ABSTRACT
The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.
Subject(s)
Obesity/genetics , Proteins/chemistry , Proteins/genetics , Adaptor Proteins, Signal Transducing , Alternative Splicing/genetics , Alternative Splicing/physiology , Animals , Base Sequence , Brain Chemistry/physiology , Chromosome Mapping , Cloning, Molecular , Exons/genetics , Gene Expression/physiology , Genetic Variation , In Situ Hybridization , Insulin Resistance/genetics , Mice , Mice, Obese , Molecular Sequence Data , Mutation/genetics , Polymerase Chain Reaction/methods , RNA, Messenger/metabolism , Sequence Homology, Amino AcidABSTRACT
Bacteriophage N4 virion RNA polymerase transcription of double-stranded promoter-containing DNAs requires supercoiled template and E. coli single-stranded DNA-binding protein (EcoSSB); other single-stranded DNA-binding proteins cannot substitute. The DNA determinants of virion RNA polymerase binding at the promoter comprise a small template-strand hairpin. The requirement for EcoSSB is surprising, since single-stranded DNA-binding proteins destabilize hairpin structures. DNA footprinting of EcoSSB on wild-type and mutant promoters indicates that EcoSSB stabilizes the template-strand hairpin owing to the hairpin-loop sequences. Other single-stranded DNA-binding proteins destabilize the promoter hairpin, explaining the specificity of EcoSSB activation. We conclude that EcoSSB activates transcription by providing the appropriate DNA structure for polymerase binding. The existence of small hairpins stable to single-stranded protein binding suggests a novel mechanism that provides structural determinants for specific recognition in single-stranded DNA transactions by an otherwise nonspecific DNA-binding protein.
Subject(s)
DNA-Binding Proteins/physiology , DNA-Directed RNA Polymerases/genetics , Escherichia coli/physiology , Podoviridae/genetics , Base Sequence , DNA Footprinting , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , DNA, Single-Stranded/genetics , DNA, Viral/genetics , Deoxyribonuclease I/metabolism , Escherichia coli/chemistry , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Viral/physiology , Molecular Sequence Data , Nucleic Acid Conformation , Promoter Regions, Genetic/genetics , Repetitive Sequences, Nucleic Acid , Transcription, Genetic/geneticsSubject(s)
DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , DNA-Directed RNA Polymerases/isolation & purification , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/metabolism , Podoviridae/enzymology , Trans-Activators/metabolism , Transcription, Genetic , Base Sequence , Chromatography, Affinity/methods , Cloning, Molecular , DNA-Directed RNA Polymerases/genetics , Escherichia coli/genetics , Phosphorus Radioisotopes , Plasmids , Podoviridae/genetics , Polymerase Chain Reaction , Promoter Regions, Genetic , Radioisotope Dilution Technique , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Templates, Genetic , Uridine Triphosphate/metabolism , Virion/enzymology , Virion/geneticsABSTRACT
The complete sequence of the polycystic kidney disease gene (PKD1) and its transcript have been described. The predicted protein is not a member of a previously described gene family, but contains several structural motifs that are present in proteins of known function. Most of these domains are present in the extracellular parts of proteins involved in interactions with other proteins and carbohydrates. The PKD1 gene product also contains potential transmembrane sequences. The molecule is likely to be involved in cell-cell or cell-matrix interactions, which is consistent with the different manifestations of polycystic kidney disease.
