Growth of mycobacterium bovis (BCG) in T lymphocyte-depleted mice. BCG Montreal (10-6 viable bacilli) injected intravenously into adult thymectomized, irradiated, and bone marrow-reconstituted (THXB) C57Bl times C3H F1 hybrid mice induced a progressive systemic infection which killed 95% of the animals within 60 days. Control mice infected with this dose of BCG did not die. The infected THXB mice failed to develop detectable levels of tuberculin hypersensitivity although they did show considerable Arthus (3 h) reactivity. The BCG-infected THXB mice lost weight progressively, and the root spleen and root lung indices increased substantially as the infection proceeded. None of the THXB mice developed an antibacterial immune response to the systemic BCG infection, and this was reflected by the continued persistence of macroscopic lung granuloma in these animals. The BCG-infected control mice developed as many surface tubercles as did the THXB animals, but the granulomas rapidly regressed in size and numbers in the normal mice. The lung changes correlated with the amount of tritiated thymidine incorporated by the lung cells in the later stages of the BCG infection. T cell depletion depressed the early splenic peak normally seen in BCG-infected controls, but, on the other hand, there was a progressive increase in lung counts in the THXB mice as the infection progressed and this late peak was not seen in the control animals. The significance of these findings is discussed in relation to the development of antituberculous immunity by BCG-infected mice.


Control of salmonellosis pacifarin biosynthesis by iron. Enterobacter cloacae strain SS4-56 produced salmonellosis pacifarins when grown in an iron-depleted synthetic medium to which 3.16 X 10-7 g-atom of iron had been added per liter, but did not do so when grown in iron-depleted medium supplemented with 3.16 X 10-4 g-atom of iron per liter. The addition of 3.16 X 10-4 g-atom of ferric iron per liter to a pacifarin-containing culture supernatant fluid had no significant effect upon the ability of the active pacifarins present, when administered per os, to protect mice from an otherwise fatal infection produced by a sequential injection of avirulent and virulent strains of Salmonella typhimurium.


Phagocytosis of Cryptococcus neoformans by alveolar macrophages. Guinea pig pulmonary macrophages phagocytized but did not kill nonencapsulated cells of Cryptococcus neoformans. The phagocytic process was inhibited by cryptococcal capsular polysaccharide. Pulmonary macrophages, activated by preinjecting heat-killed bacteria into intact animals, did not kill the engulfed yeast cells. Labeled cells of C. neoformans were neither killed nor cleared from guinea pig lungs 6 h postexposure. The results of our experiments indicate that during the first few hours after the lung is exposed to the infectious particle of C. neoformans the pulmonary macrophage does not function primarily to kill engulfed yeast cells. We believe that a rapid yet transient acute inflammatory response probably plays a major role in this process during the first few hours after C. neoformans enters the lung.


Correlation between phagocytic activity and metabolic response of polymorphonuclear leukocytes toward different strains of Escherichia coli. The bactericidal activity, the phagocytic capacity, and the metabolic stimulation of polymorphonuclear leukocytes challenged with different strains of Escherichia coli were studied. It was found that only two strains out of 10 tested stimulated the oxygen consumption and carbohydrate metabolism of leukocytes and were readily killed by the phagocytes. The lack of killing of the other eight strains was shown to be due to absent or poor phagocytosis rather than to resistance to intracellular killing. Evidence was presented that the surface K antigen plays an important role in conferring antiphagocytic properties to some strains of E. coli. It was suggested that K antigen acts by interfering with the early step of the phagocytic process, that is, the attachment step.


Immunosuppression by hydroxystilbamidine isethionate, a lysosome-stabilizing, anti-proteolytic, antifungal drug. Hydroxystilbamidine (HSB) is a potent suppressor of the plaque-forming cell response of mice injected with heterologous erythrocytes. HSB, given in varying doses and injection schedules, suppressed both the primary and secondary immune responses to bovine serum albumin. Apparently the effect is not simply a toxic effect on spleen cells, because there was no appreciable difference in cell numbers between control and HSB-treated mice. The effect of HSB was most apparent in the early phase of the immune respone.


Genetic basis of toxin production and pathogenesis in Vibrio cholerae: evidence against phage conversion. The pathogenicity of Vibrio cholerae strains "cured" of "Kappa-type" phage was not significantly altered relative to that of their "Kappa" lysogenic parental strains. Unlike Corynebacterium diphtheriae, the capacity of V. cholerae to produce exotoxin was not stimulated as a consequence of active phage multiplication. Toxin production in cultures in which Kappa-type phage multiplication was initiated either by inducing Kappa lysogens or by infecting naturally occurring or "cured" Kappa-sensitive strains was greatly reduced compared to normally growing control cultures. Kappa-sensitive El Tor strain Mak 757 and a Kappa lysogen derived from it did not differ in their capacity to colonize ligated rabbit ileal loops nor in their sensitivites to ultraviolet radiation, acidic pH, or osmotic shock. We conclude that Kappa-type phages do not directly affect the pathogenicity of these V. cholerae strains.


Cytotoxic effect of Mycoplasma fermentans on mouse thymocytes. The in vitro response of mouse thymocytes to various mycoplasmas was evaluated. Cultures of thymus cells from BALB mice were prepared in Earle minimal essential medium with 10 per cent fetal calf serum. After an initial drop in viability, cell populations stabilized at approximately 10-5 viable cells/ml for 3 to 5 days. Concentration of 10-6 to 10-8 colony-forming units of toxic isolates of Mycoplasma fermentans per ml killed over 50 per cent of these cells in a dose-dependent fashion. Four other mycoplasmas (M. pneumoniae, M. hominis, M. arthritidis and a nontoxic strain of M. fermentans) did not induce cytotoxicity of mouse thymocytes. Toxic isolates of M. fermentans multiplied in the presence of thymus cells as they were being inactivated. However, nonviable membrane preparations of these mycoplasma were also toxic, indicating that growth of the organisms is not a prerequisite for the toxic effect. The relevance of these findings for the isolation and identification of the membrane-associated toxic factor is discussed.


Characterization of Escherichia coli obtained from newborn calves with diarrhea. A total of 373 isolates of Escherichia coli were obtained from one or more calves with diarrhea in 155 herds during the 1974 calving season in Montana. Sixty-seven (18 percent) of the isolates representing 59 of the 155 herds were found to be enterotoxigenic as indicated by their ability to cause distention of the calf ligated intestinal segment. The 67 isolates of enterotoxigenic E. coli (ETEC) were placed in one of six different antigenic groups based upon agglutination of formolized whole cells in KO antiserum. Eighty-seven percent (58 of 67) of the ETEC had antigen 1, 2, or 3, whereas only 2.3 per cent (7 of 310) of the non-ETEC (NETEC) had antigen 1, 2, or 3. This antigen numbering system was used for convenience and is not related to any established typing system. Antigens 1, 2, and 3 do not belong to any of the O groups 1 to 157 or K groups 1 to 93 of the International Schema. Colony color or morphology of ETEC and NETEC grown on Tergitol-7 agar with triphenyltetrazolium chloride added could not be used as an indicator of enterotoxigenicity, although there was a tendency for E. coli with the smooth and mucoid colony type to be enterotoxigenic whereas rough colonies were seldom enterotoxigenic. Among ETEC isolates with antigen 1, 2, or 3, there was good correlation between colony type and antigen number. All 13 ETEC isolates with antigen 1 had the smooth colony type, 17 of 19 ETEC isolates with antigen 2 had the smooth, mucoid colony type, and all 25 isolates of ETEC with antigen 3 had the intermediate colony type. Conversely, of the 310 isolates of NETEC, none had antigen 1 and the smooth colony type; none had antigen 2 and the smooth and mucoid colony type; and only one isolate of NETEC had antigen 3 and the intermediate colony type. Sixteen of the 67 isolates of ETEC (24 percent) were motile. Fifteen of the 16 motile isolates of ETEC had the intermediate colony type, and none of the ETEC with smooth or smooth and mucoid colonies were motile.


Interactions between Mycoplasma pneumoniae and guinea pig complement. The "toxic" effect of guinea pig serum (GPS) on Mycoplasma pneumoniae cells was tested under various conditions, using rounding and killing of the cells as test systems. Both activities could be inhibited by heat inactivation (56 C, 30 min). Killing required both Ca2+ and Mg2+, rounding only Mg2+. Both activities were temperature dependent and no rounding or killing occurred at 4C. Incomplete complement sequences with natural of artificial defects in C1, C4, or C6 resulted in lost or reduced killing. The rounding activity was only slightly affected. Anti-C3 antiserum blocked both phenomena; incubation of GPS with 10 mg of inulin per ml reduced the rounding activity, and the same treatment of GPS deficient in C4 inhibited rounding totally. Properdin factor D was shown to be necessary for rounding by GPS, with defects in either C1 or C4. By immune adherence bound C3b could be demonstrated on M. pneumoniae cells after GPS treatment, no antibodies against M. pneumoniae could be found in GPS by immune fluorescence. The results give evidence for complement being the toxic factor in GPS. Efficient killing requires the intact complement sequence. Furthermore, M. pneumoniae cells are able to activate the alternate pathway of complement. Activation of this pathway results in rounding of the cells, which are partly able to recover after this reaction. Biological consequences for the mycoplasmas are death or damage and possibly opsonization, even in the absence of specific antibodies. The host, too, is possibly affected by products of the reaction. The interaction of M. pneumoniae and complement could be involved in the early stages of the development of M. pneumoniae disease.


Opsonin-reversible resistance of Mycoplasma pneumoniae to in vitro phagocytosis by alveolar macrophages. Several species of mycoplasmas are responsible for respiratory disease in animals and man. As yet, little is known about the interaction of these pathogens with alveolar macrophages, one of the primary components of pulmonary resistance to infections. The present study was undertaken to develop an in vitro model to examine this organism-cell interaction, using a human pathogen, mycoplasma pneumoniae, and normal guinea pig alveolar macrophages. During a 24-h incubation of M. pneumoniae with a monolayer of macrophages, mycoplasmas were found to attach directly to the surface of the cells without inducing significant phagocytosis. Ultrastructurally, the organisms appeared bound to the cell membrane by their characteristic attachment organelles. Only after the addition of specific anti-mycoplasma serum were cells able to engulf attached and surrounding organisms. These data suggest that the interaction of M. pneumoniae and alveolar macrophages is a potentially important aspect of disease pathogenesis, and immune factors which might alter this interaction merit further examination.


Calf ligated intestinal segment test to detect enterotoxigenic Escherichia coli. The ligated intestinal segment test in the young calf was utilized to establish the enterotoxigenicity of approximately 600 Escherichia coli isolates obtained from fecal specimens intestinal contents of calves with diarrheal disease. One hundred and fifty isolates were routinely tested in a single calf. There was little problem with false-positive reactions. False-negative reactions normally occurred only if the isolate was tested in the posterior 3 m of the small intestine. The ligated small intestine of the calf was distended by E. coli isolates of bovine, porcine, and human origin. Use of the calf ligated intestinal segment test is recommended to determine the enterotoxigenicity of E. coli isolates of bovine origin.


Variations in pulmonary antibacterial defenses among experimental animals. Intrapulmonary killing of Staphylococcus aureus proceeded at equal rates in guinea pigs, hamsters, rats, and mice. In the lung of guinea pigs and hamsters, Proteus mirabilis was killed at virtually the same rate as S. aureus as compared with half the rate of inactivation in the lungs of rats and mice.


Antibody and cell-mediated immunity in phytohemagglutinin-treated chickens. The effects of phytohemagglutinin (PHA) on humoral and homograft immunity in young chickens were studied. Intravenous (IV) injections of PHA stimulated the antibody production while intraperitoneal (IP) injections suppressed the antibody production. IP injected PHA failed to prolong the survival of the homografts. The route and time of PHA administration must be considered when evaluating the influence which PHA exerts on homograft survival and hemagglutinin production in the chiicken. The results obtained in this study suggest that there may be two different target cell lines involved in PHA's action. The reduced antibody production following IP injections of PHA may be due to its interference with the peritoneal macrophages and T cells and in the case ov IV administration PHA may be activating both macrophages and T cells. Possibly, there may be a difference in the effective PHA concentration after IP versus IV administration to account for different responses observed.


Effect of carrageenan on the development of hypersensitivity (Schistosoma mansoni egg) and foreign body (divinyl-benzene copolymer beads and bentonite) granulomas. The effect of carrageenan on the development of hypersensitivity and foreign body granulomas was examined. Repeated intraperitoneal injections of the polysaccharide caused a strong suppression of the primary, hypersensitivity granulomas forming around the eggs of Schistosoma mansoni worms in the lungs of mice, but had no effect on the secondary, enhanced egg granulomas in the previously sensitized animals. Carrageenan treatment virtually abolished the granulomatous reactions forming around plastic beads or bentonite particles. It is concluded that carrageenan exerted its suppressive effect not through macrophage toxicity but rather by affecting plasma factors which regulate vascular permeability and cellular traffic during the process of inflammation.


Effect of concanavalin A on immunologic abnormalities of New Zealand (NZB/W) mice. The administration of Concanavalin A (Con A) to young New Zealand (NZB/W) mice has been previously found to render them tolerable to BGG. The corresponding mechanism is unknown but is consistent with other observations that Con A is capable of activating a suppressor T cell population. We have further explored this possibility by giving New Zealand mice Con A from 1 month until 11 months of age and monitoring several known immunologic abnormalities. Con-A-treated mice, in contrast to littermate controls, were found to have lower anti-DNA levels, less proteinuria, a better response to primary sheep red cell immunization and normally rejected skin allografts.


Intracellular localization of the migration inhibitory factor (MIF) in a long-term human lymphoid cell line. Subcellular fractions of the human lymphoid cell line PGLC-33H were obtained by N2 cavitation and differential centrifugation. The purity of the fractions was assessed by the use of the following marker enzymes: beta-glucuronidase for the lysosomal-intermediate fraction; a nonspecific esterase for the microsomal fraction; and LDH for the supernatant fraction. These subcellular fractions were studied for MIF activity utilizing human lymphoid cells from established lines as target cells. MIF activity was most consistently found in the microsomal fraction. Also, MIF activity was closely associated with the relative specific activity of exterase. No such correlation with MIF activity could be demonstrated for beta-glucuronidase or LDH.


Drug-induced tolerance: selective induction with immunosuppressive drugs and their synergistic interaction. Drug-induced tolerance to polyinosinic-polycytidylic acid was studied in NZB/W mice. A single injection of cyclophosphamide (CY) or methotrexate (MTX) induced tolerance when given 24 h after antigen, whereas azathioprine (AZ) and methylprednisolone (MP) did not. However, MP and MTX each synergized with a nontolerogenic dose of CY. In addition, MP and AZ further reduced the tolerance induced by low but tolerogenic doses of CY. Drug combinations in the induction of tolerance may have wide application in the fields of clinical immunology and transplantation.


Rosette formation with sheep erythrocytes--a possible T-cell marker in the pig. A proportion of pig lymphocytes form rosettes with sheep erythrocytes. Factors affecting their demonstration have been investigated, and a standard technique defined. Rosette-forming lymphocytes lacked surface immunoglobulin detected by immunofluorescence and formation of rosettes was not inhibited by anti-immunoglobulin or anti-PLA sera, but was by anti-thymus serum. Of 18 species' erythrocytes tested only sheep, Barbary sheep and Mouflon erythrocytes formed rosettes in similar percentages. Fetal sheep erythrocytes formed no rosettes at 6o days of gestation and developed adult levels by term. Rosettes were formed by the majority of thymus cells, by only few bone marrow cells and by intermediate proportions of cells in other lymphoid tissues correlating with the probable order of T cell content. In pig fetuses, thymus contained postnatal levels of rosette-forming cells by 69 days, when such cells were not detected in other tissues. These data support the contention that SRBC rosettes are formed by T lymphocytes.


Subcellular fractions from dermis and epidermis in contact sensitization of guinea pigs to 1-chloro-2,4-dinitrobenzene. Subcellular fractions were prepared from the epidermis of guinea pigs which had been painted with 1-chloro-2,4 dinitrobenzene 14 h previously. The microsomal fraction showed less activity than other fractions in the MIF test. Similarly, the microsomal fraction from the dermis of skin irritated with croton oil before DNCB painting was relatively inactive. However, the microsomal fractions from the epidermis and more especially from the dermis of skin painted with DNCB while undergoing allergic contact dermatitis from pentadecylcatechol showed significant MIF activities.


Use of immunofluorescent localization in the normal guinea pig eye to differentiate three autoantisera. Three distinct immunofluorescent patterns were demonstrated in normal guinea pig eyes by autoantisera prepared to three different ocular tissue preparations. Anti-U serum (antiserum to ureal homogenate) exhibited specific fluorescence in the area of Bruch's membrane. Anti-P serum (antiserum to a suspension of the sedimentable portion of retina homogenate) exhibited specific fluorescence in the outer segments of the photoreceptor cells. Anti-S serum (antiserum to retinal homogenate extract) demonstrated specific fluorescence in the outer portion of the retina extending from the outer plexiform layer to the outer segments of the photoreceptor cells. The ability of anti-S and anti-P sera immunofluorescent activities to cross species lines was demonstrated.


Observations on rabbit thymocytes and peripheral T cells. II. Rosette formation with rabbit erythrocytes. The affinity of rabbit thymocytes and a proportion of lymphocytes for homologous and autologous erythrocytes has been investigated. Thymocytes were found to rosette strongly with untreated, washed erythrocytes, although reactions on peripheral lymphocytes from blood and lymph nodes were weaker. Enzyme treatment of the erythrocytes was used in attempts to improve reactions with peripheral lymphocytes. While neuraminidase-treated cells showed no increased reactivity, erythrocytes treated with papain were more firmly bound and reacted with greater numbers of blood and lymph node cells. Evidence was obtained to show that peripheral lymphocytes which have an affinity for papain-treated erythrocytes belong to the thymus-derived T cell population.


Shared antigenicity between chicken brain and bursa of fabricius. Antisera to chicken brain, thymus and bursa of Fabricius were prepared in rabbits and rendered organ-specific by appropriate absorptions. Brain was shown by immunofluorescence and immune adherence to have common antigenicity with the bursa of Fabricius but not with the thymus.


[Theories of cognitive disorder in schizophrenia based on psychological experiments (author's transl)]. Over the last few years, psychological experiments have provided a major contribution to research in schizophrenia. Historical, practical and theoretical grounds led to investigations mainly in the field of cognitive disorder. Independent psychological theories of schizophrenia emerged from the body of experimental results, at first restricted to mental performance of the patients but later generalized to center on the total problem of schizophrenia. In accordance with psychiatric thinking, psychologists favoured cognitive deficit as the essential schizophrenic characteristic which would possibly generate the basic symptomatology. Apart from methodological considerations, the main question arising is if this feature is truly typical of schizophrenia. The problem of the different concepts of schizophrenia has to be considered in this connection. A survey of theories of schizophrenia which are sufficiently consolidated by psychological experiments reveals that two lines of theoretical reasoning exist: one based on specific cognitive disorder, and the other where this is not the case. Encompassed by the first classification are the considerations of Bannister, Rodnick and Germezy, Shakow, McReynolds, Silvermann, L.J. Chapman, Payne, Mednick, and Epstein, as well as the interference theories (with the exception of Callaway's version)..


A plea for a discipline of health and medical evaluation. There is an expanding need for quantitative information to evaluate health and medical care procedures. Existing methods for designing and carrying out evaluation studies need to be improved to provide better techniques for answering questions on efficacy of health and medical procedures, and in providing a more adequate information base on which to set healthy policy. This paper reviews some of the problems in the design and conduct of evaluation research, and makes a plea for creation of a special discipline to support and develop the field of health and medical care evaluation.


Gamma-ray-induced thymine damage in the DNA in coliphage phi chi 174 and in E. coli. The radiation chemical reactivity of thymine residues has been compared for single-stranded phi chi 174 DNA free in solution and in the intact bacteriophage, as well as for native E. coli DNA in solution and inside the cell. The gamma-ray-induced release of [3H]H2O from thymine-methyl[3H] was measured to assess the reactivity of the thymine methyl group. Formation of ring-saturated products of the 5,6-dihydroxy-dihydrothymine type (see article) was determined by an alkali-acid degradation assay. It was observed that the formation of that the formation of [3H]H2O was suppressed by a factor of 9 in intact phi chi 174 bacteriophage compared to phi chi 174 DNA, whereas the formation of (see article) was only slightly suppressed in the phage. For E. coli cells the formation of [3H]H29 was reduced 790-fold compared with free E. coli DNA, and (see article) formation was reduced 140-fold. The extents of survival after gamma-irradiation of phi chi 174 phage plaque-forming ability and E. coli colony-forming ability were also determined. Under the conditions used in these experiments, a linear relationship was observed between the loss of biological activity and the formation of thymine damage for both phi chi 174 phage and E. coli cells.


Carotid endarterectomy utilizing the exclusion clamp. Carotid endarterectomy is useful in the treatment of the uncomplicated pre-stroke patient. Use of the exclusion clamp makes possible a short period of carotid occlusion without the use of either internal or external shunts. Forty-two patients were operated on using this technic.


Hydroureter and hydronephrosis of pregnancy presenting as acute obstetric or surgical emergencies. The unusual clinical course of three cases of dilatation of ureters and renal pelvises in pregnancy was reported. The presenting symptom in each case was acute abdominal pain, which was at first attributed to other surgical or obstetric emergencies like premature separation of placenta, acute appendicitis and acute hydramnion.


Cornea-damaging proteases of Serratia marcescens. Fractionation of the culture supernatant fluids of Serratia marcescens, strain BG, by ammonium sulfate precipitation, isoelectric focusing, ion-exchange chromatography, hydroxyapatite adsorption chromatography, and gel filtration failed to separate the rabbit cornea-damaging activity and the in vitro protease activity of the preparations. Two proteases having similar molecular weights (44,000), estimated by gel filtration, and isoelectric points of approximately 5.0 and 5.3 were obtained free of detectable amounts of other known extracellular serratia enzymes. Heating a mixture of the two proteases for 15 minutes at 60 degrees C. resulted in complete loss of protease and cornea-damaging activities. Production of protease and cornea-damaging activities was inhibited by ammonium sulfate. The results support the conclusion that extracellular proteases produced in vitro by S. marcescens can elicit rapid and extensive damage to the rabbit cornea.


Fluorescein diffusion in the human optic disc. The characteristics of the transcapillary transfer of fluorescein dye in the optic disc of healthy individuals has been studied. A diffusible fluorescein dye and a nondiffusible reference substance, indocyanine green (ICG), which was assumed to remain in the capillaries, were injected into the circulatory system. The time courses of the concentrations of the two dyes in the optic disc were determined by simultaneously recording the fluorescence intensity of fluorescein and the infrared absorption by ICG with a fundus reflectometer. The difference between the fluorescein concentration curve and the reference ICG curve is a measure of the accumulation of fluorescein in the disc tissue. Our measurements indicate that fluorescein dye does not diffuse across the capillaries in the optic disc. The accumulation of fluorescein in the disc only starts at about one minute after the injection and seems to be due to diffusion of the dye from the surrounding choroid. The time constant of this diffusion process was found to be approximately one minute.


Bilateral slipped upper femoral epiphysis: a rare manifestation of renal osteodystrophy. Case report with discussion of its pathogenesis. Bilateral slipped upper femoral epiphysis is a rare manifestation of renal osteodystrophy. A case of bilateral slipped femoral epiphysis in an 18-year old male suffering from chronic renal failure due to oligomeganephronic renal hypoplasia with profound signs of renal osteodystrophy is presented. Serum growth hormone levels were high, while those of urinary 17-ketosteroids were decreased. Following subtotal parathyroidectomy, the progression of the process leading to slipped epiphysis was halted with closure of the epiphyses. The patient was subsequently treated with chronic hemodialysis for several months, after which successful renal transplantation was performed. The pathogenesis of renal osteodystrophy leading to slipped epiphysis is discussed and attention drawn to the fact that bilateral slipped femoral epiphysis may be the first clinical sign of chronic renal insufficiency.


The management of urologic complications after kidney transplantation. Urologic complications occurred in four (20%) of 20 kidney transplants performed in 18 patients. The complications encountered were acute hemorrhage from the distal end of the ureter, a calyceal fistula, a ureteral stricture with associated fungus infection, and obstructive uropathy. In two of the four cases it was necessary to sacrifice the transplant.


On the history of basic fuchsin and aldehyde-Schiff reactions from 1862 to 1935. The nature of products formed by aldehydes and Schiff's reagent, whether they are sulfonic or sulfinic acid compounds, has been the subject of much discussion. It seems therefore timely to review early studies of aldehyde-Schiff reactions, including the history of pararosanilin and related dyes. Dyes of the basic fuchsin group have been studied extensively since 1862, and their triphenylmethane structure was established in 1878. The currently used structural formulas were introduced around the turn of the century. Reactions of basic fuchsin with aldehydes, with and without addition of SO2, were investigated by Schiff in the 1860's i.e. before the structure of these dyes was known. In 1900 Prud'homme showed that the reaction products of basic fuschsin, sodium bisulfite and formaldehyde are alkylated and sulfonated derivatives of the parent compound; further chemical studies indicated attachment of the sulfonic acid group to the carbon atom of the aldehyde. Prud'homme's findings were repeatedly confirmed during the following decades. Wieland and Scheuing were apparently unaware of these studies and introduced the sulfinic acid theory in 1921; furthermore, they considered substitution at two amino group of Schiff's reagent essential for formation of the colored compound. However, later chemical and spectroscopic studies showed no evidence of-N-sulfinic acids but supported the sulfonic acid theory of Prud'homme.


Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physiochemical approach to studying the unit of segregation. Physiocochemical evidence presented indicates plasmid deoxyribonucleic acid (DNA) can associate with host chromosome without linear insertion of the former into the latter. This conclusion is based on the observation that covalently closed circular (CCC) plasmid DNA can cosediment with undegraded host chromosome in a neutral sucrose gradient. When F plus bacteria are lysed under conditions that preserve chromosome, approximately 90% of CCC F sex factor plasmid (about 1% of the total DNA) is found in folded chromosomes sedimenting at rates between 1,500 and 4,000s. The remaining 10% of the CCC F DNA sediments at the rate (80S) indicative of the free CCC plasmid form. Reconstruction experiments in which 80S, CCC F DNA is added to F plus or F minus bacteria before cell lysis show that exogenous F DNA does not associate with folded chromosomes. In F plus bacteria, F plasmid is harbored at a level of one or two copies per chromosomal equivalent. In bacteria producing colicin E1, the genetic determinant of this colicin, the Col E1 plasmid, is harbored at levels of 10 to 13 copies per chromosomal equivalent; yet, greater than 90% of these plasmids do not cosediment with the 1,800S species of folded chromosome. However, preliminary evidence suggests one or two Col E1 plasmids may associate with the 1,800S folded chromosome. Based on evidence presented in this and other papers, we postulate F plasmid can link to folded chromosome because the physicochemical structure of the plasmid resembles a supercoiled region of the chromosome and, therefore, is able to interact with the ribonucleic acid that stabilizes the folded chromosome structure. Implications of this model for F plasmid replication and segregation are discussed.


Reinitiation of deoxyribonucleic acid synthesis by deoxyribonucleic acid initiation mutants of Escherichia coli: role of ribonucleic acid synthesis, protein synthesis, and cell division. The dnaA and dnaC genes are thought to code for two proteins required for the initiation of chromosomal deoxyribonucleic acid replication in Escherichia coli. When a strain carrying a mutation in either of these genes is shifted from a permissive to a restrictive temperature, chromosome replication ceases after a period of residual synthesis. When the strains are reincubated at the permissive temperature, replication again resumes after a short lag. This reinitiation does not require either protein synthesis (as measured by resistance to chloramphenicol) or ribonucleic acid synthesis (as measured by resistance to rifampin). Thus, if there is a requirement for the synthesis of a specific ribonucleic acid to initiate deoxyribonucleic acid replication, this ribonucleic acid can be synthesized prior to the time of initiation and is relatively stable. Furthermore, the synthesis of this hypothetical ribonucleic acid does not require either the dnaA of dnaC gene products. The buildup at the restrictive temperature of the potential to reinitiate deoxyribonucleic acid synthesis at the permissive temperature shows rather complex kinetics the buildup roughly parallels the rate of mass increase of the culture for at least the first mass doubling at the restrictive temperature. At later times there appears to be a gradual loss of initiation potential despite a continued increase in mass. Under optimal conditions the increase in initiation potential can equal, but not exceed, the increase in cell division at the restrictive temperature. These results are most easily interpreted according to models that postulate a relationship between the initiation of deoxyribonucleic acid synthesis and the processes leading to cell division.


Polynucleotide sequence relationships among Ent plasmids and the relationship between Ent and other plasmids. Deoxyribonucleic acid-deoxyribonucleic acid hybridization studies reveal that the plasmids coding for the production of heat stable and heat labile enteroxtoxins of Escherichia coli, regardless of their origin, have a majority of their polynucleotide sequences in common, but are not related in any significant way to those plasmids coding for the synthesis of only ST toxin. The heat stable and heat labile plasmids also share a significant degree of their polynucleotide sequences with plasmids of the FI and FII incompatibility groups, but not with R factors belonging to the I, N, W, P, or X incompatibility groups.


Positive selection of mutants with deletions of the gal-chl region of the Salmonella chromosome as a screening procedure for mutagens that cause deletions. We have developed a convenient and specific positive selection for long deletions through the gal region of the chromosomes of Salmonella typhimurium and Escherichia coli. Through simultaneous selection for mutations in the two closely linked genes, gal and chlA, a variety of deletions of varying length, some extending through as much as 1 min of the chromosome, could be readily obtained. Many of these deletions resulted in the loss of a gene, which we named dhb, concerned with the ability of the bacterium to synthesize the iron chelating agent enterobactin. The selection was adapted for the screening of mutagens for their ability to generate long deletions in the bacterial deoxyribonucleic acid. Forty agents were screened for this capability. Nitrous acid, previously reported to be an efficient mutagen for this purpose, increased the frequency of deletion mutations 50-fold in our system. Three others, nitrogen mustard, mitomycin C, and fast neutrons, were shown to increase the frequency of long deletions between five- and eightfold. The remainder were found to be incapable of generating these deletions.


Assay and regulation of S-adenosylmethionine synthetase in Saccharomyces cerevisiae and Candida utilis. A simple and sensitive assay for S-adenosylmethionine (SAM) synthetase is described which depends on the quantitative separation of the product, [14CH3]S-adenosylmethionine, from the substrate, L-[14CH3]methionine, on a Bio-Rex 70 column. L-Methionine protects the enzyme during preparation of cell extracts by sonic treatment but causes repression of enzyme activity during growth of Candida utilis. The presence of 5 mM methionine in the growth medium repressed SAM synthetase specific activity threefold compared to the specific acitivity of the enzyme isolated from cells grown in unsupplemented medium. Conversely, the presence of methionine in the growth medium resulted in an 80-fold increase in the intracellular concentration of SAM as compared to the Sam accumulated intracellularly in unsupplemented cultures.


Autolytic enzymes in hyphae of Aspergillus nidulans: their action on old and newly formed walls. Walls, purified from hyphae of the ascomycete Aspergillus nidulans, autolyzed on incubation and liberated glucose, mannose, galactose, N-acetylglucosamine, and soluble oligosaccharides. Digestion proceeded at linear rates until approximately 3% of the wall polymers had been hydrolyzed and then slowed markedly. The change in the rate of autolysis was not due to loss of enzyme activity but was caused by the disappearance of a fraction of the wall which was highly susceptible to digestion. Radioactive labeling showed that this fraction was the newly formed wall. The new wall was highly susceptible to enzyme action both when it was deposited at the apex in growing hyphae or when deposited laterally in hyphae treated with cycloheximide. The relations between wall modification and apical growth are discussed.


Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. A composite plasmid has been constructed in vitro from colicin E1 factor (mass of 4.2 megadaltons [Md]) and nontransmissible resistance factor RSF 1010 (mass, 5.5. Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease (RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (SmaR), which introduces a single scission in the colicin E1 factor but not in RSF 1010, convErted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin E1 factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments--an RSF 1010 Eco RI linear and the two expected products from the colicin E1 factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin E1, but colicin E1 itself was not synthesized. In contrast, colicin E1 was synthesized by cells containing simultaneously both colicin E1 factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h. whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of chloramphenicol suggests that the replicator of the colicin E1 factor is functional in the composite plasmid.


Recombination and the Escherichia coli K-12 sex factor F. Recombination between two Flac tra minus elements to give Flac tra plus recombinants was measured in Rec plus and Rec minus strains of Escherichia coli K-12. Polar tra mutations were used to increase the proportion of tra plus recombinants among the parental Flac tra minus elements transferred by complementation. The kinetics, measured in a rec plus strain, showed that recombination began about 1 h after the initiation of mating and was completed about 1 h later. Recombination was abolished in a recA minus strain, reduced by two-thirds in a recF minus strain, and unaffected in recB minus and recC minus strains. It is proposed that the part not due to the RecF pathway results from a RecBC- and RecF-independent system for formation of single-stranded joins. One such join could be followed either by transfer and a site-specific recombination event, or by a second single-stranded join and then transfer: in either case replication and inheritance of the recombinant molecule would be dependent upon the F transfer replication system. Chromosome mobilization by an F' element was normal in a recB plus recF minus strain, and was reduced only fourfold in a recB minus recF plus strain: in the latter strain, both the RecF pathway and the system for single-stranded joins may have contributed to mobilization. Measurement of post-conjugational chromosomal recombination in exponential-phase recipient cells carrying surface exclusion-deficient Flac mutants indicated that F does not itself determine a generalized recombination system able to replace the RecA plus product or the RecBC and RecF pathways.


Mapping of colicin E2 and colicin E3 plasmid deoxyribonucleic acid EcoR-1-sensitive sites. Colicin plasmids E2 and E3 (Col E2 and Col E3) deoxyribonucleic acid (DNA) has been shown to contain, respectively, two and three EcoR1 restriction endonuclease-sensitive sites. This was determined by measuring the DNA fragments generated after EcoR1 endonuclease treatment by agarose gel electrophoresis and electron microscopy. The structure of heteroduplex Col E2-col E3 DNA molecules formed from EcoR1-generated fragments permitted a localization of the EcoR1-sensitive sites on the plasmid chromosomes.


Rejoining of radiation-induced single-strand breaks in deoxyribonucleic acid of Escherichia coli: effect of phenethyl alcohol. Single-strand breaks in deoxyribonucleic acid of Escherichia coli B/r cells exposed to 20 krads of gamma radiation could be rejoined by incubation of irradiated cells in growth medium. In the presence of 0.25% phenethyl alcohol, this repair was completely inhibited although deoxyribonucleic acid and protein syntheses were suppressed only partially.


Lipid synthesis in stringent Escherichia coli: an artifact in acetate labeling of phospholipids during a shiftdown in growth rate. The use of (14C)acetate to label the phospholipids of stringent Escherichia coli, after a decrease in agitation, leads to artifacts resulting from a decrease in the specific activity of the acetyl coenzyme A pool.


Genetic modification of substrate specificity of hypoxanthine phosphoribosyltransferase in Salmonella typhimurium. Salmonella typhimurium strain GP660 (proAB-gpt deletion, purE) lacks guanine phosphoribosyltransferase and hence cannot utilize guanine as a purine source and is resistant to inhibition by 8-azaguanine. Strain GP660 was mutagenized and a derivative strain (GP36) was isolated for utilization of guanine and hypoxanthine, but not xanthine, as purine sources. This alteration was designated sug. The strain was then sensitive to inhibition by 8-azaguanine. Column chromatographic analysis revealed the altered phosphoribosyltransferase peaks for both hypoxanthine and guanine to be located together, in the same position as hypoxanthine phosphoribosyltransferase (hpt gene product) of the wild-type strain. Genetic analysis showed the sug mutation to be allelic with hpt. Therefore sug represented a modification of the substrate specificity of the hpt gene product.


Characteristics of cold-sensitive mutants of Escherichia coli K-12 defective in deoxyribonucleic acid replication. Four cold-sensitive mutants of Escherichia coli have been isolated which show a reduced ability to synthesize deoxyribonucleic acid at low temperature. The mutants also have a reduced ability to incorporate nucleoside triphosphates into deoxyribonucleic acid at low temperature in cell preparations made permeable with toluene. All four mutations are located at or near the dnaA locus on the E. coli genetic map. They are recessive to the wild-type allele and two of them can be integratively suppressed by F episomes.


Sedimentation analysis of deoxyribonucleic acid from thymine-starved Escherichia coli. During thymine starvation, strand breaks accumulate in the chromosomal deoxyribonucleic acid (DNA) of Escherichia coli. This effect occurs to a varying extent in different strains and is particularly enhanced in strains deficient in DNA polymerase I. The inhibition of ribonucleic acid or protein synthesis suppresses the accumulation of strand breaks. In a polA strain, rifampin is more effective than chloramphenicol or puromycin in suppressing strand break accumulation. To a certain extent the pehenomenon othymineless death correlates with the appearance of strand breaks. Although the killing can not be explained by the bulk of strand breaks, it is possible that some of them represent lethal events. On the basis of our observations we proposed the following model. (i) Transcription may be accompanied by single-strand breaks in DNA. (ii) DNA polymerase I is involved in the efficient repair of these breaks. (iii) Thymine deprivation results in the accumulation of unrepaired breaks. (iv) Polymerase I-mediated repair is less affected by thymine deprivation than are the alternative pathways because it closes the breaks with short patches, requiring less thymine.


F deoxyribonucleic acid transferred to recipient cells in the presence of rifampin. When Escherichia coli F+ cells resistant to rifampin and streptomycin are mated with F- cells sensitive to the antibiotics, a large fraction of F factor deoxyribonucleic acid (DNA), which is transferred to the recipient cells in the presence of either of the antibiotics, can be recovered as covalently closed, circular double-stranded DNA, as in the control mating in the absence of antibiotic. Similar results were obtained in the presence of chloramphenicol or chloramphenicol plus rifampin. It is suggested that the transferred single-stranded F DNA can be converted to the covalently closed, circular double-stranded form in the absence of protein synthesis, and that rifampin-sensitive transcription is not required for the conversion.


F deoxyribonucleic acid superinfected into phenocopies of donor strains. When F+ donor cells of Escherichia coli are conjugated with F-, F+, or Hfr recipients under the conditions of phenocopy mating, the male recipients are found capable of accepting the F episome as effectively as the F- recipients. The F deoxyribonucleic acid (DNA) superinfected into the male recipients is converted to the covalently closed, circular duplex form, as in the F- recipients. It is also found that the synthesis of the strand complementary to the transferred single strand and its subsequent conversion to the covalently closed, circular duplex occur effectively in male recipients as well as in female recipients. Under these mating conditions, F-ilv+ episome superinfected to F+ and Hfr cells is diluted out during growth, whereas F-ilv+ transferred into F-cells is replicated and established in almost all progeny cells. These results suggest that the incompatibility of the F episome is not due to the reduction in the rate of the conversion of transferred single-straned F DNA to covalently closed, circular duplex, but, rather, to an inhibition of further replication of the covalently closed, circular F DNA.


Mapping of the fabD locus for fatty acid biosynthesis in Escherichia coli. fabD mutants of Escherichia coli contain a thermolabile malonyl-coenzyme A-acyl carrier protein transacylase which causes defective fatty acid synthesis and temperature-sensitive growth. By conjugation and P1 transduction the fabD locus has now been mapped at min 24, between pyrC and purB and close to cat. The order of sites is tentatively given as pyrC, cat, fabD, and purB, though the orientation of cat and fabD could be reversed. The possible relationship of fabD with another mutation lying in this region and also affecting acid synthesis is discussed. In the course of these studies we also confirmed the location of the fabA gene, determined that poaA lies between fabA and pyrC, and inadvertently found that the pyr mutation in strain AT3143 is probably pyrF and not pyrC.


Vinylglycolate resistance in Escherichia coli. Escherichia coli K-12 vinylglycolate-resistant mutants have been isolated and characterized. Two of the mutants, JSH 150 and JSH 151, have been determined to be double mutants, lacking both membrane-bound L-and D-lactate dehydrogenases. The lactate transport system is intact in all strains; both radioactive lactate and vinylglycolate are actively taken up. Likewise, the phosphoenolypyruvate-dependent phosphotransferase system for hexose uptake is active. Vinylglycolate, previously shown to inhibit the phosphoenolpyruvate-dependent phosphotransferase system, has very little effect in the double mutants. The extent of vinylglycolate inhibition in other mutants seems directly related to the activity of the lactate dehydrogenases. This indicates that vinylglycolate is oxidized to 2-keto-3-butenoate before inactivating the phosphoenolpyruvate-dependent phosphotransferase system. These results were found in whole cells and confirmed in isolated membrane vesicles.


Kinetics of induced and repressed enzyme synthesis in Saccharomyces cerevisiae. Our previous work has shown that both induction, after addition of inducer, and loss of ability to produce allophanate hydrolase, after removal of inducer, proceed more rapidly than expected from the reported half-life of messenger ribonucleic acid in Saccharomyces cerevisiae. As a basis of rectifying these observations, we have characterized induction and repression of allophanate hydrolase synthesis and find that: (i) induction of the hydrolase begins immediately upon addition of inducer, (ii) once induction has been initiated removal of inducer does not result in immediate loss of synthetic capacity, (iii) induction of the capacity to produce hydrolase can occur in the absence of protein synthesis, (iv) the half-life of hydrolase synthetic capacity increases if protein synthesis is inhibited, (v) allophanate hydrolase itself is not degraded upon removal of inducer, and (vi) induction and repression of allophanate hydrolase synthetic capacity likely occurs at the level of transcription.


Escherichia coli K-12 mutant forming a temperature-sensitive D-serine deaminase. A single-site mutant of Escherichia coli K-12 able to grow in minimal medium in the presence of D-serine at 30 C but not at 42 C was isolated. The mutant forms a D-serine deaminase that is much more sensitive to thermal denaturation in vitro at temperatures above but not below 47 C than that of the wild type. No detectable enzyme is formed by the mutant at 42 C, however, and very little is formed at 37 C. The mutant enzyme is probably more sensitive to intracellular inactivation at high temperatures than the wild-type enzyme. The mutation lies in the dsdA region. The mutant also contains a dsdO mutation, which does not permit hyperinduction of D-serine deaminase synthesis.


Isolation and characterization of D-serine deaminase constitutive mutants by utilization of D-serine as sole carbon or nitrogen source. Mutants constitutive for D-serine deaminase (Dsdase) synthesis were isolated by utilizing D-serine as sole nitrogen or carbon source in the chemostat. This method generated only regulatory constitutive (dsdC) mutants. The altered dsdC gene product in these strains is apparently able to bind D-serine more efficiently than the wild-type dsdC+ gene product--a selective advantage. Constitutive synthesis of Dsdase in all of these dsdC mutants is extremely sensitive to catabolite repression, and catabolite repression is reversed by the addition of D-serine. Of the 15 mutants generated by this method, none are suppressible by supD, supE, or supF. Mutations to a low level of constitutivity (maximal specific activity of 9) occur much more frequently than mutations to a high level (maximal specific activity of 79). High level constitutive synthesis of Dsdase results from the synthesis of an altered dsdC gene product--not from loss of ability to form the dsdC product. Dsdase synthesis is not regulated by the nitrogen supply in the medium, as nitrogen starvation does not result in the derepression of Dsdase synthesis.


Positive control in the D-serine deaminase system of Escherichia coli K-12. Two new types of D-serine deaminase (Dsdase)-negative mutants have been isolated and characterized. The first fails to synthesize a functional dsdC gene product as a result of dsdC- (regulator negative) mutations. The mutations lie in the dsdC region, are cis and trans recessive to dsdC+, and give rise to revertants of novel regulatory phenotype. The second class consists of Dsdase-negative lysogens in which the phenotype is the result of the integration of lambdac1857 Sam7 into the dsdC region. Lambda lysates derived from two of the Dsdase-negative lysogens can transduce the structural gene for Dsdase (dsdA) but not the dsdC region. The dsdC+ gene product had no repressor effect on constitutive synthesis in a strain containing a dsdO (initiator constitutive) and a dsdC- mutation. These and other findings indicate that control of Dsdase synthesis is strictly positive. The partial trans effect of the dsdC+ gene product on constitutive synthesis in dsdCc (regulator constitutive) strains can thus be explained by "subunit mixing" between active dsdCc subunits and dsdC+ subunits which are inactive in the absence of the inducer, D-serine. The order of genes in the dsd region is supN-dsdC-dsdP-dsdA-aroC.


Solubilization of Escherichia coli nitrate reductase by a membrane-bound protease. Nitrate reductase extracted from the membrane of Escherichia coli by alkaline heat treatment was purified to homogeneity and used to prepare specific antibody. Nitrate reductase, precipitated by this antibody from Triton extracts of the membrane, contained a third subunit not present in the purified enzyme used to prepare the antibody. Nitrate reductase precipitated by antibody from alkaline heat extracts was composed of peptide fragments of various sizes. These fragments were produced by a membrane-bound protease which was activated by alkaline pH and heat. It is the action of this protease that releases the enzyme from the membrane, as shown by the observations that protease inhibitors decreased the amount of solubilization of the enzyme, and the enzyme remaining in the membrane after heating showed much less proteolytic cleavage than that which was released.


Anaerobic cytochrome b1 in Escherichia coli: association with and regulation of nitrate reductase. Nitrate reductase solubilized from the membrane of Escherichia coli by alkaline heat treatment was purified to homogeneity and used to prepare specific antibody. Nitrate reductase, precipitated by this antibody from Triton extracts of the membrane, contained a third subunit, not present in the purified enzyme used to prepare the antibody. This third subunit was identified as the cytochrome b1 apoprotein. This cytochrome is bound to nitrate reductase from wild-type E. coli in a ratio of 2 mol of cytochrome per mol of enzyme complex. In mutants unable to synthesize heme, this cytochrome b1 apoprotein is not bound to nitrate reductase. In these same mutants, the enzyme is overproduced and accumulates in the cytoplasm. The absence of cytochrome also affects the stability of the membrane-bound form of the enzyme.


Synthesis of nitrate reductase components in chlorate-resistant mutants of Escherichia coli. Specific antibody to purified nitrate reductase from Escherichia coli was used to identify enzyme components present in mutants which lack functional nitrate reductase. chlA and B mutants contained all three subunits present in the wild-type enzyme. Different peptides with a broad range of molecular weights could be precipitated from chlCmutants, and chlE mutants contained either slightly degraded enzyme subunits or no precipitable protein. No mutants produced significant amounts of cytoplasmic enzyme. The chlA and B loci are suggested to function in the synthesis and attachment of a molybdenum-containing factor. The chlC locus is suggested to be the structural gene for nitrate reductase subunit A and chlE is suggested to be involved in the synthesis of the cytochrome b1 apoprotein.


Effects of methotrexate on intraperiplasmic and axenic growth of Bdellovibrio bacteriovorus. The intraperiplasmic growth rate and cell yield of wild-type Bdellovibrio bacteriovorus 109J, growing on Escherichia coli of normal composition as the substrate, were not markedly inhibited by 10-3 M methotrexate (4-amino-N10-methylpteroylglutamic acid). In contrast, the growth rate and cell yield of the mutant 109Ja, growing axenically in 0.5% yeast extract +0.15% peptone, were strongly inhibited by 10-4 and 10-3 M methotrexate. Thymine, thymidine, and thymidine-5'-monophosphate, in increasing order of effectiveness, partially or completely reversed the inhibition. E. coli depleted of tetrahydrofolate and having an abnormally high protein/deoxyribonucleic acid (DNA) ratio was obtained by growing it in the presence of methotrexate. B. bacteriovourus grew at a normal rate on these depleted E. coli cells but with somewhat reduced cell yield. Mexthotrexate (10-3 M) inhibited intraperiplasmic growth of bdellovibrio on the depleted E. coli somewhat more than it inhibited growth on normal E. coli, but the effects were small compared with inhibition of axenic growth of the  mutant. Total bdellovibrio DNA after growth on the depleted E. coli in the presence or absence of methotrexate exceeded the initial quanity of E. coli DNA present. Thymidine-5'-monophosphate (10-3 M) largely reversed the inhibition and increased the amount of net synthesis of DNA. The data are consistent with the prediction that intraperiplasmic growth of B. bacteriovorus should be insensitive to all metabolic inhibitors that act by specifically preventing synthesis of essential monomers. The data also indicate that B. bacteriovorus possesses thymidylate synthetase, thymidine phosphorylase, and thymidine kinase, and has the potential to carry out de novo DNA synthesis from non-DNA precursors during intraperiplasmic growth. The results also suggest that methionyl tRNAfMet is not required for initiation of protein synthesis by B. bacteriovorus.


Utilization of nucleoside monophosphates per Se for intraperiplasmic growth of Bdellovibrio bacteriovorus. During growth of Bdellovibrio bacteriovorus on Escherichia coli, there was a marked preferential use of E. coli phosphorus over exogenous orthophosphate even though the latter permeated into the intraperiplasmic space where the bdellovibrio was growing. This preferential use occurred to an equal extent for lipid phosphorus and nucleic acid phosphorus. Exogenous thymidine-5'-monophosphate competed effectively with [3H]thymine residues of E. coli as a precursor for bdellovibrio deoxyribonucleic acid; exogenous thymidine competed less effectively and thymine and uridine not at all. A mixture of exogenous nucleoside-5'-monophosphates equilibrated effectively with E. coli phosphorus as a phosphorus source for B. bacteriovorus; the nucleotide phosphorus entered preferentially into bdellovibrio nucleic acids. A comparable mixture of exogenous nucleosides plus orthophosphate had only a small effect on utilization of E. coli phosphorus by B. bacteriovorus, as did orthophosphate alone. A mixture of exogenous deoxyriboside monophosphates equilibrium effectively with E. coli phosphorus as a phosphorus source for bdellovibrio growth; the phosphorus from this source entered preferentially into deoxyribonucleic acid. These data show that nucleoside monophosphates derived from the substrate organism are utilized directly for n-cleic acid biosynthesis by B. bacteriovorus growing intraperiplasmically. As a consequence, the phosphate ester bonds preexisting in the nucleic acids of the substrate organism are conserved by the bdellovibrio, presumably lessening its energy requirement for intraperiplasmic growth. The data also suggest, but do not prove, that the phosphate ester bonds of phospholipids are also conserved.


Incorporation of long-chain fatty acids of the substrate organism by Bdellovibrio bacteriovorus during intraperiplasmic growth. Data are presented showing that a large proportion of the fatty acids of Bdellovibrio bacteriovorus grown intraperiplasmically are derived unaltered from the fatty acids of its substrate organism. Those fatty acids of the bdellovibrio not homologous with those of the substrate organism are derived mainly by metabolic alteration of preexisting fatty acids in the latter. De novo synthesis from acetate occurs only to a small extent. These characteristics of bdellovibrio physiology are in part responsible for its minimal energy expenditure for intraperiplasmic growth. The data presented also indicate that B. bacteriovorus is capable of hydrogenating unsaturated fatty acids, of beta-oxidation of fatty acids, and of regulating the proportion of saturated and unsaturated fatty acids in the lipids.


Energy efficiency of intraperiplasmic growth of Bdellovibrio bacteriovorus. The Y-ATP (energy efficiency) of intraperiplasmic growth of Bdellovibrio bacteriovorus was determined from the distribution of radioactivity of the substrate organism ([U-14C]Escherichia coli) btween CO2 and bdellovibrio cells at the end of growth. A "best" Y-ATP value of 18.5 was obtained from single growth cycle experiments and an average value of 25.9 from multicycle experiments. Both values are much higher than the usual value of 10.5 for bacteria growing in rich media. The bases for the unusual energy efficiency for growth of B. bacteriovorus are discussed.


Molecular mechanisms of accommodation in Escherichia coli to toxic levels of Cd2+. Cells of Escherichia coli strain B develop large intracellular vacuoles and exhibit an abnormally long lag phase when inoculated into a defined medium to glucose and salts containing 3 times 10-6 M Cd2+. Early in this lag, about 95% of the cells fail to form colonies when plated on nutrient broth-NaCl-agar. Prior to the initiation of proliferation, the morphology of these cells becomes normal. They regain viability in the absence of deoxyribonucleic acid replication. The rate and extent of growth are normal once proliferation begins. This reversible phenomenon of accommodation to a growth-inhibiting concentration of Cd2+ does not appear to result from a selection of mutant cells. Cells which are proliferating in the presence of Cd2+ accumulate the ion to a very high concentration. In membranes and 31% in the cytoplasm. In unaccommodated cells, the figures are 2%, 75%, and 23%, respectively. The activity of alkaline phosphatase, a zinc-metalloenzyme which is inhibited by cadmum and is located between cell wall and membrane, is not abnormally low in accommodated cells, suggesting that the cadmim is compartmentalized in these cells. Molecular sieve chromatography of cell extracts shows that the Cd2+ is associated with two classes of macromolecules. It appears that accommodation of E. coli to the presence of Cd2+ involves exclusion of the ion from the cell and reversal of damage caused by prior exposure to the ion.


Correlation between thymineless elimination and absence of hsp II (EcoRII) specificity in N-group R factors. Examination of R factors from 12 different compatibility groups shows that thymineless elimination is apparently confined to N-group plasmids that lack the hsp II (EcoRII) restriction specificity.


Role of ribonucleic acid synthesis in replication of deoxyribonucleic acid. An experiment previously interpreted to show a ribonucleic acid requirement for propagation of deoxyribonucleic replication is reexamined and the earlier interpretation is shown to be incorrect.


Procedure for isolating mutants defective in metabolite transport or utilization. Mutants defective in uptake or utilization of a given metabolite can readily be obtained from facultative auxotrophs (for that metabolite) by penicillin enrichment under nonpermissive conditions in the presence of a low level of the required metabolite.


Stability of Escherichia coli polysomes at high hydrostatic pressure. The stability of Escherichia coli polysomes at increased hydrostatic pressure was investigated in actively growing cells, in which the initiation of transcription was blocked by rifampin. In these cells, [3-H]uridine incorporation into messenger ribonucleic acid and the subsequent degradation of the message (and therefore of polysomes) by ribonuclease could be observed. Evidence is presented that the activity of the RNases is unaffected by a pressure of 680 atm, that protein synthesis is completely inhibited at 680 atm but immediately resumes at the 1 atm rate on release of pressure, and that no degradation of messenger ribonucleic acid in polysomes occurs at 680 atm. The effects of pressure; puromycin, and chloramphenicol on polysomal degradation are discussed. These results indicate that, contrary to some previous reports, polysomes are probably stabilized by high pressures. Therefore, we consider that polysomal instability is not a factor in the inhibition of protein synthesis by high pressures.


Biochemistry and genetics of Klebsiella pneumoniae mutant strains unable to fix N2. Selected mutant strains of Klebsiella pneumoniae that are unable to fix nitrogen have been characterized according to nitrogenase component activity as well as antigenic cross-reacting material. The lesions in these strains have been mapped by transduction, and the results indicate that there are at least five genes specifically responsible for nitrogen fixation in vivo. Besides genes that specify the structure of the two nitrogenase components, there is a gene for a factor that is required for component I activity and a gene that codes for a factor possibly involved in electron transport to component II. A mutation in another site does not allow the organism to produce either of the nitrogenase components. All of these genes are co-transducible with the gene that specifics the structure of histidinol dehydrogenase.


Regulation of branched-chain aminoacyl-transfer ribonucleic acid synthetases in an ilvDAC deletion strain of Escherichia coli K-12. Valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid synthetase formation was compared in isogenic strains of Escherichia coli K-12 that differed only in that one strain carried a deletion of three genes of the ilv gene cluster, ilvD, -A, and -C. It was found that: (i) the activities of these synthetases in the deletion strain were less than those in the normal strain during growth in minimal medium supplemented with excess isoleucine, valine, and leucine, and (ii) their stability was reduced in the deletion strain during specific branched-chain amino acid limitations. The results of density-labeling experiments suggest that the in vivo stability of valyl-, isoleucyl-, and leucyl-transfer ribonucleic acid synthetases requires some product missing in the ilvDAC deletion strain.


Integration of R plasmid Rts1 to the gal region of the Escherichia coli chromosome. An R plasmid Rts1 was integrated into the gal region of the chromosome of Escherichia coli XA-7012 (galE) strain by the directed transposition technique. The integration of the Rts1 genome was confirmed mainly by conjugation studies and also by transduction experiments using phage P1. As a result, it was found that the integrated genome contained genes responsible for kanamycin resistance, conjugal transferability, and for autonomous replication. As reported previously, Rts1 is temperature sensitive in replication and inhibits the growth of the host at nonpermissive temperature. However, although a plasmid derived from the integrated Rts1 genome still demonstrates temperature sensitivity upon transfer and high level of kanamycin resistance, this plasmid no longer displays temperature sensitivity in replication and the inhibitory effect on the host. These results indicate that the temperature sensitivity of replication of Rts1 and its inhibitory effect on the host cell are due to the presence of a gene or gene cluster on the Rts1 genome and that the gene(s) is clearly discriminated from the one responsible for the temperature sensitivity of transfer.


Model for the enchancement of lambde-gal integration into partially induced Mu-1 lysogens. Temperate phage Mu-1, which is able to integrate at random in its host chromosome, is also able to mediate integration of other circular deoxyribonucleic acid, as a lambda-gal mutant unable to integrate by itself. After mixed infection with lambda-gal and Mucplus, galplus transductants are recovered that have the lambda-gal integrated in any circular permutation, sandwiched between two complete Mu genomes in the same orientation, the whole Mu-lambda-gal-Mu structure being found at any location in the bacterial chromosome. Here we show that such a lambda-gal can integrate in an induced Mu lysogen. In this case the lambda-gal is again in any circular permutation, between two Mu in the same orientation, but it is always located at the site of the original Mu prophage, and the two surrounding Mu have always the same genotype as the original Mu prophage. Active Mu replication functions are not essential for that process to occur. This suggests that bacterial replication may generate two Mu copies that in some way can regenerate a Mu attachment site that recombines with the lambda-gal. A model is presented that accounts for these observations, may be helpful for understanding some complex features of Mu development, and may possibly offer a basis for explaining spontaneous duplications.


Accumulation of the capacity of initiation of deoxyribonucleic acid replication in Escherichia coli. Several temperature-sensitive initiation mutants of Escherichia coli were examined for the ability to initiate more than one round of replication after being held at nonpermissive temperature for approximately 1.5 generation equivalents. The capacity for initiation was measured by residual synthesis experiments and rate experiments under conditions where protein synthesis and ribonucleic acid synthesis were inhibited. Results of the rate and density transfer experiments suggest that the cells may initiate more than one round of replication in the absence of protein or ribonucleic acid synthesis. This contrasts with the results of the residual synthesis experiments which suggest that, under these conditions, only one round of synthesis is achieved. These findings suggest that the total amount of residual synthesis achieved in the presence of an inhibitor may be both a function of the number of initiation events which occur and the effect of the inhibitor of protein or ribonucleic acid synthesis on chain elongation.


Temperature-sensitive recA mutant of Escherichia coli K-12: deoxyribonucleic acid metabolism after ultraviolet irradiation. A mutant of Escherichia coli K-12 temperature sensitive for genetic recombination was investigated and found to carry a mutation that could be cotransduced with cysC and hence could be in the recA gene. To determine whether recA+ can complement this mutation, matings were carried out at 35 and 40 C between Hfr donors that transfer recA+ or recA1 early and recipients carrying wild-type or mutant alleles. It was found that recA+ but not recA1 complements this mutation in zygotic temporary partial diploids. The mutant allele was accordingly designated recA44. A transductant carrying recA44 behaved normally at low temperatures but more like recA- strains at high temperatures with respect to recombinant colony formation in Hfr matings, cell survival, and deoxyribonucleic acid (DNA) synthesis after ultraviolet irradiation, cellular DNA breakdown, and prophage induction when lysogenic for lambda. Alkaline sucrose sedimentation studies of DNA from recA44 cells showed that short DNA molecules synthesized immediately after ultraviolet irradiation increased in molecular weight during subsequent incubation at 32 C but not at 45 C. Hence, recA+ is required for this molecular weight increase. Cells exposed to ultraviolet light synthesized DNA that remained of low molecular weight during a 40-min incubation at 32 C. This material increased in molecular weight in recArut not in recA44 cells during subsequent incubation at 45 C. Thus, the availability of recA+ during the first 40 min at 32 C after irradiation did not obviate the need for recA+ in the subsequent phases of this post-replication repair process.


Dose dependence of the excision of ultraviolet-induced pyrimidine dimers from nuclear deoxyribonucleic acids of haploid and diploid Saccharomyces cerevisiae. The yield of ultraviolet-induced dimers is similar for a fixed dose in both haploid and diploid Saccharomyces cerevisiae. The excision of these photo-products from the nuclear deoxyribonucleic acids of cells of both ploidies after ultraviolet incident doses of 2 times 10-3 to 4 times 10-3 ergs/mm2 decreased with the corresponding increasing dose. Postirradiation incubation in saline followed by a further incubation in nutrient medium increases the excision as compared to that seen in either nutrient medium or saline alone. Previous data regarding both pyrimidine dimer removal and the survival of haploid and diploid cells after ultraviolet irradiation and either immediate or delayed plating are discussed.


Strain of Escherichia coli with a temperature-sensitive mutation affecting ribosomal ribonucleic acid accumulation. A mutant of Escherichia coli has been isolated that has a temperature-sensitive mutation that results in specific loss of ribosomal ribonucleic acid (RNA) synthesis and some reduction in messenger RNA synthesis. When the strain was grown in glucose medium at a restrictive temperature, RNA accumulation ceased, but both messenger RNA and protein synthesis continued for an extended time. Because carbon metabolism was slowed drastically when strain AA-157 was placed at the restrictive temperature, this phenotype can be compared with carbon depletion conditions present during diauxic lag. However, the phenotype of mutant AA-157 differs from shift-down conditions in that guanosine-3',5'-tetraphosphate levels are unaffected; therefore, a different site is affected. This mutant strain (AA-157) thus shows many characteristics similar to an aldolase mutant previously reported (Böck and Neidhardt, 1966). However, the mutation occurred in a different position on the E. coli genetic map, and furthermore, aldolase was not temperature sensitive in strain AA-157. In this paper we present a study of macromolecular biosynthesis in this mutant.


Adenylate energy charge in Saccharomyces cerevisiae during starvation. Bakers' yeast cells, Saccharomyces cerevisiae, if grown aerobically on ethanol or if grown aerobically on glucose and allowed to pass into stationary phase, with utilization of accumulated ethanol, maintain a normal value (0.8 to 0.9) of the adenylate energy charge during prolonged starvation. In contrast, cells grown anaerobically on glucose and cells in the early stages of aerobic growth on glucose exhibit a rapid decrease of energy charge if transferred to medium lacking on energy source. These results suggest that functional mitochondria or enzymes of balance of adenine nucleotides during starvation. Yeast cells remain viable at energy charge values below 0.1, in marked contrast to results previously obtained with Escherichia coli. In other respects, the engery charge responses of yeast to starvation and refeeding are generally similar to those previously reported for E. coli.


Bacteriophage resistance in Escherichia coli K-12: general pattern of resistance. Resistant mutants were isolated to 42 virulent bacteriophages in one strain of Escherichia coli K-12 and tested for resistance or sensitivity to a set of 56 bacteriophages. Most of the mutants fell into 11 groups with respect to their resistance patterns. It was possible to classify the bacteriophages broadly, according to the variety of mutants that were resistant to them.


Regulation of ribosomal protein synthesis in Escherichia coli B/r. The differential synthesis rate of ribosomal protein (r-protein), alpha-r (synthesis rate of r-protein divided by synthesis rate of total protein), was measured during the cell division cycle. It was observed that alpha-r remained essentially constant and was not measurably affected by duplication of the r-protein gene cluster (i.e., str-spc region) during the process of chromosome replication. It was further observed that the rate of total protein synthesis and r-protein synthesis increased continuously and uniformly during the entire cell cycle. This gene dosage independence of the synthesis rate of r-protein was similar to that observed earlier for the synthesis of ribosomal ribonucleic acid (rRNA). These observations indicate that the synthesis rates of the protein and RNA components of the ribosome are coordinately balanced during the entire cell division cycle and are not significantly perturbed by duplication of the r-protein or rRNA genes. Furthermore, this balanced synthesis insures that neither free rRNA nor free r-protein accumulate in appreciable amounts during balanced growth.


The deoxyribonucleic acid unwinding protein of Escherichia coli. Properties and functions in replication. The DNA unwinding protein of Escherichia coli (Sigal, N., Delius, H., Kornberg, T., Gefter, M., and Alberts, B. (1972) Proc. Nat. Acad. Sci. U.S.A. 69, 3537-3541) has been purified to homogeneity by a simple procedure which utilizes its stability to heating. The protein is an asymmetric tetramer of 18,500 dalton subunits which binds preferentially to single-stranded DNA at a ratio of one protein molecule per 32 nucleotides. Binding to DNA is complete in less than 10 s at 0 degrees while release of the protein from single-stranded DNA is relatively slow even at 37 degrees. A simple functional assay for unwinding protein depends on its essential role in the conversion of phage G4 single-stranded DNA to the replicative form. Unwinding protein stimulates initiation of replication of all single-stranded phage DNAs. Approximately 300 copies of unwinding protein are present per cell, as estimated by antibody titration, an amount sufficient to cover substantial lengths of DNA in several replicating forks.


Reactive half-cystine peptides of the secretory component of human exocrine immunoglobulin A. On the basis of previous work the two forms of human secretory component, namely that which is covalently bound as a part of the exocrine immunoglobulin A molecule and the free form, are probably different states of the same protein. From autoradiographs of trypic peptide maps of bound and free secretory components which were radioactively alkylated after partial reduction, it was concluded that the same half-cystines in each are sensitive to reduction. in the present work the easily reduced half-cystines of the bound and free secretory components have been studies in more detail. In each form there are two such half-cystines. In the case of bound secretory component they provide the linkage to the remainder of the exocrine immunoglobulin A molecule. Peptides from the sensitive half-cystines were isolated from tryptic-peptic digests of free secretory component and sequenced. By diagonal electrophoresis these two peptides were shown to be joined in an intrachain disulfide bridge. Therefore, it is proposed that the exocrine immunoglobulin A molecule becomes fully assembled when a single, reactive intrachain disulfide bridge in free secretory component rearranges to yeild two interchain bridges with dimeric serum-type immunoglobulin A. This process is thought to occur within the epithelial lining cells of mucous membranes.


Small ribonucleic acid molecules produced during ribosome biosynthesis in Saccharomyces cerevisiae. A 5.8 S ribosomal RNA (rRNA) is found noncovalently bound to the 25 S RNA in the 60 S subunit of yeast ribosomes. Both molecules appear to be derived from the 27 S RNA, a ribosomal precursor species (Uden, S. A., and Warner, J. R. (1972) J. Mol. biol. 65, 227-242). We have detected two other RNA species by pulse labeling which may also be produced during 27 S RNA processing. The first is approximately 5.9 S and appears by kinetics of synthesis to be a direct precursor of 5.8 S rRNA. The second species is about 7 S, is absent from uniformly labeled RNA preparations, and is produced coincidentally with the 5.8 S rRNA. Both the 5.9 S RNA and the 7 S RNA are not produced in mutants defective in ribosome biosynthesis (Hartwell, L. H., McLaughlin, C. S., and Warner, J. R. (1970) Mol. Gen. Genet. 109, 42-56) which are unable to make the 25 S-5.8 S rRNA complex from 27 S RNA. Thus, the maturation of 27 S RNA appears to involve the production of a 7 S RNA, which could be either a precursor or a nonconserved RNA region, and a 5.9 S RNA, most of which is trimmed to give the mature 5.8 S rRNA in the 25 S-5.8 S rRNA complex of 60 S subunits.


The activity of oligonucleotides containing guanosine 5'-triphosphate in protein synthesis. I. The interaction of protein synthesis elongation factor I with cytidylyl (5'-3')-guanosine 5'-triphosphate. The interaction of protein synthesis elongation factor 1 (EF-1) from wheat embryos and elongation factor Tu from Escherichia coli with cytidylyl(5'-3')guanosine 5'-triphosphate(pppGpC) has been studied. The dinucleotide 5'-triphosphate interacts strongly with EF-1 as evidenced by its capacity to inhibit the binding of [3H]GTP to the factor. The analogs pGpC and GpC do not interfere with GTP binding to EF-1 but guanosine 5'-triphosphate cyclic 2',3'-monophosphate and ppGpC are also potent inhibitors. The binding of the dinucleotide 5'-triphosphate to EF-1 was also demonstrated directly by the nitrocellulose retention method and by Sephadex G-50 fractionation using a radioactive analog iodinated with 125I in the 5 position of the cytosine of pppGpC. The dinucleotide triphosphate can replace GTP in the formation of a ternary complex EF-1-aminoacyl-tRNA-GTP and in its requirement for the binding of aminoacyl-tRNA to ribosomes catalyzed by EF-1. The absolute requirement for GTP in an in vitro polypeptide-synthesizing system can also be met by pppGpC and by guanosine 5'-triphosphate cyclic 5',3'-monophosphate. The bacterial factor EF-Tu differs drastically from eukaryotic EF-1 in its nucleotide specificity since EF-Tu only interacts slightly (if at all) with pppGpC. The low inhibition of [3H]GTP binding to EF-Tu by pppGpC could be due to a slight contamination in the latter compound.


Methylation and processing of transfer ribonucleic acid in mammalian and bacterial cells. The relationship between the methylation and processing of tRNA in both bacterial and mammalian cell systems was investigated by assessing the methylation of an existing population of precursor-tRNAs in the absence of tRNA synthesis. When the synthesis of tRNA in Escherichia coli B (rifampicin) and human KB cells (actinomycin D) was inhibited with the appropriate antibiotic, the incorporation of [3H[methyl groups into tRNA (via [methyl-3H]methionine labeling) rapidly declined with time and was essentially complete within 30 and 60 min, respectively. Although antibiotic treatment predictably reduced the incorporation of methyl groups into tRNA, it also resulted in significant changes in the distribution of the type of methylated products formed. Thus, for KB cells the marked increases in the per cent of radioactivity incorporated into 2'-0-methylribose derivatives, N2-methylguanine, and 3-methylcytosine of tRNA preparation pre-chased with actinomycin D for progressively longer periods of time prior to labeling with [methyl-3H]methionine led to the interpretation that these methylated constituents were formed predominantly during the late stages of tRNA maturation. Similarly, progessive and marked decreases in 1-, 7-, and N2, N2-methylguanine, and moderate decreases in 1-methyladenosine, 5-methylcytosine, and 5-methyluracil revealed that these methylated products were formed primarily during the early and intermediate stages of maturation, respectively. Similar analysis of E. coli B methylation products indicated that the bulk of methyl groups incorporated into the base moieties of tRNA (1- and 7-methylguanine, 2- and N6-methyladenine, and 5-methyluracil) occurred prior to the formation of 2'-0-methylribose derivatives. Additional evidence is presented which negates the possibility that an ancillary action of these antibiotics was the inhibition of specific tRNA-methyl-transferase enzymes.


Evidence for the allosteric regulation of glycogen synthesis in the intact Escherichia coli cell. Agreement of the values of the parameters of the Hill equation fitted to data for glycogen synthesis in vivo with the abailable values obtained in vitro with adenosine diphosphoglucose synthetase. In various nutrient-limited cultures of either Escherichia coli W4597(K) or G34 a 10-fold range of rates of glycogen synthesis is observed while the energy charge values (0.86 plus or minus 0.01) and glucose 6-phosphate levels are essentially the same in each condition. The steady state level of fructose 1,6-diphosphate in these cultures varies from experiment to experiment as a function of the observed rate of glycogen synthesis. These data were fitted to the Hill equation by a nonlinear regression analysis and the statistically most probable values obtained for the Hill coefficient (n), A0.5, and V were, respectively, 2.08, 0.82mM, and 1030 mumol/g of protein per hour. The values of the first two parameters agree well with values available at energy charge 0.85 for the in vitro synthesis of ADPG by the ADPG synthetase of E. coli. When the difference in the glucose 1-phosphate concentration used in the studies in vitro from the apparent glucose 1-phosphate concentration in vivo (estimated from the glucose 6-phosphate levels) is considered, the in vitro value of V (1140 mumol of ADPG synthesized per g of protein per hour) is quite similar to the value of V (1030 mumol of glucose incorporated into glycogen per g of protein per hour) for glycogen synthesis in vivo. The close agreement of the values of the parameters of the Hill equation for glycogen synthesis in vivo to the values obtained for ADPG synthesis in vitro provides the most quantitative evidence yet obtained that allosteric regulation of bacterial glycogen synthesis functions in vivo.


Cleavage of Nonglucosylated Bacteriophage T4 deoxyribonucleic acid by Restriction Endonuclease Eco RI. DNAs lacking the glucosyl modification (Glc-) and additionally lacking the 6-methylaminopurine (N6-methyladenine) modification (Glc-, MeAde-) were prepared from appropriate T4 mutants. These DNAs were cleaved by the purified restriction endonuclease Eco TI from Escherichia coli. Normally modified DNA (Glc+, MeAde+) was not attached. The Eco RII and the hemophilus enzymes Hin dII and Hin dIII do not attack Glc-, MeAde- T DNA, possibly due to the presence of 6-hydroxymethylcytosine. Eco RI produces approximately 40 specific fragments from Glc- DNA ranging in molecular weights from 0.3 to 10.5 X 10-6.


Escherichia coli ribosomal ribonucleic acids are not cut from an intact precursor molecule. Ribonuclease III-deficient strains of Escherichia coli accumulate a "30 S" RNA species. Kinetic analysis of label incorporated into rRNA species shows, however, that 30 S RNA is not the major precursor to the 16 S and 23 S RNA species is in 7- to 10-fold excess over that into 30 S RNA. The 30 S RNA species turns over with a half-life of about 2.5 min, which could account for no more than one-tenth of the incorporation into 16 S plus 23 S RNA. Thus, under these conditions RNase III-strains of E. coli do not cut rRNAs from an intact tandem precursor molecule but rather from the elongating nascent transcript, possibly by an alternate pathway not involving RNase III.


Observations on the induction of bone in soft tissues. Using bone decalcified with 0-6 N hydrochloric acid as an inducing agent, the inductive capacity of different soft tissue sites was investigated. Muscle and fascia regularly permitted the induction of bone, while spleen, liver and kidney suppressed bone induction. Bone formation could be induced in these organs if living autologous fascia was implanted together with the inducing agent; while bone formation was inhibited when living autologous spleen tissue was implanted with the inducing agent to normally favourable sites. The administration of systemic heparin and the diphosphonate ethane-1-hydroxyl, 1-diphosphonic acid (EHDP) suppressed bone induction. It is suggested that for bone induction to occur in soft tissues, three conditions must be present: 1) an inducing agent; 2) an osteogenic precursor cell; and 3) an environment which is permissive to osteogenesis. The presence of osteogenic inhibitors in spleen, liver and kidney is postulated.


Posterior psoas transfer and hip instability in lumbar myelomeningocele. Seventy-two posterior psoas transfer operations performed in forty-four children with lumbar myelomeningocele were reviewed one to eight years after operation in an attempt to assess its value. Muscle charting, an objective recording of the child's walking ability, and radiographic examination of the hips were done. Hip stability was improved: 49 per cent were stable at the time of psoas transfer and 94 per cent at review. Functional results depend mainly on the level of neurological activity present: 57 per cent of the children had an acceptable functional result. Usually, posterior psoas transfer should be done as soon after the age of nine months as the child's condition will allow. Over the age of two years it should be restricted to children with activity in the third and fourth segments of the lumbar cord.


The basal apparatus. Mass isolation from the molluscan ciliated gill epithelium and a preliminary characterization of striated rootlets. The basal apparatus, consisting of an array of interconnected basal bodies bearing bifurcating striated rootlets encompassing a nucleus, has been isolated from hypertonically deciliated columnar gill epithelial cells of the bay scallop Aequipecten irradians through gentle lysis with Triton X-100. The rootlets, 8-10 mum in length, were not easily preserved with conventional electron microscope fixatives, suggesting that the extent of their contribution to cellular architecture has been somewhat underestimated, even though Englemann described many of the structural details of the basal apparatus in 1880. The striated rootlets were soluble at high but not at low pH, in 2 M solutions of sodium azide and potassium thiocyanate but not sodium or potassium chloride, in 1% deoxycholate but not digitonin, and in the denaturing solvents 6 M guanidine-HC1, 8 M urea, and 1% sodium dodecylsulfate at 100 degrees C. The protein found consistently when rootlets were solubilized migrated on SDS-polyacrylamide gels as a closely spaced doublet with apparent molecular weights of 230,000 and 250,000 daltons. This unique protein, distinct from tropocollagen or various muscle components, has been named ankyrin because of the rootlet's anchor-like function in the cell.


Messenger RNA metabolism of animal cells. Possible involvement of untranslated sequences and mRNA-associated proteins. The past several years have seen a virtual revolution in the study of eukaryotic mRNA. Among the notable recent achievements are the positive identification of mRNA precursors in HnRNA, the enumeration of the DNA sequences from which mRNA is transcribed, and the finding that mRNA in cultured cells is much more stable than was previously believed. One of most far-reaching discoveries has been the finding that mRNA in eukaryotes contains poly A. This discovery, aside from providing a powerful tool for mRNA isolation, has generated a large body of research into the properties and metabolism of poly A itself. In addition, the finding of a poly A-associated protein has given a renewed stimulus to the study of proteins associated with mRNA. This review is devoted to a discussion of these and related achievements, and some of their implications


Mitosis in the cellular slime mold Polysphondylium violaceum. Myxamebas of Polysphondylium violaceum were grown in liquid medium and processed for electron microscopy. Mitosis is characterized by a persistent nuclear envelope, ring-shaped extranuclear spindle pole bodies (SPBs), a central spindle spatially separated from the chromosomal microtubules, well-differentiated kinetochores, and dispersion of the nucleoli. SPBs originate from the division, during prophase, of an electron-opaque body associated with the interphase nucleus. The nuclear nevelope becomes fenestrated in their vicinity, allowing the build-up of the intranuclear, central spindle and chromosomal microtubules as the SPBs migrate to opposite poles. At metaphase the chromosomes are in amphitelic orientation, each sister chromatid being directly connected to the corresponding SPB by a single microtubule. During ana- and telophase the central spindle elongates, the daughter chromosomes approach the SPBs, and the nucleus constricts in the equatorial region. The cytoplasm cleaves by furrowing in late telophase, which is in other respects characterized by a re-establishment of the interphase condition. Spindle elongation and poleward movement of chromosomes are discussed in relation to hypotheses of the mechanism of mitosis.


Ultrastructural immunoperoxidase demonstration of autologous albumin in the alveolar capillary membrane and in the alveolar lining material in normal rats. The location of autologous serum albumin within the alveolar-capillary membrane was studied in the rat under physiological conditions using antialbumin antibodies labeled with peroxidase. Albumin was detected in the lung interstitium, and in numerous pinocytic vesicles within endothelial cells and type I alveolar epithelial cells. The immunoreaction was also positive at the level of plasmalemmal membranes of both cell types and in the alveolar lining material.


Thermal chromatography of lysine-specific transfer ribonucleic acid from Escherichia coli B. Two Escherichia coli B tRNALys isoacceptors have been separated by thermal chromatography on hydroxyapatite demonstrating the potential of this approach in the fractionation of single-stranded polynucleotides. Elution temperatures were reproducible and depended primarily on the cation present and its concentration. Cesium phosphate was a more effective eluent than sodium phosphate. The integrity of the polynucleotide following thermal chromatography was examined by ultracentrifugation in a 60% dimethyl sulfoxide solvent, electrophoresis on polyacrylamide gels containing urea and oligonucleotide mapping. Adsorption to hydroxyapatite did not noticeably increase phosphodiester bond breakage compared with that in solution. Minimal, if any, rupture of covalent linkages was detected under the conditions of preparative separation of the isoacceptors. Several observations suggest that only one tRNALys was transcribed by the E. coli B cell.


Assessment of pituitary gonadotropin reserve using luteinizing hormone-releasing hormone (LRH) in states of altered thyroid function. Menstrual irregularities commonly accompany hyper- and hypofunction of the thyroid gland. Investigation of the pituitary-ovarian axis was undertaken in 14 hyperthyroid and 8 hypothyroid patients. The pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) responses to 100 mug luteinizing hormone-releasing hormone (LRH) was assessed. These responses were found to be similar to matched control groups in hyperthyroid premenopausal women with and without menstrual disturbances and in hyperthyroid men. The postmenopausal hyperthyroid women had an exaggerated LH response, although the FSH response was comparable to that of the control group. These results indicate normal pituitary LH and FSH reserve in hyperthyroidism. In hypothyroidism a gradation of gonadotropin response to LRH was found. In 2 of the patients there was an inadequate LH response in the presence of a normal FSH response. This demonstrates that in some cases of primary hypothyroidism there is limited pituitary LH reserve.


Release of colony-stimulating activity from thymus-derived lymphocytes. Colony-stimulating activity (CSA) is essential for in vitro differentiation of bone marrow cells into colonies of granulocytes and mononuclear cells. While blood monocytes and macrophages are a major source of CSA, recent studies have indicated that CSA may be produced by lymphocytes responding to immunologic stimulation. Lymphocytes, purified from spleens and thymuses of mice by glass wool columns, were incubated in CMRL-1066 medium with fetal calf serum in vitro. Lymphocytes from the thumus and spleen released CSA when cultured in vitro, with peak levels of CSA observed after 7 days of incubation. Stimulation of cultures with phytohemagglutinin, concanavalin A, or pokeweed mitogen resulted in a 2-5-fold increase in CSA release, with peak levels of CSA released after 4 days of incubation. Thymus-dependent lymphocytes were responsible for the release of CSA from unstimulated and mitogen-stimulated cultures, since the incubation of these cultures with rabbit anti-mouse T cell sera abolished their ability to release CSA. Anti-mouse B cell sera had no effect on the ability of lymphocyte cultures to release CSA. These studies suggest that thymocytes and thymus-derived lymphocytes can release CSA in vitro and may be responsible for the increase in CSA observed in certain immunologic reactions.