Showing papers on "Lambda phage published in 1995"

Degradation of sigma 32, the heat shock regulator in Escherichia coli, is governed by HflB.

Abstract: The heat shock response in Escherichia coli is governed by the concentration of the highly unstable sigma factor sigma 32. The essential protein HflB (FtsH), known to control proteolysis of the phage lambda cII protein, also governs sigma 32 degradation: an HflB-depleted strain accumulated sigma 32 and induced the heat shock response, and the half-life of sigma 32 increased by a factor up to 12 in mutants with reduced HflB function and decreased by a factor of 1.8 in a strain overexpressing HflB. The hflB gene is in the ftsJ-hflB operon, one promoter of which is positively regulated by heat shock and sigma 32. The lambda cIII protein, which stabilizes sigma 32 and lambda cII, appears to inhibit the HflB-governed protease. The E. coli HflB protein controls the stability of two master regulators, lambda cII and sigma 32, responsible for the lysis-lysogeny decision of phage lambda and the heat shock response of the host.

Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes

Abstract: Listeria monocytogenes bacteriophages A118, A500 and A511 are members of three distinct phage groups with characteristic host ranges. Their endolysin (ply) genes were cloned and expressed in Escherichia coli as demonstrated by the conferred lytic phenotype when colonies of recombinant cells were overlaid with a lawn of Listeria cells. The nucleotide sequences of the cloned DNA fragments were determined and the individual enzymes (PLY118, 30.8 kDa; PLY500, 33.4 kDa; PLY511, 36.5 kDa) were shown to have varying degrees of homology within their N-terminal or C-terminal domains. Transcriptional analysis revealed them to be 'late' genes with transcription beginning 15-20 min post-infection. The enzymes were overexpressed and partially purified and their individual specificities examined. When applied exogenously, the lysins induced rapid lysis of Listeria strains from all species but generally did not affect other bacteria. Using hydrolysis of purified listerial cell walls, PLY511 was characterized as an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and shows homology in its N-terminal domain to other enzymes of this type. In contrast, PLY118 and PLY500 were shown to represent a new class of cell wall lytic enzymes which cleave between the L-alanine and D-glutamate residues of listerial peptidoglycan; these were designated as L-alanoyl-D-glutamate peptidases. These two enzymes share homology in the N-terminal domain which we propose determines hydrolytic specificity. Highly conserved holin (hol) gene sequences are present upstream of ply118 and ply500. They encode proteins of structural similarity to the product of phage lambda gene S, and are predicted to be membrane proteins which form pores to allow access of the lysins to their peptidoglycan substrates. This arrangement of conserved holin genes with downstream lysin genes among the siphoviral lysis cassettes explains why the cytoplasmic endolysins alone are not lethal, since they require a specific transport function across the cell membrane.

Virus DNA packaging: the strategy used by phage lambda.

Abstract: Phage lambda, like a number of other large DNA bacteriophages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit-length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is co-ordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the lambda DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric lambda DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP-driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN to complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endonuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post-cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNu1, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in lambda has aspects that differ from other lambda DNA transactions. Unlike the site-specific recombination system of lambda, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complex, and unlike the DNA replication system of lambda, the same protein machinery is used for both initiation and translocation during lambda DNA packaging.

bor gene of phage lambda, involved in serum resistance, encodes a widely conserved outer membrane lipoprotein.

Abstract: bor is one of two recently identified genes of phage lambda which are expressed during lysogeny and whose products display homology to bacterial virulence proteins. bor is closely related to the iss locus of plasmid CoIV,I-K94, which promotes bacterial resistance to serum complement killing in vitro and virulence in animals. bor has a similar in vitro effect. We show here that the bor gene product is a lipoprotein located in the Escherichia coli outer membrane. We also find that antigenically related proteins are expressed by lysogens of a number of other lambdoid coliphage, in cells carrying the cloned iss gene, and in several clinical isolates of E. coli. These results demonstrate that bor sequences are widespread and present a starting point for mechanistic analysis of bor-mediated serum resistance.

Rate of translocation of bacteriophage T7 DNA across the membranes of Escherichia coli.

Abstract: Translocation of bacteriophage T7 DNA from the capsid into the cell has been assayed by measuring the time after infection that each GATC site on the phage genome is methylated by cells containing high levels of DNA adenine methylase. Methylation at GATC sites on T7 DNA renders both the infecting genome and any newly synthesized molecules sensitive to the restriction enzyme DpnI. In a normal infection at 30 degrees C, translocation of the T7 genome into the cell takes between 9 and 12 min. In contrast, translocation of the entire phage lambda genome or of a T7 genome ejected from a lambda capsid can be detected within the first minute of infection. Entry of the leading end of the T7 genome occurs by a transcription-independent mechanism that brings both Escherichia coli and T7 promoters into the cell. Further translocation of the genome normally involves transcription by the RNA polymerases of both E. coli and T7; the rates of DNA translocation into the cell when catalyzed by each enzyme are comparable to the estimated rates of transcription of the respective enzymes. A GATC site located between the early E. coli promoters and the coding sequences of the first T7 protein made after infection is not methylated before the protein is synthesized, a result supporting the idea (B. A. Moffatt and F. W. Studier, J. Bacteriol. 170:2095-2105, 1988) that only certain proteins are permitted access to the entering T7 DNA. In the absence of transcription, the genomes of most T7 strains do not completely enter the cell. However, the entire genome of a mutant that lacks bp 3936 to 808 of T7 DNA enters the cell in a transcription-independent process at an average overall rate of 50 bp per s.

Primary structure and functional analysis of the lysis genes of Lactobacillus gasseri bacteriophage phi adh.

Abstract: The lysis genes of the Lactobacillus gasseri bacteriophage phi adh were isolated by complementation of a lambda Sam mutation in Escherichia coli. Nucleotide sequencing of a 1,735-bp DNA fragment revealed two adjacent coding regions of 342 bp (hol) and 951 bp (lys) in the same reading frame which appear to belong to a common transcriptional unit. Proteins corresponding to the predicted gene products, holin (12.9 kDa) and lysin (34.7 kDa), were identified by in vitro and in vivo expression of the cloned genes. The phi adh holin is a membrane-bound protein with structural similarity to lysis proteins of other phage, known to be required for the transit of murein hydrolases through the cytoplasmic membrane. The phi adh lysin shows homology with mureinolytic enzymes encoded by the Lactobacillus bulgaricus phage mv4, the Streptococcus pneumoniae phage Cp-1, Cp-7, and Cp-9, and the Lactococcus lactis phage phi LC3. Significant homology with the N termini of known muramidases suggests that phi adh lysin acts by a similar catalytic mechanism. In E. coli, the phi adh lysin seems to be associated with the total membrane fraction, from which it can be extracted with lauryl sarcosinate. Either one of the phi adh lysis proteins provoked lysis of E. coli when expressed along with holins or lysins of phage lambda or Bacillus subtilis phage phi 29. Concomitant expression of the combined holin and lysin functions of phi adh in E. coli, however, did not result in efficient cell lysis.

Restriction alleviation and modification enhancement by the Rac prophage of Escherichia coli K-12.

Abstract: Bacteriophage lambda encodes an antirestriction function, RaI, which is able to modulate the activity of the Escherichia coli K-12 restriction and modification system, EcoKI. Here we report the characterization of an analogous function, Lar, expressed by E. coli sbcA mutants and the hybrid phage lambda reverse. E. coli sbcA mutants and lambda reverse both express genes of the Rac prophage, and we have located the lar gene immediately downstream of recT in this element. The lar gene has been cloned in an expression plasmid, and a combination of site-directed mutagenesis and labelling of plasmid-encoded proteins has enabled us to identify a number of translational products of lar, the smallest of which is sufficient for restriction alleviation. Lar, like RaI, is able both to alleviate restriction and to enhance modification by EcoKI. Lar, therefore, is functionally similar to RaI and the nucleotide sequences of their genes share 47% identity, indicating a common origin. A comparison of the predicted amino acid sequences of Lar and RaI shows only a 25% identity, but a few short regions do align and may indicate residues important for structure and/or function.

Identification and cloning of the gene encoding penicillin-binding protein 7 of Escherichia coli.

Abstract: Penicillin-binding protein (PBP) 7 of Escherichia coli is a poorly characterized member of the family of enzymes that synthesize and modify the bacterial cell wall. The approximate chromosomal position of the gene encoding this protein was determined by measuring the expression of PBPs during lytic infection of E. coli by each of the 476 miniset members of the Kohara lambda phage genomic library. Phages lambda 363 and lambda 364, encompassing the region from 47.7 to 48 min of the chromosome, overproduced PBP 7. One open reading frame, yohB, was present on both these phages and directed the expression of PBPs 7 and 8. The predicted amino acid sequence of PBP 7 contains the consensus motifs associated with other PBPs and has a potential site near the carboxyl terminus where proteolysis by the OmpT protein could occur, creating an appropriately sized PBP 8. The PBP 7 gene (renamed pbpG) was interrupted by insertion of a kanamycin resistance gene cassette and was moved to the chromosome of E. coli. No obvious growth defects were observed, suggesting that PBP 7 is not essential for growth under normal laboratory conditions.

Functional analysis of the ffh-trmD region of the Escherichia coli chromosome by using reverse genetics.

Abstract: We have analyzed the essentiality or contribution to growth of each of four genes in the Escherichia coli trmD operon (rpsP, 21K, trmD, and rplS) and of the flanking genes ffh and 16K by a reverse genetic method Mutant alleles were constructed in vitro on plasmids and transferred by recombination to the corresponding lambda phage clone (lambda 439) and from the phage clone to the E coli chromosome An ability to obtain recombinants only in cells carrying a complementing plasmid indicated that the mutated gene was essential, while an ability to obtain recombinants in plasmid-free cells indicated nonessentiality In this way, Ffh, the E coli homolog to the 54-kDa protein of the signal recognition particle of mammalian cells, and ribosomal proteins S16 and L19 were shown to be essential for viability A deletion of the second gene, 21K, of the trmD operon reduced the growth rate of the cells fivefold, indicating that the wild-type 21-kDa protein is important for viability A deletion-insertion in the same gene resulted in the accumulation of an assembly intermediate of the 50S ribosomal subunit, as a result of polar effects on the expression of a downstream gene, rplS, which encodes ribosomal protein L19 This finding suggests that L19, previously not considered to be an assembly protein, contributes to the assembly of the 50S ribosomal subunits Strains deleted for the trmD gene, the third gene of the operon, encoding the tRNA (m1G37)methyltransferase (or TrmD) showed a severalfold reduced growth rate Since such a strain grew much slower than a strain lacking the tRNA(m(1)G37) methyltransferase activity because of a point mutation, the TrmD protein might have a second function in the cell Finally, a 16-kDa protein encoded by the gene located downstream of, and convergently transcribed to, the trmD operon was found to be nonessential and not to contribute to growth

Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit.

Abstract: The RecBCD enzyme of Escherichia coli promotes recombination preferentially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity. Employing phage lambda red gam crosses, phage T4 2- survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximately 2 h. It is concluded that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a chi-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that chi sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis- and trans-acting stimulators of RecBC-dependent recombination.

Interaction between the phage HK022 Nun protein and the nut RNA of phage lambda.

Abstract: The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and N bind to the same surface of boxB RNA, their respective complexes interact differently with RNA polymerase, inducing transcription termination or antitermination, respectively.

Enhanced activity of the bacteriophage lambda PL promoter at low temperature.

Abstract: The response of the early phage lambda PL promoter to temperature was investigated. Experiments with lacZ reporter gene fusions demonstrated that the activity of the phage lambda PL promoter is inversely dependent on temperature. The bacterial DNA-binding protein integration host factor (IHF) further enhances lambda PL promoter activity at low temperature, although no apparent changes in the cellular level of IHF protein were observed at the different temperatures. IHF protein binds DNA in vitro more avidly at low temperatures. In vitro transcription assays further revealed that the temperature response of PL is the result of an intrinsic property of the promoter as well as its activation by IHF.

The Escherichia coli heat shock response and bacteriophage λ development

Abstract: The Escherichia coli/bacteriophage lambda genetic interaction system has been used to uncover the existence of various biological machines. The starting point of all these studies was the isolation and characterization of E. coli mutants that blocked lambda growth, and the corresponding lambda compensatory mutations. In this manner, the lambda N-promoted transcriptional anti-termination machine was discovered composed of the NusA/NusB/NusE/NusG host proteins. In addition, the DnaK and GroEL chaperone machines were discovered composed of DnaK/DnaJ/GrpE and GroES/GroEL heat shock proteins. The individual members of the DnaK and GroEL chaperone machines have been conserved throughout evolution in both function and structure. Their biological roles include a direct involvement in lambda DNA replication and morphogenesis, the protection of proteins from aggregation, the disaggregation of various protein aggregates, the manipulation of protein structure and function, as well as the autoregulation of the heat shock response. The evolution of lambda to extensively rely on the status of the heat shock response of E. coli is likely linked to its lytic versus lysogenic choice of lifestyle. The bacteriophage T4 gp31 protein has been purified and shown to substitute for many of GroES' co-chaperonin activities.

Bacteriophage-mediated gene transfer systems capable of transfecting eukaryotic cells

Abstract: Lamboid bacteriophage capable of specifically interacting with and delivering nucleic acid molecules to eukaryotic cells are disclosed. Such bacteriophage-derived gene transfer systems target one or more specific receptors on eukaryotic cells, for instance by incorporating mutant tail fiber proteins or by incorporating known ligands for specific eukaryotic receptors into lambda phage. Also disclosed are methods for identifying and producing modified bacteriophage tail fiber polypeptides capable of specifically interacting with eukaryotic transmembrane proteins. Methods of treating diseases using such gene transfer systems are also disclosed.

Enhanced activity of the bacteriophage λ PL promoter at low temperature

Abstract: We found that the activity of the phage λ PL promoter is inversely dependent on temperature. Both in vivo and in vitro transcription assays revealed that the rather unique temperature response of PL is a sum of an intrinsic property of the promoter and its activation by integration host factor. We also found that at low temperature, phage λ can lysogenize efficiently but cannot complete the lytic cycle. We hypothesize that by sensing the low environmental temperature the PL promoter plays a role in determining the direction of phage λ development.

The old exonuclease of bacteriophage P2.

Abstract: The Old protein of bacteriophage P2 is responsible for interference with the growth of phage lambda and for killing of recBC mutant Escherichia coli. We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties. The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides.

Identification of related genes in phages phi 80 and P22 whose products are inhibitory for phage growth in Escherichia coli IHF mutants.

Abstract: Bacteriophage lambda grows in both IHF+ and IHF- host strains, but the lambdoid phage phi 80 and hybrid phage lambda (QSRrha+)80 fail to grow in IHF- host strains. We have identified a gene, rha, in the phi80 region of the lambda(QSRrha+)80 genome whose product, Rha, inhibits phage growth in an IHF- host. A search of the GenBank database identified a homolog of rha, ORF201, a previously identified gene in phage P22, which similarly inhibits phage growth in IHF- hosts. Both rha and ORF201 contain two possible translation start sites and two IHF binding site consensus sequences flanking the translation start sites. Mutations allowing lambda (QSRrha+)80 and P22 to grow in IHF- hosts map in rha and ORF201, respectively. We present evidence suggesting that, in an IHF+ host, lambda(QSRrha+)80 expresses Rha only late in infection but in an IHF- host the phage expresses Rha at low levels early in infection and at levels higher than those in an IHF+ host late in infection. We suspect that the deregulation of rha expression and, by analogy, ORF201 expression, is responsible for the failure of phi80, lambda(QSRrha+)80, and P22 to grow in IHF mutants.

DNA sequencing from single phage plaques using solid-phase magnetic capture.

Abstract: Many operations encountered in molecular cloning are labor-intensive and time-consuming. One case that is often troublesome is the subcloning of cDNA clones from lambda gt11 phage into plasmid vectors. In situations where several clones have been isolated, time could be saved by a means of assessing insert size and sequence unambiguously without subcloning, particularly where degenerate PCR or low-stringency hybridization approaches are taken to identify multiple members of a gene family. We describe a simple and reliable strategy for efficient sequencing of small amounts of lambda phage DNA, lysates or individual phage plaques. The strategy combines the advantages of universal lambda phage primers, rapid air thermal cycling, streptavidin magnetic bead capture of highly purified single-stranded templates and the unparalleled clarity of T7 DNA polymerase sequence. We routinely obtain 350-500 bases of unambiguous sequence from each reaction. It takes only hours from lifting phage plaques to finishing the sequencing reactions. The method provides an alternative to thermal cycle sequencing that has comparable sensitivity and affords sequence data of much higher clarity.

On the clustered exchanges of the RecBCD pathway operating on phage lambda.

Abstract: Lytic cycle crosses of Red- Gam- phage lambda were conducted in rec+ Escherichia coli carrying one or another plasmid with homology to lambda. Lambda x lambda recombinants and lambda x plasmid recombinants were formed by RecBCD-mediated recombination. We showed previously that the act of recombining with a plasmid alters the disposition of selected lambda x lambda exchanges. This work reports that the relationships between the lambda x plasmid and the lambda x lambda exchanges is unaltered by the removal from one lambda parent of the homology shared with the plasmid. This result supports our view that a reciprocal exchange, allowing for cointegrate formation, is associated with but mechanistically separable from a (presumably) nonreciprocal lambda x lambda exchange. The nature of this relationship is independent of lambda's Rap function, which is shown to alter the ratio of cointegrate formation (splices) to marker pick-up (patches) in lambda x plasmid recombination mediated by the RecBCD pathway.

Fluorescence Detection of DNA Using a Novel Peroxidase Substrate, 4-(4-Hydroxyphenylcarbamoyl)butanoic Acid

Abstract: λ phage DNA on nitrocellulose filters was quantified by a fluorometric assay using a biotin-labeled DNA probe and a p-acetamidophenol analog that yield a fluorescent product by enzymatic oxidation. Biotinylated λ phage DNA (Hind III digested), used as a DNA probe, was prepared by diazobiotin, which reacts at the phosphate moiety of DNA. Peroxidase-labeled avidin was bound to biotin-labeled DNA, which was hybridized with λ DNA on a nitrocellulose filter. The DNA was detected using a fluorogenic substrate for peroxidase, 4-(4-hydroxyphenylcarbamoyl)butanoic acid (HPBA) in the presence of hydrogen peroxide. The assay permits the determination of λ phage DNA as low as 0.1pg by a flow-through system.

Ternary complex formation on leaderless phage mRNA

Abstract: The phage Lambda pRM promoter-derived cI mRNA and phage P2 gene V mRNA are transcribed beginning with the A residue of the AUG start codon. Using lacZ fusion analysis we have assessed the effects of alterations in the immediate downstream coding region on the translational efficiency of these mRNAs. Mutations, including deletions of the putative downstream box of either cI or gene V mRNAs, showed no significant reduction in expression of the different lacZ fusions. Primer extension inhibition analysis suggests a role of ribosomal protein S1 in cI mRNA recognition.

A novel Ti‐plasmid‐convertible λ phage vector system suitable for gene isolation by genetic complementation of Arabidopsis thaliana mutants

Abstract: A new lambda phage vector system, lambda TI, has been constructed to facilitate genetic complementation of higher plant mutations. The lambda TI vectors are stable, and by using the Cre-lox site-specific recombination, are automatically convertible into Ti-plasmid binary vectors which are capable of expressing genes in higher plants. Two lambda TI vectors were constructed: (i) lambda TI1, which can generate a Ti-plasmid that contains the cauliflower mosaic virus (CaMV) 35S promoter and is suitable for the expression of cDNA in transformed plants and (ii) lambda TI2, which can generate a Ti-plasmid with the multicloning site (MCS). cDNA and genomic libraries, which were constructed from the cruciferous plant Arabidopsis thaliana in these lambda TI vectors, can be probed by large DNA fragments of more than 100 kb, such as yeast artificial chromosomes (YACs), enabling the direct screening of the clones in the chromosome region containing a specified genetic locus. These libraries will certainly become powerful tools for the genetic complementation of Arabidopsis mutant phenotypes by quickly providing transformation-competent clones.

An improved method for generating subtracted cDNA libraries using phage lambda vectors

Abstract: We have used a biotin-based subtraction procedure to enrich a potato tuber cDNA library for sink-specific clones. The method uses single-stranded phagemids with directional inserts as both driver and target. We modified the XZAP II vector for the directional cDNA cloning and for subsequent subtractive hybridization. This improved method of subtractive hybridization circumvents many problems facing standard protocols. The present method uses the advantage of X-phage cloning which makes it possible to establish libraries from limited amounts of tissue. In vivo excision of single-stranded phagemids containing the cDNA inserts was performed to provide an unlimited source of DNA for biotinylation, hybridization and subtraction. This ability to produce unlimited quantities of DNA for subtraction is advantageous over other methods which require large amounts of RNA as the starting point (2,7,8). The phage vector system is more convenient than a plasmid/colony system (3,6,9) when the original library is to be re-screened for additional clones. The XZAP II vector was modified by introducing an adaptor (EX1: 5'-AAT 1AT CTC GAG GGC CCG ATC GGC CGA ATT CGT-3' annealed to EX2: 5'-I CGA ACG AAT TCG GCC GAT CGGGCC CTC GAG AT-3'; TIB MolBiol, Germany, Berlin) that destroys the original EcoRl and Xhol sites but contains both restriction sites in inverted orientation flanking a central Sfil site. The resulting vector was designated XPAZ II. Total RNA from sink (growing) and source (sprouting) tubers was isolated according to (4) and poly A RNA was purified by chromatography on oligo-d(T)-cellulose (Pharmacia, Type 7). cDNA libraries were constructed using Uni-ZAP cDNA synthesis system (Stratagene). The cDNAs from sink and source tubers were cloned into XZAP II and XPAZ II, respectively (Fig. 1). Both libraries had a complexity of 3 x 10 p.f.u. with -90% recombinant clones. The average size of the inserts were in the range of 1 kb. From both libraries, single stranded (ss) circular DNA were generated by in vivo excision and single-stranded DNA was isolated (6). Single-stranded DNA from source tubers was biotinylated with 1 |ig/|il long-arm Photoprobe® biotin (Vector Laboratories, Burlingame, UK; 5). Typically, 2.5 |ig ssDNA from the sink tuber library was hybridized to 20-30 (J.g of biotinylated ssDNA from source tuber, in 20 ̂ 10.75 M NaCl, 50 mM HEPES pH 7.6, 10 mM EDTA and 0.1% SDS at 65°C for 20 h under mineral oil (Sigma). Subsequently, the mineral oil was removed and the mixture was incubated with 100 |ig vectrex-avidin (Vector Laboratories) for 5 min at room temperature. Streptavidin-biotin-DNA complexes were precipitated by amsink tuber cDNA library XZAPII (sense) source tuber cDNA library XPAZII (an ti sense)

Knowledge-based simulation of regulatory action in lambda phage

Abstract: We have developed a knowledge-based, discrete-event simulation system to simulate proteins-regulated genetic action in lambda phage. Lambda phage is a kind of virus which infects Escherichia coli (E. Coli). Specifically, we simulate the decision between two developmental pathways, that is, lytic growth and lysogenic growth on such conditions as mutation. The novelty of this work is the employment of two different levels of abstraction in a genetic model for the purpose of achieving greater precision. Our model is composed of a roughly abstracted level for the noncritical parts which constitute most parts of our model, and a precisely abstracted level for the critical parts. In the former level, our model is a discrete-event simulation in qualitative representation on a knowledge-based system. In the latter level, it is based on reaction formulae in quantitative representation. >

Repair of phage lambda DNA damaged by near ultraviolet light plus 8-methoxypsoralen.

Abstract: Treatment of phage λ with 8-methoxypsoralen plus near ultraviolet light (PUVA) and its subsequent infection and growth on various mutant and non-mutant hosts were investigated. A number of Escherichia coli DNA repair-deficient mutants, particularly those deficient in genes producing proteins known to participate in interstrand crosslink repair, were used as hosts to assess the roles of these gene products in the activation of phage affected by PUVA. Results show that puvA, uvrA, uvrD, recA, recO, sulA and recN of E. coli are involved in the repair process. Based on the data presented it is proposed that phage λ DNA is repaired, following PUVA damage, using the recombinational repair process. This may be in agreement with the recombinational model of the repair of E. coli DNA.