ABSTRACT
There are three known single-strand DNA-specific exonucleases in Escherichia coli: RecJ, exonuclease I (ExoI), and exonuclease VII (ExoVII). E. coli that are deficient in all three exonucleases are abnormally sensitive to UV irradiation, most likely because of their inability to repair lesions that block replication. We have performed an iterative screen to uncover genes capable of ameliorating the UV repair defect of xonA (ExoI-) xseA (ExoVII-) recJ triple mutants. In this screen, exonuclease-deficient cells were transformed with a high-copy E. coli genomic library and then irradiated; plasmids harvested from surviving cells were used to seed subsequent rounds of transformation and selection. After several rounds of selection, multiple plasmids containing the rnt gene, which encodes RNase T, were found. An rnt plasmid increased the UV resistance of a xonA xseA recJ mutant and uvrA and uvrC mutants; however, it did not alter the survival of xseA recJ or recA mutants. RNase T also has amino acid sequence similarity to other 3' DNA exonucleases, including ExoI. These results suggest that RNase T may possess a 3' DNase activity capable of substituting for ExoI in the recombinational repair of UV-induced lesions.
Subject(s)
DNA, Single-Stranded/physiology , Escherichia coli Proteins , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/radiation effects , Exodeoxyribonucleases/physiology , Exoribonucleases/physiology , Bacterial Proteins/genetics , Deoxyribonucleases/physiology , Exodeoxyribonucleases/genetics , Gene Library , Genes, Bacterial , Genetic Testing , Genotype , Models, Biological , Mutation , Plasmids/genetics , Recombination, Genetic , Suppression, Genetic , Transformation, Genetic , Ultraviolet RaysABSTRACT
The timeless protein (TIM) is a central component of the circadian pacemaker machinery of the fruitfly Drosophila melanogaster. Both TIM and its partner protein, the period protein PER, show robust circadian oscillations in mRNA and protein levels. Yet the role of TIM in the rhythm generation mechanism is largely unknown. To analyze TIM function, we constructed transgenic flies that carry a heat shock-inducible copy of the timeless gene (tim) in an arrhythmic tim loss-of-function genetic background. When heat shocked, TIM levels in these flies rapidly increased and initiated a molecular cycle of PER accumulation and processing with dynamics very similar to the PER cycle observed in wild-type flies. Analysis of period (per) mRNA levels and transcription uncovered a novel role for TIM in clock regulation: TIM increases per mRNA levels through a post-transcriptional mechanism. Our results suggest positive as well as negative autoregulation in the Drosophila circadian clock.
Subject(s)
Circadian Rhythm/physiology , Drosophila Proteins , Drosophila melanogaster/physiology , Insect Proteins/physiology , ARNTL Transcription Factors , Animals , Animals, Genetically Modified , Basic Helix-Loop-Helix Transcription Factors , Circadian Rhythm/genetics , Dimerization , Drosophila melanogaster/genetics , Gene Expression , Genes, Insect , Homeostasis , Hot Temperature , Insect Proteins/chemistry , Insect Proteins/genetics , Models, Biological , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Period Circadian Proteins , Phosphorylation , Protein Conformation , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trans-Activators/genetics , Trans-Activators/physiology , Transcription, GeneticABSTRACT
Screens for zygotic lethal mutations that are associated with specific maternal effect lethal phenotypes have only been conducted for the X chromosome. To identify loci on the autosomes, which represent four-fifths of the Drosophila genome, we have used the autosomal "FLP-DFS" technique to screen a collection of 496 P element-induced mutations established by the Berkeley Drosophila Genome Project. We have identified 64 new loci whose gene products are required for proper egg formation or normal embryonic development.
