Bacterial strains, phage and plasmids
LM2489 is a rough derivative of P. syringae pv. phaseolicola HB10Y (HB)[1] and was used as the primary host for plating Φ2954, Φ12 and Φ6.
Plasmid pLM1454 is a derivative of the cloning vector pT7T3 19U (GenBank: U13870.1). It was used for the cloning of cDNA copies of phage DNA produced by RTPCR.
Media
The media used were LC and M8 {Sinclair, 1976 #80}. Ampicillin plates contained 200 mg of ampicillin per ml in LC agar.
Enzymes and Chemicals
All restriction enzymes, T4 DNA ligase, T4 DNA polymerase, T4 polynucleotide kinase, Klenow enzyme, and Exonuclease BAL-31 were purchased from Promega, New England Biolabs and Boehringer Gmbh, Mannheim.
Preparation of pure virions of Φ2954
Bacteriophage Φ2954 was harvested from soft LB agar plates. The soft agar was spun at 7000 rpm for 10 minutes at 4°C. 0.5 M NaCl and 10% PEG-6000 was added the supernatant liquid to precipitate the phage. The suspension was centrifuged; the pellet was resuspended in 0.5 ml of buffer B overnight at 4°C. Buffer B is composed of 10 mM KHPO4, 1 mM MgCl2 and 200 mM NaCl, pH 7.5.
The resuspended Φ2954 was then spun at 28,000 rpm for 70 minutes in a zone gradient of 10-30% Renocal in 200 mM Tris-HCl pH8, 200 mM NaCl, 1 mM MgCl2. The phage band was isolated and treated with PEG to precipitate the virions. The pellet was resuspended in 30 μl of the Tris buffer and extracted with phenol, ethanol precipitated and resuspended in 5 μl of DNA buffer.
Preparation of cDNA. PolyA tailing
The RNA was denatured by boiling for 5 minutes and rapidly cooling with dry ice/ethanol. 5× polyA-polymerase buffer was added to the RNA along with ATP and yeast polyA polymerase (Amersham). The mixture was incubated at 30°C for 1 minute and transferred to ice and the reaction stopped with EDTA. The polyA-RNA was then extracted with phenol/chloroform and precipitated and resuspended in water.
First strand synthesis
1 μl of phosphorylated oligo dT was added to 10 μl of polyA-RNA. After 5 minutes at 70°C the sample was cooled on ice for 5 minutes. Then 4 μl of 5× first strand buffer, 3 μl H2O, 40 u RNase inhibitor (RNasin) and 30 u AMV reverse transcriptase was added and incubated at 42°C for 1 hour. All products needed for the first and second strand synthesis were provided by the Promega cDNA kit (Universal Riboclone cDNA Synthesis System). The reaction products were stored at -70°C overnight.
Second strand synthesis
After thawing the reverse transcribed RNA, 40 μl 2.5 × second strand buffer, 37.6 μl H2O, 0.8 u RNaseH and 23 u E. coli DNA polymerase I was added. After the second strand synthesis proceeded for 3 hours at 16°C, the E. coli DNA polymerase I was inactivated at 70°C for 10 minutes. Then T4 DNA polymerase was added for 10 minutes at 37°C to blunt the ends of the cDNA. The sample was then treated with phenol/chloroform, ethanol precipitated and resuspended in 2.5 μl H2O.
Preparation of the vector used for cloning
pLM1454 was cut with HincII, dephosphorylated with shrimp alkaline phosphatase and then purified by electrophoresis, electroeluted, precipitated and resuspended in 20 μl TE buffer. The ligation mixture was composed of 2.5 μl Φ2954 cDNA, 0.5 μl vector, 0.5 μl 10 × ligation buffer, 0.5 μl 10 mM ATP and 2.5 u T4 DNA ligase. All products are provided by the Promega cDNA kit. Incubation was overnight at16°C. The ligation mixture was used to transform super competent Epicurean E. coli (Stratagene). The cells were resuspended in 100 μl SOC medium and plated out on LC plates with 40 μg/ml X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) and 200 μg/ml Ampicillin. White colonies were picked and small DNA preparations were made. The plasmids were cut with restriction enzyme PvuII and promising candidates were sequenced first with M13 primers and then with oligonucleotides prepared on the basis of the sequence found. At the point where it seemed that the ends of the segments were identified, we prepared cDNA copies by using RTPCR with oligonucleotides having sequences found in the first copies found. Sequencing was done at the New Jersey Medical School Sequencing Facility. The sequences of segments L, M and S were deposited in GenBank with respective accession numbers of [GenBank: FJ608823, FJ608824 and FJ608825].
Preparation of complete cDNA plasmids
The cDNA pieces were assembled to form complete copies of the three genomic segments. In many cases, the connections could be made by using unique restriction sites made evident by the sequencing project. The ends of segments were prepared by using oligonucleotides with convenient restriction sites as primers for PCR reactions. Five plasmids were prepared, pLM3496, pLM3497, pLM3697, pLM3698 and pLM3691. They contain exact complete copies of genomic segments S and M in plasmid pT7T3 19U and three variants of segment L sequence. The sequences start at the first nucleotide of the SP6 RNA polymerase transcript.
In vitro transcription with nucleocapsids
Nucleocapsids of Φ2954 were prepared from purified virions stripped of their lipid-containing membranes by treatment with two percent Triton X-100 [17]. Transcription was performed in magnesium buffers [18, 19]. Labeling was with α-32P-UTP and products were analyzed by electrophoresis in agarose gels.