Structural and ultrastructural alterations, exfoliative cytology and enzyme cytochemistry and histochemistry, proliferation kinetics, immunological derangements and other causes, and clinical associations and sequallae. Our main purpose in this review article is to present some of the vast amount of information on the subject of atrophic gastritis available at the present time. Various areas of research pertaining to this entity, including that from the author's laboratory, are reviewed in this article, encompassing histological and ultrastructural abnormalities, enzyme histochemistry, and exfoliative cytology and cytochemistry of chronic gastritis, with emphasis on the diagnostic significance of these laboratory methods. The altered cell proliferation kinetics in atrophic gastric lesions is also discussed, as well as its significance in the proliferation and reduction of glandular cells of the gastric mucosa. Furthermore, some of the more recent evidence of immunological mechanisms and other causes of chronic gastritis, such as chronic alcoholism and bile reflux, is reviewed, as well as the effects of partial gastrectomy on the gastric mucosa. The important associations and sequellae of atrophic gastritis, such as gastric ulcer, gastric polyps, and gastric cancer, are discussed in detail.


Education and training in electron microscopy. There is currently a need for diagnostic electron microscopy in both autopsy and surgical pathology. As more information emerges from the research laboratories, applied electron microscopy will grow in depth, and all large medical centers will need the expertise provided by a diagnostic electron microscopy laboratory. Many physicians other than electron microscopists find it essential to have an understanding of the contributions and limitations of electron microscopy. Education and training programs therefore must encompass not only paramedical personnel who prepare the electron micrographs and pathologists who are thoroughly trained in interpret the electron micrographs, but also other physicians and scientists who utilize the information obtained therefrom. Medical students should obtain sufficient background in normal and abnormal ultrastructure to enable them to interpret the medical literature, and the means to obtain this background should be available within the medical school curriculum. Some medical students will also desire more thorough training obtained by elective courses in electron microscopy. Pathology residents should obtain sufficient expertise during their residency to enable them to utilize the information produced by the electron microscopy laboratory. Certain pathology residents and some pathologists in practice prefer fellowships for more specialized training in electron microscopy. Four representative training programs in electron microscopy from Veterans Administration hospitals have been selected for presentation. Each emphasizes a different approach and different objectives.


Porokeratosis [correction of Parakeratosis] Mibelli and skin carcinoma: a critical review. The literature on porokeratosis [corrected] Mibelli (PM) is revised critically with respect to the development of skin carcinoma. The incidence of epidermal malignancies in PM lesions is 7% (17 of 250 cases). An influence of X-ray treatment is not excluded in 4 cases with tumors in PM.


Information processing in immunogenetic analysis. V. Some irregularities caused by simple-complex transformation of T universes. A fairly uncomplicated T4 universe does give rise to a considerably complicated simple-complex image with several complexities regularly observed together in many immunogenetic systems. The present model thus 'explains' absence of antithetical alleles, absence of 'dosage' effects, presence of considerable quantitative variations of 'one and the same antigen' and peculiar 'non-autoantibodies'. These phenomena are exemplified for the Rh system with regard to the D--Du antigens, absence of d antigens, absence of clear-cut dosage between D/D and D/D and D/d samples as well as anti-D formation in seemingly 'normal Rh(+)' individuals.


In vivo effects of rabbit anti-mouse brain serum on theta bearing lymphocytes of AKR mice. The in vivo effect of rabbit anti-AKR mouse brain-associated serum (RAMB) was determined on theta bearing lymphocytes present in the spleens and thymuses of mature C3H mice and AKR mice staged into preleukemic, leukaemic and overtly leukaemic states. Following seven daily injections of RAMB serum, the splenic plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) and the percentage of theta-bearing lymphocytes in the spleen were significantly decreased in the C3H and the preleukaemic AKR mice.  Decreases in thymic weight and thymocyte numbers were also apparent.  Determinations of theta antigen density using in vitro cytoxicity tests indicated that splenic and thymic T lymphocytes (thymus-derived) remaining in the RAMB-treated C3H and preleukemic AKR mice consisted primarily of cells bearing less of the theta surface antigen.  Histopathological studies of tissues from these two treated groups revealed cortical lymphocyte depletion in the thymus, and marginal and periarteriolar depletion in the spleen.  Leukaemic AKR mice, administered seven injections of RAMB serum, demonstrated less dramatic changes in thymus weight, histopathology and theta-bearing cell percentages when compared with the data from the preleukaemic AKR or matureC3H mice.  The results from testing overtly luekaemic AKR mice administered RAMB serum for the 7 or 9 days did not demonstrate differences from findings from groupsof overtly leukaemic control mice.  These data indicate that the in vivo activity of RAMB serum in C3H mice and preleukaemic AKR mice is directed primarily toward the less mature T-lymphocyte population.  This influence of RAMB serum is lesspronounced in leukaemic and the more overtly leukaemic mice, suggesting that a decreasedpopulation of RAMB-susceptible lymphocytes are present in these animals.


Passive transfer of unresponsiveness by lymph node cells.  Studies on adjuvant disease. Pretreatment of rats with subarthritogenic doses of mycobacterial adjuvant suppresses the arthritogenic reaction induced by this adjuvant.  This unresponsiveness can betransferred to normal syngeneic recipients by viable lymph node cells from unresponsivedonors.  Serum from the same donors did not prevent the arthritis.  Abrogation ofunresponsiveness was obtained by injecting normal lymph node cells together with the challeging inoculation and also with sensitized lymphoid cells.  In agreement with the concept of tolerance as an active immune reaction, the data suggest that in this experimental model lymphoid cells actively suppress the immune response induced by microbacterial adjuvant.  Thus, arthritis or unresponsiveness may result from the predominance of either sensitized or suppressor cells.


Complement receptor lymphocytes.  Analysis of immunoglobin on their surface and further evidence of heterogeneity. Mouse complement receptor lymphocytes (CRL) were detected by a rosetting method. The existence of an immunoglobulin class or subclass on the central rosetting cell wasstudied by immunofluorescence using rabbit antisera specific for mouse IgM, IgG1, IgG2a, IgG2b and IgA.  Combining immunofluorescence for each immunoglobulin and for T cells with cytoxic removal of T cells, CRL were demonstrated to include immunoglobulin-bearing cells of all the classes and subclasses tested (IgM being the main one), a 'null' cell subpopulation, and some T cells.  A scheme describingthe various nature of CRL is discussed and correlated with possible functions.


Electrophoretic mobility and surface immunoglobin of albumin gradient fractionated mouse spleen cells. Spleen cells from normal CBA mice, containing B and T lymphocytes, cyclophosphamide-treated CBA mice, containing almost exclusively T lymphocytes, and athymic nudemice, containing only B lymphocytes, were fracitonated by differential flotation in adiscontinuous albumin gradient.  In all three cases, four density fractions were regularly obtained.  The electrophoretic mobility (EPM), which allows distinction betweenslow-EPM (B) cells and fast-EPM (T) cells and the presence of surface immunoglobulins (sIg) detectable by direct immunofluorescence, characterizing B lymphocytes, were investigated on these fractions.  Both B and T cells were recovered throughout the gradient, but in different proportions.  Thus, B cells (slow-EPM, sIg-bearing) were enriched in the light density fractions while T cells (fast-EPM)were more numerous in the denser fraction.  The mean EPM of slow-moving cells decreased, whereas that of fast-moving cells increased, as their buoyant density increased.  Less nude spleen (B) cells were found to bear sIg in the light density fractions than in the denser fractions.  These findings suggest the existence of lymphocyte subpopulations with distinct physicochemical properties which might represent stages in the maturation and differentiation of B- and T-cell lineages.


Heat-stable Escherichia coli enterotoxin production in vivo. Hysterectomy-derived, colostrum-deprived piglets were infected with enterotoxigenic Escherichia coli on day 4 of life. Samples of feces and intestinal contents were collected and tested in infant mice for enterotoxic activity. Positive enterotoxic responses were observed in mice given filtrates of feces and intestinal contents from piglets infected withe enterotoxigenic E. coli known to produce heat-stable enterotoxin but not heat-liabile enterotoxin in vitro. It is concluded that heat-stable enterotoxigenic E. coli induce diarrhea by production of heat-stable enterotoxin in vivo.


Cellular location of alpha-hemolysin in Staphylococcus aureus. Protoplast fractionation techniques were used to prepare subcellular fractions of three strains of Staphylococcus aureus for the analysis of alpha-hemolysin activity. More than 90% of the cellular hemolytic activity was localized in the periplasm, only a trace was detected in the cytoplasm, and none was found in either plasma membranes or mesosomes. This cellular portion constituted approximately 4 to 10% of the total hemolytic activity of the culture. The erythrocyte lysis spectrum and Sephadex G-100 filtration studies indicated that the cellular hemolysin closely resembles the extracellular form.


Prevention of Streptococcus mutans infection of tooth surfaces by salivary antibody in Irus monkeys (Macaca fascicularis). Four irus monkeys (Macaca fascicularis) were immunized with Streptococcus mutans 6715 killed cells and cell products by injection in the vicinity of the major salivary glands and by instillation into the parotid glands via the ducts. After immune group and a sham-immunized control group of monkeys were infected orally with viable strain 6715 organisms. Dental plaque samples were taken at intervals from the buccal and lingual grooves of the first permanent molars. These samples were evaluated for recovery of strain 6715 by cultural methods. In addition, individual samples were taken from 10 representative tooth surfaces and were evaluated by indirect immunofluorescent staining for strain 6715. Results showed that immune monkeys had fewer infected surfaces and fewer organisms on the infected surfaces than the control animals. These studies indicate that salivary antibody to cariogenic streptococci inhibits implantation of these organisms in dental plaque and may be protective against dental caries.


Antibody-mediated elimination of malaria parasites (plasmodium berghei) in vivo. An infective preparation of extracellular blood forms (FP) of Plasmodium berghei was used to study some aspects of the interaction between protective antibodies and malaria parasites. FP but not infected erythrocytes (IRBC) were shown by the fluorescent antibody technique to be coated by antibodies after in vitro incubation with immune serum. Preincubation of both FP and IRBC with immune serum followed by their washing did not result in enhanced elimination of the parasites in vivo. However, FP preincubated with immune serum and subsequently washed were eliminated more efficiently than FP preincubated with normal serum if the preparations were injected with some immune serum. Such an increase in the efficiency of elimination was not detected with similarly pretreated IRBC. It is thus probable that protective antibodies acted in vivo against extracellular parasites rather than against parasites in erythrocytes. The interaction between parasites and antibodies may be of a highly reversible nature, and washing of the in vitro-treated parasites may cause elution of antibody from the sensitized parasites so that the amount of antibody on the parasite falls below the critical level required for in vivo elimination.


Days beyond recall: subsistence homesteading in the rural south, circa 1920. Accented in some 100 semi-structured interviews with Southern aged poor is the bitter struggle to survive. People worked long hours for little, bartering farm produce for the few store-bought necessities. Primitive housing, education and medicine sufficed. Both black and white poor suffered brutal exploitation, despite which they review their life with satisfaction. We hypothesize that, to maintain mental health, it is better to idealize the unalterable past. Several global assessments of "morale" are discussed. It is also suggested that this journal become a clearing house for questions that measure psychic wellbeing in the aged more naturally and by indirection than do scales in current use.


Model of an oculomotor subsystem.

Model of an oculomotor subsystem. One of the complex control systems present in the human body is the oculomotor control system. There are two major modes of ocular movements, viz. saccadic and smooth pursuit. More knowledge about the neuromuscular mechanisms controlling these eye movements is obtained if experiments are carried out on subjects whose two types of eye movements are differentially affected due to disorders at their higher centres of control. Eye movements of patients with a novel form of Heredo-familial Spinocerebellar Degeneration have been measured and quantified. These have been compared with those of normal subjects. Subsequent analysis indicates a continuous fourth order transfer function for the smooth pursuit eye-in-skull movements in the horizontal plane with head stationary.


An algorithm for on-line, real-time computer detection of ECG changes. An algorithm for detection of ECG changes based on the Fourier transform is presented. The realisation of the algorithm was programmed on a minicomputer and could detect both large and subtle changes in an ECG waveform. The cardiac arrhythmias used in the study included normal sinus rhythm; premature atrial contraction; premature ventricular contraction; first, second, and third degree blocks; atrial and ventricular fibrillation and ventricular tachycardia. The algorithm was used to detect rhythm changes, but it is possible that, using it, a unique specification for a waveform at any frequency could be obtained.


Temperature-sensitive mutants of murine leukemia virus. V. Impaired leukemogenic activity in vivo. The in vivo biologic activities of a clonal isolate of the Rauscher strain of murine leukemia virus and several conditional lethal mutants with impaired replicative functions at the non-permissive temperature were investigated. The parental virus induced a rapidly developing lymphoid leukemia in a high percentage of NIH Swiss mice inoculated as newborns. Mice inoculated in parallel with temperature-sensitive mutant viruses showed no evidence of disease. The implications of these findings with regard to the requirements for type-C virus replication to permit oncogenic transformation of lymphoid cells in vivo are discussed.


Comparative clinical study of doxycycline and minocycline. Doxycycline and minocycline were given intravenously to patients with a serious underlying disease who presented a pulmonary infection or a wound infection. Both therapies were easy to administer and well tolerated. However they should not be used in the initial therapy since resistant strains may occur. The clinical and bacteriological effectiveness of doxycycline and minocycline were similar. In pneumococcal pulmonary infections, the rates of favorable clinical response to doxycycline and minocycline were 73% and 76% respectively. In infections caused by bacteroides sp.(mainly infections of wounds), doxycycline or minocycline resulted in a 75% rate of favorable clinical responses and in a 71% rate of favorable bacteriological responses. Adverse side effects were rare and of minor importance with the exception of bacteriological colonization by doxycycline-resistant or minocycline-resistant microorganisms. This complica-tion occurred in 39% of the patients who were treated with these drugs and resulted in clinical superinfection in 8% of the patients.


[The influence of Adipex Neu (beta-aminoisobutylbenzene as polysterol sulfonate) on lipolysis and glucose tolerance in humans]. In 16 obese patients, the effect of beta-amino-isobutyl-benzol as polysterol-sulfonate on free fatty acids, triglycerides and i.v. GTT was compaired with that of a placebo. The question was to be answered if beta-amino-isobutyl-benzol worsens the GTT and if the lipolytic effect outranges lipolysis during fasting. No statistically significant change of the GTT, free fatty acids and triglycerides could be found between the beta-amino-isobutyl-benzol period and the placebo period.


Double-blind study of a total liver extract in patients with hepatic dysfunction. Forty hospitalized male patients with hepatic functional deficit were treated i.m. for 3 weeks with a total liver extract or placebo. The study was a double-blind. Statistical analysis (Student's t test, analysis of variance. Mann-Whitney test and Fisher's probability test) showed that liver extract therapy was more effective than placebo in improving liver function as indicated by changes in clinical features(digestive disorders, constipation, hepatomegaly) and colloidal and other laboratory tests, e.g. plasma cholesterol, prothrombin time, red cell count and hemoglobin concentration. An overall clinical assessment showed that 60% of patients treated with the liver extract were improved. In addition, no local or general side effects were observed. Present results are in favour of an hepatoprotective activity of a total liver extract.


Herpetiform pemphigus, a variable pattern of pemphigus. A  patient with the clinical features of dermatitis herpetiformis and the histological features of pemphigus, subcorneal dermatosis and dermatitis herpetiformis did not respond to treatment with pyridine or sulfones. Systemic steroids and azothioprine were effective. Immunofluorescence studies demonstrated circulating pemphigus antibodies and IgG bound in vivo in the intercellular spaces of the epidermis.


Development of the upper urinary tract in myelomeningocele and consequent urological care. The findings in the upper urinary tract of 58 patients with MMC and neurogenic bladder dysfunction are discussed and therapeutical consequences are considered. In a group of 28 neonates, investigated within the first three months of life, only 10% showed some abnormalities of the upper urinary tract and (or) reflux, in a second group of 21 children, over the age of 2 years when first seen, the occurrence rate of dilatation of the upper urinary tract and of pyelonephritis signs was already doubled, whereas adults with operated MMC showed in 75% severe pathological changes. The situation of neonates born with MMC may be in some aspects compared to patients suffering from acute traumatic cord lesion with neurogenic bladder dysfunction. It was already shown that, in spite of neurogenic bladder dysfunction, it is possible in parapegics by appropiate and mainly conservative treatment to prevent progressive destruction of the upper urinary tract, once considered to be unavoidable. The key to better results in the children with MMC should therefore be early and prompt care and close follow-up. The importance of regular bladder emptying, low dosage, long-term antibiotic treatment and measures like forced urethral dilatation and sphincterotomy is stressed. Our results so far in neonatal urological care of children with MMC are encouraging.


Pyleonphritis as a side effect of hormonal contraception: an experimental research. In animal experiments it was proved that contraceptives may cause considerable renal changes. It is necessary to call gynecologists' attention to possible side effects; the contrapaceptives are contra-indicated in chronic pyelonephritis.


Experimental heterotopic bone transplant. We studied the reaction in rats to implants of heterotopic bone in the paravertebral region, using three types of material: bone preserved in alcohol, homologous bone preserved in alcohol associated with autogenous cancellous bone obtained from the iliac crest of the same animal, and isolated autogenous cancellous bone. Thirty rats were operated on, ten in each group. Some were sacrificed after 6, 14 and 21 days. The excised grafts were histologically studied. Animals in the third group showed new bone formation even in the first days, those in the second group presented less intense bone formation and those in the first group showed only traces of new bone tissue in the third week.


Optic nerve transection in cats: effect on retinal vessels. Unilateral optic nerve transection without damage to the intraocular circulation was performed on thirteen cats. Fluorescein angiograms, trypsin digestion, and histologic preparation of the retinas were carried out. No changes in the retinal circulation and angioarchitecture were observed. These findings were compared to those reported in humans with comparable optic nerve lesions. We conclude that optic nerve transection does not cause retinal vascular alteration and this fact may be of pertinence to posterior ocular damage in glaucoma.


[Keratohyaline and epidermal keratihization]. A review is given about the present status of keratohyalin, his composition and his origin and also about "prekeratin". The suggestions about the role of keratohyalin and prekeratin in the synthesis of epidermal horny layer are discussed.


Comparison of the reaction of cultured smooth and cardiac muscle cells and fibroblasts to specific antibodies to myosin. Immunofluorescent staining with anti-smooth or anti-striated muscle myosin was carried out for 30 minutes at room temperature (18-20 degrees C) on cultures of smooth muscle cells and fibroblasts from guinea-pig vas deferens, taenia coli and ureter, rabbit aorta and chicken gizzard and of cardiac muscle cells and fibroblasts from rat ventricle. With anti-smooth muscle myosin, smooth muscle cells showed an intense fluorescent staining in fine fibrils with an "interrupted" appearance running parallel to the longitudinal axis of the cell throughout the cytoplasm, and also in coarser, "non-interrupted" fibrils (termed here "attachment fibrils") concentrated at the surface of the cell adjacent to the glass coverslip. Fibroblasts in the same cultures showed similar, but much weaker, reactions. When anti-striated myosin was added to the smooth muscle cultures, staining of neither cell type was observed. In contrast, cardiac muscle cells in cultures of rat ventricle did not react anti-smooth muscle myosin, but gave bright fluorescent A-band staining with anti-striated myosin. Fibroblasts in the ventricle cultures were unreactive with anti-striated muscle myosin but gave the characteristic weak reaction with anti-smooth muscle myosin. Thus immunofluorescent stainig with anti-smooth muscle myosin is useful for distinguishing between isolated smooth muscle cells and fibroblasts in tissue culture.


Identification of the intestinal cell storing gastric inhibitory peptide. Small intestinal mucosal samples from man, pig and dog, were subjected to sequential or correlative silver impregnation techniques, applied to immunocytochemical preparations and at the ultrastructural level. The cell reacting with anti-GIP sera was identified as the ultrastructurally classified K cell and we propose that the term GIP cell be used in place of the latter. This cell can thus be recognized by its strong reactivity with the Sevier-Munger staining procedure, provided that the equally strongly reacting EC cell is excluded by virtue of its argentaffinity with the Masson technique.


A field study of coliform mastitis in sows. Three sows with agalactia and mastitis were compared with 3 normally lactating sows at 8, 16, 24, and 32 hours postpartum. Bacteriologic examination of milk resulted in isolation of Klebsiella pneumoniae or Escherichia coli from affected glands of all 3 agalactic sows. Milk from clinically normal glands of affected sows and from glands of normal sows was culture-negative for coliforms. Affected sows had higher rectal temperatures and lower milk yields, and pigs from affected sows had lower mean weights, but these differences were not significant. Mean total white blood cell counts and plasma protein to fibrinogen ratios were lower in mastitic sows at all 4 periods; differences were significant at certain periods in both cases. Evidence indicated that leukopenia appeared early, 8 to 16 hours postpartum, and was followed by increasing plasma fibrinogen content. Packed cell volumes and plasma concentrations of corticoid, estrone, and estradiol did not differ. Plasma progesterone content was higher in mastitic sows at all periods, but the differences were not significant.


Bovine infectious keratoconjunctivitis: carrier state of Moraxella bovis and the development of preventive measures against disease. Bovine infectious keratoconjunctivitis, which is caused by Moraxella bovis, occurs perennially in all cattle-raising areas. Disease may occur any time during the year but manifests itself often during summer when disease-enhancing factors such as ultraviolet radiation and fly population are increased. Although some recent research findings indicate that cattle can be immunized against the disease, many problems have to be overcome before the cattle industry can benefit from such immunization. Some pressing problems relate to lack of cross-protection between vaccines of different strains of M bovis, resistant or resilient carrier states, and how these factors affect vaccination regimens undertaken. Though cattle vaccinated with 1 strain are somewhat protected against subsequent challange by the same strain, protection against heterologous strains has not been demonstrated. In most herds, cattle harbor M bovis, and these carriers may serve as a source of infection.


Antibiotic A-16316-C, a new water-soluble basic antibiotic. A new water-soluble basic antibiotic named antibiotic A-16316-C was isolated together with antibiotic A-396-I and hygromycin B from a streptomyces strain identified as Streptoverticullium eurocidicus.  The properties of the antibiotic A-16316-C were similar to those of destomycin B.  But it was found that the antibiotic A-16316-C was not identical with destomycin B on the basis of NMR analysis.  On acidic degradation antibiotic A-16136-C gave N, N'-dimethyl-2-deoxystreptamine, destomic acid and D-mannose.  The gross structure for antibiotic A-16136-C was deduced from chemical reactions and spectral data.


Inhibitors of polypeptide elongation on yeast polysomes. Yeast polysomes are very active for amino acid incorporation when supplemented with elongation factors and the different components required for elongation of the polypeptide chain. This polysomal system is suitable for the study of the individual streps of the elongation cycle and to test the effect of different inhibitors. Anisomycin, trichodermin, trichodermol, trichothecin, fusarenon X, sparsomycin and blasticidin S inhibit peptide bond formation on these polysomes, whereas diphtheria toxin, pederine, cycloheximide and cryptopleurine block translocation.


Surfactant inactivation by hyperventilation: conservation by end-expiratory pressure. Hyperventilation, defined as repeated hyperinflations, for three hours in open-chested anesthetized cats increased elastic recoil and elevated minimum surface tension of lung extracts as measured on a surface film balance. Equivalent hyperventilation from an elevated lung volume did not alter the pressure-volume relationships. When a mixture of [3H]glycerol and [14C]palmitate had been injected 17 h before the three hour period of phyerventilation, an increase in the ratio of specific activity in wash to tissue lecithin occurred in the hyperventilated cats compared to controls. These findings suggest that hyperventilation promotes release of surface active material from tissue to alveolus, but the released material is inactivated. The application of 2.5 cmH2O positive end-expiratory pressure prevented the adverse effects of hyperventilation. The same increase in wash to tissue lecithin occurred during this study; since the material was appropriately surface active, we conclude that the positive end-expiratory pressure prevented its inactivation.


A new method for calibrating indicator dilution curves. A technique is proposed and experiments carried out to validate an in vivo method of calibrating ascorbate dilution curves. Cardiac output measured by the proposed method (COi) was compared to cardiac output measured by electromagnetic flowmeter (COe). COe averaged 1,281 mg/min and COi averaged 1,284 ml/min (P greater than 0.90). Correlation between the two methods was 0.92. It is concluded that the proposed method measures CO with reasonable accuracy and provides a number of advantages over the usual method of measuring blood flow by means of indicators.


Fluorescent antibody method for the detection of Salmonella: development, evaluation, and collaborative study. In preliminary studies, several commercial polyvalent fluorescent antibody (FA) preparations were evaluated for specificity and crossreactivity and an FA method was developed for the screening of Salmonella in products. Approximately 4000 product samples were tested by the FA method and the results were compared to those from the official final action AOAC method, 46.013-46.026. Only 4 FA false-negatives were found for a total of 619 confirmed positive Salmonella samples. The FA false-positive rate was 7%. The method was then subjected to a 2-phase collaborative study. In Phase I, 22 analysts tested 5 inoculated and 5 uninoculated samples of dried milk. In Phase II, 5 naturally contaminated and 5 presumably uncontaminated foods were analyzed. The study was designed to compare results from 11 analysts experienced in FA methodology with those from 11 analysts with little or no experience. Selenite cystine (SC) and tetrathionate (TT) broths were used for enrichment and both were inoculated into SC for post-enrichment. All 4 combinations (SC, TT, SC-SC, and TT-SC) were used with the FA method to determine the best technique. Results were compared to the analysis with TT and SC by the AOAC culture method. In all studies, FA analysis with SC-SC gave the highest correlation with the AOAC method. In a total of 200 samples, the experienced group found 125 AOAC positives and 127 FA positives; no FA false-negatives and only 2 false-positives were reported. The inexperienced group reported 9 FA false-negatives and 5 FA false-positives. All false-negatives occurred in only 3 of the inexperienced laboratories. These studies showed that enrichment and post-enrichment in SC gave the best FA results and that training in FA methodology is required for correlation with existing AOAC methodology. The FA method for the detection of Salmonella has been adopted as official first action.


Transport properties of merodiploids covering the dagA locus in Escherichia coli K-12. A membrane componenet of the dag transport system which serves for glycine, D-alanine, and D-serine is coded for by the dagA gene at minute 83 of the Escherichia coli chromosome. Merodiploid strains (dagA+/dagA+) show two to three times the transport activity for only those amino acids that are substrates of the dag transport system. The increased transport activity is a result of a two-to threefold increase in Vmax for amino acid uptake with little or no change in the Km value. The two- to threefold gene dose effect of the merodiploid strains is maintained even during carbon starvation, eliminating the possibility that a greater energy supply for transport activity may account for the effect. Since merodiploids which carry more than one copy of the dagA allele show a gene dose response for transport activity, we conclude that the membrane componenet of the dag transport system which is coded for by the dagA allele is present in limiting amounts.


Genetic characterization of the metK locus in Escherichia coli K-12. Three independently isolated metK mutants have been shown to have leisions lying between speB and glc near 57 min on the Escherichia coli chromosome. Two deletions result in a lack of the metC gene product but neither extends into the metK glc region. The three metK mutations are recessive to the wild-type allele carried on the KLF16 episome.


New phosphoglucose isomerase mutants of Escherichia coli. The mutants used to show that phosphoglucose isomerase, and glucose itself, are not essential components of Escherichia coli had not been characterized genetically, other than by mapping. We now describe two new pgi mutants, one amber and the other a Mu-phage insertion, presumably both complete inactivation mutations. The new mutations do not give a phenotype markedly different from those described earlier. However, they might be preferred for certain physiological studies, and we have prepared for this a new double mutant, strain DF214, with a Mu insertion in pgi and a deletion in zwf (flucose 6-phosphate dehydrogenase).


Glyceraldehyde 3-phosphate dehydrogenase mutants of Escherichia coli. We describe glyceraldehyde 3-phosphate dehydrogenase mutants of Escherichia coli. The gene (gap) is at approximately 34 min, with the transductional order gap-fadD-eda. One gap mutant is temperature sensitive and has a heat-labile enzyme. Another is amber.


Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase. Genetic studies show that Escherichia coli has three enzymes capable of phosphorylating glucose: soluble adenosine 5'-triphosphate-dependent glucokinase, which plays only a minor role in glucose metabolism; an enzyme II, called glucosephosphotransferase, with high specificity for the D-glucose configuration; and another enzyme II, called mannosephosphotransferase, with broader specificity. The former enzyme II is active on glucose and methyl-alpha-glucopyranoside, whereas the latter is active on D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, and D-mannosamine. Mutations leading to loss of glucosephosphotransferase activity and designated by the symbol gpt are between the purB and pyrC markers in a locus previously called cat. The locus of mutations to loss of mannosephosphotransferase, mpt, is between the eda and fadD genes. Mutations to loss of glucokinase, glk, are between the ptsI and dsd genes.


Specialized peptide transport system in Escherichia coli. Trileucine is utilized as a source of leucine for growth of strains of Escherichia coli K-12 that are deficient in the oligopeptide transport system (Opp). Trithreonine is toxic to E. coli K-12. Opp- mutants of E. coli K-12 retain complete sensitivity to this tripeptide. Moreover, E. coli W, which is resistant to trithreonine, can utlize this tripeptide as a threonine source and this capability is fully maintained in E. coli W (Opp-). A spontaneous trithreonine-resistant mutant of E. coli K-12 (Opp-) has been isolated that has an impaired growth response to trileucine and is resistant to trithreonine. Trileucine competes with the uptake of trithreonine as measured by its ability to relieve trithreonine toxicity in E. coli K-12. It is concluded that trileucine as well as trithreonine are transported into E. coli K-12 or W by a common uptake system that is distinct from the Opp system. Trimethionine can act as a competitor of trileucine or trithreonine-supported growth and as an antagonist of trithreonine toxicity in Opp- mutants. It is concluded that trimethionine is recognized by the trileucine-trithreonine transport system. Trithreonine, trimethionine, and trileucine are also transported by the Opp system, as they all relieve triornithine toxicity towards E. coli W and compete with tetralysine utilization as lysine source for growth of a lysine auxotroph of this strain.


Multiplicity of oligopeptide transport systems in Escherichia coli. The ability of Escherichia coli K-12 4212 to utilize a variety of oligopeptides as sources of required amino acids was examined. Triornithine-resistant mutants of this strain were oligopeptide permease deficient (Opp-) as judged by their inability to utilize (Lys)3 and (Lys)4 as sources of lysine and their resistance to the toxic tripeptide (Val)3. These same mutants were able to grow when Met-Met-Met, Met-Gly-Met, Met-Gly-Gly, Gly-Met-Gly, Gly-Gly-Met, Gly-Met-Met, Met-Met-Gly, or Leu-Leu-Leu were supplied in place of the requisite amino acid. The system mediating the uptake of these peptides, herein designated Opr I, was not able to transport N-alpha-acetylated peptides, nor the tetrapeptides Met-Gly-Met-Met, Met-Met-Gly-Met, or Met-Met-Met-Gly. Competition experiments indicated that trimethionine and trileucine enter E. coli K-12 via either Opp or Opr I. Analogous results were found using the methionine, leucine-requiring auxotroph E. coli B163. It appears that more than one oligopeptide transport system exists in E. coli and that the system mediating peptide uptake is complex.


chlD gene function in molybdate activation of nitrate reductase. chlD mutants of Escherichia coli lack active nitrate reductase but form normal levels of this enzyme when the medium is supplemented with 10-3 M molybdate. When chlD mutants were grown in unsupplemented medium and then incubated with molybdate in the presence of chloramphenicol, they formed about 5% the normal level of nitrate reductase. Some chlD mutants or the wild type grown in medium supplemented with tungstate accumulated an inactive protein which was electrophoretically identical to active nitrate reductase. Addition of molybdate to those cells in the presence of chloramphenicol resulted in the formation of fully induced levels of nitrate reductase. Two chlD mutants, including a deletion mutant, failed to accumulate the inactive protein and to form active enzyme under the same conditions. Insertion of 99-Mo into the enzyme protein paralleled activation; 185-W could not be demonstrated to be associated with the accumulated inactive protein. The rates of activation of nitrate reductase at varying molybdate concentrations indicated that the chlD gene product facilitates the activation of nitrate reductase at concentrations of molybdate found in normal growth media. At high concentrations, molybdate circumvented this function in chlD mutants and appeared to activate nitrate reductase by a mass action process. We conclude that the chlD gene plays two distinguishable roles in the formation of nitrate reductase in E. coli. It is involved in the accumulation of fully induced levels of the nitrate reductase protein in the cell membrane and it facilitates the insertion of molybdenum to form the active enzyme.


Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase. The prophage lambdac1857 was inserted into the bfe gene located near rif (the structural gene for the beta subunit of deoxyribonucleic acid [DNA]-dependent ribonucleic acid polymerase) on the Escherichia coli chromosome. Induced lysates (low-frequency transducing lysates) of such a lysogen contained defective lambda phage particles (lambdadrif+) that can specifically transduce the wild-type rif+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harboring both the wild-type and the mutant rif genes were isolated. Rec+ derivatives of these heterogenotes produce high-frequency transducing lysates that contain lambdadrif+ and normal active phages at a ratio of 1 to 2. The results of marker rescue experiments and of density determination with several transducing phages indicate that most of the late genes are deleted and replaced by a segment of the chromosomal DNA carrying the bfe-rif region. The length of the chromosomal segment seems to vary between approximately 0.5 and 0.6% of the total bacterial DNA among the three independently isolated lambdadrif+ phages. Electron microscopy of heteroduplex DNA consisting of one strand from lambdadrif+-6 and the other from lambdaimm-21 phages directly confirmed that most of the phage DNA of the "left arm" was replaced by the bacterial DNA. The heteroduplex study also demonstrated that the integration of prophage lambda into the bfe region occurred at the normal cross-over point within the phage attachment site.


D-Alanine-requiring cell wall mutant of Escherichia coli. A mutant of Escherichia coli is described whose cells show a spherical or irregular morphology, associated with leakage of beta-galactosidase and other intracellular proteins. The expression of the morphologic abnormality is most marked when the mutant is grown in rich media and is suppressed by D-alamine, D-serine, D-glutamate, or glycine supplementation. D-Alanine is the most effective amino acid supplement, half maximally supressing this anomalous property at a concentration of 75 mug/ml, as measured by the reduction in beta-galactosidase released from the cells. The mutant is more sensitive to penicillin G, D-methionine, and D-valine and it is relatively resistant to lysozyme. These phenotypic abnormalities are likewise corrected by the above supplementations. The relative rates of peptidoglycan synthesis in mutant and parent, grown under restrictive conditions, were measured both in vivo and in vitro by rates of incorporation of L-[14-D]alanine and uridine-5'-diphosphate-N-acetyl-D-[1-15C-A1-glucosamine, respectively. There is not metabolic block in the biosynthesis of uridine-5'-diphosphate-N-acetyl-muramyl-pentapeptide as shown by enzymic analysis and the lack of accumulation of uridine-5'-diphosphate-N-acetylmuramyl-peptide precursors. These preliminary studies suggest that the mutant possesses a defect in the biosynthesis of peptidoglycan although the exact lesion has not yet been established.


Ultrastructural studies of sporulation in Bacillus sphaericus. Spore septum formation in Bacillus sphaericus 9602 occurs 2 h after the end of exponential growth at one end of the vegetative cell, which retains a uniform diameter. The apparently rigid spore septum contains an inner cell wall layer which disappears when the sporulation septum "bulges" into the mother cell cytoplasm. This process occurs simultaneously with terminal swelling at the end of the cell containing the spore septum. It is suggested that the inner cell wall layer is peptidoglycan and that its dissolution and the terminal swelling are consequences of a localized autolysis. Engulfment of the forespore by membrane proliferation results in the production of a forespore surrounded by two flexible, closely apposed membranes. These membranes appear to become more rigid as a peptidoglycan-like layer appears between them, concomitant with the condensation of the forespore nucleoid into a crescent-shaped structure. After nuclear condensation, visible development of distinct cortex, primordial cell wall, and spore coat layers begin, and the forespore cytoplasm assumes an appearance similar to that of a refractile spore. The spore coats consist of an amorphous inner layer, a lamellar midlayer, and a structured outer layer. As cortex synthesis and spore coat assembly continue, exosporium development commences close to that portion of the mother cell plasma membrane which surrounds the forespore. The exosporium is lamellar and in tangential section is seen to have a hexagonal arrangement of subunits. The timing of these morphological events has the expected correlation with the appearance of unique enzyme activites required for cortex synthesis.


Derivation of glycine from threonine in Escherichia coli K-12 mutants. Escherichia coli AT2046 has been shown previously to lack the enzyme serine transhydroxymethylase and to require exogenous glycine for growth as a consequence. Strains JEV73 and JEV73R, mutants derived from strain AT2046, are shown here to be serine transhydroxymethylase deficient, but able to derive their glycine from endogenously synthesized threonine. Leucine is shown to be closely involved in the regulation of biosynthesis of glycine, to spare glycine in strain AT2046T, to replace glycine in strain JEV73, and to increase threonine conversion to glycine in a representative prototroph of E. coli. An interpretation of strains JEV73 and JEV73R as regulatory mutants of strain AT2046 is given. A hypothesis as to the role of leucine as a signal for nitrogen scavenging is suggested.


Rate stimulation of deoxyribonucleic acid synthesis after inhibition. The degree to which the rate of deoxyribonucleic acid synthesis in thy- cultures of Escherichia coli is stimulated after a period of thymine starvation is shown to be a function of the concentration of thymine present as well as of the culture doubling time. Inhibition of deoxyribonucleic acid synthesis by nalidixic acid yields comparable results. Periods of thymine starvation exceeding one doubling time appear to cause an irreversible inactivation of a fraction of the replication forks in the culture.


Dominant and semidominant mutations leading to thermosensitivity of ribonucleic acid biosynthesis in Saccharomyces cerevisiae. Different dominant thermosensitive mutations affecting the same gene were selected in Saccharomyces cerevisiae. Ribonucleic acid (RNA) synthesis decreased rapidly and markedly at 37 C in all the mutants whether they were in a homozygous or a heterozygous state. Protein biosynthesis was at first unaffected and then decreased slowly, stopping after 5 h. Measurements of RNA biosynthesis in isolated nuclei as well as in vitro activities of RNA polymerases A and B at 22 and 37 C failed to reveal any difference between mutants and the wild type. Analysis of the nature of the residual RNAs synthesized at the high temperature in the mutants showed a small relative increase in the messenger RNA fraction, but it was not sufficient to indicate a specific inactivation of RNA polymerase A activity. The results suggest an impairment in a common regulatory element for all RNA polymerases acting at the level of the initiation of transcription. Similar mutants with a semidominant phenotype were obtained in which the lesions were in two other unlinked loci.


Effect of growth rate on the amounts of ribosomal and transfer ribonucleic acids in yeast. The steady-state growth rate of Saccharomyces cerevisiae was varied by growing the cells in different media. The total amount of ribonucleic acid (RNA) per cell was found to decrease as a nonlinear function of decreasing growh rate. The RNA from cells growing in different media was analyzed by polyacrylamide gel electrophoresis. Although the amounts of both ribosomal RNA and transfer RNA decreased with decreasing growth rate, the ratio of ribosomal to transfer RNA was not constant. As the growth rate was reduced the ribosomal RNA fraction decreased slightly, whereas the transfer RNA fraction increased slightly. Thus the levels of ribosomal and transfer RNA were regulated to similar yet different extents. The levels of the different ribosomal RNA species were more closely coordinated. At all growth rates the ribosomal RNAs (including 5S RNA) were present in equimolar amounts. The rate of protein synthesis in yeast cells also decreased with decreasing growth rate. The low rates of protein synthesis did not appear to be due to limiting numbers of ribosomes or transfer RNA molecules.


Interaction of the expression of two membrane genes, acrA and plsA, in Escherichia coli K-12. The mutation acrA1, leading to acriflavine sensitivity through disorganization of the plasma membrane, is located between proC and purE on the Escherichia coli K-12 chromosome. Gene plsA has been reported to determine biosynthesis of membrane phospholipid and to be located very near acrA (1). Genes acrA and plsA fall into different cistrons and are arranged in the order proC-acrA-plasA-purE. The genes were shown to interact with each other. Introduction of acrA mutation into a plsA temperature-sensitive mutant mitigated the heat sensitivity. Plasmid (F-gal+) stability in acrA mutants was restored by introduction of the plasA mutation into the acrA cells. When an Hfr plsA donor was conjugated with an acrA recipient, or when reciprocally conjugated, the exogenotes were eliminated at high frequency during subsequent subcultivation in broth. However, the exogenotes were not eliminated in all other allelic combinations of genes acrA and plsA. When an F-gal+ plasmid was introduced into the unstable heterozygotes (acrA+plsA/acrApls1+), the plasmids were stably hosted, whereas the acrA+ plasA exogenotes were spontaneously lost at a high frequency. On the other hand, when the unstable heterozygotes carrying F-gal+ were cultured in acriflavine-containing medium, the F-gal+ plasmids were preferentially eliminated but the acrA+plasA exogenotes were not affected. The results suggest that the organization of the plasma membrane controls the recombination of the exogenotes introduced into zygotes.


Partial suppression of the phenotype of Escherichia coli K-12 dnaG mutants by some I-like conjugative plasmids. Three I-like conjugative plasmids, ColIdrd1, R144drd3, and R64drd11, which are derepressed for functions involved in conjugation, were found to suppress at least partially the phenotype of temperature-sensitive dnaG mutants of Escherichia coli K-12, as judged from the kinetics of deoxyribonucleic acid synthesis at elevated temperature in newly formed and established plasmid-containing strains. In contrast, the corresponding wild-type plasmids and three F-like derepressed conjugative plasmids, F101, R100drd1, and R1drd16, all failed to suppress. Suppression is presumably caused by a different plasmid-determined function from that which promotes survival of ultraviolet-irradiated bacteria, because both the wild-type I-like plasmids and their drd mutants protected irradiated bacteria. One possible interpretation of these results is that the product of a gene carried by certain I-like plasmids can substitute for the bacterial dnaG gene product during ongoing deoxyribonucleic acid replication.


Isolation and characterization of recessive, constitutive mutations for repressible acid phosphatase synthesis in Saccharomyces cerevisiae. Two new classes of mutants containing recessive constitutive mutations, phoT and phoU, that affect the repressible acid phosphatase (EC 3.1.3.2) in Saccharomyces cerevisiae were isolated along with many previously known phoR mutants. These loci segregated independently from each other, from the phoS gene, and from another regulatory gene, phoD, that exerts positive control for acid phosphatase synthesis. The phoR and phoU mutations showed the same genetic behavior in the double mutants, which also contained the phoS or phoD mutation. In contrast, the phoT mutation could not suppress the phoS mutation, which caused a loss of enzyme activity. Many mutant alleles of phoR and phoU were found to be temperature sensitive (ts), whereas those of phoT were not. These ts mutants were constitutive at 35 C but severely repressible at 25 C. These facts strongly suggest that both the phoR and phoU genes are cooperatively concerned with the production of the repressor, whereas the phoT gene might be involved in another mechanism distinct from that in which phoR and phoU are involved. No single mutation of phoR, phoT, or phoU result in an enzyme level comparable to that of fully derepressed enzyme activities, and the temperature sensitivity of the ts phoR and ts phoU mutations in such combinations almost disappeared. In addition to these observations, since the ts phoR phoS and ts phoU phoS double mutants showed some enzyme synthesis at 25 C under derepressing conditions, a defect in the ts mutant repressors was strongly suggested, even at 25 C.


Relationship between chromosome replication and cell division in a thymineless mutant of Escherichia coli B/r. The relationship between chromosome replication and cell division was investigated in a thymineless mutant of Escherichia coli B/r. Examination of the changes in average cell mass and DNA content of exponential cultures resulting from changes in the thymine concentration in the growth medium suggested that as the replication time (C) is increased there is a decrease in the period between termination of a round of replication and the subsequent cell division (D). Observations on the pattern of DNA synthesis during the division cycle were consistent with this relationship. Nevertheless, the kinetics of transition of exponential cultures moving between steady states of growth with differing replication velocities provided evidence to support the view that the time of cell division is determined by termination of rounds of replication under steady-state conditions.


Utilization of selected leucine peptide amides by Escherichia coli. Studies on the utilization of leucine peptide amides as a source of leucine for a leucine auxotroph showed that in general compounds with the structure leu-chi amide (where chi is any amide) are utilized as well as the free peptide, but that compounds with the structure chi-leu amide (where chi is not leucine) are used less effectively than the free peptide. Growth and enzymological experiments indicated that the lower capacity of Escherichia coli to utilize amides of the structure chi-leu amide is not a result of poor transport of these compounds, but rather the inability to rapidly liberate leucine from the amide when it is supplied to the cell in the form of a peptide. Competition studies indicated that the peptide amides enter the cell via the oligopeptide permease system.


Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria. Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.


Random replication of the stringent plasmid R1 in Escherichia coli K-12. The R-factor R1 is present in a low number of copies per genome (near unity, so-called stringent control of replication). The replication of R1 was studied in a density-shift experiment. One generation after the shift about 20% of the R1 copies had not replicated, whereas about 20% had replicated at least twice. The results are in quantitative accordance with a random replication of R1 in which the replicating molecules are taken from a cytoplasmic plasmid pool and transferred back to the pool after replication. This is analogous to the results obtained by Bazaral and Helinski (1970) and Rownd (1969) for plasmids that are present in 10 to 20 copies per genome (so-called relaxed control of replication). Hence, there seem to be no difference between stringent and relaxed plasmids with respect to selection of plasmid molecules for replication. However, we cannot tell whether all R1 copies in a cell replicate during a fraction of or throughout the cell cycle. The random selction of plasmid copies for replication has to be considered when models for control of replication are constructed.


Ribonucleic acid destruction and synthesis during intraperiplasmic growth of Bdellovibrio bacteriovorus. During growth of Bdellovibrio bacteriovorus on (2-14C)uracil-labeled Escherichia coli approximately 50% of the radioactivity is incorporated by the bdellovibrio and most of the remainder is released as free nucleic acid bases. Kinetic studies showed that 50 and 30S ribosomal particles and 23 and 16S ribosomal ribonucleic acid (RNA) of E. coli are almost completely degraded by the first 90 min in a 210- to 240-min bdellovibrio developmental cycle. Synthesis of bdellovibrio ribosomal RNA was first detected after 90 min. The specific activity and the ratio of radioactivity in the bases of the synthesized bdellovibrio RNA was essentially the same as those of the substrate E. coli. The total radioactivity of the bdellovibrio deoxyribonucleic acid (DNA) exceeded that in the DNA of the substrate E. coli cell, and the ratio of radioactivity of cytosine to thymine residues differed. Intraperiplasmic growth of B. bacteriovorus in the presence of added nucleoside monophosphates (singly or in combination) significantly decreased the uptake of radioactivity from (2-14C)uracil-labeled E. coli; nucleosides or nucleic acid bases did not. It is concluded that the RNA of the substrate cell, in the form of nucleoside monophosphates, is the major or exclusive precursor of the bdellovirbrio RNA and also serves as a precursor for some of the bdellovibrio DNA.


Induction of lactose transport in Escherichia coli during the absence of phospholipid synthesis. Induction of lactose transport and of beta-galactosidase synthesis was examined in two Escherichia coli strains that require exogenous glycerol for phospholipid synthesis and growth. No preferential inhibition of lactose transport induction was observed when phospholipid synthesis was restricted to 5 to 10% of the normal rate. We conclude that the lactose transport system does not require concurrent phospholipid synthesis for its functional assembly.


Nuclear and mitochondrial deoxyribonucleic acid replication during mitosis in Saccharomyces cerevisiae. To study nuclear and mitochondrial deoxyribonucleic acid (DNA) synthesis during the cell cycle, a 15N-labeled log-phase population of Saccharomyces cervisiae was shifted to 14N medium. After one-half generation, the cells were centrifuged on a sorbitol gradient in a zonal rotor to fractionate the population according to cell size and age into fractions representing the yeast cell cycle. DNA samples isolated from the zonal rotor cell samples were centrifuged to equilibrium in CsC1 in an analytical ultracentrifuge to separate the nuclear and mitochondrial DNA components. The amount of 14N incorporated into each 15N-labeled DNA species was measured. The extent of nuclear DNA replication per sample was obtained by measuring the amount of hybrid DNA. The percentage of hybrid nuclear DNA increased from 6 to 68% and then decreased to 44% during the cell cycle. Upon ultracentrifugation, mitochondrial DNA banded as a unimodal peak in all zonal rotor samples. Mitochondrial DNA replication could be ascertained only by the 14N level in each mitochondrial peak and not, as with nuclear DNA, by hybrid DNA level. In contrast to the nuclear incorporation pattern, the 14N percentage in mitochondrial DNA remained effectively constant during the cell cycle. Comparison of the data to theoretical distributions showed that nuclear DNA was replicated discontinuously during the cell cycle, whereas mitochondrial DNA was replicated continuously throughout the entire mitotic cycle.


Mating aggregates in Escherichia coli conjugation. Mating mixtures of Escherichia coli cells were shown to contain mating aggregates of two to 20 cells each rather than only mating pairs of two cells each. The mating aggregate size distribution shows two broad peaks, at two to four cells and at eight to 13 cells. The quantitative mating aggregate size distribution and the proportion of male cells in mating aggregates are dependent on the input ratio of male to female cells. At an input ratio of one to one, the average mating aggregate contains equal proportions of male and female cells and most of the cells involved in mating are in large aggregates of seven or more cells each. The deoxyribonucleic acid (DNA) transfer efficiency per mating aggregate cell was constant regardless of average aggregate size or of the ratio of male to female cells in the aggregate. Under optimal conditions essentially every male cell or every female cell in a mating aggregate can be involved in DNA transfer. A comparison of light microscopy, sucrose gradient centrifugation, and analysis with a modified Coulter counter indicated that the number of cells in mating aggregates is best equantitated using a modified Coulter counter.


Specificity and genetics of S-adenosylmethionine transport in Saccharomyces cerevisiae. The specificity of a transport system for S-adenosylmethionine was determined through the use of structurally related derivatives. Of the compounds tested, the analogues S-adenosylethionine and S-inosylmethionine and the naturally occurring compounds S-adenosyl-(5')-3-methylthiopropylamine and S-adenosylhomocysteine competitively inhibited uptake of the sulfonium compound. Ki values for these compounds indicate that the order of affinity for the transport protein is S-adenosylmethionine congruent to S-adenosyl-(5')-3-methyl-thiopropylamine greater than S-adenosylethionine greater than S-inosylmethionine greater than S-adenosylhomocysteins. S-adenosyl-(2-hydroxy-4-methylthio)butyric acid exerted inhibition of a mixed type. S-insoyl-(2-hydroxy-4-methylthio)butyric acid, S-inosylhomocysteine, and S-ribosylhomocysteine were without effect. On the basis of the inhibition data, the methionine-amino, adenine-amino, and methyl groups were identified as group important in the binding of S-adenosylmethionine to the transport protein. Comparison is made with the specificities of various transmethylating enzymes utilizing S-adenosylmethionine. In addition, a number of conventional and temperature-sensitive S-adenosylmethionine transport mutants were isolated and analyzed in an attempt to identify the structural character of the specific transport protein(s). The data obtained suggest that only a single gene (a single polypeptide) is involved in specific S-adenosylmethionine transport. Apparent interallelic complementation supports the assumption that the functional form of the protein is composed of two or more copies of a monomer.


Genes for ribitol and D-arabitol catabolism in Escherichia coli: their loci in C strains and absence in K-12 and B strains. Escherichia coli C strains can grow at the expense of the two natural pentitols ribitol and D-arabitol, sugar alcohols previously thought not to be utilized by E. coli. E. coli strains K-12 and B cannot utilize either compound. The genetic loci responsible for pentitol catabolism in E. coli C, designated rtl and atl, are separate and closely linked. Each lies between metG and his and is highly co-transducible with metG and with a P2 prophage attachment site. rtl and atl readily can be transduced into E. coli K-12 or B strains, in which they integrate at, or very near, their E. coli C location. Transduction also can be used to insert rtl and atl into certain E. coli K-12 F' plasmids. No recombination between E. coli C strains and either K-12 or B strains occurs within the rtl-atl genetic region after interstrain conjugations or transductions. No cryptic rtl or atl genes in K-12 or B strains can be detected by complementation, recombination, or mutagenesis. These results are consistent with the view that the rtl-atl portion of the E. coli C chromosome has no counterpart in E. coli K-12 or B and may have been obtained from an extrageneric source. Detailed biochemical and genetic comparisons of penitol utilization in E. coli and Klebsiella aerogenes are in progress. The ability to catabolize xylitol is conferred upon E. coli C strains by a mutation at or adjacent to the rtl locus, whereas in E. coli K-12 or B strains harboring rtl an additional mutation at a separate locus is required for xylitol utilization.


Intrinsic and extrinsic light responses of Salmonella typhimurium and Escherichia coli. Exposure to intense light in the region between 390 and 530 nm has been shown to have three effects on the motility of Salmonella typhimurium and Escherichia coli. Short pulses of light initiate continuous tumbling. Longer exposures to light induce smooth swimming, and prolonged exposures induced paralysis. The tumbling response is intimately connected with the chemical gradient-sensing apparatus of the bacterium and can be overcome by strong temporal gradients of attractant. Some mutants of S. typhimurium which are defective in the tumble-generating mechanism for chemotaxis are also unable to tumble in intense light. This intrinsic light effect can be mimicked by the addition of external dyes (the classical photodynamic effect), but it can be shown that the two phenomena are distinct. The extrinsic (photodynamic) effect can be inhibited by histidine or by anaerobic conditions, whereas the intrinsic effect is not. The observation that the extrinsic effect can also produce the three types of light responses listed above suggests a common pathway after an intial event on either an endogenous or an externally added photoreceptor.


Enhanced level and metabolic regulation of methionyl-transfer ribonucleic acid synthetase in different strains of Escherichia coli K-12. The methionyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli K-12 eductants carrying P2-mediated deletions in the region of the structural gene of this enzyme was investigated. No structural alteration of this enzyme was observed in three eductants examined. These were isolated from strain AB311, which had a threefold higher level of methionyl-tRNA synthetase than most haploid strains examined. In two of the three eductants studied, the level of this enzyme was twofold higher than in their parental strain regardless of growth conditions used. In contrast, isoleucyl-, leucyl-, and valyl-tRNA synthetases had similar levels in all strains examined. Like valyl-tRNA synthetase, but to a lesser extent, methionyl-tRNA synthetase was subject to metabolic regulation. Coupling between the level of methionyl-tRNA synthetase and growth rate was observed even in strains that had an enhanced level of methionyl-tRNA synthetase. These results suggest that the formation of methionyl-tRNA synthetase remains subject to metabolic regulation even when the repression-like mechanism that controls the synthesis of this enzyme is altered. In addition, we report that in the merodiploid strain EM20031, which was haploid for the valyl-tRNA synthetase structural gene and diploid for the structural genes of methionyl-tRNA synthetase and D-serine deaminase, the levels of these latter two enzymes varied to a minor yet significant extent with the phosphate concentration of the culture medium; under the same conditions, the level of valyl-tRNA synthetase remained unchanged. Moreover, no variation of the levels of these three enzymes in response to phosphate was observed in the haploid strain HfrH. These results indicate that in the merodiploid strain EM20031, which carries the episome F32, the number of episomes per chromosome varies to some extent according to the phosphate concentration of the culture medium.


Role of methionyl-transfer ribonucleic acid in the regulation of methionyl-transfer ribonucleic acid synthetase of Escherichia coli K-12. A decrease in the in vivo acylation level of methionine transfer ribonucleic acid (tRNAmet) induced by methioninyl adenylate led to a specific derepression of methionyl-transfer ribonucleic acid (tRNA) synthetase formation. This derepression required de novo protein synthesis and was reflected by overproduction of unaltered enzyme. Two different strains of Escherichia coli K-12 that have normal levels of methionyl-tRNA synthetase were examined and the derepression of methionyl-tRNA synthetase was observed in both. Moreover, for one of these strains, the relation between the level of methionyl-tRNA synthetase and deacylation level of tRNAmet was established; under the growth conditions used, when more than 25% of tRNAmet was deacylated, methionyl-tRNA synthetase formation was derepressed and the level of derepression became proportional to the amount of tRNAmet deacylated. Concomitantly, the enzyme was subject to specific inactivation as a consequence of which the true de novo rate of derepression of the formation of this enzyme was higher than that determined by measurements of enzyme activity. These studies were extended to strains AB311 and ed2, which had a constitutive enhanced level of methionyl-tRNA synthetase. In these strains no derepression of enzyme formation was observed on reducing the acylation level of tRNAmet by use of methioninyl adenylate.


Genetic control of yeast mannan structure: mapping genes mnn2 and mnn4 in Saccharomyces cerevisiae. Two mutations concerned with mannan biosynthesis in the yeast Saccharomyces cerevisiae have been mapping. The mnn2 mutation, which affects the addition to the polysaccharide backbone of the first side-chain D-mannose unit in alpha1-leads to2 linkage, was located on chromosome II linked to the centromere and the gall locus. The mn4 locus, which regulates the synthesis of mannosylphosphate groups on the mannan side chains, was placed on chromosome XI near trp3 and ural and a locus previously reported to regulate the ability of a S. diastaticus strain to bind alcian blue (Friis and Ottolenghi, 1970). The mnn4 mutant also fails to bind alcain blue, but the gene responsible for alcian blud binding in this strain segregates independently from the dye-binding locus of S. diastaticus, and therefore must be a different gene. A diploid heterozygous for mnn4 fails to bind dye, indicating dominance of this mutant genotype. The alcian blue dye binding locus dbll, reported to Friis and Ottolenghi (1970), is also dominant. Thus, there are at least two independent genes that control the formation of the mannosylphosphate units in the mannan side chains, and both have the property of dominance in the mutant form.


Class of small multicopy plasmids originating from the mutant antibiotic resistance factor R1 drd-19B2. The large mutant R-factor R1drd-19B2 gives rise to several classes of small, covalently closed circular deoxyribonucleic acids (DNAs), designated as Rsc DNAs, when harbored by the K-12 strain CRT46 which carries a dnaA mutation. The molecular weights of these DNA molecules range from 3 X 106 to 8.4 X 106. Cells arising from single colonies of CRT46-R1drd-19B2 harbor only one to two copies of the large mutant R-factor and in addition 10 to 20 copies of Rsc plasmid of a discrete size class per chromosome. The larger Rsc DNAs carry the ampicillin resistance gene. After transformation the small circular DNAs are present in Escherichia coli C in a large number of copies, up to 100 copies per chromosome. Hybridization studies between Rsc plasmids indicate that they possess common DNA sequences.


alphabeta sequence of F is IS31.

alphabeta sequence of F is IS31. Previous studies have shown that there is a deoxyribonucleic acid (DNA) segment, of length 1.3 kb and denoted as the alphabeta sequence, which occurs twice on the F plasmid at corrdinates 93.2 to 94.5/OF kb and 13.7 to 15.0F kb. In the present investigation, heteroduplexes were prepared between a phage DNA carrying the insertion sequence IS3 and suitable F-prime DNAs. The hybrids formed show that IS3 is the same as alphabeta. This result plus previous studies support the view that: (i) the insertion sequence IS2 and IS3 occur on F and, in multiple copies, on the main bacterial chromosome of Escherichia coli K-12; and (ii)these IS sequences on the main bacterial chromosomes are hot spots for Hfr formation by reciprocal recombination with the corresponding sequences of F.


Regulation of bacterial cell division: temperature-sensitive mutants of Escherichia coli that are defective in septum formation. Mutations ts2158 and ts1882, which confer temperature sensitivity of septum formation, map near leu in the region of min 2.0 to 2.1 on the Escherichia coli chromosome. These mutants stop division abruptly and grow as filaments at 42 C; when returned to 28 C, division resumes after about 30 min to produce short cells. The product of the gene defined by these mutations probably is required during all stages of septum formation rather than specifically for initiation of septation. Filaments that formed at 42 C contained incomplete constrictiions (septa). When actively dividing filaments (i.e., those incubated at 28 C until division resumed) were shifted to 42 C a second time, division again stopped abruptly and incomplete constrictions persisted during the incubation at 42 C. Filaments that were subjected to 28 C incubation for a brief time (10, 20, or 30 min) before being shifted again to 42 C did not resume division as would be expected of a strain defective in initiating septation. Mutations ts 1882 and ts2158 and recessive to the ts+ allele, which is consistent with the interpretation that these mutations cause the loss of a function. They did not complement each other and presumably represent one cistron. Mutants carrying ts1882 and ts2158 mutations were compared with a mutant defective in the ftA allele, also known to map near leu.


Discontinuous replication of colicin E1 plasmid deoxyribonucleic acid. The plasmid pML 21, which was found to contain approximately 49% of the Col E1 genome was used to determine the template origin of single-stranded deoxyribonucleic acid (DNA) fragments (4 to 32% of the Col E1 units length) associated with Col E1 dna replicative intermediates. The results of DNA hybridization competition experiments indicate that the single-stranded fragments derive from the full length of the Col E1 DNA template as expected for Okazaki fragments and the plasmid pML 21 contains the replication origin of Col E1 DNA.


Unusual sensitivity of Escherichia coli to adenine or adenine plus histidine. The W3110 strain of Escherichia coli K-12 is unusually sensitive to adenine. Inhibition of growth is relieved by a combination of thiamine and uridine (or cytidine). In the presence of histidine, inhibition is more severe and is relieved by a combination of thiamine, glycine, uridine (or cytidine), and inosine (or guanosine).


Strain-specific difference that affects the inhibition of division of Escherichia coli filaments by chloramphenicol. The Escherichia coli mutations ts1882 and ts2158 cause temperature-sensitive septum formation and result in growth of cells as long, multinucleate, nonseptate filaments at 42 C. When filaments are transferred to 28 C, they divide into short cells. Chloramphenicol, when added to cultures of filaments at the time of temperature reduction. Inhibited division of filaments when these temperature-sensitive mutations were present in the K-12 strain AB1157. However, when the ts1882 and ts2158 mutations were present in another k-12 strain, UTH4113, filaments of these strains divided in the presence of chloramphenicol.


Amino-terminal sequences of indoleglycerol phosphate synthetase of Escherichia coli and Salmonella typhimurium. The partial sequences of the first 40 residues of indoleglycerol phosphate synthetase of Escherichia coli and Salmonella typhimurium were determined, and three amino acid differences were observed among the 38 residues compared.


Analysis of bacteriophage deoxyribonucleic acid sequences methylated by host- and R-factor-controlled enzymes. Phages lambda and fd were propagated in Escherichia coli strains that have either host K-12 or the N-3 R-factor deoxyribonucleic acid-cytosine methylase activity. Pyrimidine tracts containing 3H-labeled 5-methylcytosine (MeC) were analyzed; in all cases, the major methylated sequence was 5' ... C-MeC-T ... 3'.


Effect of tsl mutations on Col E1 expression in a recA strain of Escherichia coli K-12. Spontaneous colicin production by Col E1 in a recA- strain of Escherichia coli K-12 is restored nearly to wild-type levels by the presence of tsl- second site mutation.


Hybridization of glyceraldehyde-3-phosphate dehydrogenase. 1. Glyceraldehyde-3-phosphate dehydrogenases [EC 1.2.1.12] from rabbit, pig, lobster, yeast, E. coli, and B. stearothermophilus have been subjected to hybridization in 3M NaCL. 2. Suitable mixtures of electrophoretically distinct glyceraldehyde-3-phosphate dehydrogenases were found to give five-membered by electrophoresis on cellulose acetate. 3. The thermophile enzyme did not form hybrids with any of its mesophile counterparts, presumably because it does not dissociate under the conditions used. 4. Hybridization of pig enzyme with lobster enzyme that had been inactivated by selective carboxymethylation of Cystetramer containing only one active pig enzyme subunit. Individual subunits would thus appear to express their activity independently even within hybrid tetramers formed with subunits of another species. 5. The successful hybridization of glyceraldehyde-3-phosphate dehydrogenases from evolutionarily distant sources suggests that the tertiary and quaternary structures of the enzyme have been highly conserved.


Preferential production of IgG2 anti-hapten antibody by immunization with hapten-conjugated Escherichia coli. It is well-known that immunization of guinea pigs with hapten-protein conjugates induces concomitant production of IgG1 and IgG2 anti-hapten antibodies.  However, the synthesis of antibody to 2, 4-dinitrophenyl groups (DNP) was found to be restricted to one of the IgG antibodies (IgG2 antibody) when guinea pigs were immunized through repeated intraperitoneal injections of 0.1 mg of 2, 4-dinitrophenylated Escherichia coli (DNP-E coli).  This selective induction of IgG2 anti-DNP antibody formation occurred both in the presence and absence of Freund's adjuvants, whereas a trace of IgG1 anti-DNP antibody was produced concomitantly on increasing the immunizing dose of DNP-E. coli (3.0 mg) in Freund's complete adjuvant (FAC).


Binding of aminoacyl-tRNA to ribosomes promoted by elongation factor Tu.  Studies on the role of GTP hydrolysis. The role of guanosine triphosphate (GTP) in the elongation factor Tu(EF-Tu)promoted binding of aminoacyl-tRNA to ribosomes has been investigated.  In the presence of EF-Tu and GTP, phenylalanyl-tRNA (Phe-tRNA) was bound to the A site of the poly(U).ribosome complex having N-acetylphenylalanyl-tRNA on its P site.  EF-Tu could be utilized repeatedly in this binding reaction in the presence of GTP but was used only once and remained bound to ribosomes when GTP was replaced by 5'guanylyl methylenediphosphonate (Gpp(CH2)p), a nonhydrolyzable analog of GTP.  The binylalanyl-tRNA, while little dipeptide was formed in the latter case.  However, when EF-Tu and Gpp(CH2)p bound to the ribosomal complex were released by centrifugation through 10% sucrose containing 0.2 mM GDP, the yield of the dipeptide was correspondingly increased.  It was concluded that the role of GTP in  this reaction is to facilitate the transfer of Phe-tRNA to ribosomes by virtue of the high affinity of EF-Tu.GTP for ribosomes.  The subsequent conversion of EF-Tu.GTP to EF-Tu.GDP, a form of EF-Tu with low affinity for ribosomes as well as for Phe-tRNA, resulted in the detachment of EF-Tu.  Thus, the hydrolysis of GTP seems to be required for the release of EF-Tu from ribosomes, which is necessary for peptidyl transfer and the reutilization of EF-Tu.  A freely reversible interaction between ribosome and Phe-tRNA.EF-Tu.Gpp(CH2)p complex was also demonstrated.


Effects on ribosomes of the substitution of spermidine or divalent cations for magnesium ions. The inactivation of rat liver ribosomes (polysomes) resulting from the replacement of bound magnesium by spermidine has been studied using a dialysis method. Inactivation of the ribosomes (polysomes) occurred when more than 40% of the Mg2+ originally bound to ribosomes in the presence of 2 mM Mg2+ was replaced by spermidine. The polypeptide-synthesizing activity of polysomes was retained partially when Mg2+ was replaced by Mn2+ by dialysis. E. coli 30S ribosomal subunits were more resistant to inactivation due to the replacement of Mg2+ by spermidine than the 50S subunits.


The involvement of guanosine 5-diphosphate-3-diphosphate in the regulation of phospholipid biosynthesis in Escherichia coli. Lack of ppGpp inhibition of acyltransfer from acyl-ACP to sn-glycerol 3-phosphate. The response of the Escherichia coli sn-glycerol-3-phosphate acyltransferase to guanosine 5-diphosphate-3-diphosphate (ppGpp) has been determined in vitro employing palmityl coenzyme A (CoA) and palmityl acyl carrier protein as acyl substrates. Levels of ppGpp which cause significant inhibition of enzyme activity with palmityl-CoA as substrate have no effect on enzyme activity when palmityl acyl carrier protein is employed as acyl donor. The inhibition of enzyme activity observed with palmityl-CoA as acyl substrate was dependent upon the relative concentrations of MgCl2 and ppGpp (MgCl2 to ppGpp ratio) employed. With palmityl-CoA as acyl donor, PPGpp inhibited the production of lysophosphatidic acid but not phosphatidic acid. With palmityl acyl carrier protein as acyl substrate, ppGpp had no influence upon the distribution of the reaction products.


The structure of the mutant dihydrofolate reductase from Streptococcus faecium. Amino acid sequence of peptide CNBr 7 and complete sequence of the protein. The complete amino acid sequence of the mutant dihydrofolate reductase from Streptococcus faecium var. Durans strain A has been determined by sequence analysis of peptides produced by tryptic, chymotryptic, thermolytic, and mild acid cleavage of the large peptide CNBr 7 and from previously reported studies. The sequence of the S. faecium enzyme is compared to the reported sequence of dihydrofolate reductase from Escherichia coli and the two are shown to contain two domains of substantial homology. One of these domains consists of the NH2-terminal 60 residues and is considered to contribute the dihydrofolate binding site. The second domain probably contains the dinucleotide binding structure. Comparison of the sequences of the dihydrofolate reductases with those of larger dehydrogenases of known structure failed to show any evidence for homology. Considerations of size and predictions of secondary structure also suggest that the second domain in the reductases has no structural similarity to the nucleotide binding site in the larger dehydrogenases. It is concluded that the two reductases are related, although distantly, but that they have evolved from an ancestral protein different from the primitive predecessor of the other oxidoreductases.


Primary structure of porcine pepsin. I. Purification and placement of cyanogen bromide fragments and the amino acid sequence of fragment CB5. Fragments resulting from the cyanogen bromide cleavage of reduced and aminoethylated porcine pepsin were purified. Only four of the five fragments theoretically present could be accounted for in major yield when the cyanogen bromide reaction was carried out at room temperature. The NH2-terminal fragment, CB2, contained an internal homoserine which was not cleaved to any significant extent. The amino acid sequence around this internal homoserine was determined by isolating and partially determining the sequence of an alpha-chymotryptic peptide. Cleavage at this methionine was increased by 50% when the cyanogen bromide reaction was carried out at 37 degrees. The NH2- and COOH-terminal sequences of five major fragments were determined. The placement of these fragments in the native pepsin molecule was demonstrated. The amino acid sequence of one of the fragments, CB5, was determined. This fragment contains 44 residues with an internal disulfide bridge. The COOH-terminal methionine of this fragment was connected to another 37-residue cyanogen bromide fragment of known sequence. Together these two fragments formed the COOH-terminal 81 residues of porcine pepsin.


Primary structure of porcine pepsin. II. Amino acid sequence of two cyanogen bromide fragments, CB3 and CB4. The amino acid sequences of two cyanogen bromide fragments from porcine pepsin have been determined. Fragment CB3 which represents the NH2-terminal 80 residues of pepsin was assembled from the peptides purified from proteolytic digests of this fragment using alpha-chymotrypsin, thermolysin, and staphylococcal protease. Two chymotryptic peptides were isolated from the NH2-terminal region of this fragment. One of these contains 2 extra residues, Ala-Leu-, at the NH2 terminus. This peptide is apparently derived from a different cleavage site of pepsinogen in its conversion to pepsin. The second cyanogen bromide fragment, CB4, contains 47 residues. The sequence was established from the peptides resulting from proteolytic digests using alpha-chymotrypsin, alpha-lytic protease, and thermolysin. An isoleucyl residue at position 29 of fragment CB4 appears to be absent in some molecules. This represents a structural variant of pepsin.


Primary structure of porcine pepsin. III. Amino acid sequence of a cyanogen bromide fragment, CB2A, and the complete structure of porcine pepsin. The complete amino acid sequence of porcine pepsin (EC 3.4.4.1) was constructed from the sequence of five cyanogen bromide fragments. The sequence of one of these fragments, CB2A, is reported here. The sequences of 4 other fragments are known from previous work. Porcine pepsin contains 327 residues with three structural variants. The active center aspartyl residue, which reacts with 1,2-epoxy-3-(p-nitrophenoxy)propane (Chen, K. C. S., and Tang, J. (1972) J. Biol. Chem. 247, 2566-2574), is located at residue 32. Another active site aspartyl residue, which reacts with diazo inactivators (Bayliss, R. S., Knowles, J. B., and Wybrandt, G. B. (1969) Biochem. J. 113, 377-386, IS LOCATED AT RESIDUE 215. The sequences around these 2 aspartyl residues are apparently homologous to each other. The sequences around the tryptophanyl residues at positions 39, 141, 181, and 300 are also homologous to one another. These homologous sequences could be genetic in origin. Fragment CB2A which contains 119 residues was constructed from the peptide sequences resulting from six proteolytic digestions and chemical cleavage at tryptophanyl bonds.


Effects of estrogen on gene expression in the chick oviduct. IV. Initiation of RNA synthesis on DNA and chromatin. The interaction of Escherichia coli RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6.) with chick oviduct DNA and chromatin has been studied in order to quantify the RNA polymerase binding sites and rifampicin-resistant initiation sites. The number of high affinity binding sites at saturation was calculated from the number of molecules of enzyme bound to DNA or to chromatin. The number of rifampicin-resistant initiation sites was calculated from the total quantity and the number average chain length of RNA synthesized under the conditions in which only RNA polymerase already in a stable preinitiation complex could avoid inhibition by the drug. Using an assessment of the temperature requirement for the information of this preinitiation complex, we were able to differentiate initiations at single-stranded or nicked DNA regions from initiations at double-stranded DNA regions. This method enabled us to rule out the possibility that RNA chain initiation on chromatin was due to initiation at nicked or end regions of DNA. In addition, we also demonstrated that not all RNA polymerase molecules bound to DNA or chromatin are located at a site at which RNA chain initiation can immediately begin. This methodological approach should allow investigators to monitor the individual reactions and factors involved in in vitro transcription of eukaryotic DNA and chromatin.


Effect of estrogen on gene expression in the chick oviduct. V. Changes in the number of RNA polymerase binding and initiation sites in chromatin. Estrogen administration to chicks results in an increase in the chromatin template activity of oviduct target tissue as assayed under standard in vitro assay conditions. However, the results obtained by the simple measurement of template activity may be a complicated function of the number of available RNA polymerase initiation sites, the rate of RNA chain elongation, and the rate of reinitiation. In the present study, we have measured separately the change in both the number of chain initiations as well as the rate of RNA chain propagation under conditions in which reinitiation was eliminated. Chromatin prepared from either estrogen-treated or control oviducts both supported an RNA chain elongation rate of six nucleotides per s and a chain size of approximately 700 nucleotides. Thus, both the elongation rate and size of the average product remained relatively constant following estrogen stimulation. In contrast, within 8 hours after a single injection of estrogen to unstimulated chicks, the concentration of RNA polymerase needed to saturate chromatin binding sites was increased to 150% in comparison to control values, and by 24 hours the level of polymerase bound to chromatin was twice that of the untreated control chick chromatin. With daily injections of estrogen, polymerase binding continued to rise. Coincident with the over-all increase in chromatin-bound polymerase was an increase in rifampicin-insensitive initiation sites and newly synthesized RNA chains. Unstimulated chick oviduct chromatin initiated 10,000 RNA chains/pg of DNA, while 24 hours of steroid treatment increased the number of initiated chains to 34,000 chains. These data demonstrate that the estrogen-induced increase in chromatin transcriptive activity was due to an increased number of polymerase binding and initiation sites on the chromatin template without a detectable change in the rate of RNA chain elongation.


The amino acid sequence of the insulin from a primitive vertebrate, the atlantic hagfish (Myxine glutinosa). Insulin has been isolated and purified from the islet organs of the cyclostome, Myxine glutinosa, by means of acid-ethanol extraction, fractional precipitation, and gel filtration. The complete amino acid sequence of the hormone has been determined by Edman degradation of the S-carboxymethylated or performic acid-oxidized A and B chains, and of various tryptic peptides derived from the chains. The 52-residue hagfish insulin has many structural features in common with other vertebrate insulins including the locations of the 6 half-cystine residues, the NH2-terminal 7 residues and the COOH-terminal 6 residues of the A chain, and several shorter sequences in the B chain that are known to comprise the dimer interface in porcine insulin crystals. Of the 24 residues which are invariant among the other known insulins, 23 are identical in hagfish int 16 of these sites it contains residues not previously observed in vertebrate insulins. The B chain also contains an additional COOH-terminal residue of methionine, making it 1 residue longer than the usual 30-residue mammalian B chains. Several features of the tertiary and quaternary structure of hagfish insulin, including the probable absence of a metal ion-stabilized hexameric form, are discussed on the basis of these findings. The results suggest that the conformation of the insulin molecule has been well conserved throughout the entire evolution of the vertebrates.


Initiation of protein synthesis. Binding of messenger RNA. Complexes between 30 S ribosomal subunits and fMet-tRNA are formed during incubation of 30 S subunits with fMet-tRNA and all other components for initiation of protein synthesis, except R17 bacteriophage RNA. That these complexes serve as intermediates in the binding of messenger RNA is demonstrated directly by the finding that upon addition of R17 RNA, fMet-tRNA in preformed fMet-RNA-30 S complexes preferentially enters fMet-tRNA-30 S-R17 RNA complexes. On the other hand, incubation of 30 S ribosomal subunits with R17 RNA and all other components for initiation except fMet-tRNA does not yield 30 S-R17 RNA complexes that can act subsequently as functional intermediates in the binding of fMet-tRNA: formation of fMet-tRNA-30 S-R17 RNA complexes does not occur when fMet-tRNA is added and further binding of R17 RNA to 30 S subunits is prevented by specific inhibitors. These experiments lead to an unambiguous order of events in the sequence of initiation, in which binding of fMet-tRNA to the small ribosomal subunit must occur before messenger RNA can be bound and phased correctly. Complexes between fMet-tRNA and 60 S subunits are in rapid equilibrium with the free components, and have a half-life of less than 2 min at 37 degrees. This explains why such complexes are not detected in sucrose gradients, unless they are first fixed with glutaraldehyde. Attachment of R17 RNA, however, results in formation of an fMet-tRNA-30 S-R17 RNA complex that is stabilized greatly; fMet-tRNA in this complex exchanges only very slowly with free fMet-tRNA. Initiation factor IF-3 has two functions in initiation. The first is to direct the binding of messenger RNA to the 30 S-fMet-tRNA complex. This function is not needed when initiation complex formation occurs on ApUpG triplets, in which case the second function of IF-3 is detected, that of providing free 30 S subunits for initiation. The ability of IF-3 to bind directly to R17 RNA may be related to its requirement in messenger RNA recognition. However, since IF-3 exhibits a greater affinity for the 30 S subunit than for R17 RNA, it appears that the recognition function of IF-3 is expressed while IF-3 is associated with the 30 S subunit.


Translational repression of a viral messenger RNA by a host protein. It is shown that factor i, a bacterial protein, specifically inhibits that step in the initiation of R17 bacteriophage RNA translation that involves the attachment of native R17 RNA to 30 S ribosomal subunits carrying fMet-tRNA. This inhibition by factor i is relieved by the addition of excess R17 RNA, but not by the addition of excess 30 S subunits. That R17 RNA is the only target of the inhibition is demonstrated further by the fact that in a cell-free extract containing all components for protein synthesis, factor i-mediated inhibition of exogenous R17 RNA translation can be overcome only by the addition of excess R17 RNA and not by excess cell-free extract. Upon relief of inhibition, phage coat protein synthesis is restored; enhancement of formation of other cistron products is not seen. While initiation of R17 RNA translation is blocked by factor i, chain elongation is not affected. Although foactor i inhibits the IF-3-dependent binding of R17 RNA to fMet-tRNA-30 S complexes, under conditions of initiation of protein synthesis formation of stable complexes between factor i and IF-3 could not be detected, and factor i did not interfere with the binding of IF-3 to free, native R17 RNA. Instead of affecting the function of IF-3 or ribosomes, factor i exerts its inhibition by binding to R17 RNA and acting as a translational repressor. Factor i prefers intact R17 RNA to fragments generated by autoradiolysis; its binding to R17 RNA is specific in that little competition is observed by transfer RNA, ribosomal RNA or poly(A). However, factor i has a high affinity for poly(U) sequences.