Barry Egan - Academia.edu (original) (raw)

Papers by Barry Egan

Molecular Microbiology, Feb 1, 1997

In the present study we show that the Apl protein of the temperate coliphage 186 combines, in one... more In the present study we show that the Apl protein of the temperate coliphage 186 combines, in one protein, the activities of the coliphage lambda proteins Cro and Xis. We have shown previously that Apl represses both the lysogenic promoter, pL, and the major lytic promoter, pR, and is required for excision of the prophage. Apl binds at two locations on the phage chromosome, i.e. between pR and pL and at the phage‐attachment site. Using an in vivo recombination assay, we now show that the role of ApI in excision is in the process itself and is not simply a consequence of repression of pR or pL. To study the repressive role of Apl at the switch promoters we isolated Apl‐resistant operator mutants and used them to demonstrate a requirement for Apl in the efficient derepression of the lysogenic promoter during prophage induction. We conclude that Apl is both an excisionase and transcriptional repressor.

Journal of Biological Chemistry, May 1, 1996

Journal of Biological Chemistry, Sep 1, 2000

The CII protein of bacteriophage 186 is a transcriptional activator of the helix-turn helix famil... more The CII protein of bacteriophage 186 is a transcriptional activator of the helix-turn helix family required for establishment of the lysogenic state. DNA binding by 186 CII is unusual in that the invertedly repeated half sites are separated by 20 base pairs, or two turns of the DNA helix, rather than the one turn usually associated with this class of proteins. Here, we investigate quantitatively the DNA binding properties of CII and its interaction with RNA polymerase at the establishment promoter, p E. The stoichiometry of CII binding was determined by sedimentation equilibrium experiments using a fluorescein-labeled oligonucleotide and purified CII. These experiments indicate that the CII species bound to DNA is a dimer, with additional weak binding of a tetrameric species at high concentrations. Examination of the thermodynamic linkages between CII selfassociation and DNA binding shows that CII binds to the DNA as a preformed dimer (binding free energy, 9.9 kcal/mol at 4°C) rather than by association of monomers on the DNA. CII binding induces in the DNA a bend of 41 (؎ 5) degrees. The spacing between the binding half sites was shown to be important for CII binding, insertion or removal of just 1 base pair significantly reducing the affinity for CII. Removal of 5 or 10 base pairs between binding half sites eliminated binding, as did insertion of an additional 10 base pairs. CII binding at p E was improved marginally by the presence of RNA polymerase (⌬⌬G ‫؍‬-0.5 (؎ 0.3) kcal/mol). In contrast, the binding of RNA polymerase at p E was undetectable in the absence of CII but was improved markedly by the presence of CII. Thus, CII appears to recruit RNA polymerase to the promoter. The nature of the base pair changes in mutant phage, selected by their inability to establish lysogeny, are consistent with this mechanism of CII action.

Journal of Virology, Nov 1, 1981

Virology, May 1, 1996

Retrons are unusual, reverse transcriptase-encoding elements found in bacteria. Although there ar... more Retrons are unusual, reverse transcriptase-encoding elements found in bacteria. Although there are a number of indications that retrons are mobile elements, their transposition has not been observed. The Escherichia coli retrons Ec67 and Ec86 are different retrons inserted at the same site and we have further characterized this site in search of clues to the mechanism of retron transposition. We confirm, by extending previous sequence analysis, that Ec67 and Ec86 are inserted into prophages related to coliphage 186. Comparison with the recently published sequence of the 186 96-2% region indicates that the retrons have replaced Ç180 bp of DNA between the phage cohesive end site (cos) and the transcription terminator of a phage DNA-packaging gene. These features-DNA replacement at the insertion site and the location of retron junctions near transcription terminators or DNA cleavage sites-are shared with other retrons and suggest ways in which retron transposition might have occurred.

Journal of Virology, Dec 1, 1974

Cell, Nov 1, 1981

The tyrT locus of E. coli contains a 208 bp spacer region that separates two copies of sequence e... more The tyrT locus of E. coli contains a 208 bp spacer region that separates two copies of sequence encoding tRNA,. lyr The spacer includes a 120 bp sequence that is homologous to a sequence that is repeated three times in the distal portion of the tyrT locus. The tyrT locus possesses a graded set of transcription termination sites that are spaced at 180 base intervals, corresponding to the distal repeated gene structure. The major termination site occurs within the second repeat unit, 225 bases beyond the mature tRNA sequences. In the presence of a temperature-sensitive rho protein there is increased read-through at this site to a termination site located 180 bases downstream in the third repeat and to several termination sites even further downstream. The primary native transcript, in the region distal to the second tRNA, carries the information for a low molecular weight, extremely basic protein. Although analogous coding sequences are present in the spacer and other repeat units, because of single base substitutions these sequences are pseudogenes. The parallel between the tyrT and tyrfJ gene clusters is discussed in relation to dual function transcripts that specify both tRNA and protein.

Journal of Biological Chemistry, Mar 1, 1998

Molecular Cell, Jun 1, 2004

This form of transcriptional interference is often Australia asymmetric, with a strong (the aggre... more This form of transcriptional interference is often Australia asymmetric, with a strong (the aggressive) promoter reducing the expression of a weak (the sensitive) promoter, and is used in nature to effect regulation of gene Summary expression. Examples include (1) the low expression of constitutive genes by uncontrolled convergent promot-Transcriptional interference with convergent traners, such as with the transposase gene of insertion sescription from face-to-face promoters is a potentially quence IS10 (Simons et al., 1983); (2) the serendipitous important form of gene regulation in all organisms. reduction in gene expression resulting from the inser-Using LacZ reporter studies, the mechanism of intertions of promoters into the genome, such as that caused ference was determined for a pair of face-to-face proby insertions of transposable elements or retroviruses karyotic promoters in which a strong promoter interthat contain very strong outward promoters at their geferes 5.6-fold with a weak promoter, 62 bp away. The netic boundaries (Nigumann et al., 2002), or during genopromoters were variously rearranged to test different mic cloning of transgenes (Eszterhas et al., 2002; Padimodels of interference. Terminating transcription from dam and Cao, 2001); (3) the indirect control of gene the strong promoter before it reached the weak proexpression by varying the interference from an aggresmoter dramatically reduced interference, indicating a sive promoter, via a transcription factor, e.g., the lysisrequirement for the passage of the converging RNAP lysogeny genetic switch of coliphage 186 (Dodd and over the weak promoter. Based on in vitro experiments Egan, 2002), or differential imprinting methylation sensishowing a slow rate of escape for open complexes at tivity, e.g., control of Tsix in developing extraembryonic the weak promoter and their sensitivity to head-on cells (Mlynarczyk and Panning, 2000). collisions with elongating RNAP, a "sitting duck" model Interference by convergent transcription has been inof interference is proposed and supported with in vivo vestigated in a number of E. coli and eukaryotic studies; permanganate footprinting. The model is further suphowever, the exact mechanism(s) of interference reported by the analysis of a second set of prokaryotic mains speculative (Ward and Murray, 1979; Horowitz face-to-face promoters.

Trends in Genetics, Jun 1, 2005

The term 'transcriptional interference' (TI) is widely used but poorly defined in the literature.... more The term 'transcriptional interference' (TI) is widely used but poorly defined in the literature. There are a variety of methods by which one can interfere with the process or the product of transcription but the term TI usually refers to the direct negative impact of one transcriptional activity on a second transcriptional activity in cis. Two recent studies, one examining Saccharomyces cerevisiae and the other Escherichia coli, clearly show TI at one promoter caused by the arrival of a transcribing complex initiating at a distant promoter. TI is potentially widespread throughout biology; therefore, it is timely to assess exactly its nature, significance and operative mechanisms. In this article, we will address the following questions: what is TI, how important and widespread is it, how does it work and where should we focus our future research efforts?

Journal of Virology, Dec 1, 1982

DNA synthesis in coliphage 186-infected cells was investigated. Phage 186 appeared to inhibit hos... more DNA synthesis in coliphage 186-infected cells was investigated. Phage 186 appeared to inhibit host DNA synthesis early in infection. The subsequent synthesis of phage 186 DNA was dependent on the product of 186 gene A. The product of gene B controlled both the production of late 186 proteins and the cessation of 186 DNA synthesis, and the products of genes 0 and P had no influence on 186 DNA synthesis. The product of gene P controlled host cell lysis, and the product of gene 0 may have some regulatory function.

Virology, Aug 1, 1998

The late-lytic region of the genome of bacteriophage 186 encodes the phage proteins that synthesi... more The late-lytic region of the genome of bacteriophage 186 encodes the phage proteins that synthesize the complex viral particle and lyse the bacterial host. We report the completion of the DNA sequence of the late region and the assignment of 18 previously identified genes to open reading frames in the sequence. The 186 late region is similar to the late region of phage P2, sharing 26 genes of known function: the single gene for activation of late gene transcription, 6 genes for construction of DNA-containing heads, 16 for tail morphogenesis, and 3 for cell lysis. We identified two 186 late genes with unknown function; one is homologous to previously unrecognised genes in P2, HP1, and CTX, and the other may modulate DNA packaging. The 186 late region, like the rest of the genome, lacks the lysogenic conversion genes that are carried by P2, allowing the 186 late region to be transcribed from only three late promoters rather than four. The relative absence of lysogenic conversion genes in 186 suggests that the two phages have evolved to use the lytic and lysogenic reproductive modes to different extents.

Journal of Virology, Sep 1, 1975

Ten sus mutants of the staphylococcal bacteriophage)11, each a representative from a different co... more Ten sus mutants of the staphylococcal bacteriophage)11, each a representative from a different complementation group, have been used in three-factor cross experiments. The results of these crosses indicate a circular genetic map for 011. Functional studies of the mutants have been limited to electron microscopic examinations of lysates after prophage induction (or infection). One gene is an early gene, five genes are concerned with tail formation, and three are concerned with head formation. The tenth gene is possibly a head gene. The contribution by)11 to the genomic content of the plasmid-phage hybrid 4llde has been investigated. 4llde contains most of the late genes and appears to be missing a continuous 4)11 segment that includes the early gene flanked by two late genes.

Journal of Biological Chemistry, Apr 1, 1968

Proceedings of the National Academy of Sciences of the United States of America, Jul 1, 1967

Molecular Cell, Jun 1, 2009

Journal of Molecular Biology, Nov 1, 1972

The members of a set of overlapping fragments of h DNA have been isolated. The set is divided int... more The members of a set of overlapping fragments of h DNA have been isolated. The set is divided into two groups, the right and left families. All members of the right family contain the cohesive site at the right end of X DNA; members of the left family contain the cohesive site at the left end. The members of each family differ in molecular length and this characteristic was used to order and to isolate the fragments. Each family is comparable to an ordered set of overlapping deletions and the principles of deletion mapping can therefore be applied to both chemically and genetically defined characteristics of h DNA. Mapping of mutant loci is possible because (1) all fragments in a family are active in the Kaiser-Hogness helper phage assay for genetic activity, and (2) the distance between a given locus and that end of h DNA defining the family is equal to the length of the shortest fragment which contains the locus. The lengths of such fragments were determined from their sedimentation coefficients to yield the following positions for six amber mutations, expressed as fa, the fraction of the length of whole h DNA measured from the right end: N7, 0.268; 029, O-214; PSO, O-186; 921, 0.090; R64, 0.56; R221, 0.052. The left boundary of the imna"/ imm434 non-homology region was mapped at fR = 0.252. The advantage of the mapping methods described here is that they are applicable to point mutations and to characteristics which can be defined by direct chemical analysis of the DNA. In the following paper (Champoux & Hogness, 1972), the pceition and orientation of the ten specifk sequences which generate poly(rC) binding sites and the topography of base composition in h DNA were determined.

Journal of Molecular Biology, May 1, 1973

We have examined the ability of two topological forms of coliphage λ DNA template, a linear form ... more We have examined the ability of two topological forms of coliphage λ DNA template, a linear form and a circular form, to serve as a source of λ gene function in vivo. If infecting λ DNA enters an environment containing λ gam product, or an environment deficient in recBC nuclease activity, both linear (half DNA molecule) and circular (whole DNA molecule) templates efficiently provide λ gene function. Entry of λ DNA into an environment lacking λ gam product results in loss of template potential for the linear molecule in both rec+ and recA hosts, while the template potential of the circular DNA molecule is unaffected. We conclude that (a) λ gam product protects linear λ DNA forms from attack by recBC nuclease; the Escherichia coli recA product affords no such protection for linear A DNA; (b) the role of template topology in λ messenger RNA synthesis is indirect but very essential. Although the linear molecule will suffice as template for transcription per se, circular topology is required for protection of the template from recBC nuclease activity until λ gam product protection is established.