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PubMed2005

The bacteriophage lambda DNA replication protein P inhibits the oriC DNA- and ATP-binding functions of the DNA replication initiator protein DnaA of Escherichia coli.

Under the condition of expression of lambda P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the lambda P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the lambda P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of lambda P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.

Adenosine Triphosphate↗
PubMed1979

A new host-vector system allowing selection for foreign DNA inserts in bacteriophage lambda gtWES.

An improved vector (lambda gtWES.T5-622) for EcoRI fragments has been derived from EK2 vector lambda gtWES.lambdaB' by replacing the lambda B fragment with two identical 1.1 Md fragments from the pre-early region of bacteriophage T5. The new vector has two advantages which facilitate elimination of parental-type recombinants in an in vitro recombination experiment. Firstly, the 1.1 Md insert is too small to be re-inserted into lambda gtWES in a single copy. Secondly the 1.1 Md T5 fragment carries T5 gene A3 which prevents growth of phage retaining this fragment when the Excherichia coli host carries plasmid ColIb. Thus, essentially all plaques are due to phage with donor DNA inserts and are free of T5 DNA fragments. The size usually given as the theoretical minimum size for insertion into the lambda gt series of vectors is 0.66 Md. We have shown that this size is an underestimate and that the lower limit is about 1.6 Md. A precise estimate is difficult since there is strong selection, among phage having small inserts, for those which have acquired additional genetic material by duplication of the lambda DNA.

Bacteriophage lambda↗
PubMed1981

Degradation of ribosomal RNA in bacteriophage lambda lysogens after thermal induction.

Stable RNA of Escherichia coli was extensively degraded about 40 min after thermal induction of lysogenized lambda cI857 phages at 42 degrees C. When several nuclease-deficient host cells were tested, RNase I activity in the host cells was inferred to be involved in the RNA degradation. Ribosomal structure was detectably altered before the degradation of ribosomal RNA was observed. 30S and 50S subunits began to sediment at 25-28S and 45-58S, respectively, still containing intact RNA. Nonpermissive host cells lysogenized with lambda cI857 susR produced progeny phages in normal burst size after thermal induction and then degraded stable RNA, though they were not lysed. In contrast cells lysogenized with lambda cI857 susS produced ten times more progeny phages under the same condition, but did not degrade stable RNA. These results indicate that the lambda S gene product, which acts as a positive effector of lysis, induced the degradation of stable RNA, presumably by a still uncharacterized effect on the cytoplasmic membrane.

Bacteriophage lambda↗
PubMed2000

Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics.

We describe the construction and characterization of two lambda surface displayed cDNA expression libraries derived from human brain and mouse embryo. cDNA inserts were obtained by tagged random-priming elongation of commercially available cDNA libraries and cloned into a novel lambda vector at the 3' end of the D capsid protein gene, which produced highly complex repertoires (1x10(8) and 2x10(7) phage). These libraries were affinity selected with a monoclonal antibody against the neural specific factor GAP-43 and with polyclonal antibodies that recognize the EMX1 and EMX2 homeoproteins. In both cases rapid identification of specific clones was achieved, which demonstrates the great potential of the lambda display system for generating affinity selectable cDNA libraries from complex genomes.

Amino Acid Sequence↗
PubMed1985

An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda.

We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda. hip mutants are recessive and are located near minute 20 on the linkage map. The gene product is not vital to bacterial growth, since deletion mutants are viable. The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically. Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts. Homologous recombination promoted by recA does not require hip function. In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants. Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes.

Bacterial Proteins↗
PubMed1988

Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage lambda terminase.

The large subunit of phage lambda terminase, gpA, the gene product of the phage A gene, interacts with the small subunit, gpNul, to form functional terminase. Terminase binds to lambda DNA at cosB to form a binary complex. The terminase:DNA complex binds a prohead to form a ternary complex. Ternary complex formation involves an interaction of the prohead with gpA. The amino terminus of gpA contains a functional domain for interaction with gpNul, and the carboxy-terminal 38 amino acids of gpA contain a functional domain for prohead binding. This information about the structure of gpA was obtained through the use of hybrid phages resulting from recombination between lambda and the related phage 21. lambda and 21 encode terminases that are analogous in structural organization and have ca. 60% sequence identity. In spite of these similarities, lambda and 21 terminases differ in specificity for DNA binding, subunit assembly, and prohead binding. A lambda-21 hybrid phage produces a terminase in which one of the subunits is chimeric and had recombinant specificities. In the work reported here; a new hybrid, lambda-21 hybrid 67, is characterized. lambda-21 hybrid 67 is the result of a crossover between lambda and 21 in the large subunit genes, such that the DNA from the left chromosome end is from 21, including cosB phi 21, the 1 gene, and the first 48 codons for the 2 gene. The rest of the hybrid 67 chromosome is lambda DNA, including 593 codons of the A gene. The chimeric gp2/A of hybrid 67 binds gp1 to form functional terminase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda. Protein association and disassociation reactions responsible for localized initiation of replication.

Binding of the O protein of phage lambda to the replication origin (ori lambda) results in the formation of an organized nucleoprotein structure termed the O-some. The O-some serves to localize and initiate a six-protein sequential reaction that provides for localized unwinding of the origin region, the critical prepriming step for precise initiation of DNA replication. By the use of electron microscopy of gold-tagged antibody complexes, we have defined four stages of protein association and dissociation reactions that are involved in the prepriming pathway. First, as defined previously, O protein binds to multiple DNA sites and self-associates to form the O-some. Second, lambda P and host DnaB proteins add to the O-some to generate an O.P.DnaB.ori lambda complex. Addition of the DnaK and DnaJ proteins yields a third stage complex containing DnaK, DnaJ, O, P, and DnaB. With the addition of ATP and single-strand binding protein (SSB), the P protein is largely removed, and the DnaB acts as a helicase to generate locally unwound, SSB-coated single strand DNA. Thus, the initiation of lambda DNA replication requires ordered assembly and partial disassembly of specialized nucleoprotein structures. The disassembly activity of DnaK and DnaJ may be their general role in the heat shock response.

Bacteriophage lambda↗
PubMed1977

Transcription in vitro of bacteriophage lambda 4S RNA: studies on termination and rho protein.

When bacteriophage lambdapga18 DNA is transcribed in a purified in vitro system by E. coli RNA polymerase (nucleoside triphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6), several major transcripts are synthesized. We have investigated transcriptional termination of one of these transcripts, the 4S, or "oop" RNA. Analysis by two-dimensional "fingerprinting" of T1 oligonucleotides reveals that transcription of the 4S RNA terminates at a specific site on the lambdapga18 DNA template, t-L with an efficiency of approximately 80%, i.e. 20% of transcripts are extended into larger RNAs. Addition of the E. coli protein rho to our transcription reactions has two effects: a) the efficiency of termination at the t-L site is increased to 100%; b) the number of 4S transcripts synthesized is increased by greater than 5-fold. Rho appears to stimulate 4S RNA synthesis by facilitating more rapid release of RNA polymerase from the t-L' termination site.

Binding Sites↗
PubMed1983

The SOS system of Escherichia coli in the regulation of bacteriophage lambda development.

A sequence homologous to the known SOS boxes is found in the Po promoter of phage lambda. It is suggested that the sequence found is a binding site for the LexA repressor. The mechanism of the LexA part in regulation of lambda development is discussed. It is based on the competitive transcription of the RNA encoding CII protein and the short OOP-RNA transcribed from the Po promoter.

Amino Acid Sequence↗
PubMed1984

Evidence that the cro repressor inhibits expression of the bacteriophage lambda P gene at high multiplicities of infection.

The activity of the lambda P gene product at various multiplicities of infection (m.o.i.) was examined in CI- conditions using an assay which measures the disappearance of the rapidly-sedimenting closed-circular (c.c.) form of phage DNA. When cells were infected with lambda CI857 at multiplicities of 5 phage/cell or less, between 65%-75% c.c. DNA was lost during incubation. If the multiplicity of infection was increased to 10 phage/cell or greater, a marked inhibition in the cleavage of c.c. DNA was observed. When bacteria were infected with either lambda CI857CII2002 or lambda CI857cro27 at low m.o.i., the usual 65%-75% decrease in the percentage of c.c. phage DNA occurred during incubation. In contrast, no losses in c.c. DNA were noted after infection with lambda CI857cro27susP3. At high m.o.i., the cleavage of c.c. DNA was inhibited after infection with lambda CI857CII2002, but not after infection with lambda CI857cro27. It is concluded that at high m.o.i. in CI- infections, the expression of gene P is unaffected by the CII gene product, but is inhibited by the increased intracellular levels of cro protein.

Bacteriophage lambda↗
PubMed1985

Conservation of genome form but not sequence in the transcription antitermination determinants of bacteriophages lambda, phi 21 and P22.

Comparisons are made among DNA sequences upstream from terminators in both leftwards and rightwards early operons of related coliphages lambda, phi 21 and P22. These sequences include both left and right determinants of response to phage-coded antitermination proteins, "N", as well as the N structural genes themselves. Despite almost total disparity of DNA sequence, the three genomes can be discerned to include the same elements in the same order and spacing: downstream from the early left promoter are sequentially a site of recognition for host nusA protein, a dyad symmetry "nut" essential for N function in lambda, overlapping sites for processing of the transcript by RNAase III and then the N structural genes; downstream from the cro gene on the right are sites of nusA recognition and nut dyad symmetries homologous to those on the left. Because the N proteins of lambda, phi 21 and P22 do not for the most part complement each other, a specific site of N recognition has been postulated for each N-responding operon. The nut dyad symmetry qualifies as such a site, since the loop of the left dyad in lambda is marked by mutations that block N function leftwards, and since DNA sequences here show close homology between the loops of left and right dyads for each phage, but less if not little homology for different phages.

Adult↗
PubMedDate not supplied

Cloning and expression in Escherichia coli of the TL-DNA gene 4 of Agrobacterium tumefaciens under the control of the PR promoter of bacteriophage lambda.

A plasmid was constructed that directs expression of the TL-DNA gene 4 protein in E. coli. The different steps of the construction were as follows: i) a region of gene 4 encoding the amino-terminal portion of the protein was fused in frame to DNA encoding an enzymatically active carboxy-terminal fragment of beta-galactosidase. The hybrid gene was poorly expressed from the upstream lambda PL promoter carried by the vector. ii) in order to generate an efficient procaryotic ribosome binding site, a DNA fragment carrying the lambda PR promoter with the nearby Shine-Dalgarno (SD) sequence of gene cro was placed in front of the gene 4-lacZ fusion. A recombinant plasmid, termed pGV793, that expressed efficiently a fused protein 4-beta-galactosidase was identified among the Lac+ clones. DNA sequencing analysis showed that pGV793 carried a hybrid ribosome binding site composed of the cro SD sequence, a five bp sequence and the ATG codon of gene 4. Plasmid pGV793 directed the synthesis of three polypeptides of molecular weight 132 Kd, 126 Kd and 122 Kd that carried beta-galactosidase antigenic determinants. The largest polypeptide had the expected size for the hybrid protein. The fusion proteins which accounted for about 0.5% of the total cellular proteins were purified by immunoadsorption using anti-beta-galactosidase antiserum. iii) the complete gene 4 coding sequence was reconstituted, with the lambda PR promoter in place. The resulting pGV822 plasmid expressed a polypeptide whose molecular weight 27 Kd corresponded to the expected size for the gene 4 product. The pI was about 7.

Bacteriophage lambda↗
PubMed1997

Stability of CII is a key element in the cold stress response of bacteriophage lambda infection.

Bacteria are known to adapt to environmental changes such as temperature fluctuations. It was found that temperature affects the lysis-lysogeny decision of lambda such that at body temperature (37 degrees C) the phage can select between the lytic and lysogenic pathways, while at ambient temperature (20 degrees C) the lytic pathway is blocked. This temperature-dependent discriminatory developmental pathway is governed mainly by the phage CII activity as a transcriptional activator. Mutations in cII or point mutations at the pRE promoter lead to an over-1,000-fold increase in mature-phage production at low temperature while mutations in cI cause a smaller increase in phage production. Interference with CII activity can restore lytic growth at low temperature. We found that at low temperature the stability of CII in vivo is greatly increased. It was also found that phage DNA replication is blocked at 20 degrees C but can be restored by supplying O and P in trans. It is proposed that CII hampers transcription of the rightward pR promoter, thus reducing the levels of the lambda O and P proteins, which are necessary for phage DNA replication. Our results implicate CII itself or host proteins affecting CII stability as a "molecular thermometer".

Bacteriophage lambda↗
PubMed1981

Restriction alleviation by bacteriophages lambda and lambda reverse.

Deletion analysis indicated that the phage lambda restriction alleviation gene(s) ral resides between the cIII and N genes. The Ral+ phenotype was expressed only when lambda ral+ carried a modification such that it was resistant to restriction by the host specificity system. Under these conditions, Ral function protected superinfecting unmodified phages from restriction by EcoK or EcoB but not from restriction by EcoP1. Ral-protected phage DNA was not concomitantly K and B modified, but rather received only the modification specified by the system of the restricting host. Possible mechanisms for Ral action are discussed. Of the other lambdoid phages tested, the hybrid phage lambda rev had Ral activity, whereas phi 80vir and one lambda-P22 hybrid did not. The restriction alleviation activity of lambda rev called Lar, may be the same as the activity expressed in sbcA- strains of Escherichia coli, but it was functionally separable from exonuclease VIII activity (the product of the recE gene), which is also expressed in sbcA- strains.

Bacteriophage lambda↗
PubMed1984

Integration of bacteriophages lambda and phi 80 in wild-type Escherichia coli at secondary attachment sites. I. Formation of secondary lysogens.

The family of lambdoid phages displays a varying specificity of integration into the host chromosome. The lambda phage DNA failed to get inserted at the secondary site(s) of the gal operon (frequency less than 2.6 X 10(-8) in the presence of the primary (normal) att site. By contrast, phi 80 and the lambda att80 hybrid (lambda X phi 80) became integrated into wild-type Escherichia coli at at least two secondary att sites of the btuB locus, and the latter near purE and purC as well (frequency 2 X 10(-3)-10(-4). The integration of phi 80 and lambda att80 into btuB occurred with about the same frequency as in cells in which the normal insertion site had been deleted (0.7-4.0 X 10(-6). An analysis of the secondary lysogens with the prophage in btuB showed them to be polylysogens; the additional prophage(s) was found at the primary att site. We also failed to observe the integration into other loci of phi 80 and lambda att80 with the formation of secondary monolysogens (frequency less than 0.0035 at MOI = 10(-3) or 10). It is presumed that these prophages become integrated at secondary att sites only if the primary site is occupied.

Bacteriophage lambda↗
PubMed1983

Regulation of the pR operon of bacteriophage lambda.

The E. coli lambda lysogen, OR1263, carries the fusion pR-cro-tR1-IS2-gal. The gal promoter is deleted and gal expression from pR, in the absence of the lambda antitermination factor N, is blocked by the efficient transcription terminator in IS2. Selection for Gal+ yields strains deleted for the IS2 terminator and various portions of the lambda chromosome. Analysis of these deletions reveals the following: (a) The lambda tR1 terminator is about 50% efficient. (b) In two deletions sequenced, DNA loss occurred as a result of homologous recombination between a 2- or a 4-base pair repeat. (c) By measuring the ability of lambda N product to suppress the polarity of a gal ochre mutation, we demonstrate that the N utilization site in the lambda pR operon lies between tR1 and cro. (d) The level of Cro repressor synthesized by a single copy prophage is sufficient to repress the cI maintenance promoter, prm, but is inadequate to inhibit pR.

Bacteriophage lambda↗
PubMed1993

A programmed translational frameshift is required for the synthesis of a bacteriophage lambda tail assembly protein.

Two proteins, one of 31 kDa and one of 16 kDa, are encoded by a segment of the phage lambda tail gene region that contains two overlapping reading frames, neither of which is long enough to encode the larger protein. We show that the abundant 16-kDa protein (gpG) is encoded by the upstream open reading frame, gene G. The 31-kDa protein, gpG-T, is encoded jointly by gene G and the overlapping downstream T open reading frame. gpG-T is synthesized as the result of a translational frameshift that occurs when a ribosome translating the G gene slips back by one nucleotide at a position six codons from the C terminus of the gene and thereby bypasses the G termination codon to continue on in the T open reading frame. The resulting protein shares 135 residues of N-terminal amino acid sequence with gpG, followed by 144 amino acid residues of unique sequence. The frameshift event occurs with a frequency of approximately 4% at the sequence G GGA AAG, which encodes the dipeptide -Gly-Lys- in both the zero and -1 reading frames. The frameshift frequencies of point mutants in this "slippery sequence" argue that codon-anticodon interactions with both the glycyl and the lysyl-tRNA are important for frameshifting to occur. We find no clear evidence for a pausing mechanism to enhance frameshifting, as is seen in other well-characterized frameshifts. No simple secondary structure has been predicted for the region downstream from the slippery sequence, but this downstream sequence does contribute to the frameshifting rate. Our results together with those of Katsura and Kühl show that the frameshift product, gpG-T, has an essential role in lambda tail assembly, acting prior to tail shaft assembly. The role of gpG in tail assembly is not known. We find that both gpG and the gpG-T are absent from mature virions.

Amino Acid Sequence↗