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R B Luftig

Publications and source records attributed to R B Luftig.

107 records · Page 6Linked to original sources
PubMed1974

Quantitation of endogenous C-type virion production in several murine cell lines.

Quantitation by enumeration of virion particles, measurement of absorbancy at 260 nm, and densitometry on sodium dodecyl sulfate-polyacrylamide gels has shown that mouse L-M cells yielded 7- to 10-fold less endogenous C-type virions than the parental lines, L or L929. The previously noted stimulation of L-M cell virion production by a concomitant 10% increase in fetal calf serum concentration (D. A. Kindig, R. Karp, and W. H. Kirsten, 1968), was not observed.

Ammonium Sulfate↗
PubMed1973

Preservation of erythrocyte ghost ultrastructure achieved by various fixatives.

We have evaluated the quality of ultrastructural preservation of erythrocyte ghosts achieved under various electron microscopic preparative conditions. Initially, negative staining was used to monitor gross morphology of the ghosts. Of several negative stains used (phosphotungstate, silicotungstate, ammonium molybdate, and uranyl acetate), all but uranyl acetate resulted in fragmentation of membranes and the appearance of small vesicular structures. Further, preservation of gross membrane ultrastructure was greatly enhanced when samples were fixed with either 4.0-5.0% glutaraldehyde or 1% osmium tetroxide (OsO(4)) before they were stained with uranyl acetate. We then examined the ability of these fixatives to preserve membrane fine structure, as monitored by thin-sectioning procedures. In these studies, fixation with 1% osmium tetroxide (alone or in conjunction with 5% glutaraldehyde) resulted in a trilamellar image about 95 A in width. Fixation with 5% glutaraldehyde alone provided a markedly different result. The membrane now appeared as a single line about 160 A wide with regions of varying electron density throughout. This result suggests that glutaraldehyde used alone may reveal the location of membrane proteins that are obscured or removed by OsO(4) fixation. This point would seem to be supported by the results obtained when erythrocyte membranes were extracted with 5 mM EDTA after fixation in either 5% glutaraldehyde or 1% OsO(4). While only 10% of the detectable protein was solubilized from glutaraldehyde-treated erythrocyte membranes, 85% was solubilized from OsO(4)-treated ghosts. Among these latter proteins are three that migrated on Ouchterlony double-diffusion agar plates at the same position as three known proteins with molecular weights of about 200,000. Additional studies indicated that, even during a routine pre-embedding procedure, OsO(4) led to solubilization of as much as 8 times the amount of protein as glutaraldehyde alone. Although the erythrocyte membrane has a notoriously weak association with its proteins, we feel that our studies provide a cautionary note with regard to the use of OsO(4) as a fixative in other membrane systems.

Animals↗
PubMed1973

Bacteriophage T4 head morphogenesis. Isolation, partial characterization, and fate of gene 21-defective tau-particles.

A lysozyme-detergent procedure was developed for isolation of tau-particles from cells infected by gene-21 mutants of T4 bacteriophage. These particles have a sedimentation coefficient of 440 +/- 10 S. They contain less than 1% detectable nuclease-resistant DNA, are smaller (650 x 850 A) than normal bacteriophage heads (800 x 1100 A), and exhibit two major bands on 7.5% Na dodecyl sulfate-acrylamide gels. The more prominent band (55,000 daltons) corresponds to the uncleaved, major capsid polypeptide (P23); the other band (32,000 daltons) corresponds to the gene-22 product (P22). Temperature-shift experiments with cells infected with tsN8 (gene 21) mutants were used to study the fate of tau-particles accumulated under nonpermissive conditions. 50 Min after ts N8-infected cells were shifted from the nonpermissive (41.5 degrees ) to the permissive (25 degrees ) temperature, a phage burst occurred that was 75% of that observed with wild-type phage. However, in "pulse-chase" temperature-shift experiments, the radioactive tau-particle peak only slightly decreased (by 10-14%) by 50 min after the shift, whereas an increased amount of radioactivity (about four times as much as the tau-particle decrease) appeared in phage particles. The results suggest that at least two pools of head polypeptides coexist in cells infected with gene-21 mutants. One pool is composed of head subunits assembled into tau-particles, which are mostly aberrant structures; the second pool is composed of head subunits that are incorporated into mature phage when the gene-21 product becomes functional.

Carbon Isotopes↗
PubMed1973

Physiocochemical studies on L-cell virions.

The L-cell virion (LCV) has been purified from supernatant fluids of mouse L cells grown in suspension culture. The virion is similar to other RNA tumor viruses by several criteria: (i) the density of the virion is 1.16 g/cm(3); (ii) the virion appears as a rounded membranous particle with an outer diameter of 146.7 +/- 11.8 nm, and contains knobs (7-nm diameter) over its surface; (iii) 15 polypeptides (ranging in molecular weight from 7,000 to 110,000) are detectable after electrophoresis of virion protein in sodium dodecyl sulfate-polyacrylamide gels; (iv) three species of RNA can be isolated-high molecular weight (80 to 88s) (50%), 7s (35%) and 4s (15%); (v) heat denaturation of the high-molecular-weight RNA yields a heterogeneous population of molecules (20 to 35s) as well as a 7s and 4s species. Despite the general similarity to infectious RNA tumor viruses, LCV is apparently defective as evidenced by the fact that it does not induce tumors in animals or transform normal mouse cells in vitro (Kindig and Kirsten [17]). The defective nature of the LCV might be related to the fact that assays for DNA polymerase in the virion showed only a negligible activity when compared to Rous sarcoma virus.

Adenosine↗
PubMed1973

Outer membrane of avian myeloblastosis virus.

Guinea pigs immunized intracerebrally with avian myeloblastosis virus (AMV) produced antiserum which reacted with intact virus particles in complement fixation. The antigen in question appeared to be located on the surface of the virion and could be distinguished from the type-specific virus envelope and the group-specific internal antigens of chicken leukosis-sarcoma viruses (ChiLSV). The material could be isolated by sequential treatments of AMV with bromelin, Tween 20, and freeze-thawing, and could be purified by differential centrifugation. Electron microscopy analysis indicated the presence of a component resembling the outer membrane of the particle. The antigenic determinant was designated virus membrane antigen (Vm). Further analyses revealed the presence of protein, lipid, and carbohydrate in a material having a molecular weight of about 6,000 as determined by sodium dodecyl sulfate gel electrophoresis. Serological studies suggested that the outer membranes of AMV and other ChiLSV are represented mainly by host cellular material.

Animals↗
PubMed1972

Bacteriophage T4 head morphogenesis. IV. Comparison of gene 16-, 17-, and 49-defective head structures.

Defective heads present in extracts of bacteriophage T4 gene 16, 17, or 49 mutant-infected cells have been characterized. All appeared as empty shells when examined by negative-stain electron microscopy and showed essentially the same polypeptide pattern on sodium dodecyl sulfate-acrylamide gels. However, when analyzed by several other methods, gene 16- and 17-defective heads were shown to differ markedly from phage heads present in gene 49-defective extracts. First, the gene 16- and 17-defective structures were found to possess a large number of attached tails (50%, rather than about 5%). Second, they contained less nuclease-resistant deoxyribonucleic acid (DNA) (3 versus 18% of a phage equivalent), had a smaller sedimentation coefficient (240 versus 315S), and a lighter density (1.31 vs. 1.34 g/ml) than gene 49-defective heads. Third, they were not attached to the intracellular DNA pool through a deoxyribonuclease-sensitive linkage. Finally, 8-nm diameter capsomers were clearly revealed on the surface of many gene 16- and 17-defective structures. There was a total of 305 +/- 25 capsomers per particle, which yielded an approximate molecular weight of 84 x 10(6) for these heads. The capsomers were presumably not seen on gene 49-defective heads because of the large amount (18%) of associated DNA. These results support the contention that gene 16- and 17-defective heads, in contrast to gene 49-defective structures, represent abortive rather than intermediate structures in T4 head morphogenesis.

Carbon Isotopes↗
PubMed1972

Bacteriophage T4 head morphogenesis. II. Studies on the maturation of gene 49-defective head intermediates.

An investigation into the metabolic requirements for maturation of gene 49-defective heads indicated that adenosine triphosphate energy and continued deoxyribonucleic acid (DNA) but not ribonucleic acid synthesis were needed. The fate of DNA present at restrictive temperatures (41.5 C) in tsC9 (gene 49)-infected cells was also examined. After lysis of infected cells, the 12 to 32% deoxyribonuclease-resistant DNA associated with isolated gene 49-defective heads was found to be attached to a deoxyribonuclease-sensitive complex associated with the debris. Pulsechase experiments where (3)H-thymidine was used to label the DNA at 41.5 C suggested that more DNA from this pool was present in phage recovered after rescue of the gene 49 function than could be accounted for by the deoxyribonuclease-resistant portion. Further, when these experiments were repeated with an additional density shift ((15)N(13)C-glucose to (14)N(12)C-glucose), the DNA extracted from phage rescued at 10 min after the temperature shift-down was found to be 90% conserved. These results suggest a model whereby DNA packaging into capsid precursors is separated from DNA replication and the energy from DNA synthesis provides the driving force for packaging. Pulse-chase, temperature-shift experiments with E920g (gene 66) or E920g;tsC9 mutant-infected cells showed that gene (49, 66)-defective heads, which were isolated as small, isometric-shaped unfilled heads, were a precursor to "petite" phage. This suggests that the maturation process is independent of the size and shape of the head membrane. Similar experiments with the double mutant tsC9;amN120 indicate that gene 49-defective heads can also be filled in the absence of tails.

Adenosine Triphosphate↗
PubMed1972

Bacteriophage T4 head morphogenesis. 3. Some novel properties of gene 13-defective heads.

The nature of phage precursors in gene 13-defective infected cells was studied by electron microscopy and pulse-chase isotopic labeling experiments. Our results suggest that both stable (20%) and fragile (70%) filled-head precursors accumulated in the absence of gene 13 product. Upon extraction, the fragile heads were found to lose most of their deoxyribonucleic acid and appeared unfilled with an average density of 1.34 g/cm(3) and a sedimentation coefficient of 300S. These unfilled heads differed from empty gene 13-defective heads which did not have any associated deoxyribonucleic acid and banded at an average density of 1.31 g/cm(3). Furthermore, it was found that a tsN38 (temperature-sensitive mutant in gene 13)- infected culture maintained at 41.5 C for increasing times led to a decrease in specific infectivity of 1,000S phagelike particles. Electron microscopy of these particles revealed that the decreased infectivity was due to an improper union of head and tails.

Carbon Isotopes↗