Showing papers by "Laboratory of Molecular Biology published in 1974"
TL;DR: In this paper, the authors describe methods for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm.
Abstract: Methods are described for the isolation, complementation and mapping of mutants of Caenorhabditis elegans, a small free-living nematode worm. About 300 EMS-induced mutants affecting behavior and morphology have been characterized and about one hundred genes have been defined. Mutations in 77 of these alter the movement of the animal. Estimates of the induced mutation frequency of both the visible mutants and X chromosome lethals suggests that, just as in Drosophila, the genetic units in C. elegans are large.
13,247 citations
TL;DR: A simple method for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film, which is ten times more sensitive than conventional autoradiography of isotopes with higher emission energies.
Abstract: A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X-ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5-diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to RP Royal “X-Omat” film at -70 °C. Optimal conditions for each step are described. β-particles from 3H interact with the 2,5-diphenyloxazole emitting light which causes local blackening of an X-ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C. 3000 dis. 3H/min in a band in a gel can be detected in a 24-h exposure. Similarly 500 dis./min can be detected in one week.
When applied to the detection of 35S and 14C in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 35S or 14C/min in a band in a gel can be detected in 24 h.
8,114 citations
TL;DR: Preliminary results do show less cross-linking of histones in chromatin than in solution, but crosslinked products up to pentamers are readily observed and call for further investigation.
Abstract: ciations of the histones in chromatin but says nothing of details, such as whether the F2A1 and F3 pair, which occurs as an (F2Al)2(F3)2 tetramer in solution, also occurs as a tetramer in chromatin. The most direct evidence for an (F2Al)2(F3)2 tetramer in chromatin is that a complex formed from tetramers, F2A2-F2B oligomers, and DNA gives the same x-ray pattern as chromatin (Fig. 4, upper two traces). Tetramers and F2A2-F2B oligomers are both required to give the x-ray pattern (Fig. 4, lower two traces), but Fl is not-in keeping with previous observations (3, 23 ) that removing Fl from chromatin does not affect the x-ray pattern. Further implications of these results are discussed in the accompanying article (24). We are currently studying associations of the histones in chromatin by cross-linking. There are two difficulties that do not arise in experiments on the histones in solution: the amino side chains are involved in salt linkages with the phosphate groups of DNA and are thus less available for chemical modification; and the presence of five rather than two histones complicates identification of products from molecular weights. -Preliminary results do show less cross-linking of histones in chromatin than in solution, but crosslinked products up to pentamers are readily observed and call for further investigation.
2,419 citations
TL;DR: The hydrophobic bond is the term used by Kauzmann to describe the gain in free energy on the transfer of non-polar residues from an aqueous environment to the interior of proteins.
Abstract: THE hydrophobic bond is the term used by Kauzmann1 to describe the gain in free energy on the transfer of non-polar residues from an aqueous environment to the interior of proteins. This has been accepted as one of the major forces involved in the folding of proteins. The exact origin of the energy of the hydrophobic bond is controversial2, but empirical values have been derived for 10 protein residue side chains by Nozaki and Tanford3 who measured the solubility of amino acids in the organic solvents ethanol and dioxane.
830 citations
TL;DR: Kornberg proposed that chromatin structure is based on a repeating subunit of 200 base pairs of DNA and two of each of the histones (with the exception of F1 which occurs only once per subunit).
Abstract: IN the past, the structure of chromatin has remained obscure despite the efforts of many laboratories and seemed far from a simple solution1. It has been known only that chromatin contains a repeating substructure2,3. Recently, however, Kornberg proposed4 that chromatin structure is based on a repeating subunit of 200 base pairs of DNA and two of each of the histones (with the exception of F1 which occurs only once per subunit). Furthermore, he suggested that in chromatin these subunits form a flexibly jointed chain.
655 citations
TL;DR: Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x 10(7) base pairs (20 times the genome of E. coli).
Abstract: Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x 10(7) base pairs (20 times the genome of E. coli). Eighty-three percent of the DNA sequences are unique. The mean base composition is 36% GC; a small component, containing the rRNA cistrons, has a base composition of 51% GC. The haploid genome contains about 300 genes for 4S RNA, 110 for 5S RNA, and 55 for (18 + 28)S RNA.
552 citations
TL;DR: Electron micrographs of outer doublet tubules from flagella have been analysed by methods which make use of the computed diffraction patterns of electron-microscope images, and a model for the whole doublet has been proposed.
Abstract: Electron micrographs of outer doublet tubules from flagella have been analysed by methods which make use of the computed diffraction patterns of electron-microscope images. Analysis of singlet A-tubules in the tips of flagella has led to a determination of the helical surface lattice of the A-subfibre, confirming that there are 13 longitudinal protofilaments in the tubule wall and that dimers in neighbouring protofilaments form a staggered arrangement, equivalent to the lattice with an axial periodicity of 8.0 nm predicted in earlier work. A low-resolution 3-dimensional image of the A-tubule has been reconstructed, which supports the evidence for an 8.0-nm-long heterodimer oriented along the protofilaments. The heterodimer is identified as a pair of 4.0-nm morphological units, which appear to be globular at this resolution. Filtered images have been obtained from doublet tubules which show that the B-subfibre is also made up of 8.0-nm dimers, but it differs from the A-tubule in that dimers in adjacent filaments are not in a staggered arrangement but are lined up obliquely at a shallow angle. Using the additional information about the hands of the lattices in the 2 subfibres which is presented in the accompanying paper, a model for the whole doublet has been proposed.
492 citations
374 citations
TL;DR: The theory shows how groups of Purkinje cells could learn to fire with particular frequencies, through signals from the climbing fibres, on receiving different inputs from the parallel fibres and demonstrated that in this way the cerebellum could be used to memorise complex movements, with perfect coordination between the various muscles involved in a movement.
278 citations
TL;DR: The amino-acid sequence of the alkali light chain 1 (Al) of rabbit skeletal muscle myosin has been determined and compared with the sequences of the smaller but related alkala light chain 2 (A2), and it was concluded that either A2 was a degraded fragment of A1 or the two proteins showed an exceptionally high degree of homology.
Abstract: The amino-acid sequence of the alkali light chain 1 (Al) of rabbit skeletal muscle myosin has been determined and compared with the sequence of the smaller but related alkali light chain 2 (A2). The molecular weights of the two proteins calculated from these sequence determinations are 20700 and 16500, respectively. The results show that the two proteins have identical sequences over their C-terminal141 residues. There is an additional sequence of 41 residues at the N-terminal end of A1 which accounts for the extra 4000 molecular weight. Between this additional sequence and the sequence common to both proteins are eight amino-acid residues. Comparison of the sequences of these eight residues in A1 and A2 reveals five amino-acid substitutions. Thus in spite of the very extensive homology between the two proteins, these substitutions indicate that there must be two RNA coding sequences for these light chains in rabbit fast muscle myosin. Rabbit skeletal muscle myosin contains two heavy chains of molecular weight about 200000 [l 21 and four light chains of molecular weight about 20000 [3]. Two chemical classes of light chains have been characterised by their thiol peptides [4] and reaction of myosin with 5,5’-dithiobis(2-nitrobenzoic acid) (Nbs,) selectively dissociated a substantial proportion of one of these light chains without significant effect on the myosin ATPase activity. This light chain has been termed the “Nbs, light chain” l. The remaining light chains cannot be dissociated without total loss of ATPase activity and these were termed the “alkali light chains”, since they were first shown to be released under alkaline conditions [5]. Gel electrophoresis of myosin in the presence of sodium dodecylsulphate shows the presence of three light chain components with apparent molecular weights of 25000, 18000 and 16000 [3]. The 18000-M, component corresponds to the Nbs, light chain and the other two components are classified as alkali light chains since they both contains the single thiol sequence characteristic of this class of light chains in fast muscle myosin [6,7]. The extent of sequence homology between alkali light chain 1 (Al, Mr = 25000) and alkali light chain 2 (A2, Abbreviation. Nbs,, 5,5’-dithiobis(2-nitrobenzoic acid). Nomenclature. The term “alkali light chain” is based on the observation that these proteins are dissociated from myosin in alkali [5]. Other terminology used elsewhere includes LC1(= Al) and LC3 (= A2) [26] and OL and y chains [29]. 1 The “Nbs, light chain” was previously called the “DTNB light chain” [4]. Mr = 16000) was examined by peptide mapping of tryptic digests of the two proteins. Peptides from both proteins stained selectively for arginine, tyrosine and histidine had identical mobilities in the two-dimensional maps, and all the peptides present in A2 could be found in corresponding positions in Al. Such differences as there were could be accounted for by additional peptide material in Al, though the amino-acid compositions indicated minimum molecular weights of 21000 for A1 and 17000 for A2, a difference of 4000 instead of the 8000 estimated from apparent molecular weights determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. From these results it was concluded that either A2 was a degraded fragment of A1 or the two proteins showed an exceptionally high degree of homology. The relative constancy in the amounts of A1 and A2 in rabbit myosin prepared under a variety of conditions together with observations that both components are found in myosin from chicken breast and leg muscles [S], and the fast skeletal muscle of pig, sheep and cat, make it unlikely that A2 arises as a result of adventitious proteolytic activity. Nevertheless we could not rule out a specific cleavage occurring in the polypeptide chains as a consequence of post-translational modification or during the myosin assembly process. For this reason detailed sequence analysis of both A1 and A2 has been carried out to investigate the relationship between them and show whether they represent products of different genes. Part of the results of this work have been published previously [9].
275 citations
TL;DR: D-2,3-DIPHOSPHOGLYCERATE facilitates the transfer of oxygen from human red blood cells to the tissues by lowering the oxygen affinity of haemoglobin by combining preferentially with one of the two alternative forms of Haemoglobin, namely the deoxy form.
Abstract: D-2,3-DIPHOSPHOGLYCERATE (DPG) facilitates the transfer of oxygen from human red blood cells to the tissues by lowering the oxygen affinity of haemoglobin1. It does so by combining preferentially with one of the two alternative forms of haemoglobin, namely the deoxy form, in the ratio of 1 mol per mol tetramer. Its binding site was predicted from biochemical and model building experiments and then determined directly by X-ray crystallography2. The site lies at the entrance to the central cavity between the N-termini of the β chains and is surrounded by four pairs of basic groups: the α amino group of valine 1 and the side chains of histidines 2 and 143, and of lysine 82. The basic groups are related in pairs by the molecular dyad and arranged so as to complement the acidic groups of DPG by forming seven salt bridges (Fig. 1). On oxygenation the N-termini of the β chains move apart and the cavity closes up so that the stereochemical complementarity is lost3. Oxyhaemoglobin does also bind DPG, but much more weakly, and the binding site is still unknown.
TL;DR: The energy refinement of hen egg-white lysozyme co-ordinates is described, a process in which atoms are moved to reduce the molecular potential energy and give a stereochemically acceptable set of atomic co-ords.
TL;DR: Two corollaries of this are that intermediates accumulating after the initial Michaelis complex are undesirable and also enzymes whose function is to optimize reaction rates should have evolved to exhibit KM values above those of accessible substrate concentrations.
Abstract: A simple derivation is given that the catalytic term k$\_{\text{cat}}$/K$\_{\text{S}}\dagger $ is at a maximum when the structure of the enzyme is complementary to the structure of the substrate in the transition state. In addition, at a constant substrate concentration, [S], the maximum reaction rate is obtained when k$\_{\text{cat}}$ and K$\_{\text{S}}$ are individually high so that K$\_{\text{S}}$ is greater than [S]; the overall reaction rate decreases with decreasing k$\_{\text{cat}}$ and K$\_{\text{S}}$ for K$\_{\text{S}}$ less than [S]. Two corollaries of this are that intermediates accumulating after the initial Michaelis complex are undesirable and also enzymes whose function is to optimize reaction rates should have evolved to exhibit K$_{\text{M}}$ values above those of accessible substrate concentrations. This could be achieved by an often 'distortionless' strain which consists either of unfavourable interactions in the enzyme substrate complex which are relieved in the transition state or increasingly favourable interactions in the transition state. A possible special role of the backbone NH groups in this context is discussed. The enzyme need not be complementary to the transition state of the substrate for catalysis to occur.
TL;DR: A set of non-complementing, closely linked, ethyl methanesulphonate-induced mutations in Caenorhabditis elegans specifically affects the structure and function of body-wall muscle cells but not the pharyngeal musculature.
TL;DR: The sequence of 52 nucleotides adjacent to the poly(A) of rabbit β globin mRNA has been determined and striking sequence and structural homologies exist between it and mouse immunoglobulin light chain mRNA.
Abstract: The sequence of 52 nucleotides adjacent to the poly(A) of rabbit β globin mRNA has been determined. Striking sequence and structural homologies exist between it and mouse immunoglobulin light chain mRNA. These homologies may be common to all mammalian mRNA.
TL;DR: Correlated physiological changes, and structural and functional changes in myosin, following cross-reinnervation of cat fast-twitch and slow-twitch muscles indicate that theMyosin phenotype is controlled through the nervous system.
Abstract: Correlated physiological changes, and structural and functional changes in myosin, following cross-reinnervation of cat fast-twitch and slow-twitch muscles, indicate that the myosin phenotype is controlled through the nervous system.
TL;DR: Sequence comparison between the myosin alkali light chain, troponin C and carp calcium binding protein shows that the pattern of hydrophobic residues which forms the core of calcium bindingprotein is preserved in both tropon in C and myos in alkaliLight chain.
Abstract: Sequence comparison between the myosin alkali light chain, troponin C and carp calcium binding protein shows that the pattern of hydrophobic residues which forms the core of calcium binding protein is preserved in both troponin C and myosin alkali light chain. Strong structural similarities between the proteins are suggested. The role of gene duplication in the evolution of troponin C and myosin alkali light chain is discussed.
TL;DR: Subunit I of the RNA phage-specific Qβ replicase is shown to be identical with the Escherichia coli 30 S ribosomal protein Sl by the following criteria: ability to restore Qβ RNA-directed activity of Qβ Replicase lacking subunit I, immunological cross-reactivity, and identity of the first four amino acids at the NH2 terminus.
TL;DR: Seventy six new temperature-sensitive mutants of simian virus 40 have been isolated by using a simple modification of the standard plaquing technique that permits complementation analyses to be performed readily.
Abstract: Seventy six new temperature-sensitive mutants of simian virus 40 have been isolated. A simple modification of the standard plaquing technique permits complementation analyses to be performed readily. By using this technique the new mutants have been divided into four complementation groups.
TL;DR: The three-dimensional structure of hemoglobin from the bloodworm, Glycera dibranchiata, has been determined crystallographically to a resolution of 2.5 A using three isomorphous heavy atom derivatives.
TL;DR: Judged by polysome content, the stage 42 tadpole seems to make protein about 20 times faster than the unfertilized egg, though it contains very few more ribosomes, and the relationship betweenpolysome content and the synthesis of various types of RNA is discussed.
TL;DR: A system to study enzyme evolution experimentally and it is proposed that such mutations will be acceptable only in ‘silent’ gene copies—shown here to be a frequent response to selective pressure.
Abstract: A system to study enzyme evolution experimentally has been developed. Multiple mutations are required to improve enzyme specificity and the authors propose that such mutations will be acceptable only in ‘silent’ gene copies—shown here to be a frequent response to selective pressure.
TL;DR: A crude system derived from T4-infected E. coli which will modify the α-polypeptides of purified E. Escherichia coli RNA polymerase in vitro, with results consistent with data on kinetics of α modification in vivo.
TL;DR: Evidence is presented that a particular gene may regulate lattice assembly in the body wall muscle cells of the nematode Caenorhabditis elegans, and a temperature-sensitive mutation can prevent the appearance of normal myofilament lattices in this muscle.
Abstract: THE assembly of contractile and calcium-related regulatory proteins into functioning myofilament arrays within muscle cells and the genetic control of muscle differentiation are still largely unknown processes1–2. Here we present evidence that a particular gene may regulate such lattice assembly in the body wall muscle cells of the nematode Caenorhabditis elegans. A temperature-sensitive mutation in this gene can prevent the appearance of normal myofilament lattices in this muscle without affecting the amounts of major contractile proteins present. The organised or defective adult structures once formed in mutant muscle are stable to changes in temperature.
TL;DR: Optical and computer analysis of bright field electron micrographs of the same negatively stained stacked disk specimens subjected to different electron doses shows that the stain migrates and redistributes itself over the protein surfaces during irradiation.
TL;DR: D mutants continue viral DNA replication at the restrictive temperature after preincubation at the permissive temperature and the viral DNA synthesized in D mutants under these conditions progresses in normal fashion through replicative intermediate molecules to mature component I and II DNA molecules.
Abstract: Temperature-sensitive mutants of simian virus 40 (SV40) have been classified as those that are blocked prior to viral DNA synthesis at the restrictive temperature, "early" mutants, and those harboring a defect later in the replication cycle, "late" mutants. Mutants of the A and D complementation groups are early, those of the B, C, and BC groups are late. Our results confirm earlier reports that A mutants are defective in a function required for the initiation of each round of viral DNA synthesis. D mutants, on the other hand, continue viral DNA replication at the restrictive temperature after preincubation at the permissive temperature. The length of time required for D function to be expressed at the permissive temperature-after which infection proceeds unabated on shifting of the cultures to the restrictive temperature-is 10 to 20 h. The viral DNA synthesized in D mutants under these conditions progresses in normal fashion through replicative intermediate molecules to mature component I and II DNA molecules.
TL;DR: The sequence of 33 nucleotides immediately preceding the start point of transcription of an operon in bacteriophage lambda has been determined and contains interdigitating symmetries.
Abstract: The sequence of 33 nucleotides immediately preceding the start point of transcription of an operon in bacteriophage lambda has been determined. This region includes recognition sites for lambda repressor and, probably, for other proteins. The sequence contains interdigitating symmetries.
TL;DR: It is suggested that the inability of cells to elevate their NAD+: NADH ratio at confluency is a characteristic of transformed cells and related to their defective growth control.
TL;DR: The role of cyclic AMP in fibroblasts is reviewed, especially the role of cyclingAMP in the transformation of these cells, which produces some of the abnormal properties of transformed cells.
Abstract: Publisher Summary Cyclic AMP is found in a wide variety of organisms and cells. It is a chemical switch that regulates the activity of existing enzymes and also can induce the synthesis of new proteins. This chapter reviews the role of cyclic AMP in fibroblasts, especially the role of cyclic AMP in the transformation of these cells. The transformed fibroblasts grow faster than normal cells and have a greatly increased saturation density. The transformed cells look different, generally they are less spindly and more refractile in appearance; they are less adhesive to the substratum on which they are growing and will grow in soft agar, whereas normal cells will not; they have altered surface molecules and are readily agglutinated by plant lectins; they often secrete abnormal amounts of macromolecules; and have abnormal tRNA and increased aerobic glycolysis. Cyclic AMP has an important role in regulating many properties of normal fibroblasts. It is involved in controlling cell shape, adhesiveness to the substratum, motility, growth, agglutinability by lectins, and the synthesis of certain macromolecules. As a result of the transformation by chemical carcinogens, X-rays, oncogenic viruses, or spontaneous selection, cyclic AMP levels are decreased and this decrease produces some of the abnormal properties of transformed cells. Transformation by different viruses decreases adenylate cyclase activity by diverse mechanisms because a variety of transformation factors exist.
TL;DR: In this article, the authors used the Burton (Burton & Petersen, 1960) depurination reaction to characterize the redigestion products and identify the pyrimidine residues at their 5′ and 3′ termini.