Arrangement of the single-stranded fragments in E. coli bacteriophage BF23 DNA

Bacteriophage BF23st(0) DNA was denatured with alkali and fractionated by agarose gel electrophoresis. Seven single-stranded fragments (designated Fragments I--VII) were identified as the major constituents of the phage DNA. The presence of several minor fragments which represent minor populations of the phage genome was also observed. The largest fragment (Fragment I) represents the intact strand of phage DNA, whereas the other fragments form the complementary strand. Thus, BF23st(0) DNA carries single-strand interruptions in only one strand. The arrangement of the major fragments in the nicked strand was determined by use of gamma-exonuclease and agarose gel electrophoresis. From the mode of action of this nuclease, and from the kinetics of release or disappearance of the fragments, the polarity of the fragments in BF23st(0) DNA was specified. In addition, the presence of two types of major phage populations differing in their composition of the fragments was demonstrated. One type has an additional nick (yielding Fragment IV and Fragment V) in a specific fragment (Fragment II) of other type.     

DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid.

Treatments in vivo of Escherichia coli with oxolinic acid, a potent inhibitor of DNA gyrase and DNA synthesis, lead to DNA cleavage when extracted chromosomes are incubated with sodium dodecyl sulfate. This DNA breakage has properties similar to those obtained in vitro with DNA gyrase reaction mixtures designed to assay production of supertwists: it is oxolinic acid-dependent, sodium dodecyl sulfate-activated, and at saturating drug concentrations produces double-strand DNA cleavage with a concommitant tight association of protein and DNA. In addition, identical treatments performed on a nalA mutant strain exhibit no DNA cleavage. Thus the DNA cleavage sites probably correspond to chromosomal DNA gyrase sites. Sedimentation measurements of the DNA cleavage products indicate that there are approximately 45 DNA breaks per chromosome. This value is similar to the number of domains of supercoiling found in isolated Escherichia coli chromosomes, suggesting one gyrase site per domain. At low oxolinic acid concentrations single-strand cleavages predominate after sodium dodecyl sulfate treatment, and the inhibition of DNA synthesis parallels the number of sites that obtain a single-strand scission. Double-strand breaks arise from the accumulation of single-strand cleavages in accordance with a model where each cleavage site contains two independent drug targets, one on each DNA strand. Since the nicking-closing subunit of gyrase is the target of oxolinic acid in vitro, we suggest that each gyrase site contains two nicking-closing subunits, one on each DNA strand, and that DNA synthesis requires both to be functional.     

Genetic control of whisker antigen of bacteriophage T4D

An antigenic component of T4 whiskers (short fibrils located in the region of the head—tail junction) has been reported to be under the control of gene 49 (Yanagida & Ahmad-Zadeh, 1970; Yanagida, 1972). This was based on immunological evidence using antiserum to particles of T4D adsorbed with gene 49-defective extract made with the mutant amE727. The latter phage, however, is shown here to be a double mutant bearing amber mutations in gene 49 and another gene, herein referred to as wac (whisker antigen control gene). Gene wac maps in the general region of gene 16. Evidence is presented indicating that the whisker antigen is under the control of wac and not gene 49. In wac-defective infections phage are produced that lack a protein. This protein appears by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gels to be the major component of the antigen.The tail fibers of wac-defective bacteriophage are in an open configuration under conditions in which those of wild-type phage are folded alongside the tail. Thus, the wac gene may have a role in the regulation of tail-fiber configuration.     

Conditionally lethal mutants of bacteriophage T4 defective in production of a transfer RNA

The two buried carboxyls (Asp-102 and Asp-194) in both chymotrypsin and chymotrypsinogen are ionized at pH values greater than 4.2 and may be ionized even as low as pH 3.This was demonstrated by coupling most of the surface carboxyis of the proteins by a carbodi-imide with glycinamide or semicarbazide to diminish the groups ionizing at low pH and then titrating the proton uptake on denaturation by sodium dodecyl sulphate between pH 3.0 and 4.6. At pH values greater than 4.2 all unblocked carboxyls are ionized. The proton uptake during the conformational change on denaturation was determined by a stopped-flow procedure and found to be about 2H⁺/mol between pH 3.0 and 3.6. The rate constant for the uptake of protons is the same as that for the exposure of tryptophan and lies in the tens of millisecond region.The buried negative charge at the active site appears to be mainly on Asp-102 rather than on His-57, the pK_a of which must be raised by the buried charge. This enhances its efficacy as a base catalyst in the “charge relay system”.The presence of an intact charge relay system in the inactive zymogen illustrates the importance of stereochemical fit between enzyme and substrate. Enzyme catalysis could hardly be mediated by a catalyst which is uniquely reactive in the absence of correct enzyme-substrate orientation as this would be inconsistent with its specificity.     

The mechanism of replication of phi x174 DNA. XVI. Evidence that the phi x174 viral strand is synthesized discontinuously

Chymostatin is a naturally occurring inhibitor of serine proteases that have chymotryptic-like specificity. This tetrapeptide inhibitor is produced by various species of Streptomyces bacteria. Chymostatin reacts with the serine enzyme Streptomyces griseus protease A in the crystalline state to produce an adduct, the structure of which is in agreement with hemiacetal formation between the C-terminal l-phenylalaninal residue of the inhibitor and the O^γ atom of the active Ser195 residue of S. griseus protease A. The 2.8 Å difference electron density map of the complex is also consistent with the novel structural features previously deduced spectroscopically for chymostatin; i.e. an essential (for inhibition) aldehyde function in the C-terminal l-phenylalaninal residue, an unusual arnino acid, 2-(2-iminohexahydro-(4 S)-pyrimidyl)-(S)-glycine as the third residue from the C terminus and an N-terminal amino group blocked by a (1S)-carboxyphenylethyl-carbamoyl group. There is no significant movement of the active site residues of S. griseus protease A upon complexation with chymostatin.     

Effect of rate-limiting elongation on bacteriophage MS2 RNA-directed protein synthesis in extracts of Escherichia coli

The consequences of limiting the rate of elongation of protein synthesis in vitro have been examined. The concentration of Trp-tRNA^Trp was manipulated by varying the amount of exogenously added tryptophan in extracts from an Escherichia coli mutant in which the tryptophanyl-tRNA-synthetase has a higher K_M for tryptophan. The evidence presented supports the hypothesis that variation of the rate of elongation can be a means of regulating gene expression, both directly, by slowing or accelerating the rate of protein synthesis and indirectly, by leading to varying three-dimensional structures of the messenger RNA when progress of the ribosomes is perturbed. The data can be described by assuming that if a specific transfer RNA is limiting, to a first approximation the overall rate of protein synthesis is determined by the relative rate of reading past an individual codon requiring that tRNA raised to the power of how many times that codon appears in the message. This could be explained by a model in which, with a significant probability, the ribosome stops protein synthesis prematurely at these codons, falls off the messenger RNA and is available for further rounds of protein synthesis. In agreement with other work, evidence is also presented that suggests that under the most drastic available limitation of the elongation rate, that is, starvation for a given amino acid, reading through the corresponding “hungry codon” occurs in vitro at a surprisingly high rate, possibly due to mistranslation.     

In vitro methylation of bacteriophage lambda DNA by wild type (dam+) and mutant (damh) forms of the phage T2 DNA adenine methylase.

The wild-type (dam⁺) and mutant (dam^h) forms of the bacteriophage T2 DNA adenine methylase have been partially purified; these enzymes methylate the sequence, 5/t' … G-A-Py … 3′ (Hattman et al., 1978a). However, in vitro methylation studies using phage λ DNA revealed the following: (1) T2 dam⁺ and dam^h enzymes differ in their ability to methylate λ DNA; under identical reaction conditions the T2 dam^h enzyme methylated λ DNA to a higher level than did the dam⁺ enzyme. However, the respective methylation sites are equally distributed on the l and r strands. (2) Methylation with T2 dam^h, but not T2 dam⁺ protected λ against P1 restriction. This was demonstrated by transfection of Escherichia coli (P1) spheroplasts and by cleavage with R·EcoP1. (3) T2 dam⁺ and dam^h were similarly capable of methylating G-A-T-C sequences on λ DNA; e.g. λ·dam₃ DNA (contains no N⁶-methyladonine) methylated with either enzyme was made resistant to cleavage by R·DpnII. In contrast, only the T2 dam^h modified DNA was resistant to further methylation by M·EcoP1 (which methylates the sequence 5′ … A-G-A-C-Py … 3′; Hattman et al., 1978b). (4) λ·dam₃ DNA was partially methylated to the same level with T2 dam⁺ or T2 dam^h; the two enzymes produced different patterns of G-A-C versus G-A-T methylation. We propose that the T2 dam⁺ enzyme methylates G-A-C sequences less efficiently than the T2 dam^h methylase; this property does not entirely account for the large difference in methylation levels produced by the two enzymes.     

Cis- and trans-diamminedichloroplatinum(II) binding products different tertiary structural changes on SV40 DNA

The proteases secreted into culture medium by MCF-7 breast cancer cells produced both plasminogen-dependent and -independent proteolysis, as shown by casein-polyacrylamide gel electrophoresis. All of these proteases except the largest (M_r 120,000) were retained on a benzamidine-Sepharose affinity column, a characteristic of trypsinlike proteases. Among the proteases which activated plasminogen, all except a major protease of M_r 59,000 were antigenically similar to urokinase. These urokinaselike proteases (M_r 65,000 to 25,000) were isolated on a antiurokinase-Sepharose affinity column. The findings indicate that in a stable cell line derived from a human breast cancer there are two distinct types of plasminogen activators, opening the possibility that these activator types may be modulated in separate ways.     

Regulation of C4 photosynthesis: regulation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and dephosphorylation

Pyruvate, Pi dikinase in extracts of chloroplasts from mesophyll cells of Zea mays is inactivated by incubation with ADP plus ATP. This inactivation was associated with phosphorylation of a threonine residue on a 100 kDa polypeptide, the major polypeptide of the mesophyll chloroplast stroma, which was identified as the subunit of pyruvate, Pi dikinase. The phosphate originated from the beta-position of ADP as indicated by the labelling of the enzyme during inactivation in the presence of [beta-32P]ADP. During inactivation of the enzyme up to 1 mole of phosphate was incorporated per mole of pyruvate, Pi dikinase subunit inactivated. 32P label was lost from the protein during the Pi-dependent reactivation of pyruvate, Pi dikinase.     

Gene 68, a new bacteriophage T4 gene which codes for the 17K prohead core protein is involved in head size determination

We have identified the gene for a major component of the prohead core of bacteriophage T4, the 17K protein. The gene, which we call gene 68, lies between genes 67 and 21 in the major cluster of T4 head genes. All of the genes in this region of the T4 genome have overlapping initiation and termination codons with the sequence T-A-A-T-G. We present the DNA sequence of the gene and show that it codes for a protein containing 141 amino acids with an acidic amino-terminal half and a basic carboxyl terminus. Antibodies prepared against the 17K protein were used to show that it is cleaved by the phage-coded gp21 protease during head maturation and that most of the protein leaves the head after cleavage. A frameshift mutation of the gene was constructed in vitro and recombined back into the phage genome. The mutated phages had a drastically reduced burst size and about half of the particles produced were morphologically abnormal, having isometric rather than prolate heads. Thus, the 17K protein is involved in head shape determination but is only semi-essential for T4 growth.     

Synthesis of a photoaffinity probe for the beta-adrenergic receptor.

The synthesis and characterization of a beta-adrenergic photo-affinity label, N-(-2-hydroxy-3-naphthoxypropyl)-N′ (-2-nitro-5-azidophenyl ethylenediamine, (NAP-propranolol) is described. The inhibition constants (Ki) for the NAP-propranolol inhibition of ³H-dihydroalprenolol binding and the inhibition of (?)-isoproterenol-stimulated adenylate cyclase in turkey erythrocytes are 100 nM and 19 nM respectively.     

Microheterogeneity of the glycoprotein subunit of the (sodium + potassium)-activated adenosine triphosphatase from the electroplax of Electrophorus electricus.

Isoelectric focusing of purified Na,K-ATPase on polyacrylamide gels resolved the protein into ten bands. The catalytic and glycoprotein subunits were separated by sodium dodecyl sulfate gel filtration. Isoelectric focusing of the isolated glycoprotein subunit showed that it accounted for nine of the ten bands. Part of this microheterogeneity can be attributed to variations in sialic acid content in individual bands, since removal of all of the sialic acid by neuraminidase treatment reduced the number of bands to four. It is suggested that the microheterogeneity of the glycoprotein subunit is due to post-translational modifications of oligosaccharides on a common polypeptide backbone.     

Studies on the oligomeric structure of yeast phosphofructo-kinase by means of cross-linking diimidoesters.

Intracellular cross-linking of yeast phosphofructokinase with a series of diimidoesters of different chain length resulted in the appearance of tetramers as largest cross-linked product of the enzyme subunits. The native enzyme is evidently composed of eight subunits being arranged in two tetramers α₄β₄. In the tetramers the monomers are probably assembled in tetrahedral geometry.     

Characterization of the deoxyribonuclease determined by lambda reverse as exonuclease VIII of Escherichia coli.

Gottesman et al. (1974) detected a new DNAase in Escherichia coli infected with λ reverse, a recombination-proficient substitution mutant of phage λ which is deleted for the λ recombination genes. We have purified this enzyme, using the procedure developed for the purification of exonuclease VIII (Kushner et al., 1974), a DNAase produced by E. coli K-12 strains carrying sbcA^? mutations. The λ reverse exonuclease (Exoλrev) is identical to exonuclease VIII by several criteria. The two enzymes elute at similar salt concentrations from DEAE-cellulose and DNA-cellulose; sediment at the same velocity in glycerol gradients, corresponding to a molecular weight of about 1.4 × 10⁵; migrate at the same R_F in sodium dodecyl sulfate/polyacrylamide gels, indicating a polypeptide molecular weight of 1.4 × 10⁵; exhibit maximum activity at 20 mm-Mg²⁺ and pH 8 to 9; and are much more active on double-stranded DNA than on heat-denatured DNA. Both enzymes are rendered sedimentable by antiserum against Exoλrev. This evidence supports the hypothesis that the non-λ DNA substitution in λ reverse includes recE, the structural gene for exonuclease VIII.     

Cloned genes for bacteriophage T4 late functions are expressed in Escherichia coli

We have constructed derivatives of plasmid pMB9 carrying EcoRI digestion fragments of bacteriophage T4 DNA that code for late gene functions. When Escherichia coli strains carrying these plasmids are infected with T4 amber mutants, burst sizes up to 30% of the wild-type level are obtained. Single burst experiments imply that the phage progeny result from complementation and do not depend on marker rescue. By electrophoretic and immunological techniques, we have established that the cloned T4 late genes are transcribed and translated in uninfected cells. A serum blocking assay has been used to quantitate the levels of one of the T4 gene products, gp11, before and after T4 infection. Uninfected cells containing the cloned T4 gene 11 DNA have 0.1% and mini cells have 1% of the gp11 levels per unit protein found in cells late after T4 wild-type infection. There is little or no additional gp10 and gp11 formed from the cloned genes after T4 infection.     

Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues

Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [³²P]phosphate labeling in vivo.It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.     

Characterization of microsomal electron transport components from control, phenobarbital- and 3-methylcholanthrene-treated mice. III. Improved resolution and quantitation of major components in ammonium sulfate fractions from total liver microsomes

Quantitation of microsomal components in ammonium sulfate fractions using a high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, and a comparison of these results with those from similar experiments on total liver microsomes has enabled us to identify and better characterize the interactions between microsomal electron transport components.

It was found that: (1) phenobarbital decreased the amount of one protein component of approximately 50 000 molecular weight while increasing a component of very similar molecular weight; (2) only two proteins appeared to be associated with CO binding; (3) another protein of approximately 68 000 molecular weight, one of the glycoproteins found in liver microsomes, appears to be induced by phenobarbital pretreatment; (4) the induction of NADPH-cytochrome c reductase activity after phenobarbital pretreatment is not dependent on an increase in the known NADPH-dependent flavoprotein, but rather on the increase in some component found predominately in our most soluble sub-microsomal fraction.

A very good separation of the above components was achieved by ammonium sulfate fractionation, e.g. simply on the basis of their solubility. This and the fact that the more-or-less soluble proteins were induced by phenobarbital or 3-methylcholanthrene respectively indicate that the solubility of membrane proteins plays a major role in the structure and function of microsomal membranes.