Specificity of DNA recognition in the nucleoprotein complex for site-specific recombination by Tn21 resolvase.

Resolvases from Tn3-like transposons catalyse site-specific recombination at res sites. Each res site has 3 binding sites for resolvase, I, II, and III. The res sites in Tn3 and Tn21 have similar structures at I and II but they differ at III. Mutagenesis of the Tn21 res site showed that sub-site III is essential for recombination though the sequences in III that are recognized by Tn21 resolvase are positioned differently from the equivalent sequences in the Tn3 site. The deletion of III caused a 1,000-fold drop in the rate of recombination. But other mutations at III, changing 3 or 4 consecutive base pairs, caused only 1.5- to 4-fold decreases in rate, even when the mutations were in target sequences for this helix-turn-helix protein. The reason why Tn21 resolvase has similar activities at a number of different DNA sequences may be due to the multiplicity of protein-protein and protein-DNA interactions in its recombinogenic complex. This lack of precision may be a general feature of nucleoprotein complexes.     

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

The SalGI restriction endonuclease. Mechanism of DNA cleavage.

The cleavage of supercoiled DNA of plasmid pMB9 by restriction endonuclease SalGI has been studied. Under the optimal conditions for this reaction, the only product is the linear form of the DNA, in which both strands of the duplex have been cleaved at the SalGI recognition site. DNA molecules cleaved in one strand at this site were found to be poor substrates for the SalGI enzyme. Thus, both strands of the DNA appear to be cleaved in a concerted reaction. However, under other conditions, the enzyme cleaves either one or both strands of the DNA; the supercoiled substrate is then converted to either open-circle or linear forms, the two being produced simultaneously rather than consecutively. We propose a mechanism for the SalGI restriction endonuclease which accounts for the reactions of this enzyme under both optimal and other conditions. These reactions were unaffected by the tertiary structure of the DNA.     

Analysis of HMW glutenin subunits encoded by chromosome 1A of bread wheat (Triticum aestivum L.) indicates quantitative effects on grain quality

Summary A gene encoding the high-molecular-weight (HMW) subunit of glutenin 1Ax1 was isolated from bread wheat cv Hope. Comparison of the deduced amino acid sequence with that previously reported for an allelic subunit, 1Ax2^*, showed only minor differences, which were consistent with both subunits being associated with good bread-making quality. Quantitative analyses of total protein extracts from 22 cultivars of bread wheat showed that the presence of either subunit 1Ax1 or 1Ax2^*, when compared with a null allele, resulted in an increase in the proportion of HMW subunit protein from ca. 8 to 10% of the total. It is suggested that this quantitative increase in HMW subunit protein may account for the association of 1Ax subunits with good quality.EMBL Data Library. Accession number: X61009     

EcoRV restriction endonuclease: communication between DNA recognition and catalysis.

A genetic system was constructed for the mutagenesis of the EcoRV restriction endonuclease and for the overproduction of mutant proteins. The system was used to make two mutants of EcoRV, with Ala in place of either Asn185 or Asn188. In the crystal structure of the EcoRV-DNA complex, both Asn185 and Asn188 contact the DNA within the EcoRV recognition sequence. But neither mutation affected the ability of the protein to bind to DNA. In the absence of metal ion cofactors, the mutants bound DNA with almost the same affinity as that of the wild-type enzyme. In the presence of Mg2+, both mutants retained the ability to cleave DNA specifically at the EcoRV recognition sequence, but their activities were severely depressed relative to that of the wild-type. In contrast, with Mn2+ as the cofactor, the mutant enzymes cleaved the EcoRV recognition site with activities that were close to that of the wild-type. When bound to DNA at the EcoRV recognition site, the mutant proteins bound Mn2+ ions readily, but they had much lower affinities for Mg2+ ions than the wild-type enzyme. This was the reason for their low activities with Mg2+ as the cofactor. The arrangement of the DNA recognition functions, at one location in the EcoRV restriction enzyme, are therefore responsible for organizing the catalytic functions at a separate location in the protein.     

Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene.

The cystic fibrosis transmembrane conductance regulator is encoded by the gene known to be mutated in patients with cystic fibrosis. This paper reports the cloning and sequencing of cDNAs for the murine homolog of the human cystic fibrosis transmembrane conductance regulator gene. A clone that, by analogy to the human sequence, extends 3' from exon 9 to the poly(A) tail was isolated from a mouse lung cDNA library. cDNA clones containing exons 4 and 6b were also isolated and sequenced, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-9 were cloned by PCR from mouse RNA. The deduced mouse protein sequence is 78% identical to the human cystic fibrosis transmembrane regulator, with higher conservation in the transmembrane and nucleotide-binding domains. Amino acid sequences in which known cystic fibrosis missense mutations occur are conserved between man and mouse; in particular, the predicted mouse protein has a phenylalanine residue corresponding to that deleted in the most common human cystic fibrosis mutation (delta F508), which should allow the use of transgenic strategies to introduce this mutation in attempts to create a "cystic fibrosis mouse".     

Escherichia coli alkaline phosphatase. Relaxation spectra of ligand binding

The temperature-jump technique was used to study the binding equilibrium between the Escherichia coli alkaline phosphatase dimer and 2-hydroxy-5-nitrobenzyl phosphonate in 0.1m-tris buffer, pH8.0. Three partially discrete relaxations were observed, two of which could be related to the bimolecular associations of ligand with different conformations of the enzyme and the third to the interconversion of these states. Relaxation spectra were also used to analyse the changes in the mechanism of ligand binding to alkaline phosphatase caused by increase in ionic strength. The relaxation spectrum observed after the addition of P(i) to the equilibrium mixture of phosphonate and enzyme was also studied. Difference spectroscopy indicated that both of these ligands were bound to the alkaline phosphatase dimer at the same time. These results are related to the catalytic mechanism of this enzyme, with particular reference to the role of two identical subunits in a dimeric enzyme that exhibits only one active site functioning in catalysis at any given time.     

Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme

1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of P_i to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.