Modification of the response to 8 MeV electrons of auxotrophic strains of Escherichia coli by amino-acid deprivation and rifampicin. An investigation has been made of the possibility that the accumulation of RNA enriched in messenger can influence the survival of bacteria after exposure to 8 MeV electrons. The multiple amino-acid auxotroph E. coli CP79 RCrel accumulates RNA during inhibition of protein synthesis, and the proportion of RNA present as messenger increases by a factor of 3 during a 2-hour period of amino-acid deprivation. Amino-acid withdrawal led to changes in the shape of the survival curve, which also occurred in strain CP78 RCstr, which synthesizes very little RNA when deprived of amino acids. We conclude that accumulation of RNA enriched in messenger plays little or no part in modification of the survival of irradiated bacteria. We show that rifampicin, an inhibitor of RNA synthesis, increases the sensitivity of both strains equally.


An overview of research on the history of leprosy. Part 1. From Celsus to Simpson, Circa. 1 A.D. Part 2. From Virchow to Møller-Christense, 1845-1973. This overview attempts to evaluate, in general, the results of nearly 2,000 years of writings on the history of leprosy. The ancients, although prone to confuse other skin diseases with leprosy, laid the emotional foundation for later work in superstitution and lore. Their efforts were faithfully copied for hundreds of years and provided at least an accumulation of facts and a documentation of the state of the art. The dawn of scientific medicine in the mid-nineteenth century and the following 125 years has brought an increasing specialization of research in the history of leprosy that might be usefully divided into medical, political, Biblical, instutional, and medieval aspects. Some of the most productive efforts within each of these areas are considered. The challenge of the future is to develop models of analysis and evaluation based on the findings of analysis and evaluation based on the findings of social scientific research rather than relying totally on pure narration. This is the way in which to free the history of leprosy from much of the myth and error that surrounds it.


Continuous postoperative peritoneal lavage in diffuse peritonitis using balanced saline antibiotic solution. Results of postoperative peritoneal lavage using balanced saline and antibiotic solution in 30 patients with diffuse peritonitis have been presented. The results of this study show that such lavage is a significantly useful adjunctive treatment in the management of diffuse peritonitis. It appreciably reduces the mortality and morbidity. Its adjunctive use in suitable cases along with surgery can be safely advocated in clinical practice.


The instilled fluid dynamics and surface chemistry of polymers in the preocular tear film. Using slit lamp fluorophotometry it was demonstrated that the rate of drainage of a vehicle placed in the eye increased with increasing volume and that polymer solutions increased the thickness of the precorneal tear film (PTF). By increasing the viscosity of the delivery vehicle, (e.g., a hydroxypropylmethylcellulose polymer solutions), the PTF retention of fluorescein could be increased. The increased retention was shown to be due to an increase in the tear reservoir volume provided by the more viscous solutions. The PTF retention of fluorescein in a polyvinyl alcohol (PVA) vehicle was not as viscosity dependent, although PVA did seem to produce greater initial PTF fluorescence. This suggested that PVA initially produced a thicker PTF. The PTF retention of fluorescein by five commercial solutions did not have any relation to their wetting properties. The only good correlation with fluorescein retention in the PTF measured, seemed to be the ability of different polymer solutions to stabilize a thick layer of water as measured by the spontaneous spreading of polymer molecules at the air/liquid interface on wet glass surfaces. This model was designed to simulate tear film spreading in vivo. The results suggest that different polymer solutions may produce thicker PTF's than normal by virtue of their ability to drag water with them as they spread over the ocular surface with each blink. Mechanisms by which polymer solutions may increase the thickness of the PTF are discussed.


Electrophysiologic evidence for normal optic nerve fiber projections in normally pigmented squinters. The Siamese cat, a type of albino, has a visual pathway anomaly in which too many optic nerve fibers cross at the optic chiasm, and also frequently has strabismus. The correlation of strabismus with this defect suggests that a similar pathway defect without pigmentation anomalies, may be the cause of much human strabismus. Creel, Witkop, and King have used evoked potential methods to show that such a pathway defect likely occurs in the human albino. While unpublished control experiments verified their results on human albinos, no such defect has been found in the normally-pigmented human squinter. It is concluded that the visual pathway anomaly is limited to albinism and is not a likely cause of most human strabismus.


Computer analysis of electromyographic signals from the human bladder and urethral and anal sphincters. An investigation has been made into the form of the EMG signals from the human bladder and urethral and anal sphincters. A buildup of the low frequency detrusor EMG before micturition occurs in some patients and this is more marked in patients with urgency. A good correlation occurs between the amount of EMG activity and bladder activity as assessed by the cystometrogram. Marked spike activity has been seen in the detrusor EMGs of patients with irritable bladders. The comparison of anal and urethral sphincter EMGs is of interest from the viewpoint of the anatomy of the urethral sphincter and the mechanism of micturition.


[Significance of proteolytic sperm enzymes for the fertility]. Proteolytic enzymes of the male genital tract have important functions in the reproduction process. The sperm enzyme acrosin is of particular significance for the penetration of the zona pellucida. Proteinases are involved in coagulation and liquefaction of human semen. The kallikrein-kinin system has an important role in stimulation and regulation of sperm motility. In addition, semen proteinases seem to be involved in the penetration and migration of spermatozoa through the cervix uteri. Disturbances of the proteolytic potential of human semen may be the cause of subfertility or  infertility. Specific inhibition of proteinases participating in the fertilization process could be an approach for an effective antienzymatic contraception.


[Experience with new skin sutures: synthetic, resorbable material]. A new combined technique of the excision of small and large skin tumors and of exploratory excisions is reported. Besides of the semilunar incision and the modified subcutaneous lambloid decompressing suture already formerly described, the new application consists of continuous intracutaneous suture with submerged ends. The combined method is only practicable because of the high tensile strength of the synthetic absorbable suture material Dexon. Dexon seems to be superior to all other absorbable filaments, e.g. catgut. Healing of scars is very promising with the mentioned method and the new suture material.


Starling curves as a guide to fluid management in the critically ill. Information derived from Starling curves is useful in following the course of critically ill patients. With this knowledge, the physician can better assess the efficiency of the myocardium and determine the optimal filling pressure. Thus, he is enabled to prescribe the necessary fluid and/or drug therapy to maintain the patient at his most efficient cardiac performance. The nurse who monitors cardiac output and wedge at frequent intervals can regulate fluid replacement on this basis.


Preparation of cells from suspensions for correlative scanning electron and interference microscopy. Neutrophils from bovine milk and blood platelets from dog plasma were washed in PBS, fixed in GA, dehydrated, suspended in a drop on a formvar-coated slide and immediately critical-point-dried in CO2. After coating with Pt-Pd the specimens were examined in an SEM. The same cells were then examined by interferometry (Int) in a light microscope, and the dry mass was determined. It is shown that this preparation method for both types of microscopes (SEM and Int) appears to give adequate results as far as fine surface structure (SEM-appearance) and dry mass determinations (Int) are concerned. The method has the advantage of a more precise characterization of individual particles, than would have been possible, if both methods of microscopy (SEM and Int) had been employed on the same sample, but on different specimens.


[Covering of defects in the lateral head and neck regions with shoulder flaps (author's transl)]. In many situations the head and neck surgeon is forced to excise large areas of tumor bearing skin in the lateral head and neck regions. This skin, either containing primary malignancies or tumors arising from underlying structures (such as lymph nodes, the mandible or the parotid gland), often is heavily irradiated. Consequently, free full-thickness skin grafts have limited chance of survival because of the reduced blood supply of the adjacent tissue. In these cases we use the so-called shoulder flap, as first described by Conley, to cover large defects. This flap, bases between the trapezius muscle and the scapula, has a rich blood supply, closes the defects in one stage and avoids restriction of the head. In addition, the donor site can be closed transposition of adjacent skin when possible without functional defect. Nine clinical cases are presented.


[The use of synthetic foam postnasal packing (author's transl)]. For about a year postnasal space packing made of synthetic foam has been used. This fits the contours of the epipharynx, causes no mucosal damage and is better tolerated by the patient than the traditional packing. In 50 cases of postadenoidectomy bleeding, uncontrolled epistaxes and bleeding from epipharyngeal tumours we have been convinced of the usefulness of this synthetic foam.


[Tonsils and immunology (author's transl)]. Tonsils are lymphatic organs carrying out functions of humoral and cellular immunity. They form a local immunologic barrier; they are also involved in general immunologic defence mechanisms. Tonsillectomy or the destruction of tonsils by infections does not reduce the immunoglobulin concentration and antibody titres in peripheral blood. Only the local formation of IgA antibodies against poliomyelitis antigen seems to be impaired at least for a short time. The clinical relevance of this finding is unknown. Signs of defects in immunosurveillance, which were found in some study groups resulting from an increase of the morbidity rates of Hodgkin's disease and thyroid carcinoma, must be considered seriously although there is no statistic evidence for an increase of tumor frequency after tonsillectomy. The indication for tonsillectomy must be decided on clinical findings. Signs for a reduction of local antibody synthesis or for a possible diminuation of immunosurveillance should serve to critical indication.


[The problems of dogbite injuries in the regions of the ear and the larynx of children (author's transl)]. Three characteristic dogbite injuries in the regions of the ear and the larynx of children are described, and the incidence of tissue-alteration and complication summarized. Criteria are given for appropriate wound treatment in order to achieve maximal cosmetic and functional rehabilation.


Pathogenesis of canine pyometra.

Pathogenesis of canine pyometra. Escherichia coli strains associated with pyometra in 85 of 100 bitches investigated had an affinity for urinary tract epithelium and smooth muscle as well as for progesterone-stimulated endometrium and myometrium. The adherence via K-antigen seemed to be important for colonization of the pathogen during development of pyometra. urinary tract infection was detected in 23 of 32 bitches from which urine specimens were obtained. In these bitches, a strain of E coli identical to the strain in the infected uterus was isolated. It was concluded that subclinical urinary tract infection is associated with pyometra and that the uterus will be infected at early metestrus, when receptors for E coli are developed in the endometrium and myometrium, thus enhancing the colonization of bacteria in the uterus.


Salmonellosis in a human infant, a cat, and two parakeets in the same household. Salmonellosis occurred in a human infant, cat, and 2 pet parakeets in the same household. Salmonella typhimurium var copenhagen was isolated from all 4 subjects; however, its original source was never determined. The parakeets subsequently died, and necropsy revealed enteritis, foci of hepatic necrosis, and leptomeningitis.


Localization of luteinizing hormone-releasing hormone in rat hypothalamus using radioimmunoassay. Radioimmunoassays for LH-RH were performed on frozen rat brain sections cut serially in coronal, parasagittal and horizontal planes. In some of the assays, samples were pooled from corresponding areas in different animals. A clear pattern of distribution of LH-RH rich regions emerged. Two prominent components - a caudal high curve and a rostral smaller hump - were observed, and their variable characteristics discussed. The high curve represents the arcuate-medium eminence (ME) region. Our data suggest that this region is not homogeneous, and three different subdivisions of this arcuate-ME region can be distinguished on the basis of LH-RH content. High values were obtained consistently in the arcuate-ME region, except for females in the late afternoon of dioestrus day 2, at which stage the levels in this region dropped until they were little more than base line. The rostral hump of high LH-RH activity varies both in position and amplitude. These variations are associated with (1) the sex of the animal and (2) the stage of the female cycle. In males this hump appeared in the region of the suprachiasmatic nucleus, while in dioestrous females it appeared in the medial preoptic area, rostral to the male location. Some changes in LH-RH levels are thought to be related to the stage in the female sex cycle. During the afternoon of dioestrus, the caudal high curve representing the arcuate-ME region shrank, whereas the rostral smaller hump (preoptic region) showed much higher levels. Some feed-back take-off may occur from the LH-RH released by the arcuate-ME region. Instead of synthesizing its own LH-RH, the preoptic area may concentrate some of the LH-RH released from the arcuate-ME region, thereafter initiating sexual behaviour as suggested by Moss & McCann (1973).


The binding of kasugamycin to the Escherichia coli ribosomes. The binding of kasugamycin to the E. coli ribosomes has been demonstrated by equilibrium dialysis. Kasugamycin binds to the 70S ribosomes in a molar ratio of 1:1. The association constant is approximately 6 X 10(-4) M-1. The antibiotic binds to the 30S subunit as well as to 70S ribosomes, but only slightly to the 50S subunit. The binding of kasugamycin is not significantly affected by the presence of other aminolgycoside antibiotics: streptomycin, kanamycin. or gentamicin. Kasugamycin does not bind to ribosomes derived from a kasugamycin-resistant mutant, ksgC.


Charactertization of spontaneous fosfomycin (phosphonomycin)-resistant cells of Escherichia coli B in vitro. The characteristics of spontaneous fosfomycin-resistant cells isolated in vitro were investigated. Distribution of resistance level to the drug in Escherichia coli B was thought to be broad and, for this reason, resistance to the drug seemed to develope easily in vitro. In the process of isolating the resistant cells, two groups of cells, differing in colony size, were distinguished. Smaller colony-forming cells appeared more frequently than Larger colony-forming ones. Many of the former seemed to be slow growers and decreased simultaneously in utilization of several carbohydrates. One of the Smaller colony-forming isolates was distinctly different from glp T- or uhp-.


Effective respiratory system elastance during positive-pressure breathing in supine man. Two healthy males relaxing supine on a ballistobed were mechanically ventilated at positive end-expiratory pressures (PEEP) from 0 to 19 cmH2O. Pressures at the airway opening, middle esophagus, and stomach were monitored, together with tidal volume (VT) and ballistobed displacement. The effective elastance (i.e., sum of active and passive components) of the respiratory system (E'rs) and its components--abdominal muscle (E'ab), diaphragm (E'di), and rib cage (E'rc)--were calculated. With increasing PEEP, lung compliance increased slightly, E'rc and E'di decreased linearly, and E'ab increased linearly. The combined effective elastance of abdomen and diaphragm (E'ab+di) first decreased and then increased again. The abdomen-diaphragm contribution to VT during mechanical ventilation was approximately half that of spontaneous breathing.


Strontium-85 extraction during transcapillary passage in tibial bone. Indicator dilution experiments were done to determine the extraction of 85Sr during a single passage through capillaries of the tibial diaphysis. Extraction was estimated by injection of 85SrCl2 and a nonpermeant, reference tracer, T-1824-labeled albumin, into the nutrient artery and recording of the effluent venous dilution curves (femoral vein). The mean (+/- SD) maximal instantaneous extraction was 0.53 +/- 0.08 (N = 12). Net retention after 10 min, estimated from venous curves, was 0.41 +/- 0.06 (N = 12), which appeared not substantially different from the retention estimated by direct isotope counting of the tibias for 85Sr, 0.35 +/- 0.06 (N = 12). In a second set of experiments in intact animals, tibial 85Sr extraction after intravenous injection was apparently higher, 0.53 +/- 0.28 (N = 15). Values of tibial diaphyseal blood flow, estimated from washout curves for iodoantipyrine after tibial nutrient artery injection, were 1.47 +/- 0.63 ml/min per 100 g (N = 27). The extraction was not much diminished by higher flows. The estimates of permeability-surface area product (PS) for bone capillaries did increase with flow, suggesting recruitment of more capillaries at higher flows. PS values averaged 0.63 +/- 0.29 (N = 12); we conclude that the capillary membrane is a primary barrier to the passage of 85Sr and presumably other small hydrophilic solutes.


Effect of continuous postive-pressure ventilation (CPPV) on edema formation in dog lung. The effect of CPPV on edema formation in lungs perfused at constant blood flow was studied in whole dogs and in isolated dog lungs. In intact animals, subjected to an increase in left atrial pressure relative to pleural pressure of 40 Torr, pulmonary shunts correlate inversely (r = -0.82) with the level of end-expiratory pressure (PEE). CPPV had no significant effect on total extravasation of liquid even though PEE higher than 20 Torr was effective in preventing liquid from accumulating in the airways. In isolated lobes, perfused at constant blood flow and at a venous pressure of zero, accumulation of liquid occurred when PEE was increased above 8-10 Torr.  At comparable levels of pulmonary arterial pressure, an increase in PEE resulted in lesser accumulation of liquid than when pulmonary venous pressure was elevated. Morphometric measurements revealed no significant difference in the distribution of accumulated liquid within the lung parenchyma between lobes made edematous either by raising venous pressuure or by raising PEE. It would appear that CPPV, while beneficial in improving arterial oxygen tension in pulmonary edema, does not prevent extravasation of liquid in lungs perfused at constant blood flow. High levels of PEE appear to damage the lung by favoring accumulation of liquid in the extravascular spaces of the lung.


Similarity of blood flows using indocyanine prepared in saline or distilled water. Directly measured blood flow pumped through a mixer circuit was compared to estimates of flow from indicator dilution curves derived from bolus injections of indocyanine green dye prepared in 0.9% saline (saline dye) and in triple-distilled water (3 D dye). At flows whose mean appearance time was 1.6 s, the mean error of estimate +/-SD of 2.3 +/- 5.1% using 3 D dye was significantly different (P less than 0.05) from that of 5.6 +/- 7.0% using saline dye. Essentially, there was no difference in estimates of blood flow with each dye solution when mean appearance time was increased to 3.8 s. Thus, the error, which was perhaps due to electrolyte in the concentrated indocyanine green dye (saline dye) and subsequent slowed rate of optical stabilization after dilution in plasma, is small and does not explain the overestimation by indocyanine dilution curves of blood flow in intact dogs.


Gene dosage effects in polyploid strains of Saccharomyces cerevisiae containing gua-1 wild-type and mutant alleles. Triploid and tetraploid Saccharomyces strains containing different combinations of a gua-1 mutant allele and the corresponding wild type were prepared. The cultivation of the different strains in media upon which the mutant fails to grow leads to a pronounced growth rate response to the dosage of the wild-type allele. Proportionality between the specific activity of the guanosine 5'-monophosphate synthetase and the wild-type dosage was reavealed. Inosine 5'-monophosphate dehydrogenase, the precursor enzyme in the pathway, is derepressed in a sigmoid manner when the wild-type dosage is reduced, whereas the activity of cytosine deaminase, investigated as a reference enzyme, is less affected.


Genetic analyses of an amber mutation in Escherichia coli K-12, affecting deoxyribonucleic acid ligase and viability. Genetic analyses of an Escherichia coli K-12 mutant possessing the amber mutation lig-321 were carried out. This mutant is defective in deoxyribonucleic acid (DNA) ligase and conditionally lethal. We constructed strains harboring an F'lig+ or F'lig-321 plasmid. Genetic complementation analyses were done by using these plasmids and by constructing a lig-4/F'lig-321 merodiploid. It was shown that lig-321 does not complement lig-4, unless the former is suppressed by an amber suppressor. The same was found to be the case between lig-321 and lig-ts7. Transductional mapping of lig-321, by a four-factor cross, revealed that lig-321 is very closely linked to lig-4. The frequency of recombinants between the two alleles was not unreasonable for assuming that they arose by intragenic recombination. The lig-4 and lig-ts7 alleles are known to reside in the structural gene for DNA ligase, in which lig-321 may also be located.


Genetic loci responsible for incompatibility on a co-integrate plasmid, R100-1. An R plasmid, R100-1, was mapped previously (Yoshikawa, 1974) by transduction from an integratively suppressed Hfr strain to a recipient with a mutation in gene dnaA. By this method various types of transductants of plasmid R100-1 that exist autonomously or in the integrated state were obtained. Seventy-one such transductants were used in the present study to map gene inc, which is responsible for incompatibility. The results obtained can be explained by either of the following: (i) R100-1 has only a single gene or gene cluster (inc) despite previous work suggesting that this plasmid is a co-integrate of two replicons; (ii) R100-1 possesses more than one inc locus located between the repA and tra loci.


Production of cells without deoxyribonucleic acid during thymidine starvation of lexA- cultures of Escherichia coli K-12. When thymidine-requiring lexA- strains were starved for thymidine, the kinetics of survival were similar to those of a nearly isogenic lexA+ strain. The size distribution of cells in the lexA- and lexA+ cultures were, however, quite different. Whereas most of the cells in the starved lexA+ cultures grew into long filamentous forms (longer than 4.0 mum), many of the lexA- cells were found to have a normal rod shape (4.0 mum or shorter). It was shown that lexA- cells undergo more divisions during thymidine starvation than lexA+ cells. Furthermore, using an autoradiographic method to analyze deoxyribonucleic acid (DNA) distribution in the starved cells, we demonstrated that cells without DNA are produced in both normal and starved lexA- cultures at a much higher frequency than in lexA+ cultures. Some of these cells may be produced by breakdown of DNA, but we favor the hypothesis that they result from an abnormal cell division process. Since lexA mutations are dominant, we conclude that a diffusible product decreases the synthesis or activity of an inhibitor of cell division in lexA- strains when DNA synthesis is blocked by thymidine starvation.


Ribosomal distribution in a polyamine auxotroph of Escherichia coli. The distribution of ribosomal particles has been studied in a polyamine-deficient mutant of Escherichia coli by sucrose gradient centrifugation analysis. Lysates from starved cells contained less 70S monomers and 30S subunits but more 50S particles than those prepared from bacteria supplemented with putrescine. The addition of the polyamine to putrescine-depleted cells induced a rapid change of the ribosomal profile. A similar effect could be obtained in vitro by equilibrium dialysis against a polyamine-containing solution. The ribosomal pattern obtained from starved bacteria was specific for polyamine deficiency. We conclude that the changes in ribosomal profiles upon restoration of putrescine levels in previously starved cells denote a shift of the equilibrium between 30S-50S couples and ribosomal subunits.


Compatibility of pTM89, a new F-like R factor, and of derivative plasmids. pTM89, an fi+ R factor that controls the production of repressed F-type pili, is incompatible with plasmids belonging to the FII and P groups. The results of P1 transduction show that all of the resistance markers of pTM89 are part of a single replicon, which also includes RTF. When the compatibility of different derivative plasmids was investigated, it was found that they fall into two classes. Those of the first class have lost the compatibility of pTM89 for the P group but are still incompatible with FII group, whereas those of the second class are compatible with plasmids of both groups. Plasmids of the latter class that are also compatible with each other and, therefore, apparently lack any determinant for compatibility are genetically stable and self-transmissible. It appears, therefore, that compatibility between plasmids cannot be explained by the hypothesis of competition for a maintenance site.


Translocation of the tetracycline resistance determinant from R100-1 to the Escherichia coli K-12 chromosome. Pairs of normally incompatible derivatives of R100-1 (one ChlS TetR, the other ChilR TetS) were forced to coexist in a recA host by selection for ChlR TetR cells. After many generations stable derivatives were isolated. The analysis of none independent stabilization experiments showed that in each case TetR was translocated from the plasmid to the chromosome of the host. No evidence for the joint integration of other plasmid genes (those controlling transfer, antibiotic resistance, incompatibility, or origin of transfer replication) was obtained. One of the chromosomal TetR determinants was mapped close to metE.


Acid-soluble degradation products of ribonucleic acid in Escherichia coli and the role of nucleotidases in their catabolism. The fate of the internally formed nucleotides resulting from the degradation of ribonucleic acid was studied. Prelabeled Escherichia coli cells were submitted to carbon starvation, and the acid-soluble products were separated by thin-layer chromatography. It was determined that free bases constitute some 75% of the end product, the balance consisting of nucleoside diphosphates, 5'-nucleoside monophosphates, 3'-nucleoside monophosphates, and nucleosides. The majority of degradation products, including phosphorylated derivatives, were excreted into the medium. The amount of products in the pool remained constant. The soluble products formed by E. coli mutants lacking either 5'-nucleotidase (Ush-) or 3'-nucleotidase (Cpd-) were compared with those produced by the parental strain with both enzymes. The results obtained indicated that 5'-nucleotidase is involved in the degradation of internally foromed nucleotides but that 3'-nucleotidase takes no part in the process.


Symbiosis-independent and symbiosis-incompetent mutants of Bdellovibrio bacteriovorus 109J. Symbiosis-independent (Sin) mutants were isolated from the symbiosis-dependent and symbiosis-competent (Sdcomp+) Bdellovibrio bacteriovorus 109J. Independently isolated Sin mutants were examined for their symbiosis competence and most were found to be comp+. Bdellovibrios comp- were selected from the Sincomp+ mutants. The Sincomp+ bdellovibrios are always at a selective disadvantage, either against Sincomp- bdellovibrios (in organic medium) or against Sdcomp+ bdellovibrios (in buffer with Escherichia coli cells).


Interference of dna ts mutations of Escherichia coli with thymineless death. Thermosensitive mutants of Escherichia coli in which deoxyribonucleic acid replication is inhibited at high temperature have been assayed for their response to thymine deprivation at permissive and nonpermissive temperatures. The survival rate of mutants in which initiation of replication is defective at 42 C (dnaA and dnaC-D) is much higher at nonpermissive temperatures than at 32 C. By contrast, thymineless death is not hindered at 42 C in two elongation mutants (dnaE and dnaG). Bacterial strains belonging to the dnaB class exhibit both types of behavior. These results are in accordance with the model linking thymineless death to a disruption of an active replication fork. We discuss the possibility that the dnaB protein takes part in this process of cell decay.


Uroporphyrin-accumulating mutant of Escherichia coli K-12. An uroporphyrin III-accumulating mutant of Escherichia coli K-12 was isolated by neomycin. The mutant, designated SASQ85, was catalase deficient and formed dwarf colonies on usual media. Comparative extraction by cyclohexanone and ethyl acetate showed the superiority of the former for the extraction of the uroporphyrin accumulated by the mutant. Cell-free extracts of SASQ85 were able to convert 5-aminolevulinic acid and porphobilinogen to uroporphyrinogen, but not to copro- or protoporphyrinogen. Under the same conditions cell-free extracts of the parent strain converted 5-aminolevulinic to uroporphyringen, coproporphyrinogen, and protoporphyrinogen. The conversion of porphobilinogen to uroporphyrinogen by cell-free extracts of the mutant was inhibited 98 and 95%, respectively, by p-chloromercuribenzoate and p-chloromercuriphenyl-sulfonate, indicating the presence of uroporphyrinogen synthetase activity in the extracts. Spontaneous transformation of porphobilinogen to uroporphyrin was not detectable under the experimental conditions used [4 h at 37 C in tris(hydroxymethyl)aminomethane-potassium phosphate buffer, pH 8.2]. The results indicate a deficient uroporphyrinogen decarboxylase activity of SASQ85 which is thus the first uroporphyrinogen decarboxylase-deficient mutant isolated in E. coli K-12. Mapping of the corresponding locus by P1-mediated transduction revealed the frequent joint transduction of hemE and thiA markers (frequency of co-transduction, 41 to 44%). The results of the genetic analysis suggest the gene order rif, hemE, thiA, metA; however, they do not totally exclude the gene order rif, thiA, hemE, metA.


trans-Recessive mutation in the first structural gene of the histidine operon that results in constitutive expression of the operon. The first enzyme for histidine biosynthesis, encoded in the hisG gene, is involved in regulation of expression of the histidine operon in Salmonella typhimurium. The studies reported here concern the question of how expression of the histidine operon is affected by a mutation in the hisG gene that alters the allosteric site of the first enzyme for histidine biosynthesis, rendering the enzyme completely resistant to inhibition by histidine. The intracellular concentrations of the enzymes encoded in the histidine operon in a strain carrying such a mutation on an episome and missing the chromosomal hisG gene are three- to fourfold higher than in a strain carrying a wild-type hisG gene on the episome. The histidine operon on such a strain fails to derepress in response to histidine limitation and fails to repress in response to excess histidine. Furthermore, utilizing other merodiploid strains, we demonstrate that the wild-type hisG gene is trans dominant to the mutant allele with respect to this regulatory phenomenon. Examination of the regulation of the histidine operon in strains carrying the feedback-resistant mutation in an episome and hisT and hisW mutations in the chromosome showed that the hisG regulatory mutation is epistatic to the hisT and hisW mutations. These data provide additional evidence that the first enzyme for histidine biosynthesis is involved in autogenous regulation of expression of the histidine operon.


Genetic and physical characteristics of an enterotoxin plasmid. We are engaged in the genetic and physical characterization of an enterotoxin (Ent) plasmid, Ent P307, which contains genes for the production of a hear-labile and a heat-stable enterotoxin. We are using an Escherichia coli K-12 strain, 711 (P307), constructed by S. Falkow, which contains no other plasmids besides Ent P307. Our genetic studies have shown that the plasmid is incompatible with the sex factor F, both in the integrated (Hfr) and the autonomous (F-prime) state. Ent P307 can thus be assigned to incompatibility group FI. An R factor, R386, which belongs to the same incompatibility group, was also found to be incompatibile with Ent P307, whereas five other R factors belonging to different incompatibility groups were compatible with Ent P307. In the presence of Ent P307, conjugal transfer and sensitivity to a male-specific phage of a derepressed F-like R factor, R1drd19, were repressed. Ent P307 is, thus, finO+. Presumably, it also causes repression of its own transfer genes since conjugal transfer of Ent P307 could not be demonstrated. Unlike F, it does not restrict the growth of female-specific phage phiII. From physical studies on extracted deoxyribonucleic acid, the molecular weight of Ent P307 was determined to be 54 X 10(6). By electron microscope heteroduplex analysis, the plasmid was found to be homologous with F in four regions, encompassing about half of its length. One long region and two short ones contain genes for conjugal transfer; the other short region carries genes for replication and incompatibility.


Fine-structure genetic map of the cysB locus in Salmonella typhimurium. A genetic map of the cysB region of the Salmonella typhimurium chromosome was constructed using bacteriophage P22-mediated transduction. Strains bearing delta (supX cysB) mutations were employed to divide this regulatory locus into 12 segments containing a total of 39 single-site mutations. Twenty-five of these single-site mutations were further ordered by reciprocal three-point crosses. The results do not support the concept of multiple cistrons at cysB and suggest that the abortive transductants previously observed in crosses between certain cysB mutants were due to intracistronic complementation. The prototrophic cys-1352 mutation, which causes the constitutive expression of the cysteine biosynthetic enzymes, was found to lie within the cysB region itself. It is bracketed by mutations, which lead to an inability to derepress for these enzymes and result in auxotrophy for cysteine.


Association of nascent ribosomal ribonucleic acid with polyribosomes in Escherichia coli. Sedimentation distributions of rapidly labeled ribonucleic acid (RNA) in extracts prepared from cells labeled for 60 s or less with [3H]uridine showed that the immature ribosome precursor particles themselves have an RNA precursor which sediments with polyribosomes and 70s ribosomes. In extracts prepared from cultures labeled for 30 s, the fraction of labeled RNA which did not sediment with polyribosomes was much less than the fraction which was ribosomal RNA (rRNA). Rapidly labeled RNA associated with polyribosomes had a nucleotide base composition consistent with the presence of rRNA, and was shown by RNA-deoxyribonucleic acid hybridization to contain 30 to 40% rRNA. Nascent rRNA is thus associated with polyribosomes during its transcription. The sedimentation distribution of rapidly labeled 16s and 23s rRNA showed that these molecules are separated during transcription and associated with different polyribosomes.


Genes affecting coliphage BF23 and E colicin sensitivity in Salmonella typhimurium. Rough strains of Salmonella typhimurium were sensitive to coliphage BF23. Spontaneous mutants resistant to BF23 (bfe) were isolated, and the trait was mapped using phage P1. The bfe gene in S. typhimurium was located between argF (66% co-transducible) and rif (61% co-transducible). The BF23-sensitive S. typhimurium strains were not sensitive to the E colicins. Cells of these rough strains absorbed colicin, as measured by loss of E2 or E3 killing units from colicin solutions and by specific adsorption of 125I-colicin E2 to bfe+ cells. Sensitivity to colicins E1, E2, and E3 was observed in a S. typhimurium strain carrying the F'8 gal+ episome. This episome complemented the tolB mutation of Escherichia coli. We conclude that the bfe+ protein satisfies requirements for adsorption of both phage BF23 and the E colicins. In addition, expression of a gene from E. coli, possibly tolB, is necessary for efficient E colicin killing of S. typhimurium.


Unbalanced growth and the production of unique transfer ribonucleic acids in relaxed-control Escherichia coli. The unique leucine-, arginine-, valine-, and phenylalanine-specific transfer ribonucleic acids (tRNA's) produced in relaxed-control (rel-) Escherichia coli during leucine or arginine starvation are chromatographically similar to those produced by chloramphenicol treatment. The major unique rel- leucine-specific and phenylalanine-specific tRNA's are heterogeneous, accumulate with time of starvation, and can account for up to 70% of the respective amino acid acceptor activities. The changes which occur in the isoacceptor profiles for tRNALeu and tRNAPhe as a function of starvation time suggest that the unique species are undermodified precursors to the major isoacceptor species observed in nonstarved cells. Analyses of the isoacceptor patterns of tRNA from cells recovering from starvation suggest that the unique species of tRNALeu and tRNAPhe may not be normally occurring precursors. When leucine-starved cells were incubated in fresh, fully supplemented medium, the major unique tRNALeu and tRNAPhe appeared to be converted to normal species only slowly or not at all. The results are consistent with the view that some of the events in the post-transcriptional modification of tRNA may occur in an ordered sequence. An examination of the subcellular distribution of the unique leucine and phenylalanine tRNA's revealed that these species occur on the ribosome at about the same frequency as the major, normally occurring isoacceptor species. This result provides additional evidence of a precursor-product relationship for the unique and normal tRNA's and further indicates that there is no discrimination against the unique species by the ribosome.


Derepression and repression of the histidine operon: role of the feedback site of the first enzyme. Thiazolealanine, a false feedback inhibitor, causes transient repression of the his operon previously derepressed by a severe histidine limitation in strains with a wild-type or feedback-hypersensitive first enzyme but not in feedback-resistant mutants. Since experiments reported here clearly demonstrate that thiazolealanine is not transferred to tRNAHis, it is proposed that this "transient repression" is effected through the interaction of thiazolealanine with the feedback site of the enzyme. Experiments in the presence of rifampin indicate that this thiazolealanine-mediated effect is exerted at the level of translation. We conclude that histidine (free), in addition to forming co-repressor, also represses the operon at the level of translation through feedback interaction with the first enzyme of the pathway (adenosine 5'-triphosphate phosphoribosyltransferase). Rates of derepression in feedback-resistant strains are roughly half of those observed in controls, suggesting a positive role played by a first enzyme with a normal but unoccupied feedback site. Some feedback-resistant mutants, in contrast to the wild type, were unable to exhibit derepression under histidine limitation caused by aminotriazole.


Deoxyribonucleic acid modification by intermediate-type modification mutants of Escherichia coli K-12 and B. The modification of bacteriophages grown on r-m+/- restriction and modification mutants of Escherichia coli K-12 or B appears to be related to the number of restriction-specific sites in the viral genome. Bacteriophage fd and its mutant U1 fd, which carry two and one B-specific sites, respectively, are not modified in vivo by rB-mB+/- mutant strains. In vitro treatment of fd RF-B+/- deoxyribonucleic acid (DNA) or U1 fd RF-B+/- DNA by endo R-Eco B results in cleavage of the substrate DNA. Lambda bacteriophage, after growth in r-m+/- mutant host strains (lambda-K+/- or lambda-B+/-), is partially protected from in vivo degradation by wild-type homospecific strains. Its efficiency of plating on these strains is approximately 10(-2). However, a hybrid phi80-lambda phage which carries only one K-specific site (sklambda-1) is not modified by rK-mK+/- strains. Labeled DNAs from lambda-B+/- and lambda-K+/- phages were used as substrates for endo R-Eco B and endo R-Eco K nucleases. Zonal centrifugation analysis of the products of the reactions indicate that rK-mK+/- mutants do not protect lambda DNA from in vitro degradation by endo R-Eco K. In contrast, rB-mB+/- mutants appear to partially protect lambda DNA from attack by endo R-Eco B.


Excision of bacteriophage lambda from a site in the arabinose B gene. A lambda lysogen with the prophage inserted into the arabinose B gene of Escherichia coli strain K-12 has been prepared. Induction of the phage from this lysogen yields viable phage at a frequency 4 X 10(-6) that found for induction of lysogens with phage inserted at the normal attachment site. Over 30% of the phage particles induced from the insertion in ara are arabinose-transducing phage. The excision end points of 62 independently isolated, nondefective araC-transducing phage containing less than the entire araC gene were genetically determined and were found to be randomly distributed through the araC gene. The amount of arabinose deoxyribonucleic acid contained on four selected transducing phage was determined by electron microscopy of deoxyribonucleic acid heteroduplexes, providing a physical map of the araC gene. The efficiency with which these phage transduce araC and araB point mutations was found to be approximately proportional to the homology length available for recombination.


Alterations in membrane function in an Escherichia coli mutant tolerant to colicins Ia and Ib. An Escherichia coli mutant (tolI) previously shown to be tolerant to colicins Ia and Ib is defective in several functions of the bacterial cytoplasmic membrane. When compared with its parental strain, X36, whole cells of tolI show reduced rates of respiration with succinate, malate, or lactate as the substrate but near-normal rates with glucose or glycerol. Cell membrane preparations prepared from tolI cells exhibit reduced succinate and D-lactate oxidase activity but elevated levels of reduced-form nicotinamide adenine dinucleotide (NADH) oxidase. tolI cells have reduced levels of succinate and D-lactate dehydrogenase but normal levels of NADH dehydrogenase. Glycerol-grown tolI cells and membrane vesicles prepared from such cells are defective in the active transport of several amino acids and thiomethyl-beta-D-galactoside; however, they accumulate higher levels of alpha-methylglucoside when compared with X36 whole cells or vesicles. Although tolI cells adsorb less colicin Ia at high colicin concentrations than do X36 cells, it is shown that the adsorption of an Ia molecule to tolI cells has a lower probability of eliciting cell death than does Ia adsorption to strain X36 cells. It is concluded that a single mutation can lead to an alteration in several aspects of cytoplasmic membrane function and colicin I sensitivity.


Isolation and characterization of a new temperature-sensitive cell division mutant of Escherichia coli K-12. A new temperature-sensitive mutant strain of Escherichia coli K-12 which forms filaments at 42 C has been described. The mutant, Y16, maintained growth and deoxyribonucleic acid synthesis at 42 C. The resulting multinucleate filaments gradually lost their viability at 42 C but could be recovered, even after 240 min of incubation, upon return to 30 C. Septation was resumed and growth was promptly re-established at normal rates. Recovery still took place in the presence of chloramphenicol added to the culture at the time of temperature shift from 42 to 30 C. A study has been made of the effects of adenine and various nucleosides on cultures of strain Y16 as compared with another filament-forming mutant, T44 tif-. Adenine (75 mug/ml), known to promote filamentation of strain T44 tif-, prevented the development of filaments and the loss of viability in cultures of Y16. Recovery of septation after temperature shift in cultures containing adenine presented a pattern similar to that found with the adenine-less cultures. Protection afforded by adenine at 42 C could be reversed by the addition of guanosine plus cytidine (100 mug/ml each). The effects of high concentrations of adenine and nucleosides on strain Y16 thus are the reverse of those observed with mutant T44 tif-. However, whereas tif-1 mutation promotes prophage induction at restrictive temperatures, no modification could be detected in the process of prophage induction in cultures of the lambda-lysogenic derivative of Y16 at 42 C, be it spontaneous or ultraviolet-mediated induction. The osmolarity increase afforded by 1% NaCl added to the medium did not alter the phenotype characteristics of strain Y16. The mutation has been mapped between argG and bgl. A close linkage has been observed between ftsH and argG, thereby locating the new mutation near 61 min on the map of E. coli chromosome, a previously undescribed region involved in cell division. The evidence reported indicates that strain Y16 differs in several respects from the already descirbed strains of the same class.


Sporulation in D-glucosamine auxotrophs of Saccharomyces cerevisiae: meiosis with defective ascospore wall formation. Mutants that require exogenous D-glucosamine for growth were isolated from Saccharomyces cerevisiae X2180-1A after ethyl methane sulfonate mutagenesis. Class A auxotrophs fail to grow on yeast extract-peptone-dextrose and minimal media, whereas class B auxotrophs grow on minimal medium and readily revert to grow on yeast extract-peptone-dextrose medium. Class B auxotrophs are suppressible by a class of suppressors distinct from nonsense suppressors, and their properties suggest that they are defective in a regulatory function. All 23 mutants studied were recessive and allelic, and they define a new gene designated gcn1. An analysis of a class A auxotroph revealed that it lacked L-glutamine:D-fructose 6-phosphate amidotransferase (EC 2.6.1.16) activity and indicates that GCN1 codes the amino acid sequence of this enzyme. The finding that all mutants were allelic indicates that the amidotransferase is the only enzyme responsible for D-glucosamine synthesis in S. cerevisiae. The occurrence of allelic complementation and media-conditional mutants suggests that the amidotransferase is a multimeric enzyme with an activity subject to metabolic control. Diploids homozygous for gcn1 fail to complete sporulation. They proceed through meiosis normally, as judged by the occurrence of meiotic recombination, the production of haploid nuclei, and the formation of multinucleate cells visible after Giemsa staining. However, the formation of glusulase-resistant ascospores is blocked, and deformed spores lacking the electron-dense outer layer characteristic of the normal spore wall are observed by electron microscopy. Cells that acquire the ability to synthesize D-glucosamine, because of gene conversion during meiosis, complete sporulation in a normal fashion. Thus, the GCN1 gene product appears to be synthesized late in sporulation and may prove to be a useful developmental landmark for the termination of ascospore development.


Timing and function of chitin synthesis in yeast. A temperature-sensitive mutant of Saccharomyces cerevisiae, L-2-42, is blocked at 37 C at a stage of the cell cycle prior to septum formation. When single cells of the mutant are allowed to bud at 37 C in a medium containing tritiated glucose, a large incorporation of radioactivity into chitin takes place. Thus, the synthesis of chitin, the major component of the primary septum, is initiated in a phase of the cell cycle which precedes septum closure. This early period of chitin synthesis is not required for emergence and growth of buds because, in the wild type, budding takes place normally in the presence of concentrations of polyoxin D that effectively and specifically prevent chitin formation. However, at a later time a majority of these cells lyse, presumably because of the inability to form a septum. Polyoxin D also prevents the appearance of enhanced fluorescence at the junction between mother cell and bud, as observed in the presence of a brightener. Therefore, the fluorescence is due to chitin and its presence at the base of very early buds indicates that chitin synthesis begins at or shortly after bud emergence. A scheme for chitin synthesis and primary septum formation which embodies these and other results is presented.


Ultrastructural visualization of surface carbohydrate structures on mycoplasma membranes by concanavalin A. Surface carbohydrates of Mycoplasma mycoides var. capri were made visible by the cytochemical staining procedure with concanavalin A, horseradish peroxidase, and diaminobenzidine.


Unusual membranous structures in minicells and minicell-producing strains of Escherichia coli. Minicell-producing strains of Escherichia coli K-12 seem to produce extra membranous material yielding internal cross membranes, "piggy-back" minicell forms, and unusual vesicles.


Genetic map location of the Escherichia coli dnaG gene. The dnaG locus of Escherichia coli K-12 has been mapped at about 60 min on the genetic map by three-factor crosses using P1 transduction. In crosses selecting for dnaG+, the cotransduction frequency with the tolC marker is 15% and that with the uxaC marker is 49%. The gene order is tolC dnaG uxaC.


Balanced production of 30S and 50S ribosomal proteins after a nutritional shift-up. The synthesis of bulk ribosomal protein (r-protein) after a nutritional shift-up in Escherichia coli B/r was examined. It was found that the molar ratio of the net synthesis rates of 30S and 50S r-protein remains constant during the transition period after the shift-up and equal to the preshift ratio. The implications for the control of ribosome synthesis are discussed.


Transfectability of rough strains of Salmonella typhimurium. Cells of rough (but not smooth) strains of Salmonella typhimurium become competent for transfection by phage P22 deoxyribonucleic acid after treatment with 0.1 M CaCl2. The yield of infectious centers is about 10(-8) per genome equivalent of deoxyribonucleic acid. However, different sorts of rough strains vary in their ability to become competent in a fashion that can be correlated with the level of the genetic block in cell wall lipopolysaccharide synthesis. The most amenable strains are blocked by defects in the addition of galactose units I and II of the lipopolysaccharide by the inability to synthesize uridine 5'-diphosphate-galactose (galE point mutants and gal deletion mutants). Strains blocked only in the addition of galactose I, glucose I, or heptose II have low levels of transfectability, whereas strains with either more complete or more deficient lipopolysaccharide core are not competent for transfection. When normal lipopolysaccharide synthesis is restored either genetically or by furnishing exogenous galactose (galE point mutants that can still use it), the cells are not longer competent for transfection.


Effects of growth conditions on thymidine nucleotide pools in Escherichia coli. The cellular levels of thymidine nucleotide derived from [3H]thymine or [3H]thymidine were followed under various environmental conditions with a thymine-requiring mutant of Escherichia coli. It was shown that the pool sizes varied greatly with the growth conditions; that is, with growth temperature, inhibition of DNA synthesis of replacement of thymine with thymidine. In the strain used here, the level of compound X, presumably dTDP-sugar, was very much higher than those of other thymidine nucleotides. It is suggested that the conversion of thymine to thymidine is rate-limiting, while the conversions of thymidine to dTMP, and of dTMP to dTDP are more rapid than other steps in the salvage pathway of thymidine nucleotide.


Transport of sugars and amino acids in bacteria. XIII. Mechanism of selective inhibition of the active transport reactions for proline, leucine, and succinate by zinc ions. A mutant (S-1) of Escherichia coli which lacks succinate dehydrogenase [EC 1.3.99.1] but has normally succinate transport system, and various oxidase activities other than succinate, was isolated from the strain U24. Using these strains studies were made on the effects of zinc ions on the active transport reactions for succinate proline, and leucine. Zinc ions inhibited succinate transport reaction and succinate oxidation by intact cells. The active transport reaction for proline, which was supported by endogenous energy source in intact cells, was slightly inhibited by zinc ions. This inhibition was not reversed by large excess of magnesium ions. The initial rates of active uptakes of proline were increased by exogenous energy sources such as succinate and glycerol and zinc ions greatly inhibited these. However, D-lactate dependent proline uptake was enhanced slightly by the presence of zinc ions. It was found that zinc ions at a high concentration enhanced the steady level of proline accumulation in cells. This mechanism was studied in detail and we concluded that zinc ions inhibited completely the exchange-exit reaction for proline. Effects of zinc ions on the active transport reaction for leucine were rather simple when compared with these for proline. Zinc ions inhibited strongly the initial rates of leucine uptakes which were driven by endogenous and exogenous energy sources. The ions also inhibited the exchange-exit reaction.


Transport of sugars and amino acids in bacteria. XV. Comparative studies on the effects of various energy poisons on the oxidative and phosphorylating activities and energy coupling reactions for the active transport systems for amino acids in E. coli. The effects of various energy poisons on oxidation of respiratory substrate, synthesis of cellular ATP, and energy transformation reaction in intact Escherichia coli cells were studied systematically. Various mutants were, therefore, used in which specific functions in the energy-transducing reactions were defective or altered. The energy poisons examined were: sodium azide. DPPA and azidebenzenes which are inhibitors of respiratory-chain phosphorylation, SF6847, and CCCP which are known to be uncouplers, zinc sulfate which is an inhibitor for certain dehydrogenases, and sodium arsenate and sodium fluoride which are inhibitors of glycolytic synthesis of ATP. The preferential inhibitions occurred in the oxidation reactions with certain respiratory substrates by energy poisons used. DPPA inhibited glycerol oxidation much more strongly than succinate oxidation. However, DPPA could  inhibit the oxidation of both glycerol 3-phosphate and succinate by membrane fraction strongly while the oxidation of NADH and D-lactate slightly. It inhibited glycerol 3-phosphate dehydrogenase [EC 1.1.2.1] strongly as well as succinate dehydrogenase [EC 1.3.99.1],.but not D-lactate dehydrogenase of membrane fraction. MAB and other azidebenzene derivatives inhibited succinate oxidation preferentially. SF6847 and CCCP inhibited succinate oxidation strongly, while sodium azide inhibited it weakly and these three poisons were less inhibitory for glycerol oxidation. DPPA, sodium azide, SF6847, and CCCP inhibited the synthesis of ATP coupled with respiration but not with glycolysis. Zinc sulfate inhibited the cellular ATP synthesis coupled with either respiration or glycolysis.


Enzyme-linked immunoassay. I. Novel method for synthesis of the insulin-beta-D-galactosidase conjugate and its applicability for insulin assay. Pork insulin was subjected to mercaptosuccinylation and then coupled to beta-D-galactosidase [EC 3.2.1.23] from Escherichia coli using N,N'-o-phenylenedimaleimide. The competitive binding of the conjugate and insulin to anti-insulin antibody was tested. Results showed that formation of an insulin-beta-D-galactosidase conjugate could be used for immunoassay of insulin.


Formation of a binary complex between elongation factor G and guanine nucleotides. The interaction of the polypeptide chain elongation factor G (EF-G) from E. coli with guanine nucleotides was investigated using the hydrophobic dye, 1-anilino-8-naphthalensulfonic acid. It was found that the fluorescence intensity of the hydrophobic dye elicited in the presence of EF-G was diminished markedly by addition of GTP, and to a lesser extent, by addition of GDP. Direct evidence for the formation of the binary complexes, EF-G-GTP and EF-G-GDP, was provided by gel filtrations of EF-G on Sephadex G-25 columns equilibrated with buffers containing radioactive GTP and GDP, respectively.


In vitro transcription of the Escherichia coli histidine operon primed by dinucleotides. Effect of the first histidine biosynthetic enzyme. Initiation of transcription of the Escherichia coli histidine (his) operon in vitro has been analyzed. The DNA of a specialized transducing phage, ø80dhis, was used as a template, and his RNA was measured by RNA/DNA hybridization. Taking advantage of the fact that E. coli RNA polymerase cannot initiate transcription when the nucleoside triphosphates are present at very low (5 muM) concentration, his RNA initiation was primed by dinucleoside monophosphates. It has been found that his RNA synthesis can be stimulated by one of the three dinucleotides CpA, ApA, and ApG. Under these conditions, it is the initiation of his RNA synthesis that is stimulated. Stimulation of his RNA synthesis by the three dinucleotides apparently occurs at a single initiation site, as judged by the nonadditivity of the effects of the three dinucleotides. This was further confirmed by the effect of phosphoribosyltransferase (the first enzyme of histidine biosynthesis, which specifically represses the synthesis of his RNA) on ApA primed RNA synthesis. Addition of his protein results in a sharp decrease of his RNA synthesis, with no effect whatsoever on the levels of RNAa transcribed from other regions of the template. Our data suggest that the 5' -terminal sequence of his RNA made in vitro is ApApG and that the base immediately preceding this sequence is C.


A novel function of Escherichia coli transfer RNA nucleotidyltransferase. Biosynthesis of the C-C-A sequence in a phage T4 transfer RNA precursor. The biosynthesis of the phage T4-coded proline and serine transfer RNA species proceeds through a precursor RNA containing both tRNA sequences. Neither tRNA sequence in the precursor RNA contains the 3'-terminal C-C-A common to all mature tRNAs. Seidman and McClain ((1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1491-1495) have proposed that the C-C-A sequence is added to serine tRNA while it is still part of the large precursor RNA. In the present work, I show that, in vitro, a purified preparation of Escherichia coli tRNA nucleotidyltransferase (EC 2.7.7.25) accurately synthesized 3'-terminal C-C-A in the serine tRNA portion of the precursor RNA. This result establishes a role of tRNA nucleotidyltransferase in the biosynthesis of the phage T4 serine tRNA. The finding that tRNA nucleotidyltransferase utilizes the large precursor RNA as a substrate represents a novel function of the enzyme.


Acylation of sn-glycerol 3-phosphate in Escherichia coli. Study of reaction with native palmitoyl-acyl carrier protein. The sn-glycerol-3-phosphate acyltransferase activity of Escherichia coli has been assayed using native palmitoyl-acyl carrier protein as the acyl donor. This substrate was synthesized by a plant chloroplast system which utilized E. coli acyl carrier protein. The properties of the acyltransferase as assayed with palmitoyl-acyl carrier protein were similar to those observed using palmitoyl-CoA as the acyl donor. This finding suggested that single enzyme catalyzed transfer of acyl groups from either thioester to sn-glycerol 3-phosphate. This hypothesis was tested by assay of two classes of E. coli mutants which have altered sn-glycerol-3-phosphate acyltransferases. Both classes (plsA and plsB) of mutants have similarly altered activities as assayed with either palmitoyl-acyl carrier protein or palmitoyl-CoA. These results indicate that the same acyltransferase enzyme (or enzyme system) catalyzes the incorporation of both thioester substrates into phospholipid. Other experiments have shown that the acyltransferase of a plsB mutant was abnormally thermolabile only when palmitoyl-CoA was the acyl donor in the reaction. No thermolability was observed with palmitoyl-acyl carrier protein as acyl donor. The thermolability observed with palmitoyl-CoA is attributed to the detergent properties of this substrate. In agreement with Lueking and Goldfine (Lueking, D. R., and Goldfine, H. (1975) J. Biol. Chem. 250, 4911-4917), we found that guanosine-5'-diphosphate-3'-diphosphate (ppGpp) inhibits the acyltransferase only when palmitoyl-CoA was the acyl donor. No inhibition was observed when the acyltransferase was assayed with palmitoyl-acyl carrier protein in the presence of ppGpp. Incubation of the enzyme with ppGpp to assay results in a profound inhibition of acyltransfer from palmitoyl-CoA but has no effect on the incorporation of acyl groups from palmitoyl-acyl carrier protein.


Salt effects in the glutathione-facilitated reactivation of reduced bovine pancreatic ribonuclease. The rate of regeneration of reduced RNase by glutathione was examined in the presence of several added substances: substrate, phospholipid, other proteins, bacterial ribosomes, and neutral salts. Of these, only neutral salts showed substantial effects. K2HOP4 and (NH4)2SO4 strongly accelerated regeneration, the alkali chlorides showed moderate acceleration or inhibition, while LiBr and KSCN strongly inhibited. The t1/2 for regeneration in 1 M Pi is 4 min compared to 75 min in the absence of Pi; in 0.5 M KSCN t1/2 greater than 100 min. The pattern of specific salt effects is similar to a Hofmeister series. There is a strong parallel between the pattern of specific salt effects on the kinetics of RNase regeneration and the pattern of effects of the same salts on the equilibrium stability of biopolymers. This suggests that the role of salts in the regeneration is to stabilize or destabilize rate-limiting folding intermediates. Pi-accelerated glutathione regenerations showed a broad temperature optimum from 30-37 degrees. In strong contrast with the virtual concentration independence of the Pi-free controls, with Pi = 1 M, both rates and yields of RNase activity were decreased markedly at [RNase] greater than 2 x 10(-6) M. Phosphate and pyrophosphate showed additive, and in some cases, synergistic accelerations. These results suggest that specific ion binding occurs in addition to general solvent effects.


Catalysis of a step of the overall reaction by the alpha subunit of Escherichia coli succinyl coenzyme A synthetase. The isolated alpha subunit or succinyl-CoA synthetase from Escherichia coli is capable of catalyzing one step of the overall reaction, namely its own phosphorylation by the substrate ATP. The data presented herein also suggest that the binding sites for other substrates (succinate, succinyl-CoA) are located either on the beta subunit or comprise part of both subunit types. From these observations and from our earlier finding that the two subunits species are necessary for the overall reaction, we propose that the active site is assembled at or close to the point of contact of the two subunits in the native alpha2beta2 enzymic structure.


Inhibition of DNA replication in Escherichia coli by cyanide and carbon monoxide. The inhibition of DNA replication in aerobically growing Escherichia coli by cyanide or carbon monoxide occurs within about 20 s at 15 degrees, as previously reported by Cairns and Denhardt (Cairns, J., and Denhardt, D.T. (1968) J. Mol. Biol. 36, 335-342). This rapid inhibition can be explained by the nearly complete depletion of both intracellular ATP and deoxynucleoside triphosphates which occurs during the time that replication stops. There is probably no direct effect of carbon monoxide on any of the enzymes involved in replication because this reagent has no effect on replication rate or ATP level in anaerobic cells. These cells produce ATP by glycolysis. The inhibition of replication by cyanide, a highly reactive compound, appears to be more complex since anaerobically growing cells can still be completely inhibited, although higher concentrations are required than for aerobically growing cells. The sensitivity of anaerobic cells to cyanide is probably due to the ability of this highly reactive compound to react nonspecifically with many proteins and other molecules.


Lifetime and rotational relaxation time of dansylgalactoside bound to the lac carrier protein. The results presented in this paper demonstrate that the excited state lifetime, anisotropy, and rotational relaxation time of 2'-(N-dansyl)aninoethyl 1-thio-beta-D-galactopyranoside (DG2) increase when the probe is bound specifically to the lac carrier protein in "energized" Escherichia coli membrane vesicles. Although the probe also binds nonspecifically to the vesicle membrane, such binding is independent of the lac carrier protein and is unaffected by "energization" of the vesicles. The experiments provide further evidence that the dansylgalactosides are useful probes for the beta-galactoside transport system and support the hypothesis that the changes in dansylgalactoside fluorescence observed on "energization" of membrane vesicles reflect changes in the binding of the probe.


Conformation of deoxynucleoside triphosphate substrates on DNA polymerase I from Escherichia coli as determined by nuclear magnetic relaxation. A unique conformation of deoxynucleoside triphosphate substrates bound to Escherichia coli DNA polymerase I has been determined by nuclear magnetic resonance techniques. The effects of Mn(II) bound at the active site of the enzyme on the longitudinal (T1p-1) and transverse (T2p-1) relaxation rates of the alpha, beta, and gamma phosphorus atoms and 5 protons of enzyme-bound thymidine 5'-triphosphate (dTTP) were measured at 40.5 MHz (31P), 100 and 220 MHz (1H). From frequency dependence of T1p-1, a correlation time of 7 X 10(-10) s and Mn(II) to proton distances of 10.4, 9.9, 10.3, 10.8, and 8.4 A were calculated for the --CH3, H6, H'1, H'2, and H'4 protons. The calculated Mn(II) to phosphorus distances of 4.2, 4.8, and 3.2 A for the alpha, beta, and gamma phosphorus atoms indicates that Mn(II) corrdinates directly only with the gamma-phosphoryl group and that a puckered triphpsphate conformation exists for the enzyme-bound dTTP. This differs from the binary Mn(II)-dTTP complex in which alpha, beta, and gamma phosphoryl coordination occurs, and a thymine-deoxyribose torsion angly (chi) about the glycosidic bond of 40 degrees is detected. The eight manganese-substrate distances on the enzyme are fit by a unique Mn-dTTP conformation, with a torsion angle equal to 90 degrees, indistinguishable from that found for a deoxynucleotidyl unit in double helical DNA-B. Hence, binding to DNA polymerase appears to adjust the conformation of dTTP for Watson-Crick basepairing. Similarly, the binding of Mn-dATP to DNA polymerase I increased the distances from Mn(II) to the H2, H8, H'1, and H'4 protons of dATP but the adenine-deoxyribose torsion angle of 90 degrees was preserved. Such preorientation of substrates could facilitate incorporation of the complementary nucleotide. When positioned within the DNA structure, the conformation of enzyme-bound Mn-dTTP requires an inline nucleophilic attack on the alpha phosphorus with Mn(II) promoting pyrophosphate departure.


Active transport in Escherichia coli B membrane vesicles. Differential inactivating effects from the enzymatic oxidation of beta-chloro-L-alanine and beta-chloro-D-alanine. Isolated membrane vesicles from Escherichia coli B grown on DL-alanine and glycerol carry out amino acid active transport coupled to a membrane-bound D-alanine dehydrogenase (Kaczorowski, G., Shaw, L., Fuentes, M., and Walsh, C. (1975) J. Biol. Chem. 250, 2855). Certain L-amino acids can also energize solute transport by conversion to their D isomers via an alanine reacemase. Both D-chloroalanine and L-chloroalanine initially drive amino acid and methyl-beta-thiogalactose uptake. The D isomer however causes rapid inactivation of both dehydrogenase-coupled transport and the phosphotransferase system. Transport functions can be protected by dithiothreitol which is postulated to act as a scavenging nucleophile. This inactivation by the D isomer is time-dependent and irreversible not only for proline transport but also for alpha-methylglucoside uptake. Unlike the D isomer, beta-chloro-L-alanine does not inactivate transport. L-Chloroalanine is not racemized to the D isomer but rather undergoes a racemase catalyzed beta elimination of chloride ion to produce pyruvate. Pyruvate can subsequently be oxidized to stimulate active transport. This pyridoxal phosphate-dependent racemase is inactivated by low concentrations of D-chloroalanine but the L isomer can only cause inactivation at a 40-fold higher concentration and longer times of exposure. The D-alanine dehydrogenase-catalyzed oxidation product of D-chloroalanine is chloropyruvate, and this keto acid is hypothesized to be the inactivating species of transport for the following reasons. Chloropyruvate has been isolated from D-chloroalanine oxidation but not from oxidation of the L isomer. Chlorolactate which can be oxidized to chloropyruvate (via membrane-bound lactate dehydrogenases) also causes inactivation of transport in E. coli K-12 membrane vesicles. Mutants having diminished lactate dehydrogenase activity show a slower rate of inactivation with chlorolactate. Moreover, synthetic chloropyruvate irreversibly inactivates both active transport of proline and phosphotransferase system-dependent group translocation of alpha-methylglucoside. The effects of D- and L-chloroalanine and chlorolactate on transport in membrane vesicles are also seen in whole cells.


Active transport in Excherichia coli B membrane vesicles. Irreversible uncoupling by chloropyruvate. In the accompanying report (Kaczorowski, G., Shaw, L., Laura, R., and Walsh, C. (1975) J. Biol. Chem. 250, 8921-8930), we have shown that the oxidation of beta-chloro-D-alanine by a membrane-bound D-alanine dehydrogenase results in the inactivation of both dehydrogenase-coupled and P-enolpyruvate-dependent active transport in membrane vesicles. We have also demonstrated that chemically prepared chloropyruvate has the same inactivating effects on transport. In this report, we show that in addition to abolishing hexose and proline uptake, chloropyruvate inhibits lactose and several other amino acid uptake systems to different extents, although proline transport is the most severely inhibited. The degree of transport inactivation also depends on whether the keto acid is added exogenously or is generated by the D-alanine dehydrogenase. Chloropyruvate treatment does not inhibit D-alanine dehydrogenase, D-lactate dehydrogenase of the passage of electrons to oxygen by the membrane cytochrome chain. However, alanine racemase and pyruvate oxidase (to a lesser extent) are inactivated by this keto acid. Treatment of vesicles with chloropyruvate does not affect the establishment of maintenance of a membrane potential, however, this does inhibit solute transport in response to an artificially induced potential. If chloropyruvate is added at any point during a time course of proline transport, there is an instantaneous blockade of amino acid uptake suggesting that the proline carrier can no longer translocate solute across the membrane. Upon examining the functionality of the carrier protein after exposure to chloropyruvate, there is no appreciable difference in efflux or exchange properties as compared to untreated controls. Therefore chloropyruvate does not block proline passage through the membrane, but rather appears to interfere with the ability of the proline carrier to sense the membrane potential. The beta-halo keto acid does not then uncouple respiration from energization of the membrane but does interfere with the ability of the energized membrane state to be used for the transport of most solutes.


Template properties of bacteriophage T4 vegetative DNA. I. Isolation and characterization of two template fractions from gently lysed T4-infected bacteria. The synthesis and template properties of T4 vegetative DNA were studied. The DNA-containing material in lysates of cells taken 20 min past T4 infection sediments in sucrose gradients as two major components. Both fractions function as templates for amino acid incorporation in a DNA-dependent in vitro system (coupled transcription-translation). The slower sedimenting activity is not present in uninfected cells and appears in wild type T4-infected cells only after 12 min at 30 degrees, shortly after DNA synthesis starts. It is dependent for its activity on an added S-30 fraction from either uninfected or T4-infected cells and is completely inhibited by deoxyribonuclease or rifampin. On a weight basis the slower sedimenting template is about 30 to 70% as active as mature T4 DNA when supplemented with S-30 extracts from uninfected cells. The spectrum of proteins synthesized in response to the slower sedimenting template is different from that produced in response to mature T4 DNA. In contrast to mature DNA, this template is capable of directing the synthesis of material that precipitates with antiserum directed against whole T4 particles. Thus, it appears capable of directing the synthesis of mRNA for phage structural proteins, i.e. late proteins. The faster sedimenting component is about 8-fold less active for stimulating amino acid incorporation than mature DNA. Significant amounts of RNA polymerase are associated with this DNA in active transcription complexes, yet polyacrylamide gel electrophoresis of the proteins synthesized in response to this fraction show a pattern that resembles the early proteins made from mature T4 DNA in extracts from uninfected cells.


Template properties of bacteriophage T4 vegetative DNA. II. Effect of maturation and DNA-arrest mutations. The DNA in gently lysates of T4-infected Escherichia coli cells sediments in sucrose gradients as two major components; the slower sedimenting component is designated as the S-5 fraction and the faster sedimenting component as the pad fraction. The distribution of these fractions in lysates of cells infected with T4 maturation-defective and DNA-arrest mutants was determined, and their template activities were compared in a DNA-dependent amino acid-incorporating system. The S-5 DNA template was found to be completely absent in E. coli B cells infected with a T4 maturation-defective mutant (gene 55). On the other hand, DNA sedimenting as the S-5 component is greatly increased, while that sedimenting as the pad component is virtually absent in nonpermissive cells infected with a DNA-arrest mutant (gene 46). The S-5 fractions prepared from cells infected with a DNA ligase mutant (gene 30) and a gene 30 gene 46 double mutant are reduced in their ability to stimulate amino acid incorporation compared to similar preparations from cells infected with wild type T4 or a gene 46 mutant. Moreover, the template activity of partially purified replicative DNA prepared from cells infected with phage-carrying mutations either on gene 30 or in both genes 46 and 56 (dCTPase) is lower than that of DNA obtained from cells infected with wild type phage. The polypeptide products of reaction mixtures programmed with several of the mutant DNAs were found to be qualitatively different from polypeptides synthesized in response to either mature DNA or replicative DNA prepared from cells infected with wild type phage. These data suggest that the expression of phage DNA may be significantly influenced by physical changes in the DNA arising from abnormal replication.


Mutants of Escherichia coli defective in membrane phospholipid synthesis. Effect of cessation of net phospholipid synthesis on cytoplasmic and outer membranes. The effect of cessation of net phospholipid synthesis on the cytoplasmic and outer membranes of Escherichia coli was investigated in a mutant strain defective in the first enzyme of phospholipid synthesis, the sn-glycerol-3-phosphate (glycerol-P) acyltransferase. The glycerol-P (glycerol) auxotropic phenotype of this strain resulted from an altered membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of the parental activity. When net phospholipid synthesis was halted during glycerol deprivation, both soluble and cell envelope protein synthesis continued. Fractionation of the membranes derived from glycerol-supplemented and glycerol-deprived cultures by isopycnic banding in sucrose gradients revealed that both the cytoplasmic and outer membranes of the deprived culture banded at higher buoyant densities. The protein/phospholipid ratio of both the cytoplasmic and outer membranes increased approximately 60% during the period of glycerol deprivation. The distribution of two cytoplasmic membrane activities, NADH oxidase and 1-acylglycerol-P acyltransferase, and an outer membrane activity, phospholipase A1, showed that the total membranes derived from glycerol-deprived cultures were separated cleanly into cytoplasmic and outer membrane fractions. Both cytoplasmic and outer membrane proteins were synthesized and integrated into their respective membranous structures when net phospholipid synthesis was halted. Hence, the biosynthesis of membrane phospholipid and membrane protein are not tightly coupled. Further, these data suggest that cellular control mechanisms exist which maintain the protein content of both membranous structures below the point where they are saturated with protein.


Binding of MET-TRNAf and GTP to homogeneous initiation factor MP. Homogeneous initiation factor MP forms a stable complex with Met-tRNAf which binds to nitrocellulose filters in the absence of ribosomal subunits. Complex formation is rapid at 0 degrees and the rate of reaction is stimulated 20-fold by GTP when freshly prepared initiation factor MP is used. Under optimal assay conditions, a 1:1:1 stoichiometry for initiation factor MP, GTP, and Met-tRNAf is indicated, based on a molecular weight for initiation factor MP of 180,000. Kinetic analysis of ternary complex formation suggests an ordered reaction sequence with binding of GTP followed by binding of Met-tRNAf. However, binding of GTP appears to produce an unstable state which leads to rapid inactivation of initiation factor MP in the absence of Met-tRNAf. Formation of a stable binary complex of initiation factor MP and Met-tRNAf occurs in the absence of GTP. The binary complex cannot subsequently bind GTP. While storage of initiation factor MP at 0 degrees for several weeks has no effect on the rate or extent of Met-tRNAf binding in the presence of GTP, the rate of binary complex formation is increased 10-fold. The binary and ternary complexes appear to bind to 40 S ribosomal subunits with equal efficiency.


Kinetic analysis of ribonucleic acid chain initiation by Escherichia coli Ribonucleic acid polymerase bound to DNA. The kinetics of the RNA chain initiation reaction carried out by RNA polymerase bound to the initiator region of a DNA template have been analyzed. Initiation proceeds in a two-substrate reaction in which the initial binary complex (enzyme-DNA) is transformed into a ternary complex (enzyme-DNA-RNA) by formation of a dinucleoside tetraphosphate and release of inorganic pyrophosphate. In this reaction RNA polymerase serves as a reactant rather than acting catalytically. The concentration of the reacting binary complex decreases throughout the reaction; hence steady state approximations cannot be used. Kinetic equations for an ordered two-substrate reaction are derived. These are most useful for the special case of reaction in the presence of an inhibitor of initiation, such as rifampicin. Equations for the latter instance are solved exactly with recourse to the steady state approximation. It is found that measurements of the extent of the initiation reaction determined at different inhibitor and substrate concentrations can give information about the initiation reaction analogous to that obtained in standard steady state kinetic analysis. This theory is applied to the experimental study of the initiation reaction carried out by Escherichia coli RNA polymerase. It is found that the inhibitor rifampicin, which blocks the initiation reaciton, acts by binding to the same form of the binary complex as the nucleoside triphosphate substrate (ATP or GTP) which is incorporated into the 5' terminus of nascent RNA molecule. The binding of the 5'-terminal nucleoside triphosphate to the enzyme appears to be rate-limiting for the initiation reaction under standard assay conditions. Initiation appears to follow an ordered reaction mechanism; however, the order of addition of the two substrates is still uncertain.


Effectiveness of various unsaturated fatty acids in supporting growth and respiration in Saccharomyces cerevisiae. The saturated fatty acid auxotroph of Saccharomyces cerevisiae, KD115, was used to determine the efficiency of various unsaturated fatty acids in supporting growth. The efficiency, as the number of cells produced per fmol of unsaturated fatty acid, ranged from zero for a number of acids to over 26 cells per fmol of eicosapentaenoic acid. Efficiencies tended to be higher for acids with fewer carbons or more double bonds. In a series of positional isomers of cis-octadecenoic acid, the delta9 isomer had the greatest efficiency (12 cells per fmol). Exogenous oleic acid was taken up and incorporated into cellular lipid early in the growth of the cells. Further growth proceeded with a decrease in the relative content of oleate in lipids until a minimum value of 9 mol % was reached at stationary phase. The initial concentration of supplemental acid did not affect the final mole % value. Other unsaturated fatty acids reached limiting values of mole % in phospholipid at stationary phase that were characteristic for the acid used. When cells were grown with glycerol as the carbon source, the efficiencies of most acids in supporting growth were one-third to one-fifth the value with glucose and the final mole % of supplement acid in phospholipid at stationary phase was two to five times greater. Apparently, mitochondrial energy transduction necessary for glycerol utilization requires higher levels of unsaturated fatty acids in membrane lipids than do extramitochondrial functions. The respiratory rate of mitochondria was not decreased at lower levels of oleic or palmitoleic acid in lipids, although respiratory control was lower when the mole % of unsaturated fatty acid was lower. Mitochondria from cells supplemented with eicosaenoic acid were found to have both low respiration and respiratory control. The decreased respiration of these mitochondria coincided with a decreased cytochrome content, not a decrease in respiration per mol of cytochrome.


Requirements for unsaturated fatty acids for the induction on respiration in Saccharomyces cerevisiae. Unsaturated fatty acids provided during the release from glucose repression were shown to be essential for derepression of respiration in an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae (KD115). Cells derepressed in the presence of oleic acid contained three to six times as much cytochrome per cell as those derepressed in the absence of unsaturated fatty acid or those derepressed with eicosaenoic acid. The delta9 isomer was the most efficient of the cis-octadecenoic acid isomers in supporting that increase, and eicosaenoic acid supported an increase at only 15% the rate observed with oleic acid. Derepression, even in the presence of oleic acid, proceeded only after a lag of 3 hours. When glucose was removed prior to the addition of oleate, the lag was reduced by the time of the preincubation with glycerol. This result suggests that some processes necessary for increased respiration can proceed in the absence of an added unsaturated fatty acid, but these processes apparently require certain levels of unsaturated acids in the pre-existing lipids, since they occurred in cells whose membranes contained 50 mol % oleate, but not in cells containing only 20 mol %. These processes leading to eventual increased respiration were inhibited by cycloheximide but not chloramphenicol, suggesting that protein synthesis on cytoplasmic ribosomes but not mitochondrial ribosomes were required. Derepression in the absence of oleate for 3 hours lessened the inhibition or respiration induction by ethidium bromide. This result indicates that the transcription of mitochondrial DNA necessary for the induction of respiration may have occurred in the absence of added unsaturated fatty acid, but that some subsequent event required added esterified unsaturated fatty acid.


Interaction of tetraiodofluorescein with aspartate transcarbamylase and its isolated catalytic and regulatory subunits. The interaction of the dye tetraiodofluorescein with native aspartate transcarbamylase and its isolated subunits has been investigated by both binding and activity measurements at 4 and 23 degrees. At room temperature low concentrations of tetraiodofluorescein activate the native enzyme, but high concentrations inhibit the enzyme's activity. At the low temperature the native enzyme is inhibited by all concentrations of dye. Isolated catalytic subunit is very effectively inhibited at both temperatures. For the native enzyme these results are explained by 18 tetraiodofluorescein sites of approximately equal affinity (K = 7.3 X 10(-5) M) on each enzyme hexamer: one class of six sites at the nucleoside triphosphate effector binding sites is responsible for the activation, a second class which competes with the substrate carbamylphosphate causes the inhibition, and a third class does not interact with either the effectors or the substrates. Measurements of tetraiodofluorescein binding to isolated regulatory, catalytic, and p-hydroxymercuribenzoate-inactivated catalytic subunits support the above assignments. This scheme of tetraiodofluorescein binding sites successfully predicts the changes in the tetraiodofluorescein-aspartate transcarbamylase difference spectrum induced by the addition of various ligands. The activity changes induced by the dye are explained if the binding of a single tetraiodofluorescein molecule to one of the six regulatory sites activates all six of the catalytic sites, while while a dye molecule binding to any one of the catalytic sites inactivates only that catalytic site.


Transport of purines and deoxyadenosine in Escherichia coli. The characteristics of adenine, guanine, hypoxanthine, xanthine, and uracil uptake in Escherichia coli B show that each base is transported by a specific system. The data support the concept that the transport of guanine, hypoxanthine, xanthine, and uracil function without direct involvement of the respective purine or pyrimidine phosphoribosyltransferase enzymes. Uracil phosphoribosyltransferase is not demonstrable in E. coli B, and large differences are observed in the inhibitory effects of heterologous purines on the uptake of guanine, hypoxanthine, and xanthine as compared to the corresponding inhibitory effects reported for the soluble purine phosphoribosyltransferase enzymes of E. coli B. Additional evidence is provided by the low Km values determined for the transport of adenine, guanine, hypoxanthine, and xanthine relative to the corresponding Km values for the phosphoribosyltransferase enzymes. Data are presented indicating that adenine may be transported without participation of adenine phosphoribosyltransferase. The stimulatory effect of glucose, the inhibitory effect of KCN, and the high intracellular to extracellular concentration gradients of the bases produced in the presence of glucose provide evidence that the transport processes are energy-dependent. The Km values for transport of the purines and uracil range from 10(-7) M to 5 X 10(-7) M. Characteristics of adenine and uracil uptake are similar in E. coli B, E. coli K-12, and a showdomycin-resistant mutant of E. coli B. Adenosine and deoxyadenosine are transported in E. coli B by independent transport systems. Adenine or hypoxanthine does not share the adenosine or deoxyadenosine transport systems as evidence by the mutual lack of competition of free bases and nucleosides on transport. The transport systems for deoxyadenosine and adenosine are defective in the mutant.


Sequence variability and structure of D-glyceraldehyde-3-phosphate dehydrogenase. The amino acid sequences of pig muscle and of yeast glyceraldehyde-3-phosphate dehydrogenase are compared with the three-dimensional structure of the lobster muscle enzyme. Residues in sheet and helical regions, on the exterior and interior, in subunit and domain interfaces, as well as residues in the active site have been examined for evolutionary conservation. The residues in the first (NAD binding) domain (1-147) are less conserved than residues in the second (catalytic) domain (148-334) probably because there are fewer internal residues and fewer residues involved in interactions between subunits. Residues in subunit interface are conserved to a significantly greater extent than others, and those involved in catalysis are conserved most of all. Patterns of residues in helices and sheets follow those found for other proteins.


Cross-linking studies on the 50 S ribosomal subunit of Escherichia coli with methyl 4-mercaptobutyrimidate. The 50 S ribosomal subunits of Escherichia coli were incubated with methyl 4-mercaptobutyrimidate and the formation of intermolecular protein:protein disulfide bonds was promoted by oxidation. Cross-linked proteins were analyzed by diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Three pairs of cross-linked proteins were identified: L2-L7,L12; L5-L7,L12; and L17-L32. The significance of the results in relation to the location of sites for factor binding, peptidyltransferase, and GTP hydrolysis is discussed.


Regulation of mitochondrial protein synthesis at the polyribosomal level. Polysomes consisting of two to eight monosomes were isolated from yeast mitochondria by lysing the mitochondria with Triton X-100 and centrifugation in a 20 to 40% linear sucrose gradient. When yeast spheroplasts were pulse-labeled with [3H]-Leucine in the presence of cycloheximide to block cytoplasmic protein synthesis, radioactivity which was trichloroacetic acid-precipitable was present mainly in the polysome region. Incorporation of leucine was blocked by erythromycin, a specific inhibitor of mitochondrial protein synthesis. Release of radioactivity to the top of the gradient resulted from treating labeled polysomes with either puromycin or ribonuclease (in the latter case with the breakdown of polysomes), indicating that the radioactivity was present in nascent polypeptide chains. Yeast cells were grown in chloramphenicol for 3 hours and in fresh medium for 1 hour and then pulse-labeled with either [3H]leucine or [14C]formate. Three parameters showed a 2-fold increase in cells grown in chloramphenicol prior to pulse labeling: the polysome to monosome ratio, the amount of labeled precursor incorporated into proteins, and the rate of polypeptide chain initiation as judged by the formation of fMet-puromycin. Conversely, these parameters were all decreased approximately 50% in cells treated with cycloheximide prior to pulse labeling. Mitochondria were also isolated from cells previously grown in chloramphenicol or cycloheximide and incubated in vitro with [3H]leucine under optimal conditions. Acid-precipitable radioactivity in the polysome region was increased 3-fold in mitochondria from cells grown previously in chloramphenicol and decreased 75% in those grown in cycloheximide. Furthermore, chain initiation was deomonstrated in the isolated mitochondria by formation of fMet-puromycin. The rate of chain initiation in vitro was increased 2-fold in mitochondria isolated from chloramphenicol-treated cells.


A revised preparation of yeast (Saccharomyces cerevisiae) pyruvate kinase. A revised preparation of pyruvate kinase from saccharomyces cerevisiae is reported. By purifying this cold-labile enzyme at room temperature, an improved recovery and specific activity was obtained. More than 350 mg of pure enzyme with a specific activity of 350 to 400 units/mg at 30 degrees were obtained from a pound of fresh yeast. The last step of the preparation, passage of the enzyme over Sephadex G-100, was required to remove a contaminating protease. The molecular parameters of the new preparation are: molecular weight, 209,000; four subunits of identical size; E 280 nm, 0.51; pI 6.6; and pH optimum, 6.28. Kinetic parameters are: Km for P-enolpyruvate and ADP, 0.09 and 0.18 mM in the presence of saturating Fru-1,6-P2, and 1.8 and 0.34 mM in the absence of Fru-1,6-P2; Ka for Fru-1,6-P2, 0.014 mM. No free NH2-terminal amino acid could be detected. Amino acid composition was determined and compared with other pyruvate kinase preparations.


Specific binding of Excherichia coli chain Initiation factor 2 to fMet-tRnafMet. A stable Escherichia coli IF-2-fMet-tRNAfMet complex is formed upon incubation of IF-2 (prokaryotic initiation factor) with fMet-tRNAfMet is the presence of 50 mM Tris-HCl, pH 7.0, 100 mM NH4Cl, and 7 mM 2-mercaptoethanol. The complex thus formed is retained on a Millipore filter and is assayed accordingly. Complex formation does dot require GTP, is unstable in the presence of 5 mM Mg2+, and is specific for fMet-tRNAfMet. Other amino acyl-tRNAs or deacylated tRNAs do not form such a complex with IF-2. A crude ribosomal high salt wash preparation contains other protein factors which bind unspecifically to RNAs under the above binding conditions. One of these factors elute similarly to IF-1 on DEAE-cellulose chromatography. Extensively purified IF-1 and IF-3 show weak and unspecific RNA-binding activities. The RNA-protein complex formed in each of the above cases, like the IF-2-fMet-tRNAfMet complex, is retained on Millipore filter and is sensitive to Mg2+.


Studies on arginyl-tRNA synthetase from Escherichia coli B. Dual role of metals in enzyme catalysis. Studies carried out in arginyl-tRNA synthetase from Escherichia coli indicate that metals may have two functional roles in the catalytic mechanism. Complete metal activation is observed when MgCl2, MnCl2, CoCl2, or FeCl2 is present at a concentration (5.0 mM) in excess of the total ATP concentration (2.0 mM). When CaCl2 is substituted for MgCl2, activity is not observed unless a small amount (0.1 mM) of MgCl2, MnCl2, CoCl2, FeCl2, or ZnCl2 (unable to produce activity alone at 5.0 mM) is added. A model, based on kinetic data, is proposed in which the enzyme possesses a site for free metal, which, when filled, lowers the Km for all three substrates (arginine, tRNAArg, and metal-ATP) and increases the Vmax of the reaction.


Serratia arthritis in heroin users.

Serratia arthritis in heroin users. Septic arthritis due to Serratia species was seen in four users of intravenous heroin. In all cases, the organism was cultured from joint aspirates. Both the clinical presentation and the involvement of the sacro-iliac and knee joints were notably similar to the Pseudomonas septic arthritis encountered in other heroin users. All four patients responded satisfactorily to therapy although one had a residual flexion contracture of the knee. Twelve previously reported cases of Serratia arthritis are reviewed.


The management of infants with scoliosis. This is a study of children who first attended as infants with either progressive infantile idiopathic scoliosis or congenital scoliosis. All had a pattern of scoliosis in which early and damaging deterioration is inevitable. The infants were treated from before the age of three, initially by plaster casts and then a Milwaukee brace, followed at about the age of ten by correction and fusion. The cases were then observed to the end of growth or near that point. In the main study there were twelve cases, six of progressive infantile idiopathic scoliosis and six of congenital scoliosis, which were followed through this long period. Only one of the twelve had a curve worse at the end of growth compared with the initial radiograph as an infant; this one curve had increased only 16 degrees in almost as many years. Although small, the series does show that it is nearly always possible to control even the most serious scoliosis in an infant, if it is tackled early and unremittingly. There are supportive studies of children who have partially completed this regime, and interim results in a newer group of children with spina bifida and scoliosis.