Preparation of single-stranded E1A-oncogene DNA fragments from simian adenovirus SA7 by endonuclease hydrolysis recombinant phage M13 SS-DNA complexed with oligonucleotides, containing restriction sites]

The ss-DNA of the (+) and (-) chains of Ela DNA fragment was obtained by hydrolysis of the recombinant bacteriophages M13 mp8G and mp9G (where G is 1-1750 bp:, E1a region of oncogene SA7) in complexes with the 16 bp oligonucleotides containing AluI and BspRI sites of restriction and sequences complementary to E1a SA7. The obtained fragments overlap the E1a zones associated with the immortalizing potential of SA7.     

Subcloning of DNA fragments of the simian adenovirus SA7 oncogene in bacteriophage M13

The XmaI/PstI and XmaI DNA fragments of adenovirus SA7 oncogene and the adjacent region (16.7% of the physical map of SA7 left end DNA) were recloned in M13 bacteriophages mp8 and mp9 in order to obtain the singlestranded fragments EIa and EIb from the DNA region of monkey adenovirus SA7 located on the recombinant plasmid pASP carrying the DNA APstI fragment including the adenovirus SA7 oncogene.     

Characterization of DNA-protein complexes from simian adenovirus SA7.

DNA-protein complexes prepared from purified simian adenovirus SA7 virions and from lytically infected monkey kidney cells exhibited similar properties when compared with respect to size by sucrose gradient centrifugation, to configuration by electron microscopy, and to susceptibility to a variety of treatments by electron microscopy and electrophoresis in agarose gels.     

Transforming activity and the oncogenicity of simian adenovirus SA7 DNA]

The transforming and oncogenous activity of uncleaved DNA of simian adenovirus SA7 (AdSA7) and the products of its restriction by endonucleases R. Bam HI and R. SalI was studied. It was shown that uncleaved virus DNA transformed the rat kidney cells and rat embryo fibroblasts and induced tumors in newborn hamsters. AdSA7 DNA, hydrolysed by R. Bam HI, posessed the transforming activity. The mixture of DNA fragments, obtained after hydrolysis by R. SalI was oncogenous in hamsters.     

Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami

DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improved by reprogramming the RCA in E. coli.     

Preparation and characteristics of a new transformed cell line G11 by transfection of NIH/3T3 mouse fibroblasts with DNA from the SA7 oncogene of simian adenovirus

Murine fibroblasts NIH 3T3 were transfected with the plasmid pASP containing simian adenovirus oncogene insertion. Focus forming transformants were cloned with a final dilution technique and a new cell line G11 was created as a result. Transformed status of this cell line is evidenced by changes in morphology, specific cytochemical and adhesion properties, ability to grow in semisolid agar and FCS concentration growth independence. Presence of intact integrated E1a-region of adenovirus SA7 oncogene was shown by blot-hybridization technique. Transformed status of G11 cells can be explained by integration of SA7 oncogene, that is evidenced indirectly by the increased resistance to heat shock.     

Production of a cell line of embryonic rat fibroblasts after transfection with the DNA from the early region of simian adenovirus SA7

Transfection of plasmid DNA, containing coding sequences for the early region of simian adenovirus SA7, into primary cultures of the rat embryonic fibroblasts results in the establishment of a stable cell line REF-1. The cells have been passed more than 40 times (about 160 doublings) without reaching the critical stage. Their growth is substrate- and concentration-dependent.     

Insertion of the Tn3 transposon into the genome of the single-stranded DNA phage M13.

The transposable genetic element Tn3, which carries an ampicillin (Ap) resistance determinant, has been translocated from a ColE1-Apr plasmid, RSF2124, to the genome of the filamentous single-stranded DNA phage M13. The site orientation of the inserted element has been determined for one such phage, M13::Tn3-15. The insertion is within the intergenic space separating genes 2 and 4 and containing both the viral strand and complementary strand origins. The lengths of both the filamentous phage and the duplex replicative form (RF) DNA are 1.7--1.8 times those of M13 phage and replicative form DNA. Both plaque formation and transduction of sensitive cells to ampicillin resistance by M13::Tn3-15 are sensitive to purified antibodies to the M13 major coat protein.     

Using iodinated single-stranded M13 probes to facilitate rapid DNA sequence analysis--nucleotide sequence of a mouse lysine tRNA gene.

From a recombinant lambda phage, we have determined a 387 bp sequence containing a mouse lysine tRNA gene. The putative lys tRNA (anticodon UUU) differs from rabbit liver lys tRNA at five positions. The flanking regions of the mouse gene are not generally homologous to published human and Drosophila lys tRNA genes. However, the mouse gene contains a 14 bp region comprising 13 A-T base pairs, 30-44 bp from the 5' end of the coding region. Cognate A-T rich regions are present in human and Drosophila genes. The coding region is flanked by two 11 bp direct repeats, similar to those associated with alu family sequences. The sequence was determined by a "walking" protocol that employs, as a novel feature, iodinated single-stranded M13 probes to identify M13 subclones which contain sequences partially overlapping and contiguous to an initially determined sequence. The probes can also be used to screen lambda phage and in Southern and dot blot experiments.     

New data on the type of structural changes in DNA of simian adenovirus SA7 microinjected into mouse zygotes

The structure of the transgene has been analysed in a new series of experiments on the transfer of adenovirus SA7 DNA into the mice zygotes by microinjection technique. The previous data on SA7 DNA elimination from the genomes of different organs (sceletal muscles, heart, tail) have been confirmed and detailed for the F0 and F1 generations of transgenic animals. The left end of adenoviral genome has been shown to be predominantly transfered after microinjections of SA7 DNA into the mice zygotes.     

A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2.

In this paper we describe the synthesis and cloning of a short segment of DNA complementary to the region immediately adjacent to the EcoRI insertion site in the single-stranded bacteriophage vector M13mp2. This segment is useful as a "universal" primer for DNA sequencing by the dideoxynucleotide chain termination method; the template can be any DNA species cloned in M13mp2 or its derivatives. The primer has been cloned into the tetracycline resistance gene of plasmid pBR322 as one strand of a 26 bp EcoRI/BamHI fragment. This fragment may be readily prepared from an EcoRI + BamHI restriction digest of the parent plasmid (designated pSP14) by a simple size fractionation.     

Specificity of ionizing radiation-induced mutagenesis in the lac region of single-stranded phage M13 mp10 DNA.

M13 mp10 single-stranded phage DNA was irradiated with 60 Co gamma-rays, and transfected into Escherichia coli. One hundred and sixteen mutant clones having lesions in the lac insert were selected, and mutational sites were examined by DNA sequence analysis. Fourteen out of the 15 nucleotide changes thus detected were base substitutions, and the rest was a base addition. Transitions and transversions were almost equal in number. Mutational events were observed at cytosine residues more frequently than at other residues, and the predominant base change was a C ---- T transition. Possible roles in gamma-ray-induced mutagenesis played by the misincorporation of dAMP owing to radiolytic derivatives of cytosine residues and/or formation of apurinic/apyrimidinic sites are discussed.     

Mutagenic specificity of imidazole ring-opened 7-methylpurines in M13mp18 phage DNA

The most abundant lesion formed in DNA upon modification with methylating agents 7-methylguanine, under alkaline conditions is converted into 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua). We have previously shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversions. We have analyzed which lesions lead to these mutations. We compared mutagenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethylsulfate and NaOH after selective elimination of damaged bases from molecules used for transfection into SOS-induced E. coli. Partial elimination of Fapy-7MeGua from phage DNA performed by its digestion with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions. Selective depurination of methylated bases (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde as demonstrated by HPLC analysis of [3H]MNU alkylated phage DNA used as a probe, caused a dramatic, 9-fold decrease of A-->G transitions. Alkali-catalysed rearrangement of 7MeAde was followed by HPLC analysis of [3H]MNU alkylated poly(A) and poly(dA). After incubation of these oligonucleotides in NaOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new peaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy-7MeAde as observed for Fapy-7MeGua. Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde. Fapy-7MeGua was at least an order of magnitude less mutagenic, but in SOS-induced cells it gave rise to G-->T and G-->C transversions.     

A family of hypervariable minisatellites detected by means of a sequence derived from phage M 13

A new family of "hypervariable minisatellites" has been identified in the genome of man and of a variety of animal species using as a probe a DNA segment isolated from the M 13 bacteriophage. This finding provides a new series of hyperpolymorphic genetic markers and renders the "DNA-fingerprinting" methodology available to every molecular biology laboratory.