[Urological treatments before and after kidney transplantations].

The repair of pre-existing bladder sphincter lesions in patients excluded from kidney transplant programs, and the recovery of grafts threatened by ureteral complications, are favourable influences in establishing equilibrium between dialysis and transplantation. In a series of 74 kidney transplants, two patients were able to receive grafts: one after the reconstruction of an ileal tube and the other after bladder diverticulectomy and resection of the bladder neck. Two other grafts, complicated by ureteral necrosis, were able to be conserved following a uretero-ureterostomy in the first case, and a psoic bladder in the second. These repair operations are discussed from three points of view: incidence, procedures, and complications. Advances made in transplanting kidneys encourage its use in patients who were previously excluded from receiving transplants because of bladder sphincter lesions. These lesions can be the cause of a renal insufficiency, or those associated with the original kidney disease. This group of patients represents 3 to 5% of the population of patients on permanent dialysis who respond to the other criteria for inclusion in the lists of potential receivers of kidney transplants: some of them could benefit from the graft if their lesions were treated by the standard urological methods. Furthermore, 5 to 8% of those with kidney transplants could lose the grafted kidney, which is immunologically tolerated, because of urological complications. As with patients in the first category, they also could obtain benefit from repair procedures on the urinary tract. A total of 74 kidney grafts were performed in the Sheba medical Center between March 1971 and July 1977: two patients were able to benefit from preventive urological procedures before transplantation: two others with grafts developed ureteral complications and were benefited by therapeutic procedures rarely used in kidney transplantation cases.


Tick-borne relapsing fever in Colorado. Historical review and report of cases.

Since 1915 the front range of the Colorado Rocky Mountains has been postulated as a focus of endemic tick-borne relapsing fever. However, the disease has rarely been identified: only two cases have been reported in Colorado since 1944. Three sporadic cases in 1977--tightly grouped geographically and temporally--prompted an epidemiologic review. Tick-borne relapsing fever should be considered in the differential diagnosis of recurrent paroxysmal fever--with or without known presence of ticks--whenever exposure in an endemic area is part of a patient's history.


A natural resource. Prevalence of cadaver organs for transplantation and research.

To determine the prevalence of potential cadaver kidney, eye, and research-tissue donors in a tertiary referral hospital, the charts of 718 patients who died were studied. We found a potential kidney-donor rate of 2.4 per 100 hospital deaths, a potential eye-donor rate of 45.4 per 100 deaths, and a potential pancreas-donor rate of 26.5 per 100 deaths. In the study year, 20 potential cadaver kidney donors were projected, theoretically meeting the needs of the transplantation center. Recognized potential eye and pancreas donors were remarkably numerous. Effective education, surveillance, and procurement programs are necessary to salvage these potential donor tissues.


Combined amphotericin B-flucytosine therapy in Aspergillus pneumonia.

The incidence of aspergillosis is increasing while the prognosis remains dismal. Standard single-drug therapy with amphotericin B offers unacceptably low cure rates. A patient with Aspergillus pneumonia was treated with a synergistic combination of amphotericin B and flucytosine. A prompt clinical response and progressive resolution of the infection were observed. Combination therapy appeared to decrease the dose of amphotericin B needed for effective therapy. It may provide improved results with decreased toxic effects.


Penicillin levels in blood and CSF achieved by treatment of syphilis.

Treponemicidal levels of penicillin in the CSF were not obtained following intramuscular (IM) injections of 600,000 IU of penicillin G procaine along with 2% aluminum monostearate (PAM). Treponemicidal levels of penicillin in the CSF were achieved in 31 patients by probenecid, 500 mg/6 hr by mouth, with penicillin G, 500,000 IU by IM injection, and by probenecid every six hours by mouth and IM injections of penicillin G procaine once a day in two of three patients following injections of 600,000 IU, in four of five following 1.2 million IU, and in three (now all nine) following 2.4 million IU.


Risk of pneumococcal infections in renal transplant patients.

Because of the recent licensing of pneumococcal vaccine, the risk of pneumococcal infections on a renal transplant service was determined. The overall risk of infection was 7% (14 of 197 patients) over a six-year period, or 1% per year. In patients with functioning allografts, this represented a risk of 28 infections per 1,000 patient-years of follow-up. Clinically, the major presentations of pneumococcal infection were pneumonia in the patient who was rejecting his kidney and pneumococcemia of sudden onset that was often fatal. Five of the pneumococci isolated from bacteremic patients were typed; three of the isolates were types that are in the vaccine. The frequency and seriousness of pneumococcal infections in renal transplant patients suggest that pneumococcal vaccine should be given to these patients, probably before transplantation.


Influence on blood pressure of renal isografts between spontaneously hypertensive and normotensive rats, utilizing the F1 hybrids.

The F1 hybrids (F1) of spontaneously hypertensive rats (SHR) and Wistar rats (W), whose kidneys were transplanted from SHR at the age of 10 weeks and 20 weeks, showed significant elevation of blood pressure (BP) for 11 weeks after the transplantation. In F1 with W or F1 kidneys BP was decreased near to the normal level. F1 whose kidneys were transplanted from SHR or W showed low renin activity both in plasma and the kidney. It is suggested that BP of SHR is probably determined by the renal pro-hypertensive factor(s) other than renin influencing on sympathetic nerves through central nervous systems.


Change of P-Q intervals of the electrocardiogram in the rat hearts sensitized with the killed group A streptococci.

Prolongation of P-Q interval in the anesthetized rat was observed by repeated injection of killed group A streptococci. The prolongation was clearly recognized at about the 11th week after the first injection, but afterwards P-Q interval recovered to the normal level in spite of continuous injection of killed streptococci. His bundle electrogram recorded from isolated heart revealed the prolongation of A-H interval in the treated rat. Moreover, the transmembrane action potential in the atrioventricular nodal region of treated rat was slightly deteriorated, but the action potentials in the other cardiac muscles were not changed. It was deduced from the above results that P-Q prolongation was transiently brought about by the injection of killed group A streptocci and that deterioration of muscle in the atrioventricular nodal region might take a main part in the P-Q prolongation.


Membrane receptors of human erythrocytes for bacterial lipopolysaccharide (LPS).

The binding specificity of bacterial lipopolysaccharide (LPS) was investigated by inhibition experiments of the binding of 3H-labelled Escherichia coli lipopolysaccharide (LPS) AND 3H-labelled Salmonella minnesota R595 glycolipid and lipid A to human erythrocytes using various glycoproteins as inhibitors. PAS-1 glycoprotein and band-3 glycoprotein of human erythrocyte membranes exerted strong inhibitory activity. To characterize membrane receptors for LPS, solubilized membranes of human erythrocytes were subjected to affinity chromatography with an affinity adsorbent prepared by coupling S. minnesota R595 glycolipid to activated Sepharose 4B. Band-3 and PAS-1 glycoproteins were identified as major receptor sites.


Heterophile antibodies in the sera from renal transplant recipients.

Microcytotoxicity tests with the sera from renal transplant recipients against cultured B-cell lines were performed by using guinea pig complement. Out of 106 sera, 18 sera showed strong cytotoxicity against cultured cell lines. The same results were obtained by using normal human serum as a complement source. These sera did not show any cytotoxicity against normal peripheral T- and B-lymphocytes. On the other hand, the sera containing the cytotoxicity against cultured B-cell lines were found to have high-titered heterophile antibodies against bovine red blood cells (BRBC) when hemolysis in agar gel with guinea pig complement was used. The sera without cytotoxicity did not have high-titered heterophile antibodies. The sera with cytotoxicity against cultured B-cell lines reacted in the same way against both human tonsilar lymphocytes cultured with the medium containing fetal calf serum and human serum. Therefore, the antigenicity of cultured cells was not affected by the fetal calf serum in the culture medium. After absorption with BRBC, the sera lost their cytotoxicity against cultured cells. Hemolysins in the sera were almost completely removed after absorption of the sera with cultured cells. From these results, it seemed apparent that the sera from renal transplant recipients contained heterophile antibodies which reacted with the cells of some cultured B-cell lines and tonsilar lymphocytes.


Progressive and fatal infection with attenuated Mycobacterium bovis (BCG) in nude mice.

Congenitally athymic nude mice (nu/nu) were infected intravenously with Mycobacterium bovis BCG Japanese strain under specified pathogen-free (SPF) or germ-free (GF) conditions. SPF euthymic litter mates (nu/+) serving as controls were found to tolerate the infection well, while SPF nu/nu mice following infection of 3 X 10(7) organisms died by week-36. Animals having received a very small dose (3 X 10(0) of organisms and their non-infected cage mates showed no evidence of infection at week 37 post-infection. Time-course observations carried out on SPF and GF nu/nu mice following infection with 10(5) or 10(6) organisms revealed that the number of organisms in the liver and spleen reached 10(6) to 10(7) viable units per organ at week 12 and this level was maintained for 50 weeks post-infection. Bacillary counts in the kidney and lung increased progressively and reached a level of 10(7) to 10(8) at the terminal stage of infection. In the liver, spleen and lymph nodes of nu/nu mice, granulomas were noted 12 weeks postinfection. The granulomas were composed of macrophages and accompanied by slight infiltration of lymphocytes, plasma cells and a small number of polymorphonuclear leucocytes. In later infection stages, small aggregations of pigmented macrophages packed with acid-fast bacilli were present in the liver, spleen and lymph nodes. Lesions with large foci of bacilli-laden macrophages developed progressively in the kidney, lung and subcutaneous and periosteal connective tissues. Periosteal granulomatous lesions, sometimes accompanied by exudation, intruded occasionally into the bone marrow, resulting in extensive granulomatous osteomyelities.


The pathogenesis of ovarian neoplasia.

Currently in the United States intra-abdominal neoplasia involving or originating in the ovary is responsible for the death of 11,000 women per year, or one every 50 minutes. Historically, as well as can be documented, although benign ovarian cysts were common, malignant neoplasia was rare. Furthermore, similar lesions arising in or adjacent to the testes are essentially unknown. The uniqueness of the tumors is appreciated by the recognition of the common cell of origin, but there are many different histologic varieties of neoplasia that may arise therefrom. The differences in incidence between the sexes and the apparent significance of irritants to which the female pelvic cavity may be exposed suggest that such proliferating agents may ascend from the vagina through the patent fallopian tube and into the pelvic and/or the general abdominal cavities. Since little improvement has been made in the overall five-year survival rate during the past 25 years (in spite of the widespread use of chemotherapeutic and other treatment modalities), possibly our basic thrusts should be directed at defining carcinogenic processes and thus preventing the development of such lethal disease.


The behavior of bacteria: on the mechanism of sensory transduction in bacterial chemotaxis.

The mechanism of bacterial chemotaxis is beginning to be understood. At the receptor end, we have considerable knowledge about the molecular properies of chemoreceptors. At the effector end, we know that flagella rotate and that the direction of rotation is determined by attractants and repellents, although we do not yet know the molecular features of the motor and the gear shift. Between the receptors and the effectors is a system for integrating the sensory information and transmitting a message to the flagella. This system, sensory transduction, somehow involves methylation of membrane proteins and probably a change in membrane potential, but further details of how the mechanism works remain to be elucidated.


[Comparative evaluation of cardiac output in the acute period of macrofocal myocardial infarct according to the methods of dye dilution technic and echocardiography].

Seventy-eight simultaneous studies of the stroke volume value by the method of dye dilution and by echography were conducted in 28 males on the first to tenth day of macrofocal myocardial infarction. Despite the satisfactory correlation of the stroke volume values obtained by both methods in the whole group (r=0.71), the authors note a difference in the correlation depending on the localization of infarction and the kinetic condition of the diseased and the intact zones. The authors believe that the echocardiogrphic method is not reliable in determining the volumetric values in patients with macrofocal myocardial infarction since it misrepresents the true value of the stroke volume though it reflects the general trend of changes in the hemodynamic indices. Echocardiography yields essential supplementary information on the state of the left ventricular pumping function and the mechanisms of its compensation in the acute period of myocardial infarction.


[Hemodynamic disorders in the acute period of myocardial infarct according to the radiocardiographic data].

Changes in central hemodynamics in patients suffering from acute macrofocal and transmural myocardial infarction with or without circulatory insufficiency were studied by radiocardiography. Cardiac output was found to be increased in the first 24 hours of the disease in persons without circulatory insufficiency as compared to the control group, which may be linked with marked activation of the sympatheticoadrenal system. In the groups of patients with circulatory insufficiency the cardiac output during this period was diminished. In most patients myocardial infarction was preceded by marked changes in teh myocardium. Diminution of hemodynamic values was recorded in all groups of patients on the third day, which was most conspicuous in cases with circulatory insufficiency.


[Language of physicians' ECG conclusions].

The elaboration of a standardized language for expressing the ECG diagnosis is of great practical importance and is necessary for the creation of a system of automatic treatment of the electrocardiosignal. A language is suggested for the ECG findings according to the changes in the ECG contour and disorders of the cardiac rhythm and conduction. It is based on the syndromal approach which makes it possible in analysis of the ECG to only distinguish definite syndromes without dictating a narrow diagnostic interpretation on the doctor. A list of 56 ECG diagnoses according to the character of the ECG contour and 60 elementary diagnoses relating to disorders of cardiac rhythm and conduction is given. The suggested language embraces all clinically important ECG syndromes and may be used in the elaboration of a system of automatic analysis and accepted as the foundation of a standardized language for expressing the ECG findings in practical electrocardiology.


Estimation of relative glomerular capillary surface area in normal and hypertrophic rat kidneys.

The quantity of antiglomerular baSEMent membrane antibodies (antiGBM) binding to the glomeruli of rats 4 hr after i.v. injection of 660 microgram of antiGBM was used as a measure of relative glomerular capillary surface area (Sr). Intact immature and adult rats (N = 27) weighing 46 to 440 g were studied to assess the effect of normal growth on Sr. Young adult rats (N = 36) were studied at 0, 8, 15, and 22 days following uninephrectomy or sham operation to assess the effect of hypertrophic kidney growth on Sr. Bound antiGBM increased from 95 microgram to approximately 350 microgram as rats grew from 46 to 200 g; further growth was associated with no further growth was associated with no further increases in bound antiGBM. In contrast, there was no progressive increase in Sr following uninephrectomy or sham operation despite as 45% increase in kidney weight at 22 days over the comparable kidney in the sham-operated rats (1.32 +/- SEM 0.06 g vs. 0.91 +/- SEM 0.01 g, P less than 0.0001). Thus, increases in GFR during early normal kidney growth parallel anatomic increases in Sr, but increases in GFR with later growth or with compensatory hypertrophy in young adult rats are not accompanied by changes in Sr.


[Alpha 1 antitrypsin deficiency in childhood].

The author deals with the clinical importance of the alpha-1-antitrypsin deficiency in form of a survey with tabular representation of own cases. Alpha-1-antitrypsin as an inactivator of proteolytically effective enzymes essentially participates in the localised effect of proteinases. A genetically determined decreased alpha-1-antitrypsin serum level (= alpha-1-antitrypsin deficiency) by this causally participates in the pathogenesis of certain hepatopathies and in the pulmonary emphysema appearing already at the carly adult age. Within differential-diagnostic considerations an alpha-1-antitrypsin deficiency should be excluded in: 1. etiologically unclear cholestasis in infancy, 2. etiologically unclear hepatopathy in childhood, and in 3. early emphysema.


[Delayed wound dehiscence with traumatic aphakia after keratoplasty (author's transl)].

A description is given of two patients who had a severe wound dehiscence with traumatic aphakia after perforating kertoplasty. In both cases, after vitrectomy and resuturing the transplant remained clear in its central part (visuel accuity 0.7 p). Details of the operative technic are discussed.


[Erosive-ulcerative type of herpes simplex of the lids (author's transl)].

In 14 patients the diagnosis of an erosive-ulcerative type of herpes simplex of the lids was made. It is characterized by erosions of the intermarginal portion of the lid or by erosions or ulcers of the skin, especially close to the lid margin. A combination of both clinical forms is possible. Characteristic herpetic vesicles have been observed only in one case, being situated in close proximity to the erosive lesion of the lid margin. The visualisation of erosions located at the intermarginal portion of the lid can be facilitated by staining with fluorescein. The lack of vesicular eruptions impedes a proper diagnosis; hence one should search for the history of recurrent herpetic infections. A presumptive diagnosis can be confirmed by the immunofluorescence technique. For treatment, topical application of virustatic and antibiotic ointment is recommended.


[Cytomorphologic findings in the diagnosis of intraocular lymphocytic tumors (author's transl)].

Fifteen cases with haemolytic changes in the region of the eye, i.e. ten cases of lymphocytic sarcoma, two cases of M. Hodgkin, two cases with reticulosarcoma and one of acute lymphatic leukosis are presented. With cytologic punctions of the eyelids and of the retrobulbar tissue, typical cytomorphologic findings for above mentioned haemoblastoses were obtained.


[Ophthalmological themes in baroqueiconography in north-west Croatia (author's transl)].

The northwest Croatia is distinguished through valuable master-pieces of exuberant baroque art. Cultural comprehensions and spirit of time are registered in numerous sacred and profane monuments of art. It is also understandable, that actualities of medicine are reflected in art, too. Small and big plastic-arts, pictures and frescoes are iconographic material of what ophthalmological themes we try to analyse to conceive knowledges about health and sick eyes in that time, about regional pathology and social aspects of eye diseases, also thus to get to learn ophthalmological problems of that time.


[Report on renal transplant patients. Ocular changes due to renal disease and immunosuppressive therapy (author's transl)].

51 patients with renal transplants were examined ophthalmologically 31,1 (1--77) months after the transplantation. 80,4 p. c. showed ocular complications: cataract formation in 43,1 p. c. of the patients examined and increased intraocular pressure values between 22 and 30 mm Hg in 3 patients are to be attributed to the systemic immunosuppressive therapy. Further ocular changes were recurrent subconjunctival haemorrhages due to increased vascular rigidity, calcium phosphate deposits in the conjunctiva due to persistant secondary hyperparathyroidism and fundus changes (pigmentary irregularities in the foveal regions, narrow arterial vessels). Although marked arterial hypertension was observed in 21 patients after the transplantation, no signs of hypertensive retinopathy could be found. Despite the high incidence of ocular complications after renal transplantation the risks of immunosuppressive therapy must be considered as tolerable: cataract formation and increased intraocular pressure do not impair the positive effect of renal transplantation on ocular functions. Regular ophthalmological control examinations of renal transplant patients are advisable.


[Pilocarpin eye drops with a novel Pilocarpin polymeric salt (author's transl)].

A new pilocarpin salt was obtained by chemically linking pilocarpin to a polymere substance. By galenic processing to the respective emulsion, eye drops containing this pilocarpin salt were produced, which enable a retarded pilocarpin delivery in vitro. With respect to glaucoma therapy, clinical tests showed that frequency of application can considerably be reduced. In the present investigations on a collective of 30 normal volunteers the effect of a commercial aqueous pilocarpin solution on the IOP, pupillary diameter and accommodation was compared with that of the novel pilocarpin drops.


[Prospective controlled clinical trials in surgery. controversial issues in motivation and performance (author's transl)].

Controversial aspects in prospective controlled trials in surgery can be detected at various stages in the course of this research work, such as in motivation, planning and performance as well as in assessment and evaluation of the results. Especially the introduction of control groups, randomization and "blindness" of patients, operators and doctors running the follow-up are subjects of a controversial discussion. Subthreshold arguments for defenders and eniers of the trial are taken from different theories of science, such as empirism and rationalism which are more effective in the daily routine work than expected. Provided that convincingly only therapeutic and diagnostic procedures are compared which were considered as equi-effective and reliable to the same extent according to the best and most complete knowledge available it is ethically justified to perform controlled randomized trials. It should, however, not be expected that they (always) produce differences between treatment of high degree of difference. The aim of the controlled trials is not the introduction of sensational novelties into medicine, but the abolition and prevention of unnecessary and unworthy modes.


Studies on salmonellosis in the house mouse, Mus musculus.

Salmonellae were isolated from the faeces from 17 of 170 (10%) wild house mice. Salmonella typhimurium was isolated from 10, S. typhimurium, var. Copenhagen from 2, S. thompson from 1, and S. muenchen from 4. It was concluded that house mice could be a reservoir of infection and play an important role in human and animal salmonellosis.


Studies on Tyzzer's disease: a long-term study of the humoral antibody response in mice, rats and rabbits.

Mice treated with an antigen prepared from livers infected with Bacillus piliformis developed antibodies to the microorganism which reached a peak on the 7th day and disappeared within 40 days: antibody titres in experimentally-infected mice remained at a high level throughout life. The antibody titres in naturally-infected mice, rats and rabbits ramined positive throughout life and followed the same pattern as that of the experimentally-infected mice.


Studies on Tyzzer's disease: transplacental transmission of Bacillus piliformis in rats.

Clinically healthy rats with antibodies to Bacillus piliformis were given prednisolone in the last week of pregnancy. B. piliformis was demonstrated in the livers of their offspring. None of the dams or the young rats showed clinical signs of disease. Antibodies to B. piliformis were found in the young rats at birth, and presisted for several months. The importance of potential transplacental infections when attempting to establish colonies free from B. piliformis in discussed.


Enzyme-linked immunosorbent assay for the detection of antibodies to pneumonia virus of mice in rat sera.

An enzyme linked immunosorbent assay (ELISA) was established to detect rat serum antibodies to pneumonia virus of mice (PVM). Vero cells infected with the virus were absorbed to the surface of microplates, and the presence of antibodies in the sample evaluated were demonstrated by the subsequent use of goat anti-rat immune globulin G conjugated to peroxidase and by specific substrate. The results indicated that antibodies against pneumonia virus of mice could be detected in rat serum with the ELISA procedure. The results were reproducible, and the method was approximately eight times more sensitive than the hemagglutination inhibition procedure.


The effects of selected antimicrobials on glucose transport in the rat intestine.

To determine whether oxytetracycline hydrochloride and the sodium salt of ampicillin have any adverse effects on the rat intestine, enteric enzyme levels and glucose transport rates were measured in vitro in rats. The intestinal transport of glucose did not differ significantly between control animals and those pretreated with ampicillin. For animals pretreated with oxytetracycline, the transport rates were significantly lower than those for the control group. The difference between the ampicillin and oxytetracycline groups, however, was not statistically significant. No significant differences in enteric levels of sucrase and maltase activity were found between any of the groups. The possibility that some antimicrobial agents may interfere with the absorption of nutrients suggested the need for caution in using these drugs in experimental animals.


Induction of extraglomerular renal damage in experimental chronic serum sickness. I. Histologic and immunofluorescence findings.

Histologic and immunofluorescence studies were performed in nine rabbits after daily administration of human serum albumin at a fixed dosage for periods up to 26 weeks. All but one of the rabbits studied by immunofluorescence showed IgG, C3, and fibrinogen deposits in the renal glomeruli and in the tubules or peritubular tissues. In most cases, human serum albumin could also be identified in the deposits. In two rabbits killed at week 10, kidney pathology was minimal; in one of the rabbits killed in the early phases of the disease and in all rabbits killed after week 20, there were obvious pathologic changes affecting glomeruli, tubules, and/or interstitium. Tubulointerstitial changes were absent in only one of the rabbits killed at late stages of human serum albumin administration, in spite of evidence of tubular deposition of immune complexes. These observations suggest that tubulointerstitial damage is at least as frequent as glomerular damage during the induction of chronic serum sickness.


Squamous-cell carcinoma of the pilonidal sinus: report of a case and review of the literature.

Squamous-cell cancer developing in chronic pilonidal sinuses is extremely rare. Thousands of pilonidal cysts and sinuses are operated upon each year, and malignancy is almost never encountered. Only 26 cases of malignancy arising in pilonidal sinuses are reported in the world literature. We report and document a squamous-cell cancer arising in a chronic pilonidal sinus and also review the world literature.


Surgical repair of criss-cross heart with straddling atrioventricular valve.

The criss-cross heart is a rare anomaly in which systemic and pulmonary bloodstreams cross at the atrioventricular (AV) level. Although diagnosed now with increasing frequency, successful surgical repair of associated anomalies has been reported only once. Straddling AV valve is another rare cardiac anomaly which only recently is being diagnosed consistently preoperatively and corrected surgically. This is the first report of successful repair of cardiac anomalies in a patient with the combination of straddling AV valve and criss-cross AV relationships. The uneventful postoperative course and good result on follow-up in this patient are gratifying and suggest that this combination of anomalies now can be repaired with results as good as those for less complex forms of congenital heart disease.


Intercostal pleuroperitoneal hernia.

The patient described in this report had the initial symptoms and signs of a fractured right ninth rib followed soon by severe signs of trauma to the local chest wall. In the ensuing 5 months sequella suggesting an intercostal hernia gradually developed. Further studies indicated that this hernia consisted of a posterior pleural and anterior peritoneal component. At operation the diaphragm was found to have torn away from its costal attachments. In addition to repairing the intercostal pleural hernia, we recommend that a strip of Marlex mesh be fixed to the inner costal surfaces to form a continuous and durable new attachment for the diaphragmatic margin prior to the repair of the peritoneal component. Determining a probable explanation for the mechanism of the injury was helpful in understanding the reconstruction procedure.


Control of immunologic abnormalities associated with aging.

In an attempt to encourage more intensive studies on the control of immunologic abnormalities associated with aging, the six different approaches which have been attempted are reviewed briefly. They are as follows: (a) internal body temperature control, (b) tissue ablation, (c) dietary manipulation, (d) genetic manipulation, (d) genetic manipulation (e) cell therapy and (f) chemical therapy. The first four are preventive and the latter two are restorative in approach. Many of these studies are very preliminary, but overall, the findings are most encouraging. These studies should resolve the extent to which decline in immunologic vigor with age influences the disease pattern of aging individuals and on their life expectancy.


Development of immune competence.

Present evidence indicates that the precursors of B- and T-cells can be found in the extra-embryonic tissues four days after implantation (day 5) and that by the following day (day 10) certain of the B-cell precursors have differentiated to the stage of the antigen-binding cell. Cells able to secrete antibody are not detected, however, until the later stages of pregnancy. B-cell differentiation has been shown to advance in a stepwise manner through several compartments, and the early stages of maturation are independent of thymic or T-cell regulation. The thymic rudiment appears by the 12th day of pregnancy and the reticuloepithelial tissue is quickly colonized by T-stem cells which migrate from the fetal liver. Within 4 days these cells respond to PHA, recognize and respond to allogeneic antigens, and begin to seed to the peripheral lymphoid tissues. During the latter stages of pregnancy and until about the sixth week after birth T-cell mediated suppressor activity predominates. T-cell killer function can be detected in the neonatal thymus shortly after birth, but this activity increases slowly in the peripheral lymphoid tissues. T-cell helper activity increases slowly after birth as suppressor activity declines. The adult levels of helper-suppressor function are approached about six to eight weeks after birth.


Current status of age altered enzymes: alternative mechanisms.

The occurrence of inactive enzyme molecules in a variety of tissues and animal species has been shown to be of a general nature. The levels of inactive enzyme molecules found in old animals were produced by amino acid analogs in young animals. These levels have been shown to be initially detrimental but subsequently the young system shows recovery by efficiently disposing of the analog-modified proteins. In old animals this disposal is considerably less efficient. Evidence is presented which suggests that post-translational modifications of proteins are the main cause of enzyme inactivation in old animals. Amino acid substitutions and modifications involving charge differences apparently do not contribute significantly to this phenomenon.


The formation of altered enzymes in aging animals.

Evidence is accumulating which supports the idea that the alteration of enzymes is due to post-synthetic modifications rather than to sequence changes. Although unequivocal proof is still lacking, the idea that the changes in old enzymes are due to a subtle denaturation rather than to covalent changes is strengthened by recent work. That altered enzymes may result from a slowed protein turnover in old organisms appears to be a viable hypothesis.


Longevity, stability and DNA repair.

The functional capacity of a cell, tissue, organ, or organism is dependent upon its ability to maintain the stability of its unit components. The higher the differentiated state of the system, the greater the amount of stability required to maintain that state as a function of time. Stability can be achieved via either redundancy or repair. Redundancy while easily achievable in biological systems is both costly and limited by thermodynamic considerations. Repair, in its general sense, has no such limitations. Repair at the cellular and macromolecular level is multiple in its forms and varies as a function of species, tissue, and stage of the cell cycle. The repair of DNA damage is a dynamic process with many components and subcomponents, each interacting with one another in order to achieve a balance between individual stability and evolutionary diversity. Thus, between internal and external factors which damage DNA and the subsequent expression of alterations in the functional stability of DNA lie the multi-functional pathways which attempt to maintain DNA fidelity. A strong correlation between ulta-violet light induced excision or pre-replication repair, as measured by autoradiogrphy and maximum species lifespan has been reported within different strains of the same species, between related species (e.g. Mus musculus and Peromyscus leucopus), between five orders of mammals, and most recently within members of the primate family. As has been demonstrated by the authors and others, differences in excision repair between species and tissues may relate to the turning off of portions of the repair processes during embryogenesis. Regardless of why such correlations exist or the nature of their mechanisms, it is naive to either assert or deny a causal role for DNA repair in longevity assurance systems. For example, while species-related differences in DNA repair may reflect the turning off of such repair processes during fetal development this does not mean that rates of accumulation of DNA damage are not altered by such changes. Indeed, such a phenomena might well explain the rapid evolution of lifespan within the primates without a concurrent input of new genes.


Molecular clocks, molecular profiles, and optimum diets: three approaches to the problem of aging.

It has been hypothesized that the deamidation of glutaminyl and asparaginyl residues serves as a molecular clock for many biological processes including protein turnover, development, and aging. At present, this hypothesis has passed some experimental tests which are necessary but not sufficient for its acceptance. The current state of evidence about deamidation as a molecular clock is discussed. In addition, since the molecular biology of aging, especially in humans, is only partly understood, it is of value to develop quantitative, empirical measures of physiological human age and to use these measures to evaluate alternative human living conditions, especially easily adopted alternatives like variations in diet. This may allow some decrease in the suffering and loss from human aging until such time as molecular biology provides superior and more intellectually satisfying answers. An empirical system which consists of quantitative measurement of several hundred human chemical constituents followed by computerized pattern recognition is described. It is hoped that this system will eventually become an aid in the minimization of the rate of human aging through changes in diet and other factors.


The role of membrane structure and function in cellular aging: a review.

A survey of the available theoretical and experimental evidence is given regarding the cell membrane properties, such as the membrane fluidity, potassium permeability, etc., which are involved in the regulation of intracellular ion contents. These properties of the membrane undergo some age-dependent alterations in numerous cells studied so far, resulting in an increase of the intracellular potassium content. This phenomenon has a serious inhibitory effect on the rates of enzymatic catalyses involved in the protein synthesis, and may explain the decreased protein synthetic capacity of the old cells. Data are presented indicating that some changes in the membrane permeability are also involved in the malignant transformation of the cells; however, these are of opposite direction as compared to aging of cells. The "membrane hypothesis" of aging based on these facts seems to be a useful experimental approach to the problem of cellular aging.


Connective tissues--possible implications of the temporal changes for the aging process.

Two of the "old" mono-cause theories of aging have temporal changes in the connective tissues, especially those in collagen, as main events. The crosslinking of collagen is stable under physiological conditions shortly after the formation of fibrils and the additional physico-chemical stability attained later on probably does not influence the physiological functions of the tissues significantly. The temporal changes in collagen seen as increased thermal stability, decreased solubility and increased mechanical stiffness are discussed in relation to the underlying structural changes. It is concluded that the increased stability of the collagen (mainly type I) in the locomotive system and skin are not "true" aging phenomena. It is possible that the changes in the connective tissues of the lungs and kidneys contribute to the decrease of function with age. The "normal" increase of stiffness of type I collagen may contibute to the increased compliance and residual volume of the aging lung. Our present knowledge of the structure and function of collagens and ground substances in various basement membranes does not permit an evaluation of the role of connective tissues in the age changes in the alveolo--capillary complex, the glomeruli and the exchange between tissues and capillaries in general.


Aging, genetics, and the environment: potential of errors introduced into genetic information transfer by metal ions.

Metal ions, which are introduced into living organisms from the environment, are required in every aspect of genetic information transfer. However, the "wrong" metal ion, or even the "right" metal ion in the wrong concentration, can produce deleterious information transfer effects. Metal ions react with nucleic acids in many different ways, and virtually all of these reactions effect major changes in the structure of the nucleic acids. Cellular metal ion concentrations change with age. The effects of such concentration changes on genetic information transfer suggest the possibility that metal ions can influence the aging process. A specific example of such influence is the accumulation in brain cells of aluminum ions, which may have a relationship to Alzheimer's disease.


The effect of aging on sister chromatid exchange.

The advent of the bromodeoxyuridine(BrdU)-differential staining techniques has greatly facilitated the detection of sister chromatid exchanges (SCE). These SCE have been demonstrated to be an accurate reflection of DNA damage both in vitro in cultured cells and in vivo in mouse and rate bone marrow and spleen cells. In this review, we examine the effect of cellular aging on both baseline and mutagen-induced SCE levels. In all systems examined, aging did not appear to significantly affect the baseline levels of SCE. However, in human fibroblast cultures we have found a significant decrease in the levels of mutagen-induced SCE as a function of both in vitro passage level (in vitro aging) and the age of the cell culture donor (in vivo aging). In addition we have found a similar decrease in mutagen-induced SCE levels in both mouse and rat bone marrow cells and mouse spleen cells where examinations were performed entirely in vivo. Diminished mutagen-induced SCE levels were obtained with a wide variety of agents including mitomycin-C, cyclophosphamide, adriamycin, ethyl methanesulfonate and N-acetyl-2-acetoxyamino-fluorene. These decreased SCE levels were accompanied by increased frequencies of chromosomal aberrations in the older cell populations. If SCE represents a form of DNA repair as has been suggested by several investigators, our finding would indicate impaired DNA repair occurring in old cells.


Cellular replication and aging.

Controversy exists concerning the effect of aging on replicating cell systems. This review summarizes a number of studies which indicate that both in vivo and in vitro, cell replication is significantly altered during aging. In vitro, studies of both human lymphocytes and fibroblasts indicated that a number of replication kinetic parameters are influenced by the age of the cell donor. In vivo, the application of the bromodeoxyuridine-(BrdU)-differential chromatid staining techniques to the analysis of cellular replication kinetics has permitted us to demonstrate that cellular replication is also significantly diminished with aging in mouse and rat cell populations. Therefore, both in vivo and in vitro in human as well as rodent cell populations, the rates of cellular replication are significantly decreased with cellular aging.


Evolution of human longevity: a critical overview.

Evolution of longevity of the ungulates, carnivores and primates is reviewed. Special emphasis is focused on recent evolutionary history of longevity along the hominid ancestral-descendant sequence leading to modern man. Maximum life span potential (MLP) or the change in MLP is predicted in extinct species by (1) a phylogenetic analysis of the MLP of present living species and (2) an empirical equation using brain and body weight estimates from fossils. Both of these methods indicate MLP generally increased during mammalian evolution and at an extremely fast rate during the appearance of the hominid species. These results suggest that relatively few genetic alterations were necessary during the recent evolutionary history of man to significantly extend his innate ability to maintain mental and physical health. Much evidence indicates these genetic alterations principally involve regulatory genes, which control a conserved set of structural genes. Evolution of longevity in man could therefore be a result of simple changes in temporal and quantitative expression. Whether these genetic alterations result from mutational changes and/or chromosomal rearrangement cannot yet be evaluated.


Activities of salivary myoepithelial cells: a review.

Historical developments concerning salivary myoepithelial cells have been outlined and structural features of the cells have been described. Evidence from structural and functional studies supports the belief that myoepithelial cells usually have a dual innervation by parasympathetic as well as sympathetic nerves, and impulses from both types of nerve cause the cells to contract. Functional assessment using salivary flow phenomena and intraluminal pressure changes have been used to determine, so far as is possible, the effects of myoepithelial contractions. In some instances tissues have been examined structurally after such experimental procedures. These investigations indicate that salivary myoepithelial activity 1. Speeds up the outflow of saliva 2. Reduces luminal volume 3. Contributes to the secretory pressure 4. Supports the underlying parenchyma 5. Helps salivary flow to overcome increases in peripheral resistance. However, beyond a certain point, this may lead to sialectatic damage of striated ducts with increase in glandular permeability. Myoepithelial activity may also help to expel parenchymal cell contents in certain instances.


[Ionising rays and laryngeal carcinomas (author's transl)].

Review of the literature and report of a new care of laryngeal cancer after irradiation of a benign lesion of the neck. These cases obviously become rare since benign lesions are no longer irradiated. Today the risk of inducing a second carcinoma by a successful irradiation of the first tumor becomes more important. A study of 109 patients, irradiated for laryngeal carcinoma and surviving with no evidence of disease for a period of at least 5 years has been performed. 8 of these patients developed a second primary in the previously irradiated area after 7-15 years. These second carcinomas are not rare if one considers that most patients with laryngeal carcinoma are 60-70 years old and therefore the life expectance on an average is low. These facts should be taken into consideration when deciding between surgical or radiation therapy in younger patients with high life expectance.


[Remarks about otoplasty and avulsion of the auricle (author's transl)].

I. Correction of the prominent ear. A modification of the Mustarde-technique is reported. We mill with a diamonddrill two rims, one above and a second beneath the new anthelix. So we are able to form anthelix and helix to get a smooth contour. II. Restoration of superior helix. 1. Using the method reported by Cronin (1953) it is necessary to plan the reconstructed part a little bit larger than the normal contour. By shrinking of the skin and resorption of the incorporated cartilage we get normal proportions half a year later. 2. Using the method by Crikelair (1956) we took the auriculo-cephalic flap as a composite graft from the concha. The defects could be closed primarily without using a free graft. III. Replantation of the amputated auricle. We used the method, reported by Arfai (1963). Four operations were necessary to get a satisfying result.


[Foley catheter technique as an alternative to Bellocq pack (author's transl)].

233 cases with posterior nasal pack are compared, two third being treated with Foley catheter technique. Using the Bellocq technique there are 47% of bleeding at intervals and 9% of bleeding while using the Foley catheter. Local and general complications are 3.5 times higher in Bellocq technique than in Foley catheter use.


[The reconstructive use of bipedicled chest flap in cancer surgery of the neck (author's transl)].

The reconstructive use of bipedicled chest flap in cancer surgery of the neck is described. This flaps have been found extremely useful in cases of surgical treatment of postlaryngectomy tracheal stomal recurrences.


Fine structure of the 21S ribosomal RNA region on yeast mitochondria DNA. I. Construction of the physical map and localization of the cistron for the 21S mitochondrial ribosomal RNA.

1. We have used restriction enzyme analysis of petite mtDNAs to construct a detailed physical map of the 21S region on the mtDNA of the Saccharomyces cerevisiae strain JS1-3D. The map covers a segment of about 20,000 bp, on which the recognition sites of the enzymes HapII, HindII, HindIII, Sa1I, XhoI and HhaI have been localized (22 sites in total). This map has been checked in various ways against the independently constructed overall physical map of the mtDNA of strain JS1-3D. In addition, we have constructed a physical map with a resolution of about 200 bp of a HapII fragment of 1850 bp long, which carries the loci omega, RIB-1 and probably RIB-2. 2. The 21S rRNA hybridizes with the five adjacent HindII + III fragments TD9, DT19, TD15, DT14 and TT1, which lie in that order on the physical map of the 21S region. Of these, the two non-adjacent fragments TD9 and DT14 show a much stronger hybridization with 21S rRNA than DT19, TD15, and TT1. 3. The fragment DD5 (= DT19 + TD15) and part of DT14 belong to a sequence of about 1000 bp, which is absent from Saccharomyces carlsbergensis mtDNA. Although DD5 and DT14 show (very weak, respectively stronger) hybridization with 21S rRNA, the 1000 bp insert probably does not code for the 21S rRNA: the 21S rRNA of S. carlsbergensis comigrates with the 21S rRNA of JS1-3D on polyacrylamide gels under denaturing conditions. 4. Fragment DT14 hybridizes with the HindII + III fragment TD9, which shows the strongest hybridization with 21S rRNA. The presence of these sequence homologies has hampered the precise mapping of the 21S rRNA cistron. Our results are compatible, however, with the hypothesis that the sequences, coding for 21S rRNA, are located on HindII + III fragments that are not adjacent on JS1-3D mtDNA, namely TD9, DT14 and TT1.


Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA. II. The organization of sequences in petite mitochondrial DNAs carrying genetic markers from the 21S region.

We have investigated the organization of sequences in ten rho- petite mtDNAs by restriction enzyme analysis and electron microscopy. From the comparison of the physical maps of the petite mtDNAs with the physical map of the mtDNA of the parental rho+ strain we conclude that there are at least three different classes of petite mtDNAs: I. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA. II. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA with a terminal inverted duplication. III. Mixed repeats of an (almost) continuous rho+ mtDNA segment. In out petite mtDNAs of the second type, the inverted duplications do not cover the entire conserved rho+ mtDNA segment. We have found that the petite mtDNAs of the third type contain a local inverted duplication at the site where repeating units can insert in two orientations. At least in one case this local inverted duplication must have arisen by mutation. The rearrangements that we have found in the petite mtDNAs do not cluster at specific sites on the rho+ mtDNA map. Large rearrangements or deletions within the conserved rho+ mtDNA segment seem to contribute to the suppressiveness of a petite strain. There is also a positive correlation between the retention of certain segments of the rho+ mtDNA and the suppressiveness of a petite strain. We found no correlation between the suppressiveness of a petite strain and its genetic complexity. The relevance of these findings for the mechanism of petite induction and the usefulness of petite strains for the physical mapping of mitochondrial genetic markers and for DNA sequence analysis are discussed.


Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA. III. Physical location of mitochondrial genetic markers and the molecular nature of omega.

1. We have determined the physical location of mitochondrial genetic markers in the 21S region of yeast mtDNA by genetic analysis of petite mutants whose mtDNA has been physically mapped on the wild-type mtDNA. 2. The order of loci, determined in this study, is in agreement with the order deduced from recombination analysis and coretention analysis except for the position of omega+: we conclude that omega+ is located between C321 (RIB-1) and E514 (RIB-3). 3. The marker E514 (RIB-3) has been localized on a DNA segment of 3800 bp, and the markers E354, E553 and cs23 (RIB-2) on a DNA segment of 1100 base pairs; both these segments overlap the 21S rRNA cistron. The marker C321 (RIB-1) has been localized within a segment of 240 bp which also overlaps the 21S rRNA cistron, and we infer on the basis of indirect evidence that this marker lies within this cistron. 4. In all our rho+ as well as rho- strains there is a one-to-one correlation between the omega+ phenotype, the ability to transmit the omega+ allele and the presence of a mtDNA segment of about 1000 bp long, located between sequences specifying RIB-3 and sequences corresponding to the loci RIB-1 and RIB-2. This segment may be inserted at this same position into omega- mtDNA by recombination. 5. The role which the different allelic forms of omega may play in the polarity of recombination is discussed.


Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis.

Hybrid ColE1 plasmids called ColE1-coslambda-qua A or ColE1-coslambda-gal can be efficiently tranduced into various E. coli K-12 cells through packaging into lambda phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E. coli K-12 mutants. (1) The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-coslambda-guaA. (2) Pre-existing hybrid ColE1 plasmids had no effect on the frequency of lambda phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. (3) ColE1-coslambda-guaA and ColE1-coslambda-gal DNAs could temporarily but not stably co-exist in E. coli K-12 recA cells. (4) The presence of ColE1-coslambda-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-coslambda-guaA about 7-fold. (5) The same ColE1-coslambda-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA+ gene function(s) and suggest that ColE1 plasmid itself provides norecA+-like functions.


Cis- and trans-activity of P22 antirepressor protein against c-repression specified by the closely related Salmonella phages L and Px1.

The ant products of Salmonella phage P22, synthesized by its immI region, releases when acting in cis replication inhibition for phages P22 and L. When ant product acts trans on a coinfecting immunity sensitive phage (Thomas-Bertani-experiment as test for release of replication inhibition) full replication ensues only if both superinfecting phages are homologous in the specificities of their immC and immI regions. If these regions are heterologous, differing in immC, immI or in both, the replication of the phage expected to be complemented by ant is inhibited. This inhibition is observed in both L- and Px-lysogenic bacteria and can be released in case of ant- amber phage by action of ant in cis in su+ lysogenic bacteria.


Nucleotide sequence of the recognition site of the B-specific restriction modification system in E. coli.

Two sB mutations in the genome of bacteriophage fd were located by sequence analysis in the fd sequence at positions 971 and 6341. Base changes at or close to these positions in phage M13 and in phage fl am 124 also correlate with a loss of sensitivity to B restriction. From the sequence homology between the sequences at the two sB sites the recognition signal for the E. coli B restriction/modification enzzyme is predicted to be: 5' TGA---8N---TGCT 3'  3' ACT---8N---ACGA 5'.


The contruction and replication properties of hybrid plasmids composed of the r-determinant of R100.1 and the plasmids pCRI and pSC201.

We have cloned the entire r-determinant of the antibiotic resistance plasmid R100.1 on the plasmic vectors pCR1 and pSC201. We find that the hybrid plasmids segregate from cultures in which replication of the vector is blocked. This suggests that the r-det is not capable of autonomous replication.


The isolation and genetic characteristics of lambda transducing phages of the uvrA+ and uvrC+ genes of E. coli K12.

Lambda transducing phages carrying the excision repair genes uvrA+ and uvrC+ were selected from a pool of lambda phages carrying EcoR1 fragments of E. coli DNA. These phages and also lambdauvrB+ (obtained from Gottesman) were used to make lysogens of excision-defective strains carrying uvrA-, uvrB- or uvrC-. Lambda uvrA+ was found to transduce strains carrying uvrA- but not those carrying uvrB- or uvrC-, to normal ultraviolet resistance. Similarly, lambdauvrB+ and lambdauvrC+ were found to complement only the corresponding uvr- allele. The lambda transducing phages were co-transduced with gal+ by P1 phage into lysogenic gal- recipients, and presumably were integrated at the normal prophage site.


Rifampicin supersensitivity of rho strains of E. coli, and suppression by sur mutation.

Escherichia coli strains with mutations rho-115, rho-ts15, rho-101 (psu-1) or rho-102 (psu-2) are more sensitive ("supersensitive") to rifampicin than isogenic parent strains, as measured by growth rate in broth and colony forming efficiency on solid media with 5, 10, or 20 microgram of rifampicin per ml. There is no change in sensitivity of rho mutants to the antibiotics penicillin, erythromycin, chloramphenicol, or the detergent desoxycholate. The rho-101 or rho-102 mutations confer rifampicin supersensitivity at 32 degrees C but not 42 degrees C. Mutants of a rho-115 strain that have lost polarity suppression can be isolated by selection for rifampicin resistance. This phenotype, Sur, is not due to reversion of the original rho gene mutation but to a second mutation perhaps in the gene for rho protein or the gene for the beta subunit of RNA polymerase. One class of Sur mutation, occurring in rho-115 cells isolated as resistant to 20 microgram of rifampicin per ml, is co-transducible with the marker ilv, and the gene order is rbs-ilv-sur-38. A model suggested by this map position is that the mutations rho-115 and sur-38 define the domain of rho protein which interacts with the beta subunit of RNA polymerase.


In vitro replication of a DNA fragment containing the vicinity of the origin of E. coli DNA replication.

The restriction nuclease cleavage pattern of E. coli DNA synthesized in vitro in the cellophane membrane system (Schaller et al., 1972) is similar to the one obtained after labelling E. coli in vivo. This is shown for exponentially growing cells and for cells synchronized by amino acid starvation followed by thymine starvation. In synchronized cells a piece of some 180 kilobase pairs is labelled containing oriC and neighbouring regions at 82 min on the genetic map of E. coli. A pulse label in vitro is incorporated into the same piece of DNA, but the center of this region, i.e. the EcoR1 fragment of 8.6 kbp length which contains the oriC region (Marsh and Worcel, 1977; v. Meyenburg et al., 1977; Yasuda and Hirota, 1977) is missing.


The control region of the F sex factor DNA transfer cistrons: physical mapping by deletion analysis.

A technique has been developed which allows the isolation of random deletions extending from unique restriction enzyme sites in plasmid DNA molecules. The method involves transformation of E. coli cells with linear plasmid DNAs generated by restriction enzyme cleavage. We have used this technique to map DNA transfer genes in the tra control region of F sex factor DNA. Deletions within EcoRI fragment f6 of F DNA have been isolated and used to assign physical locations to tra genes by a combination of genetic complementation tests, restriction enzyme analysis, DNA heteroduplexing and the analysis of the proteins synthesised in minicells and in vitro. Deletion analysis has also allowed the identification of the traK gene product.


Reversal of protection by light of the ethidium bromide induced petite mutation in yeast.

An intermediate in the ethidium bromide (EB) induced petite mutation pathway may be destabilized by daylight light to cause a reversion to the normal grande phenotype. Starved cells preincubated in the dark for up to 6 h with 100 microgram/ml EB could be reverted to grandes after one hour of light exposure, whereas similarly treated cells maintained in the dark expressed the petite mutation in more than 80 percent of the population. In addition, the production of petite mutants by EB in buffer could be prevented if cell suspensions were exposed to light immediately upon the addition of EB. Photoreversal of the EB-derived petite mutation in growing cells was less efficient presumably because the availability of an energy source caused a continuation of mutation events beyond the light revertible step to a non-reversible fixation of the mutation. Cells treated with EB in growth media at 4 degrees C were more responsive to light protection and reversal of the mutation. This may be due to the cold inhibition of an enzyme which comes into play beyond the light sensitive step in the mutation pathway.


Mechanisms of recombination by the RecBC and the RecF pathways following conjugation in Escherichia coli K12.

The recombinational processes directed by the RecBC and the RecF pathways following conjugation in E. coli have been compared. The viable recombinant products of the RecF pathway show a higher incidence of mismatch correction, higher percentage of heterogeneous clones produced by single ex-conjugants and a much slower rate of integration and segregation compared to the RecBC pathway. There are reasons to suspect that the product of recB and recC genes may be necessary for conversion of the single stranded donor DNA in the zygote to double stranded DNA. Theoretical considerations suggest that an exchange involving only one strand of DNA may be a much slower process, with more stringent homology requirement for the entire exchanged segment, than a double strand exchange of a comparable length; the latter should be much faster, with stringent homology requirements for only the terminal regions of the exchanged segments. It is suggested that the RecF pathway mainly mediates replacement of relatively long stretches of single strands of recipient DNA by the corresponding strands of donor DNA while the RecBC pathway mediates exchange of mostly double stranded DNA between the donor and the recipient; in addition, the RecBC pathway may also catalyze the integration of very small segments of single strands of the donor DNA. A model based on the above basic hypothesis is described. It is further suggested that the enzymes exonucleaseV and exonucleaseI Control the relative yields of the recombinants produced by the two pathways by regulating the supply of the donor substrates required by these pathways; the former diverts the potential substrate of the RecF pathway (single stranded DNA) to the duplex substrates of the RecBC pathway while the latter destroys the substrates of the RecF pathway, especially in absence of exonucleaseV.


Cis-dominant regulatory mutations affecting the expression of GABA permease in Aspergillus nidulans.

In Aspergillus nidulans expression of the gabA gene, the probable structural gene for the gamma-amino-n-butyrate (GABA) permease, is controlled by induction, via the intA gene, ammonium repression, mediated by the areA gene, and probably carbon catabolite repression. Regulatory mutations, tightly linked to gabA, were selected by reverting an areAr-2 strain on GABA as nitrogen source. These mutations, gabI-1, gabI-2, and gabI-3 result in increased gabA expression and are cis-dominant in their effects on the gabA gene. Mapping data show that the regulatory mutations map on one side of all gabA- alleles tested.


Concerted repression of the synthesis of the arginine biosynthetic enzymes by aminoacids: a comparison between the regulatory mechanisms controlling aminoacid biosyntheses in bacteria and in yeast.

It has been shown that in bacteria, besides specific regulatory mechanisms, the synthesis of aminoacid biosynthetic enzymes is also controlled by the endogenous aminoacid pool. The latter regulates the intracellular level of ppGpp, a positive effector of RNA messenger transcription. A similar regulatory control exists in yeast but does not appear to involve the same general effector. This was established by the observation that derepression of the enzymes belonging to several aminoacid biosynthetic pathways follows aminoacid starvation or tRNA discharging. We now report the repression of the arginine pathway by the total aminoacid pool. New mutations affecting the repressibility of the arginine enzymes as well as enzymes belonging to other aminoacid biosyntheses, when cells are grown in the presence of an excess of aminoacids, were identified.


Genetics and biochemistry of the peptidoglycan-associated proteins b and c of Escherichia coli K12.

To collect information on synthesis and regulation of the peptidoglycan-associated pore-forming outer membrane proteins b and c, mutants resistant to phages Me1 and TuIa were analyzed. Genetic analysis showed three linkage groups, corresponding with the genes tolF (phenotype b-c+), meoA (phenotype b+c-) and ompB (phenotypes b-c-, b-c+, b++c- and b++c+/-). It has recently been described that also a b+c- phenotype can occur in the latter linkage group [Chai, T., Foulds, J., J. Bacteriol. 130, 781-786 (1977)]. Among ompB (b-c+)/meoA (b+c-) double mutants strains were found with the b+c- phenotype, showing that ompB is not the structural gene for protein b. Studies on purified proteins b and c showed profound differences between the two proteins with respect to the electrophoretic mobility of fragments obtained by treatment with cyanogen bromide, trypsin and chymotrypsin. The amino acid in position three of the amino-termini of proteins b and c, isolated from isogenic strains, were identified as isoleucine and valine respectively. Both the genetic and biochemical results are consistent with a model recently published [Ichihara, S., Mizushima, S., J. Biochem. (Japan) 83, 1095-1100 (1978)] which predicts that tolF and meoA are the structural genes for the proteins b and c respectively and that ompB is a regulatory gene whose product regulates the levels of both proteins.


meoA is the structural gene for outer membrane protein c of Escherichia coli K12.

The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Me1 resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptor-complex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.


Template specificity of Qbeta and SP phage RNA replicases as studied by replication of small variant RNAs.

Template specificity of two RNA-dependent RNA polymerases (Qbeta and SP RNA replicases) was examined using "variant RNAs" as template. Three variant RNAs, one (8S) generated by Qbeta replicase and two (6S and 5.2S) generated by SP replicase, were isolated from the reaction mixtures incubated in the absence of exogenous template RNA. All these RNAs were found to be active as template for both Qbeta and SP replicases, though homologous RNA exhibited activities about three times higher than heterologous RNA with either enzyme, in agreement with the results obtained in phage RNA-dependent reactions. In these reactions, faithful replication of variant RNA was observed, and the amount of RNA synthesized was in a many-fold excess over the template RNA added. We also found that the heterologous RNA-dependent reactions were suppressed by increasing the concentration of salts or decreasing the concentration of substrates. Under such conditions, replication of heterologous variant RNA was almost completely suppressed, while the amount of homologous variant RNA synthesized was only reduced to 50% of that synthesized under the standard conditions. Thus the template specificity of the two RNA replicases seems to be expressed more strictly in these replication systems.


Assembly of the mitochondrial membrane system: two separate genes coding for threonyl-tRNA in the mitochondrial DNA of Saccharomyces cerevisiae.

1. Mitochondria of Saccharomyces cerevisiae contain two tRNA's that are acylated with threonine. The two isoaccepting species (tRNA1Thr and tRNA2Thr) can be separated by reversed-phase chromatography on RPC-5. 2. A cytoplasmic mutant has been isolated which lacks tRNA1Thr but has normal levels of tRNA2Thr. This mutation was previously shown to map between the oxi 1 and oxi 2 loci on mitochondrial DNA. 3. tRNA1Thr and tRNA2Thr hybridize to wild type mitochondrial but not nuclear DNA and are capable of partially competing with each other. Hybridization of each species to different segments of mitochondrial DNA isolated from p- clones indicate that there are two threonyl tRNA genes. One gene is located between oxi 1 and oxi 2 and codes for tRNA1Thr. The second gene codes for tRNA2Thr and is near the cap locus. 4. Binding assays to E. coli ribosomes indicate that tRNA2Thr recognizes the threonine triplet ACA and may also recognize the other three triplets but with a much lower efficiency. None of the four codons for threonine stimulate the binding of tRNA1Thr to the ribosomes.


Production of petites by cell cycle mutants of Saccharomyces cerevisiae defective in DNA synthesis.

Mutations in two genes (cdc8 and cdc21) required for nuclear and mitochondrial DNA synthesis in Saccharomyces cerevisiae result in a 6- to 11-fold increase in the rate of mitotic segregation of petites at the permissive temperature. The defect in DNA replication and the increased rate of petite production result from the same mutation since the two phenotypes cosegregate and corevert. Most of the petites isolated from strains carrying mutations in cdc8 and cdc21 contain mtDNA. Therefore, the petites do not result simply from an underreplication of mitochondrial DNA. The mutation rates for nuclear and mitochondrial genes are the same in cdc8, cdc21 and their wild-type parent. Therefore the petites are unlikely to result from an increase in the rate of base pair substitution.


Evidence for co-transcription of the RNA polymerase genes rpoBC with a ribosomal protein gene of escherichia coli.

The adjacent genes rpoB and rpoC code for the beta and beta' subunits of RNA polymerase in Escherichia coli, and are cotranscribed in the order given. The nearest known genes to rpoB are rplL and rplA,J,K, which code for ribosomal proteins, and which are transcribed in the same direction as the polymerase genes. It has been suggested that rpoBC may be distal elements of a larger operon including these ribosomal genes. To test this possibility we have cloned a segment of DNA, derived by endoR. HindIII digestion from the rpoBC-transducing bacteriophage lambdarifd18, in the replacement vector NMlambda761. The structure of the lambdarpoBC bacteriophages so produced is such that the inserted DNA can be transcribed from lambda promoters, allowing us to confirm that it carries intact rplL, rpoB, and rpoC genes. We have studied these bacteriophages as lysogens in rec+ and rec bacteria, and by infection of UV-irradiated bacterial strains in which lambda promoters are either repressed or active. The results indicate that the cloned DNA contains at most a very weak promoter for the above genes, in contrast to that present in the larger segment of bacterial DNA carried by lambdarifd18. We have in the same way cloned the adjacent bacterial HindIII-fragment of lambdarifd18 DNA, and have found that it displays vigorous autonomous expression of the tufB, rplA, and rplK genes. We conclude that rpoB and C are obligatorily co-transcribed with rplL, from a promoter located outside the DNA segment cloned in lambdarpoBC. We discuss the evidence for the existence of a regulatory site, rpoU, located between rplL and rpoB.


Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. IV. Mutants with increased levels of leucyl- or seryl-tRNA synthetase.

Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile leucyl-tRNA synthetase and seryl-tRNA synthetase were selected for growth at 40 degrees C. Among these, strains were found with increased levels of both thermolabile synthetases. Two distinct genetic loci were found responsible for enzyme overproduction. leuR, located near xyl, causes elevated levels of leucyl-tRNA synthetase; while serR, located near leu, causes elevated levels of seryl-tRNA synthetase.


DNA sequence of the transposable element IS1.

The nucleotide sequence of an IS1 element recently transposed into the lacI gene is reported. This sequence is nearly identical to one previously reported for another IS1 element (Ohtsubo and Ohtsubo, 1978). The implications of this similarity are discussed. The sizes of potential polypeptides encoded in the IS1 DNA have been determined and possible roles for these peptides in the illegitimate recombination events mediated by the element are considered.


ColE1 plasmid mobility: essential and conditional functions.

Sequences essential for the conjugal transfer of ColE1 can be divided into a cis-acting site and a region encoding trans-acting products. Each of these was successively cloned into a non-transmissible plasmid vector. The resulting chimera was transmissible by the conjugative plasmids F'lac,pro (incFI) and R64drd11 (incIalpha). The sequences encoding colicin E1, immunity, and incompatibility were absent from this chimera: therefore they are not essential for the conjugal transmission of the ColE1 plasmid. In contrast to ColE1, however, the same chimera was deficient in conjugal transfer initiated by R751 (incP) and R388 (incW). This suggests that ColE1 sequences other than those cloned in the chimeric plasmid are necessary for its mobilization by R751 and R388. Three such regions were revealed by screening a series of ColE1 insertion mutants for transfer by R751 and R388. Two of these regions encode no other known function while the third is encoded by a region which overlaps the gene for colicin E1 itself.


Physical mapping of the Xba I, Hinc II, Bgl II, Xho I, Sst I, and Pvu II restriction endonuclease cleavage fragments of mitochondrial DNA of S. cerevisiae.

A detailed molecular dissection of the yeast mitochondrial genome can be made with restriction endonucleases that generate site-specific cuts in DNA. The ordering of restriction fragments provides the basis of the physical mapping of mitochondrial transcripts and antibiotic resistance (antR) loci, and is a means of analyzing the molecular organization of mtDNA of petite and mit- deletion mutants. We have previously mapped the sites in the mtDNA of yeast strain MH41-7B recognized by the endonucleases Eco RI, Hpa I, Hind III, Bam HI, Sal I, Pst I, and Hha I, providing a total of 41 cleavage sites. We have now mapped the sites recognized by the endonucleases Xba I, Hinc II, Bgl II, Pvu II, Xho I, and Sst I, which make 6, 13, 5, 6, 2, and 2 cuts, respectively. Fragment maps for each of these endonuclease sites were derived by analysis of the products of double-enzyme digests and by hybridization of 3H-cRNA probes transcribed from low-kinetic-complexity petite mtDNAs to restriction fragments generated by various combinations of enzymes.


Genetic control of formation of very fast sedimenting DNA of bacteriophage T4.

Formation of very fast sedimenting DNA (VFS-DNA) in cells of Escherichia coli infected with phage T4 carrying a defect in gene 49 was differentially affected by a secondary mutation in gene 30 or 46; a mutation of gene 46 markedly reduced formation of VFS-DNA, whereas that of gene 30 did not.


Physical mapping of the yeast mitochondrial genome: derivation of the fine structure and gene map of strain D273-10B and comparison with a strain (MH41-7B) differing in genome size.

(1) We have derived a fine-structure map of the 70 kb mitochondrial genome of the yeast S. cerevisiae, strain D273-10B, and compared it with our previous maps for strain MH41-7B. Restriction fragment maps for 56 enzyme recognition sites for 13 endonucleases, Eco RI, Hpa I, Bam HI, Hha I, Hinc II, Xba I, Hind III, Bgl II, Pvu II, Sal I, Pst I, Sst I, and Xho I, have been derived. We have used several methods to obtain these maps: (a) Four enzymes (Sal I, Sst I, Xho I, Pst I), each of which cuts D273-10B mtDNA at a single site, were employed to localize and orient fragments from multi-site enzyme digests that are cleaved by the single-site enzyme. (b) Radioactively labeled probes (rRNA or copy RNA [cRNA] transcribed from simple-sequence petite mtDNA) were hybridized to restriction fragments from different digests for identification of fragments which share common sequences. (c) The products of double or triple enzyme digests were identified for mapping and confirmation of the localization of restriction sites. (2) The antibiotic-resistant (antR) loci for erythromycin (E), chloramphenicol (C), paromomycin (P), and oligomycin (OI, OII) were positioned on the physical restriction map by hybridization of 3H-labeled cRNA transcribed from simple-sequence petite mtDNAs that retain a single genetic antR marker to appropriate restriction fragments bound to nitrocellulose filters. (3) Mitochondrial transcripts (21s rRNA, 14s rRNA, and tRNAs) labeled with 125I were hybridized to restriction fragments for identification of the corresponding coding sequence. (4) The gene order and localization of the antR loci and mitochondrial transcripts are as follows: C(0-1.5u)-tRNA I(0-21.5u)-P(29-36.6u)-tRNA II(29-46.4u)-14s rRNA(36-38.3u)-OII(60.3-62.5u) - tRNA III(73-76u) - OI(78.6-83.0u) - tRNA IV(82.5-83.0u) - E(94.2-98.6u) - 21s rRNA (94.2-99.4u). (5) The DNA fine structure and gene map of the 70 kb D273-10B mtDNA were compared to the map of the larger MH41-7B (76 kb) mtDNA. There are 56 restriction sites on D273-10B and 67 sites on MH41-7B for the 13 enzymes studied. The additional restriction sites are largely accounted for by the presence, in MH41-7B, of two sets of sequences, "A" (2.7 kb) and "B" (3.0 kb), located on either side of the OII marker. The remainder of the fragments map is remarkably similar for the two strains. The distances separating the antR loci and the mitochondrial transcripts are very similar except in the two regions surrounding OII.


In vitro system for the replication of the mini R1 factor Rsc11.

The in vitro synthesis of the mini R1-factor, Rsc11, was achieved using a soluble Escherichia coli cell-extract system. Triton X-100 lysates of the K12 strain 1101 (Rsc11) fractionated by Sephadex G25 chromatography supported the incorporation of labeled deoxyribonucleotides into covalently-closed circular (30S) and open-circular (25S) plasmid DNA as well as other molecules of various sizes. DNA synthesis required the presence of all four ribonucleotides and was rifampicin sensitive. Pulse-chase experiments indicated that this reaction is discontinuous. A dependence on ATP and sensitivity to nalidixic acid suggested this system capable of the replicative synthesis of Rsc11 DNA. Density-shift analysis confirmed this. In addition to hybrid, fully-heavy plasmid supercoils were synthesized indicating that more than one round of replication was completed. Approximately one-third of the molecules available to the system participate in this reaction.


Control of bacteriophage lambda repressor establishment transcription: kinetics of l-strand transcription from the y-cII-oop-O-P region.

The kinetics of lambda l-strand repressor establishment RNA synthesis were measured from the y-cII region of induced tof- prophage. The activity of the repressor is epistatic to the expression of gene tof coding for the antirepressor (Tof). The activity of Tof, is epistatic to the expression of repressor gene cI transcription from Prm and the expression of repressor establishment transcription from a site 600 to 800 nucleotides upstream from Prm. Three modes of l-strand rex-cI-tof-y-cII-oop transcription occur: (a) Prm promoted cI-rex mRNA synthesis from noninduced prophage, (b) coordinate lit and oop synthesis from induced tof+ prophage and (c) establishment transcription from induced tof- prophage. The synthesis or stability of oop RNA is much reduced from induced tof-, compared with tof+ prophage. The oop transcription from tof- prophage is not coordinate with RNA synthesis from the y-cII interval. The y-cII-(oop) portion of the establishment transcript appears more unstable than the translated downstream copy of genes rex-cI. The initiation of any repressor establishment transcription requires the products of lambda genes cIII, cII, P and Escherichia coli genes dnaB, dnaG, but not actual lambda DNA synthesis. This result demonstrates that common factors, i.e. replication gene products, are required for the initiation of establishment transcription, lambda replication and lit, oop RNA synthesis; and explains why cIII+ cI+ cII+ replication defective phage lysogenize poorly at low multiplicities of infection. The cIII and cII products were shown to act after an earlier replication initiation or activation event. Repressor establishment transcription and repressor mRNA synthesis from Prm (from induced cI- tof-, cIII- cI- tof- or cI- tof- cii- prophage) are amplified by gene dosage. The extent of lysogenization of E. coli by lambda cIII-, cII- or replication minus mutants, defective for initiation of establishment synthesis, is attributed to gene dosage dependent transcription from Prm. The mechanism by which Tof inhibits the initiation of establishment transcription does not appear to require repression of RNA synthesis from PL and PR. RNA synthesis from these promoters is blocked by renaturation of the repressor 5 min after induction, before establishment transcription is detected; however, establishment RNA synthesis measured between 12-13 min after induction, i.e. 7 min after renaturation of the repressor, is only partially reduced.


The DNA repair capability of cdc9, the Saccharomyces cerevisiae mutant defective in DNA ligase.

The cell cycle mutant, cdc9, in the yeast Saccharomyces cerevisiae is defective in DNA ligase be deficient in the repair of DNA damaged by methyl methane sulphonate. On the other hand survival of cdc9 after irradiation by gamma-rays is little different from that of the wild-type, even after a period of stress at the restrictive temperature. The mutant cdc9 is not allelic with any known rad or mms mutants.


Spontaneous degradation of pRD1 DNA into unique size classes is recA dependent.

The his and nif genes of the P1 type plasmid pRD1 were lost at a high frequency in a recA+ but not in a recA- Escherichia coli host during growth in a non-selective medium. 92% of the His- Nif- segregants after 6 subcultures retained the genetic markers of the precursor plasmid RP4, while the remainder lost all of the pRD1 markers with the concomitant loss of ccc-DNA. Plasmids purified from the His- Nif- segregants resembled RP4 in the physical and genetic properties examined. The contour length of pRD1 DNA molecules from a recA- strain was 49 micrometer corresponding to a molecular weight of 101 Mdals and the buoyant density was 1.715 g/cm3. In contrast, the contour lengths of plasmid molecules isolated from a recA+ host carrying pRD1 fell into 3 size classes of 49, 19 and 2 micrometer corresponding to molecular weights of 101, 39 and 4 Mdals respectively and two DNA species of buoyant density 1.715 and 1.719 g/cm3 were observed.


DNA synthesis in UV-irradiated Escherichia coli K-12 strains carrying dnaA mutations.

It has been found that UV irradiation of dnaA mutants of E. coli K-12 enables the initiation of DNA synthesis at a temperature restrictive to these mutants. The UV-induced DNA synthesis is dependent on protein synthesis and on a transcriptional event at a time when protein synthesis is no longer required. In contrast to dnaA mutants UV irradiation fails to induce DNA synthesis in the two other initiation mutants dnaC2 and dnaB252. DNA synthesis at the restrictive temperature is initiated also when the tif-1 phenotype is expressed in the dnaA46 tif-1 double mutant. Possible mechanisms of the observed capability of dnaA mutants to synthesize DNA at the restrictive temperature after UV irradiation or under conditions of tif-1 expression are discussed.


The regulation of hexokinase and phosphoglucomutase activity in Aspergillus nidulans.

The levels of glucose-6-phosphate and 6-phosphogluconate dehydrogenase in wildtype cells of Aspergillus nidulans varied with the carbon and nitrogen source. In general, hexokinase activity did not vary with carbon or nitrogen source. The ammonium derepressed mutant amrA1 had only 50% of the wildtype level of hexokinase. Phosphoglucomutase activity was low in wildtype cells grown with nitrate, but high in cells grown with ammonium when glucose was the carbon source. A non-inducible mutant, nirA-1, in the regulatory gene for nitrate reductase, had high phosphoglucomutase activity when grown with nitrate or ammonium. A constitutive mutant nirAc1, in the regulatory gene for nitrate reductase had low phosphoglucomutase activity when grown with nitrate or ammonium. The mutants nir-1 and nirAc1 are recessive and semi-dominant respectively for abnormal phosphoglucomutase activity.


Transposition of DNA inserted into deletions of the Tn5 kanamycin resistance element.

Tn5-trp hybrid transposons have been constructed by insertion of a trpPOED Hind III fragment into an in vivo Tn5 internal deletion mutant or by substitution of trp for the internal Tn5 Hind III fragment. These hybrids are called, respectively, Tn409 and Tn410. Both Tn409 and Tn410 will transpose into lambda in the presence of a complementing Tn5 element. In the absence of a wild Tn5, lysogens carrying R1162::Tn409 and R1162::Tn410 plasmids will yield lambdatrp phages at less than six per cent of the complemented frequency. This reduction indicates that Tn409 and Tn410 lack a diffusible transposition function provided by wild Tn5 elements. However, the formation of lambdatrp phages without complementation is real. Most of these transducing particles contain Tn409 and Tn410 still linked to the carrier R1162 plasmid. This observation suggests that uncomplemented Tn409 and Tn410 elements mediate the formation of lambda-transposon-plasmid cointegrate structures. Thus, the missing transposition function may be involved in resolving these cointegrate structures to the final lambda::Tn409 or lambda::Tn410 product.


Molecular cloning of the 4.2 Md EcoRI fragment of Aspergillus nidulans mitochondrial DNA.

The 4.2 Md EcoRI fragment of Aspergillus nidulans mitochondrial DNA was cloned using the plasmid pBR 332 as vector and E. coli as host. Hitherto unknown sequence of HindIII sites within this region of mitochondrial genome was established.


Expression of Escherichia coli tryptophan operon in Rhizobium leguminosarum.

RP4-trp hybrid plasmid containing Escherichia coli whole tryptophan operon was conjugatively transferred from E. coli to Rhizobium leguminosarum strains carrying mutations in different trp genes, converting their Trp- phenotype to Trp+. That the phenotype change of the R. leguminosarum cells was due to the presence of the E. coli tryptophan operon was verified by the isolation of RP4-trp hybrid plasmid from the R. leguminosarum conjugant cells, and by re-transfer of RP4-trp plasmid by conjugation back to E. coli trp and Pseudomonas putida trp strains. Enzymatic activities of anthranilate synthetase and beta subunit of tryptophan synthetase in crude extracts of R. leguminosarum cells containing RP4-trp plasmid were much higher than that of the wild-type cells and were not repressed by the presence of tryptophan in the culture medium.


Genetic evidence for the nature, and excision repair, of DNA lesions resulting from incorporation of 5-bromouracil.

Escherichia coli mutants defective in DNA uracil N-glycosidase (ung-) or endonuclease VI active against apurinic/apyrimidinic sites in DNA (xthA-) exhibit enhanced sensitivity towards 5-bromodeoxyuridine relative to the wild type strain, pointing to involvement of these enzymes in repair of bromouracil-induced lesions in DNA. Mutants defective in DNA polymerase I, either in polymerizing activity (polAl-) or (5' leads to 3')-exonuclease activity (polA107-) exhibit unusually high sensitivity (including marked lethality) in the presence of 5-bromodeoxyuridine. The results indicate that DNA polymerase I, and its associated (5'--3')-exonuclease activity, are involved in repair of bromouracil-induced lesions and are not readily replaced, if at all, by DNA polymerases II and III. Thermosensitive mutant in DNA ligase gene (lig ts7) shows high sensitivity towards 5-bromodeoxyuridine at 42 degrees C indicating the role of the enzyme in repair of bromouracil-induced lesions in DNA. Involvement of DNA uracil N-glycosidase, and endonuclease active against apurinic/apyrimidinic sites in recognition and repair of 5-bromouracil-induced damage permits of some inferences regarding the nature of this damage (lesions), in particular dehalogenation of incorporated bromouracil to uracil residues.


Non-random distribution of transduction termini in transductants from the integrated R plasmid, R100-1.

Tra+ and tra- derivatives of drug resistance plasmid, R100-1, were isolated by phage P1 from an Hfr donor with integrated R100-1 and then analyzed by complementation tests with tra- point mutants of Flac. Tra+ derivatives of R100-1 carrying tetracycline resistance alone and those carrying all six drug-resistrance genes could support transfer of tra- point mutants of Flac except Flac traJ, whereas all of tra- derivatives of R100-1 failed to complement any one of tra- point mutants of Flac. This suggests that these tra- derivatives of R100-1 carrying tetracycline resistance gene are deleted for all the transfer genes impaired in the Flac point mutants tested. We assume a "hot point", probably a specific base sequence similar to an IS element, at the left of the tetracycline gene (Fig. 1) becomes a transduction terminus in transduction of the integrated R100-1 by phage P1. Complementation analysis of tra- derivatives carrying five resistance genes except the tetracycline gene led us to a supposition that a gene(s), probably analogous to traJ of the F plasmid, is located on R100-1 near the tetracycline gene which plays an important regulatory role for self-transfer as well as for the complementation of tra- Flac mutants.


The regulation of RNA synthesis in yeast IV. Synthesis of double-stranded RNA.

A study has been made of the regulation of the synthesis of Pl double-stranded (ds) RNA, the genome of the yeast virus-like particle. When yeast protein synthesis is prevented by starvation for a required amino acid or by addition of cycloheximide, the rate of Pl dsRNA synthesis is reduced markedly. During nitrogen starvation the synthesis of Pl dsRNA persists but is accompanied by the degradation of pre-existing molecules. This degradation appears to require the induction of new enzymes and it is likely that the breakdown products are used to enable the cell to complete its division cycle. However, all of the copies of the VLP genome are not degraded in this process, some are conserved and can replenish the amount of Pl dsRNA on return to growth conditions. The controls which must operate on Pl dsRNA synthesis are discussed and compared with those exerted on nuclear RNA synthesis in yeast.


Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis.

From a collection of 8,000 transposon-insertion mutants of Escherichia coli K12 we identified two mutations, trg-1::Tn5 and trg-2::Tn10, that simultaneously eliminate chemotactic response to ribose and galactose, two attractants recognized by independent receptors. We show that these transposon-insertions confer a Trg phenotype, indicating that this specific pattern of tactic defects is a null phenotype. The two mutation sites are cotransductionally linked to an extend consistent with placement in the same gene. The Trg phenotype of a family of deletion mutants produced by curing trg-2::Tn10 implies that trg is a single gene. Experiments with appropriate F-primes and Hfr's locate the trg locus at approximately 31 min on the linkage map, with a marker order: pyrF-rac-(P.O. 43)-trg-man. We also found one trg mutant whose Trg phenotype was not linked to a transposon-insertion but is probably the result of a mutator activity in the parent strain. Selection of transposon-insertions near, but not in trg allowed demonstration of a very close linkage between the spontaneous trg-3 and the transposon-generated trg's, indicating all three mutations are probably in the same gene. In our manipulations of transposon-insertions we found that Tn5 had a tendency to translocate from its initial site of insertion while Tn10 was relatively stable. The trg-product is probably a chemotactic signal transducer, which interacts directly with two independent receptor proteins and transmits information to the central chemotactic machinery.


Mutants in yeast affecting ethidium bromide induced rho- formation and their effects on transmission and recombination of mitochondrial genes.

A series of mutants called ebi, less inducible by ethidium bromide than the parental strain for the rho+ leads to rho- mutation have been isolated after E.M.S. mutagenesis. Some of the ebi mutants also show an important accumulation of rho- cells, in the absence of ethidium bromide. Ebi mutations are nuclearly inherited as shown by meiotic segregation. The effects of these mutants on the transmission and recombination of mitochondrial genes among the diploid progeny of crosses have been studied. Some of the ebi mutants show a non coordinated transmission of the oli1 mitochondrial marker with respect to other mitochondrial markers unexpected for homosexual crosses. This bias which is independent from omega will be discussed in relation to the segregation and recombination. No significant decrease of the frequency of recombinants has been detected.


Study of a suppressor of mutT in Escherichia coli.

A suppressor mutation of E. coli mut T has been isolated and mapped in the lue--ace(E/F) region.


Cloning of the replication gene P of bacteriophage lambda: effects of increased P-protein synthesis on cellular and phage DNA replication.

A restriction fragment of lambdaDNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/lambdaE complement lambdaPam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/lambdaE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of lambdaDNA proceeds normally under these conditions. Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type lambda (lambdawt), acquire the ability to replicate lambdaPam80 phage but not lambdawt when they are transformed with a plasmid carrying the lambdaP gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of lambdawt phage when infected at a high multiplicity. lambdaPam80 phage does not multiply in these cells.


The influence of certain growth conditions on the phosphatase activity of Streptococcus mutans grown in batch and continuous culture.

Acid phosphatase activity was detected in Streptococcus mutans strain NCTC 10832, and both acid and alkaline phosphatase in strains 2M2 and K1R. In batch culture, activity was maximal by mid exponential phase for 2M2 and at the end of this phase for NCTC 10832. Alkaline, but not acid, phosphatase activity of 2M2 and K1R increased when the inorganic phosphate in the medium was low; this was considered due, at least partly, to inducible or derepressible enzymes. In continuous culture, acid phosphatase activity of NCTC 10832 varied with the sugar substrate. The activity was increased by cell disruption and the degree of this increase for cells grown on different sugars parallelled the amounts of extracellular, insoluble polysaccharide produced on those sugars. Activity was highest for glucose-grown whole cells and for sucrose-grown disrupted cells.


Stability and maturation of E. coli ribosomal RNA.

The kinetics of rRNA maturation were investigated in a rifampicin permeable strain of E. coli during exponential growth in glucose minimal medium. The method used involves isotopic labelling of rRNA, and separation of precursor and mature forms by gel electrophoresis. The maturation of both 16s and 23s rRNA was found to follow first order kinetics. The mean life time of the precursors was found to be about 1.5 min. In glucose minimal medium all pulse label in precursors was recovered in mature rRNA, i.e. nascent rRNA is stable.