Alteration of plasmid DNA-mediated transformation and mutation induced by covalent binding of benzo[a]pyrene- 7,8-dihydrodiol-9,10-oxide in Escherichia coli

Plasmid-mediated transformation and mutagenesis induced by (±)-trans- benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BP-DEI) in recipient Escherichia coli (E. coli) have been studied. Because plasmid DNA is used, the system is entirely free from direct toxic effects of BP-DEI on the recipient cells. Plasmid pK0482 DNA, which has two dominant genes, β-lactamase (amp-r) and galactokinase (galK) was modified with BP-DEI prior to its transformation of E. coli N99, AB1157, AB2463(recA^?) and AB1886(uvrA^?). Transformants were selected by ampicillin resistance and mutations were analyzed simultaneously by the altered expression of the galK gene. (1) Approx. 3 molecules of BP-DEI per molecule of pK0482 DNA decreased the transformation efficiency to 37% in AB1157 and the mutation frequency in this strain was proportional to the amount of BP-DEI covalently bound to pK0482 DNA. (2) In AB1886(uvrA^?) a 37% transformation efficiency was produced by only 1 molecule of BP-DEI per molecule of pK0482 DNA, and the mutation frequency in this strain was higher than in AB1157. (3) In AB2463(recA^?), the transformation efficiency was similar to that obtained with AB1157, but mutagenesis was clearly suppressed. (4) Polyacrylamide gel patterns of restriction digests of the pK0482 mutated at the galK gene were indistinguishable from those of the unmutated plasmid DNA.     

Actin acetylation in Drosophila tissue culture cells

In a permanent cell line derived from Drosophila embryos, cytoplasmic actin is produced as an unstable precursor, which is subsequently converted to a stable form. This conversion results in a reduction in isoelectric point, with no apparent change in molecular weight. The conversion involves an enzymatic acetylation, and results in an insensitivity to aminopeptidase digestion, suggesting N-terminal blockage. Both the acetylated and unacetylated actins can participate in the assembly of F-actin, but with different efficiencies.This work was supported by a grant from the NIH (GM 22866).     

Structure of cellulose II hydrate

A hydrate of cellulose II can be formed by swelling Fortisan fibers in hydrazine and then washing in water. The hydrate is stable at 93% relative humidity and has a monoclinic unit cell with dimensions a = 9.02 Å, b = 9.63 Å, c = 10.34 Å, and γ = 116.0°; the space group is P2₁. The unit cell contains disaccharide sections of two chains and approximately four water molecules. The structure was refined using the LALS method, based on 10 observed and 10 unobserved reflections. An antiparallel arrangement of adjacent chains was assumed, since this occurs in cellulose II (the starting material), and the hydrate also reverts to cellulose II on dehydration. Refinement of the positions and side-chain conformations of the chains shows that the chains are stacked in the same way as in cellulose II, and the hydrate is formed by insertion of water molecules between the stacks. However, all efforts to arrange the water molecules in crystallographically regular positions led to unsatisfactory agreement between the observed and calculated intensities. These results suggest an irregular arrangement of the water molecules, which was modeled using water-weighted atomic scattering factors. The analysis resulted in two refined models with relative chain staggers of ～ +c/4 and ～ -c/4, which are indistinguishable in terms of the x-ray agreement. Our preference is for the +c/4 model, for which the stacks of chains are analogous to those in cellulose II.     

Macrophage-T cell interactions. III. Production from T cells, under the influence of macrophages, of a factor which complexes Ig and antigen.

Soluble complexes of Ig and antigen have been detected in the serum of mice within 6 hr after immunization. Such complexes are taken up by a subpopulation of T cells. We present evidence which suggests that the complexes are formed through the mediation of a factor released from T cells, tentatively called Ig-antigen complexing factor or IACF. IACF is produced as a result of a macrophage/T-cell interaction, when macrophages are present in an optimal proportion in relation to T cells (4%). Particulate or aggregated substances stimulate macrophages to release a mediator which subsequently acts on Fc receptor-negative T cells to produce IACF. Free-SH groups are important for the activity of the macrophage mediator. Mercaptoethanol and l-cysteine can also release IACF from T cells in the absence of macrophages. Protein synthesis is necessary for the production of this factor, the activity of which is abolished by trypsin digestion. It is postulated that the complexes of Ig and antigen formed under the influence of IACF represent a mechanism of presentation of antigen to T cells.     

INTRACELLULAR PHYTOCHROME DISTRIBUTION AS A FUNCTION OF ITS MOLECULAR FORM AND OF ITS DESTRUCTION

The immunocytochemically observed intracellular redistribution of phytochrome as a function of its molecular form is described by utilizing color photomicrography. The reversible change from a diffuse to a discretely localized distribution following photoconversion of the red-absorbing Pr form to the far-red-absorbing Pfr form observed with etiolated oat (Avena sativa L., cv. Garry) coleoptile parenchyma cells is not seen with etiolated wheat (Triticum sativum L., cv. unknown), barley (Hordeum vulgare L., cv. Harrison), or rye (Secale cereale L., cv. Balbo). Whether redistribution in these latter cases does not occur or is below the limit of detection is not known. Upon continuous actinic irradiation, phytochrome, which is discretely localized as Pfr, rapidly disappears by both immunocytochemical and spectral assay. However, after about 90 min irradiation, a new association of phytochrome with nuclei is evident which is more pronounced after 4 or 8 h of irradiation. With longer irradiation times there is a total loss of antigenically detectable phytochrome at the resolution employed in these experiments.     

delta5 desaturation of fatty acids in L-M cells

L-M cells grown in a lipid-free medium containing ¹⁴C-labeled 9,12-linoleic acid incorporated most of this acid into glycerolipids as linoleic acid. Only a small amount (3%) was elongated to eicosadienoic acid. No Δ6 desaturation occurred. When the cells were incubated with ¹⁴C-labeled 8, 11, 14-eicosatrienoic acid, 22% of the activity was found in 5,8,11,14-eicosatetraenoic acid. Treatment of the cells for 24 hr with N-isopropylethanolamine, a choline analog, depressed this desaturation reaction to about 60% of control values. The identity of the tetraene product was established by two different chromatographic analyses of the fatty acid methyl esters. Location of the double bond at position C-5 was determined by ozonolysis and subsequent reduction of the ozonides to aldesters followed by gas-liquid chromatography. These results prove that L-M cells have a Δ5 desaturase and an elongation enzyme converting 18:2 to 20:2, but lack a Δ6 desaturase.     

Purification and characterization of two isozymes of 3-phosphoglycerate kinase from the mouse.

Two isozymes of 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), designated PGK-A and PGK-B, were purified from separate extracts of muscle and testicular tissue of DBA/2J mice, respectively. A similar procedure was used to purify the corresponding isozymes from C57BL/6J mice in order to make inter-strain comparisons. The purification involved the use of affinity chromatography with an 8-(6-aminohexyl)amino-ATP-Sepharose column and DEAE-Sephadex chromatography. Lactate dehydrogenase isozyme LDH-X was also co-purified from extract of mouse testes by this two-step procedure. The same isozyme isolated from either mouse strain was found to be identical in physical and biochemical properties. Both isozymes are monomeric as determined by gel filtration chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Furthermore, the isozymes have similar molecular weights, of 47 000 +/- 2000 and exhibit similar Km values for both coenzymes and substrate, as well as temperature dependence of enzyme activity. However, it was observed that the B isozyme is more labile than the A isozyme by denaturation at high temperature, urea and acidic pH.