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J R Sadler

Publications and source records attributed to J R Sadler.

27 records · Page 2Linked to original sources
PubMed1976

Recovery of operator DNA binding activity from denatured lactose repressor.

Lactose repressor monomer subunits, prepared with sodium dodecyl sulfate, have been renatured-reassociated to yield tetrameric repressors active in binding to operator DNA. The majority of moleculesin renatured repressor populations have operator binding properties indistinguishable from the stock, undenatured repressors from which they were prepared. Renaturation of a mixture of monomers from wild-type (Q) and a tight binding repressor yields a population of active repressors containing molecules with operator binding characteristics intermediate to those of the "parental" repressor types.

Binding Sites↗
PubMedDate not supplied

Variants of a cloned synthetic lactose operator. I. A palindromic dimer lactose operator derived from one stand of the cloned 40-base pair operator.

Starting with one strand of the 40-bp synthetic operator (Sadler et al., 1978), we have constructed and cloned a 66-bp, palindromic DNA segment with the following sequence (Formula: see text), where the horizontal arrows indicate the locations of the two 21-bp "core" operator sequences in this segment and the vertical arrow designates the dyad axis of symmetry. Upon denaturation and rapid renaturation, each strand of this fragment forms a hairpin molecule still retaining an EcoRI cohesive end. Two hairpin molecules can be joined with T4 DNA ligase to form a duplex DNA molecule having no ends (dumbbell form A). Denaturation and rapid renaturation of dumbbell A yields a mixture of two dumbbell forms: dumbbell A which is a substrate for Eco RI, and a new form, dumbbell B, which is not a substrate. Each of the conformations of this DNA fragment have been purified and all are active in binding lactose repressor in vitro.

Base Sequence↗
PubMedDate not supplied

Cloning and characterization of the natural lactose operator.

A 55-bp DNA segment carrying the wild-type lactose operator sequence has been cloned. Its sequence is: (Formula: see text). With the exceptions of the bases at positions 19 and 41, 26 and 34, and 28 and 32, the sequence is a perfect inverted repeat about base pair 30. This segment was obtained from the wild-type lactose promoter and operator region of lambda h80dlac phage DNA by a combination of in vitro and in vivo steps. Up to four direct-repeat copies of this segment have been cloned in plasmid pMB9 and pBR325. Repressor affinity for this 55-bp fragment does not differ significantly from that for a 40-bp synthetic operator fragment cloned previously, even though the 55-bp fragment contains the complete set of sequence symmetries associated with the natural operator, whereas the 40-bp fragment does not. An improved procedure for operator purification is described: this was used to prepare 14 mg of the 55-bp fragment over a 2-month period.

Bacteriophage lambda↗