The Gene Construction Kit: a new computer program for manipulating and presenting DNA constructs

The Gene Construction Kit is a new tool for manipulating and displaying DNA sequence information. Constructs can be displayed either graphically or as formatted sequence. Segments of DNA can be cut out with restriction enzymes and pasted into other sites. The program keeps track of staggered ends and notifies the user of incompatibilities and offers a choice of ligation options. Each segment of a construct can have its own defined thickness, pattern, direction and color. The sequence listing can be displayed in any font and style in user defined grouping. Nucleotide positions can be displayed as can restriction sites and protein sequences. The DNA can be displayed as either single- or double-stranded. Restriction sites can be readily marked. Alternative views of the DNA can be maintained and the history of the construct automatically stored. Gel electrophoresis patterns can be generated and can be used in cloning project design. Extensive comments can be stored with the construct and can be searched rapidly for key words. High quality illustrations showing multiple editable constructs with added graphics and text information can be generated for slides, posters or publication.     

荧光标记的染色体原位杂交及其在人类基因组研究中的应用

FISH技术是80年代开始发展起来的一种新的定位技术.在人类基因组研究中得到了广泛的应用.通过中期染色体的FISH可以进行SCP,Cosmid和YAC的染色体定位,嵌合克隆的鉴别;通过间期核的FISH可以在50kb的分辨率下进行基因作图;最新的研究进展已可以进行伸展的染色质丝(chromatin fibre)的FISH,直接测量基因的长度,从而达到高精度基因作图的目的.总之,随着FISH技术本身的发展,它将在人类基因组研究中发挥更大的作用.     

RnaViz, a program for the visualisation of RNA secondary structure.

RnaViz is a user-friendly, portable, windows-type program for producing publication-quality secondary structure drawings of RNA molecules. Drawings can be created starting from DCSE alignment files if they incorporate structure information or from mfold ct files. The layout of a structure can be changed easily. Display of special structural elements such as pseudo-knots or unformatted areas is possible. Sequences can be automatically numbered, and several other types of labels can be used to annotate particular bases or areas. Although the program does not try to produce an initially non-overlapping drawing, the layout of a properly positioned structure drawing can be applied to a newly created drawing using skeleton files. In this way a range of similar structures can be drawn with a minimum of effort. Skeletons for several types of RNA molecule are included with the program.     

Protein φ and ψ dihedral restraints determined from multidimensional hypersurface correlations of backbone chemical shifts and their use in the determination of protein tertiary structures

The chemical shifts of the backbone atoms of proteins can be used to obtainrestraints that can be incorporated into structure determination methods. Eachchemical shift can be used to define a restraint and these restraints can besimultaneously used to define the local, secondary structure features. Theglobal fold can be determined by a combined use of the chemical shift basedrestraints along with the long-range information present in the NOEs ofpartially deuterated proteins or the amide–amide NOEs but not from suchlimited NOE data sets alone. This approach has been demonstrated to be capableof determining the overall folding pattern of four proteins. This suggeststhat solution-state NMR methods can be extended to the structure determinationof larger proteins by using the information present in the chemical shifts ofthe backbone atoms along with the data that can be obtained on a small numberof labeled forms.     

Demiarcs,creaons and genons

Useful insights into the representation of natural systems can be gained by decomposing directed graphs (digraphs) into elementary components. Arcs of digraphs can be split into male demiarcs (outarcs) which leave vertices and female demiarcs (inarcs) which enter demiarcs. Likewise, a vertex can be split into an input perceiving side called the creaon and an output generating side called the genon. Digraphs can be regarded as being hierarchically organized because each vertex in a level-1 digraph can be expanded into a level-2 digraph. In general, each vertex of a level-i digraph can be expanded into a level-(i+1) digraph. Arcs of a level-i digraph can be regarded as bundles of level-(i + 1) arcs which are split at the vertex boundary. These elementary graphical components are shown to be useful for depicting input-output systems such as organisms, ecosystems and societies.     

Shave excision of superficial solar skin lesions

Shave excision is a useful technique for the treatment of superficial solar lesions. The most common of these is the solar keratosis, which can be a red or gray scaling lesion. This can at times be difficult to differentiate from the superficial basal cell carcinoma or squamous cell carcinoma or their more infiltrating types. Shave excision gives the advantage of a histopathologic report. Lesions found to be infiltrating or in danger of recurrence can then be further excised. The cosmetic appearance of the healed shave excision site is generally quite good, although it can be paler than the surrounding skin. If this is likely to be a problem, the shave graft can be applied, as with deeper shave excisions. A thin shave graft also can be used to repigment pale scarred areas. A series of 1313 shave excised lesions is analyzed.     

Harming the non-conscious

Peter Singer has argued that nothing done to a fetus before it acquires consciousness can harm it. At the same time, he concedes that a child can be harmed by something done to it when it was a non-conscious fetus. But this implies that the non-conscious fetus can be harmed. The mistake lies in thinking that, since existence can be intrinsically bad for a being only if it is conscious, it can be harmed only if it is conscious. In fact, its being harmed only implies that it could have been conscious (and led a good life).     

Modification of commerical and custom-built slab gel electrophoresis units for two-dimensional polyacrylamide gel electrophoresis

Many commercial and custom-built slab gel electrophoresis units can be modified to function as two-dimensional polyacrylamide gel electrophoresis units with the insertion of Plexiglas adapters. These adapters can be made for about $50 a pair and can be used for either temporary or permanent modification of the slab gel units. The physical dimensions of the adapters can be varied to permit great flexibility in the diameter of cylinder gels and the thickness of slab gels that can be run together. For example, proteins from 6-mm cylinder gels can be easily separated on 1-mm slab gels, which can then be dried for autoradiography.     

The use of amplified fragment length polymorphism (AFLP) in the isolation of sex-specific markers

Sex identification is a problem in research and conservation. It can often be solved using a DNA test but this is only an option if a sex-specific marker is available. Such markers can be identified using the amplified fragment length polymorphism (AFLP) technique. This is usually a taxonomic method, as it produces a DNA fingerprint of 50-100 PCR bands. However, if male and female AFLP products are compared, sex-specific markers are confined to the heterogametic sex and can rapidly be identified. Once a marker is found, AFLP can be used to sex organisms directly or the marker can be sequenced and a standard PCR test designed.     

Rapid multivariate analysis and display of cross-reacting antibodies on human leukocytes.

We present an application which can rapidly determine the binding patterns of monoclonal antibodies on mixed populations of cells simultaneously in a single rapid analysis. It is an application of the tube identifier parameter (TIP) system which can provide fully correlated list-mode data of the entire patient phenotype in a single file. Using the phenogram analytical display, we are able to determine the cross-reacting antibodies for an entire antibody panel for each cell type. This information can be displayed in a single plot. Using light scatter gating to select different populations of lymphocytes, monocytes, and neutrophils, phenograms can be simultaneously generated. This provides a directly comparable means of displaying the positive and negative binding characteristics of each antibody on each cell population. Any marker combination that is abnormal will be identifiable in the phenogram. Additionally, by plotting the fluorescence distributions of each marker beside one another (termed overview), quantifiable differences in intensity can be determined. There are 3 major benefits of the proposed analysis. By using the TIP concept, several sets of antibodies can be compared simultaneously. Any light scatter gate can be used and this gate can be changed on one histogram or plot, yet apply to the total analysis. Data analysis is particularly rapid since the entire phenotype of a patient can be evaluated by performing a single rapid analysis.     

VP7/IFNα-2b融合蛋白防治轮状病毒腹泻的可行性

轮状病毒(RV）外壳蛋白VP7可用作抗原;通过构建植物表达载体,将该抗原基因导入植物细胞,最终在植物细胞中获得抗原蛋白的表达;这种蛋白能保持自然免疫特性,口服能诱发粘膜免疫反应,从而达到对RV的预防作用。干扰素作为一种广谱的抗病毒蛋白质,能对RV引起的肠炎起治疗作用。本提出利用干扰素与轮状病毒表面抗原融合蛋白用于防治轮状病毒腹泻的构想,并对其可行性作了探讨。     

VISUALIZING MULTIVARIATE SELECTION

Recent developments in quantitative-genetic theory have shown that natural selection can be viewed as the multivariate relationship between fitness and phenotype. This relationship can be described by a multidimensional surface depicting fitness as a function of phenotypic traits. We examine the connection between this surface and the coefficients of phenotypic selection that can be estimated by multiple regression and show how the interpretation of multivariate selection can be facilitated through the use of the method of canonical analysis. The results from this analysis can be used to visualize the surface implied by a set of selection coefficients. Such a visualization provides a compact summary of selection coefficients, can aid in the comparison of selection surfaces, and can help generate testable hypotheses as to the adaptive significance of the traits under study. Further, we discuss traditional definitions of directional, stabilizing, and disruptive selection and conclude that selection may be more usefully classified into two general modes, directional and nonlinear selection, with stabilizing and disruptive selection as special cases of nonlinear selection.     

Fiber optic array biosensors

Optical fiber arrays provide a powerful substrate for creating high-density sensing systems that can address a variety of biological problems. The fiber substrate can be used to create femtoliter wells that can be loaded with individual beads to create high-density arrays for multiplexed screening and analysis. In addition, living cells can be loaded into the arrays, and their individual responses monitored over long time periods, enabling functional screening of biologically active compounds. Adherent cells can be attached to the fiber substrate to provide a rapid method for observing cell migration and for screening anti-migratory compounds. Finally, individual enzyme molecules can be loaded into the array wells enabling single molecule detection via enzyme-catalyzed signal amplification.     

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.     

Monitoring biotransformations in polyamide fibres

The enzymatic hydrolysis of polyamide fibres yields amino and carboxylic groups. These groups can be found in solution treatments as polyamide monomers and soluble oligomers. The amino groups can also be found at the surface of the fibres as end group chains. In this paper we report two methods to quantify the formation of these groups as a result of the enzymatic action. Soluble amino groups can be quantified with 2,4,6-trinitrobenzenesulfonic acid (TNBS), which yields a coloured complex which can be determined spectrophotometrically. The amino groups on the fibre surface can be quantified by reaction with a wool reactive dye and determination of colour intensities after a dyeing procedure below the glass transition temperature of polyamide.     

Visual analysis of trabecular bone structure

Acquiring image data of bone biopsies by a micro-CT scanner is today a common technique. The amount of data to be assessed is huge. The task to assess quantitative measures requires a concise visualization. We present visualization techniques that can be used interactively on state-of-the-art PCs and demonstrate how the frontier can be pushed further. A skeletonization process is applied to the image of the bone to create the central surface. After triangulation this surface can be renderd at interactive frame rates. When the surface is additionally colored by local measures (mean grey value of image data, local thickness) the overall structure and details can be recognized at the same time. This can facilitate the exploration of the biopsy and can help finding special features.     

Industrial strain improvement: Mutagenesis and random screening procedures

Industrial strain improvement plays a central role in the commercial development of microbial fermentation processes. In recent years new procedures such as rational screening and genetic engineering have begun to make a significant contribution to this activity but mutagenesis and selection - so-called ‘random screening’ - is still a cost-effective procedure, and for reliable short-term strain development is frequently the method of choice. The current practice of strain improvement by mutagenesis and selection is a highly developed technique drawing on the latest advances from a wide range of scientific and technical disciplines. Mutagenic procedures can be optimized in terms of type of mutagen and dose, mutagen specificity effects can be taken into account and mutagenesis itself can be enhanced or directed in order to obtain the maximum frequency of desirable mutant types among the isolates to be screened. Screens can be designed to allow maximum expression of the desirable mutant types and the application of statistically-based screening procedures will maximize the probability of detecting them. Automated procedures can be developed using robotics and microprocessors to increase the numbers of isolates that can be processed through a screen per unit time. The relationship between screening and production conditions can be organized so as to minimize the probability of improved isolates selected by the screen failing to scale up.     

Enhancement of tomogram interpretability using the locked self-rotation function

The interpretation of cryo-electron tomograms of macromolecular complexes can be difficult because of the large amount of noise and because of the missing wedge effect. Here it is shown how the presence of rotational symmetry in a sample can be utilized to enhance the quality of a tomographic analysis. The orientation of symmetry axes in a sub-tomogram can be determined using a locked self-rotation function. Given this knowledge, the sub-tomogram density can then be averaged to improve its interpretability. Sub-tomograms of the icosahedral bacteriophage phiX174 are used to demonstrate the procedure.     

Differentiation genes: are they primary targets for human carcinogenesis?

In spite of extensive research in molecular carcinogenesis, genes that can be considered primary targets in human carcinogenesis remain to be identified. Mutated oncogenes or cellular growth regulatory genes, when incorporated into normal human epithelial cells, failed to immortalize or transform these cells. Therefore, they may be secondary events in human carcinogenesis. Based on some experimental studies we have proposed that downregulation of a differentiation gene may be the primary event in human carcinogenesis. Such a gene could be referred to as a tumor-initiating gene. Downregulation of a differentiation gene can be accomplished by a mutation in the differentiation gene, by activation of differentiation suppressor genes, and by inactivation of tumor suppressor genes. Downregulation of a differentiation gene can lead to immortalization of normal cells. Mutations in cellular proto-oncogenes, growth regulatory genes, and tumor suppressor genes in immortalized cells can lead to transformation. Such genes could be called tumor-promoting genes. This hypothesis can be documented by experiments published on differentiation of neuroblastoma (NB) cells in culture. The fact that terminal differentiation can be induced in NB cells by adenosine 3',5'-cyclic monophosphate (cAMP) suggests that the differentiation gene in these cells is not mutated, and thus can be activated by an appropriate agent. The fact that cAMP-resistant cells exist in NB cell populations suggests that a differentiation gene is mutated in these cancer cells, or that differentiation regulatory genes have become unresponsive to cAMP. In addition to cAMP, several other differentiating agents have been identified. Our proposed hypothesis of carcinogenesis can also be applied to other human tumors such as melanoma, pheochromocytoma, medulloblastoma, glioma, sarcoma, and colon cancer.     

Development of DNA-mediated transformation systems for plants

The genetic engineering of plants by DNA-mediated gene transfer requires that efficient transformation systems be developed. Considerable progress has been made in manipulating the Ti plasmid of Agrobacterium tumefaciens as a vehicle for delivery of foreign genes into protoplasts of dicotyle-donous plants. Part of the Ti plasmid, the T-DNA, can be incorporated into the genome of the host cell; the T-DNA can carry a foreign DNA sequence which co-integrates with it; under normal conditions, the tumorigenic-causing portion of the T-DNA can be inactivated so that transformed protoplasts can be regenerated and T-DNA with an inserted foreign gene can be stably maintained during regeneration, meiosis and gamete formation. A foreign gene has yet to be expressed in regenerated plants although a T-DNA gene for opine synthesis can function in regenerates. Developing a more ubiquitous transformation system for monocotyledons is further from fruition. Based on transformation systems for simple eukaryotic organisms, it is reasonable to expect that a DNA vector which is capable of amplifying a novel plant gene and which contains both a drug resistance marker to facilitate the selection of transformed plant protoplasts and a species-specific autonomously replicating sequence to ensure the stable maintenance of the input gene in the recipient cell can be constructed.