Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu.

PERSPECTIVES AND SUMMARY . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 ORIGINS OF CONSERVED GENE REGULATORS . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 211 Steroids . . . . .. . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . .... . . . . . . .. . . . . . . . .. . . . . . . .. . 211 Steroid Receptors .... " . . . . . . . . . . . . . . . . . . . . . " . . . . . . . . . . . . . . . . . . . . . . . . "" . . . . . . . . . . . . . . . . . . . . . . " 211 FUNCTIONAL ASPECTS OF RECEPTOR STRUCTURE" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Activation and Intracellular Localization ... . ". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Steroid Binding .. ...... , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " . . . . . . . . . . . . . . . . . . , 214 Nonspecific DNA Binding . .. . ..... ....... . ....... " . . . . . . . . .. . . . . . . . . . . . . . . . . " 215 Functional Heterogeneity 216 SELECTIVE RECEPTOR:DNA INTERACTIONS ... . . .... .. .... . .... . 21 7 Receptor Binding at Genomic Sites of Action . " . . . . . . . " 218 Detection of Specific DNA Binding Sites """""",,, , , """""'" . . . . . . . . . . . . . . . . . . . . . . . 21 8 DNA Sequences Bound by Steroid Receptors .. . ......... . ..... . ..... . ..... . ... , 221 LOCALIZATION OF STEROID RESPONSE ELEMENTS (REs) . .... " . . . . . . . . . 222 Deletion Mapping .. . ..... ......... . ....... . " . . . . . . . . . . . . . . . . . . . . . ""." . . .... . . . 223 RE-Promoter Fusions .... ......... . " . . . . . . . . . . . . . . . . . ". . . . . . . . . . . . . . . . . . 223 In Vitro Mutagenesis" ...... . ........ ......... .. " . . . . . . . . . . . . . . . . . "" . . . . . . " . . . . . . . . . . . . . . . . " 224 TRANSCRIPTIONAL ENHANCEMENT BY RECEPTOR:RE COMPLEXES " " "'" ' ' 224 Glucocorticoid-Dependent Enhancement 225 Detection and Characterization of Enhancers. . .. . . . . . . .... . . .... . . . . . . .. . . . . . . .... . . ... . . . . 225 EFFECTS OF RECEPTOR BINDING ON GENOME STRUCTURE ... " . . ". . . . . . . . . . . . . . 227 Cytological and Nuclease Probes .. . . """""" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " . . . . . . . . . 227 Chromosomal Protein Alterations 229 DNA-Structure Alterations 230 New Experimental Approaches . . . . . ... """ . . ,, .. " . . . . ,,". . . . . . . . . . . . . . . . . . . . . . . . . 232 HIGHER-ORDER CONTROL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 233 Gene Networks ..... ...... ........ ....... . " . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ". . . . . . . . . 234 Multifactor Regulation ...... ... ,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

Steroid Receptor Regulated Transcription of Specific Genes and Gene Networks

A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

/pdf/an-efficient-recombination-system-for-chromosome-engineering-4acmootzcr.pdf

An efficient recombination system for chromosome engineering in Escherichia coli

https://mmbr.asm.org/content/mmbr/47/2/180.full.pdf

Linkage map of Escherichia coli K-12, edition 7.

http://zhiyuan.sjtu.edu.cn/file/course/20131031102853_Zhuang%20Yuan-Lecture%201-optional%20reading.pdf

The V(D)J Recombination Activating Gene, RAG-1

A conserved zinc binding domain in the largest subunit of DNA-dependent RNA polymerase modulates intrinsic transcription termination and antitermination but does not stabilize the elongation complex.

Inhibition of a Transcriptional Pause by RNA Anchoring to RNA Polymerase

The repressor of the galactose operon of Escherichia coli has been partially purified and identified as a protein. Induction of a lysogen in which λ was linked to the bacterial galR and lysine genes resulted in a large increase in the production of the gal repressor. Single-step purification by affinity chromatography, using the ligand p-aminophenyl-β-D-thiogalactoside linked to beaded agarose, provided a convenient method of separating the gal repressor from other DNA-binding proteins. Binding of gal repressor to λpgal[32P]DNA was studied by assay of binding to a nitrocellulose filter. Interaction between gal repressor and λpgal DNA showed a high degree of specificity; the dissociation constant of the complex was estimated to be 1.0 × 10-12 M. Unlabeled λpgal DNA competed for binding to gal repressor, but λDNA and λh80dlac DNA did not. Fucose and galactose, which function as inducers of the galactose operon in vivo, produced one-half maximal inhibition of gal repressor-λpgal DNA binding at concentrations of 5 × 10-5 M.

Isolation of the Gal Repressor

We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth.

DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu

Integrative recombination of bacteriophage lambda occurs by two sequential, reciprocal strand exchanges at specific positions within the attachment sites. Both exchanges are promoted by the lambda Int protein; the first forms a Holliday structure, and the second resolves it to recombinant products. Recombination requires sequence homology within the 7 bp 'overlap' region that separates the two points of strand exchange. To see if homology promotes the second strand exchange, we constructed attachment site Holliday structures by annealing DNA strands and then assayed Int-promoted resolution. Holliday structures corresponding to strand exchange between sites with homologous overlap regions were efficiently resolved to give mixtures of recombinants and parents. Holliday structures corresponding to exchanges between heterologous sites fell into two classes. Members of the first class, in which heterology limited but did not completely prevent migration of the branchpoint within the overlap region, were resolved efficiently and preferentially to parental molecules. We propose that resolution to recombinants occurs only if homology allows branch migration from the first to the second exchange site. Members of the second class, in which heterology constrained the branchpoint within an Int binding site, were resolved poorly. We suggest that Holliday structures that have a branchpoint within an Int binding site are poor substrates for Int.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1989.tb03543.x

Robert A. Weisberg

Papers

A conserved zinc binding domain in the largest subunit of DNA-dependent RNA polymerase modulates intrinsic transcription termination and antitermination but does not stabilize the elongation complex.

Inhibition of a Transcriptional Pause by RNA Anchoring to RNA Polymerase

Isolation of the Gal Repressor

DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu

Mutations of the phage lambda attachment site alter the directionality of resolution of Holliday structures.