Bacteriophage Lambda Genome Research Articles

Precise mass measurement of a double-stranded 500 base-pair (309 kDa) polymerase chain reaction product by negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) has been used to determine the mass of a double-stranded 500 base-pair (bp) polymerase chain reaction (PCR) product with an average theoretical mass of the blunt-ended (i.e. unadenylated) species of 308 859.35 Da. The PCR product was generated from the linearized bacteriophage Lambda genome which is a double-stranded template. Utilization of ethanol precipitation in tandem with a rapid microdialysis step to purify and desalt the PCR product was crucial to obtain a precise mass measurement. The PCR product (0.8 pmol/µL) was electrosprayed from a solution containing 75% acetonitrile, 25 mM piperidine, and 25 mM imidazole and was infused at a rate of 200 nL/min. The average molecular mass and the corresponding precision were determined using the charge-states ranging from 172 to 235 net negative charges. The experimental mass and corresponding precision (reported as the 95% confidence interval of the mean) was 309 406 +/- 27 Da (87 ppm). The mass accuracy was compromised due to the fact that the PCR generates multiple products when using Taq polymerase due to the non-template directed 3'-adenylation. This results in a mixture of three PCR products with nearly identical mass (i.e. blunt-ended, mono-adenylated and di-adenylated) with unknown relative abundances that were not resolved in the spectrum. Thus, the experimental mass will be a weighted average of the three species which, under our experimental conditions, reflects a nearly equal concentration of the mono- and di-adenylated species. This report demonstrates that precise mass measurements of PCR products up to 309 kDa (500 bp) can be routinely obtained by ESI-FTICR requiring low femtomole amounts. Copyright 1999 John Wiley & Sons, Ltd.

Decreased imino proton exchange and base-pair opening in the IHF-DNA complex measured by NMR

Integration Host Factor, IHF, is an E. coli DNA binding protein that imposes a substantial bend on DNA. Previous footprinting studies and bending assays have characterized several recognition sequences in the bacterial and lambda phage genome as unique in the way they are bound by IHF. We have chosen one of the lambda phage sites, H1, for study because it presents a small yet sequence-specific substrate for NMR analysis of the complex. A 19 base-pair duplex, H19, corresponding to the recognition sequence at the H1 site was constructed by isotopically labeling one of the strands with 15N. (1H, 15N) heteronuclear NMR experiments aided in assigning the imino proton resonances of the DNA alone and in complex with IHF. The NMR results are consistent with a mode of binding observed in the recent crystal structure of IHF bound to another of its sites from the lambda phage genome. Additionally, the dramatic change that IHF imposes on the imino proton chemical shifts is indicative of a severe deviation from canonical B-DNA structure. In order to understand the dynamic properties of the DNA in the complex with IHF, the exchange rates of the imino protons with the solvent have been measured for H19 with and without IHF bound. A drastic reduction in exchange is observed for the imino protons in the IHF bound DNA. In the DNA-protein complex, groups of adjacent base-pair exchange at the same rate, and appear to close more slowly than the rate of imino proton exchange with bulk water, since their exchange rate is independent of catalyst concentration. We infer that segments of the double helix as large as 6 bp open in a cooperative process, and remain open much longer than is typical for opening fluctuations in naked duplex DNA. We discuss these results in terms of the specific protein-DNA contacts observed in the crystal structure.

Identification and characterization of the fis operon in enteric bacteria.

The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage lambda genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens, E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence for H. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) preceding fis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from -53 to +27 and the region around -116 containing an ihf binding site. Both beta-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.

Identification of new Fis binding sites by DNA scission with Fis-1,10-phenanthroline-copper(I) chimeras.

The chimeric nuclease Fis-OP has been used to identify novel Fis binding sites. Tethering the chemical nuclease OP-Cu+ to position 73 of the protein with a newly developed longer acetyl-beta-alanylamino spacer has facilitated the localization of two high-affinity Fis binding sequences in a 3 kb pUC19 plasmid. The shorter acetamido linker has allowed the chimeric nuclease to locate two strong Fis binding sites in the 50 kb phage lambda genome. All four sites reside in biologically interesting loci and have been confirmed by gel-retardation and DNase I footprint analyses. A newly discovered site resides in the lac operon of Escherichia coli. The binding of Fis to this site may antagonize repression by the LacI repressor. These studies demonstrate the feasibility of applying chimeric chemical nucleases to the task of identifying functional protein binding sites of biological interest within genomes without any assumption about their sequence preference.

Targeted mutagenesis of DNA using triple helix-forming oligonucleotides linked to psoralen.

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Here we use a 10-base triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve site-specific, targeted mutagenesis in an intact, double-stranded lambda phage genome. Site-specific triplex formation delivers the psoralen to the targeted site in the lambda DNA, and photoactivation of the psoralen produces adducts and thereby mutations at that site. Mutations in the targeted gene were at least 100-fold more frequent than those in a nontargeted gene, and sequence analysis of mutations in the targeted gene showed that 96% were in the targeted region and 56% were found to be the same T.A to A.T transversion precisely at the targeted base pair. The ability to reproducibly and predictably target mutations to sites in intact duplex DNA by using modified oligonucleotides may prove useful as a technique for gene therapy, as an approach to antiviral therapeutics, and as a tool for genetic engineering.

Sequence of a variant Shiga-like toxin type-I operon of Escherichia coli O111:H −

PCR amplification was used to screen faecal isolates of Escherichia coli from a 12-month-old boy with haemolytic uraemic syndrome for the presence of Shiga-like toxin (SLT)-encoding genes. One isolate, belonging to serotype O111:H-, was positive for SLT-I by this method. UV induction indicated that the strain was lysogenic for a lambdoid bacteriophage, but this did not encode the toxin. Southern hybridization analysis of chromosomal DNA revealed that the SLT-I gene was located on an 8.5-kb EcoRI fragment. SLT-I was further localized to within a 3.0-kb SphI-EcoRI fragment. A separate subclone contained a 3.75-kb HindIII fragment, 1.18 kb of which was common to both. Nucleotide sequence analysis of derivatives of these clones revealed that the SLT-I A subunit gene from E. coli O111:H- differed from the previously published sequences for SLT-I by 5 bp [resulting in two amino acid (aa) changes]. It was more closely related to the gene encoding the A subunit of the Shiga toxin from Shigella dysenteriae type 1, from which it differed by 3 bp (resulting in one aa change). The DNA sequence of the B subunit-encoding gene was identical to that of the other two toxins. The region of DNA upstream from the SLT-I of E. coli O111:H- contained an IS element, as well as a region with strong homology to a portion of the genome of bacteriophage lambda.

Expression of the Rz gene and the overlapping Rz1 reading frame present at the right end of the bacteriophage lambda genome

The Rz lysis gene of bacteriophage lambda was cloned into the expression vectors, pT7-3 and pT7-7. The recombinant plasmids expressed either a protein of an unexpected 6.5-kDa size (pT7-3H and pSB54) or two proteins of 6.5 and 17.2 kDa (pBH21). The 6.5-kDa protein alone did not complement the lysis defect of the lambda Rz mutant; hence, this protein was not the Rz gene product. Complementation observed as a result of pBH21 expression thus can be ascribed to the 17.2-kDa protein, which agrees with the size based on the nucleotide sequence of Rz. The 6.5 kDa is a product of an open reading frame entirely encompassed within the Rz sequence and denoted by us Rz1. Both proteins were detectable only by autoradiography, which may mean that the genes are expressed at low rates. Polyclonal anti-Rz antibodies (Ab) were obtained by rabbit immunization with a synthetic polypeptide corresponding to an antigenic determinant of Rz defined by a computer program. The Ab reacted with the 17.2-kDa protein resulting from pBH21 expression, as well as with the 17.2-kDa protein present in the induced Escherichia coli W3350(lambda cI857Sam7) lysate.

Specificity of recognition sequence forEscherichia coli primase

We have surveyed the frequency of each of 64 trinucleotide permutations at every nucleotide frame located from 1 to 15 nucleotides upstream of primer RNA-DNA transition sites mapped within a 1.5 kb region of the bacteriophage lambda genome and a 1.4 kb region of the Escherichia coli genome. We have demonstrated that in both systems initiation of DNA synthesis strongly correlates with a CAG sequence located 11 nucleotides upstream of the DNA start sites. Based on the examination of various reports of the priming reaction catalyzed by E. coli primase in vivo and in vitro, we propose that (i) E. coli primase itself recognizes a 3'GTC 5' sequence on the template strand, (ii) DnaB helicase releases the specificity of E. coli primase and, (iii) the consensus recognition sequence for E. coli primase associated with DnaB helicase is 3'PuPyPy 5'.

Automated DNA sequencing methods involving polymerase chain reaction.

Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

Stochastic models for heterogeneous DNA sequences.

The composition of naturally occurring DNA sequences is often strikingly heterogeneous. In this paper, the DNA sequence is viewed as a stochastic process with local compositional properties determined by the states of a hidden Markov chain. The model used is a discrete-state, discrete-outcome version of a general model for non-stationary time series proposed by Kitagawa (1987). A smoothing algorithm is described which can be used to reconstruct the hidden process and produce graphic displays of the compositional structure of a sequence. The problem of parameter estimation is approached using likelihood methods and an EM algorithm for approximating the maximum likelihood estimate is derived. The methods are applied to sequences from yeast mitochondrial DNA, human and mouse mitochondrial DNAs, a human X chromosomal fragment and the complete genome of bacteriophage lambda.

Bacteriophage lambda site-specific recombination proceeds with a defined order of strand exchanges.

Previous work has established that integration of the genome of bacteriophage lambda into the chromosome of its bacterial host proceeds via two independent strand exchanges, which make and then resolve a Holliday-structure intermediate. We find that a phosphorothioate substitution at the site of exchange in one strand of a recombination site depresses the yield of Holliday structures much more than a similar substitution in the other strand. Furthermore, we show that the Holliday structures that accumulate in unblocked reactions have all been made by recombination of one particular pair of strands. We conclude that there is a strong bias in the choice of strands that initiate crossing-over. Excision, the recombination reaction that excises the integrated prophage, exhibits the same bias as integration. This proves, at least at the level of strand exchange, that excision is not the simple reversal of integration. We have altered the relative orientation of parts of the phage attachment site, attP, to demonstrate that the strand-exchange bias is determined not by the local environment around the point of exchange in the core of attP but by more distant elements in its arms. This suggests that the order of the strand exchanges is dictated by an asymmetry in the way that the nucleosome-like structure that forms at attP brings the bacterial site, attB, into juxtaposition prior to strand exchange. Finally, we use the altered attP to show that homology between attP and attB is most critical when it is adjacent to the point of strand exchange.

Recognition of ill-defined signals in nucleic acid sequences

A set of programs has been developed for the definition and handling of nucleic acid sequence consensus information. The sequences of known genetic control signals are combined in a matrix. The origins and positions of the signals are recorded. Old matrices can be updated dynamically: new signals are included and obsolete ones deleted. Matrices of several different types are computed optionally. Several of these matrices can be combined to find possible new signals. The use of matrices allows the exact quantification of signal qualities. The described programs are part of a program library named GENEXPERT. Application examples given are the search for tRNA genes and the search for promoters in the bacteriophage lambda genome.

RNA-primed intitiation sites of DNA replication in the origin region of bacteriophage λ genome

Using DNA molecules synthesized in the early stage of lambda phage infection, deoxynucleotides at the transition sites from primer RNA to DNA synthesis have been mapped in the 1.5 kbase area of the lambda phage genome containing the genetically defined replication origin (ori lambda). Sites in the 1-strand (the polarity of the 1-strand is 5' to 3' from the left to the right direction of the lambda phage genetic map) were distributed both inside and outside of the ori lambda, whereas the sites in the r-strand (the strand in the opposite polarity) were mainly distributed more than three hundred nucleotides apart from the ori lambda to the right. A CPuPu sequence was found at -12 to -10 region of transition sites of the r- and the 1-strands in the frequency of 80% and 70%, respectively, and over 60% of the CPuPu sequences were CAG. Properties of the transition sites are discussed in relation to the primer synthesis.

An open reading frame in the Escherichia coli bacteriophage lambda genome encodes a protein that functions in assembly of the long tail fibers of bacteriophage T4

Assembly of the long tail fibers of the Escherichia coli bacteriophage T4 requires the catalytic action of two auxiliary proteins. It was found that a gene of the entirely unrelated phage lambda codes for a protein which can substitute for one of these T4 polypeptides, protein 38. The lambda gene was designated tfa (tail fiber assembly). Protein 38 consists of 183 residues, and the Tfa protein consists of 194 residues; the two polypeptides are about 40% homologous. Although the tfa gene is dispensable for the growth of phage lambda, these results indicate that it may have a function in lambda morphogenesis.

Pseudogene in the genome of bacteriophage lambda?

We find a region in the non-coding part of bacteriophage lambda genome that codes for the conserved fold which repressors and other proteins use for specific DNA binding. The region is involved in a long open reading frame exceeding one kilobase and is read in the same frame as gene A in the opposite strand. The putative translation product of this open reading frame has a highly ordered secondary structure with a predominance of alpha helices, which is typical of repressors. In addition, codon usage in this frame suggests a protein-coding region. However, there is a TGA stop codon located between the putative gene start point and the region coding for the DNA binding fold. It thus appears that bacteriophage lambda had one more DNA binding protein, perhaps repressor, in the past that was inactivated by a mutation.

Large palindromes in the lambda phage genome are preserved in a rec+ host by inhibiting lambda DNA replication.

A large palindrome carried by phage lambda has been shown to prevent growth of the phage on a rec+ strain of Escherichia coli. The phage do form plaques on recBC sbcB strains, but the palindrome is not stable--deletions that either destroy the palindrome or diminish its size overgrow the original engineered palindrome-containing phage. We have prepared stocks of lambda carrying a palindrome that is 2 X 4200 base pairs long. These phage stocks are produced by induction of a lysogen in which the two halves of the palindrome are stored at opposite ends of the prophage and are of sufficient titer (10(9) phage per ml) to enable one-step growth experiments with replication-blocked phage. We find that the large palindrome as well as a lesser palindrome of 2 X 265 base pairs are recovered intact among particles carrying unreplicated chromosomes following such an infection of a rec+ host. We propose that DNA replication drives the extrusion of palindromic sequences in vivo, forming secondary structures that are substrates for the recBC and sbcB gene products.

Mutational analysis of integrase arm-type binding sites of bacteriophage lambda: Integration and excision involve distinct interactions of integrase with arm-type sites

Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome ( attP) and the Escherichia coli genome ( attB) results in a prophage flanked by the hybrid recombinant sites attL and attR. Each att site contains sequences to which proteins involved in recombination bind. Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int “arm-type” binding sites located within attP, attL and attR. Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int. Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm type sites within attP are required for efficient integrative recombination. Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm type sites in attL and attR. These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm type sites.

Resolution of synthetic att-site Holliday structures by the integrase protein of bacteriophage lambda.

Site-specific recombination of the bacteriophage lambda genome into and out of the host bacterial genome is postulated to involve the formation of Holliday structure intermediates by reciprocal single-strand exchanges. Synthetic analogues of the predicted recombination intermediates are resolved in vitro by the protein product of the lambda int gene. Some of the structural features and reaction conditions for this genetic recombination can now be defined.

Evidence that a nucleotide sequence, "boxA," is involved in the action of the NusA protein.

We report the isolation of a mutation, boxA1, in the nutR region of the phage lambda genome. The nutR region, located downstream of the pR promoter, includes the site nutR where the lambda N protein is thought to act to render subsequent transcription termination-resistant. We have previously suggested that the boxA sequence, 5'CGCTCTTA3' (or its RNA analog), located 8 bp promoter-proximal to nutR, might be the recognition site for the E. coli host factor, NusA, which has been shown to be necessary for N action. The boxA1 mutation, an A:T to T:A transversion, results in a changed boxA sequence upstream of nutR, CGCTCTTT. This change is necessary for lambda to effectively use the NusA of Salmonella typhimurium, a NusA function not normally active with the N product of lambda. Other lambdoid phages with unique N functions and nut sites that are normally active with the NusA of Salmonella have boxA sequences with the terminal three Ts. Moreover, sequences closely resembling boxA have been found near transcription termination sequences in E. coli operons where NusA has been shown to be involved in termination. These findings identify boxA as an important recognition signal for the NusA protein.

Molecular structures of packageable ColE1 hybrids and the generation of deletion mutants from one of the hybrids.

ColE1 derivatives carrying cohesive end sites of lambda phage genome (= cos lambda) can be packaged within lambda phage particles. The DNA structure of the prototype ColE1-cos lambda derivative named pKY2257 was studied because of its potential usefulness in various fields in molecular biology. pKY2257, which carries an intact galactose operon of E. coli, is a convenient replicon to detect Tn3 translocation. It was found that one of the PK2257::Tn3 derivatives, pKY2113, generated various small plasmids in E. coli. The molecular structures of some of these deletion mutants were compared with each other and with those of parental plasmid DNAs by heteroduplex analysis and restriction enzyme digestion. A possible mechanism, which seems to be unique to this kind of deletions, is discussed on the basis of the present results.