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1.
Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon 总被引:3,自引:0,他引:3
Nerina R. Harborne Lesley Griffiths Stephen J. W. Busby Jeffrey A. Cole 《Molecular microbiology》1992,6(19):2805-2813
Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon. Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC 1.6.6.4). A series of plasmids has been constructed in which each of the open reading frames has been fused in-phase with the beta-galactosidase gene, lacZ. Rates of beta-galactosidase synthesis during growth in different media revealed that nirB, -D, -E and -C are transcribed from the FNR-dependent promoter, p-nirB, located just upstream of the nirB gene: expression is co-ordinately repressed by oxygen and induced during anaerobic growth. Although the nirB, -D and -C open reading frames are translated into protein, no translation of nirE mRNA was detected. The cysG gene product is expressed from both p-nirB and a second, FNR-independent promoter, p-cysG, located within the nirC gene. No NADH-dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild-type bacteria. Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing. NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme. 相似文献
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On the binding of tRNA to Escherichia coli RNA polymerase. Interactions between the core enzyme, DNA and tRNA 总被引:7,自引:0,他引:7
We have investigated the interplay between the binding of tRNA and DNA to core RNA polymerase. We show that the monomer core enzyme can bind stably to either DNA or tRNA, whereas the dimer core can fix both DNA and tRNA in a stable ternary complex. We have examined the kinetics of the exchange between DNA and tRNA bound to the core enzyme. DNA bound to monomer core can be rapidly displaced by tRNA without prior dissociation of the core from the DNA. Similarly tRNA bound to the core can be displaced by DNA without prior dissociation of the tRNA. We confirm the result of Hinkle and Chamberlin [J. Mol. Biol. 70, 157-185 (1972)] that, in contrast, the core enzyme must first dissociate from one DNA molecule before it can transfer to another DNA. As this dissociation is very slow we suggest that, in vivo, the tRNA can act as a 'porter' providing the core enzyme with a more kinetically favourable path to transfer from one DNA site to another. 相似文献
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Domain structure, stability, and interactions of human complement C1s-: characterization of a derivative lacking most of the B chain 总被引:1,自引:0,他引:1
A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain. 相似文献
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Space biomagnetics 总被引:1,自引:0,他引:1
Douglas E. Busby 《Origins of life and evolution of the biosphere》1968,1(1):23-63
Astronauts who venture from their spacecraft onto the lunar surface and the surfaces of our neighboring planets will be exposed for a few hours in duration to magnetic-field intensities which are markedly less than that of the earth's field. The intensities of magnetic fields to which they will be exposed while inside their spacecraft can be stated only after completing a detailed survey of the contribution made to these fields by the functioning electronic components of spacecraft. Assessment of individuals regularly working in and exposed continuously for 10 days to magnetic fields less than 100 gammas in intensity indicate that extremely low-intensity magnetic fields encountered during a nominal Apollo moon mission should not affect astronaut health or performance. Careful physiological and psychological observations first on higher primates, then on man exposed to such fields for more prolonged periods of time must be carried out before this conclusion can be drawn for longer exposures.Recent technological advances in propulsion and radiation protection have made it possible that astronauts might also be exposed intermittently to high-intensity, relatively low-gradient magnetic fields during space missions. The duration of such exposures could range from less than an hour if an activated magnetohydrodynamic engine must be serviced, to several days if pure magnetic or plasma-radiation shielding is used for astronaut protection from solar flare radiation. From past experience with personnel who enter high-intensity magnetic fields for brief periods of time in their work, magnetic-field exposures while servicing magnetohydrodynamic engines should not be hazardous to astronauts. On the other hand, past exposures of man and sub-human systems to high-intensity magnetic fields do not indicate whether or not astronauts who are exposed for up to several days to currently estimated magnetic-field intensities associated with pure magnetic or plasma-radiation shielding could suffer impairment of their health or performance. This answer can be obtained only by carefully conducted experiments which closely simulate such exposures, and look closely for physiological, psychological and pathological changes, especially in exposed higher primates, before assessing the response of man to such exposures.Magnetic force is animate or imitates life; and in many things surpasses human life, while this is bound up in the organick body.Prepared under Contract NASr-115 at The Lovelace Foundation for Medical Education and Research, Albuquerque, N.M., U.S.A. 相似文献
8.
Transcription activation at Class II CAP-dependent promoters 总被引:25,自引:8,他引:17
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10.
Tobias I. Baskin Catherine H. Busby Larry C. Fowke Margaret Sammut Frank Gubler 《Planta》1992,187(3):405-413
Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.Abbreviations BrdU
5-bromodeoxyuridine
- DTT
dithiothreitol
- EGTA
ethylene glycol-bis(-aminoethyl ether)
- N,N,N,N
tetraacetic acid
We thank Ann Cork for technical assistance, Professor B.E.S. Gunning (Australian National University) and Drs. A.R. Hardham (A.N.U.) and R.E. Williamson (A.N.U.) for intellectual and material support, Dr D. McCurdy (A.N.U.) for the purified anti-actin antibody, and Professor B. Stone (La Trobe University, Melbourne, Australia) for generously providing the anti-callose antibody. We also thank the Electron Microscopy Unit of A.N.U. for the use of facilities. L.C.F. gratefully acknowledges financial support from the National Sciences and Engineering Research Council of Canada. 相似文献