Yeast transformation process studied by fluorescence labeling technique

A new method based on fluorescence imaging and flow cytometry was developed to investigate the transformation process of Saccharomyces cerevisiae AY. Yeast and fluorescent-labeled plasmid pUC18 were used as models of cells and DNA molecules, respectively. Binding of DNA molecules to yeast cell surfaces was observed. Factors influencing DNA binding to cell surfaces were investigated. It has been found that poly(ethylene glycol) (PEG) could induce DNA binding to yeast surfaces, while Li(+) showed a weak effect on the binding. When both Li(+) and PEG were used, synergetic effect occurred, resulting in the binding of pUC18 to the surface of more yeast cells compared with that in the presence of PEG or Li(+) only. It was also confirmed that heat shock, Li(+), and PEG all can increase the permeability of yeast cells. This simple method is helpful for understanding the process of yeast transformation and can be used to investigate the interaction of DNA with cell surfaces.     

Chromosome fibers studied by a spreading technique

Chromosomes and interphase nuclei can be spread on the surface of water in a simplified Langmuir trough. Interphase nuclei of Triturus erythrocytes display fibers with a diameter of about 250–300 Å. Very similar fibers are seen in metaphase chromosomes of cultured human cells. Fibers from grasshopper spermatocyte chromosomes (prophase) are more variable in diameter, and many fibers thinner than 200 Å extend laterally from the chromosome. In the grasshopper spermatocyte, fibers align in parallel to form plates. It is suggested that the 250–300 Å fibers may represent an inactive state of the chromosome material, and that only the thinner fibers are involved in RNA synthesis. The 250–300 Å fibers may result from the folding or coiling of a thinner fiber having the approximate dimensions of the nucleohistone molecule.     

A photographic ageing technique used on warthog

The ratios of tusk-length: snout-width from a series of warthog (Phacochoerus aethiopicus) photographs were calculated and plotted against the date the photographs were taken. Regression lines, as well as the 95% confidence intervals, were calculated from the photographs and plotted. These regression lines fell into four distinct groups, representing Age Classes II through V. The average degree of error in the tusk-length: snout-width ratio was 3-01 units, a figure well within the above 95% confidence intervals.     

Structure of lipoprotein (a) studied by spin-labeling

The potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate causes a 2-fold increase in 1,2-diacylglycerol levels within 15–30 min in cultured chick embryo differentiated myoblasts. The weak tumor promoter 12-O-decanoylphorbol 13-acetate was 250 times less effective and the non-promoter 4-α-phorbol 12,13 didecanoate was ineffective at producing this response. During subcellular fractionation, the stimulated portion of the diacylglycerol was distributed similarly to the plasma membrane fraction. Evidence is presented that this diacylglycerol originates from pre-existing lipid rather than from $\text{de}\text{novo}$ synthesis. Possible implications of these findings with regard to the inhibition of myoblast fusion by the tumor promoter are discussed.     

用发光酶基因(luxAB)标记法研究慢生花生根瘤菌的竞争结瘤能力

luxAB基因标记是一种新型基因标记技术,在很多研究领域都有着良好的应用前景。研究通过三亲本杂交将luxAB基因成功地向慢生型花生根瘤菌进行了转移,并获得了一株带LuxAB基因标记的菌株Cspr7-1。对Cspr7-1进行性状、标记基因的遗传稳定性检测,结果表明,LuxAB基因不仅能有效表达,而且性状稳定。在无氮水培条件下进行标记菌株与土著根瘤菌的竞争结瘤试验。结果证实,Cspr7-1在植物根系上的占瘤率平均达到61.3％,比土著根瘤菌的竞争结瘤能力强,而且Cspr7-1在主根上的侵染能力远较侧根上的强,平均高出22.3％-39.6％。     

The mutagenic effect of benzene,toluene and xylene studied by the SCE technique

In experiments in vitro, neither benzene, toluene nor xylene changed the number of sister-chromatid exchanges (SCEs) or the number of chromosomal aberrations in human lymphocytes. Toluene and xylene caused a significant cell growth inhibition which was not observed with benzene in the same concentrations.     

Na-cellulose formation in a single cotton fiber studied by synchrotron radiation microdiffraction

A cotton fiber was kept under slight tension and exposed locally to a stream of aqueous 1 N NaOH microdrops of 50 microm diameter. The resulting "macrodrop" of about 300 microm size was at the origin of the formation of Na-cellulose I domains extending about 550 microm from the center of the macrodrop along the fiber. The phase transformation zone between cellulose I and Na-cellulose I was mapped by scanning synchrotron radiation microdiffraction using a 300 nm x 300 nm beam. A stitching technique was used to limit radiation damage. Subsequent exposure of the NaOH containing macrodrop to a stream of H2O or HCl microdrops converted part of the Na-cellulose I back into cellulose I.     

Oligonucleotide hybridization studied by a surface plasmon diffraction sensor (SPDS)

A novel label-free biosensor concept based on surface plasmon-enhanced diffraction by micro- patterned interfaces was applied to the study of hybridization reactions of target DNA oligonucleotides (15mers and 75mers) from solution to probe DNA oligonucleotides attached via streptavidin to the sensor surface. The self-referencing and quadratic signal amplification mechanism of the sensor allowed highly sensitive detection of the hybridization process. Association and dissociation processes of DNA targets could be recorded in real time and used for the quantification of their binding affinities, which differ considerably with a single base pair mismatch. An equilibrium titration approach was also applied in order to obtain the binding affinities for 15mer targets, yielding similar affinity values. The hybridization efficiencies were found to be higher for the 15mers than for the 75mers, although the latter contained the same recognition sequences. The hybridization efficiency was shown to depend on the probe density and reached nearly 100% for the 15mer fully complementary targets at a probe density of ~1.2 × 10¹² molecules/cm². Using the assay as an end-point determination method, the lowest detectable coverage of a 15mer oligonucleotide was at least ~1.1 × 10¹¹ molecules/cm². The diffraction sensing concept offers a completely novel way to integrate a reference channel in large-scale, label-free screening applications, to improve the stability and to enhance the sensitivity of microarray read-out systems.     

The mutagenic effect of benzene, toluene and xylene studied by the SCE technique.

In experiments in vitro, neither benzene, toluene nor xylene changed the number of sister-chromatid exchanges (SCEs) or the number of chromosomal aberrations in human lymphocytes. Toluene and xylene caused a significant cell growth inhibition which was not observed with benzene in the same concentrations.     

The effect of anaesthetics on protein conformation in membranes as studied by the spin-labelling technique

The effect of general and local anaesthetics was studied on the membrane components (proteins and lipids) of frog sciatic nerve using the spin-labelling technique. A rapid and effective method was developed to incorporate fatty acid labels into the lipid regions and to attach the maleimide spin labels to the thiol sites of the proteins of the membrane. On the basis of the rotational correlation time of the attached maleimide spin labels, at least three different protein domains were identified. One part of the labels has a preferred orientation with respect to the axis of the nerve fibre. The effect of halothane and of local anaesthetics such as lidocaine and tetracaine, which influence primarily the lipid regions of the membrane, is efficiently transferred to the spin-labelled membrane proteins via strong lipid-protein interaction. The results support the concept that the architecture and the physiological activity of the membrane-bound proteins are sensitive to changes in the physical state of membrane lipids.     

Uptake of nitrogen by rice studied with a 15N point-placement technique

The main objective of the work described in this paper was to investigate the usefulness of a point-placement technique for investigating nitrogen-use by irrigated rice in a tropical lowland environment. The technique was used to deliver a small quantity of urea ¹⁵N to a specific location adjacent to the roots of a rice plant in a few minutes. The amount of ¹⁵N delivered was equivalent to 4–8 h of the daily nitrogen uptake requirement of a rice plant. The ¹⁵N was placed in a gelatin capsule attached to a wooden handle and the capsule was inserted adjacent to the roots at the soil water interface or at 5 cm soil depth beneath the rice plant. The gelatin dissolves in a few minutes. The plants were harvested at varying intervals through to crop maturity and their ¹⁵N content measured. The uptake of ¹⁵N followed an exponential pattern and it took approximately two weeks to reach a maximum of about 65% of the initial quantity supplied. The time to reach maximum uptake was unexpectedly slow and a simple theory was developed to better understand the processes governing the uptake and loss of the ¹⁵N. There were no differences between the applications made at the soil surface and those made 5 cm below the surface. The technique worked very well for tracking the fate of nitrogen acquired by the plant at different growth stages. For example, 14 days after label application at 10 days after transplanting (DAT), 66% of the labeled N acquired by the plant was found in the leaves, 26% in the stems and sheaths, and 8% in the roots. About 37% of the label supplied on 10 DAT was located in the grain at maturity suggesting the technique can be used to follow the nitrogen dynamics of irrigated rice crops.     

A photographic technique for investigating the splashing of water drops on leaves

The formation of splash from water drops falling onto bean leaves was investigated by multiple exposure flash photography. A relatively simple technique was developed which recorded up to four images at 2 ms intervals, of a splash on one photograph. The images were recorded on colour reversal film, and the flash sequence was colour coded to aid the interpretion of the superimposed images. Analysis of the photographs allowed measurement of the initial speed and angle of ejection of different sized splash droplets. Droplets were ejected at speeds of up to about 10 m s^-1although most speeds were less than about 3 m s^-1, and small droplets tended to travel faster than large ones. Droplets were ejected at all angles but the majority were ejected almost horizontally; there was little correlation between droplet size and angle of ejection. Deposition patterns estimated from calculated droplet trajectories compared well with measured ones.     

Encapsulation of enzymes by radiation technique

Summary A new preparative technique for the encapsulation of enzymes by radiation polymerization was studied using cellulase, 2-hydroxyethyl methacrylate, and tetraethylene-glycol diacrylate. The enzyme activity of the immobilized enzyme capsules varied with the thickness of the membrane and with the size of the capsule; optimum size and thickness giving high enzyme activity were 0.5 – 2 mm and 50 – 200 m, respectively. The durability of the capsules was examined by continmuous enzyme reaction.