The mechanism of regulation of fibroblastic cell replication. III. A central role for nutrient limitation: a conditioning effect due to serum imprinting of the cell response

Haploid Saccharomyces cerevisiae cells of mating type a, but not α, produce and secrete a diffusible substance, designated a factor. The a factor transiently arrests cells of mating type α, but not a, at a very early stage of the cell cycle, prior to budding and to the initiation of DNA synthesis. While the cells are arrested at this stage, few, if any, of the functions required for the ensuing cell cycle are carried out. This stage of the cell cycle coincides with the stage at which α factor, produced by cells of mating type a, specifically arrests cells of mating type a [2]. It seems probable that the reciprocally acting a and α factors together provide the mechanism by which haploid cells are synchronized to the appropriate stage of the cell cycle as a prelude to conjugation.     

Liquid scintillation counting of 14C- and 3H-labeled samples on solid supports: a general solution of the problem

The data presented demonstrate the potential of surfactant-fortified scintillation cocktails in overcoming many of the problems encountered in the quantitation of radioactivity on solid supports. Using a broad range of representative ionic and polar biochemical compounds, the in vial elution and quantitative recovery of ³H and ¹⁴C-labeled components from a wide assortment of common solid support media has been demonstrated. This methodology in combination with zero elution counting systems and in some circumstances gelled suspension counting should combine to overcome most of the problems associated with determination of isotopic activity on such solid support media.     

A simple assay for methionine adenosyltransferase using cation exchange paper and liquid scintillation spectrometry

A rapid, sensitive and simple procedure for the assay of l-methionine-S-adenosyltransferase based on the use of phosphocellulose ion-exchange paper is presented. The analytical procedure may be generally useful for all enzymes where there is a large cationic charge difference between the substrates and products. An application of a simpllified counting procedure for radioisotopes on solid supports is presented.     

The information content of DNA and evolution

The information content of DNA molecules has been calculated for various organisms using experimentally measured codon frequencies as well as those estimated theoretically. A direct relationship between the information content and the evolutionary rank of the organism is observed. The biological and physical significance of information content in the context of evolution is discussed.     

Immunological studies on glucose 6-phosphate dehydrogenase of rat liver

Glucose 6-phosphate dehydrogenase (G6PD) was purified from the supernatant fraction of rat liver to a homogeneous preparation by a specific elution with substrate. A specific antibody against the purified enzyme was prepared in rabbits and was shown to completely inhibit the enzyme activity and precipitate the enzyme protein of liver supernatant. With this antiserum, liver supernatants with varying specific G6PD activities obtained under several experimental conditions and supernatants from other tissues examined all formed single precipitin lines, which fused with each other in the Ouchterlony double-diffusion system. Three interconvertible microheterogeneous forms of G6PD in liver, supernatant were immunologically indistinguishable from each other. The G6PDs in participate fractions of liver were, however, distinct from the supernatant enzyme both in inhibition of the enzyme activity and in formation of precipitation by the specific antiserum. Liver supernatant G6PD, which was inactivated with various reagents or by heating, showed a simultaneous loss of ability to form precipitin line. Aggregation and disaggregation of the dehydrogenase to the tetramer and monomer, respectively, also resulted in loss of immunological reactivity. The increase in G6PD activity in the cytoplasm of carbon tetrachloride-treated or glucose casein-refed rat liver was accompanied by a proportional increase in the quantity of immunologically reactive G6PD protein.     

Fatty acids diffusion in lecithin multilayers: hydration and PH effects.

The diffusion of the sodium salt of monocarboxylic fatty acids, from formate to stearate, has been studied as a function of water content and pH in lecithin--water lamellar phases. Evolution of the diffusion coefficients with increasing chain length reflects the different localizations of fatty acids in the system. From formate to butyrate, which are mainly restricted to the hydrophilic layer of the phase, diffusion rates decrease rapidly. From butyrate to stearate, fatty acids (anchored at the hydrophilic--lipophilic interface) undergo lateral diffusion and then the decrease of D with increasing chain length is much slower. The diffusion of stereate is already comparable to the diffusion of the lecithin molecule itself. The diffusion rates strongly depend upon phase hydration and pH: it is shown that both parameters control the fatty acid ionization. The variations in diffusion rates observed may be ascribed to the fact that, depending upon their state of ionization, fatty acids assume a different localization and therefore experience different interactions in the lamellar system.     

Intermediates of chromosomal DNA replication in Escherichia coli

The product of bacteriophage T4 gene 63 has two activities, one which catalyzes the attachment of tail fibers to base plates during morphogenesis (TFA) and one which catalyzes the joining of single-stranded polynucleotides (RNA ligase). The only phenotype attributed to mutations in gene 63 is a defect in attachment of tail fibers leading to fiberless T4 particles. However, it is suspected that TFA and RNA ligase are unrelated activities of the same protein since they have very different requirements in vitro.We have isolated new mutants which have lost the RNA ligase but have retained the TFA activity of the product of gene 63. These mutants exhibit defects in T4 DNA replication and late gene expression in some strains of Escherichia coli. This work allows us to draw three conclusions: (1) the TFA and RNA ligase activities are unrelated functions of the gene 63 product making this the prototype for a protein which has more than one unrelated function; (2) the RNA ligase is probably involved in DNA metabolism rather than RNA processing as has been proposed: (3) the RNA ligase and polynucleotide 5′ kinase 3′ phosphatase of T4 perform intimately related functions.     

Comparison of the human,canine and swine E apoproteins

A comparative study of the E apoprotein isolated from the d<1.02 lipoproteins of human, canine and swine plasma revealed that the various apo-E preparations had similar molecular weights (37,000–39,000) and had similar amino acid compositions in that glutamic acid, alanine, leucine and arginine were present in high concentrations. The various preparations showed partial immunochemical cross-reactivity, demonstrating significant sequence homology between the species. However, determination of the amino-terminal amino acid sequence by automated Edman degradation showed each apo-E was different, demonstrating that the amino-terminal portion of the E apoprotein was a variable region of this protein.     

On the computation of the tertiary structure of globular proteins. III. Inter-residue distances and computed structures

An analysis of geometrical models for computing the tertiary structure of globular proteins from the primary structure is presented. The roles of initial configuration, input information on inter-residue distances and the errors in this information are delineated. It is shown that for local information like that on secondary structure, the calculated structure is very sensitive to errors and to the initial configuration. Thus, such information is far from adequate for predicting the tertiary structure. On the other hand, global information on all the inter-residue distances is quite insensitive to errors. A semi-empirical method is presented to estimate these distances and the calculated structures are given for two proteins—pancreatic trypsin inhibitor and parvalbumin. These structures have good resemblances to those determined by X-ray diffraction. A strategy for further refinement of the method is indicated.     

Plasmid replication in DNA Ts mutants of Bacillus subtilis.

In an attempt to increase our understanding of plasmid replication in Bacillus subtilis we determined the effect of various dna Ts mutations [Gass, K. B., and Cozzarelli, N. R. (1973). J. Biol. Chem. 248, 7688–7700; Gross, J. D., Karamata, D., and Hempstead, P. G. (1968). Cold Spring Harbor Symp. Quant. Biol.33, 307–312; Karamata, D., and Gross, J. D. (1970). Mol. Gen. Genet.108, 277–287] on pUB110 replication. pUB110 is a kanamycin resistance plasmid originally isolated in Staphylococcus aureus and introduced into B. subtilis by transformation. At temperatures nonpermissive for chromosomal DNA synthesis dnaA13, dnaB19, dnaC6, dnaC30, dnaD23, dnaE20, and dnaI102 permit replication of the plasmid. In several cases this “amplification” continues until approximately equal amounts of plasmid and chromosomal DNA are present. dnaG34, dnaH151, dnaF133, mut-1, and polC26 affect both pUB110 and host DNA synthesis at nonpermissive temperatures. The last three mutations are known to affect the activity of DNA polymerase III (PolIII). When polC26 is incubated at a nonpermissive temperature, there is an accumulation of plasmid DNA with a density on EtBr-CsCl gradients intermediate between that of covalently closed circular (CCC) and open circular DNA. pUB110 can replicate in a strain which is deficient in DNA polymerase I (PolI). Finally, chloramphenicol (Cm) inhibits the replication of pUB110 as well as of chromosomal DNA.     

DNA replication in asynchronous and synchronous ehrlich ascites cells in different conditions of growth

Ehrlich ascites tumour cells were labelled for DNA fibre autoradiography within the peritoneal cavity of a tumour-bearing mouse. The generation and the evaluation of the autoradiographic patterns is described and discussed. To study possible changes of the autoradiographic patterns during a natural S phase the labelling was performed in the mouse or in culture with asynchronous cells which were afterwards separated into synchronous subpopulations by zonal centrifugation. The subpopulations obtained were characterized by flow cytofluorometry in connection with the thymidine labelling index. We compared the DNA fibre autoradiographic patterns of several synchronous and asynchronous cell populations growing in the mouse or under different conditions in culture: The replicon size distributions of all populations examined were virtually the same. The fork movement rate was found to depend mainly on the metabolic condition of the cells. In culture it was significantly slower than in the mouse although a shortened S phase and therewith an increased DNA synthesis rate occurred. During a natural S phase it increased slightly, at most, while the DNA synthesis rate was considerably enhanced at the end of S. The changes in the rate of total DNA synthesis cannot account for the changes in the rate of chain growth. We conclude that the DNA synthesis rate is regulated almost exclusively by changing the replicon initiation frequency, while the fork movement rate is limited by the actual metabolic condition of the cells.     

On the computation of the tertiary structure of globular proteins II.

The semi-empirical approach proposed earlier (Y?as et al., 1978) to compute the tertiary structures of globular proteins is here amplified and developed further. Using, as input, information on sequence and certain averages of interatomic distances which can be semi-empirically estimated, structures have been computed for pancreatic trypsin inhibitor, lysozyme and staphylococcal nuclease which resemble those determined by X-ray diffraction methods. The approach used is compared and contrasted with others proposed recently.     

The error catastrophe hypothesis with reference to aging and the evolution of the protein synthesizing machinery.

Recent investigations of the thermodynamics of protein denaturation, in particular of pressure effects, have questioned the fundamental importance, hitherto assumed, of hydrophobic interactions in the native conformations of proteins. The volume changes observed on protein denaturation are incompatible with the volume changes estimated on the basis of volume effects observed in low molecular weight model systems of the aliphatic groups. In the present paper the model systems generally considered are critically discussed. It is concluded, that solutions of low molecular weight alkanes may not be any adequate models of aliphatic groups in proteins. Studies of more appropriate model systems suggest that the volume changes to be expected, when buried aliphatic groups of proteins are exposed to water, are small and positive, and mainly due to structural changes of the water. These volume changes are in accordance with the volume changes actually measured of protein denaturation, and the latter volume effects are taken as supporting evidence of the importance of hydrophobic interactions in protein confonriations.     

Two components of cysteine oxidase in rat liver

Purified cysteine oxidase in rat liver is composed of two distinct proteins. These proteins are able to be fractionated by DEAE-cellulose column chromatography. It appears that one of them is a catalytic protein named protein-B having tightly bound iron as a prosthetic group, while the other is either a modifier or activating protein named protein-A. Protein-B is found to exist in both an active and an inactive form. Inactive protein-B is activated by incubation with substrate cysteine under anaerobic condition. Activated protein-B alone exhibited an extremely low catalytic activity but in the presence of protein-A remarkable increase in activity was observed.     

Isolation and sequence determination of a peptide located in or near the active site of bovine muscle pyruvate kinase

Bovine muscle pyruvate kinase was inactivated by treatment with trinitrobenzenesulfonic acid; approximately one trinitrophenyl group was incorporated per subunit. ADP or Mg-ADP decreased the rate of inactivation but Mg⁺⁺ alone or phosphoenolpyruvate had no effect. The inactivated protein was treated with trypsin and the trinitrophenylated peptide isolated by gel filtration. Homogeneity of the isolated peptide was shown by high voltage electrophoresis and high pressure liquid chromatography. Amino acid analysis and sequence determination revealed the presence of an acidic peptide 34 amino acids long and containing ?-trinitrophenylated lysine.