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Biomedical subjects

D Kennell

Publications and source records attributed to D Kennell.

48 records · Page 3Linked to original sources
PubMed1972

Exclusion of bacteriophages by T2 ghosts.

T2 ghosts do not exclude T4, T7, or lambda-induction in Escherichia coli which survive ghost infection. Latent periods are extended, probably by the temporary inhibition of protein synthesis.

Bacterial Proteins↗
PubMed1972

Polypeptide synthesis by extracts from Escherichia coli treated with T2 ghosts.

Infection of Escherichia coli B in inorganic salts-glycerol with a multiplicity of deoxyribonucleic acid-less T2 "ghosts" just sufficient to block all protein synthesis results in both viable and killed bacteria. We enriched for the viable cells by a combination of lysozyme treatment and filtration and measured the in vitro capacity of their extracts to synthesize polypeptides. Without added template ribonucleic acid (RNA), such "ghost extracts" incorporate amino acids (endogenous synthesis) at approximately one-half the rate as do extracts from uninfected bacteria. However, they are unable to use added synthetic or natural template RNAs for peptide synthesis. Some activity can be observed but only at high concentrations of Mg(2+). These results suggest that ghost infection may result in a blockage of ribosomes during translation. Mixing experiments show that the incapacity of ghost extracts to translate added template RNA is due to a defect in the ribosomes.

Amino Acids↗
PubMed1970

Inhibition of host protein synthesis during infection of Escherichia coli by bacteriophage T4. II. Induction of host messenger ribonucleic acid and its exclusion from polysomes.

Two gene clusters on the Escherichia coli chromosome were induced at early times after T4 infection when >99% of the cells were infected: the lactose (lac) operon and prophage lambda. Their messenger ribonucleic acid (mRNA) was detected by hybridization to phi80 dlac deoxyribonucleic acid (DNA) and lambdaDNA, respectively. Synthesis of host mRNA could be initiated during the first few minutes after T4 infection, although no beta-galactosidase activity could be detected. Hybridization analyses of selected fractions from sucrose gradients revealed that most of this lac mRNA induced at very early times of T4 infection was not associated with ribosomes. In contrast, virtually all lac mRNA in uninfected bacteria was associated with polysomes. This exclusion affected all host mRNA; about 70% of E. coli(3)H-mRNA, labeled from 2 to 3 min after T4 infection, was excluded from polysomes. Infection even reduced the yield of beta-galactosidase from lac mRNA induced before infection. Gradients from rifampicin-inhibited cells showed the normal growth of lac mRNA polysomes; in contrast, T4 infection prevented growth of the preinduced lac polysomes. It is concluded that T4 infection interferes within seconds with the reassociation of ribosomes to host mRNA.

Animals↗
PubMed1970

Inhibition of host protein synthesis during infection of Escherichi coli by bacteriophage T4. 3. Inhibition by ghosts.

Deoxyribonucleic acid (DNA)-less T2 "ghosts" were prepared by osmotic shock and purified by KBr density gradient centrifugation. Escherichia coli B was treated with these ghosts in inorganic salts-glycerol medium to see which features of phage infection could be elicited by ghosts. At a multiplicity that was just sufficient to block induction of beta-galactosidase (EC 3.2.1.23), 89% of the bacteria were killed and the rates of ribonucleic acid (RNA) and DNA synthesis were about 10 to 15% of normal. However, protein synthesis was almost completely blocked but resumed after 30 min. During this period, it was possible to induce messenger RNA (mRNA) from the lactose operon, although this mRNA could not be translated into active beta-galactosidase. These results suggest to us that the viable cells surviving ghost infection synthesize nucleic acids at close to a normal rate but are temporarily blocked in protein synthesis. The continued formation of untranslated host mRNA mimics the pattern of bacterial synthesis just after whole-phage infection, and is consistent with the interpretation that the immediate block in the initiation of host translation by these viruses is due to their attachment.

Bacterial Proteins↗
PubMed1968

Inhibition of host protein synthesis during infection of Escherichia coli by bacteriophage T4. I. Continued synthesis of host ribonucleic acid.

The ribonucleic acid (RNA) synthesized at specified intervals during infection of Escherichia coli K-12 by bacteriophage T4 was hybridized to denatured E. coli or T4 deoxyribonucleic acids (DNA). The reactions were performed under conditions that maximized the yield and at RNA/DNA inputs such that excess DNA sites were available for all RNA species. Most of the RNA synthesized at any time during the first 3 min of infection was host-specific. The fraction declined rapidly as infection progressed; host RNA represented about half that made between 3 and 4 min. It is unlikely that this represented RNA synthesized by bacteria that had escaped infection, as judged by the kinetics of adsorption and killing as well as by the rapid inhibition of beta-galactosidase induction after infection. The nature of the host RNA was also examined. Part of the RNA synthesized during infection of cells rendered sensitive to actinomycin was stable in the presence of this inhibitor. This RNA was essentially all host-specific and it sedimented as ribosomal and transfer RNA; most of the ribosomal RNA was incorporated into 30S and 50S ribosomes. Hybridization analyses suggested that unstable E. coli messenger RNA was also synthesized for several minutes after infection; the proportion of unstable to stable host RNA synthesized appeared to be similar in infected and uninfected cells. Thus, it is concluded that significant amounts of E. coli RNA are synthesized during the first minutes of T4 infection. Host messenger RNA initiated after infection may not be translated into enzymes; alternatively, it is conceivable that continued bacterial messenger RNA synthesis only reflects the completion of transcription of operons whose reading was initiated prior to infection.

Bacterial Proteins↗
PubMed1967

Magnesium starvation of Aerobacter aerogenes. I. Changes in nucleic acid composition.

Aerobacter aerogenes incubated in a medium containing all factors necessary for exponential growth except Mg(++) continued to synthesize nucleic acids and proteins for more than 70 hr, provided the major carbon source was in excess at all times. After 24 hr of Mg(++) starvation, deoxyribonucleic acid content in the culture had increased 10-fold. In contrast, the viable-cell count increased only about threefold during the first few hours and then remained approximately constant for the subsequent 70 hr. After specified intervals of Mg(++) starvation, extracts of the bacteria, or ribonucleic acid (RNA) purified from them, was centrifuged through gradients of sucrose to separate transfer RNA from ribosomal components. After correcting for losses, we obtained the following results. (i) There was a progressive rise in the content of transfer RNA competent to accept amino acids and during starvation it remained completely stable. (ii) In contrast, the contents of normally sedimenting ribosomal RNA and ribosomal subunits (30 and 50S) remained approximately constant for more than 24 hr. This did not result from stability of ribosomes made prior to starvation together with an inhibition of synthesis of new particles. Rather, ribosomes were continually breaking down and being replaced by an equivalent number of new ones. (iii) The breakdown of ribosomes appeared to be sequentially ordered; polysomes yielded 70S monomers, which then gave 30 and 50S particles, and these disintegrated to smaller units and finally to acid-soluble products. (iv) Furthermore, the particles derived from breakdown do not appear to exchange with subparticles on the path of assembly. Thus, ribosome decay was age-dependent and ribosomal RNA molecules had a minimal life expectancy of 90 min; however, some survived much longer.

Bacterial Proteins↗
PubMed1967

Magnesium starvation of Aerobacter aerogenes. II. Rates of nucleic acid synthesis and methods for their measurement.

The rates of synthesis of Aerobacter aerogenes nucleic acids were estimated during incubation of the bacteria in a Mg(++)-free medium. Deoxyribonucleic acid (DNA) synthesized during Mg(++) starvation, or in the preceding exponential growth, remained acid-precipitable for 2.5 hr before breaking down to acid-soluble products during a period of many hours. Rates of DNA synthesis were calculated by correcting the net amounts of DNA per milliliter to values that would have appeared had there been no decay. After the first few hours, this rate was constant, the amount of DNA present at the start of Mg(++) starvation being synthesized every 130 min. Rates of synthesis of total ribonucleic acid (RNA) were established in two ways: (i) by measurements of the incorporation of exogeneous uracil and glucose carbon into RNA, and (ii) by the accumulation of transfer RNA (tRNA), since this component is stable during Mg(++) starvation. After the first few hours, this rate was constant, the amount of RNA present at the start of Mg(++) starvation being synthesized about every 120 min. Fractionation by gradient centrifugation revealed that at all times of starvation the ratio of newly synthesized tRNA-rRNA was the same as it was during exponential growth. Furthermore, newly synthesized ribosomal RNA (rRNA) became a part of polysomal structures. Thus, in the absence of Mg(++), DNA, tRNA, and rRNA were synthesized in the same relative proportions as during exponential growth, at rates close to one-half the instantaneous rates of synthesis in the bacteria growing exponentially at the start of starvation.

Carbon Isotopes↗
PubMed1967

Magnesium starvation of Aerobacter aerogenes. 3. Protein metabolism.

The metabolism of the ribosomal and soluble protein components of Aerobacter aerogenes was examined during its incubation in a Mg(++)-deficient medium. Bacteria were exposed to leucine-H(3) during the exponential growth period preceding Mg(++) starvation, and extracts were prepared after intervals of starvation and were centrifuged through gradients of sucrose to separate ribosomal from soluble proteins. Ribosomal proteins synthesized during the preceding exponential growth were slowly lost from the ribosomes; after 8 hr of starvation, few, if any, sedimented with ribosomes. Losses of total protein, together with the known rate of ribosome decay during Mg(++) starvation, suggested that these ribosomal proteins are ultimately degraded to acid-soluble products and account for all protein lost by the starving cells. These conclusions were supported by studies of Mg(++) starvation in a uracil-requiring strain of A. aerogenes: during uracil starvation a smaller fraction of the proteins synthesized were ribosomal, and the fraction of protein which subsequently decayed during Mg(++) starvation was correspondingly less. During recovery from Mg(++) starvation, proteins, lost from disintegrated ribosomes, were not detectably reutilized into new particles even before their degradation to acid-soluble products was complete. Synthesis of soluble proteins continued for more than 24 hr of starvation at a rate per milliliter close to 45% of the instantaneous rate per milliliter of the exponentially growing bacteria at the time Mg(++) was removed. This value agreed with that found previously for synthetic rates of deoxyribonucleic acid, transfer ribonucleic acid, and ribosomal ribonucleic acid during starvation relative to rates during exponential growth.

Bacterial Proteins↗
PubMed1967

Magnesium starvation of Aerobacter aerogenes. IV. Cytochemical changes.

A number of cytochemical changes were revealed by microscopic observations of Aerobacter aerogenes populations starving for Mg(++). During the first few hours, while the synthesis of deoxyribonucleic acid (DNA) was paralleled by an increase in viable bacteria, the cells became progressively smaller. Subsequently, the number of viable cells in the culture remained constant in spite of continuing DNA synthesis, and the cells progressively elongated into filamentous forms. During this time, a second population of very small bacteria could be identified. These cells, whose number increased progressively, were inert with respect to (i) growth or reproduction when returned to a complete medium and (ii) biosynthetic activity as judged by autoradiographic estimation of uracil-H(3) incorporation into nucleic acids. When observed by electron microscopy, many thin sections from bacteria that had been starved of Mg(++) for 20 hr appeared to be almost devoid of ribosomal particles. Thionine staining indicated that the inert cells contain DNA. Furthermore, the rate of DNA synthesis in the culture corresponded to the rate of accumulation of inert cells, suggesting that their presence can account for the difference between total DNA and viable count.

Autoradiography↗