Cleavage of head and tail proteins during bacteriophage T5 assembly: selective host involvement in the cleavage of a tail protein

Electrophoresis studies showed that at least three phage-specified proteins undergo proteolytic cleavage during the development of bacteriophage T5. One of these proteins has a molecular weight of about 135,000 and the product of this cleavage reaction is a minor component of the T5 tail, having a molecular weight of about 128,000. All of the tail-defective T5 mutants studied in this report failed to induce this cleavage reaction under restrictive conditions. This reaction also failed to occur in Escherichia coli groEA639 and groEA36 infected with wild type T5. Examination of lysates of infected groE cells in the electron microscope revealed the presence of filled and empty heads as well as tubular head structures, but no tails were detected. The filled heads were able to combine with separately prepared T5 tails in vitro to form infectious phage particles. Therefore, propagation of T5 in these groE mutants is prevented primarily by a specific block in tail assembly. A T5 mutant, T5?6, was isolated, which has the capacity to propagate in these groE hosts. The gene locus in T5?6 was mapped.The second T5 protein which is cleaved has a molecular weight of 50,000 and is related to head morphogenesis. Treatment of infected cells with l-canavanine (50 μg/ml) inhibited cleavage of this polypeptide. Only small quantities of the major head protein (32,000 mol. wt) were produced in these treated cells. Treatment with canavanine lead to production of tubular heads. The major protein component of partially purified tubular heads has a molecular weight of 50,000. Cells infected with T5 amber H30b, a mutant defective in head gene D20, does not produce the 50,000 and 32,000 molecular weight proteins. These findings suggest that the 50,000 molecular weight protein undergoes cleavage to form the major head polypeptide. A third T5 protein is cleaved to form a minor head component with a molecular weight of 43,000 and its cleavage is linked to that involving the major head protein.     

E. coli RNAase P has a required RNA component

RNAase P has been partially purified from three thermosensitive strains of E. coli and the thermal inactivation characteristics of each preparation have been determined. The RNAase P preparations from two of these mutant strains, ts241 and ts709, and the wild-type strain have been separated into RNA and protein components. Various mixtures of the reconstituted components have been checked in vitro for complementation of their thermal sensitivity properties. The protein component of RNAase P from ts241 and the RNA component of RNAase P from ts709, respectively, account for the thermal sensitivity of the rnaase P from the two strains. The amount of the RNA component of RNAase P is lower in ts709 than in ts241 or the wild-type parent, 4273. RNAase P partially purified from a revertant of the third mutant strain, A49, which maps at or near the ts241 mutation, has an altered charge when compared to the RNAase P from the parent strain, BF265. We conclude that mutations which affect either the protein or RNA component of RNAase P can confer thermal sensitivity on the enzyme both in vivo and in vitro.     

A secondary attachment site for bacteriophage lambda in trpC of E. coli.

We have determined the nucleotide sequence of a secondary lambda attachment site in trpC. Direct sequence analysis of lambdatrp transducing phage DNA fragments carrying the two prophage attachment sites reveals a 6 nucleotide homology in the crossover region which is a subset of the 15 nucleotide core sequence in the primary lambda attachment site: GCTTTTTTATACTAA. This 6 nucleotide sequence is also present in the intact trpC genome at the attachment site, as shown by analysis of trpC mRNA spanning this region.     

Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studied by use of a fluorescent lanthanide

The acetylcholine receptor isolated from $\text{Torpedo ocellata}$ binds about 10 moles of a fluorescent lanthanide, terbium, per mole α-bungarotoxin-binding site, a process which is accompanied by a fluorescence enhancement (λexcitation 295 nm, λemission 546 nm) which allows detection of receptor-Tb³⁺ complexes at μM concentrations. In presence of calcium two types of terbium-binding site are revealed, both with terbium dissociation constants of 18 ± 0.5 μM. About 60% of the sites bind calcium with an apparent dissociation constant of 1.1 ± 0.1 mM. Sites which interact with calcium also interact with activators of neural transmission, carbamylcholine and decamethonium, but not with the inhibitors, d-tubocurarine and α-bungarotoxin. Whether the displacement of calcium by chemical mediators is directly responsible for activator-induced changes in ion permeability of neural membranes is an important question raised by our experiments. The results show that fluorescent lanthanides can be an important tool in such studies.     

The relationship between concentration of prostaglandin E and rates of cell replication

Phorbol esters act synergistically with phytohemagglutinin (PHA) and Concanavalin A to promote DNA synthesis in bovine lymphocytes. Studies of this response indicate that phorbol esters are useful tools for elucidating the cellular processes that are related to the action of mitogens.     

Specificity of codon recognition by Escherichia coli tRNALeu isoaccepting species determined by protein synthesis in vitro directed by phage RNA.

Codon-anticodon recognition and transfer RNA utilization for the leucine tRNA isoaccepting species of Escherichia coli have been studied by protein synthesis in vitro directed by sequenced bacteriophage MS2 RNA. We have added radioactive Leu-tRNA^Leu isoaccepting species as tracers, rather than use a tRNA-dependent system, since in the presence of an excess of non-radioactive leucine, there is no transfer of radioactive leucine from one isoaccepting species to another. MS2-specific peptides containing leucine residues encoded by known codons were isolated and identified, and the relative abilities of the Leu-tRNA^Leu isoaccepting species to transfer leucine into these peptides compared. Sequenced tRNA₁^Leu and sequenced tRNA₃^Leu are of roughly equal efficiency in their ability to recognize CUC and CUA codons, while tRNA₃^Leu is highly preferred for the CUU codon; tRNA₄^Leu and tRNA₅^Leu both recognize UUA and UUG codons, with tRNA₄^Leu slightly preferred for the UUA codon. We conclude that: (1) wobble is greater than permitted by the wobble hypothesis; (2) there is still some discrimination in the third code letter, and that the CUX₄ (CUC, CUA, CUU, CUG) portion of the leucine family of six codons is not read by a simple “two out of three” mechanism; (3) a Watson-Crick pair (C · G) between codon and anticodon does not appear to be preferred over an unorthodox pair (C · C) in the wobble position; (4) a standard wobble pair (U · G) between codon and anticodon is preferred over an unorthodox pair (U · C); and (5) the extensive wobble observed in the CUX₄ leucine codon series is not paralleled in the UUX₄ leucine (UUG, UUA) and phenylalanine (UUU, UUC) codon series, where mistranslation would be the consequence of such wobble.     

Immunofluorescent and autoradiographic analysis of the relationship between the presence of histone H5 and DNA synthesis in developing chick erythroid cells

Simultaneous detection of histone H5 by indirect immunofluorescence and of [³H]thymidine incorporation by autoradiography on the same preparations of developing erythroid cells have been used to precisely define the extent of correlation between the loss of nuclear activity and the presence of histone H5. It was found that from day 3–12 of embryonic life there are two successive waves of double-labelled cells. At some stages, as many as 30% of the cells which incorporate [³H]thymidine also contain histone H5. Thus, the simple presence of H5 cannot be sufficient to cause nuclear inactivation. A kinetic analysis of the appearance and disappearance of [³H]thymidine-labelled cells, containing histone H5, and cells which are positive for both markers is presented. The result is consistent with the interpretation that the appearance of H5 in the first wave of double labelled cells occurs just before the erythroid cells become metabolically inactive. These observations modify the concept that histone H5 functions uniquely or solely as a template repressor.     

Interaction of far- and near-ultraviolet radiation. The occurrence of photo-augmentation and photo-recovery in cultured mammalian cells

Guided by the phenomena of photo-augmentation and photo-recovery, which have been described with respect to the induction of erythema in human skin, experiments were undertaken with cultured mammalian cells to study whether irradiation with far- and near-ultraviolet radiation results in an interaction at the cellular level with respect to cell survival and induction of mutations. Evidence was found for both photo-augmentation and photo-recovery. Photo-augmentation (more than an additive effect) was observed for cell survival when the long-wave ultraviolet irradiation (UVA) preceded the short-wave ultraviolet irradiation (UVB). Photo-recovery (less than an additive effect) was observed for cell survival if the UVA was given after or simultaneously with the UVB. The latter effect, however, was strongly influenced by dose: doses of UVA higher than 20 000 J/m2 no longer lead to photo-recovery in cell survival. For mutation induction, reduction in mutant frequency appears indicated for both combinations of UVA and UVB and for high and low doses of UVA.     

Isolation and characterization of acetylornithine delta-transaminase of wild-type Escherichia coli W. Comparison with arginine-inducible acetylornithine delta-transaminase.

The enzyme that catalyzes the reversible conversion of N-acetylglutamic γ-semialdehyde and l-glutamate to α-N-acetyl-l-ornithine and α-ketoglutarate, acetylornithine δ-transaminase, has been isolated in homogeneous form and crystallized from both the wild-type and the arginine-inducible strains of Escherichia coli W. The molecular weight of the wild-type transaminase is 119,000 while the molecular weight of the arginine-inducible enzyme is 61,000. However, the arginine-inducible acetylornithine δ-transaminase is not a breakdown product of the wild-type, arginine-repressible transaminase. Analysis of crude extracts of the wild-type and arginine-inducible strains by varying the acrylamide concentration in polyacrylamide disc gel electrophoresis showed that arginine-inducible and wild-type transaminases differed in ionic charge. Immunochemical analysis of the two transaminases showed that neither enzyme would cross-react with antibodies prepared against its counterpart. Treatment of the two enzymes with sodium dodecyl sulfate, followed by disc gel electrophoresis revealed that both transaminases were composed of 31,000-dalton subunits. Tryptic digestion of the two transaminases showed that nearly identical peptides were present. The overall data suggest that the wild-type and inducible transaminases were products of two different structural genes. The two transaminases have different molecular weights, ionic charges, and antigenic determinants, but both are composed of similar molecular weight subunits and show a high degree of similarity in amino acid content and peptide composition.     

Interaction of Triton X-100 with particulate cardiac guanylate cyclase: comparison between Mg2+- and Mn2+-supported enzyme activities in particulate. detergent-solubilized and detergent-insoluble fractions

Guanylate cyclase activity was determined in a 1000g particulate fraction derived from rabbit heart homogenates using Mg²⁺ or Mn²⁺ as sole cation in the presence and absence of Triton X-100. With Mg²⁺, very little guanylate cyclase activity could be detected in the original particulate fraction assayed with or without Triton, or in the particulate fraction treated with varying concentrations of Triton (detergent-treated mixture) prior to enzyme assay. However, the detergent-solubilized supernatants as well as the detergent-insoluble residues (pellets) derived from detergent-treated mixtures possessed appreciable Mg²⁺-supported enzyme activity. With Mn²⁺, significant enzyme activity was detectable in the original particulate fraction assayed without Triton. Much higher activity was seen in particulate fraction assayed with Triton and in detergent-treated mixtures; the supernatants but not the pellets derived from detergent-treated mixtures possessed even greater activity. The sum of enzyme activity in pellet and supernatant fractions greatly exceeded that of the mixture. When the pellets and supernatants derived from detergenttreated mixtures were recombined, measured enzyme activities were similar to those of the original mixture. With Mg²⁺ or Mn²⁺, the specific activity of guanylate cyclase in pellet and supernatant fractions varied considerably depending on the concentration of Triton used for treatment of the particulate fraction; treatment with low concentrations of Triton (0.2–0.7 μmol/mg protein) gave supernatants showing high activity whereas treatment with relatively greater concentrations of the detergent (>0.7 μmol/mg protein) gave pellets showing high activity. The relative distribution of guanylate cyclase in pellet and supernatant fractions expressed as a function of Triton concentration during treatment (of the particulate fraction) showed that 50 to 80% of the recovered enzyme activity remained in supernatants at low detergent concentrations whereas 50 to 80% of the recovered activity resided in the pellets at higher detergent concentrations. Inclusion of excess Triton in the enzyme assay medium did not alter the specific activity profiles and the relative distribution patterns of the cyclase in pellet versus supernatant fractions. The results demonstrate the inherent potential of cardiac particulate guanylate cyclase to utilize Mg²⁺ in catalyzing the synthesis of cyclic GMP. However, it appears that some factor(s) endogenous to the cardiac particulate fraction severely impairs the expression of Mg²⁺-dependent activity; Mn²⁺-dependent activity is also affected by such factor(s) but apparently less severely. Further, the results suggest that previously reported activities of cardiac particulate guanylate cyclase, despite being assayed with Mn²⁺ and in the presence of Triton X-100, represent underestimation of what otherwise appears to be a highly active enzyme system capable of utilizing physiologically relevant divalent cation such as Mg²⁺.     

Replacement of the B protein requirement of the E. coli quinolinate synthetase system by chemically-generated iminoaspartate

Two proteins (A and B) from $\text{Escherichia}\text{,}\text{coli}$ are required for $\text{in}\text{,}\text{vitro}$ synthesis of the NAD⁺ precursor, quinolinate, from L-aspartate and dihydroxyacetone phosphate. The requirement for B protein and L-aspartate in this system can be replaced by millimolar concentrations of oxaloacetate and ammonia if they are added together. This finding supports the concept that the B protein (L-aspartate oxidase) functions to form iminoaspartate which is condensed with dihydroxyacetone phosphate by the A protein to form quinolinate.     

5-Bromodeoxyuridine inhibition of differentiation. Kinetics of inhibition and reversal in myoblasts

This paper describes experiments on the kinetics of inhibition of muscle differentiation in vitro in the presence of 5-bromodeoxyuridine (BrdUrd) and the recovery phenomena that occur when such inhibited cells are permitted growth in normal medium. The studies consist of a quantitation of cell fusion in the presence of the analog and during recovery in its absence coupled with simultaneous studies on changes in buoyant density of cellular DNA. We find that if myoblasts are exposed to BrdUrd during the last doubling before cell fusion would normally occur, most cells do not differentiate, but as many as 18% of the cells can fuse in spite of the incorporation of BrdUrd into their nuclei. These nuclei contain approximately the amount of BrdUrd expected for a full round of DNA synthesis. Studies on the rate of recovery of inhibition of cell fusion following one generation in BrdUrd reveal that after one doubling of inhibited cells in the presence of normal medium. fusion reaches about 50% of the control value; after two doublings it reaches 75% of control value; and after 2.5 doublings of reversal, recovery is essentially complete. We find that both the degree of inhibition after approximately one round of BrdUrd incorporation and the rate of cell differentiation after two generations of reversal are consistent with a model which assumes that BrdUrd “sensitivity” resides on single pair of chromosomes and that inhibition occurs in a dominant fashion if approximately 30% or more of the thymidine is replaced by BrdUrd in the readout strand of either chromosome.