Pathogenesis of inflammatory periodontal disease. A summary of current work. Chronic periodontitis, a common disease of microbial origin, is the major cause of tooth loss in adult humans. The disease serves as a convenient experimental model for analysis of many aspects of chronic inflammation. A consideration of currently available data has permitted the formulation of a new concept of the pathogenesis of this disease. The gingival tissues respond within 2 to 4 days to a beginning accumlation of microbial plaque with a classic acute exudative vasculitis which we have termed the initial lesion. This response, which includes loss of perivascular collagen, is comparable to that elicited in most other tissues subjected to acute injury and may be a consequence of the elaboration and release of chemotactic and antigenic substances by microbial plaque. Within 4 to 10 days, the early lesion develops. It is characterized by a dense infiltrate of lymphocytes and other mononuclear cells, pathologic alteration of fibroblasts, and continuing loss of the connective tissue substance. The structural features of the early lesion are consistent with those expected in some form of cellular hypersensitivity, and a mechanism of this kind may be important in the pathogenesis. The early lesion is followed by the established lesion which develops within 2 to 3 weeks and is distinguished by a predominance of plasma cells in the absence of significant bone loss. The established lesion, which is extremely widespread in humans and in animals, may remain stable for years or decades, or it may become converted into a progressive destructive lesion. Factors causing this conversion are not understood. In the advanced lesion, plasma cells continue to predominate although loss of the alveolar bone and periodontal ligament, and disruption of the tissue architecture with fibrosis are also important characteristics. The initial, early, and established lesions are sequential stages in gingivitis and they, rather than the advanced lesion which is manifest clinically as periodontitis, make up the major portion of inflammatory gingival and periodontal disease in humans.


Cystadenoma and cystadenocarcinoma of the pancreas. Cystadenoma and cystadenocarcinoma comprise the majority of the true neoplastic cysts of the pancreas. Five cystadenomas and two cystadenocarcinomas of the pancreas were found in a review of the clinical records of the University of Minnesota Hospitals and the Minneapolis Veterans Administration Hospital. The report of these cases serves to review the clinical and pathological features of these neoplasms. These rare neoplastic cysts occur predominantly in women and are not associated with a history of trauma, alcoholism, or pancreatitis. They produce symptoms by compression of neighboring organs and are slow growing, often attaining large size. With complete excision the results of surgical treatment are usually good.


Antiperistaltic bowel segment for prevention of ileoproctostomy diarrhea. Persistent diarrhea accompanied by water, electrolyte, and nutritional depletion and skin excoriation can occur following subtotal colectomy with ileoproctostomy. Attempting to improve the course of these patients, an experimental study using dogs was undertaken to evaluate the use of antiperistaltic ileal segment in the management of ileoproctostomy diarrhea. Subtotal colectomy with ileoproctostomy was performed in dogs. One animal died from weight loss and massive diarrhea with water and electrolyte depletion. The surviving dogs had water and electrolyte imbalance and lost an average of 30.6% of their body weight. A second group of animals was treated identically except that an antiperistaltic ileal segment was placed one foot proximal to ileorectal anastomosis. These dogs maintained their weight and electrolyte and water balance. The stools of these animals became solid, as opposed to the watery diarrhea of the animals with ileoproctostomy only. Ileoproctostomy diarrhea can be successfully controlled by the use of antiperistaltic ileal segment.


The contribution of atrial contraction to right heart function before and after right ventriculotomy. Experimental and clinical observations. Effective atrial contraction was abolished by simultaneous atrial and ventricular (A-V) pacing in an acute canine preparation. At constant cardiac output, aortic pressure, and heart rate, only a small rise (1.4 mm. Hg) in mean right atrial pressure was observed before vertical right ventriculotomy, but a larger rise (9.5 mm Hg) occurred after ventriculotomy (p less than 0.01). Right heart failure with tricuspid regurgitation was induced after ventriculotomy by volume overload and A-V pacing. Restoration of atrial contraction (sequential A-V pacing) eliminated regurgitation and lowered average mean right atrial pressure from 22 to 4 mm. Hg (p less than 0.001). After right ventriculotomy, loss of atrial contraction at a constant right atrial pressure resulted in a 42 per cent reduction in cardiac output. In 8 patients who had had right ventriculotomies, abolition of effective atrial contraction by A-V pacing caused an average reduction in cardiac output of 22 per cent, whereas cardiac output fell only 5 per cent in 5 control patients (p less than 0.01).


Function of lower lung lobe autograft in experimental animals. The changes in ventilation, gas exchange, blood gases, and acid-base balance, as well as the compensatory capacity of the autotransplanted lung lobe, were studied in 30 dogs with an uncomplicated course following autotransplantation of the lower lobe of the left lung. The studies demonstrated that in the early postoperative period some deterioration in ventilation and gas exchange is noted in the autografted lobe which results in the development of arterial hypoxemia and metabolic acidosis. A normalization of the ventilation and gas exchange is observed in the beginning of the late postoperative period (3 months after surgery). As judged by the functional test with the exclusion of the right (intact) lung from respiration, the compensatory capacity of the autografter left lower lobe remained reduced for a long time after surgery. The author noted no restoration of the Hering-Breuer reflex on the side of the autografted lung lobe, the maximum follow-up period being 7 1/2 years.


Pulmonary pathology in acute respiratory insufficiency: lung biopsy as a diagnostic tool. Forty-two patients underwent open-lung biopsy during the early phase of acute respiratory insufficiency. Correlation between the gross appearance of the lung at operation and the microscopic findings was good. Although only fair correlation was found between lung and tracheal cultures, the findings of two positive cultures in the lung only was of utmost importance. Biopsying multiple areas from the same operation showed identical pathology in 86 per cent of cases. The mortality rate of open-lung biopsy was zero; the morbidity rate was 4 per cent. The over-all survival rate of acute respiratory insufficiency (ARI) due to trauma was 39 per cent; that of pneumonia, 11 per cent. In 17 (33 percent) patients specific diagnoses and/or specific therapies were employed as a direct result of the biopsy or the thoracotomy. The incidence and prognostic implications of fibrosis and microthromboembolism are presented and discussed. Open-lung biopsy has been extremely safe and valuable in characterizing and managing ARI.


Double-outlet right ventricle. A collective review with a surgical viewpoint. Double-outlet right ventricle (DORV) should be classified according to its various subsets. Van Praagh's symbolic terminology provides an appropriate mechanism for doing that. Each of these subsets is described in terms of its pathology, clinical characteristics, cardiac catheterization findings, and operative repair. The operative repair for each is different. The presence or absence of continuity between the semilunar and atrioventricular valves is unimportant from the standpoint of operation and should not be a criterion for classification.


Cardiac output in infants and children after open-heart surgery. Green dye measurements of cardiac index (in liters per minute per square meter) were compared in 32 infants and 58 children, 6 and 24 hours after normothermic cardiopulmonary bypass. Cardiac index was significantly lower in infants under one year of age, as compared to older infants and children. This parameter was also lower in patients with total anomalous pulmonary venous drainage, truncus arteriosus, and tetralogy of Fallot. Five infants who died had 6-hour cardiac index determinations of 2.0 L./min./sq. M. Only 2 of 27 survivors had cardiac indices below this level. Simultaneously recorded heart rate and central venous pressure did not correlate with cardiac index. Systolic blood pressure was significantly lower in infants in whom the cardiac index was less then 2.0 L./min./sq. M. (p less than 0.01). However, in individual situations, determination of systemic blood pressure lacked specificity in predicting outcome. Measurements of cardiac index are helpful in the management of infants and children following intracardiac repair by (1) assessing the hemodynamic status, (2) providing a useful indication of the cardiocirculatory effects of therapeutic interventions, and (3) predicting outcome.


Bone marrow lymphocytes.

Bone marrow lymphocytes. Some aspects of bone marrow lymphocyte production are reviewed. Attention is drawn in particular to fundamental histogenetic differences in the production of B and T lymphocytes. B lymphocyte precursors are transitional cells.


Soluble proteins of human bronchogenic carcinomas. Solid-phase affinity chromatography has been used to search for tumor-associated proteins of bronchogenic carcinomas. All of the apparently normal proteins were removed from extracts of squamous cell carcinoma, large cell carcinoma, and adenocarcinoma of the lung. The remaining soluble proteins were partially characterized as to heat stability, approximate molecular size, electrophoretic mobility, and biologic function. These tumor-associated proteins were not tumor-specific by current definition. Neither lung cancer patients' lymphocytes nor serum proteins were specifically reactive with these tumor-associated proteins.


Stimulation-produced analgesia.

Stimulation-produced analgesia. Recent experimental and clinical reports have indicated that analgesia can be produced by stimulation techniques. The mechanism of stimulation-produced analgesia has been proposed to involve neurophysiologic and neurohumoral inhibitory effect at the level of the spinal cord, brain stem, thalamus, and cortex. Research in this area will improve the chance of understanding pain mechanisms and providing effective methods for pain control.


The spectrum of von Willebrand's disease revisited. We have examined nine patients with presumed von Willebrand's disease who present the spectrum of that disorder. Two had findings that would be accepted generally as diagnostic of von Willebrand's disease, and seven had variations of the usual pattern. The commonest variation was the combination of borderline and variable levels of coagulant Factor VIII, commensurate levels of Factor VIII-related antigen, and low levels of ristocetin-Willebrand factor.


The use of radioisotope methods in pharmacologic and toxicologic studies. By the use of radioisotopes, some pharmacologic studies can be carried out with less perturbation of physiologic processes than with other methods. Some isotope methods used at the Chair of Pharmacology of the Medical Academy in Lódź include determination of capillary circulation in the heart and skeletal muscle, minute output and stroke volume of the heart, renal clearance of 125J hippurate, and levels of some hormones and enzymes.


An experimental analysis of liver development. Liver differentiation results from cellular interactions between the mesoderm and the endoderm. The presumptive hepatic endoderm is determined to differentiate into parenchymal cells from the 4-5 somite stage under the influence of the precardiac mesoderm. Later on, the endodermal cells proliferate and give rise to the hepatic buds from which strands of glandular cells are derived. The mesenchymal liver component stimulates both proliferation and differentiation of the endodermal cells. Its action is, however, not specific since the same effect is obtained with all the mesenchymes derived from the lateral plate mesoderm. The characteristics of the mesenchymal liver component were studied. In both quail and chick a cholinesterase activity was detected in the mesenchymal liver cells, which show an extensive development of the granular ergastoplasmic reticulum and of the Golgi apparatus. A close contact between endodermal and mesenchymal cells seems to be required for the cell interactions to occur. In monolayer cultures, glycogen synthesis ceases in the hepatocytes which after some days become fibroblastic in appearance. However, such a dedifferentiation is not irreversible. If reassociated with hepatic mesenchyme the fibroplastic cultures can recover their ability to synthesize and store glycogen.


[Problems in the surgical treatment of sliding hiatal hernia (author's transl)]. The aim of surgical treatment of hiatus hernia is well defined: restoration of the esophago-gastric closing mechanism to prevent further reflux of gastric contents and gastric juice into the esophagus. The main problem to realize this demand lies in the variety of discussed closing mechanisms. Interaction of different mechanisms influencing the "anti-reflux barrier" as well as hormonal and neural regulation of the lower esophagus are not yet examined sufficiently enough. Benefits and hazards of several types of operations for repair of hiatal hernia are discussed before the background of the small but slowly growing understanding of the esophago-gastric closing mechanism.


[A new organ perfusion system: Successful preservation of kidney function for 72 hrs (author's transl)]. A new preservation system is described. In this unit 17 dog kidneys were nonpulsatile perfused with hypothermic human albumin for 72 hrs and then transplanted. All kidneys started immediately to function and the PAH and inulin clearances attained normal values at least at the 7th postoperative day. The quality of the immediate function is referred to the applied perfusion pressure (20 mm Hg) and the applied temperature of the perfusion medium (7.5 degrees C). During preservation substrate utilization (oxygen, unesterified fatty acids) and the accumulation of toxic metabolic products (ammonia, uric acid) were controlled in the perfusate. Their importance concerning to the limitation of preservation time is discussed.


[Automation of cardiological studies of the population]. To conduct large-scale medical examinations of the population with a view to detecting cardiovascular affections an experimental automated complex has been set up. The program of the examination to be instituted is largely drawn up conformable to recommendation of the WHO. In the complex are envisaged collection and registration of the data on the patient, biochemical analyses of the blood and urine, compilation of the case-history, anthropometric measurements and determination of the arterial blood pressure, ECG recording, veloergometric load test measurements, along with spirometric ones and the measurement of the subcutaneous fat layer thickness. The processing of the information obtained is done at the computation under "off-line" conditions.


[Automatic analysis of electrocardiograph signals in the diagnosis of myocardial infarct]. At present the work of automatization of the processes of the ECG analysis for diagnosing myocardial infarction with the help of computer technique is going forward on an ever broader scale. For an automatic identification of the focal changes syndrome on the ECG, which in the clinical practice is considered to be tantanount to myocardial infarction, the use of a special diagnostic device (SDD) is proposed. The SDD are comparatively cheap and simple in operation. The process of diagnosing provides for utilization of medical experience, but, at the same time, eliminates shortcomings inherent in systems that reproduce the physicians' manipulations in establishing the diagnosis of myocardial infarction with the help of an electrocardiogram. The choice of the SDD was made by stimulating it with an all-purpose computer. The results of clinical trials of the SDD conducted at specialized clinics bear proof to great possibilities of the automatic electrocardiographic diagnostics.


Glucagon responses to hypoglycemia in adrenalectomized man. In order to examine the possibility that epinephrine is involved in either the mediation or modulation of the enhanced glucagon release during glucopenia, glucagon responses to insulin-induced hypoglycemia were evaluated in four men with bilateral adrenalectomy in comparison with ten normal men. In the adrenalectomized patients, the mean nadir of plasma glucose and the rate of ascent to baseline levels were indistinguishable from those observed in normal subjects. Similarly, glucagon responses in the adrenalectomized group were not different from those encountered in the normal volunteers. We conclude that epinephrine does not contribute significantly to the augmented glucagon release during abrupt glucopenia in normal man and is not necessary for normal recovery from hypoglycemia.


[Technics for isolation of ATP from microorganisms]. A technique for isolation of ATP from microorganisms is suggested. The technique is based on increasing permeability of the cell membrane for substances of low molecular weight during dehydration. Treatment of the dry biomass with boiling water gives a higher yield of ATP from the cells of eukaryote and prokaryote microorganisms as compared with other techniques, and permits to register the amount of APT at the moment of experiment.


[Effect of cultivation conditions on the electrophoretic spectrum of various dehydrogenases in Saccharomyces cerevisiae]. The activity and electrophoretic spectrum of glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, and malate dehydrogenase in Saccharomyces cerevisiae 14 depended on the conditions of cultivation, i. e. the sources of carbon and their concentration, the content of nitrogen, and aeration of the medium. A heterogeneous tris-buffer protein fraction was isolated after disintegration of the cells; the dehydrogenases were represented by several molecular forms whose number, electrophoretic mobility, and activity depended on the conditions of cultivation.


[Bactericidal activity of serum and chemotherapy in sensitive and resistant exciter (author's transl)]. Comparing examinations with Ampicillin sensitive  and resistant bacteria-strains show that the bactericidal activity of serum is dependent on the bacteria-strains, on the Ampicillin sensitivity of the particular exciter and on the number of bacteria/ml (germ count). Bactericide effect could always be obtained with sensitive strains as a result of additional chemotherapy. With several resistant strains a bactericide effect could not be obtained in this case the continuous optimal Ampicillin addition was the decisive factor. Because of the extremely complicated process of the bactericide one should not make general conclusions from the individual experimental results.


[Indanyl Carbenicillin in the treatment of urinary tract infections due to difficult urological organisms (author's transl)]. Fifty in-patients suffering from chronic urinary tract infections with underlying urological diseases received the oral Carbenicillin preparation Indanyl Carbenicillin (short term Carindacillin) for 10 days in a dosage of 1 g q.i.d. The therapy results were objectified by blood and urine tests performed prior to and after treatment. Indanyl Carbenicillin led to a sterile urine in nearly 60% of the patients; in about 85% of the cases the primary organism was eliminated. The drug was particularly active against difficult organisms such as Proteus, Klebsiella and Pseudomonas. The results obtained in this study are somewhat surprising as the dosage used produces bactericidal levels of the active substance only in the cavity system of the efferent urinary tract but not in the tissue. No effect on blood picture or liver function was observed. Side effects occurred in 8 patients, they disappeared with continued therapy or after discontinuation of the drug. Considering the good efficacy observed in this study and because of the possibility of oral administration, Indanyl Carbenicillin appears to be suitable also for out-patient treatment of chronic urinary tract infections due to difficult urological germs.


Passive oral immunization with bovine immunoglobulins: enterpathogenic Escherichia coli from infants and bovine anti-E. coli lactoserum assayed in the rabbit ileal loop model. The effect of immune bovine lactoserum (BLS) antipolyvalent enteropathogenic Escherichia coli on bacterial growth, viability and bacteria-induced fluid accumulation was examined in rabbit ileal loops. Human enteropathogenic E. coli strains 0125:K70 (B15), 0111:K58 (B4) and 055:K59 (B5) (1-3 X 10(9) per inoculum) induced secretion of 4-6 ml fluid per 10 cm loop. This effect was inhibited effectively by BLS (corresponding to 50 mg IgG 1 per loop) while the viability of bacteria counts decreased 2-25 fold compared with bovine serum albumin. E. coli 026:K60 (B6), 0126:K71 (B16) and 0127:K63 (B8) caused moderate secretion (2-3 ml/10 cm loop) that was significantly neutralized by BLS. E. coli 086:K61 (B7) and 0128:K67 (B12) did not give positive loops. The fluid secretion was shown to be dose dependent for E. coli 0125:K70 (B15) over the range from 2.5 X 10(9) to 8 X 10(7) bacteria/loop. The titration of the effect of BLS on fluid secretion caused by the same strain revealed a dose dependent decrease. The best inhibition was obtained with 100 mg BLS/loop, the highest dose tested.


[Antibody differentiation in human toxoplasmosis experiments on model cases (author's transl)]. Human sera with antibodies against Toxoplasma gondii were investigated-before and after gel filtration (Sephadex G 200)-by IgM-and IgG-specific immunofluorescent reactions as well as well by complement fixation tests.-In sera from patients with "latent" infections IgM-antibodies could not be demonstrated neither by the IgM-IFT-nor the CFT technique, although IgG antibodies with IFT titres between 1:50 qnd 1:200 were present.-In sera from patients in the "acute" stage of infection both IgM and IgG antibodies were always found. In some cases the IgG fraction had complement fixing properties only, although IgM antibodies were present as demonstrable by the IFT technique. -These observations leed to the possibility of immunologic differentiation between IgM-IFT and CF-antibodies. The role of the different antibodies in the serodiagnosis of human toxoplasmosis is discussed.


recA+-dependent inactivation of the lambda repressor in Escherichia coli lysogens by gamma-radiation and by tif expression. When gamma lysogens of E. coli are induced by gamma-radiation the gamma repressor, as measured by its specific binding to gamma DNA, is rapidly inactivated by a recA+-dependent process which does not require new protein synthesis. This rapid inactivation is similar to inactivation of repressor by expression of the temperature sensitive E. coli mutation tif. In contrast, induction by UV irradiation or mitomycin C treatment requires new protein synthesis and there is a lag before the repressor is inactivated (Tomizawa and Ogawa, 1967; Shinagawa and Itoh, 1973).


Transversion mutagenesis in bacteriophage T4. Transversion mutations can be distinguished from transition mutations by the use of special tauII mutants of bacteriophage T4. Methyl methanesulfonate did not induce reversion of the tester mutants along transversion or transition pathways from A:T1 base pair sites, nor along transversion pathways from G:C base pair sites. Ethyl methanesulfonate and N-methyl-N-nitrosourea, however, induced both transversions and transitions at an A:T base pair site; no transversions were detected at G:C-sites. Mn++ induced transversions and transitions at both A:T-and G:C-sites. The influence of temperature-sensitive gene-43 DNA polymerase mutator and antimutator mutations on the reversion of the tauII tester mutants was measured: some gene-43 mutants differentially influenced different pathways of reversion. Studies of thymineless mutagenesis demonstrated A:T-site transversion mutations. A synergistic interaction between thymineless mutagenesis and the gene-43 mutator, tsL56, was used to demonstrate thymineless mutagenesis at one site where it was not detected in the presence of the wild type polymerase.


Zygote heterogeneity and uniparental inheritance of mitochondrial genes in yeast. A number of different crosses between strains of Saccharomyces cerevisiae differing in mitochondrial genotype are analyzed with respect to the extent to which individual zygotes transmit mitochondrial genes from one parent or the other. Many crosses produce two or more distinct classes of zygotes in this respect. Some crosses produce a high frequency of uniparental zygotes, which transmit mitochondrial genes  exclusively or nearly so from one parent. Such zygotes cannot be accounted for in terms of an unequal input of mitochondrial DNA molecules from the two parents; they indicate that mitochondrial DNA from one parent is selectively replicated, or mitochondrial DNA from the other parent is selectively destroyed, in the zygote. Multiple zygote classes, and uniparental zygotes, are seen in studies of mitochondrial and chloroplast inheritance in other organisms, and may have a common explanation.


Three-marker extranuclear mitochondrial crosses in Aspergillus nidulans. Two- and three-point extranuclear crosses have been carried out via heterokaryons involving the three extranuclear mitochondrial markers of Aspergillus nidulans: (oliA1), (cs67) and (camA112). All three markers appear to be located on a single functional mitochondrial genome. Recombination between all three pairs of extranuclear markers appears to be equally frequent, suggesting a lack of genetic linkage. An important feature of these results is the variable and often marked non-equality of frequency of reciprocal classes of recombinants.


Two new genes controlling the constitutive acid phosphatase synthesis in Saccharomyces cerevisiae. Two new classes of mutants, phoF and phoG, lacking the constitutive acid phosphatase activity, were isolated. They both complemented each other and the phoC mutation. No linkage was detected among these three complementary genes.


IS1 and IS2 mutations in the ribosomal protein genes of E. coli K-12. The insertion mutations I15 and I16 in the ribosomal protein gene cluster at 64 min in Escherichia coli are identified as IS1 and IS2 using electron microscope heteroduplex analysis.


The restriction of the recombination of mitochondrial DNA molecules in the zygotes of Saccharomyces cerevisiae. Crosses were made between strains carrying a nuclear control factor (NC) and strains classified with respect to their omega allele (omega+ or omega-). The characteristic asymmetrical transmission was always observed (as was seen) in crosses not involving the omega factor. The analysis of functional recombinants in a cross involving an NC factor has indicated that the absence of the omega effect may be caused by a restriction in the zygote of the recombination of mitochondrial DNA molecules.


Biogenesis of mitochondria. 43. A comparative study of petite induction and inhibition of mitochondrial DNA replication in yeast by ethidium bromide and berenil. The action of ethidium bromide and berenil on the mitochondrial genome of Saccharomyces cerevisiae has been compared in three types of study: (i) early kinetics (up to 4 h) of petite induction by the drugs in the presence or absence of sodium dodecyl sulphate; (ii) genetic consequences of long-term (8 cell generations) exposure to the drugs; (iii) inhibition of mitochondrial DNA replication, both in whole cells and in isolated mitochondria. The results have been interpreted as follows. Firstly, the early events in petite induction differ markedly for the two drugs, as indicated by differences in the short-term kinetics. After some stage a common pathway is apparently followed because the composition of the population of petite cells induced after long-term exposure are very similar for both ethidium bromide and berenil. Secondly, both drugs probably act at the same site to inhibit mitochondrial DNA replication, in view of the fact that a petite strain known to be resistant to ethidium bromide inhibition of mitochondrial DNA replication was found to have simultaneously acquired resistance to berenil. From consideration of the drug concentrations needed to inhibit mitochondrial DNA replication in vivo and in vitro it is suggested that in vivo permeability barriers impede the access of ethidium bromide to the site of inhibition of mitochondrial DNA replication, whilst access of berenil to this site is facilitated. The site at which the drugs act to inhibit mitochondrial DNA replication may be different from the site(s) involved in early petite induction. Binding of the drugs at the latter site(s) is considered to initiate a series of events leading to the fragmentation of yeast mitochondrial DNA and petite induction.


Alteration of ribosomal proteins in revertants of a valyl-tRNA synthetase mutant of Escherichia coli. The work described in this paper was done to see whether the partial suppression of temperature-sensitive aminoacyl-tRNA synthetase mutations by ribosomal mutations is restricted to the aminoacyl-tRNA synthetase mutation which was used for the selection of the suppressor strains or whether the ribosomal mutations can also suppress mutations of other aminoacyl-tRNA synthetases. It is shown that a mutation in ribosomal protein S5 which was selected for suppression of an alanyl-tRNA synthetase mutation (alaS-3) can also partially compensate the temperature-sensitivity of two valyl-tRNA synthetase mutants and of another alanyl-tRNA synthetase mutant. Furthermore, revertants of a temperature-sensitive valyl-tRNA synthetase mutant were isolated and screened for alterations in ribosomal proteins by electrophoretic and immunochemical methods. Alterations in at least two proteins, S8 and S20, were clearly observed among the mutants. The alteration in protein S8 renders the growth of this strain severely cold-sensitive. Presence of the mutation in protein S8 is strictly correlated with suppression of temperature-sensitivity. The S8 mutation maps between strA and spc on the Escherichia coli chromosome. Five suppressor strains have quantitatively or qualitatively altered ribosomal proteins S20. In one strain no S20 protein could be detected at all, employing different electrophoretic and immunological methods. All five suppressor mutations map in the thr-leu region of the E. coli chromosome, i.e. in an area where the alteration of protein S20 in two alaS suppressor strains has been localized previously.


Analysis of ribosomal proteins in streptomycin resistant and dependent mutants isolated from streptomycin independent Escherichia coli strains. Mutants resistant to (Str-R) or dependent on streptomycin (Str-D) were isolated from several streptomycin independent (Str-I) strains of Escherichia coli. From 90 of these mutants ribosomes were isolated and the ribosomal proteins analyzed by two-dimensional polyacrylamide gel electrophoresis. The results which are summarized in Tables 1-4 led to the following conclusions: a) The phenotype (Str-R) or Str-D) of the mutants isolated from the Str-I strains strongly depends on the parental strain. b) No other ribosomal proteins than S4, S5 and S12 seem to be altered by mutations leading to dependence on, independence from or resistance to streptomycin. c) The S4 proteins of the analyzed mutants belong to three groups. The ratio  between the groups depends more on the origin of the mutants than on their phenotype. d)Eight new types of altered S4 proteins were detected. It is very likely that many, if not all, of the altered S4 proteins originated by frame shift mutations. e) Some of the mutants differ from the wild type by alterations in three ribosomal proteins (S4, S5 and S12). The alteration in one protein, S4, apparently compensates for that in another protein, S5, in such a way that the original phenotype is expressed. These mutants are therefore an excellent tool for studies at the molecular level on the interaction of ribosomal components within the particle.


Isolation and properties of a conditionally lethal bacteriophage lambda mutated in the chi region. A thermosensitive lambda phage mutant was isolated which can grow at high temperature only in the presence of the lambda chi gene product supplied in trans. This mutation tn was mapped within the chi region, and lambda tn phage expressed the pRoR-OP operon only poorly at high temperature. Effects of the tn mutation on expression of other operons were also examined and compared with those observed with cro27 or some tof mutations.


Regulation of the ilv 1 multifunctional gene in Saccharomyces cerevisiae. The ilv 1 gene in S. cerevisiae codes for a regulatory protein involved in depression of the ilv 2 and ilv 3 genes as well as a biosynthetic enzyme, threonine deaminase. 2. The ilv 1 gene does not autogenously regulate its catalytic product threonine deaninase. 3. Regulation of the ilv 2 and ilv 3 gene products involve different aporepressors than regulation of the ilv 1 gene product. 4. The ilv I multifunctional gene in S. cerevisiae may be a duplication and fusion of a bacterial like ilv 1 gene where ilv 1 catalytic and regulatory function have been differentially conserved.


Cytogenetic demonstration of mitotic chromosomes in the yeast Saccharomyces cerevisiae. A cytogenetic study of the nuclear material of the yeast Saccharomyces cerevisiae as it appears during mitosis was undertaken using a technique which combines light and electron microscopic examination of spheroplasts. Condensation of chromatin into separate chromosomes is observed. The most frequent chromosome number counted is 18.


Cooperative control of translation fidelity by ribosomal proteins in Escherichia coli. I. Properties of ribosomal mutants whose resistance to neamine is the cumulative effect of two distinct mutations. Two spontaneous mutants of Escherichia coli strain KMBL-146 selected for resistance to the aminoglycoside antibiotic neamine show severe restriction of amber suppressors in vivo. Purified ribosomes from the mutant strains exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin. Biochemical analysis shows that the mutants each have two modified 30S ribosmal proteins, S12 and S5. In agreement with these results, genetic analysis shows that two mutations are present, neither of which confers resistance to neamine by itself; the mutation located in gene rpxL (the structural gene for protein S12) confers streptomycin dependence but this dependence is suppressed in the presence of the second mutation, located in gene rpxE (the structural gene for protein S5).


Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli. II. Localization of amino acid replacements in proteins S5 and S12 altered in double mutants resistant to neamine. Protein S5 and S12 were isolated from 30S ribosomal subunits of two E. coli mutants highly resistant to the antibiotic neamine, and of the parental strain. Proteinchemical analyses on these proteins led to the following results: a) In protein S5 the arginine residue in peptide T2 of the parental strain is replaced by glycine in one (nea 314) or serine in the other (nea 319) of the two mutants. b) In protein S12 The proline residue in peptide T15 of the parental strain is replaced by leucine in mutant nea 314 and by glutamine in mutant nea 319. Comparison of these results with those obtained in earlier studies on other mutants with altered ribosomal proteins revealed that the amino acid replacements in neamine resistant mutants and in "revertants" from streptomycin dependence occur at the same amino acid positions of proteins S5 and S12. Therefore it is likely that both types of mutants belong to the same class.


Biosynthesis of RNA polymerase in Escherichia coli. II. control of RNA polymerase synthesis during nutritional shift up and down. As an effort to elucidate the control of quality and quantity of the DNA-dependent RNA polymerase in Escherichia coli, the rate of synthesis of the individual subunits was determined during shift-up and -down of nutrients. When the strain B/r grown in a succinate medium was imposed to a shift-up by adding a mixture of glucose and amino acids, rapid rise was observed of the differential rates of the synthesis of alpha, beta and beta' subunits, the constituents of core enzyme, leading to the increase of core polymerase concentration. The differential rates decreased thereafter to the level characteristic of the post-shift rate of cell growth. Compared to the strain B/r, the adaptation was slow in the strain K12 W3350. On the other hand, upon transfer of the strain B/r from a glucose-amino acids medium to a glucose medium lacking amino acids, the differential rate of core polymerase synthesis decreased rapidly and then regained the rate characteristic of the new growth rate. Similar control was also observed on the rate of ribosomal protein synthesis suggesting the coordinate expression of genes for the core polymerase subunits and ribosomal proteins. Thus, the intracellular concentration of RNA polymerase as well as of ribosomes might be one of the most important factors that affect the rate of bacterial growth. The rate of alpha subunit synthesis, however, exhibited little change during the shift-up but a considerable decrease was observed during the shift-down.


Control of gene expression in bacteriophage lambda: suppression of N mutants by mutations of the antirepressor. The lysogenization and induction properties of phages lambdasusN7CI857Ai7 and lambdasusN53cro27 are described. Both phages, at 32 degrees kill little, but show only a moderate frequency of lysogenization whether an amber suppressor is present or absent in the host bacterium. In  the latter case, lysogens for lambdasusN7CI857Ai7 or lambdasusN53CI857cro27 can exist in two different regulatory states, here called P r- and Pr+. The Pr+ phase is characterized by phage release and cell death at 40 degrees; conversely, cells in the Pr- phase are similarly killed but release no or very little phage. Pr- is the phase usually obtained at lysogenization. Each phase may be transmitted at 32 degrees for an unlimited number of generations, however, shifts to the opposite phase take place from time to time with a low probability. Two previously described antirepressor defective mutants. Ai7 and cro27, were found to suppress specifically the growth defect caused by an amber mutation in gene N. This suppression is observed in non-suppressing hosts at 40 or 42 degrees. Apparent revertants of N- mutants were shown to be often (80%) caused by a second mutation, in the Ai gene (also called tof, cro and fed). All the revertants so far examined appeared to be recessive. Lambda phages bearing a double amber mutation in gene N did not acquire full N independence by the acquisiton of an Ai mutation; this could be achieved, however, in the presence of a CII mutation. The above findings are discussed in terms of a direct interaction between the N, Ai and CII products.


Persistence and decay of thermoinducible error-prone repair activity in nonfilamentous derivatives of tif-1, Escherichia coli B/r: the timing of some critical events in ultraviolet mutagenesis. Ultraviolet (UV) mutagenesis in E. coli is associated with a UV-inducible type of error-prone postreplication repair ("SOS" repair) which, in tif-1 strains, is thermo-inducible in coordination with other recA+ lexA+-dependent inducible functions, including filamentous growth. Mutants of E. coli B/r tif-1 strains have been isolated which retain thermoinducibility of SOS repair activity, but lack the thermosensitivity caused by filamentous growth at 42 degrees C. These strains have been used to determine: the kinetics of decay at 30 degrees C of thermally induced ability to enhance UV mutagenesis; the kinetics of thermal enhancement of spontaneous and UV-induced mutability at 42 degrees C, and the kinetics of decay at 30 degrees C of susceptibility to thermal enhancement of spontaneous and UV-induced mutability. Mutations from tryptophane requirement to prototrophy (Trp- to Trp+) were scored. UV doses were 0.2 J/M2 for excision repair-deficient (Uvr-) and 2J/m2 for Uvr+ strains. The results support the following conclusions. 1) thermally induced SOS repair activity decays at 30 degrees C to about 25% of its maximum level in 45 min, and is no longer detectable after 90 min. 2) Thermal enhancement of UV mutability occurs at sites produced primarily (perhaps exclusively) before completion of the first post-irradiation cell division. 3) UV-induced sites susceptible to thermally induced SOS repair are stable at 30 degrees C in cells not containing the error-prone repair system, and are refractory to constitutive error-free repair for at least 2-3 hours. 4) UV produces a potentially mutagenic type of photoproduct in DNA which can, without interacting with another UV lesion, provide a site susceptible to SOS repair, but which is not a sufficient signal for SOS induction. 5) 50-70% of the SOS-mutable SOS-noninducing UV photoproducts are photoreversible pyrimidine dimers. The results are discussed in relation to current models of UV mutagenesis and induction of UV-inducible functions.


Strains of Escherichia coli diploid for the chromosomal origin of DNA replication. F' strains of E. coli have been isolated which are merodiploid for various chromosomal segments between 66 and 78 minutes. Strains diploid for the chromosomal DNA between the genes bgl and mtl grow slowly, have a reduced DNA/mass and an increased cell size. These properties could result if the chromosomal replication origin and a second, extrachromosomal, copy of the origin (located in this case on the F') were to compete for a substance required to initiate replication. We therefore suggest that these strains are diploid for the chromosomal origin of replication and that, therefore, the origin is located between bgl and mtl, that is between 71 and 73 minutes on the E. coli chromosome.


The role of Chi mutations in the Spi- phenotype of phage lambda: lack of evidence for a gene delta. Lambda red- gam- makes a small plaque on P2 lysogens (the partial Spi- phenotype). It has been proposed that inactivation of an additional gene delta, mapping in the recombination region, makes the plaque bigger (the full Spi- phenotype) (Zissler et al., 1917b). The present paper demonstrates that the Chi mutation in lambda (Stahl et al., 1975) confers upon red- gam- phage the full Spi- phenotype and that the deletion of the region of the chromosome attributed to delta does not. It appears unnecessary to invoke a gene delta in the Spi- phenotype.


Selection of spontaneous mutants by inositol starvation in yeast. A new method for the routine isolation of mutations of spontaneous origin in the yeast, Saccharomyces cerevisiae, is reported. The technique is based on the observation that inositol auxotrophs die when deprived of inositol. However, if macromolecular synthesis is inhibited, most of the cells survive. Appropriate manipulation of inositol requiring mutants can therefore result in the selective survival of cells possessing mutations which affect macromolecular synthesis. Since reversion to inositol prototrophy can be a major source of interference in efficient selection, a haploid double mutant strain has been constructed which reverts to inositol prototrophy with a frequency estimated to be several orders of magnitude lower than the expected frequency of single, spontaneous mutational events. Using this strain, enrichment in excess of 10,000 fold has been obtained for various classes of auxotrophic mutants. Spontaneous temperature sensitive mutants have also been obtained.


The organization of genes in yeast mitochondrial DNA II. The physical map of EcoRI and HindII + III fragments. 1. We have isolated large fragments of the mtDNA of the yeast Saccharomyces carlsbergensis and digested these with restriction endonucleases. The digestion products were separated by electrophoresis in agarose gels. 2. Endonucleases EcoRI, HindII + III, HpaI, HindIII and HapII yield 9, 11, 6, 0 and greater than 80 fragments, respectively. 3. By analysis of partial digestion products and by redigesting the fragments obtained with one endonuclease with a second, we have established the order of all EcoRI and HindII + III fragments. The map is circular and its contour length is 22.1 +/- 0.35 mum, in good agreement with earlier estimates of the size of yeast mtDNA, using electron microscopy and renaturation kinetics. 4. A comparison of the fragmentation pattern of mtDNAs from S. carlsbergensis and various strains of Saccharomyces cerevisiae with endonuclease HindII + III suggests that the overall gene order is similar.


Genes coding for the structure of the acid phosphatases in Saccharomyces cerevisiae. The phoE locus, one of the loci in which mutations lack the activity for repressible acid phosphatase, was found to be the structural gene for the enzyme by examining the enzymic characteristics of repressible acid phosphatase activity using cell extracts prepared from the leaky phoE mutants, the PHOE revertants and the PHOE recombinants between the different phoE mutants. Other evidence which strongly suggests that the phoC locus is coding for the constitutive acid phosphatase was obtained by a similar investigation. Although the phoC and phoE loci are tightly linked, they were separable by meiotic recombination.


A mutant of Escherichia coli auxotrophic for organic phosphates: evidence for two defects in inorganic phosphate transport. An inorganic phosphate transport mutant has been isolated as a sn-glycerol-3-phosphate auxotroph and characterized genetically. Two lesions are responsible for the transport defect. One lesion, pst, is located at minute 74 of the E. coli genetic map while the other lesion, pit, is located at minute 68. All "K10" strains that were examined carry the pit lesion. Evidence is presented that the glycerol phosphate and hexose phosphate transport systems are not important inorganic phophate transport systems. The mapping data indicate that the genetic distance between malA and xyl is greater than that now allowed.


Induction of RNA polymerase synthesis in Escherichia coli. Studies on the rate of synthesis of the beta and beta' subunits of RNA polymerase in haploid strains of Escherichia coli K12 containing poorly-suppressed rif degrees am mutations provide conclusive evidence that synthesis of at least these two subunits is regulated.


Location on the chromosome of Escherichia coli of genes governing purine metabolism. Adenosine deaminase (add), guanosine kinase (gsk) and hypoxanthine phosphoribosyltransferase (hpt). Genes coding for enzymes functioning in purine salvage pathways have been located on the chromosome of Escherichia coli. The gene add encoding adenosine deaminase was located by transduction at 31 min, the gene order was established to be man-uidA-add-aroD. A deletion covering man-uidA-add was obtained. The gene gsk encoding guanosine kinase was cotransducible with purE and shown to be located at 13 min. The gene hpt encoding hypoxanthine phosphoribosyltransferase was cotransducible with tonA indicating a location at 3 min. The location of the gene gpt encoding guanine (xanthine) phosphoribosyltransferase in the proA-proB region was confirmed.


Transmission and recombination of extranuclear genes during sexual crosses in Aspergillus nidulans. Three extranuclear mitochondrial mutations in Aspergillus nidulans, (oliA1), (camA1) and (cs67), were used as markers in sexual crosses to provide information on the frequencies of transmission and recombination of the mitochondrial genome. Any individual perithecium contained ascospores of only one extranuclear genotype. Using mono-, bi- and trifactorial crosses it was found that all three markers could be recovered from the progeny, although the transmission frequencies were different for each marker. This bias was present irrespective of the nuclear background or the presence of selective agents in the medium on which the cross was established. These findings enable a series of "transmission strength" to be established, as shown below:-- (camA1) greater than (cs67, camA1) greater than (+) = (cs67) greater than (oliA1, cs67) greater than (oliA1) greater than (oliA1, camA1). However, the numbers of recombinants isolated were so variable as to make this form of analysis unsuitable for mapping the mitochondrial genome.


The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control. With the use of neutral sucrose sedimentation techniques, the size of unirradiated nuclear DNA and the repair of double-strand breaks induced in it by ionizing radiation have been determined in both wild-type and homozygous rad52 diploids of the yeast Saccharomyces cerevisiae. The number average molecular weight of unirradiated DNA in these experiments is 3.0 X 10(8)+/-0.3 Daltons. Double-strand breaks are induced with a frequency of 0.58 X 10(-10) per Daltonkrad in the range of 25 to 100 krad. Since repair at low doses is observed in wild-type but not homozygous rad52 strains, the corresponding rad52 gene product is concluded to have a role in the repair process. Cycloheximide was also observed to inhibit repair to a limited extent indicating a requirement for protein synthesis. Based on the sensitivity of various mutants and the induction frequency of double-strand breaks, it is concluded that there are 1 to 2 double-strand breaks per lethal event in diploid cells incapable of repairing these breaks.


Mitochondrial genetics. XI. Mutations at the mitochondrial locus omega affecting the recombination of mitochondrial genes in Saccharomyces cerevisiae. 1. A series of CS revertants has been selected from various strains (both omega+ and omega-) carrying a CR mitochondrial mutation at the RIB1 locus. The properties of mitochondrial recombination exhibited by these CS revertants in various crosses, have been examined systematically. The omega allele of the CS revertants has been defined in crosses with omega+ and omega- tester strains using two criteria: the polarity of recombination and a new criterium called relative output coefficient. We found that mutations of omega appear frequently associated with the mutations at the RIB1 locus selected from omega- strains but not with those selected from omega+ strains. A new allelic form of omega (omega n) which had not been found amongst wild type yeast strains is characterised. Similarly omega n mutation was found frequently associated with CR mutants at the RIB1 locus selected from omega- CS strains but not with those selected from omega+ CS strains. The omega n mutants, and the omega+ and omega- strains, explain the groups of polarity previously observed by Coen et al. (1970). 2. Main features of mitochondrial crosses with omega n strains (omega+ x omega n, omega- x omega n and omega n x omega n) are analysed. Recombination is possible between the different mitochondrial genetic markers. No high polarity of recombination is observed and the frequency of recombinants are similar to those found in homosexual crosses (omega+ x omega+ and omega- x omega-). A striking property, observed for the first time, exists in crosses between zota+ omega n CS strains and some zota- CREO mutants: the zota- CREO are unable to integrate by recombination their CR allele into the zota+ mit-DNA of omega n CS strains while being capable of integrating it into omega+ CS or omega- CS genomes. 3. It is proposed that the omega locus is the site of initiation of non reciprocal recombination events, the omega+/omega- pairing specifically initiates the non-reciprocal act while omega+/omega n or omega-/omega n pairings do not. 4. The molecular nature of the omega n mutation and its bearing on the structure of the omega locus are discussed. It is suggested that omega n mutations correspond to macrolesions (probably deletions) of a segment of the mit-DNA covering the omega and RIB1 loci. If omega n is a partial deletions of the omega- sequence the omega+ could be an additionnal deletion of the omega n sequence. 5. The occurrence of spontaneous CR and ER mitochondrial mutations has been analysed by the Luria and Delbrück fluctuation test in omega- and omega n isonuclear strains. Results of these tests indicate that an intracellular selection of resistant copies preexisting the action of the anttibiotic occurs.


I.n vitro transcription of adenovirus 2 DNA. I. Characterization of promoters for E. coli RNA polymerase. E coli RNA polymerase holoenzyme is able to recognize transcription initiation sites on Adenovirus 2 DNA that are functionally indistinguishable from promoters for the enzyme on phage DNAs. The complexes formed between the polymerase and the DNA at these sites can exist in two states-either as I (initiation) complexes, from which rapid RNA chain initiation is not possible, or as RS (rapid starting) "rifampicin resistant" complexes, from which rapid RNA chain initiation can occur. When transcription is limited to that initiated from stable, rifmapicin-resistant pre-initiation complexes, initiation is strictly dependent on the presence of sigma factor; in addition, the frequency of initiation exhibits sigmoidal dependence on the temperature at which pre-initiation complexes are allowed to form, with a transition temperature of 26-28 degrees C. The average half-time for initiation of RNA chains from sites on Ad 2 DNA is shown to be comparable to half-times for initiation of RNA chains from promoters on T7 and lambda DNAs. At saturating levels of enzyme, the half-times are 0.6, 0.9, and 1.6 sec for lambda b2, Ad 2 and T7 DNAs, respectively. The existence of efficient, phage-like promoters for E coli RNA polymerase on Ad 2 DNA suggests to us that such promoters may be closely related functionally and spatially to promoters for mammalian RNA polymerases.


In vitro transcription of adenovirus 2 DNA. II. Quantification and localization of promoters for E. coli RNA polymerase. We estimate that E. coli RNA polymerase is able to form stable, rifampicin-resistant, pre-intiation complexes with Adenovirus 2 DNA at three to six binding sites. The number of RNA chains initiated from such complexes has been determined form the incorporation of gamma-32P-ATP and -GTP at two rifampicin concentrations (7 mug/ml and 24 mug/ml) and after pre-incubation at either 25 or 37 degrees C. The total number of RNA chains initiated ranges from 2.6 per Ad 2 DNA molecule at a rifampicin concentration of 24 mug/ml and pre-incubation temperature of 25 degrees C, to 5.4 per Ad 2 DNA molecule at a rifampicin concentration of 7 mug/ml and pre-incubation temperature of 37 degrees C. Efficient initiation with GTP occurs only after pre-incubation at 37 degrees C whereas initiation with ATP is equally as efficient at either pre-incubation temperature. Promoters for initiation with ATP have been localized to the leftmost 58% of the Ad 2 DNA molecule, defined by the EcoR.RI restriction endonuclease fragment A; promoters for initiation with GTP are located on the remaining 42% of the Ad2 DNA molecule. It is likely that on Adenovirus 2 DNA each RNA chain is initiated from a unique binding site which constitutes a seperate promoter for E. coli RNA polymerase.


Isolation and characterization of lambdadargECBH transducing phages and heteroduplex analysis of the argECBH cluster. Transducing lambda bacteriophages have been isolated which carry the divergently transcribed argECBH operon of E. coli K12 and various portions of the adjacent ppc and bfe chromosomal regions. They were recovered from lysates prepared by the procedure of Schrenk and Weisberg using a Ppc+ Arg+ Bfe+ strain carrying a deletion of the usual attachment site of lambda. Heteroduplex DNA mapping of these lumbdadarg and of the phi 80 darg isolated by B. Konrad indicates that the two kinds of phages carry the arg cluster in opposite orientations, a situation favorable for the isolation of argECBH DNA. A physical map of the ppc argECBH bfe region including 2 unusual attachment sites of lambda has been constructed. The localization of the end points of certain arg deletions provides a useful reference framework for the currently pursued mapping of mutations affecting the control of divergent transcription and for the location of restriction enzyme cleavage sites in the arg region.


Stimulation in trans of synthesis of E. coli gal operon enzymes by lambdoid phages during low catabolite repression. The infection of E. coli cells with different lambdoïd prophages triggers a stimulation of galactokinase synthesis when cells are grown in a medium giving rise to a mild catabolite repression (tryptone broth) with an inducer of the gal operon (fucose). These results show that during phage infection (or induction) some factor acting in trans is produced which is able to overcome efficiently catabolite repression of the kinase cistron. Using different strains of lambdapbio252 (pam, qam, "hl), lambdapbio256Hl and lambdaNNS7 we have concluded that the factor is the N gene product which is known for its anti- p(rho) action. Studies of the whole gal operon in the same conditions show that epimerase unlike transferase and galactokinase is practically insensitive to catabolite repression by tryptone broth and that viral development has a low effect on it. This indicates that there is an internal modulation of gal operon expression. A mRNA termination site sensitive to the p factor is known in the gal operon between galE and galT. Another site weaker than this one might exist between galE and operator-promoter region.


Isolation and genetic characterization of the nitA mutants of Escherichia coli affecting the termination factor rho. Taking advantage of the Spi (sensitivity to P2 interference) phenomenon, bacterial mutants seemingly resistant to phage lambdasusNnin5, but sensitive to phage lambdaspi, were isolated from a strain of E. coli K12 carrying no nonsense suppressor and lysogenic for P2. A class of these mutants, designated nitA (N-independent transcription), is described here. Upon infection of the nitA mutants with a trp transducing phage lambdasusN7N53ptrp46 which carries the E. coli trpE and D genes in the CIII-att region of the lambda genome, formation of anthranilate synthetase (ASase, a complex protein of trp E and D gene products) was clearly demonstrated. In contrast, no ASase formation was observed in the parent nitA+ strain under the same conditions. The synthesis is subject to "turn off" control, and is completely repressed by the CI repressor of phage lambda. The nitA cells lysogenic for lambdaCI857susN7N53 are killed by thermal induction much more efficiently than the parent cells lysogenic for the same phage. The nitA mutants support the growth of lambdasusN7N53byp much better than the parent. These results suggest that the nitA mutation permits the early leftward and rightward transcription of the lambda genome in the absence of the N gene product. On the E. coli genetic map, nitA is located between ilv and metE, nearer to ilv. The mutant allele is recessive to the wild-type allele. The present evidence, together with results of biochemical investigations to be reported, suggests that nitA is a gene specifying the transcription termination factor rho.


Isolation of rec- mutants from an F-prime merodiploid strain of Escherichia coli K-12. This paper describes a method of screening mutagenised populations of an E. coli galA/galB F- prime merodiploid for mutants defective in recombination. The method relies on scoring colonies on Eosin-Methylene Blue agar that have fewer than normal numbers of Gal+ papillae. With a suitable choice of gal- mutations most of the papillae arise by recombination and some of those colonies with less than normal numbers prove to be defective in some aspect of recombination or DNA repair. In addition to strains carrying mutations that can be ascribed to known loci, several novel mutant phenotypes were identified.


RNA polymerase mutants of Escherichia coli. III. A temperature-sensitive rifampicin-resistant mutant. Temperature-sensitive mutants of Escherichia coli that are unable to grow at high temperature can be obtained among those selected for resistance to streptovaricin or rifampicin at low temperature (Yura et al., 1973). One of these mutants (KY5323) that was supposed to carry a single mutation affecting both rifampicin resistance and temperature sensitivity was further investigated. Using purified RNA polymerase preparations obtained from the mutant and the wild type, it was found that the activity for RNA chain elongation is more sensitive to heat treatment than that for RNA chain initiation or DNA binding, and that the mutant enzyme is significantly more labile than the wild-type enzyme with respect to RNA chain elongation, when heat treatment is carried out at high salt concentration. These results, taken together with those of the enzyme reconstitution experiments, strongly suggest that the beta subunit of the polymerase is directly involved in both RNA chain initiation and elongation reactions. Enzyme reconstitution experiments using isolated subunits derived from the mutant and the wild-type polymerases demonstrate that the alteration of beta subunit is primarily responsible for both rifampicin resistance and thermolability of the mutant enzyme. In addition, the results suggested the apparent alteration of both beta and alpha subunits in this mutant. Extensive transduction experiments provided genetic evidence that are consistent with the view that the strain KY5323 carries a second mutation affecting the beta subunit, beside the primary mutation affecting the beta subunit. The hypothetical beta subunit mutation seems to modify quantitatively the rifampicin resistance caused by the beta subunit mutation.


Expression of a valine-resistant acetolactate synthase activity mediated by the ilv O and ilv G genes of Escherichia coli K-12. A strain carrying the ilv0603 mutation has been isolated in E. coli K-12 and its characteristics were found to be very similar to those previously reported by Ramakrishnan and Adelberg (1965a) for other ilv0 mutants. The strain carrying the ilv0603 mutation is resistant to valine inhibition (Valr) and we show that this resistance depends on the expression of a newly recognized gene, ilvG, which is located at min 75, between ilvE and ilvD on the E. coli K-12 map. The ilvG gene causes the expression of a Valr acetolactate synthase, which is detectable only when the ilv0603 mutation is also present in cis on the same chromosome. Under these conditions the Valr acetolactate synthase activity is eluted, on a hydroxylapatite column, at an ionic strength slightly lower than that required for elution of the remaining acetolactate synthase activity (sensitive to valine inhibition). The Valr peak is missing in a strain carrying an ilvG (amber) mutation.


Mitochondrial DNA in yeast recombination and subsequent modification following mating between a Grande and a suppressive Petite. The fate of mitochondrial DNA, following mating between a grande and suppressive petite of Saccharomyces cerevisiae, has been followed for up to 60 generations. The buoyant density of the mitochondrial DNA was seen to change in a manner explicable by a combination of recombination and subsequent modification phenomena whilst the suppressivity of the petite zygotic clones always remained high. These findings are consistent with current models of mitochondrial DNA metabolism in which petite strains have been observed to undergo deletion and reamplification of certain parts of their genomes.


The depression of endolysin synthesis in bacteria infected with high multiplicities of phage lambda. The effect of multiplicity of infection was studied in Escherichia coli with lambda phage, using phage endolysin as an example of a late gene product. A very sensitive endolysin assay method was used so that the initiation time of endolysin synthesis could be more accurately determined. It was observed that high multiplicity of infection (1) increases the rate of lysogenization, (2) progressively delays lysis time, and (3) significantly delays and reduces the synthesis of endolysin in lamdacIII+ cII+ -infected cells. The extent of delay and reduction in endolysin synthesis increases with increasing multiplicity. In contrast, lamdacIII67cII68-infected cells show no delay in endolysin synthesis at high multiplicity of infection when compared with the lamdacIII+ cII+ -infected cells. The results suggest that (1) the expression of cIII and cII genes is multiplicity dependent, (2) high multiplicity of infection enhances the expression of the cIII and cII genes, and (3) the expression of the cIII and cII genes interferes with the expression of the late genes. A model to explain how the expression of the cIII and cII genes interferes with the expression of the late genes is proposed.


Biosynthesis of Escherichia coli RNA polymerase subunits upon release of rifampicin inhibition. Upon release of rifampicin inhibition of Escherichia coli cells, the initiation of transcription will resume. The sequential resumption of the synthesis of proteins after release of rifampicin inhibition reflects the genetic order and size of the corresponding transcriptional units. We have used this approach to analyze whether the genes for alpha and sigma are on the same transcriptional unit as the genes for beta and beta', employing a method, which allowed us to measure the amounts of RNA polymerase subunits, alpha, beta, beta' and sigma in crude extracts. We have found that the alpha and sigma subunits are synthesized concurrently with the beta subunit in the rifampicin restart experiment, which suggests that the genes for alpha and sigma belongs to different transcriptional units.


Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit. Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apriion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974). Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes. Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30S ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur. Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.


Effects of different alleles of the E. coli K12 pol A gene on the replication of non-transferring plasmids. The effects of eight different polA alleles on the replication of six different non-transferring enterobacterial plasmids have been tested. Using phage P1CM transduction, different allelic polA mutations were introduced into E. coli K12 strains carrying one of several antibiotic resistance plasmids. Plasmid stability in the transductants was examined by testing clones for drug resistance after growth under various conditions. From the results, the R factors may be divided into three different classes. One plasmid is only affected by PolA conditions which inhibit host cell growth, 3 plasmids (from the same compatibility group) are unstable under conditions in which the cells are severely deficient in DNA polymerase I and two other plasmids (compatible with each other and with the other 4) are immediately lost from such transductants and are unstable in a number of others. Furthermore, the plasmids which are most dependent on DNA polymerase I have been shown to replicate in the presence of chloramphenicol and therefore typigy a class of plasmids which includes bacteriocinogenic factors such as ColE1 and CloDF13, resistance determinant RSF1030 and the E. coli 15 minicircular plasmid.


Characterisation of plasmids coding for the restriction endonuclease EcoRI. The properties of two plasmids coding for the CcoRI restriction and modification enzymes are described. Both plasmids are non auto-transferring (NTP) but can be mobilised by transfer factors. Strains carrying NTP13 produce colicin E1 and the EcoRI enzymes. This plasmid has a molecular weight of 6 X 10(6) daltons and is present as approximately 12 copies per chromosome. The second plasmid, NTP14, was detected after mobilisation of the EcoRI plasmid with the R factor RI-19. NTP14 codes for ampicillin resistance, synthesis of the EcoRI enzymes and colicin E1. The molecular weight of NTP14 is 10.7 X 10(6) daltons and there are about 14 copies per chromosome. DNA-DNA reassociation experiments were performed to determine the interrelationships of NTP13, NTP14, ColE1 and the R factor R1-19. NTP13 and NTP14 continue to replicate when cellular protein synthesis is inhibited by the addition of chloramphenicol.


Radiation sensitivity of a mutant of Escherichia coli K-12 associated with DNA replication: evidence for a new repair function. The isolation and properties of a new radiation sensitive mutant of Escherichia coli K-12 are described which shows a correlation between radiation sensitivity and replication of irradiated DNA. The mutation, called rer, is located between arg B and pur D loci. The mutant, when grown in tryptone broth after irradiation, is sensitive to UV and lambda-rays and incorporates little or no 3H-thymidine but in minimal glucose-salts medium both the radiation sensitivity and incorporation of 3H-thymidine remain identical to that of the parent strain. Studies with a temperature sensitive double mutant rer dnaC show that 1 hr incubation of irradiated cells at 42 degrees C before their transfer to 30 degrees C results in higher survival as compared to their incubation at 30 degrees C only. It is suggested that rer controls the replication of irradiated DNA and thus regulates the coordination between replication and repair of DNA.


R factor-mediated tetracycline resistance in Escherichia coli K12. Dominance of some tetracycline sensitive mutants and relief of dominance by deletion. Strains of Escherichia coli K12 heterozygous for the R100-1 tetracycline resistance region were constructed. They carried the wild-type Tetr genes in the chromosome and single site Tets mutations on plasmids. Some heterozygotes could not express tetracycline resistance fully after induction. The mutant tet allele was thus partially dominant. When heterozygotes carrying the dominant tet mutant were plated on agar containing 20 mg/ml tetracycline, mutants which grew normally occurred at a frequency of 1-4 X 10(-4). Analysis of these dominance relief mutants showed that in 53/56 isolates the dominant tet allele was lost forming either Tra+ or Tra- deletion mutants of the plasmid. The mutation frequency was not affected either by the host chromosomal recA mutation or by the temperature of growth of the culture.


Pyrimidine nucleoside analogues as inducers of pyrimidine nucleoside catabolizing enzymes in Salmonella typhimurium. Various structural analogues of cytosine and uracil nucleosides were tested as potential inducers of the nucleoside catabolizing (cyt) enzymes in Salmonella typhimurium. Some analogues, e.g. 5'-O-alkyl cytidines and uridines, resistant to catabolic enzymes, were as effective as the natural inducers cytidine and uridine; but etherification of one of the cis 2' or 3'hydroxyls fully abolished activity, pointing to a requirement of an intact ribose cis-glycol system for activity. A uridine analogue in the syn conformation, 6-methyluridine, a good substrate for uridine phosphorylase, was inactive as an inducer. The behavior of various other analogues, in relation to their structure, conformation and substrate properties, indicated the absence of any correlation between inducing activity and substrate susceptibility. The overall findings are consistent with conclusions derived from genetic experiments. The active analogues apparently act via similar pathways, and probably affect the same regulatory mechanism(s) as the natural inducers.


Immunopathology of sympathetic ophthalmia. The long held notion that sympathetic ophthalmia represents an autoimmune reaction to uveal pigment is no longer tenable. Pigmentation influences the histopathologic picture of sympathetic ophthalmia but no evidence supports the role of uveal pigment as inciting antigen. Several recent studies have confirmed our initial report of the participation of cellular hypersensitivity to ocular tissues in the pathogenesis of this disease. Both clinical and experimental studies implicate retinal tissue as being more immunogenic than uveal antigens. The recent histopathologic observation that eosinophils concentrate near the choriocapillaris suggests that outer retina and retinal pigment epithelium should not be overlooked as a potential source of the stimulating antigen in sympathetic ophthalmia.


[Infectivity of different forms of lambda bacteriophage DNA in transfection of calcinated Escherichia coli]. Infectivity of linear lambdaDNA molecules is proved to be about a hundred times higher in calcinated E. coli K12 (lambai434) than in E. coli K12(lambda-): the levels of transfection were 1-3-10(7) and 1-2-10(5) infective centers per 1 mug DNA, respectively. In E. coli JC 5743 rec B21 defective for exonucleases I and V the level of transfection was 1-3-10(6). High infectivity of linear lambdaDNA in lysogenic cells cannot be explained by a helping effect of phage particles spontaneously liberated by these cells. It can be caused by recombinations of inserted lambdaDNA molecules with prophage or by the low activity of some nucleases in the lysogenic cells. Covalently closed and "Hershey" ring forms of lambdaDNA penetrate the calcinated cells as readily as linear molecules do but the infectivity of the former ones is proved to be very low.


The role of uveal and retinal antigens in experimental autoimmune ocular pathology. Guinea pigs immunized with retina antigen, located by immunofluorescence in the photoreceptor cell layers, develop retina-specific immune responses and autoimmune uveoretinitis. Ocular pathology is characterized by cellular infiltration and destruction of the photoreceptor cells subsequent to inflammation of the iris, ciliary body and choroid. Guinea pigs immunized with homologous choroid antigen, localized by immunofluorenscence in the area of Bruch's membrane or pigment epithelium, developed choroiditis, subretinal exudation and loss of photoreceptor outer segments. The pathology is associated with immune responses directed primarily against antigens recovered with choroid, but which demonstrated partial cross reactions with outer segment antigens. Possible relationship of uveal and retinal antigens is discussed.


Prostaglandins and inflammation in the eye. Traumatic injury of the eye promotes the release of prostaglandins E2 and F2alpha from the iris and other tissues. These induce vasodilation, increased capillary permeability and an increase in protein content of the aqueous. They are leukotactic. With an influx of leukocytes PGE1 appears in the aqueous having been synthesized by these cells. Infectious agents also attract leukocytes and sensitized lymphocytes characterize inflammation of both allergic and infectious origin. The cascade of molecular and cellular events seen in ocular inflammation of various origin seem ultimately to result in a reaction largely mediated by prostaglandins. Effective therapy should be directed at preventing their synthesis (synthetase inhibitors), interferring with their action once synthesized (receptor blockers), and inhibiting the migration of leukocytes into the eye.


Studies on the mechanism of translocation in ribosomes. IV. The role of ribisomal proteins S12 in translocation. It is shown that ribosomes, the 30S subparticles of which are reconstituted without protein S12, read out poly(U) and synthesize polyphenylalanine in the absence of protein elongation factors (EF-T and EF-G) and GTP, i.e. perform "non-enzymatic" translation. On the contrary, ribosomes, the 30S subparticles of which are reconstituted with protein S12, do not display "non-enzymatic" translation without its activation with parachloromercuribenzoate. This means that a complete removal of protein S12 from the ribosome, as well as its damage with para-chloromercuribenzoate, leads to the unblocking of the potential ability of ribosomes for spontaneous ("non-enzymatic") translocation. The presence of intact protein S12 in the ribosome prevents spontaneous (EF-G-GTP-independent) translocation. A suggestion is made that the intact protein S12 forms an additional contact between the ribosomal subparticles and thus participates in the ribosomal mechanism of translocation by affecting the locking-unlocking of the subparticles.


[Phage T4 partial diploids obtained with the method of DNA interrupted injection. II. The mapping technique based on the physical size measurement of the diploid region]. Populations of the E. coli B mixedly infected with the whole chromosome of the amber mutant in respect to two genes under consideration and with a chromosome fragment of the phage T4 "wild" type are examined. The fragment length with which both of the amber mutants defect functions are compensated is determined to measure physical distance between the genes. Distances so obtained do not depend on recombination frequencies and are shown to be additive and "complementary". The latter property means that the sum of the smaller and "complementary", larger distances between two genes on the circular map equals the complete linkage group quantity. Moreover, a "complementary", larger distance is measured directly and independently on a smaller one, whereas current mapping technique fail to do so. The method enables to evaluate a size of rather long phage T4 gene region, for example gene 43, i. e. the structural DNA polymerase gene. Phage T4 genes 34 and 35 are shown to have a common start region for transcription and translation, i. e. they belong to the same operon. In consequences the circular map of physical distances between phage T4 genes is constructed.


Histoincompatibility and allogeneic corneal grafting between inbred strains of rats. To study corneal allograft reactions in antigenically controlled models, our method for interlamellar grafting was applied in two rat inbred strain combinations, the one with intermediate and the other with strong (RtH-1) histoincompatibility. In syngeneic controls, no rejection of grafts applied near the limbus could be observed clinically and histologically, but all allografts were rejected; the speed and intensity of the reaction depend on the degree of histoincompatibility. Systemic sensitization following corneal grafting took place in both strain combinations as indicated by accelerated rejection of subsequent skin grafts.


The role of tissue typing in corneal transplantation. The ABO relationships of 150 corneal transplant recipients and their donors were evaluated as were IgG and IgM isoagglutinins in recipients receiving grafts from ABO-incompatible donors. The HL-A antigens of 43 corneal transplant recipients and their donors were determined as were the cross-matches of donor lymphocytes in selected cases. No relationships of significance between mismatched donors and recipients or presence of antibody in recipients against antigens of the donor for either ABO or HL-A systems were found. It was concluded that in this series histocompatibility mismatches could not be shown to be a factor in corneal transplant failure.


[Studies on the mechanism of translocation in ribosomes. V. Comparison of the effect of antibiotic inhibitors of ribosomes on "enzymatic" and "non-enzymatic" translation]. A comparison of the effect of 11 antibiotic inhibitors of protein synthesis on "enzymatic" and para-chloromercuribenzoateactivated "non-enzymatic" translation was made. It was found that fusidic acid which inhibits protein synthesis by blocking the action of one of the elongation factors (EF-G) did not suppress functioning of the "non-enzymatic" system. On the contrary, all the antibiotics blocking different functional sites of the ribosome itself such as tetracycline, chloramphenicol, erythromycin, edeine, thiostreptone, viridogrisein, spectinomycin, streptomycin, kanamycin and neomycin, were shown to inhibit the "non-enzymatic" translation. The data obtained corroborate the idea that the molecular mechanism which the ribosome utilized during "non-enzymatic" translation are similar or identical to the mechanism of normal "enzymatic" translation. It was found that the "non-enzymatic" working ribosome is more sensitive to all the antibiotics as compared to the ribosome synthesizing peptide with the participation of elongation factors (EF-T and EF-G). This suggests that the elongation factors increase the level of general resistance of the working ribosome against very different hindrances.


Immunologic protection of rabbit corneal allografts with heterologous 'blocking' antibody. Immunologic protection of corneal allografts. Exchange penetrating keratoplasties were performed in NZW rabbits using conreas soaked in heterologous 'blocking' antibody (guinea pig anti-rabbit gamma-globulin) under various experimental situations and immunogenic stimuli. Results suggest that the antibody can protect the rabbit graft from weak rejection stimuli possibly by blocking the afferent arc of the immunologic reflex, but cannot protect the graft from presensitization, or nonocular sensitization. The use of a blocking antibody in human transplantation would be simple and of slight risk to a graft recipient.


[A method of comparison of DNA polynucleotide sequences]. The results of a comparative analysis of the kinetic curves of reassociation of DNA of closely related species of Microtus (Microtus arvalis, Microtus oeconomus oeconomus, Microtus oeconomus Chachlovi) are presented. A five power polynomial was used as a mathematical model of the kinetic DNA reassociation curves. The values of polynomial coefficients and the theoretical value, both with statistical deviations dispersion, have been estimated with the help of a standard computer programme. The modified Fischer criterium was used for comparing the model kinetic curves of reassociation of the above species and of subspecies of Microtus, and to estimate the significance of the difference between two random means. A pair-wise comparison of the model curves shows that the similarity of the DNA polynucleotide sequences in two subspecies is less expressed than in two species of Microtus. A method of comparison of DNA nucleotide sequences is suggested on the basis of mathematical models of kinetic curves of DNA reassociation.


[Approach to a priori calculation of stable forms of proteins]. Basing on semi-empirical conformational analysis an approach is suggested to the calculation of stable forms of protein molecules. The physical model proposed implies: 1) that the most stable conformation of a protein corresponds to its native state, 2) that interactions of three types which determine conformational states of every residue should be treated separately, 3) that the protein molecule is highly compact structure with interactions of different kinds compensating each other, 4) that interactions of the three types contribute differently to the formation of the spatial structure of a globule. These interactions are: 1) those of peptide chain atoms within a particular residue, 2) interactions of the residue with the nearest neighbours in the chain, 3) interactions with residues which are remote in the chain but close in the globule. The approach proposed allows to separate the conformational analysis of a protein into a number of subsequently solved problems. The scheme of the calculation of stable forms is presented.


Immunology of the vitreous body.

Immunology of the vitreous body. The vitreous is feebly antigenic and is characterized by the absence of gamma-globulins and immunocompetent cells. Moreover, it is isolated from the aqueous humour by a mucopolysaccharide molecular filter. It represents an incomplete and primitive immunological system, reacting like an embryonic tissue and exhibiting only phagocytosis.


Immunological aspects of viral uveitis.

Immunological aspects of viral uveitis. The study of the immunological aspects of viral uveitis deals with virology as well as ophthalmology. Viral uveitis can be the result of direct invasion of the uvea by viral particles, or possibly by immune complexes, and even autoimmune reactions can play a role. The diagnosis will be made, besides the rare finding of viruses, by measuring circulating antibodies in serum and aqueous. The evolution of the disease and its therapy raise many questions involving the humoral and the cellular immune systems.


Some remarks on the immunopathology of toxoplasmic uveitis. Data obtained from prospective studies of congenital toxoplasmosis and from serological examination of patients with uveitis suggest that ocular toxoplasmosis is mostly congenital in origin. It is mainly related to the immunological clearance of residual cysts persisting after a subclinical congenital toxoplasmosis.


Demonstration of cell-mediated immunological reactivity to transplantation antigens in uveo-retinal tissues. Lymph node cells from inbred rats, sensitized against allotransplantation antigens, were tested for reactivity against uveo-retinal tissues, using a two-step leucocyte migration inhibition test: (1) These tissues induced positive reactions indicating the presence of transplantation antigens. (2) This was true for strong and weaker histoincompatibility systems. (3) Quantitatively, uveo-retinal tissues were somewhat less reactive than lymphocytes as antigen.


Experimental lens-induced granulomatous endophthalmitis. Experimental lens-induced granulomatous endophthalmitis is an experimental autoimmune disease that is histopathologically identical to the human disease known as phacoanaphylactic endophthalmitis or lens-induced uveitis. The capacity to develop experimental disease after lens injury can be passively transferred to virgin animals with hyperimmune serum. Fluorescein-labeled anti-IgG and anti-C'3 antisera bind to the lenses of appropriately sensitized animals after lens injury but not to the injured lenses of unsensitized control animals. Cobra Venom factor which inhibits C'3 prevents the development of lens-induced granulomatous uveitis in appropriately sensitized animals. Hypersensitized animals develop a massive Arthus-like reaction. On the basis of these observations we feel that experimental lens-induced granulomatous endophthalmitis is an immune complex disease. Because the experimental disease is histopathologically identical to human lens-induced uveitis or phacoanaphylactic endophthalmitis we believe an immune complex mechanism of immunopathogenesis is important in phacoanaphylactic endophthalmitis.


History of immunology of uveitis.

History of immunology of uveitis. The history of immunology of uveitis is given from the point of view of the author. Immunological studies on clinical cases of uveitis and later experimental uveitis in rabbits using the fluorescent antibody technique of COONS are shortly discussed. Later studies on viral diseases in collaboration with Dr. Martenet are mentioned.


Hydroxyurea.

Hydroxyurea. Hydroxyurea (HU) is an anti-leukaemia and anti-tumour drug which has also found limited application in the treatment of dermatological disorders. It is a potent inhibitor of DNA synthesis in many organisms from viruses to man and in cell culture systems. To a lesser degree it can also inhibit RNA and protein synthesis. It is anti-mitotic and cytotoxic depending on the concentration used, the duration of exposure, and the sensitivity of the organism. In most cells HU is active mainly in the S-phase of the cycle and because of the easy reversibility of its action it has been used as a synchronising agent in cell cultures with some success. There is conflicting evidence about the ability of HU to act as an inhibitor of the natural DNA repair mechanisms in cells exposed to radiation or alkylating agents. HU has, however, been shown to induce chromosome damage in a number of organisms including man. It is clearly teratogenic in mammals and can cross the placental barrier at least in rats and the golden hamster, but it is believed to have only a limited mutagenic activity. Studies involving a direct comparison of the action of HU with other compounds suggest that the = C--NHOH moiety is responsible for its biological activity.


Theobromine and theophylline.

Theobromine and theophylline. Theobromine and theophylline have a limited therapeutic use and in addition they occur in plants used in the preparation of a number of widely consumed drinks. Thus most of the population must be exposed to both compounds. Chromosome abnormalities are caused by both theobromine and theophylline in plant cells and in mammalian cells in culture, and both have anti-mitotic activity. While they are fairly potent mutagens in Escherichia coli and other lower organisms the rather scanty available evidence suggests that they are not mutagenic in mammals. The difference in mutagenic activity may be due to the reported inability of E. coli to demethylate these compounds, a process which occurs readily in mammals including man. The structure-activity relationships of these compounds are complex but the available evidence suggests that methylation at position 1 is the most important for both mutagenic activity and the anti-mitotic effect while methylation at position 3 is of most importance in the action on chromosomes.


The lethal effect on bacteria of dimethylnitrosamine used without an activating agent. Survival curves have been obtained for various strains of bacteria treated with dimethylnitrosamine (DMN) solutions of different concentrations. The results are compatible with the conclusion that DMN kills the bacteria by an attack on their deoxyribonucleic acid (DNA) and the damage so caused can be repaired by the same systems that repair damage created by ultraviolet light or X radiation. No activating agent was added to the DMN solutions and under these circumstances their activities were in proportion to the squares of their concentrations. It is suggested that this is because two molecules of DMN plus one of oxygen produce two carbonium ions, two hydroxyl ions, two formaldehyde molecules and two nitrogen molecules.


Inactivation and mutation of yeast cells by hydrogen peroxide. It has been found that inactivation and mutation to respiratory deficiency of yeast cells can be induced by hydrogen peroxide treatment. However, considerable inconsistencies were encountered in measuring these responses. The inconsistencies were found to arise from differences in the abilities of cells in different metabolic states to destroy H2O2 and in the intrinsic sensitivities of cells in different growth states. Parallels are drawn with previous inactivation data from bacterial studies suggesting reparable lesions in DNA provide a useful model to explain H2O2-induced inactivation, but it is not yet possible to eliminate alternative interpretations. Mutation induction in stationary phase cells is strongly depressed at high "doses" of H2O2 (or low cell survival), as is found with a variety of other agents.


Two mutations which confer temperature-sensitive radiation sensitivity in the yeast Saccharomyces cerevisiae. X-ray survival curves for two mutations, rad54 and rad55, in the yeast Saccharomyces cerevisiae are presented. These mutations confer temperature sensitive X-ray sensitivity; that is rad54 and rad55 strains display a wild type X-ray survival response at permissive temperatures and a radiosensitive X-ray survival response at restrictive temperatures. The survival response of cells which were shifted from a permissive to a restrictive temperature or vice versa at various post-irradiation times indicates that repair and fixation of X-ray induced lesions is largely complete three hours after X-irradiation. Experiments to determine the utilization sequence of the rad54 and rad55 gene products in the repair of X-ray induced damage suggest that the two products are required in an interdependent manner.


The gene dosage effect of the rad52 mutation on X-ray survival curves of tetraploid yeast strains. The mutation rad52 in the yeast Saccharomyces cerevisiae confers sensitivity to X-rays. The gene dosage effect of this mutation on X-ray survival curves of tetraploid yeast strains is shown. With increasing number of rad52 alleles, both a decrease in the survival for a given dose and a decrease in the survival curve shoulder width are observed. The generation of such a family of survival curves using three different mathematical models is discussed.


Effect of caffeine on recovery from DEB-induced cell inactivation in UV-sensitive mutants of Saccharomyces cerevisiae. The UV-sensitive yeast mutants rad3 and rad6 are highly sensitive to diepoxybutane (DEB) as compared with the RAD strain. The two mutants show differential response to liquid holding (LH) after exposure to DEB and UV. The survival of rad3 increases markedly after DEB and decreases after UV. Caffeine significantly affects LH recovery of DEB-treated RAD strain, slightly decreases recovery of rad3 and has almost no effect on survival of rad6. When DEB-treated cultures are plated immediately on caffeine-containing medium, survival of rad3 decreases more significantly than that of the RAD strain, whereas survival of rad6 is only slightly decreased as compared with the untreated cultures. Possible mechanisms of recovery from DEB-induced cell damage are discussed.


Genetic effects of formaldehyde in yeast. I. Influence of the growth stages on killing and recombination. In random cultures, stationary phase cells of Saccharomyces cerevisiae are more resistant to killing induced by formaldehyde than are exponentially growing cells. It is shown that this compound induces intra- and intergenic recombination in this eucaryotic organism. In synchronized populations the lag and G1 phases demonstrate the higher resistance to both killing and induction of recombinants by formaldehyde whereas maximal sensitivity occurs during the end of G2 and/or the mitotic division. This pattern in contrast with that found after treatments by ionizing or ultraviolet radiations.


A cytogenetic study in women who had used oral contraceptives and in their progeny. A study has been made of the chromosome numbers and characteristics of leucocytes cultured from mothers (a total of 77) who had taken one or more types of oral contraceptive, and their babies (108), and compared with similar numbers of control subjects. When the total cytogenetic results from test and control mothers were compared, no significant differences in frequency or type of chromosome abnormalities was observed. However, with certain types of oral contraceptive, minor differences in the numbers of chromosome abnormalities were found between test and control groups and the significance of these findings are discussed. The only significant abnormalities observed in the babies from mothers taking oral contraceptives were slight increases in the numbers who had cells with less than 46 chromosomes and who exhibited chromosomal gaps. The number of abnormal cells was significantly higher in babies born to mothers who had been taking norethynodrel (Conovid) (4.6% abnormal cells in test babies; 2.0% in controls).


Use of snail enzyme for the selection of glucose-and mannose-negative mutants in Saccharomyces cerevisiae. The relationship between growth and sensitivity of Saccharomyces cerevisiae cells to snail enzyme led us to work out the conditions for using this enzyme to isolate mannose-negative and glucose-negative mutants. The technique is based on a prior growth on a glycerol medium in which any cell becomes resistant to snail enzyme, followed by a period of growth in a medium in which only the wild-type cells can grow, thereby becoming sensitive to the enzyme. This technique was very effective in both tested cases. This result suggests that the method described might also be applied to isolate other kinds of mutant.


Spontaneous and induced mutability or frameshift strains of Salmonella typhimurium carrying uvrB and polA mutations. Three strains of Salmonella typhimurium carrying frameshift mutations affectin g the histidine genes (hisC3076, hisD3052 and hisC207) showed increased sensitivity to mutagenesis by ICR-191 (as judged by measuring back mutation to prototrophy), if they were made deficient in excision repair by deleting the uvrB gene. One frameshift strain, hisC3076, also showed increased sensitivity to mutagenesis by ICR-191 when it carried either of two different polA alleles, whereas the hisD3052 and hisC207 frameshifts reduced sensitivity to mutagenesis in the presence of these alleles. Studies of spontaneous back mutation to prototrophy revealed significant mutator effects of the polA1 mutation on reversion of the hisD3052 frameshift and of the polA3 mutation on reversion of the hisC3076 frameshift. Other smaller mutator effects of the polA alleles on complex interactions between different polA alleles and different frameshift mutations, it is tentatively suggested that deletion frameshifts may arise mainly during DNA replication, while addition frameshifts may arise mainly during post-replication repair.