Preparation of specific antisera to digoxin using hapten-[C-17]- and [C-12]-bovine serum albumin conjugates.
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Animals↗
Biomedical subjects
S Mizusawa
Publications and source records attributed to S Mizusawa.
Transcription of the sulA gene and repression by LexA.
The Escherichia coli sulA gene product is a highly unstable protein, whose synthesis in response to DNA damage is associated with an inhibition of septation. Genetic evidence as well as sequence information suggests that the sulA gene is part of the E. coli SOS system and is induced after DNA damage. We have constructed a plasmid carrying only the sulA gene; this plasmid is stable only when it contains an amber mutation in the sulA structural gene. Using fragments of this plasmid, we have carried out in vitro transcription experiments and demonstrated one major start site for RNA transcription. We have mapped this initiation point to an adenylate residue 30 nucleotides before the protein start. Purified LexA protein completely abolishes this transcription, in agreement with the prediction made from the genetic and sequence information previously available.
Bacterial Proteins↗
Protein degradation in Escherichia coli: the lon gene controls the stability of sulA protein.
Escherichia coli lon mutants are defective in the ATP-dependent proteolysis of abnormal proteins. The mutants are also sensitive to ultraviolet light (UV) in that septation is inhibited after exposure to UV. sulA mutations, isolated as suppressors of UV sensitivity unlinked to lon, do not affect proteolysis but allow septation to occur after DNA damage. We have confirmed the hypothesis that the product of the sulA gene is degraded by lon proteolysis. If sulA (the product of sulA) is a UV-inducible division inhibitor, as suggested by a variety of experiments, lon (the product of lon) may regulate cell division by regulating the half-life of sulA. We cloned the sulA gene in a bacteriophage lambda vector from a plasmid carrying the ompA region of E. coli. An 18-kilodalton polypeptide was identified as the product of the sulA gene. Pulse-chase labeling demonstrated that the half-life of the sulA protein is 1.2 min in lon+ cells and 19 min in lon- cells. This work demonstrates that lon proteolysis affects the stability of a native E. coli protein.
Bacteriophage lambda↗
A bacteriophage lambda vector for cloning with BamHI and Sau3A.
A phage lambda cloning vector has been constructed which contains a single site for the restriction endonuclease BamHI. Since Sau3A and Bg/II produce the same cohesive ends as BamHI, this vector can also be used to clone DNA fragments generated with either of these enzymes. We have used this vector to construct an Escherichia coli library using partial digestion with Sau3A. This vector will be most useful for applications requiring genetic analysis of cloned E. coli genes.
Bacteriophage lambda↗