Isolation and characterization of nonchemotactic CheZ mutants of Escherichia coli

The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ(1-189) peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.     

Nucleotide sequence of the yeast regulatory gene GAL80

The GAL80 gene in Saccharomyces cerevisiae encodes a negative regulatory protein for the set of inducible genes involving metabolism of galactose and melibiose. We have determined the nucleotide sequence of GAL80 and its flanking regions and assigned the 5' end of its mRNA to the sequence. The deduced coding sequence for GAL80 protein contains 1305 nucleotides and the calculated molecular weight of the peptide chain is 48309. The 5' end of the GAL80 mRNA maps about 67 nucleotides upstream from the translation initiating ATG. We have also determined the nucleotide sequence of uninducible alleles GAL80S-0, GAL80S-1 and GAL80S-2, and found single base substitution in each of these mutant genes which would lead to alteration of amino acid in GAL80 protein.     

Molecular analysis of the flagellar switch protein FliM of Salmonella typhimurium.

Defects in the chemotaxis proteins CheY and CheZ of Salmonella typhimurium can be suppressed by mutations in the flagellar switch, such that swarming of a pseudorevertant on semisolid plates is significantly better than that of its parent. cheY suppressors contribute to a clockwise switch bias, and cheZ suppressors contribute to a counterclockwise bias. Among the three known switch genes, fliM contributes most examples of such suppressor mutations. We have investigated the changes in FliM that are responsible for suppression, as well as the changes in CheY or CheZ that are being compensated for. Ten independently isolated parental cheY mutations represented nine distinct mutations, one an amino acid duplication and the rest missense mutations. Several of the altered amino acids lie on one face of the three-dimensional structure of CheY (A. M. Stock, J. M. Mottonen, J. B. Stock, and C. E. Schutt, Nature (London) 337:745-749, 1989; K. Volz and P. Matsumura, J. Biol. Chem. 266:15511-15519, 1991); this face may constitute the binding site for the switch. All 10 cheZ mutations were distinct, with several of them resulting in premature termination. cheY and cheZ suppressors in FliM occurred in clusters, which in general did not overlap. A few cheZ suppressors and one cheY suppressor involved changes near the N terminus of FliM, but neither cheY nor cheZ suppressors involved changes near the C terminus. Among the strongest cheY suppressors were changes from Arg to a neutral amino acid or from Val to Glu, suggesting that electrostatic interactions may play an important role in switching. A given cheY or cheZ mutation could be suppressed by many different fliM mutations; conversely, a given fliM mutation was often encountered as a suppressor of more than one cheY or cheZ mutation. The data suggest that an important factor in suppression is a balancing of the shift in switch bias introduced by alteration of CheY or CheZ with an appropriate opposing shift introduced by alteration of FliM. For strains with a severe parental mutation, such as the cheZ null mutations, adjustment of switch bias is essentially the only factor in suppression, since the attractant L-aspartate caused at most a slight further enhancement of the swarming rate over that occurring in the absence of a chemotactic stimulus. We discuss a model for switching in which there are distinct interactions for the counterclockwise and clockwise states, with suppression occurring by impairment of one of the states and hence by relative enhancement of the other state. FliM can also undergo amino acid changes that result in a paralyzed (Mot-) phenotype; these changes were confined to a very few residues in the protein.     

In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants.

Bacterial chemotaxis results from the ability of flagellated bacteria to control the frequency of switching between smooth-swimming and tumbling episodes in response to changes in concentration of extracellular substances. High levels of phosphorylated CheY protein are the intracellular signal for inducing the tumbling mode of swimming. The CheZ protein has been shown to control the level of phosphorylated CheY by regulating its rate of dephosphorylation. To identify functional domains in the CheZ protein, we made mutants by random mutagenesis of the cheZ gene and constructed a series of deletions. The map position and the in vivo and in vitro activity of the resulting gain- or loss-of-function mutant proteins define separate functional domains of the CheZ protein.     

Cloning and characterization of the O-methyltransferase I gene (dmtA) from Aspergillus parasiticus associated with the conversions of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigmatocystin in aflatoxin biosynthesis.

O-Methyltransferase I catalyzes both the conversion of demethylsterigmatocystin to sterigmatocystin and the conversion of dihydrodemethylsterigmatocystin to dihydrosterigmatocystin during aflatoxin biosynthesis. In this study, both genomic cloning and cDNA cloning of the gene encoding O-methyltransferase I were accomplished by using PCR strategies, such as conventional PCR based on the N-terminal amino acid sequence of the purified enzyme, 5' and 3' rapid amplification of cDNA ends PCR, and thermal asymmetric interlaced PCR (TAIL-PCR), and genes were sequenced by using Aspergillus parasiticus NIAH-26. A comparison of the genomic sequences with the cDNA of the dmtA region revealed that the coding region is interrupted by three short introns. The cDNA of the dmtA gene is 1,373 bp long and encodes a 386-amino-acid protein with a deduced molecular weight of 43,023, which is consistent with the molecular weight of the protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal half of the deduced protein exhibits 76.3% identity with the coding region of the Aspergillus nidulans StcP protein, whereas the N-terminal half of dmtA exhibits 73.0% identity with the 5' flanking region of the stcP gene, suggesting that translation of the stcP gene may start at a site upstream from methionine that is different from the site that has been suggested previously. Also, an examination of the 5' and 3' flanking regions of the dmtA gene in which TAIL-PCR was used demonstrated that the dmtA gene is located in the aflatoxin biosynthesis cluster between (and in the same orientation as) the omtA and ord-2 genes. Northern blotting revealed that expression of the dmtA gene is influenced by both medium composition and culture temperature and that the pattern correlates with the patterns observed for other genes in the aflatoxin gene cluster. Furthermore, Southern blotting and PCR analyses of the dmtA gene showed that a dmtA homolog is present in Aspergillus oryzae SYS-2.     

The Mutation Bronze-Mutable 4 Derivative 6856 in Maize Is Caused by the Insertion of a Novel 6.7-Kilobase Pair Transposon in the Untranslated Leader Region of the Bronze-1 Gene

The Ds-controlled allele, bz-m4 Derivative 6856 [bz-m4 D6856], is reported to have an altered temporal- and tissue-specific pattern of gene expression. We have cloned this allele and have characterized it at the molecular level. The mutation was caused by the insertion of a complex transposon-like structure 36 base pairs downstream from the Bz mRNA cap site. The insert is 6.7-kbp long. Ds elements, each approximately 2 kbp in length, are at both ends of the insert. The sequence between the Ds elements is a partial duplication of flanking sequences from the 3' end of the Bz gene. These data suggest that Ds initially inserted near the 3' end of the gene and mobilized adjacent sequences as it transposed.     

The nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae: a potential adenine nucleotide binding amino acid sequence and a nonessential acidic carboxyl terminal region.

The RAD3 gene of Saccharomyces cerevisiae is required for excision of pyrimidine dimers and is essential for viability. We present the nucleotide sequence of the RAD3 protein coding region and its flanking regions, and the deduced primary structure of the RAD3 protein. In addition, we have mapped the 5' end of RAD3 mRNA. The predicted RAD3 protein contains 778 amino acids with a calculated molecular weight of 89,779. A segment of the RAD3 protein shares homology with several adenine nucleotide binding proteins, suggesting that RAD3 protein may react with ATP. The twenty carboxyl terminal amino acids of RAD3 protein are predominantly acidic; however, deletion of this acidic region has no obvious effect on viability or DNA repair.     

Isolation and characterization of the rat proenkephalin gene

The rat proenkephalin gene has been isolated by molecular cloning and characterized by DNA-sequence analysis. The gene exhibits a structural organization similar to that of the human gene. The nucleotide sequence encoding the biologically active opioid peptides which are generated from the proenkephalin precursor as well as the 3' untranslated region of the mRNA are found on a large exon at the 3' end of the gene (Exon III). The nucleotide sequence encoding the N terminus of the mature protein and its signal peptide are located on Exon II while Exon I encodes the 5' untranslated region of the mRNA. The nucleotide sequence of these exons and their flanking regions has been determined and compared to the human proenkephalin gene. Analysis of the nucleotide sequence homology between the human and rat proenkephalin gene reveals the presence of highly conserved regions within both the coding and noncoding portions of the genes. Enkephalin-coding sequences as well as 5' flanking sequences appear to be the most highly conserved. The importance and possible function of these sequences are discussed.     

Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions

The primary sequence of the major heat shock gene of D. melanogaster, that for the 70,000 protein, has been determined. One of the reading frames is devoid of stop codons for over 2000 bp. The region between the first ATG and the first stop codon encodes a protein of molecular weight 70,270. The 5' end of the messenger RNA was localized in the DNA sequence by two independent methods. The 5' flanking sequences of three distinct 70K genes were also determined. Extensive homology in the primary sequences extends about 500 bp upstream from the ATG, which is the presumptive initiation of protein synthesis. Each 70K gene has the putative promoter sequence TATAAATA about 325 bp upstream from this ATG. A heptanucleotide sequence identified as the capping site for other messengers is found 24-30 bp downstream from the ends of the A-T-rich sequence. A 12 bp sequence with dyad symmetry begins 23 bp upstream from the beginning of the above A-T-rich sequence.     

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by S1 nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the leu-5 mutation had been mapped before. We show that the leu-5 strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the leu-5 strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in leu-5 extracts, the leu-5 strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.