Viral DNA in transformed cells: II. A study of the sequences of adenovirus 2 DNA in nine lines of transformed rat cells using specific fragments of the viral genome

³²P-labeled adenovirus 2 DNA was treated with restricting endonuclease from Escherichia coli strain RY-13 (Yoshimori, 1972) (EcoRI) or restricting endonuclease from Hemophilus parainfluenzae (Hpa I) and the resulting fragments of DNA were separated by gel electrophoresis. The kinetics of renaturation of each of the fragments and of complete adenovirus 2 DNA were measured in the presence of DNA extracted from nine lines of adenovirus 2-transformed rat cells and from control cells. Six of the transformed cell lines contained viral DNA sequences homologous to two of the seven Hpa I⁴ fragments and to part of one of the six EcoRI fragments. From the order of the fragments formed by EcoRI and Hpa I on the adenovirus 2 map we conclude that these cell lines contain only the segment of viral DNA that stretches from the left-hand end to a point about 14% along the viral genome. Thus, any viral function expressed in transformed cells must be coded by this small section of viral DNA. The three remaining lines of adenovirus 2-transformed rat cells are more complicated and contain not only the sequences from the left-hand end of the viral DNA, but also other segments of the viral genome. However, no adenovirus 2-transformed rat cell contained DNA sequences homologous to the complete viral genome.     

Trans-ovum transmission of a cytoplasmic polyhedrosis virus of Heliothis virescens (Lepidoptera: Noctuidae)

Adults of Heliothis virescens infected with a cytoplasmic polyhedrosis virus (CPV) produced healthy offspring when their eggs were surface sterilized with either 15% formaldehyde or 0.2% sodium hypochlorite solution. Larvae from infected parents (1) cultured on a vitamin-deficient medium, (2) exposed to cold treatment (5°C, 24 hr), or (3) as progeny of adults from diapaused infected pupae, produced the same number of infected individuals as larvae reared in the customary way. Field studies indicated that the percent of CPV infection in larvae originating from virus-infected parents was density dependent.     

Analysis for possible linkage between the loci for the Waardenburg syndrome and various blood groups and serological traits

Summary Linkage was sought between the Waardenburg syndrome locus and the loci for various genetic markers segregating in a single family. Close linkage was shown to be unlikely with the loci for Rh, MN, Ag, ADA, HL-A, and Gm. Evidence obtained is consistent with the possibility of linkage with the locus for the AB0 blood group, but study of additional families will be required to provide a definite answer.

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Zusammenfassung In einer Familie wurde nach Genkopplung zwischen dem locus für das Waardenburg-Syndrom und verschiedenen genetischen Markern gefahndet. Für die loci für Rh, MN, Ag, ADA, HL-A und Gm wurde enge Kopplung als unwahrscheinlich erwiesen. Dagegen lassen die Daten die Annahme einer Kopplung mit dem AB0-locus zu. Für eine endgültige Entscheidung müßten zusätzliche Familien untersucht werden.

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Research supported by grants No. HD 04134, HL 09011, and HL 08630 from the National Institutes of Health.     

Superoxide Dismutase in Bacillus popilliae, a Catalaseless Aerobe

Bacillus popilliae, a cytochrome-containing aerobic organism that lacks catalase and peroxidase, was examined for superoxide dismutase activity. The activity was quite high relative to a wide variety of organisms previously surveyed and was induced by oxygen. No correlation could be made between superoxide dismutase activity and the unexplained death of this organism after completion of exponential growth.     

Studies on the mechanism of freezing damage to mouse liver. IV. Effects of ultrarapid freezing on structure and function of isolated mitochondria

Mouse liver mitochondria isolated in 0.25 m sucrose were subjected to progressively increasing cooling rates by quench-thaw from liquid nitrogen, isopentane at ?155 °C, and liquid propane at ?185 °C. Structural damage, assessed by electron microscopy and by quantitation of supernatant protein, increased progressively with the cooling rate. Oxidative phosphorylation (with succinate as substrate) was destroyed at all three cooling rates, while acceptorless respiration (succinoxidase) showed a progressive increase with cooling rate, suggesting uncoupling. The succinate cytochrome c reductase system showed no functional damage. Dimethyl sulfoxide, 10–20% by volume, markedly improved structural preservation of the mitochondria, but did not restore oxidative phosphorylation, and further increased the degree of uncoupling.Upon resuspending the mitochondria in 0.15 m KCl prior to quench-thaw, the succinate cytochrome c reductase system displayed an optimal recovery after isopentane quench-thaw, with a sharp decline at still higher cooling rates, as had been encountered in tissue slice experiments, suggesting a compartmental ice-transition in mitochondria over this range of cooling rates. Structurally, however, the KCl-resuspended mitochondria were equally and maximally disrupted by all three quench-thaw procedures. Sixty percent of the mitochondrial protein was extruded into the supernate, far above the levels released from sucrose-suspended mitochondria by quench-thaw and significantly above the 45% released by sonication. Compared to isotonic KCl, isotonic sucrose was thus providing full cryoprotection for the reductase complex and moderate protection for mitochondrial structure. The discrepancies among the several structural and functional indicators of mitochondrial damage leave little possibility that a single compartmental ice-transition, occurring over this range of cooling rates, could provide a coherent explanation for freezing damage to liver mitochondria.     

SPHEROPLAST FORMATION AND ASSOCIATED ULTRASTRUCTURAL CHANGES IN A SYNCHRONOUS CULTURE OF ANACYSTIS NIDULANS TREATED WITH LYSOZYME1,2

Cells of Anacystis nidnlans were grown in synchronous culture using a light-dark alternation to obtain synchronization. Two synchronous cycles were obtained with, decay of synchrony beginning with the third cycle. Cells of various ages in the growth cycle were treated with lysozyme to form spheroplasts. The percentage of spheroplast formation varied with age of the cells. After extended periods of lysozyme treatment, up to 90% of the cells of all ages showed spheroplast formation. Some cells were resistant to the action of lysozyme regardless of age or length of treatment. An ultrastructure study of the spheroplast was made. The electron-dense inner layer of the cell wall was removed by the action of lysozyme on the glucosamine residues of the cell wall, indicating true spheroplast formation. The photosynthetic apparatus became more pronounced with extended treatment with lysozyme.     

The association of chloroplast peripheral reticulum with low photorespiration rates in a photorespiring plant species

Summary A peripheral reticulum occurs in mesophyll chloroplasts of the pentose cycle plantDactylis glomerata L. (orchardgrass). This structural feature was previously thought to occur primarily in the chloroplasts of tropical grasses and other species utilizing the C₄-dicarboxylic-acid photosynthesis pathway. Since the peripheral reticulum is seen in a selection ofD. glomerata which has a low rate of photorespiration, but not in a selection which has a high rate of photorespiration (Carlsonet al., 1971), photorespiratory rates may be dependent in part on the presence or absence of a chloroplast peripheral reticulum.Cooperative investigations of the University of Florida and the Crops Research Division, Agricultural Research Service, U.S. Department of Agriculture. Trade names are mentioned for clarity and do not imply endorsement of products by the U. S. Department of Agriculture. Journal Series No. 3813 of the Florida Agricultural Experiment Station.     

Organization of the MAL loci of Saccharomyces. Physical identification and functional characterization of three genes at the MAL6 locus

Summary We have physically and functionally identified three genes at the MAL6 locus of Saccharomyces carlsbergensis. Using multicopy yeast plasmid vectors, we have subcloned various segments of the entire MAL6 locus. The functional characterization of the MAL6 subcloned regions was determined by (1) analyzing biochemically the levels of MAL-encoded proteins (maltase [-D-glucosidase, E.C. 3.2.1.20] and maltose transport protein) in cells transformed with various MAL6 subclones, and (2) testing the ability of the subclones to complement the maltose fermentation defects of well characterized Mal^– mutants in the highly homologous MAL1 locus. The physical homology between MAL6 and MAL1 is in part demonstrated by the gene disruption of MAL1 using subcloned MAL6 DNA sequences. The results demonstrate that the MAL6 locus is a complex of at least three genes: MAL6R, MAL6T and MAL6S. These genes specify, respectively, a regulatory function, a maltose transport activity (presumably the maltose permease) and the structural gene for maltase. The functional organization of the MAL6 locus is thus identical to that which we had previously determined by mutational analysis for the MAL1 locus.     

Binding of hydrolases from wheat aleurone to concanavalin A- and wheat-germ agglutinin-sepharose

We have studied the ability of hydrolases (acid phosphatase and glycosidases) from the aleurone layers of resting wheat grains to interact with Con A- and WGA-Sepharose as a way to examine their glycoprotein nature. Aliquots (6–85% depending on the enzyme) of all the enzymes interacted with Con A-Sepharose. The major part of α-mannosidase activity (85%) was present in this form. Aliquots (2–20% depending on the enzyme) of the following four enzymes, β-galactosidase, α-mannosidase, β-N-acetylglucosaminidase and acid phosphatase, interacted with WGA-Sepharose. All the enzymes were found in forms which were unable to interact with either lectin. No forms of hydrolases interacting with both lectins were found in the crude extract. The specific activities of most of the enzymes recovered from the lectin-Sepharose gels were greater than those measured in the crude extract. In particular, the highest specific activities were found for β-N-acetylglucosaminidase and β-galactosidase recovered from WGA-Sepharose. Different lectin-binding forms of hydrolases were compared with respect to pH optimum and stability under various conditions (heat and guanidine hydrochloride treatments). The lectin-binding pattern of the hydrolases released in the incubation medium by the aleurone layers was similar to that reported above for the enzymes extracted from these tissues, suggesting that none of the hydrolase forms found in the aleurone layers is selectively released during incubation of these tissues.