LYSOGENY BY MS2 PHAGE. ANALYSIS OF A RECOMBINANT PLASMID CONTAINING MS2 RNA-LIKE SEQUENCE

Introduction. Dispite of RNA-containing phages being among the best studied biosystems, some of their properties, especially phage-host inter­ actions, have not been completely investigated. One of the problems is a cause of multiple resistant cells development in E. coli cultures infected by RNA-containing phages in the conditions not favorable for quick cell division. In some previous papers concerning RNA-containing phages bio­ logy this phenomenon has been discussed from the point of view of pos­ sible preexisting mutants selection. Nevertheless, such an interpretation does not seem to be completely convincing because of large scale phage resistance development. In our previous paper [1] we have shown that more than 1 % of infected bacterium offspring receives MS2-resistance marker which may not be due to pre-existing mutants selection, We interpret such a fact as a non-direct proof of direct interactions bet­ ween phage genome and cell host DNA; out interpretation has been con­ firmed by non-stability of primary MS2-induced mutations and derivative forms segregation from these ones [2]. In order to prove that these mu­ tants properties are caused not by a persistent MS2 infection but due to phage genome integration into the host cell chromosome we have created a genetic library of EcoRI fragments of a segregant obtained by us — a lysing E. coli AB 259 Hfr 3000 mutant [3]. A total RNA preparation iso­ lated from plasmid-forming cells has been hybridised both with pMS27 DNA containing MS2 phage cDNA and with a part of this cDNA without vector sequence. The results obtained prove phage specific RNA to be transcripted from chromosomal DNA of the MS2-induced E. coli mutant; so we have really demonstrated that MS2 phage is able to establish true lysogeny as a state based on physical integration of phage and cellular DNA's. In the present paper we present our first data on our study of cloned host cell DNA fragment having the goal to determine how large is MS2-specific region and which is its localisation on the genetic map of our recombinant plasmid; we aim also to find there some recognition sites for several popular restrictive enzymes.

Introduction.Dispite of RNA-containing phages being among the best studied biosystems, some of their properties, especially phage-host inter actions, have not been completely investigated.One of the problems is a cause of multiple resistant cells development in E. coli cultures infected by RNA-containing phages in the conditions not favorable for quick cell division.In some previous papers concerning RNA-containing phages bio logy this phenomenon has been discussed from the point of view of pos sible preexisting mutants selection.Nevertheless, such an interpretation does not seem to be completely convincing because of large scale phage resistance development.In our previous paper [1] we have shown that more than 1 % of infected bacterium offspring receives MS2-resistance marker which may not be due to pre-existing mutants selection, We interpret such a fact as a non-direct proof of direct interactions bet ween phage genome and cell host DNA; out interpretation has been con firmed by non-stability of primary MS2-induced mutations and derivative forms segregation from these ones [2].In order to prove that these mu tants properties are caused not by a persistent MS2 infection but due to phage genome integration into the host cell chromosome we have created a genetic library of EcoRI fragments of a segregant obtained by us -a lysing E. coli AB 259 Hfr 3000 mutant [3].A total RNA preparation iso lated from plasmid-forming cells has been hybridised both with pMS27 DNA containing MS2 phage cDNA and with a part of this cDNA without vector sequence.The results obtained prove phage specific RNA to be transcripted from chromosomal DNA of the MS2-induced E. coli mutant; so we have really demonstrated that MS2 phage is able to establish true lysogeny as a state based on physical integration of phage and cellular DNA's.In the present paper we present our first data on our study of cloned host cell DNA fragment having the goal to determine how large is MS2-specific region and which is its localisation on the genetic map of our recombinant plasmid; we aim also to find there some recognition sites for several popular restrictive enzymes.
Materials and methods.P 1 a s m і d s.Recombinant plasmid pL34 has been constructed by us using non-replicative Лр-fragment of pCVl6 plas mid and described in the previous paper [3] as pL26 plasmid.A strain containing pCV16 plasmid [4}' has been received from the Institute of Biochemistry and Physiology of Micro-organisms (Russian Academy of Sciences, Pushtchino-na-Okie, Moscow district, Russian Federation).A strain carrying pMS27 plasmid [5,6] is a friendly present of Dr R. Devos (Gent University, Belgium).
Nutrient media.We have mostly used 0.6% and 1.2% ami no peptide (AP) containing agar and AP-containing broth.Plasmid iso lation, restriction analysis, and blot-hybridisation have been perfected according to [7]; physical mapping of plasmid DNA sequences has been made with mutual and single digestion approaches [8].
Results and discussion.To determine the size of a recombinant DNA plasmid fragment carrying MS2-\\ke sequence and its localisation in the f 2 3  recombinant plasmid we have used physical mapping and blot-hybridi sation.Our probes were both the whole pMS27 plasmid containing MS2 cDNA and a fragment of this DNA copy without vector sequence.The results of an experiment with EcoRI and PstI treatment of pL34 and pCV16 plasmids are shown in the Fig. 1, a.
It is clear from the Fig. \,b that the labelled pMS27 having been used as a probe gives hybridisation with all the fragments of both plas mids (pCV16 and pL34).It may be due to pCV16 origin, i. e. to the pre sence of some homologies with pBR322 sequences in the vector part of pMS27.In the next blot-hybridisation experiment (see Fig. l,c) where cDNA copies of phage MS2 RNA have been used hybridisation lines cor respond to pCV16 fragments of the size 5.1 kbs and to pL34 fragmentsas large as 5.1 and 4.8 kbs in single and double treatments.The smaller pCVJ6 fragments (their sizes are about 1.1 and 0.7 kbs) corresponding to Ар-fragment of the total size 1.8 kbs do not hybridise with pMS27 fragment, i. e. a region of the recombinant plasmid able to hybridise with pMS27 fragment is not a part of pCV16 plasmid.Our data obtained in the experiments of recombinant and vector plas mids hybridisation with pMS27 plasmid carrying MS2 cDNA and with this cDNA copy permit us to conclude that the sequence homoloo-ical to MS2 RNA is situated in the large pL34 fragment (5.1 kbs) limited by Pstl sites.The hybridisation observed with pCV16 DNA is thought to be most probably due to random short sequences, pCV16 DNA beino-nonhybrisidable both with MS2 RNA and also total RNA isolated from a strain containing a recombinant plasmid and being able to hybridise both with labelled pMS27 plasmid and with its fragment [3] These data sug gest that on the contrary to recombinant plasmid pCV16 plasmid con-tains no sequence coding MS2-like RNA synthesis and forming hybrids with such RNA.So the hybridisation observed in pCV16 experiments may be explained by a presence of a random sequence or of a sequence with the biological function analogue to lysogenic phage integration site in host cell chromosome.Electrophoregrams of the recombinant plasmid af ter PstI, BamHI, Hindlll, and EcoRI restrictive digests as well as blothybridisation of fragments obtained with pMS27 fragment are demonstra ted in the Fig. 2.
Our results lead to the conclusion that ЛІ52-1іке sequence in most probably localised inside the fragment as large as 3.9 kbs limited by PstI and BamHI cleavage sites.The localisation of this fragment is shown on the recombinant plasmid map given in the Fig. 3.
The undoubtful hybridisation of this fragment with a fragment of MS2 cDNA fragment and almost total coincidence of its size (3.9 kbs) with MS2 RNA size (3.569kbs) [9] permit to think that MS2-like sequence integrated into pL34 recombinant plasmid is localised inside this fragment.It should be noted, however, that this region contains no EcoRI and Sail sites detected inside MS2 cDNA [10].A Sail site situated inside of Pstl-BamHI fragment (see Fig. 4) is localised not far from one of its termini corresponding in its localisation to none of two Sail sites detected inside DNA copy of the phage RNA Fig. 4. Electrophoregram of pL34 plasmid with corresponding lanes: l -pL34 DNA+Pstl+BamHI; 2 -pL34 DN A+Pstl+ +BamHI+PvuII; 3 -pL34 DNA+Pstl+BamHI+BgUI; 4 -pL34 DNA+Pstl+BamHI+Sall; 5 -lambda phage DNA-f +EcoRI+HindIII (marker lane) [10].So the region of recombinant plasmid being correspondent to MS2 sequence according to hybridisation experiments is probably a fragment of the structure more complex than a double-stranded one.Such a con clusion is not contradictory to our previous results demonstrating that labelled recombinant plasmid DNA fails to hybridise with MS2 RNA [3].The absence of recombinant plasmid DNA and MS2 RNA hybridisation accompanied by MS2-like RNA synthesis in plasmid-containing cells may be most probably explained by a hypothesis that this plasmid contains a plus-chain of MS2 RNA-like DNA or even MS2 RNA and has a threestranded structure in the fragment of the size about 3.9 kbs.Our point of view is the last version is also probable because of being able to ex plain our negative results obtained when we have attempted to detect some restrictive sites inside pL34 plasmid Pstl-BamHI fragment with si ze of 3.9 kb, these ones being undoubtfully present in the DNA copy of MS2 RNA.