A graphical user interface for quantitative imaging and analysis of electrophoretic gels and autoradiograms.

DNA/GUI (DNA Graphical User Interface) is an interactive software system for rapid and efficient analysis of images of the types used in genome mapping, such as autoradiograms and electrophoretic gels. Images are digitized using a commercially available charge-coupled-device (CCD) camera system and analyzed on a graphics workstation using a menu-driven user interface. DNA/GUI features automatic lane and band detection, simultaneous display of multiple images and a unique spatial-normalization algorithm. Images and their associated data are archived and easily available for later recall. Preliminary results indicate that DNA/GUI is a useful tool in the analysis and comparison of images used in a variety of applications such as genetic-linkage analysis and DNA restriction mapping. The interactive display software is based on the X Window System and is therefore readily portable to a variety of graphics workstations.     

A laser densitometer for selective spot analysis on dot blots and two-dimensional gel autoradiograms

We describe an inexpensive densitometer, employing a small HeNe laser and an IBM-compatible personal computer that performs accurate measurements of selected spots on two-dimensional gel autoradiograms or chromatograms with an accuracy and a sensitivity equal or superior to those of many commercial instruments. Our open-table design allows the operator to visually monitor the scanning process in room lighting, and provides great flexibility in both the size and the nature of items to be scanned. The instrument has two moving parts (a prism and a small motor). A commercially available software package (ASYST) acquires digital data, graphs the data on the TV monitor, converts the data to optical density or to radioactive incorporation (cpm), subtracts background, integrates peak areas, and stores data on disk. The total time for these operations is 20-30 s per spot. The instrument has a dynamic range of 0.25 to 3.0 OD units and can measure a 10,000-fold range of 14C or 35S isotope concentrations on autoradiograms. The complete device can be assembled with a hobbyist's knowledge of electronics, moderate programming abilities (no machine language required), and a cost of less than $3000, not including the IBM PC.     

A comparative polyacrylamide gel electrophoretic study of arginase in vertebrate tissues

The electrophoretic behaviour of arginase in the tissue extracts of rat, beef, lizard and frog was studied by bidirectional polyacrylamide gel electrophoresis. The enzyme from rat liver and submaxillary gland migrated to the cathode with the activity concentrated in a single peak. Arginase from beef liver emerged as a single peak of anodal migration with a significant shoulder in the sample gel. Frog liver and kidney enzymes also appeared as single peaks with a distinct anodal movement. The activity in mammalian kidney and lizard liver and kidney resolved into two peaks of anodal migration suggesting the presence of two isoenzymes of arginase in these tissues.     

Spatial normalization of one-dimensional electrophoretic gel images

A strategy for using processed, digitized images of one-dimensional electrophoretic gels to facilitate the analysis of large sets of overlapping clones is described. The images are acquired from fluorescently stained gels or from transilluminated gel photographs using a cooled, solid-state charge-coupled device camera. By employing sets of bands in the size-standard lanes as reference points, all the gel images are spatially normalized to a common reference template. After normalization, lane images from different gels can be compared as though the gels had been electrophoresed under identical, uniform-field conditions. Applications of this procedure to the analysis of a large set of overlapping lambda clones from chromosome VII of Saccharomyces cerevisiae and to the estimation of fragment sizes are illustrated.     

H-2 antigens as genetic markers: A two-dimensional gel electrophoretic study

Direct immunoprecipitation and two-dimensional (2D) gel electrophoresis have been used to identify and characterize genetic variation of theH-2K andH-2D regions. Using inbred strains of mice and alloantisera, haplotype-specific polypeptides were defined for five differentH-2 haplotypes. Specific immunoprecipitates prepared from strains of different haplotypes were applied to 2D gels in pairwise combinations to determine whether peptides specific to one haplotype can be distinguished from peptides specific to another. Those haplotype-specific peptides that migrate to unique positions on 2D gels with respect to the positions occupied by haplotype-specific peptides of another haplotype are useful as biochemical genetic markers. Cross-reactivity amongK- andD-region antigens of different haplotypes was identified on 2D gels and found to correlate well with existing data based on serological cross-reactivity. An anti-mouse 2-microglobulin serum was found to be a useful general reagent for immunoprecipitating haplotype-specific H-2 antigens to permit their visualization on 2D gels.Abbrevations used in this paper NP-40 nonidet P-40 - 2D two-dimensional - SDS sodium dodecyl sulfate - IEF isoelectric focusing     

Quantitative evaluation of gel electrophoretic patterns by videodensitometry

Videodensitometry based on television technique has been shown to be suitable for recording gel electrophoretic patterns. Its speed of operation is about 60 times as fast as that of conventional densitometers, whereas the patterns recorded are practically the same.     

Caldesmon: anomalous electrophoretic behaviour in polyacrylamide gel

In SDS gels caldesmon (Mr = 140 kDa) and myosin light chain kinase (Mr = 130 kDa) migrate as a closely separated doublet. When glycerol is added to the gel caldesmon is characterized by an anomalous migration. In fact under this latter condition, the distance between caldesmon and myosin light chain kinase is enhanced by two-three times. The nature of putative caldesmon and myosin light chain kinase was confirmed by physicochemical, enzymatic and immunological methods.     

Lymphokines which influence electrophoretic mobility of macrophages.

Supernatants of blood lymphocytes from immunized guinea pigs after incubation with purified protein derivative (PPD) were fractionated on Sephadex G-75. The fractions were tested for activity to change the electrophoretic mobility of macrophages. Dependent on incubation time, three different regions of activity with an average molecular weight of 13 000, 40 000 and more than 100 000 were estimated.     

A pitfall in the computer-aided quantitation of autoradiograms

Computer-aided quantitation of autoradiograms is now available as a result of recent developments in optical scanners and microcomputers. Data expressed as optical density values, however, are based on the unverified assumption that optical density and radioactivity density are linearly correlated. This article demonstrates the need to construct a calibration curve which should be used to calculate radioactivity density values more precisely.     

A computer program for displaying two-dimensional gel electrophoresis data

A computer program is described which facilitates comparison between the pattern of spots seen on different two-dimensional polyacrylamide electrophoresis gels. Essentially, the position of each spot is replotted on a graph by the computer using its molecular weight and isoelectric point as coordinates. An intensity factor is also assigned to each point by the operator which will determine the size and shape of the final plotted spot on the computer drawn figure. The resulting plot makes it more feasible to compare patterns of spots between independently run two-dimensional electrophoresis gels.     

Two-dimensional gel electrophoretic method for mapping DNA replicons.

We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems.     

A neutral pH acrylamide gel electrophoretic system for histones and other basic proteins.

A method has been devised whereby measurement of the acid-labile sulfide content of spinach subchloroplast particles is free of interference resulting from the presence of Triton X-100. Quantitative extraction into an organic solvent allows accurate detection of micromolar concentrations of sulfide.     

autoinfer1.0: a computer program to infer biogeographical events automatically

autoinfer is a computer program for biogeographical inference based on nested clade analysis. To reduce the obscurity caused by manual inference, we defined geographically concordant clades and intermediate geographical areas between two clades. The program will perform most of the inferences automatically with a minimum of input from the user. We believe that autoinfer will save much time for the user compared with using the inference key by hand and, furthermore, will reduce the errors of inference resulting from different criteria in deduction.     

Polyacrylamide gradient gel electrophoretic studies of residual catalase in acatalasemia

Erythrocyte catalse in a Japanese-type acatalasemia and a normal control subject was separated by chromatofocusing with or without prior partial purification with DEAE-cellulose. Fractions were analyzed by polyacrylamide gradient gel electrophoresis for catalse activity and protein stain. Chromatofocusing revealed no marked difference in pI values between normal and acatalasemic catalases with or without partial purification. In the gel electrophoresis, molecular weights were also similar; two bands of catalase activity with molecular weights of 290,000 and 350,000 for the acatalasemia and of 280,000 and 360,000 for the normal control were found in the partially purified preparations. The molecular weight of normal catalase in untreated hemolysate was 250,000. Normal catalse was identified as protein bands on polyacrylamide gradient gel after fractionation of hemolysate by chromatofocusing. A more sensitive method for protein stain is still required for demonstration of residual catalse protein on the gel.     

Purification of the photoaffinity-labeled glucagon receptor by gel electrophoretic methods

Rat liver plasma membrane glucagon receptor has been purified with a yield of 0.01% to an estimated homogeneity of 32-60%, using a 2-stage electrophoretic procedure. SDS-solubilized membrane proteins labeled by the photoaffinity-agent, Ne-4-azidophenylamidinoglucagon (APA-glucagon), were separated by polyacrylamide gel electrophoresis in SDS-containing buffers. Gel slices corresponding to the molecular weight of the receptor were excised, electrophoretically extracted and concentrated. The concentrate was subjected to isoelectric focusing on Sephadex to yield a purified product in which the photoaffinity-labeled receptor, with a molecular weight of 56K and a pI' of 5.9, is the sole major component.