In vivo 5'' terminus and length of the mRNA for the proton-translocating ATPase (unc) operon of Escherichia coli.

The promoter for the proton-translocating ATPase (unc) operon of Escherichia coli was localized by using a plasmid promoter-screening vector system. S1 nuclease analysis, using the appropriate single-stranded DNA probe from this promoter region and in vivo mRNA, revealed that the 5' end of the in vivo unc mRNA initiates with a guanine residue 73 bases before the start of the proposed gene 1 or 474 bases before uncB. An in vivo unc mRNA species of approximately 7,000 nucleotides in length which initiates in the unc promoter region was shown to exist by RNA-DNA hybridization analysis. This unc mRNA species (based on DNA sequence analysis) is sufficient in length to contain all nine genes, gene 1 and uncBEFHAGDC. That gene 1 is cotranscribed with the unc genes was confirmed by using hybridization probes containing the promoter-proximal (gene 1) or -distal gene (uncC). No strong internal promoters within the unc operon were revealed with either the promoter-screening vector system or the RNA-DNA hybridization analysis. The 5' terminus and the length of the unc mRNA were found to be identical in cells grown either aerobically or anaerobically. The level of unc operon expression, as assayed with the unc promoter plasmid, did not significantly differ when cells bearing the plasmid were grown either aerobically or anaerobically.     

Proton leakiness caused by cloned genes for the F0 sector of the proton-translocating ATPase of Escherichia coli: requirement for F1 genes.

To study expression of uncG, the gene coding for the gamma subunit of the Escherichia coli proton-translocating ATPase, deletions were made in the intergenic region between uncA, the gene coding for the alpha subunit, and uncG. Two deletions which fused uncA and uncG coded for alpha-gamma fusion polypeptides which were synthesized well both in vitro and in vivo, demonstrating that uncG expression is normally controlled by nucleotides in the intergenic region. Multicopy plasmids carrying these fusion genes and the genes for the other subunits of the ATPase had a harmful effect on the growth of E. coli. The effect was overcome by N,N'-dicyclohexylcarbodiimide, indicating that the cells probably leaked protons. The deleterious effect was eliminated by making a nonpolar deletion in the upstream F0 gene uncB, or by cloning each of the uncA-uncG fusion genes onto a separate plasmid, removed from the F0 genes, thus demonstrating that the fusion genes were not primarily responsible for the proton permeability. A plasmid which carried F0 genes and the gene for the delta subunit caused deleterious proton leakiness in unc+ cells but not in cells from which the unc operon was deleted. The proton leakiness caused by these different plasmids was therefore due to the production of a leaky F0 proton channel and required the presence of F1 genes. The results support a model for ATPase assembly in which F1 genes or polypeptides are involved in the formation or opening of the F0 proton channel.     

Catabolite repression of the H(+)-translocating ATPase in Vibrio parahaemolyticus.

Cells of Vibrio parahaemolyticus grown in the presence of glucose showed reduced (by about 40%) oxidative phosphorylation. With this observation as a basis, we examined the effect of glucose on the level of H(+)-translocating ATPase. The addition of glucose to the growth medium reduced the specific activity and the amount of the H(+)-translocating ATPase in membrane vesicles of V. parahaemolyticus. These reductions were reversed by adding cyclic AMP (cAMP) to the growth medium. We cloned some parts of the unc genes encoding subunits of the H(+)-translocating ATPase of V. parahaemolyticus by means of the polymerase chain reaction. Using an amplified DNA fragment, we carried out Northern (RNA) blot analysis and found that glucose reduced the mRNA level of the H(+)-translocating ATPase gene by about 40% and that cAMP restored it. We determined the DNA sequence of the unc promoter region of V. parahaemolyticus and found a consensus sequence for the cAMP receptor protein-cAMP-binding site. Such a sequence was also found in the promoter region of the unc operon of Vibrio alginolyticus but not in its counterpart in Escherichia coli. We observed a similar reduction in the level of ATPase due to glucose in V. alginolyticus. In E. coli, however, reductions in the ATPase and the unc mRNA levels were not observed. Thus, the unc operon is controlled by cAMP-regulated catabolite repression in V. parahaemolyticus and V. alginolyticus but not in E. coli. Catabolite repression of the unc operon in V. parahaemolyticus is not severe.     

Use of lacZ fusions to measure in vivo expression of the first three genes of the Escherichia coli unc operon.

We have constructed in-frame lacZ protein fusions to the first three genes of the Escherichia coli unc operon, which codes for the subunits of the proton-translocating ATPase. We have used these constructions to measure the relative in vivo expression of these genes. The second and third genes, uncB and uncE, which code for the a and c subunits of the F0 sector, were expressed at relative levels of approximately 1:10, although the measured expression of uncB depended upon how much of the gene was fused to lacZ. These rates compared favorably with the relative numbers of a and c subunits (a1:c10) in the purified F1F0 complex. The in vivo expression of uncI, the first gene of the operon, was very low, at best 10 to 20 times less than the expression of uncB.     

Developmental regulation of myelin basic protein expression in mouse brain

Developmental regulation of myelin basic protein expression in mouse brain has been examined by comparing the myelin basic protein coding potential of mRNA in vitro with the accumulation of myelin basic protein-related polypeptides in vivo. In vitro translation of mRNA isolated from mouse brain generated eight myelin basic protein-related polypeptides with apparent molecular weights of 34K, 30K, 29K, 26K, 21.5K, 18.5K, 17K, and 14K. A similar set of eight myelin basic protein-related polypeptides with corresponding molecular weights was identified in vivo when total brain proteins were analyzed by immunoblotting. Each of the myelin basic protein-related polypeptides shows a characteristic developmental profile in terms of mRNA level and rate of accumulation implying a complex developmental program of myelin basic protein gene expression with regulation and modulation at several different biosynthetic levels.     

Characterization of the H(+)-pumping F1F0 ATPase of Vibrio alginolyticus.

The F1F0 ATPase of Vibrio alginolyticus was cloned from a chromosomal lambda library. The unc operon, which contains the structural genes for the ATPase, was sequenced and shown to have a gene organization of uncIBEFHAGDC. The sequence of each subunit was compared with those of other eubacterial ATPases. The V. alginolyticus unc genes exhibited greater similarity to the Escherichia coli unc genes than to any of the other bacterial unc genes for which the sequence is available. The ATPase was expressed in an E. coli unc deletion strain, and the ATP hydrolytic activity was characterized. It has a pH optimum of 7.6 and is stimulated by the addition of Triton X-100 or any of a variety of salts. The recombinant F1F0 was purified 30.4-fold and reconstituted into proteoliposomes. This enzyme catalyzed the pumping of protons coupled to ATP hydrolysis as measured in fluorescence quenching experiments but would not pump Na+ ions under similar conditions.     

Order and orientation of genic sequences in circular mitochondrial DNA fromSaccharomyces exiguus: Implications for evolution of yeast mtDNAs

Mapping of sequences specifying the large and small ribosomal RNAs and six polypeptides in the circular 23.7 kbp mitochondrial DNA of Saccharomyces exiguus has shown that these genes have the same orientation and that a 5 gene cluster is common to this DNA and the 18.9 kbp mtDNA from Torulopsis glabrata. Included in the preserved region are juxtaposed sequences specifying ATPase subunits 6 and 9 which have the same order and orientation as analogous genes in the Escherichia coli unc operon. The above data, together with knowledge that these two sequences are dispersed in larger yeast mtDNAs, leads us to suggest that larger forms are derived from a smaller ancestral molecule that would have had some resemblance to the mtDNAs of S. exiguus and T. glabrata.     

Use of lac fusions to measure in vivo regulation of expression of Escherichia coli proton-translocating ATPase (unc) genes.

In-frame fusions to lacZ were constructed in two adjacent genes of the unc operon of Escherichia coli, uncA and uncG, which code for the alpha and gamma subunits of the proton-translocating ATPase. After each fusion was moved into the E. coli chromosome, measurement of beta-galactosidase activities from single-copy genes showed that uncA was expressed significantly better in vivo than was uncG, but the relative expression dependent on the chromosomal location of each fusion and the presence or absence of other unc genes.     

Purification and characterization of a β-galactosidase from Sclerotinia sclerotiorum

The DNA coding for the eight structural genes and uncI of the sodium dependent ATPase of Propionigenium modestum has been cloned and sequenced. Based on sequence homology, the genes were determined to appear in the order uncBEFHAGDC as in several other bacterial species. Minicell experiments revealed that plasmids containing the P. modestum DNA expressed those ATPase polypeptides in Escherichia coli. These were very similar in molecular mass to those obtained from the purified ATPase of P. modestum. No membrane-bound ATPase activity was observed in E. coli unc deletion strains containing the P. modestum ATPase genes. Amino acid alignments which were done with the Fo subunits revealed only a few conservative changes in the highly conserved regions of the polypeptides.     

Assembly of a functional F0 of the proton-translocating ATPase of Escherichia coli

We have investigated both structural and functional assembly of the F0 portion of the Escherichia coli proton-translocating ATPase in vivo. Fractionation of E. coli minicells containing plasmids which code for parts of the unc operon shows that each of the F0 peptides a, b, and c insert into the cytoplasmic membrane independent of each other and without the polypeptides which form the F1 portion of the complex alpha, beta, gamma, delta, and epsilon. Assays of membrane energization indicate that, while formation of a functional proton channel requires the presence of all three F0 polypeptides a, b and c, they are not sufficient. Synthesis of both the alpha and beta subunits of the F1 are required for formation of a functional proton channel.