Cloning and functional characterization of a eucaryotic DNA photolyase gene from Neurospora crassa.

We cloned a genomic fragment of a photolyase gene from Neurospora crassa by polymerase chain reaction using synthesized oligonucleotide primers designed from the most conserved amino acid sequences among photolyases of various organisms. Using the cloned fragment as a hybridization probe we isolated a genomic fragment and cDNA clones encoding the complete photolyase gene of this organism. The amino acid sequence of the photolyase deduced from the determined nucleotide sequence indicates a protein consisting of 615 amino acid residues (Mr 69,971), which is most similar to that of Saccharomyces cerevisiae. Like yeast photolyase it contains a protruding amino terminus which is missing in photolyases of bacterial origin. Comparison of amino acids sequences among six photolyases suggests that the Neurospora crassa photolyase is more similar to photolyases of pterin type than those of deazaflavin type.     

Cloning, sequence, and expression of the pantothenate permease (panF) gene of Escherichia coli.

Pantothenate permease, the product of the panF gene, catalyzes the sodium-dependent uptake of extracellular pantothenate. The panF gene was isolated from an Escherichia coli genomic DNA library and subcloned into multicopy plasmids. Increased copy number of the panF+ allele resulted in increased rates of pantothenate uptake and a significant increase in the steady-state intracellular pantothenate concentration. Despite the higher levels of pantothenate, the utilization of pantothenate for coenzyme A formation was not elevated, indicating that pantothenate kinase activity is the dominant regulator of coenzyme A biosynthesis. DNA sequencing of the panF gene revealed the presence of a single open reading frame that encoded a hydrophobic protein with a molecular weight of 51,992. Sequence analysis predicts that pantothenate permease is an integral membrane protein possessing 12 hydrophobic membrane-spanning domains connected by short hydrophilic sequences. The predicted topological profile of pantothenate permease is similar to that of other membrane carriers that catalyze cation-dependent symport.     

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli.

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Cloning, nucleotide sequence, and characterization of mtr, the structural gene for a tryptophan-specific permease of Escherichia coli K-12.

The mtr gene of Escherichia coli K-12 encodes an L-tryptophan-specific permease. This gene was originally identified through the isolation of mutations in the 69-min region of the chromosome, closely linked to argG. Cells with lesions in mtr display a phenotype of 5-methyltryptophan resistance. The mtr gene was cloned by using the mini-Mu system. The amino acid sequence of Mtr (414 codons), deduced by DNA sequence analysis, was found to be 33% identical to that of another single-component transport protein, the tyrosine-specific permease, TyrP. The hydropathy plots of the two permeases were similar. Possible operator sites for the tyrosine and tryptophan repressors are situated within the region of DNA that is likely to be the mtr promoter.     

The lysP gene encodes the lysine-specific permease.

Escherichia coli transports lysine by two distinct systems, one of which is specific for lysine (LysP) and the other of which is inhibited by arginine ornithine. The activity of the lysine-specific system increases with growth in acidic medium, anaerobiosis, and high concentrations of lysine. It is inhibited by the lysine analog S-(beta-aminoethyl)-L-cysteine (thiosine). Thiosine-resistant (Tsr) mutants were isolated by using transpositional mutagenesis with TnphoA. A Tsr mutant expressing alkaline phosphatase activity in intact cells was found to lack lysine-specific transport. This lysP mutation was mapped to about 46.5 min on the E. coli chromosome. The lysP-phoA fusion was cloned and used as a probe to clone the wild-type lysP gene. The nucleotide sequence of the 2.7-kb BamHI fragment was determined. An open reading frame from nucleotides 522 to 1989 was observed. The translation product of this open reading frame is predicted to be a hydrophobic protein of 489 residues. The lysP gene product exhibits sequence similarity to a family of amino acid transport proteins found in both prokaryotes and eukaryotes, including the aromatic amino acid permease of E. coli (aroP) and the arginine permease of Saccharomyces cerevisiae (CAN1). Cells carrying a plasmid with the lysP gene exhibited a 10- to 20-fold increase in the rate of lysine uptake above wild-type levels. These results demonstrate that the lysP gene encodes the lysine-specific permease.     

Cloning and transcriptional analysis of the segmentation gene fushi tarazu of Drosophila

In the course of studying the Antennapedia (Antp) locus, we found that one of the 3' Antp exons has weak cross-homology to another gene affecting segmentation, fushi tarazu (ftz; meaning "not enough segments"), which is 30 kb to the left of Antp. Homozygous ftz- embryos die before hatching and lack alternate body segments. The reduced number of segments results from the fusion of the anterior portion of one segment with the posterior portion of the next segment. The ftz gene encodes a single 1.9 kb poly(A)+ RNA expressed exclusively from the early blastoderm to gastrula stages of embryonic development. The structure of the ftz gene has been analyzed by S1 nuclease mapping and by restriction mapping of a cDNA clone. The ftz gene consists of two exons, and it is the 3' exon that cross-hybridizes with the 3' exon of Antp. The role of ftz in cell determination is discussed.     

Mistranslation in a eucaryotic organism.

Previous work from our laboratory has demonstrated that a subclass of the aminoglycoside antibiotics, those containing the drug fragment paromamine, stimulates mistranslation in cell-free protein-synthesizing systems derived from eucaryotic cells. We report here experiments which show that the ciliate Tetrahymena thermophila (formerly T. pyriformis, syngen 1) is sensitive to the paromamine-containing aminoglycoside antibiotics. The drugs are active with respect to growth inhibition, inhibition of protein synthesis in the whole organism, inhibition of protein synthesis in vitro and the stimulation of mistranslation in cell-free protein-synthesizing systems. Because of their misreading properties, these drugs may be useful in isolating and propagating strains carrying mutations which can be translationally suppressed (that is, missense and nonsense mutations).