text: 1 . two functional origins of replication , one from pbr322 and the other from pakijl , 2 . one or more dna segments that confer resistance to at least one antibiotic when transformed into a sensitive host cell , said host cell being susceptible to transformation , cell division , and culture . 3 . one or more restriction endonuclease sites located in portions of the cloning vector non - essential for replication or expression of drug resistance . these are cloning sites for the in vitro insertion of foreign dna . the vectors of the present invention are initially constructed by ligating the origin of replication - containing bglii restriction fragment of pakijl ( molecular size : 4 . 10kb ) to a partially bamhi - digested preparation of pij39 ( molecular size : 6 . 26 kb ). the ligation mix was introduced by transformation into competent cells of escherichia coli k12 w5445 ( feitelson ( 1985 ) j . gen . micro . 131 : 2431 ), and transformants selected by their resistance to ampicillin . forty - eight ampicillin resistant clones are screened by rapid plasmid isolation techniques for plasmids larger than pij39 . four clones contain recombinant plasmids of the desired composition , having a molecular size of 10 . 36kb , the sum of the two reactant plasmids . subsequent restriction mapping with bamhi and hindiii placed the four pcll4 plasmids into three classes . a clone was isolated which contains the plasmid identified as pcll4 whose restriction map appears in fig1 . therefore , three out of the four possible sites and orientations of the ligation of pakijl / bglii to the pij39 / bamhi partial digest are recovered . this plasmid is selected for further analysis and manipulation because of the convenient locations of its restriction sites , and deposited as atcc 67 , 447 . to test whether the pakijl - derived portion of pcll4 would enable the plasmid to function in streptomyces , dna is introduced by transformation into protoplasts of streptomyces lividans , with selection for thiostrepton resistance . surprisingly , there is a high efficiency of transformation of streptomyces lividans using escherichia coli - derived pcll4 . this result proves that the origin of replication isolated from the actinomadura plasmid , pakijl , functions in streptomyces , and provides an experimentally convenient method to further localize the functional origin of replication . as shown in fig1 there are two in vitro deletions made in pcll4 in order to test for non - essential dna . the first ( δ1 ) is a 1 . 99 kb hindiii deletion extending 346 base pairs into pbr322 and 1 . 64 kb into the pakijl sequence . the small pbr322 deletion is not expected to result in any phenotypic effect , as the tetracycline resistance gene already is inactivated and this region is known to contain the tetracycline resistance gene promoter ( widera , et al . ( 1978 ) mol . gen . genet . 163 : 301 ). however , removal of 40 % of the pakij sequence could easily have eliminated the origin of replication , and thus prevented successful transformation of streptomyces lividans . escherichia coli ( atcc 67 , 447 )- purified pcll4 is digested to completion with hindiii , and ligated at low dna concentration to favor circularization of the 8 . 36 kb fragment . the mixture is introduced by transformation into competent escherichia coli cells , ampicillin - resistant colonies selected , and sixteen are screened for plasmids smaller than pcll4 . fifteen out of sixteen clones contain plasmids which have a molecular size of 8 . 36 kb . digestion of one plasmid preparation with hindiii confirms the expected structure . the resulting plasmid , pcll7 , which is shown in fig1 is able to transform streptomyces lividans protoplasts to thiostrepton resistance with high efficiency , and proves that the 1 . 64 kb deleted region from the hindiii site to the bglii site in pakijl is not necessary for replication . in a manner similar to that used to construct and test pcll7 , the second deletion ( δ2 ) is made in vitro to pcll4 by complete digestion with kpni and self circularization . this results in about a 890 bp deletion near the middle of the pakijl sequence , and slightly overlaps with the previously described hindiii deletion in pcll7 . again , fifteen out of sixteen ampicillin - resistant escherichia coli transformants contain plasmids smaller than pcll4 , and have only one site for kpnl . the resulting plasmid , pcll8 , has the expected molecular size of 9 . 4 kb , but is completely unable to transform streptomyces lividans protoplasts to thiostrepton resistance . this result suggests that the small kpni deletion eliminates sequences essential to replication in streptomyces , and narrows down the origin of replication of pakijl to between the hindiii site and overlaps the kpni site . this interpretation is further supported by the insertional inactivation of replication of pcll7 in streptomyces by cloning at the bamhi site removed in pcll8 ( see below ). thus , the origin of replication has been localized to a sequence of less than 2 . 46 kb ( hindiii - bglii ), and possibly as small as 730 bp ( hindiii - kpni ). a highly desirable feature of a cloning vector is the ability to easily distinguish , by a genetic test , clones carrying recombinant dna from those which simply contain intact vector molecules . this is usually done by constructing plasmid vectors containing two drug resistance genes ; cloning into one of the markers inactivates it , but selection can still be applied for the other marker . this is the principle of insertional inactivation , an example of which is found with pbr322 ( bolivar , et al ., supra ). plasmid pij39 has a bamhi thiostrepton resistance fragment which inactivated the plasmid tetracycline resistance gene , but selection can still be exerted in escherichia coli because of the intact ampicillin resistance gene ( bla ). in streptomyces , a very useful gene to employ for insertional inactivation is mel , which codes for tyrosinase . tyrosinase catalyzes the conversion of tyrosine to black melanin pigment . there are three useful cloning sites within the mel fragment : sphi , ssti , and bglii . the function of the gene is destroyed when dna is inserted into it at any of these sites and the transformed colonies can easily be identified by their color . transformants harboring the unaltered vector plasmid containing mel form black colonies when grown on tyrosine - containing regeneration plates because they can convert tyrosine into melanin ; recombinant plasmids produce colonies of white cells . the strategy for simultaneously deleting a bamhi site in pcll7 and introducing the mel gene is shown in fig2 . there are two possible sites of insertion using partial bamhi digests of pcll7 : one at the junction of the tsr ( thiostrepton resistance gene ) fragment and pbr322 , and the other in the pakijl sequence previously suggested to be essential for replication in streptomyces . gel - purified dna from pij702 ( katz , et al . supra ) containing the 1 . 55 kb bcli fragment encoding the mel gene is ligated to partially bamhi - digested pcll7 , and introduced by transformation into a competent escherichia coli . a total of 156 ampicillin - resistant escherichia coli clones are screened for plasmid dna larger than pcll7 ; four have the predicted size of 9 . 91 kb . one clone ( pcll9 ) was deposited as atcc 67 , 449 . both physical ( southern blot hydridization analysis ) and genetic ( retransformation into escherichia coli ) data suggest that the plasmids pcll4 and pcll7 at least partially integrate into the chromosome of streptomyces lividans . these results suggest that the vectors will prove useful for the stable insertion of foreign genes into streptomycetes and other actinomycetales . a portion of the plasmid paverl may also be ligated to pij39 using the procedures set forth herein above to obtain a novel plasmid that may be used as described herein . all of the referenced applications and publications are hereby incorporated by reference .