Showing papers on "Gel electrophoresis published in 1995"

The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells

Abstract: Single cell gel electrophoresis is a sensitive method for detecting DNA strand breaks. Cells embedded in agarose are converted to nucleoids by treating with detergent and high salt. DNA breaks render the nucleoid DNA susceptible to extension by electrophoresis, forming 'comets'. We find that when DNA breakage resulting from H2O2 treatment is examined, freshly isolated normal human lymphocytes are relatively resistant compared with transformed human cells. When incubated after treatment with H2O2, HeLa cells repair most strand breaks within 1 h, and a substantial fraction of the oxidised pyrimidines (detected by converting them to DNA breaks with endonuclease III) within 4 h. However, lymphocytes are less proficient at repair; during incubation for 4 h after treatment with H2O2, no detectable removal of endonuclease III-sensitive sites is seen. While the addition of deoxyribonucleosides promotes completion of repair of UV damage by lymphocytes, it has no significant effect on repair of oxidative damage.

Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.

Abstract: A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.

Ultra-high-speed DNA sequencing using capillary electrophoresis chips.

Abstract: DNA sequencing has been performed on microfabricated capillary electrophoresis chips. DNA separations were achieved in 50 x 8 microns cross-section channels microfabricated in a 2 in. x 3 in. glass sandwich structure using a denaturing 9% T, 0% C polyacrylamide sieving medium. DNA sequencing fragment ladders were produced and fluorescently labeled using the recently developed energy transfer dye-labeled primers. Sequencing extension fragments were separated to approximately 433 bases in only 10 min using a one-color detection system and an effective separation distance of only 3.5 cm. Using a four-color labeling and detection format, DNA sequencing with 97% accuracy and single-base resolution to approximately 150 bases was achieved in only 540 s. A resolution of greater than 0.5 was obtained out to 200 bases for both the one- and four-color separations. The prospects for enhancing the resolution and sensitivity of these chip separations are discussed. This work establishes the feasibility of high-speed, high-throughput DNA sequencing using capillary array electrophoresis chips.

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis

Abstract: Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

Preparation of megabase‐size DNA from plant nuclei

Abstract: A novel technique has been developed for the preparation of high molecular weight (HMW) DNA from plant nuclei. This technique involves physical homogenization of plant tissues, nuclei isolation, embedding of the nuclei in low-melting-point agarose microbeads or plugs, and DNA purification in situ. This technique is simple, rapid, and economical, and the majority of the DNA prepared is over 5.7 Mb in size. The genomic DNA content of the HMW DNA prepared by this technique is enriched by at least threefold and the chloroplast DNA content is reduced by over twofold relative to that prepared from plant protoplasts by existing methods. The DNA is readily digestible with different restriction enzymes and partial digestions of the DNA could be reproducibly performed. This method has been successfully used for the preparation of HMW DNA from a wide range of plant taxa, including grasses, legumes, vegetables, and trees. These results demonstrate that the DNA prepared by this technique is suitable for plant genome analysis by pulsed-field gel electrophoresis and for the construction of yeast and bacterial artificial chromosomes.

DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis

Abstract: Myocardial tissue taken from 19 autopsy cases of myocardial infarction were examined both by the nick and labeling method (NELM) and by DNA agarose gel electrophoresis in order to demonstrate the localization of cells with fragmented DNA and to confirm the internucleosomal cleavage of DNA biochemically. The nuclei corresponding to those with the histological features of acute myocardial infarction in hematoxylin and eosin (H&E)-stained sections were stained strongly positive with the nick end labeling method. Myocardial cells corresponding to those with nick end labeling method-stained nuclei, on the other hand, had mostly pyknotic and karyolytic nuclei and some unremarkable nuclei, even nuclear ghosts, and showed degenerated cytoplasm, including contraction band necrosis in H&E-stained preparations. The agarose gel electrophoresis of DNA extracted from the corresponding areas mentioned above showed the ladder pattern of internucleosomal cleavage characteristic of apoptosis. The present study revealed that infarcted myocardial cells with nuclear outlines, even nuclear ghosts, showed a distinct DNA fragmentation with the ladder pattern of internucleosomal cleavage. It is concluded from this study that the damaged myocardial cells of acute myocardial infarction represent a coagulation necrosis having the biochemical nature of apoptosis.

Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation.

Abstract: A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.

Instrument and method for the sequencing of genome

Abstract: Improved techniques are provided for DNA sequencing, and particularly for sequencing of the entire human genome. Different base-specific reactions are utilized to use different sets of DNA fragments from a piece of DNA of unknown sequence. Each of the different sets of DNA fragments has a common origin and terminates at a particular base along the unknown sequence. The molecular weight of the DNA fragments in each of the different sets is detected by a matrix assisted laser absorption mass spectrometer to determine the sequence of the different bases in the DNA. The methods and apparatus of the present invention provide a relatively simple and low cost technique which may be automated to sequence thousands of gene bases per hour, and eliminates the tedious and time consuming gel electrophoresis separation technique conventionally used to determine the masses of DNA fragments.

Purification and characterization of two potent heat-stable protein inhibitors of protein phosphatase 2a from bovine kidney

Abstract: Two heat-stable protein inhibitors of protein phosphatase 2A (PP2A), tentatively designated I1PP2A and I2PP2A, have been purified to apparent homogeneity from extracts of bovine kidney. The purified preparations of I1PP2A exhibited an apparent M(r) approximately 30,000 and 250,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-300, respectively. In contrast, the purified preparations of I2PP2A exhibited an apparent M(r) approximately 20,000 and 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200, respectively. The purified preparations of I1PP2A and I2PP2A inhibited PP2A with 32P-labeled myelin basic protein, 32P-labeled histone H1, 32P-labeled pyruvate dehydrogenase complex, 32P-labeled phosphorylase, and protamine kinase as substrates. By contrast, I1PP2A and I2PP2A exhibited little effect, if any, on the activity of PP2A with 32P-labeled casein, and did not prevent the autodephosphorylation of PP2A in incubations with the autophosphorylation-activated protein kinase [Guo, H., & Damuni, Z. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504]. The purified preparations of I1PP2A and I2PP2A had little effect, if any, on the activities of protein phosphatase 1, protein phosphatase 2B, protein phosphatase 2C, and pyruvate dehydrogenase phosphatase. With 32P-labeled MBP as a substrate, kinetic analysis according to Henderson showed that I1PP2A and I2PP2A were noncompetitive and displayed a Ki of about 30 and 25 nM, respectively. Following cleavage with Staphylococcus aureus V8 protease, I1PP2A and I2PP2A displayed distinct peptide patterns, indicating that these inhibitor proteins are the products of distinct genes. The N-terminal amino acid sequences of the purified preparations indicate that I1PP2A and I2PP2A are novel proteins.

Identification and purification of a new staphylococcal enterotoxin, H

Abstract: A staphylococcal enterotoxin which elicited an emetic response in monkeys but did not share antigenic determinants with any of the identified enterotoxins was identified and purified from Staphylococcus aureus FRI-569. The emetic activity of this new enterotoxin was neutralized only by antibodies specific to it and not by antibodies to enterotoxins A, B, C, D, and E or toxic shock syndrome toxin 1. Immunodiffusion assays did not detect cross-reactivity between this new and all the other identified enterotoxins. The purification procedure involved removal of the enterotoxin from culture supernatant fluids by batch adsorption with CG-50 resin, CM-Sepharose FL ion-exchange chromatography, and Sephacryl 100 HR and Bio-Gel P-30 gel filtration. The molecular weight of this enterotoxin, 27,300, determined by gel filtration on Sephacryl 100 HR agreed with the molecular weight, 28,500, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The apparent migration of this enterotoxin determined by SDS-PAGE did not shift in the presence of a disulfide reducing agent, indicating that it is composed of a single-chain protein. The N-terminal amino acid sequence of the enterotoxin was determined to be Glu-Asp-Leu-His-Asp-Lys-Ser-Glu-Leu-Thr-Asp-Leu-Ala-Leu-Ala-Asn-Ala-Tyr- Gly- Gln-Tyr-Asn-His-Pro-Phe-Ile-Lys-Glu-Asn-Ile, which did not match the N-terminal sequences of any known proteins. The isoelectric point of the enterotoxin determined by isoelectric focusing was about 5.7.(ABSTRACT TRUNCATED AT 250 WORDS)

The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay.

Abstract: Single cell gel electrophoresis under alkaline conditions is a technique used to detect primary DNA damage in individual mammalian cells. Cells embedded in agarose on microscope slides are subjected to lysis, unwinding of DNA and electrophoresis at high pH. After staining with a fluorescent dye, cells with DNA damage display increased migration of genetic material from the cell nucleus. The damage is quantified by measuring the displacement between the genetic material of the nucleus ('comet head') and the resulting 'tail'. The torsional moment of the tail ('tail moment') has been suggested to be an appropriate index of induced DNA damage in considering both the migration of the genetic material as well as the relative amount of DNA in the tail. In the present paper it will be shown that the moment of inertia ('tail inertia'), a not previously described tail parameter, provides a more precise description of the distribution of individual DNA fragments within the tails. The tail inertia was also found to be the most sensitive indicator of the DNA damage induced in peripheral lymphocytes from mice given a single intraperitoneal injection of cyclophosphamide (150 mg/kg b.w.). It is concluded that the tail inertia is an important complement to other tail parameters when looking for damage of DNA with the single cell gel electrophoresis assay.

Purification and characterization of different molecular forms of prostate-specific antigen in human seminal fluid.

Abstract: We have developed a new procedure for purifying prostate-specific antigen (PSA) from human seminal fluid. The method is based on ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration, and anion-exchange chromatography. It can be completed within 2 days with a recovery of intact PSA of 30%. By anion-exchange chromatography, five isoforms of PSA (A, B, C, D, and E) can be separated. The major form (PSA-B) consists of the intact enzyme, as shown by the occurrence of only one band of 33 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, and by amino acid sequencing, which reveals only one amino-terminal sequence corresponding to the reported amino-terminal sequence of intact PSA. The specific absorbance of 1 g/L PSA-B at 280 nm was 1.61, and 80% of the PSA-B formed a complex with alpha 1-antichymotrypsin, indicating that it is enzymatically active. Three cleaved forms of PSA with different nicking sites and low enzymatic activity were separated from intact PSA by ion-exchange chromatography. In addition, we isolated a glycosylation variant, PSA-A, which showed a higher isoelectric point (pI = 7.2) than PSA-B (pI = 6.9) but similar enzymatic activity; this form accounts for 5-10% of total PSA. After treatment with sialidase, PSA-A and B had the same isoelectric point value (pI = 7.7).

The major proteins of wheat endosperm starch granules

Abstract: Wheat starch contains two classes of associated proteins: proteins which are embedded within the granule and loosely associated surface proteins. The characterisation of the major proteins that are embedded in the granule are described. Gel electrophoresis on the basis of size resolved these proteins into five bands of molecular weights 60, 75, 85, 100 and 105 kDa. These polypeptides were demonstrated to be within the granule by their resistance to proteinase K digestion when granules were ungelatinised. The N-terminal sequences of these polypeptides are reported. The most prominent polypeptide is the 60 kDa granule-bound starch synthase. The N-terminal sequence obtained from the 75 kDa polypeptide shows homology to rice soluble starch synthase. The 85 kDa band was resolved into at least two types of polypeptides, one of which reacted with polyclonal antiserum to the maize branching enzyme IIb. The 100 and 105 kDa polypeptides were located only in the granule and are related, on the basis of N-terminal sequence similarity and cross-reactivity to monoclonal antibodies. SDS-PAGE and monoclonal antibody cross-reactivity experiments suggest that the 100 and 105 kDa polypeptides are absent from starch granules from all other species examined, including other cereals. It is speculated that all the major granule proteins are involved in starch biosynthesis.

Identification of macrophage and stress-induced proteins of Mycobacterium tuberculosis.

Abstract: Using phosphorimager technology to quantitate differences in protein expression, we have investigated the modulation of protein synthesis by Mycobacterium tuberculosis in response to intracellular residence in human macrophages and, for comparison, in response to various stress conditions during extracellular growth. Proteins of M. tuberculosis growing intracellularly in human THP-1 cells and extracellularly in broth were labeled with [35S]methionine; during intracellular growth, host cell protein synthesis was inhibited with cycloheximide. The metabolically labeled proteins were separated by two-dimensional gel electrophoresis and quantitatively analyzed. Intracellular residence in macrophages induced a profound change in the overall phenotype of M. tuberculosis. The expression of at least 16 M. tuberculosis proteins was induced (at least a twofold increase compared with growth in broth) and 28 proteins repressed (at least a twofold decrease). Many of the phenotypic changes in protein expression induced during intracellular growth occurred during extracellular growth in response to stress conditions including heat-shock, low pH, and H2O2. However, the pattern of induced and repressed proteins was unique to each stress condition. Of the 16 macrophage-induced proteins, 6 were absent during extracellular growth under both normal and stress conditions. Such proteins are potential virulence determinants and/or they may be important in the cell-mediated and protective immune response to M. tuberculosis infection.

Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands

Abstract: We have used an in vitro selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of selection. Rounds of photocrosslinking selection were alternated with rounds of selection for RNA sequences with high affinity to Rev. This iterative, dual-selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. In vitro selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.

Handbook of Molecular and Cellular Methods in Biology and Medicine

Abstract: Isolation and Purification of DNA Isolation and Purification of RNA Extraction and Purification of Proteins/Enzymes Preparation of Nucleic Acid Probes Electrophoresis, Blotting, and Hybridization cDNA Libraries Genomic DNA Libraries DNA Sequencing PCR Techniques and Applications DNA Fingerprinting DNA Mutagenesis In Vitro Inhibition of Gene Expression by Antisense DNA and RNA DNA Footprinting and Gel Retardation Assay Translation of mRNA(s) In Vitro and Analysis of Proteins by Gel Electrophoresis Gene Transfer and Expression in Animals Gene Transfer and Expression in Plants Plant Cell Culture Plant Tissue Culture Microscopy: LM, SEM, TEM, Confocal and Applications Bioseparation Techniques and Their Applications Preparation of Monoclonal and Polyclonal Antibodies against Specific Protein(s)

Solubilization and partial characterization of extensin fragments from cell walls of cotton suspension cultures. Evidence for a covalent cross-link between extensin and pectin

Abstract: Extensin, a major hydroxyproline (Hyp)-rich glycoprotein in walls of cultured cells of dicotyledonous plants, is very difficult to solubilize. To learn about the nature of the insolubilization, we have tested the ability of a variety of selective hydrolytic methods, and combinations of them, to liberate extensin or fragments of extensin from suspension-culture cell walls. After the complete deglycosylation of cotton (Gossypium hirsutum L.) walls, trypsinization solubilized 80% of the Hyp. The sequences of three abundant peptides were: (a) serine-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-serine-Hyp-Hyp-lysine, (b) serine-Hyp-Hyp-Hyp-Hyp-valine-lysine, and (c) serine-Hyp-Hyp-serine-alanine-Hyp-lysine. After a sequential treatment of walls with endopolygalacturonase, cellulase, -73 degrees C anhydrous hydrogen fluoride solvolysis, and ammonium bicarbonate extraction, only sugars indicative of rhamnogalacturonan I and protein remained insoluble. Trypsin treatment of this residue liberated 50% of the Hyp. A significant proportion of rhamnogalacturonan-associated sugars co-solubilized and co-purified along with the extensin fragments following the trypsinization. By sodium dodecyl sulfate gel electrophoresis and gel filtration, the glycopeptides fell into two classes. One class contained distinctly sized molecules with relative molecular weights in the range of 4,000 to 24,000. The other class did not enter the resolving gel and was hetero-disperse. After complete deglycosylation by a 0 degrees C anhydrous hydrogen fluoride treatment, the first class was little affected in its electrophoretic mobility, whereas the larger heterogeneous material mostly entered the separating gel. After further trypsinization of the deglycosylated peptides and analysis by capillary zone electrophoresis, the peptides in both size classes were shown to contain the sequences described above. From our observations we suggest that cotton extensin becomes insolubilized into cell walls in part by pectin-protein cross-links in addition to the protein-protein (or protein-phenolic-protein) cross-links that have been repeatedly suggested.

Identification of endo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase as cluster-dispersing enzymes in Staphylococcus aureus.

Abstract: Two proteins which are capable of dispersing cell clusters of Staphylococcus aureus have been purified from a S. aureus FDA209P culture supernatant. Both of them were found to have bacteriolytic activity. From the elution profile of column chromatography and Western blot (immunoblot) analysis, one of them was identified as a 51-kDa endo-beta-N-acetylglucosaminidase (GL). The other was a 62-kDa protein on the basis of sodium dodecyl sulfate gel electrophoresis. Analysis of the peptidoglycan fragments following treatment with the 62-kDa protein indicated that this protein is an N-acetylmuramyl-L-alanine amidase (AM). In vitro studies of cluster dispersion activities using S. aureus mutant strains Lyt66 or S. aureus Wood46 grown as clusters demonstrated that these two enzymes act synergistically to disperse clusters into single cells. Antiserum against the 51-kDa GL cross-reacted with the 62-kDa AM, and S. aureus FDA209P grown in the presence of anti-51-kDa-GL immunoglobulin G induced giant clusters. Clusters induced by anti-51-kDa GL and by Cibacron blue F3G-A were dispersed by coincubation with the 51-kDa GL and the 62-kDa AM. Western blot analysis demonstrated that the 51-kDa GL and the 62-kDa AM were missing in culture supernatants of S. aureus Lyt66, Wood46, and RUSAL2 (Tn551 autolysin-defective mutant), which grow in clusters. These results strongly suggest that the 51-kDa GL and 62-kDa AM are involved in cell separation of daughter cells after cell division.

Analysis of T cell receptor-gamma gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products. Correlation with tumor-specific sequences.

Abstract: We describe a modified denaturing gradient gel electrophoresis (DGGE) procedure with a 40-nucleotide GC clamp in the polymerase chain reaction to improve resolution in amplifying T cell receptor-gamma (TCR-gamma) rearrangements. DNA from 46 cases of lymphoblastic leukemia/lymphoma, 5T cell lines, 2 B cell lines, 7 normal lymphocytes, and 3 cases of Hodgkin's disease was amplified by polymerase chain reaction. In addition, 20 cases of paraffin-embedded T cell lymphomas and 5 cases of reactive hyperplasia were also studied. Clonal TCR-gamma rearrangements were identified on DGGE by the presence of a predominant band. Results obtained from 5 T cell lines and 12 lymphoblastic leukemia/lymphomas containing known TCR-gamma gene rearrangements revealed 100% concordance in detecting clonal rearrangements between DGGE and traditional Southern blot analysis. Of the remaining 34 lymphoblastic leukemia/lymphoma cases studied by DGGE alone, 30 were positive. DGGE analysis of 10 lymphoblastic leukemia/lymphoma cases with known group IV gamma to J gamma 1 or J gamma 2 rearrangement sequences confirmed that the electrophoretic migration was dependent on the tumor-specific rearranged TCR-gamma sequence. In addition, 17 of 20 cases of paraffin-embedded T cell lymphomas were positive by DGGE, 6 of which had the clonal population also identified in fresh tissue DNA. DGGE analysis of GC-clamped polymerase chain reaction products can provide a way to more accurately detect TCR-gamma clonality of lymphoid tumors and can be applied to archival tissues.

Characterization of a 100-kilodalton binding protein for the six serotypes of coxsackie B viruses.

Abstract: Viral infection of host cells primarily depends on binding of the virus to a specific cell surface protein. In order to characterize the binding protein for group B coxsackieviruses (CVB), detergent-solubilized membrane proteins of different cell lines were tested in virus overlay protein-binding assays. A prominent virus-binding protein with a molecular mass of 100 kDa was detected in various CVB-permissive human and monkey cell lines but was not detected in nonpermissive cell lines. The specificity of CVB binding to the 100-kDa protein on permissive human cells was substantiated by binding of all six serotypes of CVB and by competition experiments. In contrast, poliovirus and Sendai virus did not bind to the 100-kDa CVB-specific protein. A fraction of HeLa membrane proteins enriched in the range of 100 kDa showed functional activity by transforming infectious CVB (160S) into A-particles (135S). In order to purify this CVB-binding protein, solubilized membrane proteins from HeLa cells were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by elution of the 100-kDa protein. Amino acid sequence analysis of tryptic fragments of the CVB-binding protein indicated that this 100-kDa CVB-specific protein is a cell surface protein related to nucleolin. These results were confirmed by immunoprecipitations of the CVB-binding protein with nucleolin-specific antibodies, suggesting that a nucleolin-related membrane protein acts as a specific binding protein for the six serotypes of CVB.