Regulation of the in vitro synthesis of the alpha-peptide of beta-galactosidase directed by a restriction fragment of the lactose operon. |
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Authors: | H Kung M Tainsky H Weissbach |
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Institution: | Roche Institute of Molecular Biology, Nutley, New Jersey, 07110 USA |
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Abstract: | The regulation of the synthesis of the N-terminal portion of the β-galactosidase molecule (α-peptide) has been investigated using DNA fragments of the lactose operon as template. DNA fragments of about 789 base pairs were isolated after endonuclease (Hin II) digestion of either λplac5, λh80dlacps or λh80dlacUV5 phage DNA or DNA from the recombinant plasmid PMC3. The regulation of the expression of these fragments is similar to that observed for the synthesis of β-galactosidase using total phage or plasmid DNA as template, indicating that the regulatory regions on the fragments are intact and functional. Thus, the synthesis of the α-peptide required an inducer due to the presence of repressor in the S-30 extract used. In addition a dependency on adenosine 3′,5′-cyclic monophosphate (cAMP)1 for α-peptide synthesis was obtained with the fragments isolated from λplac5 and λh80dlacps DNAs, whereas little effect of cAMP was seen with the fragment isolated from λh80dlacUV5 phage DNA or PMC3 plasmid DNA containing a UV5 promotor region. However, a significant difference in the effect of guanosine-3′-diphosphate-5′-diphosphate (ppGpp) was observed. With the total phage DNA as template, ppGpp resulted in a 2–4 fold stimulation whereas with the fragment, or PMC3 plasmid DNA, directed synthesis of the α-peptide no significant stimulation by ppGpp was seen. |
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