Sequence analysis of mitochondrial DNA in a mouse cell line resistant to chloramphenicol and oligomycin.

A mouse L-cell line, designated 111-OB3, is described which is resistant to two drugs, chloramphenicol and oligomycin. The cells contain two types of mitochondrial DNA molecules, in roughly equal proportions, which differ in that one is cleaved by endonuclease EcoRI at a novel site within the coding sequence for subunit 6 of the mitochondrial ATPase (ATPase-6). Sequence analysis reveals that the cleavage site was created by a single transversion which predicts a replacement of valine in the wild-type ATPase-6 by glutamic acid. The replacement occurs in a hydrophobic amino acid sequence which is highly conserved in mouse, human, and bovine proteins. The position of the replacement is similar to a substitution observed in one class of yeast mutants resistant to oligomycin. Both of the mitochondrial DNA molecules in 111-OB3 also have a single nucleotide change in the gene encoding the large (16S) rRNA. These observations are consistent with the hypothesis that oligomycin resistance in mammalian cells can be cytoplasmically determined and can result from alterations in ATPase-6. The appearance of the mutation before selection in oligomycin suggests a model for the origin of mitochondrial mutations in mammalian cells.     

Active transport of l-aspartic acid in Neurospora crassa

Transport of l-aspartic acid in Neurospora crassa conidia is shown to be mediated by neutral and general amino acid transport systems. The transport activity is dependent on pH and results in accumulation of l-aspartic acid against a gradient. Mutants deficient in transport of l-aspartic acid are described.     

Fragments of human γG immunoglobulins produced by a porcine pancreatic esterase

The effect of a porcine pancreatic esteroproteolytic enzyme on human IgG has been described. A sequential breakdown of the molecule occurs. The first cleavage results in the formation of an F′c fragment together with an F(ab)₂ fragment. A subsequent proteolysis of the F(ab)₂ fragment liberates two Fab fragments. Each fragment has been characterized by its antigenic properties, molecular weight, and sulfhydryl content.     

Passive Electrical Properties of Microorganisms: V. Low-Frequency Dielectric Dispersion of Bacteria

Very high dielectric constants have been observed for bacteria at low frequencies. High dielectric constants such as these can be explained by a theory which has been developed for the low-frequency dielectric dispersion of porous charged particles and which has been tested successfully through measurements with ion exchange resins. The bacterial cell wall is electrically similar to an ion exchange resin. Observations show that the theory provides a quantitative explanation for the low-frequency dielectric dispersion of bacteria.     

Filtering rate of the cladoceran,Daphnia pulex as a function of body size,light and acclimation

Daphnia pulex were raised under nine light intensities (0, 1.7, 3.5, 7, 14, 28, 55, and 110 ft-c), polarized light (6.6 ft-c), and four wavelength ranges.Light intensity significantly affected the relationship between filtering rate and body size of unacclimated animals. The b values were lowest at 7 and 14 ft-c and they increased above 7 ft-c as light intensity increased. There were significant differences among b values and adjusted means for unacclimated animals. Acclimation to their respective conditions resulted in some significant differences between b values and adjusted means. For acclimated animals there were no significant differences among b values but there were some differences among adjusted means. The filtering rates of unacclimated and acclimated animals were lower at 14 ft-c while 1.7 and 3.5 ft-c were generally stimulatory and the effect was more pronounced in larger animals. Light intensities above 28 ft-c tended to suppress the filtering rate of small unacclimated animals and stimulate filtering rate in larger unacclimated animals. After acclimation, intensities above 7 ft-c did not affect the filtering rate of either small or large animals.The effect of polarized light on filtering rate was inseparable from the effect of light intensity, however, acclimation to polarized light resulted in a significantly higher b value.There were no significant effects of wavelength among b values for unacclimated animals and the adjusted mean for blue wavelengths was significantly higher than for violet wavelengths. There were no significant differences among b values or adjusted means for acclimated animals. Acclimation to their respective wavelengths did result in some significant differences between b values and adjusted means. Except for the effects of blue wavelengths on unacclimated animals and red wavelengths on acclimated animals, the effects of wavelengths are inseparable from the effects of light intensity.This paper is part of a Ph.D. Thesis submitted to the Faculty of Graduate School of The University of Kansas.Supported in part by a NSF Summer Traineeship and a grant from the Research Corporation to St. Olaf College, Northfield, Minnesota 55057.     

OBSERVATIONS ON THE RELATIONSHIP OF THE GOLGI APPARATUS TO WALL FORMATION IN THE MARINE CHRYSOPHYCEAN ALGA, PLEUROCHRYSIS SCHERFFELII PRINGSHEIM

The role of the Golgi apparatus in wall formation of vegetative cells of a marine chrysophyte, Pleurochrysis scherffelii, is described. Wall fragments are synthesized within the cisternae of the Golgi apparatus. A single Golgi apparatus is always located at the cell periphery, and the distended cisternae are oriented toward the cell surface. A highly-ordered body found near the inflated cisternae is associated with spherical, membrane-bounded bodies which may be involved in the progressive degeneration of cisternal membranes which release wall fragments. Protoplast movement has been detected by time-lapse cinephotomicrography and is correlated at the ultrastructural level with change in positions of the Golgi cisternae. Wall-synthesizing capacity is greatest during transverse wall formation. Senescent cells lack a Golgi apparatus with inflated cisternae. In addition, wall fragments are not present in the Golgi cisternae at this stage. Zoosporogenesis results in a temporary loss of the wall-forming capacity of the Golgi apparatus; this activity then resumes with the formation of a different morphological entity, the scale. Preliminary quantitative measurements of the turnover capacity of the Golgi apparatus have been made. From these data it has been determined that between 41 and 82 Golgi generations are required to synthesize the cell wall of an actively growing cell; this estimate indicates that approximately one cisterna is produced every 2 min, provided the cell generation time is 3 days. The time-lapse cinephotomicrographic data confirm that the rate of production of Golgi cisternae is at least one cisterna every 2 min.     

Assaying Actual Hematein Content of Commercial Hematoxylins and Hemateins

Hematein-free hematoxylin (HFH) was prepared by a modification of the procedure of Palmer and Lillie (Histochemie, 5: 44-54, 1965). Fifty mg of HFH were dissolved in 5 mg of ethylene glycol and then 45 nil of an aqueous solution of 2.25 gm KAl(SO₄)₂. 12H₂O and 5.445 mg KIO₃ were added. Since this amount of KIO₃ would be sufficient to oxidize 25 mg of HFH to hematein we have termed this half-oxidized hematoxylin (HOH). The peak absorbance (560 nm) of this purple solution remained constant for at least a week. With omission of the KIO₃ the solution was colorless. A curve was constructed by plotting absorbance against concentration of hematein in HOH at various dilutions. For analyses of hematein content of commercial hematoxylins 50 mg of sample and 100 mg of hydroquinone were dissolved in 5 ml of ethylene glycol and then 45 ml of a 5% solution of KAl(SO₄)₂. 12H₂O were added. The addition of the hydroquinone stabilized the absorbance for about 5 min. The hematein content could then be calculated by comparing the observed absorbance with the standard curve. Eleven samples of hematoxylin certified by the Biological Stain Commission had hematein concentrations varying from 0.01 to 0.43%. For analyses of the available hematein content of commercial hemateins, 50 mg of sample were dissolved in 10 ml of ethylene glycol, then 45 ml of water and 45 ml of 5% KAI(SO₄)₂. 12H₂O added. The hematein content could then be calculated by comparing the observed absorbance with the standard curve. In 9 samples of hematein from 4 different sources the active hematein content varied from 19 to 97%.     

Efficiency of a Multitest System (Enterotube) for Rapid Identification of Enterobacteriaceae

Enterotube, a multiple-test system which combines nine biochemical tests useful in the identification of members of the family Enterobacteriaceae, was tested and compared with the PathoTec test system in the identification of gram-negative bacilli isolated from clinical specimens. It was found that both the Enterotube and the PathoTec systems rapidly and accurately defined the position of the organisms in the major groups of the family Enterobacteriaceae. Discrepancies were noted between the results of citrate tests on Simmons' citrate-agar and in the Enterotube, as well as between Simmons' citrate-agar and the PathoTec citrate test. It was concluded that the Enterotube system provides a simple, reliable, and rapid method for the presumptive identification of Enterobacteriaceae. The major advantage of the Enterotube is that all tests are done simultaneously by inoculation from a single isolated colony.