Cos Site Research Articles

A portable DNA sequence carrying the cohesive site ( cos) of bacteriophage λ and the mob (mobilization) region of the broad-host-range plasmid RK2: a module for the construction of new cosmidst

A Polylinker DNA sequence carrying the cos site of bacteriophage λ and the mob ( oriT) region of the IncP group plasmid RK2 was constructed. This composite Polylinker has EcoRI sites at both termini and also unique sites for ClaI, HindIII, PstI and XbaI. The cos-mob region is portable with the use of EcoRI or a combination of EcoRI with ClaI, HindIII or XbaI. Another cos-mob cassette was also constructed from which the cos-mob region can be lifted with HindIII, ClaI or either of these enzymes in combination with others. These cos-mob cassettes can be used in constructing new cosmids that can be mobilized into a variety of Gram-negative bacteria. Using one of these cassettes we have constructed a small IncW group cosmid (11.1 kb) that was mobilizable into Escherichia coli, Rhizobium spp. and Alcaligenes eutrophus at high frequency.

Nucleotide sequence and properties of the cohesive DNA termini from bacteriophage HP1 c1 of Haemophilus influenzae Rd

The termini of the mature DNA of phage HP1 c1 of Haemophilus influenzae Rd have been characterized by DNA ligation, nucleotide sequencing, and deoxynucleotide incorporation experiments. A hybrid plasmid containing the joined phage termini (the cos site) inserted into pBR322 has been constructed. The phage DNA has cohesive termini composed of complementary 5 ' single-stranded extensions which are seven residues long. The left cohesive terminal extension consists only of pyrimidines and the right only of purines. When the ends of the phage are joined, the terminal sequences constitute the central 7 bp of an 11 bp sequence containing only purines on one strand and pyrimidines on the other strand. This oligopyrimidine/oligopurine sequence does not possess rotational symmetry. A 10-bp sequence and its inverted repeat are located approx. 20 bp to the left and right of the fused ends.

Double cos site vectors: simplified cosmid cloning

A new vector for construction of cosmid libraries is described. Cosmid c2XB contains restriction sites for use in the insertion of foreign DNA and two λ cos sites separated by a blunt-end restriction site. The presence of two cos sites on a single plasmid eliminates the need to prepare two separate cosmid arms, and the internal blunt-end restriction site prevents cosmid concatemerization. Thus, a double restriction-enzyme digestion is sufficient to prepare the vector for subsequent ligation with DNA fragments which are dephosphorylated to prevent their self-ligation. The use of this vector system allows efficient cosmid cloning (1 × 10 5 colonies per μg insert DNA) and eliminates background due to vector self-ligation. Furthermore, the procedure is so rapid as to eliminate the need to amplify cosmid libraries for storage and reuse. Also described is a cosmid vector for use in construction of cosmid libraries which are to be introduced into cultured eukaryotic cells. This vector contains the Herpex simplex virus thymidine kinase (HSV tk) gene as a selectable marker and a retroviral long terminal repeat (LTR) region as an enhancer sequence.

Structure and inherent properties of the bacteriophage lambda head shell: IV. Small-head mutants

Missense mutants of bacteriophage lambda that produce small proheads were found among prophage mutants defective in the major head protein gpE. Measurements of the sedimentation coefficient and molecular weight of the small proheads showed that they have the T = 4 structure composed of 240 molecules of gpE instead of the wild-type T = 7 structure composed of 420 molecules of gpE. When the phage mutants were grown in groE mutants of Escherichia coli, they produced small unprocessed proheads, which contained a smaller number (about 60) of the core protein (gpNu3) molecules than normal unprocessed proheads, which contain about 180 molecules of gpNu3. This shows that the major head protein determines the size of not only the shell but also the core of unprocessed proheads. These mutants by themselves produce very few mature small-headed phage particles, partly because the λ, DNA molecule, whose cos sites are separated at a distance of 48,500 bases, is too long to be packaged into the small proheads. However, the small proheads can package shorter DNA in vivo and in vitro at somewhat reduced efficiency, if the length or a multiple of the length between the cos sites of the DNA is 13,000 to 19,000 bases.

A third defective lambdoid prophage of Escherichia coli K12 defined by the λ derivative, λ qin111

We describe the isolation and characterization of a new Q-independent substitution mutant of lambda, lambdaqin111, which differs from other characterized Q-independent lambda phages. This mutant defines a new lambda-like prophage in the bacterial chromosome, as seen by homologous recombination between lambdaqin111 and the host DNA and by DNA/DNA hybridization methods. Genetic and electron microscopy data show that this new prophage carries, at least, genes analogous to Q-S-R of lambda and also a cos site functionally identical to lambda cos. It is located near 34 min on the Escherichia coli K12 map, i.e. in the same region but at a different site from the defective Rac prophage.

Bacteriophage lambda DNA packaging: scanning for the terminal cohesive end site during packaging.

Bacteriophage lambda packages the DNA of the related phage 21 poorly [Hohn, B. (1975) J. Mol. Biol. 98, 93--106]. To understand the nature of the packaging defect, the interaction of the cohesive end site (cos) specific for phage 21 (cos phi 21) with phage lambda terminase has been investigated. The ability of lambda terminase to cleave cos phi 21 was studied in vitro; lambda terminase cleaved cos phi 21 only 1% as well as it cleaved the phage lambda cohesive end site (cos lambda). In vitro packaging experiments showed that the lambda and 21 packaging specificities observed in vivo are also found in vitro. The cos cleavage reaction was modified so that competition experiments could be performed; these experiments showed that cos phi 21 was unable to bind lambda terminase, thus identifying the nature of the defect. Previous work [Feiss, M., Fisher, R. A., Siegele, D. A., Nichols, B. P. & Donelson, J. E. (1979) Virology 92, 56--67] has shown that the base pairs giving lambda or 21 packaging specificity are at the left end of the chromosome, outside the 22-base-pair symmetry region that includes the annealed cohesive ends. Therefore, terminase binding to cos requires interactions with base pairs to the Nu1 side of the cohesive end symmetry segment. The evidence supports the proposition that cos consists of adjacent sites for binding of terminase and for nicking by terminase. Because cos phi 21 can be cut by lambda terminase to terminate DNA packaging, it is proposed that the terminase that binds and nicks at the initial cos site is brought into contact with the terminal cos site by the packaging process. Terminase recognizes and nicks the cohesive end sequence of the terminal cos without requiring the binding site.

Complementation analysis of Agrobacterium tumefaciens Ti plasmid mutations affecting oncogenicity.

A wide host range cosmid vector has been constructed by insertion of the lambda cos site into the plasmid pRK2501. This cosmid, which is maintained in Agrobacterium tumefaciens and is compatible with the Ti plasmid, has been used to make a clone bank of the A. tumefaciens pTiA6 plasmid. Several pTiA6 cosmids have been used to complement Tn5-induced Ti plasmid mutations. Five avirulent mutations which map outside of the region of the plasmid maintained in plant tumours (T-DNA) could be complemented in a trans orientation. Two mutations which are located on a single HpaI restriction fragment outside of the T-DNA, as well as three mutations which map within the T-DNA region, could not be complemented in a trans orientation in a REC- host.

Gene shuttling: moving of cloned DNA into and out of eukaryotic cells.

Successful shuttling of cloned DNA in eukaryotic cells should allow isolation of expressed genes. We tested the utility of cosmids for moving DNA into and out of eukaryotic cells. The unique cleavage of DNA at the cos site by the terminase function of lambda was exploited to maintain the linkage between the vector and inserted gene sequences, a prerequisite for successful rescue of the transforming DNA from high molecular weight DNA of the eukaryotic transformant. A cosmid recombinant containing the HSV thymidine kinase gene and a lambda recombinant containing the chicken thymidine kinase gene were used to test the feasability of this method. It was found that these recombinants can be rescued with high efficiency from DNA of HAT-resistant cells.

Construction and use of SPP1v, a viral cloning vector for Bacillus subtilis

A unique BamHI restriction site has been inserted into a nonessential region of the genome of a deletion mutant of phage SPP1. Construction of this phage, designated SPP1v, required the in vitro conversion of a BclI site to a BamHI site. SPP1v has been used as a vector phage to clone BamHI, BglII and BclI-generated restriction fragments of DNA. A direct selection for recombinants has been developed. Transfection with SPPlv requires intact, genomic-length molecules, and cleavage with BamHI destroys the transfecting ability of this DNA. Recombinants in which the BamHI site has been destroyed by ligation to BglII or BclI-generated fragments of DNA become resistant to BamHI digestion after ligation and are active in transfection. Cloning of DNA containing BamHI sites has been accomplished by using the enzyme Bst15031 to methylate Bam HI sites before insertion, and so to protect them during the BamHI digestion used to select against vector molecules. The in vitro construction of SPPlv generated XmaIII sites directly adjacent to, and on both sides of the inserted BamHI site. This permits precise excision of cloned DNA even when cloning destroys the BamHI insertion site. Restriction-enzyme generated fragments of DNA in the size range of 0 to 4 Md have been cloned, including a full-length copy of plasmid pUB110, almost the complete sequence of plasmid pBR322, and a sequence of DNA that carries the λ cos site.

Protein synthesis induced by infection with packaged λdv plasmid

Plasmid λdv or imm 21dv DNA was joined to a λ arm having a cos site. This recombinant plasmid can be packaged in a λ head, and used to infect Escherichia coli K12 cells. The injected DNA molecules become plasmids in cells. By adding these particles to uv-irradiated uvrA cells, the packageable λdv or imm 21dv plasmids can be induced to synthesize proteins coded by genes on the plasmid genome. The packageable plasmid system is thus suitable for studying on synthesis and regulation of plasmid-coded biopolymers. Analyses of the dv-coded proteins in gel electrophoreses revealed that among several genes carried on the dv plasmid genome, only those genes that are members of the pRoR- tof- cII- O- P operon can be expressed. Evidence has been presented to show that expression of this operon, which is directly correlated with replication of the genome, is only partially allowed in cells perpetuating the dv plasmid. These observations are discussed in connection with the autorepressor model ( D. E. Berg, 1974, Virology 62, 224–233; K. Matsubara, 1976, J. Mol. Biol. 102, 427–439) that genetically accounts for the control mechanism of plasmid replication.

Construction and characterization of a cohesive end site mutant of bacteriophage λ

A cohesive end site mutant ( cos1) of bacteriophage λ has been isolated following in vitro manipulations of λ DNA, including removal of the cohesive ends by digestion with S1 nuclease. The resulting mutation is a 0.4-kilobase deletion which starts at cos, as determined by heteroduplex studies, and extends to the right into the Nu1 gene. Mature λ chromosomes are normally generated by cleavage of two cos sites: Packaging proceeds in an A gene to R gene direction from an initial cos site to a terminal cos site. The cos1 mutation inactivates cos function at both the initial and the terminal cleavages. Aside from the Nu1 defect, expression of all the morphogenesis genes by λ cos1 is normal. Complementation experiments give results expected for a cos mutant: The cos1 mutation results in a cis-specific packaging defect.

A Comprehensive Molecular Map of Bacteriophage LAMBDA

Physical and genetic mapping of deletion mutations has been correlated with the available molecular sizes of the λ gene products and the DNA base sequence to construct a comprehensive molecular map of the phage λ genome. The physical length of the DNA making up the left arm from the cos site through gene J is not sufficient to account in a nonoverlapping manner for all the proteins of the sizes reported to be coded, especially in the Nu1− C region. In the right arm all the coding capacity has not been accounted for, and it appears to be oversaturated only in the gam-ral region. The positions of several IS and Tn elements, and of restriction endonuclease cleavage sites are specified.

Construction and some properties of packageable plasmid F

A derivative of plasmid F which is packageable in lambda phage coat was constructed using techniques of in vitro recombination. This plasmid is composed of three DNA fragments generated by restriction enzyme EcoRI: a miniF fragment (fragment f5 of F'lac) which is able to replicate autonomously, a DNA fragment from Staphylococcus plasmid that carries the beta-lactamase gene, and a portion of guaA (B) transducing lambda phage DNA carrying lambda cohesive ends (cos site) along with almost all the late genes but devoid of all those genes and sites that are needed for replication, regulation, and recombination. The hybrid plasmid has a molecular weight of 2.7 x 10(7) daltons, about 84% size of lambda phage genome, and can be packaged in lambda coat when helper phage replicates in the plasmid-carrier cell. The packaged plasmid and the helper lambda phage particles are separated by CsCl density gradient centrifugation. The replication characteristics of the recombinant plasmid are all those of F including the copy number, incompatibility, and curing with acidine orange. The packaged plasmid is injected into an F- cell and establishes a plasmid state with normal efficiency. In F+ or Hfr cells, the resident F factor hinders this process.

Packaging of the bacteriophage lambda chromosome: Dependence of cos cleavage on chromosome length

Packaging of the bacteriophage lambda chromosome is polarized, proceeding from an initial cohesive end site ( cos) in an A to R direction to a terminal cos site. We have performed helper packaging studies which indicate that the frequency of cleavage of the terminal cos site is dependent on the length of the chromosome being packaged. The helper packaging experiments involved infection of repressed lysogens containing a packagable chromosome by a heteroimmune helper. The prophage at which packaging terminated contained a cos duplication segment, so that failure to terminate packaging by cleavage of the first cos site results in packaging of a cos duplication chromosome. The frequency of packaging of chromosomes with the duplication decreases with increasing chromosome length, leading to the conclusion that cos cleavage depends on chromosome length. This result is most compatible with DNA packaging models in which cos cleavage follows packaging. No dependence of cleavage on the length of the distal duplication segment was found, indicating that extensive one-dimensional diffusion of DNA does not occur during packaging.

Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage λ: “cosmids”

A plasmid which contains a cos site of λ and can be packaged into lambda bacteriophage particles is termed a “cosmid”. Such plasmids can be used as gene cloning vectors in conjunction with an in vitro packaging system. The properties of a new series of cosmids based on the ColE1 replicon are described, including small temperature-sensitive plasmids which have lost mobilisation functions and carry no IS sequences. Amongst these plasmids are vectors for XmaI, BglII, BamHI, HindIII, PstI, KpnI, SalI and EcoRI. It is demonstrated that by using cosmids in particular size ranges these plasmids provide a high efficiency cloning system which yields essentially only hybrid clones without resort to a second selection or screening step, and without prior modification (e.g. phosphatase) treatment of the DNA. Attempts were made to optimise the cloning properties of the cosmid system. An Escherichia coli “gene bank” was obtained with an efficiency of 5·10 5 clones per μg of E. coli DNA, and in which any particular unselected marker may be found in about one out of every 400 clones. It was demonstrated that deletion of mobilisation functions leads to loss of ability to form relaxation-complex without affecting copy number or segregation properties of the temperature-sensitive derivatives. The vectors are amplifiable in chloramphenicol to make up about 50% of the total cellular DNA.

178-Nucleotide sequence surrounding the cos site of bacteriophage lambda DNA.

A nucleotide sequence of 61 nucleotides at the left end and 117 nucleotides at the right end of DNA from bacteriophage lambdacI857Sam7 was determined by the Maxam and Gilbert method. A perfect inverted repeat sequence of 10 nucleotides is near the left end, and one of 15 nucleotides is near the right end. DNA from another closely related lambda strain, lambdacI857prm116Sam7, has about 10% divergence in the sequence of the first 110 nucleotides at the right end and has a 17-member perfect inverted repeat sequence.

Packaging of coliphage lambda DNA: I. The role of the cohesive end site and the gene A protein

The cohesive ends of the DNA of bacteriophage λ particles are normally formed by the action of a nuclease on the cohesive end sites ( cos) of concatemeric λ DNA (reviewed by Hohn et al., 1977). The nuclease also cuts the cos site of an integrated prophage, and DNA located to the right is preferentially packaged into phage particles. This process occurs with approximately the same efficiency and rate in a single lysogen as in a tandem polylysogen. Thus, the rate of cos cutting does not increase when the number of cos sites per molecule increases, an hypothesis that has been proposed to explain why cohesive ends are not formed in circular monomers of λ DNA. We propose instead that the interaction of Ter with cos is influenced by the configuration of the DNA outside of cos during packaging, and that this configuration is different for circular monomers than for other forms of λ DNA. A model that gives rise to such a difference is described. We also found that missense mutations in the λ A gene changed the efficiency of packaging of phage relative to host DNA. This was not the case for missense mutations in several phage genes required for capsid formation. Thus, the product of gene A plays a role in determining packaging specificity, as expected if it is or is part of the nuclease that cuts λ DNA at cos.

Packaging of coliphage lambda DNA: II. The role of the gene D protein

The gene D protein (p D) of coliphage λ is normally an essential component of the virus capsid. It acts during packaging of concatemeric λ DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site ( cos). In this report we show that cos cutting and phage production occur without p D in λ deletion mutants whose DNA content is less than 82% that of λ wild type. D-independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted λ and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of p D; and (2) D-independence cannot be attributed to loss of a specific segment of the phage genome. p D-deficient phage resemble p D-containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept p D. p D appears to act by stabilizing the head against disruption by overfilling with DNA rather than by changing the capacity of the head for DNA. This is shown by the observation that the amount of DNA packaged by a “headful” mechanism, normally in excess of the wild-type chromosome size, is not reduced in the absence of p D. In fact, p D is required for packaging headfuls of DNA. This implies that a mechanism exists for preventing the entry of excess DNA into the head during packaging of concatemers formed by deletion mutants, and we suggest that this is accomplished by binding of cos sites to the head. The above results show that p D is not an essential component of the nuclease that cuts λ concatemers at cos during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of p D. Yet, p D is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to account for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of p D leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. p D promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of cos duplication mutants (Emmons, 1974).

Insertion of DNA carrying ribosomal protein genes of Escherichia coli into Charon vector phages.

DNA fragments from lambdaspc1 and lambdafus2, carrying ribosomal protein genes from Escherichia coli, were inserted into lambda phage vectors Charon 3 and Charon 4. Eight of the resulting clones were characterized by agarose gel electrophoresis of EcoRI digests, analytical CsCl equilibrium centrifugation, and electron micrographic analysis of heteroduplexes. In each case, the identity, order, and orientation of each cloned fragment was determined. In all, 8 of the 12 EcoRI fragments of lambdafus2 were cloned in various arrangements. In the accompanying paper, genes for 15 ribosomal and related proteins and three bacterial promoters were detected in these phages. In addition, four of the hybrid phages carried fragments of lambda-DNA including the phage origin of replication (ori), the late promoter, PR', and the cohesive ends (cos site) in both orientations. The latter phages yield a circularly permuted collection of DNA molecules.

Symmetrical nucleotide sequences in the recognition sites for the ter function of bacteriophages P2, 299 and 186

The 3′-terminal sequences of DNA from the temperate coliphages P2, 299 and 186 have been determined from analyses of radioactive oligonucleotides in DNAase digests of DNA preparations that had been labelled at their 3′ termini. Together with the sequences previously determined for the 5′ cohesive ends of DNA from these phages, the results define a sequence of 33 base-pairs in the region of the ends of these molecules. If these sequences are written with the cohesive ends of the DNA base-paired, they form a duplex region called cos . The cos sequences for the three DNAs are very similar, but not identical, and an axis of 2-fold rotational symmetry can be drawn through their central pair of bases. In each case, two blocks of sequences flanking the paired cohesive ends (i.e. at the 3′ termini of the mature DNA molecule) were symmetrically disposed about this axis, but most of the 19 base-pairs within the cohesive region were non-symmetrical. The symmetrical parts of these sequences may be involved in recognition of the cos site by the phage ter system, which is responsible for formation of the cohesive ends of the mature DNA molecules.