控温条件下C3、C4草本植物碳同位素组成对温度的响应

采取人工控制实验,探讨了6种C3、C4草本植物在昼/夜温度指标为20/12℃!36/28℃的范围内植物碳同位素组成(δ13C)及其对温度变化的响应,并结合植物比叶面积(SLA)、胞间CO2浓度(ci)与环境CO2浓度(ca)的比值、碳同化率(净光合速率Pn/胞间CO2浓度ci)等光合生长指标对植物δ13C的影响进行了分析。结果表明:所有C3、C4植物样品的δ13C值分别变化在-28.3‰!-32.1‰和-14.4‰!-17.6‰之间;在C3植物中,油菜δ13C值分布范围最集中,位于-31.1‰!-32.1‰之间;C4植物中,谷子δ13C值分布范围最窄。在控制的温度范围内,3种C3植物的平均δ13C值随温度升高而显著变低,而C4植物δ13C平均值与温度呈先增大后减小的抛物型关系,但线性回归结果未达到显著水平(P0.05)。单个植物种的δ13C值对温度的响应不同,茄子、高粱的δ13C值与温度呈线性负相关,其它4种植物与温度均呈二次抛物线关系,这可能与不同植物种具有不同的光合最适温度以及植物δ13C分馏对温度变化的敏感程度不同有关。     

温度和无机碳浓度对龙须眼子菜（Potamogeton pectinatus）碳同位素分馏的影响

温度和无机碳浓度是沉水植物碳同位素分馏的重要影响因素.通过在16、19、22、25、28、31℃ 6个温度条件和溶解无机碳浓度(DIC)为0.0001mol·L-1和0.0001mol·L-1两种条件下培养沉水植物--龙须眼子菜(Potamogeton pectinatus),获得了不同条件下生长的龙须眼子菜植物样品,随后进行了定量和碳同位素组成(δ13CP)分析.分析结果表明,随生长温度的升高,龙须眼子菜碳δ13CP均升高,具有明显的温度相关性(R2＞0.90).在DIC浓度为0.0001mol·L-1时,随着生长温度的升高, δ13CP从-14.83‰增加至-13.47‰;而在DIC浓度为0.001mol·L-1时, δ13CP从-18.56‰增加至-15.15‰.同一生长温度条件下, DIC浓度对δ13CP也有明显影响, δ13CP随DIC浓度增高而降低.在不同生长温度条件下, DIC浓度对δ13CP的影响大小随生长温度的增高而降低,在温度为16℃时,两个DIC浓度下的δ13CP相差3.73‰;而在温度为31℃时,这一差值减少为1.68‰.同时,通过建模进行计算,获得了不同温度下龙须眼子菜(P. pectinatus)碳同位素分馏与初始DIC浓度的关系,并对有关的参数的意义进行了探讨.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

溶液中d(AT)_6的构象转化

 本文报道了溶液中d(AT)_6构象研究的结果。探讨了d(AT)_6的物化特征。d(AT)_6在0.05mol/L NaCl和4.5 mol/L NaCl中的UV和CD谱是典型的Z-和B-型DNA谱,UV熔化曲线形状依赖于盐的浓度。当盐浓度低于0.05 mol/L NaCl时,曲线有负斜率;而盐浓度大于0.05mol/L NaCl时,曲线的斜率变为正,即随盐浓度增加,T_m值增加。在链浓度的依赖性基础上,计算了两种构象的△H和△S。     

煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较

目的:在生物浸出中,微生物群落结构分析有着重要意义,而群落分析的基础是提取纯度高、损失少的基因组DNA。为了解决这一问题,本实验通过比较两种较常用的DNA提取方法,煮沸裂解法和试剂盒法,寻找一种灵敏、快速、经济实用的制备浸矿细菌基因组DNA的方法。方法:分别用煮沸裂解法和试剂盒法提取6种浸矿菌的基因组DNA,从所提取的基因组DNA浓度、纯度、回收率和对PCR扩增反应的影响方面比较了两种方法的提取效果;用两种方法来处理不同浓度梯度的一种菌,通过实时定量PCR来比较两种方法的灵敏性。结果:相同处理量(108个)的革兰氏阳性菌(1株)、革兰氏阴性菌(4株)、古菌(1株)经两种方法提取的基因组DNA差异较大,煮沸裂解法所得的6组基因组DNA更纯,其OD260/OD280的值更接近1.8-2.0(纯DNA的OD260/OD280在1.8-2.0之间),前者所提DNA回收率最大可达后者的16.7倍;煮沸裂解法只需较少菌(102个)便能让实时定量PCR检测到所提DNA模板浓度,比试剂盒法灵敏。结论:两种方法提取的基因组DNA均可用于后续的PCR扩增,此外,前者提取的DNA浓度随细菌浓度增加而呈线性增大,而后者随菌浓度增大,所提DNA量增加有限,因此,在生物浸出中微生物基因组DNA的提取可直接采用简单快速的煮沸提取法,为实验节约成本和时间。     

对人眼晶状体α－晶体蛋白聚集体的准弹性激光散射研究

以准弹性激光散射技术,研究了人眼晶状体内α－晶体蛋白聚集体的扩散系数和流体力学半径,以及其弥散性随温度和浓度的变化。所研究的α－晶体蛋白用分离的方法分别取自成人和胚胎眼晶状体。研究结果表明,人眼α－晶体蛋白在一定浓度和温度下可形成聚集体,且其聚集体半径随α－晶体蛋白的浓度近乎线性地增大,随温度的增加而变小。对α－晶体蛋白溶液（５０ｍｍｏｌ／Ｌ磷酸脂缓冲液）的弥散度分析表明,溶液中有两种不同粒径的散     

脲对果菠萝蛋白酶活力与构象的影响

本文用荧光光谱,紫外差示光谱和CD谱研究果菠萝蛋白酶在不同浓度的脲溶液中的构象及酶活力的变化情况。酶的荧光强度随脲浓度增大而明显增加,8mol/L脲使荧光强度增强65％,发射峰出现红移。差示谱表明在232nm和288nm出现二个正峰,它们均随脲浓度增大而加剧,前者与主链构象变化有关,而后者与生色基团(Trp、Tyr)的微环境变化相关。CD谱表明:天然酶在208nm和225nm处有二个负峰,脲变性后,225nm的负峰基本上不随脲浓度增大而变化,但208nm峰则明显发生变化并逐渐出现红移,6mol/L以上此峰则完全消失。     

盐分和温度对盐节木种子萌发的影响

采用多因素交叉试验设计,探讨了不同盐分类型、盐分浓度以及温度对盐节木(Halocnemum strobilaceum)种子萌发的影响。结果表明:15~25℃范围是盐节木种子萌发的最适温度范围,5~15℃和25~35℃的变温不利于种子萌发;最适温度下,单盐和土盐浓度分别小于0.96%和0.6%(电导率小于9.375dS/m)时可促进种子萌发,当浓度分别高于0.96%和0.6%(电导率大于9.375dS/m)时,单盐处理下的种子萌发率随浓度升高而降低,土盐处理下的种子萌发率随浓度升高表现为先升高后下降,但2种盐处理下的平均种子萌发率差异不显著(P>0.05),非最适温度下,2种盐处理下的平均种子萌发率差异显著(P<0.05)。由此得出:盐分浓度和温度是影响盐节木种子萌发的关键因素,土盐对种子萌发的抑制作用较单盐的小。     

cDNA微阵列制作的优化

为了优化筛检cDNA微阵列中靶基因的最适长度、浓度及点样溶液的种类,设计持家基因betaactin和GAPDHRT PCR3对引物,产物长度在189～1078bp之间,以乙肝病毒DNA片段为阴性对照,扩增纯化后分别溶于3×SSC、50%DMSO及0.5mol/L碳酸盐缓冲液(pH=9.0)中,调整浓度分别为0.5μg/μL、1.0μg/μL和1.5μg/μL,比较上述不同条件的杂交结果。结果表明,杂交具有较好的特异性,阴性对照(乙肝病毒)和空白对照(点样溶液)均未见杂交信号;3种长度的同一靶基因杂交信号强度无明显差别(betaactinP=0.378;GAPDHP=0.866);3种点样溶液中以50%DMSO杂交信号最好,较强且均匀一致(P=0.0001),其余2种差异不显著(P=0.142);3种浓度靶基因杂交信号差异不显著(P=0.648),浓度高者信号略强。短片段靶基因(200bp左右)可获得与长片段靶基因(1000bp以上)一样较好的杂交信号,点样溶液以50%DMSO效果最好,靶基因浓度为0.5μg/μL时即可得到较好的杂交结果。     

BSA与α-MnO2及δ-MnO2间的界面吸附作用

比较研究了牛血清白蛋白(BSA)与α-MnO2和δ-MnO2的界面吸附作用及其影响因素。结果表明,BSA在两种MnO2颗粒物表面有明显的吸附,且δ-MnO2比α-MnO2对BSA的吸附能力略强。pH3.8~8.0范围内,BSA在α-MnO2和δ-MnO2上的吸附率随pH的升高而减小,pH3.8条件下,α-MnO2上的吸附率为88.2%,δ-MnO2上的吸附率为94.0%。BSA在α-MnO2和δ-MnO2上的吸附量均随BSA浓度的增加而增大,吸附率随NaCl浓度的增加而减小。BSA在α-MnO2上的吸附具有很高的不可逆性,δ-MnO2上的吸附完全不可逆。吸附过程中BSA发生解螺旋作用,引起结构熵增大。     

Role of small loops in DNA melting

Short melted regions less than 100 base pairs (bp) in length are rarely found in the differential melting curves (DMC) of natural DNAs. Therefore, it is supposed that their characteristics do not affect DNA melting behavior. However, in our previous study, a strong influence of the form of the entropy factor of small loops on melting of cross-linked DNAs was established (D. Y. Lando, A. S. Fridman et al., Journal of Biomolecular Structure and Dynamics, 1997, Vol. 15, pp. 141-150; Journal of Biomolecular Structure and Dynamics, 1998, Vol. 16, pp. 59-67). Quite different dependencies of the melting temperature on the relative concentration of interstrand cross-links were obtained for the loop entropy factors given by the Fixman-Freire (Jacobson-Stockmayer) and Wartell-Benight relations. In the present study, the influence of the entropy factor of small loops on the melting of natural DNAs, cross-linked DNAs and periodical double-stranded polynucleotides is compared using computer simulation. A fast combined computational method for calculating DNA melting curves was developed for this investigation. It allows us to assign an arbitrary dependence of the loop entropy factor on the length of melted regions for the terms corresponding to small loops (less than tau bp in length). These terms are calculated using Poland's approach. The Fixman-Freire approach is used for long loops. Our calculations have shown that the temperature dependence of the average length of interior melted regions (loops) has a maximum at T approximately T(m) (T(m) is the DNA melting temperature) in contrast to the dependence of the total average length of melted regions, which increases almost monotonously. Computer modeling demonstrates that prohibition of formation of loops less than tau base pairs in length does not markedly change the DMC for tau < 150 bp. However, the same prohibition strongly affects the average length of internal melted regions for much smaller tau's. The effect is already noticeable for tau = 1 bp and increases with tau. A tenfold increase in the entropy factor of all loops with length less than tau bp causes a noticeable alteration of the DMC for tau > or = 30 bp. It is shown that DMCs are identical for the Wartell-Benight and for the Fixman-Freire (Jacobson-Stockmayer) form of the loop entropy factor. However, for low degree of denaturation, the average length of internal melted regions is 40% lower for the Wartell-Benight form due to the fluctuational opening of short AT-rich regions less than 10 bp in length. The same calculations carried out for periodical polynucleotides demonstrate a much stronger difference in melting behavior for different forms of entropy factors of short loops. The strongest difference occurs if the length of stable GC-rich and unstable AT-rich stretches is equal to 30 bp. However, the comparison carried out in this work demonstrates that the entropy factor of short loops influences melting behavior of cross-linked DNA much stronger than of unmodified DNA with random or periodical sequences.     

Electric birefringence of restriction enzyme fragments of DNA: optical factor and electric polarizability as a function of molecular weight

The electric birefringence of restriction enzyme fragments of DNA has been investigated as a function of DNA concentration, buffer concentration, and molecular weight, covering a molecular weight range from 80 to 4364 base pairs (bp) (6 × 10⁴–3 × 10⁶ daltons). The specific birefringence of the DNA fragments is independent of DNA concentration below 20 μg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent. At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field at which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law region the rise of the birefringence is accurately symmetrical with the decay for fragments ≤ 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated from a 1/E extrapolation of the high field birefringence data is ?0.028, independent of molecular weight; if a 1/E² extrapolation is used, the optical factor is ?0.023. The induced polarizability, calculated from the Kerr constant and the optical factor, is proportional to the square of the length of the DNA fragments, and inversely proportional to temperature. Saturation curves for DNA fragments ≤ 161 bp can be described by theoretical saturation curves for induced dipole orientation. The saturation curves of larger fragments are broadened, because of a polarization term which is approximately linear in E, possibly related to the saturation of the induced dipole in high electric fields. This “saturated induced dipole” is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp sample is decreased 6°C in an electric field of 8 kV/cm, because the lower charge density of the coil form of DNA makes it more stable in an electric field than the helix form.     

Ribosomal RNA genes ofPhaseolus coccineus V. Relationship between rDNA phenotype and somatic differentiation

Summary Size variations in the intergenic spacer of ribosomal DNA were detected between individual plants of openly pollinatedPhaseolus coccineus. Eleven days after sowing, two plant samples were examined: slowly developing plants with a length less than 40 cm; and fast developing plants with a length greater than 70 cm. The two samples were characterized by different plant weight and, at maturity, by highly distinctive seed yield. They also exhibited distinct patterns of protein expression as analyzed by 2-D electrophoresis. In particular a 38 kDa protein, related to malate dehydrogenase on the basis of its N-terminal sequence, was present at higher concentration and higher activity levels in fast developing plants. Intergenic spacer length variants were detected in both samples at approximately 180 bp intervals. More than one spacer length variant was present in each individual plant. At least 13 different intergenic spacer hybridization patterns were in fact detected: some patterns occurred equally in both slowly and fast developing samples while the majority of patterns was significantly different between the two samples.Abbreviations FDP fast developing plants - IGS intergenic spacer - MDH malate dehydrogenase - rDNA SLV spacer length variant of ribosomal DNA - SDP slowly developing plants     

Influence of fragment size on DNA quantitation using DNA-binding proteins and a sensor-based analytical system: applications in the testing of biological products.

A novel immunoassay system which rapidly quantifies picogram levels of total DNA was characterized with respect to the effects of DNA length. Nine chromatographically purified HaeIII restriction fragments of phi X174 were tested. Assay performance was found to be dependent on both the amount and length of DNA present in the sample. DNA fragments longer than 100 base pairs (bp) could be quantitatively detected with this system. Fragments shorter than 100 bp inhibited assay performance and thus could be detected through the use of inhibition studies; however, only qualitative information could be obtained. DNA fragments approximately 10 nucleotides in length had no apparent effect on assay performance. The size of the binding site (number of bases) required for each DNA-binding protein to bind to a nucleic acid fragment is suggested as an explanation for the observed influence of DNA size on assay performance. The total DNA assay was used in conjunction with a Pharmacia FPLC system to characterize the size distribution and amount of DNA in two partially purified biopharmaceutical samples. The results indicate that the majority of residual DNA in these samples is less than 600 bp in length. This technique can be used to rapidly determine the DNA size distribution in an in-process or final product biopharmaceutical sample. This data can then be used in process design and optimization for removal of residual DNA in biological products.     

The label free DNA sensor using a silicon nanowire array

Biosensors based on silicon nanowire (Si-NW) promise highly sensitive dynamic label free electrical detection of various biological molecules. Here we report Si-NW array electronic devices that function as sensitive and selective detectors of as synthesized 2D DNA lattices with biotins. The Si-NW array was fabricated using top-down approach consists of 250 nanowires of 20 μm in length, equally spaced with an interval of 3.2 μm. Measurements of photoresistivity of the Si-NW array device with streptavidin (SA) attached on biotinylated DNA lattices at different concentration were observed and analyzed.. The conductivity in the DNA lattices with protein SA shows significant change in the photoresistivity of Si-NW array device. This Si-NW based DNA sensor would be one of very efficient devices for direct, label free DNA detection and could provide a pathway to immunological assays, DNA forensics and toxin detection in modern biotechnology.     

Electrical conduction through nerve and DNA

The aim of the present study was to analyse electric resistivity at different ambient temperatures between 300 to 20K in the frog sciatic nerve and salmon sperm DNA. When the electrical contacts were leaned just into the sciatic nerve, an increase of the sciatic nerve resistivity was observed for 240 K < T < 300 K and a rise of electrical conductivity was apparent below 240 K. This dependence is generally associated with a semiconductor behaviour. Once the sciatic nerve temperature was driven below 250K, the resistivity abruptly decreased and then at temperatures lower than 234 K, it remained constant and close to one tenth of its ambient temperature value. By contrast, when the electrical contacts were leaned into Salmon sperm DNA, the resistivity remained constant between 300K to 20K, showing a high electrical stability at low temperature. Thus, we report the existence of a new form of electric conductivity in the sciatic nerve at low ambient temperature, which in turn has many electric similarities with inorganic or organic superconductors, whereas temperature failed to alter DNA electrical properties until 20K.     

High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature

Pulsed-field gel electrophoresis (PGF) subjects DNA alternately to two electrical fields to resolve DNA ranging from 10,000 base pairs (10 kb) to 10,000 kb in size. The separations are quite sensitive to a variety of experimental variables. This makes it critical to have a wide range of reliable size standards. A technique is described for preparing mixtures of bacteriophage DNA oligomers that span a size range from monomer to more than 30-mer. The relationship between size and mobility of oligomers of different bacteriophage DNA monomers is generally self-consistent. Thus, these samples can serve as primary length standards for DNAs ranging from 10 kb to more than 1500 kb. They have been used to estimate the size of the chromosomal DNAs from various Saccharomyces cerevisiae strains and to test the effect of gel concentration and temperature on PFG. DNA resolution during PFG is slightly improved in agarose gels with small pore sizes, in contrast to continuous electrophoresis where the opposite is observed. PFG mobility is surprisingly sensitive to changes in the running temperature.     

Influence of DNA length on spermine-induced condensation. Importance of the bending and stiffening of DNA

Using DNA restriction fragments of 258 to 4362 base-pairs, we have investigated the influence of the DNA length on the condensation process induced by spermine, with the aid of electric dichroism measurements. The 258- and 436 bp fragments condensed into rod-like particles, while the fragments of 748 bp or more condensed into torus-shaped particles. Our results suggest that a DNA molecule longer than the circumference of the toroids observed previously (680 bp) is required to serve as a nucleus for the growth of the condensed particles. The toroids were more stable in the electric field than the rod-shaped particles, suggesting that rapid fluctuations of the bound spermine counterions can provide one of the main attractive forces yielding to the condensation process. Relaxation time data for the 436 bp fragment revealed that the structure of DNA was altered at a spermine concentration as low as one-tenth of that required for condensation: the DNA became bent in the presence of spermine. Moreover, the field strength dependence of the relaxation times, as well as the fitting of the decay curves at 12.5 kV/cm, showed an increase of the stiffness of the DNA double helix upon spermine addition. We estimated that, in the case of DNA condensation by spermine, a decrease in the measured persistence length may occur, irrespective of the DNA flexibility, owing to the bending of the DNA molecule.     

Influence of DNA length on spermine-induced condensation: Importance of the bending and stiffening of DNA

Using DNA restriction fragments of 258 to 4362 base-pairs, we have investigated the influence of the DNA length on the condensation process induced by spermine, with the aid of electric dichroism measurements. The 258- and 436 bp fragments condensed into rod-like particles, while the fragments of 748 bp or more condensed into torus-shaped particles. Our results suggest that a DNA molecule longer than the circumference of the toroids observed previously (680 bp) is required to serve as a nucleus for the growth of the condensed particles. The toroids were more stable in the electric field than the rod-shaped particles, suggesting that rapid fluctuations of the bound spermine counterions can provide one of the main attractive forces yielding to the condensation process. Relaxation time data for the 436 bp fragment revealed that the structure of DNA was altered at a spermine concentration as low as one-tenth of that required for condensation: the DNA became bent in the presence of spermine. Moreover, the field strength dependence of the relaxation times, as well as the fitting of the decay curves at 12.5 kV/cm, showed an increase of the stiffness of the DNA double helix upon spermine addition. We estimated that, in the case of DNA condensation by spermine, a decrease in the measured persistence length may occur, irrespective of the DNA flexibility, owing to the bending of the DNA molecule.     

A 23Na-NMR study of sodium-DNA interactions in concentrated DNA solutions at low-supporting electrolyte concentration

Aqueous solutions of DNA fragments with a contour length (500 A) near the persistence length at DNA concentrations ranging from 10 to 290 mg/mL solvent and a constant supporting electrolyte concentration of 0.01 M (predominantly NaCl) were examined by 23Na-nmr spectroscopy at temperatures of 20, 40, and 60 degrees C. Over the higher portion of this concentration range (greater than 100 mg/ml) the DNA solutions undergo a complex series of transitions between different anisotropic, liquid crystalline phases (T. E. Strzelecka and R. L. Rill, Biopolymers, in press). Counterions in solutions of strong polyelectrolytes are usually described in terms of a two-state model as free or "bound" (influenced by the electrostatic field of the polyanion). The longitudinal relaxation rate (R1 = 1/T1) at all DNA concentrations decreased with increasing temperature, demonstrating fast exchange between free and bound counterions. R1 increased nearly linearly with increasing DNA phosphate/sodium ratio in the isotropic domain until the onset of anisotropic phase formation, in agreement with similar nmr studies conducted at low DNA concentrations. The value of R1,b = 194 +/- 7 Hz obtained for the isotropic phase from 10 to 100 mg DNA/mL at 20 degrees C was in agreement with values reported previously. A nonlinear increase in R1 with DNA concentration was observed upon onset of anisotropic phase formation, indicating an increase in the product of the fraction of bond ions times their relaxation rate (r.R1,b). The spectral lineshape of all isotropic samples was Lorentzian. Spectra of anisotropic samples exhibited low magnitude quadrupole splitting of less than or equal to 400 Hz correlated with appearance of a cholesteric phase with pitch approximately 2 microns. The magnitude of the quadrupole splitting decreased with increasing DNA concentration at low temperatures and increased with concentration at high temperatures. At all concentrations the quadrupole splitting decreased then increased with temperature. These temperature- and concentration-dependent changes in quadrupole splitting are consistent with an angle between the DNA helix axis and the principal component (VZZ) of the local electric field gradient tensor near the "magic angle" of 54.7 degrees.