Showing papers by "Laboratory of Molecular Biology published in 1978"
TL;DR: The experiments established the usefulness of the bybrid myeloma technique in preparing monospecific antibodies against human cell surface antigens and highlights the possibilities not only of obtaining reagents for somatic cell genetics, but also of obtaining mouse antibodies detecting human antigenic polymorphisms.
1,892 citations
TL;DR: The identification of such a cell line, Sp2/0-Ag14, is reported here the identification of a tumour cell fusion partner that makes no Ig but which can nevertheless be fused with spleen cells to obtain hybrids secreting only the specific antibody.
Abstract: FUSION of myeloma cells which grow in tissue culture with spleen cells from an immunised mouse provides a general method for obtaining cell lines (hybridomas) which make antibody of the desired specificity1–3. Hybrids derived from these myelomas make the immunoglobulin (Ig) heavy and light chains of the myeloma parent as well as the antigen-specific heavy and light chains of the spleen cell parent. In conditions in which the two heavy and two light chains associate randomly, a hybridoma would make 10 distinct Ig molecules, and the specific antibody would comprise only 1/16 of the total Ig4,5. To obtain hybridomas making only the specific antibodies requires a tumour cell fusion partner that itself makes no Ig but which can nevertheless be fused with spleen cells to obtain hybrids secreting only the specific antibody. We report here the identification of such a cell line, Sp2/0-Ag14.
1,654 citations
TL;DR: A recently characterised class of adhesive, high molecular weight glycoproteins is present on the surfaces of cells, in connective tissue matrices, and in extracellular fluids.
Abstract: A recently characterised class of adhesive, high molecular weight glycoproteins is present on the surfaces of cells, in connective tissue matrices, and in extracellular fluids. These proteins may have important roles in cellular adhesion, malignant transformation, reticuloendothelial system function, and embryonic differentiation.
1,393 citations
TL;DR: Using the plus and minus [l] and the terminator [3] methods, serious variations in the distances between consecutive nucleotide bands in regions of dyad symmetry where base-paired loop structures can form are found, and this can lead to difficulties of interpretation.
1,130 citations
TL;DR: The determination of the total 5,224 base-pair DNA sequence of the virus SV40 has enabled us to locate precisely the known genes on the genome.
Abstract: The determination of the total 5,224 base-pair DNA sequence of the virus SV40 has enabled us to locate precisely the known genes on the genome. At least 15.2% of the genome is presumably not translated into polypeptides. Particular points of interest revealed by the complete sequence are the initiation of the early t and T antigens at the same position and the fact that the T antigen is coded by two non-contiguous regions of the genome; the T antigen mRNA is spliced in the coding region. In the late region the gene for the major protein VP1 overlaps those for proteins VP2 and VP3 over 122 nucleotides but is read in a different frame. The almost complete amino acid sequences of the two early proteins as well as those of the late proteins have been deduced from the nucleotide sequence. The mRNAs for the latter three proteins are presumably spliced out of a common primary RNA transcript. The use of degenerate codons is decidedly non-random, but is similar for the early and late regions. Codons of the type NUC, NCG and CGN are absent or very rare.
1,000 citations
TL;DR: The nucleosome assembly protein has been identified and purified from eggs of Xenopus laevis and it is shown that it binds histones and DNA to form nucleosomes.
Abstract: The nucleosome subunits of chromatin are assembled from histones and DNA by an acidic protein which binds histones. The nucleosome assembly protein has been identified and purified from eggs of Xenopus laevis.
707 citations
TL;DR: All factors that lower the oxygen affinity of hemoglobin act by strengthening the salt bridges that constrain its quaternary deoxy (T) structure.
Abstract: Electrostatic effects dominate many aspects of protein behavior. When polypeptide chains fold up, most polar side chains seek the exterior, where they can be solvated. Water bound in the interior has been found between the domains of enzymes of the chymotrypsin family, and between the subunits of hemoglobin and tobacco mosaic virus protein. Assembly of this protein from disk to virus is triggered by electrostatic interactions between neighboring subunits. Lysozyme stabilizes the constellation of charges involved in the transition state of its substrate by both permanent and induced dipoles. All factors that lower the oxygen affinity of hemoglobin act by strengthening the salt bridges that constrain its quaternary deoxy (T) structure. Enzymes of thermophile bacteria owe their extra stability mostly to additional salt bridges. The rate of denaturation of hemoglobins by alkali is determined by the ionization of internal side chains with pK9s of about 12.
670 citations
TL;DR: The polypeptide chain of a TBSV subunit folds into two domains, connected by a hinge, and a flexibly-linked N-terminal arm, and RNA is also not uniquely fixed to sites on the major domains.
Abstract: The polypeptide chain of a TBSV subunit folds into two domains, connected by a hinge, and a flexibly-linked N-terminal arm. Sixty of the 180 N-terminal arms inter-digitate in groups of three, in an unexpected mode of protein association. The remaining 120 arms are not uniquely positioned with respect to the rest of the subunit. RNA is also not uniquely fixed to sites on the major domains.
654 citations
TL;DR: The complete nucleotide sequence of the DNA of bacteriophage φX174 has been determined and the amino acid sequences of the ten proteins for which the DNA codes have also been deduced.
608 citations
TL;DR: Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared and each antigenic target was analyzed by gel electrophoresis of immunoprecipitated 125I‐labeled cell surface molecules.
Abstract: Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with 125I-labeled anti-rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated 125I-labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross-inhibition of binding of different monoclonal antibodies.
It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat-stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major 125I-labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105000. Five IgG-secreting clones identify the fourth antigen, a heat-stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross-inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.
562 citations
TL;DR: Two octapeptides corresponding to the CCK-like component previously identified by RIA are purified from sheep brain, and the isolation, sequence and some properties of these molecules are reported.
Abstract: SEVERAL peptides—for example, substance P and neurotensin—are now known to occur in both endocrine cells of the gut and in central or peripheral neurones1. The significance of this dual distribution is far from fully understood, but it seems possible that the same peptide might function as both hormone and neurotransmitter. Vanderhaeghen et al. have reported that radioimmunoassays (RIA) for the antral hormone gastrin have shown activity in extracts of brain, particularly cerebral cortex, of a wide range of vertebrate species2. However, gastrin has the same COOH-terminal pentapeptide sequence as the intestinal hormone cholecystokinin (CCK) and antisera specific for the COOH terminus of gastrin usually cross-react to some extent with CCK also. We found that the pattern of cross-reactivity of boiling water extracts of brain with six different antisera resembled that of a COOH-terminal fragment of CCK more closely than gastrin, and gel filtration studies have indicated that the main component in the extracts was compatible with the COOH-terminal octapeptide of CCK (CCK8)3; these findings have since been confirmed by others4–6. We have purified from sheep brain two octapeptides corresponding to the CCK-like component previously identified by RIA, and report here the isolation, sequence and some properties of these molecules.
TL;DR: Protein subunits in the two layers of the disk of tobacco mosaic virus have very similar conformations, including salt-bridge systems and aromatic clusters within each molecule linked in a hydrophobic girdle encircling each ring.
Abstract: Protein subunits in the two layers of the disk of tobacco mosaic virus have very similar conformations. Much of the bonding between subunits is polar, including salt-bridge systems. Arginine residues play a prominent part here and elsewhere. Interactions within each layer involve groups whose contacts can be adjusted to allow the transition from disk to virus helix. Aromatic clusters within each molecule are linked in a hydrophobic girdle encircling each ring.
TL;DR: The complete sequence of chicken ovalbumin mRNA is presented and an extensive 3′ noncoding region of 637 nucleotides which may have no function that is precisely dependent on its sequence is presented.
Abstract: The complete sequence of chicken ovalbumin mRNA is presented; it is 1,859 residues long, excluding its terminal 'cap' and poly(A). The region coding for ovalbumin lies close to the 'cap' but is separated from the poly(A) by an extensive 3' noncoding region of 637 nucleotides which may have no function that is precisely dependent on its sequence.
TL;DR: In this paper, the role of glycosylation in the biosynthesis, processing and turnover of CSP, the major cell surface glycoprotein of chick embryo fibroblasts (CEF), was investigated.
TL;DR: All three polypeptides of insulin, epidermal growth factor and alpha2-macroglobulin are internalized within the same vesicles by a common pathway.
TL;DR: The potential molecular interactions and structural overlaps of these control signals apparently couple the regulation of the decision between lytic and lysogenic growth patterns by phage λ.
Abstract: ρ factor-mediated transcription termination at the tR1 terminator site of bacteriophage λ is examined. Mutations affecting the termination event are characterised. These mutations define features of the site which seem to be important to terminator function. In addition, other related transcriptional and translational regulatory elements are defined within the region surrounding the termination site. The potential molecular interactions and structural overlaps of these control signals apparently couple the regulation of the decision between lytic and lysogenic growth patterns by phage λ.
TL;DR: The alteration was shown, by restriction endonuclease analysis and electron microscopy, to be an insertion of the F attachment sequence λδ (2.8 to 8.5F), which is, therefore, an insertion sequence.
TL;DR: It is proposed that the pattern of relative rates of attack reflects the common protection or exposure of sites on the two turns of a DNA super helix which has about 80 base-pairs per turn and can be correlated with X-ray crystallographic studies.
TL;DR: Moulting of Caenorhabditis elegans has been observed by Nomarski interference contrast microscopy, and by electron microscopy of animals at selected stages, and the excretory system is not essential for moulting.
Abstract: Moulting of Caenorhabditis elegans has been observed by Nomarski interference contrast microscopy, and by electron microscopy of animals at selected stages. The wild type, cell division mutants and animals in which cells had been ablated by a laser microbeam were examined. The median lateral hypodermis, or "seam", is required for the formation of alae and for dauer larva maturation. During cuticle deposition, large Golgi bodies are seen in the seam cells. The excretory system is not essential for moulting.
TL;DR: In lymphocytes and P3 myeloma cells, cross-linking of surface Ig by the capping and patching phenomenon has been used to demonstrate the induction of a specific association between surface Ig and cellular actin.
Abstract: In lymphocytes and P3 myeloma cells, cross-linking of surface Ig by the capping and patching phenomenon has been used to demonstrate the induction of a specific association between surface Ig and cellular actin.
TL;DR: The core region also cross-links to DNA, indicating intimate interactions between this region in all the non-H1 histones with DNA, and indicates proximities for these molecules within the nucleosome.
TL;DR: The findings suggest that nuclear proteins contain in their molecular structure a signal that enables them to accumulate in the nucleus, which is absent in proteins such as actin, which are similarly abundant in both nucleus and cytoplasm.
Abstract: THE protein composition of the amphibian oocyte nucleus (germinal vesicle, or GV) is very different from that of the cytoplasm, as shown by SDS electrophoresis1–3. The oocyte nucleus is known to accumulate components such as RNA polymerase4, histones5 and factors that protect DNA from nucleases6, probably stockpiled for use during early development. Furthermore, some nuclear proteins accumulate in the germinal vesicle after microinjection into the oocyte cytoplasm, as shown by Gurdon7 for labelled histones and by Bonner8, who injected labelled total GV contents into oocytes. We describe here a study of these phenomena using a recently described high resolution method of protein analysis involving two-dimensional gel electrophoresis9. Having defined those proteins which are nuclear, cytoplasmic, or present in both cell compartments of Xenopus laevis oocytes, we micro-injected a soluble preparation of labelled GV proteins into oocyte cytoplasm and analysed their distribution in the nuclear and cytoplasmic compartments. The results show that cells have exquisitely selective mechanisms that determine the extent to which a protein will become concentrated in the nucleus. Our findings suggest that nuclear proteins contain in their molecular structure a signal that enables them to accumulate in the nucleus. This signal is absent in proteins such as actin, which are similarly abundant in both nucleus and cytoplasm.
TL;DR: The question of radiation damage at low temperatures has been investigated with the view in mind of reducing somewhat the rate at which damage occurs, relative to the damage rate at room temperature.
Abstract: When biological specimens are irradiated by the electron beam in the electron microscope, the specimen structure is damaged as a result of molecular excitation, ionization, and subsequent chemical reactions. The radiation damage that occurs in the normal process of electron microscopy is known to present severe limitations for imaging high resolution detail in biological specimens. The question of radiation damage at low temperatures has therefore been investigated with the view in mind of reducing somewhat the rate at which damage occurs. The radiation damage protection found for small molecule (anhydrous) organic compounds is generally rather limited or even non-existent. However, large molecular, hydrated materials show as much as a 10-fold reduction at low temperature in the rate at which radiation damage occurs, relative to the damage rate at room temperature. In the case of hydrated specimens, therefore, low temperature electron microscopy offers an important advantage as part of the overall effort required in obtaining high resolution images of complex biological structures.
TL;DR: The hypothesis is put forward that the mitochondrial reticulum serves as a system for transport of energy, oxygen and fatty acid residues along mitochondrial membranes over distances commensurable with the muscle fiber diameter.
TL;DR: P815 cells spontaneously shed material (exfoliate) in which actin is the major protein, and the association between actin and H–2 is stable even in the presence of detergent, indicating that protein–protein interactions exist between the actIn and H-2.
Abstract: P815 cells spontaneously shed material (exfoliate) in which actin is the major protein. The exfoliate also contains H-2. The association between actin and H-2 is stable even in the presence of detergent, indicating that protein-protein interactions exist between the actin and H-2. The exfoliate probably consists of highly purified microvilli.
TL;DR: The monoclinic crystal structure of yeast tRNA Phe is refined against a complete set of X-ray data at 2.5 A resolution, using real-space refinement and a combination of energy minimisation and crystallographic least-squares.
TL;DR: The method permits the quantitative isolation of covalently closed circular DNA from either total cellular DNA or partially purified preparations, to a degree of purity comparable with buoyant density procedures.
Abstract: A new technique has been developed for the rapid isolation of covalently closed circular DNA molecules. The procedure is a selective extraction based on differences in the partitioning of covalently closed circular DNA molecules and noncovalently closed species between phenol and water at acid pH and low ionic strength. Under the conditions described, linear as well as nicked circular DNA is extracted into phenol, while covalently closed circular DNA molecules remain in the water phase. The method permits the quantitative isolation of covalently closed circular DNA from either total cellular DNA or partially purified preparations, to a degree of purity comparable with buoyant density procedures.
TL;DR: A set of monoclonal antibodies derived by fusing P3-NS1/1-Ag4-1 myeloma cells with spleen cells from a rat immunized with mouse spleen were screened for activity against a tumor cell panel and one was found to react only with mouse embryonal carcinoma cells and no other tumor cell type tested.
TL;DR: The development events that generate the adult complement of cells occur in a period preceding the first larval moult, and during this period a class of pre-existing, juvenile motoneurones changes its pattern of connectivity.
Abstract: THE ventral nerve cord of the nematode Caenorhabditis elegans contains a linear array of motoneurones (Fig. 1) which innervate the body muscles that mediate locomotion. The adult ventral cord has about three times as many cells as that of the first stage larva. The development events that generate the adult complement of cells occur in a period preceding the first larval moult. During this period we find that a class of pre-existing, juvenile motoneurones changes its pattern of connectivity. Neuromuscular junctions are removed from ventral muscles and are reformed onto dorsal muscles. Similarly the dendritic input to these neurones changes over from the dorsal to the ventral side.
TL;DR: Three synthetic oligonucleotides were prepared to be complementary to known regions of the mouse immunoglublin light chain mRNA, and their ability to prime the transcription of complementary DNA (cDNA) was studied.