Protein synthesis and accumulation in Drosophila melanogaster imaginal discs: Identification of a protein with a nonrandom spatial distribution

Proteins from Drosophila imaginal discs and disc fragments were analyzed on two-dimensional electrophoretic gels following labeling in vitro with [³⁵S]methionine. The protein synthetic pattern in autoradiograms is very complex and parallels the pattern of protein accumulation visualized in silver-stained gels. We find no reproducible qualitative differences in the proteins synthesized or accumulated by different disc types. Additionally, analysis of the proteins synthesized by different fragments of wing and haltere discs has resulted in the identification of a polypeptide which is synthesized preferentially in homologous regions of these two imaginal discs. Scanning densitometry of our autoradiograms corroborates these findings. This protein, therefore, has some of the properties one would predict for a molecule involved in the imaginal disc positional information system.     

Low-cost two-dimensional gel densitometry

A major obstacle to full utilization of the powerful technique of two-dimensional (2-D) gel electrophoresis is the expense and complexity of quantifying the results. Using an analog-to-digital converter already present in the widely available Commodore 64 or Commodore 128 microcomputer, we have developed a 2-D gel densitometer (GELSCAN) which adds only $20.00 to the cost of the Commodore system (currently around $700.00). The system is designed to work with autoradiograms of 2-D gels. Spots of interest are identified visually and then positioned manually over a light source. A pinhole photoelectric sensor mounted in a hand-held, Plexiglas holder, or "mouse," is briefly rubbed over each spot. Maximum density of the spot is determined and its value is converted to counts per minute via an internal calibration curve which corrects for the nonlinear response of film to radiation. Local spot backgrounds can be subtracted and values can be normalized between gels to adjust for variation in amount of radioactivity applied or in exposure time. Reproducibility is excellent and the technique has some practical as well as theoretical advantages over other more complicated approaches to 2-D gel densitometry. In addition, the GELSCAN system can also be used for scanning individual bands in 1-D gels, quantitation of "dot-blot" autoradiograms and other tasks involving transmission densitometry.     

Conservation of interphase chromatin nonhistone antigens as components of metaphase chromosomes

The degree of conservation of HeLa interphase chromatin nonhistone antigens among the nonhistones of isolated metaphase chromosomes was determined with immunological procedures. Proteins were separated on SDS-polyacrylamide gels and electrophoretically transferred to diazophenylthioether (DPT)-paper, which was then overlaid with antiserum to chromatin from interphase nuclei. The bound antibodies were detected with 125I-labeled protein A. Alternatively, polyacrylamide gels were directly overlaid with antiserum and with 125I-protein A. Densitometry of autoradiograms and stained gels revealed the degree of conservation of nonhistone antigenic determinants from interphase to metaphase to be over 90% for chromatin.     

Effect of retinoic acid on the phosphorylation of endogenous proteins in HL-60 cells

HL-60 cells were incubated with [32P]-Pi in order to label endogenous phosphoproteins in situ. These were then resolved via two-dimensional electrophoresis and autoradiograms were made from the gels. A comparison of autoradiograms made from retinoic-acid-differentiated cells with those made from control cells revealed a small number of phosphoproteins whose labeling was enhanced in differentiated cells. Incubating HL-60 cells with [35S]-methionine revealed that RA induced the synthesis of one of these proteins, (53 kDa, pl 4.8) although not to a degree sufficient to account for the increased phosphate labeling observed in differentiated cells. Difluoromethylornithine (DFMO), which arrests HL-60 cell proliferation without inducing differentiation, had no effect on endogenous protein phosphorylation. Treatment of DFMO-arrested cells with retinoic acid, however, increased the phosphorylation of the proteins and resulted in differentiation of the cells. Densitometric analysis of autoradiograms made from two-dimensional gels revealed that the phosphorylation of the 53-kDa, pl 4.8 protein was significantly elevated in cells exposed to RA for as little as 12 hours, i.e., before the cells were committed to differentiate and before a significant increase in the number of phenotypically mature cells was observed. It therefore appears that this protein may be an intermediate in the retinoic-acid-induced differentiation process.     

Computer video acquisition and analysis system for biological data

The BIAS (Biological Image Analysis System) was developed to:(i) permit accurate entry and image processing of biologicaldata; (ii) minimize the need for specialized hardware; and (iii)aid in the human genome mapping and other projects. The firstmouse/cursor key-driven module was designed to be user interactiveand readily accessible to many laboratories. It contains theDRSNDS programs which automate the entering of data in a systematicformat. The types of data that can be entered utilizing thismodule are DNA — RNA gels from either a positive or negativePolaroid^TM image, autoradiograms or biotinylated images fromSouthern, Northern and dot or slot blot hybridization analyses.The image is acquired using a video camera and then digitizedfor subsequent analysis. During the analysis graphical representationsof the intermediate results are provided to assure user confidence.At any point within the program the user may obtain on-linehelp with the current task. The output displays the mol. wtof each individual component in the appropriate context. Thepresent version of the program produces results comparable witha human interpreter for some data. Band shifting and opticaldensity calculations are in a prototype form to permit evaluationof various techniques. Future work is directed at expandingthe system's capabilities to interpret data from other biologicalanalyses including DNA sequencing gels. Received on December 1, 1987; accepted on December 12, 1987     

Quantitative analysis of two-dimensional electrophoretograms.

A method for quantitative analysis of complex film density distributions in autoradiograms is described. The method is intended particularly for measuring the distribution of radioactivity among the proteins resolved by two-dimensional gel electrophoresis but should, of course, be suited to analyzing other two dimensional separations. The film density distribution is first digitized by a high speed rotating drum scanner to generate the image data array that is stored on a magnetic disk. Subsequent analysis involves: 1) data averaging, 2) detection of contours and of their locations, 3) splitting of overlapping spots, 4) conversion of film density to radioactive intensity by means of calibration films, and 5) differentiation and integration to measure the total radioactivity contained in the protein which generates a spot in the autoradiogram. The product of the analysis is a numbered contour map and a table listing coordinates and radioactivity content of each resolved spot. Coordinate transformations for comparison and matching of autoradiograms are also described. A set of utility programs print and graph the data at intermediate stages of the analysis in order to facilitate the checking of procedures and programs.     

Electrophoretic gel image analysis software for the molecular biology laboratory

We present GelReader 1.0, a microcomputer program designed to make precision, digital analysis of one-dimensional electrophoretic gels accessible to the molecular biology laboratory of modest means. Images of electrophoretic gels are digitized via a desktop flatbed scanner from instant photographs, autoradiograms or chromogenically stained blotting media. GelReader is then invoked to locate lanes and bands and generate a report of molecular weights of unknowns, based on specified sets of standards. Frequently used standards can be stored in the program. Lanes and bands can be added or removed, based upon users' subjective preferences. A unique lane histogram feature facilitates precise manual addition of bands missed by the software. Image enhancement features include palette manipulation, histogram equalization, shadowing and magnification. The user interface strikes a balance between program autonomy and user intervention, in recognition of the variability in electrophoretic gel quality and users' analytical needs.     

Core histone acetylation during lymphocyte activation

Histone acetylation has been followed in cultures of human lymphocytes, in PHA-stimulated lymphocytes and in mixed lymphocytes obtained from identical twins and from unrelated donors. A computer assisted analysis of two-dimensional gels and autoradiograms revealed that in cultured lymphocytes only H3 and H4 core histones incorporate labeled acetate and that two H3 variants greatly differ in their rate of acetate uptake.     

Acidic and basic fibroblast growth factors similarly regulate the rate of biosynthesis of rat astroblast proteins

Quiescent rat astroblasts in culture have been treated for various periods of time with acidic and basic fibroblast growth factors. Both factors elicited similar effects on the cell proliferation and glutamine synthetase activity. The rate of biosynthesis of the proteins analyzed on autoradiograms of polyacrylamide gels after two-dimensional electrophoresis was also similarly modulated by the two growth factors. These results suggest that the two fibroblast growth factors act through the same membrane receptors on rat astroblasts in culture.     

Solubilization of the plasmin receptor from human carcinoma cells

We could solubilize the plasmin receptor from the human colonic tumor cell line SW1116, using various detergents. Among these, Triton X100 and Sodium dodecyl sulfate were the most efficient. The solubilized receptor retained its plasmin binding activity, as judged by dot blot tests. After electrophoresis of the solubilisate in SDS polyacrylamide gels and transfer to nitrocellulose, specific plasmin fixation was still observed on autoradiograms as a sharp band in the 50K area. This band was not altered by treatment of the solubilisate with beta-mercaptoethanol.     

Identification of surface autoantigens which appear during spermatogenesis

Autoantigens that appear during spermatogenesis in the rabbit were identified using immunoadsorbent chromatography and SDS-PAGE. To identify cell-surface proteins, samples of freshly isolated, staged cells were labeled by the lactoperoxidase or Iodo-Gen iodination procedure and run on SDS-PAGE. Autoradiograms of the stained, dried gels were prepared. By correlating the band patterns in the SDS gels of immunocolumn and surface-labeled samples with the band patterns in the autoradiograms, it was possible to show when the autoantigenic proteins appeared on the cell surface. To further support the identification of membrane autoantigens, surface-labeled, staged cell samples were lysed in Triton X-100 and immunoprecipitated with antitestis cell autoantisera. Three types of autoantigens have been identified: (1) late class antigens that are present only on late spermatids and epididymal spermatozoa, but are intracellular in early stages, (2) early class antigens which occur on the surface of pachytene spermatocytes and are present throughout subsequent stages of development, and (3) early class, transient antigens, which appear on spermatogenic cells but are not present on epididymal spermatozoa.     

Phosphorylation of peripherin, an intermediate filament protein, in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons

Peripherin, an intermediate filament protein, described recently, is expressed in well defined neuronal populations. We studied the phosphorylation, in vivo, of this protein in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons labelled with [32P]-orthophosphate. The autoradiograms of proteins separated on two-dimensional polyacrylamide gels were compared with the Coomassie-blue stainings. The results show that peripherin occurs as a mixture of phosphorylated and non-phosphorylated isoforms, and that these forms coexist in both differentiated and non-differentiated cells. We demonstrate by cleavage at the unique tryptophan residue, a characteristic shared by most other intermediate filament proteins (IFP), that the phosphorylation sites are located on the amino-terminal half of peripherin as it is for vimentin and desmin. These results are discussed in relation to the organization of the filamentous network constituted by peripherin.     

Cell cycle variations in ADP-ribosylation of HeLa nuclear proteins

Changes in ADP-ribosylation of nuclear proteins during the HeLa cell cycle were determined. Portions of synchronized cultures were withdrawn at intervals and cells were permeabilized by resuspension in hypotonic buffer containing detergents. Nuclear proteins were radioactively labeled by incubating samples with [32P]NAD. Modified species were resolved using one-dimensional and two-dimensional polyacrylamide gel electrophoresis. Measurements of the incorporation of [32P]NAD by permeabilized cells showed that ADP-ribosylation is a significant modification throughout the cell cycle. A twofold increase was detected during S phase. Autoradiograms of one-dimensional sodium dodecyl sulfate-polyacrylamide gels revealed that many nuclear nonhistones are modified, though the major acceptors of 32P were the histones and a 116,000-Da species (poly(ADP-ribose) polymerase). The same modified proteins were present through the cell cycle, but densitometry of autoradiograms demonstrated a general increase in the level of incorporation in S phase. Autoradiograms of two-dimensional gels of nuclear proteins labeled with [32P]NAD were consistent with these results. Although nonhistones of isolated metaphase chromosomes show a substantial reduction in ADP-ribosylation, histone modification is essentially unchanged in metaphase.     

An optical comparator for measuring two-dimensional polyacrylamide gel electrophoresis records using an on-line microcomputer

A comparator which makes it possible to compare two wet gels or photographic negatives or autoradiograms through a flickering light system has been built. The system consists of two special-purpose projectors which combine the images on a digitizing platform. When the lights are switched On and off out of phase, the positions of the common components remain unchanged, whereas those that are spatially displaced appear to jump from side to side and those present in one image but not the other switch on and off. This produces a flickering image in which differences are readily seen. Commercial camera lenses were used to construct the projectors and the overall specifications for the system are given. The coordinates of both the displaced components, as well as the selected standards from the two images, are digitized and entered automatically into an on-line microcomputer. By using an iterative procedure for collecting records from several superimposable records of the gel, it is possible to compensate for the lack of total reproducibility over the whole gels. These coordinates are then normalized and superimposed on a master map through a television display using a curser to adjust the coordinates. The whole procedure can be repeated for many gels using a common reference gel in the comparator, and the result is a set of normalized coordinates which can be plotted on a single map to provide a final record of the experiments.     

Elsie 4: quantitative computer analysis of sets of two-dimensional gel electrophoretograms

We have developed and refined a system for quantitative computer analysis of two-dimensional polyacrylamide gel electrophoretograms. The system, named Elsie 4, is based on one described by Vo et al. (Anal. Biochem. 112, 258 (1981]. It is highly automated. Elsie 4 can find, and measure the intensity of, almost any spot resolvable on two-dimensional gels, including spots visible only as shoulders off larger spots and spots so close together that there is no "valley" between them. It can automatically match the spot patterns of different gels, potentially without the need for a user to provide landmark matches. The matches between paired gels let us follow the synthesis of any spot through a set of gels. Information about a group of matched spots can be obtained by referring to any spot in the group. There is generally no need for a standard or reference gel. Data for two experiments can be combined and compared by matching any gel in one experiment with any gel in the other. There are ways to automatically find possible mismatches in sets of gels. Scans and the results of the analysis can be shown on an image displayer. The programs use function libraries; this helps ensure consistency and increase portability. The programs and functions can be linked together in many ways; this lets users build custom programs for analysis of specific experiments.     

The myth of automated, high-throughput two-dimensional gel analysis

Many software packages have been developed to process and analyze 2-D gel images. Some programs have been touted as automated, high-throughput solutions. We tested five commercially available programs using 18 replicate gels of a rat brain protein extract. We determined computer processing time, approximate spot editing time, time required to correct spot mismatches, as well as total processing time. We also determined the number of spots automatically detected, number of spots kept after manual editing, and the percentage of automatically generated correct matches. We also determined the effect of increasing the number of replicate gels on spot matching efficiency for two of the programs. We found that for all programs tested, less than 3% of the total processing time was automated. The remainder of the time was spent in manual, subjective editing of detected spots and computer generated matches. Total processing time for 18 gels varied from 22 to 84 h. The percentage of correct matches generated automatically varied from 1 to 62%. Increasing the number of gels in an experiment dramatically reduced the percentage of automatically generated correct matches. Our results demonstrate that these 2-D gel analysis programs are not automatic or rapid, and also suggest that matching accuracy decreases as experiment size increases.     

High sensitivity quantification of RNA from gels and autoradiograms with affordable optical scanning.

To increase sensitivity and to improve normalization of RNA levels in Northern blot analysis, a comparatively inexpensive optical scanner was utilized for digitizing photonegatives of ethidium bromide stained gels and autoradiograms. The optical scanner captures the image with a maximum resolution of 300 dots per inch by assigning one of 256 gray levels (8-bit) to each dot in the image. With the use of the public domain NIH Image program (requires a Macintosh II and an 8-bit video card), gel or autoradiogram bands in the digitized image are selected and their average gray scale density measured. We found that the digitized image of a photonegative of a TAE (Tris-acetate/EDTA) agarose gel, loaded incrementally with 50-1500 ng total RNA, produced a linear response over a 4-fold range down to 100 ng (R2 greater than 0.950). In utilizing "quantification" gels like this, RNA samples that are too dilute or too small for traditional spectrophotometric techniques can be normalized and loaded uniformly onto subsequent Northern gels. Results from autoradiogram scans demonstrate highly linear gray scale responses over a 4-fold range of total RNA (R2 greater than 0.950) that are reproducible with different blots and probe types (e.g., riboprobe, cDNA and oligonucleotide). In addition, we describe a normalization technique using a 30-mer oligonucleotide probe for rat 28S ribosomal RNA as a measure of total RNA loaded per gel lane. Altogether, this scanning, ribosomal RNA normalization system allows the measurement of relative changes between 20% and 400% using standard autoradiographic methods.     

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.     

Specific changes in the biosynthesis and acetylation of nucleosomal histones in the early stages of embryogenesis of sea urchin.

Histones of the sea urchin Strongylocentrotus droebachiensis were labelled in vivo with [¹⁴C]protein hydrolysate and [¹⁴C]sodium acetate at the developmental stages of blastula, gastrula and pluteus. They were further resolved by electrophoresis in 15% polyacrylamide gels in the presence of 0.9 M acetic acid, 8 M urea as well as in 20% polyacrylamide gels containing the same concentrations of acetic acid and urea, but in addition, 4.3 mM Triton X-100. On comparison of electrophoretic patterns with autoradiograms it was shown that during the early stages of differentiation the synthesis of several new subfractions of some of the histones takes place, namely of two of H1, of two of H2b and of nine of H2a. At the same time the electrophoretic pattern of the arginine-rich histones remained constant, H3 consisting of three and H4 of four subfractions. The basic reason for this heterogeneity seems to be the specific post-synthetic acetylation of the histones. At the blastula stage all the newly synthesized H4 molecules are equally acetylated, while the histone H3 molecules are acetylated to varying extents. After gastrulation both H3 and H4 are also subject to a different level of acetylation.     

Selective labeling of functional groups on membrane proteins or glycoproteins using reactive biotin derivatives and 125I-streptavidin

Amino groups, sulfhydryl groups or oxidation-induced aldehydes on erythrocyte membrane proteins and/or glycoproteins, were reacted with biotinyl N-hydroxysuccinimide ester (BNHS), 3-(N-maleimido-propionyl) biocytin (MPB) or biocytin hydrazide (BCHZ), respectively. The detergent-lysed biotinylated samples were subjected to SDS-polyacrylamide gel electrophoresis, and the proteins were transferred onto nitrocellulose membranes. The blot was then incubated with a solution containing 125I-streptavidin, and processed for autoradiography. The advantages of this approach over previously reported procedures for labeling the three functional groups include the following: extremely high sensitivity; short exposure times of autoradiograms and relatively low levels of radioactivity; single-step radiolabeling procedures subsequent to processing and handling of gels and no background labeling in control samples.