNA hybrids.

Kinetic parameters of macromolecular systems are important for their function in vitro and in vivo. These parameters describe how fast the system dissociates (the characteristic dissociation time), and how fast the system reaches equilibrium (characteristic relaxation time). For many macromolecular systems, the transitions within the systems are described as a random walk through a number of states with various free energies. The rate of transition between two given states within the system is characterized by the average time which passes between starting the movement from one state, and reaching the other state. This time is referred to as the mean first-passage time between two given states. The characteristic dissociation and relaxation times of the system depend on the first-passages times between the states within the system. Here, for a one-dimensional random walk we derived an equation, which connects the mean first-passage time between two states with the free energies of the states within the system. We also derived the general equation, which is not restricted to one-dimensional systems, connecting the relaxation time of the system with the first-passage times between states. The application of these equations to DNA branch migration, DNA structural transitions and other processes is discussed.     

Sperm DNA fragmentation and oxidation are independent of malondialdheyde

Background  

There is clinical evidence to show that sperm DNA damage could be a marker of sperm quality and extensive data exist on the relationship between DNA damage and male fertility status. Detecting such damage in sperm could provide new elements besides semen parameters in diagnosing male infertility. We aimed to assess sperm DNA fragmentation and oxidation and to study the association between these two markers, routine semen parameters and malondialdehyde formation.     

Study of mechanisms of electric field-induced DNA transfection. III. Electric parameters and other conditions for effective transfection.

Electric parameters, osmolality, temperature, and pH of the suspending medium and the growth phase of cells, etc., are known to influence the efficiency of the pulsed electric field (PEF)-induced DNA transfection of cells. PEF-induced transfection of Escherichia coli JM105 by plasmid DNA PUC18, PUC19, PBR322, and PMSG has been used as a model system to establish quantitative relationships between these parameters and transfection efficiency. The main findings are summarized for experiments using unipolar square wave PEF. (a) For a given field strength (up to 6 kV/cm), the transfection efficiency (TE) was linearly dependent on the pulse width (up to 1 ms). (b) When field strength is fixed, Log [TE] correlated with the number of pulses applied. Similarly, when field duration was fixed, Log [TE] correlated with the number of pulses. (c) In the absence of MgCl2, TE showed a maximal value at 50 mM sucrose and was reduced by several fold at lower and higher sucrose concentrations. Cell survival was nearly constant in the range 1-300 mM sucrose. (d) E. coli in the early and mid-exponential growth phases was more susceptible to PEF for DNA transfection than it was in the stationary phase. (e) For a given set of electric parameters, TE was the highest at neutral pH and was greatly reduced at acidic and alkaline pH. (f) Increasing the temperature from 0 to 37 degrees C resulted in the reduction of TE by three orders of magnitude. This could reflect a rapid shrinking of pores at higher temperatures. (g) TE was inversely proportional to the square of the size of the plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of deoxyribonucleotide metabolism and its relation to DNA synthesis. Supercomputer simulation and model-system analysis.

A model system (1) was established to analyze purine and pyrimidine metabolism. This system has been expanded to include macrosimulation of DNA synthesis and the study of its regulation by terminal deoxynucleoside triphosphates (dNTPs) via a complex set of interactions. Computer experiments reveal that our model exhibits adequate and reasonable sensitivity in terms of dNTP pool levels and rates of DNA synthesis when inputs to the system are varied. These simulation experiments reveal that in order to achieve maximum DNA synthesis (in terms of purine metabolism), a proper balance is required in guanine and adenine input into this metabolic system. Excessive inputs will become inhibitory to DNA synthesis. In addition, studies are carried out on rates of DNA synthesis when various parameters are changed quantitatively. The current system is formulated by 110 differential equations.     

DNA image cytometry in the differential diagnosis of endometrial hyperplasia and adenocarcinoma

OBJECTIVE: To test the value of DNA image cytometry in the differential diagnosis of hyperplastic endometrial lesions and endometrial carcinoma on a series of 153 cases of simple hyperplasia (n = 71), complex hyperplasia (n = 28), complex atypical hyperplasia (n = 11) and endometrial adenocarcinoma (n = 43). STUDY DESIGN: Monolayer smears were prepared from three 50-micron-thick sections by a cell separation technique and were stained according to Feulgen. The DNA content of 250 epithelial cells, chosen randomly, was determined using a TV image analysis system (CM-1, Hund, Wetzlar, Germany). The DNA content of 30 lymphocytes served as an internal standard for the normal diploid value in every case. Different DNA cytometric parameters and the mean nuclear area were calculated. RESULTS: Cases of adenocarcinoma and complex atypical hyperplasia (n = 54) were defined as clinically "positive" as these patients are normally treated by hysterectomy. The remaining cases of simple and complex hyperplasia (n = 99) were interpreted as clinically "negative" as conservative therapy is usually preferred. Requesting a specificity of > 90%, high sensitivity rates were calculated for ploidy imbalance (94%), mean ploidy (91%), diploid deviation quotient (91%), DNA stemline ploidy (87%) and 2c deviation index (85%), based on suitable thresholds. Entropy (76%), 5c exceeding events (63%), mean nuclear area (48%) and 9c exceeding events (6%) revealed lower sensitivity values. 5c Exceeding events (P = .0117) and mean nuclear area (P = .0392) were helpful in differentiating between atypical hyperplasia and endometrial carcinoma as the data distribution was significantly different with the U test. CONCLUSION: Our results indicate that DNA single cell cytometry is a highly relevant tool in the differential diagnosis of endometrial lesions and could be used as a complementary diagnostic method, especially in histomorphologically difficult cases.