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W Gilbert

Publications and source records attributed to W Gilbert.

144 records · Page 8Linked to original sources
PubMed1977

Contacts between the lac repressor and the thymines in the lac operator.

We have identified important points of contact between the lac repressor and the lac operator by crosslinking the repressor to bromouracil-substituted operator. We substituted bromouracils for thymines in a 55-base-long restriction fragment containing the lac operator and labeled one or the other 5' end with 32P. Ultraviolet irradiation of this fragment produced single-strand breakds at the bromouracils. We examined breakage at each bromouracil in the sequence by denaturing the DNA and displaying the UV-generated fragments on a polyacrylamide gel. In the presence of lac repressor, UV radiation failed to break at specific sites. We attribute this to a competing reaction in which the DNA crosslinks to the repressor rather than breaking. These crosslinkable sites thus define positions at which the lac repressor protein lies close to the methyl group of a thymine in the major groove of DNA.

Base Sequence↗
PubMed1977

A new method for sequencing DNA.

DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.

Adenine↗
PubMed1977

Rous sarcoma virus genome is terminally redundant: the 5' sequence.

When Rous sarcoma virus RNA is transcribed into DNA by the reverse transcriptase, a tRNA primer is elongated into DNA. The primer is near the 5' end of the virus genome; the first major DNA made is a "run-off" product extending 101 bases from the primer to the 5' end of the template. We have studied this DNA molecule to determine the sequence of the first 101 bases at the 5' end of the Rous sarcoma virus genome (Prague strain, subgroup C). Twenty-one bases at the extreme 5' end are also at the 3' end of the virus genome (see D. E. Schwartz, P. C. Zamecnik, and H. L. Weith, this issue, pp. 994-998), and thus this virus is terminally redundant. The existence of this sequence repetition immediately suggests mechanisms by which the growing DNA copy can jump from the 5' end to a 3' end of the template and become circular. The sequence also displays a possible ribosome binding site and enough secondary structure to permit a possible 5'-5' linkage of viral RNA molecules.

Avian Sarcoma Viruses↗
PubMed1977

Mapping adenines, guanines, and pyrimidines in RNA.

The positions of adenines, guanines, and pyrimidines can be determined by partial nuclease digestion of a terminally labeles RNA molecule. In urea, at elevated temperatures, RNase T1 generates a pattern reflecting cleavage at guanines while RNase U2 cleaves only at adenine. A limited alkaline hydrolysis provides a continuum of fragments derived from breaks at every phosphodiester bond. The reaction products are electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel. An autoradiograph of the gel displays the sequence up to 100 nucleotides from the end of the molecule, although uracil cannot as yet be distinguished from cytosine. These techniques form the basis of an RNA sequencing method and are demonstrated on yeast 5.8S ribosomal RNA.

Adenine↗
PubMed1976

Construction of plasmids carrying the cI gene of bacteriophage lambda.

By techniques of recombination in vitro, we have constructed a plasmid bearing the repressor gene (cI) of bacteriophage lambda fused to the promoter of the lac operon. Strains carrying this plasmid overproduce lambda repressor. This functional cI gene was reconstituted by joining DNA fragments bearing different parts of that gene. Flush end fusion techniques, involving no sequence overlap, were necessary for the construction; in certain cases, the abutting of the DNA molecules bearing ends generated by different restriction endonucleases creates a sequence at the junction which is recognized by one of the restriction endonucleases.

Coliphages↗
PubMed1976

Preferential protection of the minor groove of non-operator DNA by lac repressor against methylation by dimethyl sulphate.

The binding of lactose repressor to non-operator DNA was studied by the modification of several DNA's, including glycosylated DNA, with dimethyl sulphate, which affects the minor and major grooves of DNA and single stranded DNA regions. The non-specific binding of the repressor to DNA protected the minor groove but apparently not the major groove of the DNA double helix against methylation and did not increase the content of single stranded DNA regions. This suggests that the repressor on binding to non-operator DNA makes contacts mainly in the minor groove of DNA and does not uncoil the DNA double helix. This is different from the interaction of the repressor with lactose operator DNA which occurs, as shown by Gilbert et al. (1), along both the major and the minor groove.

Alkylating Agents↗
PubMed1973

The nucleotide sequence of the lac operator.

The lac repressor protects the lac operator against digestion with deoxyribonuclease. The protected fragment is double-stranded and about 27 base-pairs long. We determined the sequence of RNA transcription copies of this fragment and present a sequence for 24 base pairs. It is: 5'--T G G A A T T G T G A G C G G A T A A C A A T T 3' 3'--A C C T T A A C A C T C G C C T A T T G T T A A 5' The sequence has 2-fold symmetry regions; the two longest are separated by one turn of the DNA double helix.

Base Sequence↗
PubMedDate not supplied

Detection in vivo of protein-DNA interactions within the lac operon of Escherichia coli.

Studies of the sequence-specific binding of proteins to DNA have so far relied on in vitro experiments using cloned restriction fragments containing the relevant DNA sequences. We have applied the genomic sequencing technique of Church and Gilbert to show that the interactions observed in vitro occur in vivo. We use this approach to study the binding of regulatory proteins to the lac operon in vivo and detect changes in the reactivity (inhibition or enhancement) of guanines to methylation by dimethyl sulphate caused by the proximity of proteins to the N-7 atom of these guanines. We can detect the simultaneous binding of the catobolite gene activator protein (CAP) and the Lac repressor to their specific recognition sequences, and following induction of the lac operon we observe effects that are related to RNA polymerase binding or RNA elongation. We have successfully used oligonucleotide probes as short as 17 bases to display genomic sequence.

Base Sequence↗
PubMedDate not supplied

Cell-type-specific contacts to immunoglobulin enhancers in nuclei.

The introns separating the variable and constant regions of active immunoglobulin genes contain tissue-specific transcriptional enhancer elements, DNA segments which act in cis in an orientation- and distance-independent (up to a few kilobases (kb)) manner to enhance transcription initiation at adjacent promoters. The immunoglobulin heavy-chain enhancer is active only in lymphoid cells: in transfection assays it is capable of controlling in cis transcription from the simian virus 40 (SV40) T-antigen, rabbit beta-globin and immunoglobulin gene promoters up to at least 2 kb away. Genetic deletion analysis suggests that a region of as few as 140 base pairs (bp) is sufficient for the enhancement effect. These functional characteristics and DNA sequences are conserved between mouse and man. However, it is not known whether tissue-specific proteins bind to the enhancer. Proteins that interact with DNA at specific sequences can prevent or enhance the reactions of individual guanines or adenines with dimethyl sulphate (DMS), and this property has been used to display the DNA contacts of various regulatory proteins. Here we apply this DMS strategy in experiments involving single-copy genes within intact mammalian nuclei using genomic sequencing.

Animals↗
PubMedDate not supplied

[A method for determining DNA sequence by labeling the end of the molecule and cleaving at the base. Isolation of DNA fragments, end-labeling, cleavage, electrophoresis in polyacrylamide gel and analysis of results].

We elaborate basic chemical principles and current laboratory procedures for sequencing end-labeled DNA by partial cleavage and gel electrophoresis (A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. USA, 1977, v. 74, p. 560-564). We provide step-by-step protocols for 32P-labeling DNA ends, segregating the labeled ends by cutting with a second restriction enzyme or separating strands, partially cleaving the DNA at specific bases with reagents, electrophoresing the labeled products of cleavage on sequencing gels, and interpreting sequencing band patterns. Many of these procedures have been condensed, to make them faster and easier, and some are new. We also discuss sequencing strategies, and suggest a technique which will reduce plasmid or viral DNA to a collection of singly-end-labeled fragments in one day, for efficient sequencing of these chromosomes in 250-nucleotide blocks.

Base Sequence↗