Fine Structure Mapping in Yeast with Sunlamp Radiation

The X-ray mapping procedure of Manney and Mortimer (1964) is the most widely applicable and convenient method for fine structure analysis in yeast, but suffers the disadvantage that suitable X-ray machines or gamma ray sources are very expensive. Although many other recombinogens are known, none gives a linear dose-response like X-rays and few are as convenient or give as reproducible results. Experiments with Saccharomyces cerevisiae reported in this paper show, however, that the near-ultraviolet radiation emitted by fluorescent sunlamps gives linear dose-response relations, as reproducible results as ionizing radiations, and map distances which correlate highly with those obtained by using (60)Co gamma rays. It is suggested that this convenient recombinogen may be a suitable low-cost substitute for ionizing radiations in fine structure mapping.     

Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S(0)]/v(i) (initial substrate concn./initial velocity) versus [S(0)] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S(0)]/v(i) versus [S(0)] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S(0)]/v(i) versus [S(0)] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25 degrees C is characterized by K(m) (1) (the dissociation constant of ES)=1.22+/-0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57x10(-2)+/-0.32x10(-2)s(-1), K(a) (2) (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38+/-0.06m, and k' (the rate constant for the breakdown of SES to E+P+S)=0.45+/-0.04s(-1). 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester and of alpha-N-benzoyl-l-arginine amide; K(m) (1) and k for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine amide hydrolysis and K(as) and k' for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of k(cat.) and K(m) for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (K(m)=52+/-4mm, k(cat.)=2.80+/-0.1s(-1) at pH7.0, I=0.1, 25.0 degrees C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.     

Affinity labelling of the binding site of rabbit antibody. Evidence for the involvement of the hypervariable regions of the heavy chain

The binding sites of rabbit antibodies with affinity for the haptenic group 4-azido-2-nitrophenyl-lysine have been specifically labelled by photolysis of the hapten-antibody complex. The extent of covalent labelling was 0.5-0.9mol of hapten bound/mol of antibody and, by using an immunoadsorbent, antibody with 1.3mol of hapten/mol was obtained. The antibody was specifically labelled in the binding site and the ratio of labelling of heavy and light chains was in the range 3.3-5.0. The labelled heavy chains were cleaved by CNBr treatment and after reduction and alkylation of the intrachain bonds, were digested with trypsin. Evidence is presented that two regions of the heavy chain, positions 29-34 and 95-114, together contain about 80% of the label on the heavy chain; these two regions respectively include two of the hypervariable regions of rabbit heavy chain.     

Mutant of Yeast Sensitive to 2,6-Diaminopurine

A mutant of yeast sensitive to growth inhibition by 2,6-diaminopurine (2,6-DAP) was analyzed genetically and found to be a double mutant. One gene, dap, conferred approximately 30% sensitivity to the analogue. The other, slw, potentiated the inhibition such that the double mutant dap slw was inhibited 90%. The mutation dap conferred concomitant sensitivity to a number of other purine analogues. The activity of a purine phosphoribosyltransferase with 2,6-DAP in a strain carrying dap was found to be three times higher than in the wild type. It is inferred that the mutation alters the properties of a purine phosphoribosyltransferase. A possible mechanism for the effect of slw is also discussed.     

Control of Bacteriophage-induced Enzyme Synthesis in Cells Infected with a Temperature-sensitive Mutant

The timing of "early" and "late" protein synthesis in Escherichia coli infected with T-even bacteriophage was studied with a temperature-sensitive phage mutant, T4 tsL13. This strain was completely unable to direct the synthesis of phage deoxyribonucleic acid (DNA) at 44 C because it makes a deoxycytidylate hydroxymethylase which cannot act at that temperature. However, the mutant did multiply normally at 30 C. No detectable formation of the late protein, lysozyme, occurred at 44 C, in agreement with the idea, proposed by several workers, that DNA replication is necessary for activation of late genetic functions. However, the formation of an early enzyme, thymidylate synthetase, was shut off at about 10 min, as in normal infection. This implied that separate mechanisms were responsible for cessation of early functions and activation of late ones. That the infected cell at 44 C retained the capacity for synthesis of early enzymes was shown by the fact that DNA synthesis occurred after a culture was transferred from 44 to 30 C as late as 30 min after infection. This synthesis was inhibited by chloramphenicol, indicating that de novo synthesis of an early enzyme can take place at a late period in development. It is suggested that cells infected under normal conditions maintained an appreciable rate of early enzyme synthesis throughout the course of infection.     

Teaching of Fertility Regulation in Medical Schools: A Survey in the United States and Canada, 1964

Fertility regulation is taught didactically in 82 of 94 medical school departments of obstetrics and gynecology in the United States and Canada, but students are given clinical experience in only 59 medical schools, according to a survey conducted in 1964 by a committee of the American Public Health Association. Legal prohibitions impeded teaching in 1964 in two States and in all of Canada. Nearly all schools teach that help with fertility regulation should be offered for medical and socioeconomic stress, and most teach that it should be offered routinely in premarital counselling and in the postpartum period, but only two-thirds teach that this help should be given to unmarried adults and only one-third teach that any person requesting help with fertility regulation should receive it.     

FOWLER'S BACILLUS AND ITS PARASPORAL BODY

Fowler's bacillus is one of several organisms which form a non-viable inclusion or parasporal body during the process of sporulation. This body is globular and may be as large as or larger than the spore. Its position in the cell is not random; the spore is terminal and the body paracentral, lying between the spore and the remaining vegetative cell chromatin bodies. On completion of sporulation both spore and body are contained within an exosporium. The sequence in the development of the cell structures was followed in ultrathin sections of material fixed in permanganate. When sporulation is well advanced the body begins to grow from a single crystal, then presumably as a result of some disorientation in the growth process it develops as a multicrystalline body with the lattices orientated at different angles. When the body approximates the spore in size, a lamella coat is formed and an exosporium develops which eventually encircles the body and the spore. Other lamella systems microscopically similar to those surrounding the parasporal body develop free in the cytoplasm outside the exosporium. In both of these systems the number of lamellae is variable. The spore coat of Fowler's bacillus, consisting of an outer lamella layer and an inner unresolved amorphous layer has been found microscopically identical to the spore coat of B. cereus. In both organisms the lamella layer of the spore coat consists, in contrast to the other lamella systems, of a regular number of lamellae. Physiological tests would indicate that Fowler's bacillus is a variety of B. cereus.