PLASMIDS WHICH INCLUDE PROMOTERS FOR BACTERIOCINS ADAPTED FOR EXPRESSION OF FOREIGN POLYPEPTIDES IN ESCHERICHIA COLI

Abstract: A plasmid adapted for expression of a foreign polypeptide which, when introduced into a suitable E. coli cell carrying a temperature-inducible RecA protease gene and grown under appropriate conditions, effects expression of a foreign gene inserted into the plasmid and production of the foreign polypeptide. The plasmid is a double-stranded DNA molecule which includes a 5' to 3' order the folowing: a DNA sequence which includes a temperature-inducible promoter, including its associated ribosomal binding site, which promoter is derived from a bacterial plasmid and which promoter normally directs expression of a bacteriocin selected from the group consisting of colicin and the peptide encoded by the col gene; a DNA sequence which encodes an ATG initiation codon; a DNA sequence which encodes the foreign polypeptide; a DNA sequence that is a gene which facilitates high copy number replication of the plasmid; a DNA sequence that is a gene associated with a selectable or identifiable phenotypic trait manifested when the plasmid is present in the host cell; and a DNA sequence that contains an origin of replication from a bacterial plasmid capable of autonomous replication in the host cell.

Description: PLASMIDS WHICH INCLUDE PROMOTERS FOR BACTERIOCINS ADAPT¬ ED FOR EXPRESSION OF FOREIGN POLYPEPTIDES IN ESCHERICHIA £ΩI_I Background of invention Throughout this application various publications are referenced by number within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this applica¬ tion in order to more fully describe the state of the art to which this invention pertains. Colicin El is one of the simplest and most widely used bacterial plasmids. This plasmid is a closed covalent circular DNA duplex containing 6646 base pairs. Within the genome there is a unique origin for replication, several genes involved in replication and the regulation of replication and a cluster of genes controlled by the col promoter (18, 28). The cea gene (also referred to as the col gene) of this cluster encodes the 56 kilodalton colicin protein, a bacteriocin that is toxic to many bacterial cells that do not contain the plasmid (1). Colicin kills plasmidless cells at very low concentration. E t coli cells containing the Colicin El plasmid are unusually sensitive to DNA damaging reagents such as itomycin C (19, 24, 25 and Zhang, S., Farl, A. and Zubay, G., Mitomycin Induced Lethality of ___ coli Cells Containing the ColEl Plasmid: Involvement of the kil gene. J. Bacter., submitted). As little as 0.4 g/ml of mitomycin C (MTC) will kill greater than ninety percent of the cells after one to two hours. The same cells without plasmid survive this treatment. Cells containing the plasmid are protected from the harmful effects of colicin by products of the plasmid imm gene (e.g., see references 11, 18, 23) now known to be locat¬ ed downstream of the cea gene. At very high levels of colicin even plasmid-containing cells are killed, so it was quite natural to assume that the sensitivity of such cells to MTC was due to the high levels of colicin synthesis that it stimulates. That colicin itself was not responsible for the increased drug sensitivity was first shown by Shafferman, Flashner and Cohen (24) . They observed that deletion within the cea gene induced lethality. However, a deletion which also eliminated the cea gene promoter greatly lowered drug sensitivity. Tnl insertions within the cea gene also lowered drug sensitivity to a comparable extent so that plasmid- containing cells were no more sensitive to MTC than plasmidless cells. Tnl insertions usually have a polar effect blocking downstream transcription of a gene from the point of insertion. The characteristics of the insertion and deletion mutants led to the suggestion that transcription originating from the cea gene pro¬ moter but not the colicin protein itself was essential for drug induced lethality. Derepression of the cea promoter after exposure to MTC was presumed to result in the expression of a gene in the same operon as the cea gene which in turn caused a lethal reaction. This hypothesized gene was named the kil gene. Prior to the proposal of a kil gene it was known that the imm gene, responsible for immunity to exogenous colicin, is located immediately downstream of the cea gene. Cells harboring cea , imm " plasmids usually grow poorly if at all. Inselburg found that such cells showed improved growth in liquid culture if trypsin was added to the growth medium (11) . This observation suggested that the toxic action of colicin was confined to its effect after transport outside the cell since trypsin action could not otherwise be effective. It also demonstrated most clearly that the function of the jjOfll gene was to protect the cell from colicin in the extracellular environment. In view of this function it seemed unlikely that the imm gene could in any way be responsible for drug induced killing, or to put it another way, that the imm gene could be the k L gene. Nevertheless its location imme¬ diately downstream of the cea gene did complicate the search for the JsLL gene and the selection for kil " mutants. An insertion mutation in the imm gene might eliminate drug sensitivity due to the kil gene; at the same time it might lead to lethality because of the loss of the imm gene function. Recognizing this complica¬ tion Suit, et al. (21, 25) attempted to devise assays that would permit discrimination between two forms of lethality, one resulting from insufficient imm gene product and the other resulting from high level expres¬ sion of the kil gene. They found that increased mem¬ brane permeability and the associated transport of colicin could be ascribed to one function (presumably Kil) and that immunity to extracellular colicin could be ascribed to another function (presumably Imm) . Suit, et al. isolated two particularly interesting mutant plasmids containing Tn5 insertions in the imm gene. One of these mutant plasmids gave the Cea , Kil " , Imm phenotype as evidenced by the fact that cells carrying this plasmid were not killed by low levels of MTC even though they made large amounts of colicin when exposed to the drug. The colicin made in such cells was severely inhibited in transport. Even though the in- sert in this plasmid was in the imm gene it did not appear to affect imm gene function. This is possible since it is known that the C-terminal portion of the immunity protein is not required for its function (see reference 18) . The second plasmid mutant gave a Cea + , Kil " , Imm " phenotype. Cells containing it were killed by low levels of MTC. Unlike cells carrying the wild type plasmid however, their membranes do not become highly permeable after MTC treatment and they transport the colicin they make after induction very poorly. The mutant studies of Suit, et al. were helpful in suggest¬ ing certain properties associated with the kil gene. They were confusing in that they did not establish the location of the kil gene. This is because Tn5 inser¬ tions like Tnl insertions (discussed above) tend to be polar, disrupting the gene in which they are located but also inhibiting transcription of downstream genes in the same operon. Thus in any particular mutant the kil gene could be located at the site of Tn5 insertion that gives a kil " phenotype or further downstream from the site of the insertion. Evidence suggesting the location of the kil gene in ColEl comes from studies on related plasmid CloDF13 and sequence comparisons between the two plasmids. The CloDF13 plasmid contains three clustered genes, clo, imm. and ϋ in the same operon (see figure 1 and refer¬ ence 9) . These genes are contranscribed from a promot¬ er adjacent to the clo gene. The clo and the imm genes of CloDF13 are completely different in structure from the cea and the imm genes of ColEl although they serve comparable functions. In each case the first gene, clo or cea, encodes a protein toxin (bacteriocin) whereas the imm gene encodes an immunity factor which protects plasmid-harboring cells from the toxin. The fl gene of CloDF13 encodes a 6 kilodalton protein called protein H (9, 10) . A high concentration of protein H, due either to the drug induced expression of the fl gene or to a gene dosage effect, results in the killing of bacterial cells. The gene for protein H is located immediately downstream of the imm gene in CloDFl3 (see figure 1) . Determination of the precise location of the gene in¬ volved: (1) sequence analysis of the plasmid in that region, (2) comparison of reading frames in that region which could encode a 6 kilodalton protein with the actual amino acid composition of protein H, (3) and finally showing that deletion mutations involving part of the presumptive gene result in a loss of the ability to make protein H and the absence of cell lysis associ¬ ated with exposure to MTC. A strikingly similar sequence to the H protein gene is located on the other side of the imm gene in ColEl (for relevant sequence data see reference 18 and 28) . This gene, believed to be the kil gene, is oriented so that transcription initiated from the cea gene promoter and extending through the kil gene should make productive messenger for the kil gene protein. Related to this Ebina and Nakazawa (7) showed that transcripts initiat¬ ed from the cea gene promoter are of two types; some terminate at Tl, located about 60 base pairs downstream from the cea gene while others proceed an additional 500 base pairs downstream T2 (see figure 1 and reference 20) . The latter transcript includes the presumptive coding sequences for the Kil protein. In view of the described mechanism for activation of the cea gene it should not be surprising to find that cea gene expression could also be encouraged by certain host mutations that lead ' to a depletion of the LexA protein (14) . Compatible with this others have discovered that certain mutants in either the recA gene or the lexA gene, as well as mutations in the colicin gene that will not be discussed here (8, 17), lead to high levels of colicin synthesis. This laboratory has been studying colicin synthesis in £__ coli strains that carry a tem¬ perature inducible RecA protease or a LexA mutation. In both of these strains very high levels of colicin syn¬ thesis- result when the cultures are grown at elevated temperatures. As much as 25% of the membrane free cell mass in such a culture exists as colicin protein. Suprisingly, cell death is undetectable in such cul¬ tures. This, is in contrast to the situation where colicin synthesis is induced in wild type cells by DNA- damaging reagents in which case massive cell death results. Colicin synthesis is induced by temperature induction in the recA or lexA mutant cells. Without replenishing the growth medium, the intracellular colicin level rises for several hours normally reaching a peak at about 5 to 6 hours. After this time the intracellular colicin levels drop precipitously and colicin begins to ' appear in large quantities in the extracellular fluid. This 'secretion 1 of colicin can be eliminated by introduction of a kil mutant in the plasmid. Under these conditions there is no loss of intracellular colicin even after eighteen hours of growth at elevated temperatures. The optimum level of colicin synthesis achievable is also influenced by the plasmid copy number. In plasmid mutants carrying an impaired rom gene the plasmid copy number is approximately doubled and the yield of colicin in derepressed cultures is approximately doubled. If all of the factors favoring colicin synthesis dis- cussed above are combined into one system the intracellular colicin yields approach 35% of the dry cell mass. Optimum conditions should include a growth medium containing no more than 0.1% glucose, a host E. coli cell carrying a temperature inducible RecA protease and a plasmid variant that is rom " . Ml " . Plasmid containing cells are initially grown at 30°C in a growth medium of choice (usually standard L broth with 0.1% glucose) . Sixty minutes prior to a temperature upshift nine volumes of fresh growth medium are added to the growing culture. After a temperature upshift to 41°C growth is continued for 6 to 18 hours and then the cells are collected by centrifugation. The colicin content of the culture is determined on a crude cell lysate after removal of the membrane fraction by brief centrifuga- tion. The cell free lysate is electrophoresed on a 5- 20% acrylamide gradient, the gel is stained with cooma- sie blue, a general protein stain that permits a visual estimation of the relative amounts of different proteins present. A single band of the 56 kilodalton colicin protein dominates the gel pattern. The enormous yields of colicin protein obtainable in this system, the inherent stability of the system under low temperature conditions and the simple mechanism of induction suggests that this system would provide an excellent expression vector for the synthesis of virtu¬ ally any protein whose gene could be inserted in place of the colicin gene. The complete sequence of ColEl is known yielding information about restriction endonuclease recognition sites that permit ready re- placement of the colicin gene by any gene for which comparable sequence information is available. The techniques for gene replacement are standard techniques of DNA recombinant technology. In doing a gene replace- ment for purposes of gene expression it is essential that the newly inserted gene be positioned so that the coding sequence is located at precisely the same dis¬ tance from the ribosome recognition site (Shine-Daigarno sequence) as it is for colicin. Otherwise the protein yields would be substantially reduced. Whereas this work has been focused on the advantages of using the colicin promoter of ColEl, related plasmids that encode different bacteriocins such as CloDF13 should possess equally effective promoters. Based on the usual conditions of colicin induction in nature and the function of this gene product it is clear that this type of expression vector is virtually unique. Its sole function is to produce optimal levels of a single protein without any concern for the survival of the producing cell. As far as is known this is a virtually unique phenomenon wherein most of the energy of the cell can be focused on the synthesis of a single protein. It comes close to rivaling the vertebrate erythrocyte's capacity to focus synthesis on the single protein hemo¬ globin. Conditions have been devised under which any given gene located in the colicin gene reading frame can be main- tained in a highly repressed state or induced simply by changing the growing temperature. This is a system with enormous industrial potential. Sum ary of the Invention The invention concerns a double-stranded DNA plasmid adapted for expression of a foreign polypeptide in a suitable ___ coli cell carrying a temperature inducible RecA protease gene comprising in 5' to 3' order: a DNA sequence which includes a temperature inducible promoter, including its associated ribosomal binding site, which promoter is derived from a bacterial plasmid and which promoter normally directs, expression of a bacteriocin selected from the group consisting of colicin and the peptide encoded by the col gene; a DNA sequence which encodes an ATG initiation codon; a DNA sequence which encodes the foreign polypeptide; a DNA sequence that is a gene which facilitates high copy number replication of the plasmid; a DNA sequence that is a gene associated with a selectable or identifiable phenotypic trait manifested when the plasmid is present in the host cell; and a DNA sequence that contains an origin of replication from a bacterial plasmid capable of autonomous replica¬ tion in the host cell. The plasmid of this invention may be introduced into a suitable JL * . colj host cell carrying a temperature-induc¬ ible RecA protease gene, preferably the mutant tif gene. The foreign gene, the expression of which is controlled by a temperature-inducible promoter, may be expressed under suitable conditions. Presently preferred foreign genes are those for the interferons, growth hormones and insulins, and those encoding various • hybrid polypeptides, i.e. a hybrid polypeptide encoded by a DNA sequence comprised of most of the beta-galactosidase gene with a small fragment of the chloramphenicol transacetylase gene. Preferred temperature-inducible promoters are those which control the expression of bacteriocins, such as the col promoter of the plasmid ColEl or the clo of the plasmid CloDF13. Additionally, the plasmid contains a gene which facilitates high copy number replication, preferably the mutant rom gene. Host cells containing the plasmid of this invention may be used to produce foreign polypeptides by growing the cells under suitable conditions at a temperature suffi- cient to induce enhanced expression of the polypeptide, and recovering the polypeptide so produced. Brief Description of the Figures Figure 1. Genetic regions in colicin DF13 and colicin El that contain the bacteriocin genes and other nearby genes in the same operon. . Transcripts are indicated by wavy lines with termination points at the head of the arrow. In colicin DF13, transcription of all three genes, clo, imm and fl is initiated from a promoter on the left (not shown) . This transcript terminates at T- ^ which is located between the imm and the fl gene or at T 2 which is located after the fl gene. In colicin El, transcription also .initiates from a promoter on the left (not shown) . This transcript terminates about 50 bps downstream from the cea gene at , or about 500 bps further downstream after the kil gene at T 2 in this case transcription of the jmm gene initiates from an imm specific promoter in the opposite direction. The ColEl plasmid contains 6646 base pairs. The tran¬ scription and translation start and stop sites for the three genes are given: cea gene: transcription start (5, 13), 5065; transcription stop (5) , 115 (T^) and 613 (T 2 ) ; translation start (19), 5140; translation stop (19), 60. _____ gene: transcription start (5) , same as cea gene; transcription stop (5), same as T~; translation start (inferred by compari¬ son of references 6 and 11) , 448; transla¬ tion stop (inferred by comparisons of refer- ences 6 and 11), 583. imm gene: transcription start (13), 409; transcription stop unknown; translation start (11), 400; translation stop (11), 61. Figure 2. Genetic map of ColEl. Chromosome is shown in the linear form with numbering of bases (1 to 6646) starting at the unique EcoRl cleavage site. The location of the origin of replication (ori) and of the cea, mm, and col genes is shown. pAFl, pAF2, pSZl, pSZ3 and pSZ4 are mutant plasmids containing deletions as shown. pDS1107, pAGl, pSZ5 are mutant plasmids containing inserts. All three of these plasmids of these plasmids contain a Tnl insert around bp4100. pAGl contains an additional 1030bp tandem repeat shown. Greater detail on the structure of this insert is given in figure 3. pSZ5 contains an 8 bp insert with an EcoRl restriction site at bp 479. The preparation of the mutant plasmids and the precise location of the deletion and the insertion is described in Materials and Methods. Figure 3. Structure of cea-imm boundary in pDS1107 and in pAGl which contains a 1030 bp tandem duplication of the boundary region. pDSH07 has the same structure in this region as wild type ColEl. Construction pAGl is described in Materials and Methods. Transcripts as in figure 1 are indicated by wavy lines. The cea stop indicated corresponds to the -dependent termination signal T, . The alternative T« termination signal is not shown for this transcript but further transcription beyond T, is suggested by a dashed arrow. The actual transcription termination signal for the imm gene is not known. The locus desig- nated as the jmm stop should be labeled presumptive imm stop. The DNA sequence in this region shows the dyad symmetry characteristic of a stop signal. it can be seen that leftward transcription ending at the T, stop signal ordinarily overlaps with rightward transcript terminating at the presumptive imm stop signal. This overlap is eliminated in the pAGl plasmid. Figure 4. Survival of cells in liquid broth containing Q_A g of Mitomycin C. On the ordinate the logarithmic scale indicates the ratio of viable cells at time zero to viable cells after exposure to Mitomycin C. Time of exposure to Mitomycin C is indicated on the abscissa in hours. Viable cell number is determined by spinning cells down from liquid broth and plating a suitable aliquot on nutrient agar in the absence of drug. Number of colonies after overnight incubation at 37 C is taken as an indication of the number of viable cells in the aliquot. Treated cells either contained no plasmid or plasmid as indicated. Structure of different plasmids is indicated in figure 2. Figure 5. ONPG assay of beta-galactosidase. On the ordinate activity is graphed in arbitrary units. The abscissa indicates time after induction at which an aliquot of the culture is withdrawn for assay. PAZ430 refers to the hybrid protein gene insert into our plasmid pA03 grown at 30°C. PAZ441 refers to the same plasmid grown at 30°C and then shifted to 41°C at time zero. PXZ130 and PXZ141 refer to the use of our plasmids pXT5; otherwise the meaning is the same. Figure 6. Details of the Construction of Plasmids pXZl and pAZ4. In both constructions, a 3217 bp segment containing the chloramphenicol transacetylase- β - galactosidase (CMBG) gene was isolated from a ≤m__.I, Stul double restriction enzyme digest of the pla__LZl plasmid. The pXZl plasmid was constructed from this fragment and the larger vector fragment resulting from j alll digestion of the pXT5 plasmid followed by blunt end ligation of the mixture. The pXZl plasmid was used for transfection and the resulting tet r , lac + clones were isolated. The pAZ4 plasmid was similarly constructed, except that the plasmid vector was derived from pA03. CMBG expression under control of the col promoter was verified by induction. Figure 7. Details of the Construction of pXT5 and pA03. To place the IFN-α A gene under control of the col promoter, the following constructions were made. The ColEl plasmid was isolated and digested with Xmnl. The large fragment was isolated and treated with calf alkaline phosphatase to produce a blunt-ended fragment containing the col gene and promoter. A blunt-ended fragment from pBR322 containing the tet r gene was isolated, and was ligated to the col gene fragment to produce pXT2 or pXT5, depending on the orientation of the tet r - containing fragment. pXT5 was used to construct pA03. pXT5 was first digested with Ahalll and EcoRl, and the large fragment was isolated. This fragment was treated with mung bean nuclease followed by calf alkaline phophatase to produce a blunt-ended fragment containing the col promoter. pAOl, which contains the IFN-αA gene, was digested with Hindlll and Hpal. The small fragment of this digestion was isolated and was treated with mung bean nuclease. The two fragments were then blunt-end ligated. The resulting plasmid, pA03, contains the IFN gene in the proper orientation downstream from the col promoter. Detailed Description of the invention This invention provides a double-stranded DNA plasmid adapted for expression of a foreign polypeptide which, when expressed in a suitable E_. coli cell carrying a temperature-inducible RecA protease gene, produces a foreign polypeptide. The plasmid includes in 5' to 3 1 order: a DNA sequence which includes a temperature- inducible promoter, including its associated ribosomal binding site, which promoter is derived from a bacterial plasmid and which promoter normally directs expression a of bacteriocin selected from the group consisting of colicin and the peptide encoded by the clo gene; a DNA sequence which encodes an ATG initiation codon; a DNA sequence which encodes the foreign polypeptide; a DNA sequence that is a gene which facilitates high copy number replication of the plasmid; a DNA sequence that is a gene associated with a selectable or identifiable phenotypic trait such as drug resistance, e.g., ampi- cillin or tetracycline resistance, which is manifested when the plasmid is present in the host cell; and a DNA sequence that contains an origin of replication from a bacterial plasmid e.g. ColEl, capable of autonomous replication in the host cell, e.g., E_. coli. In one embodiment, the temperature-inducible RecA protease gene is a mutant tif gene of E__ coli. Fur¬ ther, the temperature-inducible promoter in the plasmid is one which is induced by a suitable increase in the temperature of the host cell, e.g., from about 30°C to about 41°C. The inducible promoter may be the col promoter of plasmid ColEl or the clo promoter of plasmid CloDFl3. Genes encoding foreign polypeptides are well known to those skilled in the art and may be synthesized or recovered using known methods or may be obtained from depositories such as the American Type Culture Collec- tion in Rockville, Maryland, U.S.A. The foreign polypeptide may be one of a wide variety of polypeptides, e.g. interferons such as alpha or beta interferons; growth hormones such as human or bovine growth hormones or insulin. The foreign polypeptide may also be a fusion polypeptide. In a presently preferred embodiment, the polypeptide comprises a portion of beta- galactosidase gene fused to a portion of chloramphenicol transacetylase gene. The foreign polypeptide may be encoded by either a genomic DNA sequence, a cDNA sequence or . a synthetic DNA sequence encoding the polypeptide, including a synthetic sequences e ' ncoding a novel fusion polypeptide. The ATG initiation codon may be derived from the se¬ quence encoding the foreign polypeptide gene or may be derived from a sequence containing a temperature-induc¬ ible promoter, e.g., the col promoter of plasmid ColEl or the clo promoter of plasmid CloDF13 or may be derived from a synthetic linker. The gene facilitating high copy number replication of the plasmid, i.e., an increase in the average number of plasmid copies in a cell, may be a mutant rom gene, although other genes e.g. mutant genes which faciltate high copy number replication are wel known to those skilled in the art. Plasmids within the scope of this invention may be constructed or prepared using methods well known to those skilled in the art to which this invention re¬ lates. Thus, the col promoter and flanking sequences of plasmid ColEl may be recovered using known restriction enzyme cleavage sites by treatment with the correspond¬ ing restriction endonucleases under suitable conditions. Similarly, sequences encoding foreign polypeptides may be isolated, e.g., using restriction endonucleases. The sequence encoding the foreign polypeptide may be at¬ tached, in the correct reading frame, directly or through synthetic linker sequences to the segment of the plasmid containing the promoter sequence. Synthetic DNA linkers may be used to provide compatible sequences by which the sequence encoding the foreign polypeptide is joined to the sequence encoding the promoter. Ligase enzymes, e.g., T4 ligase, may be used to join the se¬ quences. In one embodiment, the genes controlled by the col promoter of plasmid ColEl (cea f imm and kil) are re- placed by the interferon coding sequence, inserted in the correct reading frame, and transfected in a tif strain by methods known to whose skilled in the art. The plasmid-containing strain is grown at about 30°C and the interferon produced by the cells collected after a period of about 6 to about 18 hours following a tempera¬ ture increase to about 41°C. Preferably, the growth medium, e.g., L broth, should contain ' no more than about 0.1% glucose, and the plasmid should include the mutant rom gene. In }__ coli cells containing the tif mutant the ColEl plasmid is stable at about 30°C. Further, tif mutants show approximately a 5,000-10,000 fold amplifi¬ cation in colicin synthesis at elevated temperatures. Similar enhancement in production of the foreign polypeptide may be achieved using the system described herein. Experimental Details First Series of Experiments Cells were grown in L broth which contains (per liter) : lOg Bactotryptone (Difco) , 5g yeast extract (Difco) , 5g NaCl, lg glucose. Colicin preparation. Colicin was prepared by minor modifications of Schwartz and Helinski (22). Its puri¬ ty was estimated by agar gel electrophoresis as greater than 90%. Colicin production. The synthesis of colicin by Ε__ coli under different conditions was assayed in several ways. The stab test is a qualitative assay used only to con¬ firm the presence or absence of a colicin-producing plasmid. The dilution drop test and acrylamide gels are semi-qualitative assay used only to confirm the presence or absence of a colicin producing plasmid. The dilution drop test and acrylamide gels are semi-quantitative assays. The double-antibody assay also is semi-quanti¬ tative. The lacuna assay gives an estimate of the number of cells making colicin. Stab test. Single colonies were touched with a tooth¬ pick and stabbed into an agar plate. The stabbed plates were placed in an incubator at the desired temperature for from 6 to 16 hours. Colonies which grew in the stabs were removed by pressing the plates down on a Kimwipe placed over a replica plate block. The plates were then overlaid at 45°C with soft agar (0.06%) con¬ taining 2% (v/v) of a fresh overnight culture of a colicin-sensitive strain. Clearings were seen within 4 hours in the growth of sensitive cells above the stabs which are producing colicin. Dilution drop test. Colicin containing cells grown in L broth. The number of cells was estimated by the OD 55Q . Comparable numbers of cells from different cultures were collected by centrifugation. The cell pellet was 5 . resuspended in 2 ml of 0.1 M KH 2 P0 4 , pH7.0 and disrupted in a French pressure cell. The lysate was centrifuged in a Sorvall ss34 rotor at 20,000 RPM for 60 min to pellet cellular debris. The supernatant was diluted in L broth in 5-fold increments. Agar plates were spread 0 with 50 1 of a fresh overnight culture of sensitive cells. 15 1 of each dilution was dropped onto the plates, which were incubated overnight. Killing units are defined as the highest dilution which gives a clearing on the plate. Estimates of killing units are accurate to about a factor of two. Lacuna test. This test measures the number of single cells making enough colicin to produce a clearing around the cell in a lawn of sensitive cells. Cells were collected and all samples were diluted to an OD 5 CQ of 0.1. Serial 1ml dilutions were made. Appropriate aliquots were plated to determine viable cells. One drop of CHC1 3 was added to each tube, and the tubes were shaken vigorously by hand for a few seconds. After 15 min, 100 1 of diluted cells (usually four different dilutions) was added to a disposable plastic tube con¬ taining 200 1 of a fresh sensitive stationary culture; to this was quickly added 2.5 ml of 0.4% soft agar at 45°C. The mixture was poured and gently spread onto a 60 mm Petri dish and incubated for at least 4 hours. The layer of agar must be as thin as possible so that colicin producers will not be buried so far beneath the surface that the region of inhibition of growth around the cell will fail to reach the surface where most of the rapidly dividing sensitive cells are located. Lacunae can be very small (leading to inaccuracy in counting) or as large as 2 mm. Large lacunae are always associated with cultures making large amounts of colicin in the experiments reported here. Double-Antibody Assay. The double antibody assay was complex and is described in considerable detail for those who would like to apply it for the detection of colicin or other bacterial proteins. Preparation of the rabbit antiserum to colicin is described elsewhere (3). The antiserum was fractionated prior to use by the following procedure. First the antiserum was diluted to an OD 280 of 20 (about 15 mg/ml) in PBS (per liter: 30 ml of 5M NaCl; 20 ml of 0.2 M NaH 2 P0 4 ; 80 ml of 0.2 M Na 2 HP0 4 ) . Saturated (NH 4 ) 2 S0 4 (SAS) at 4°C was added to make the final solution 40% saturated. The SAS was added slowly while the solution was mixed in the cold. Mixing was continued slowing for 10 min. The solution was allowed to sit for 60 min at 4°C and spun in an ss34 rotor at 10,000 RPM for 15 min. The above step was repeated twice more each time decanting the supernatant after centrifugation. About half the original protein was recovered in the final precipitate. This was re- dissolved in PBS" to a concentration of 30 mg/ml and dialyzed against PBS. Ouchterlony tests were carried out following the advice of L. Eckhardt and Reference (3) . Cells were washed in 1 ml of PBS, centrifuged for 1 min, and resuspended in H 0 by vortexing for 30 sec. 10 1 5 Δ containing about 10 cells was dropped on a slide. The slides were allowed to dry and flamed very briefly after which they were placed flat into a dessicator containing a beaker of CHCI3 for 15 min. The CHCI3 was removed and the dessicator evacuated for 15 min. To each fixed sample was added a 15 1 drop of antibody diluted 100-fold. The slides were covered with a Petri dish inside of which was taped a wet piece of filter paper (to prevent drying out of 'the sample) and allowed to sit for 30 min. Slides were washed for 30 min in PBS with occasional agitation. Each slide was dried with a paper towel around the slides of the samples, leaving the samples wet. To each sample was added 15 1 of goat anti-rabbit rhodamine-conjugated IgG (Boehringer Mann¬ heim) diluted 100-fold. The samples were covered with a wet Petri dish for 30 min. Slides were washed for at least 1 hour in PBS and dried with a towel. A small drop of Aquamount was applied to each sample and cover- slips were placed over the samples. The samples were stored at 4°C and Viewed in a Leitz Laborlux 12 micro¬ scope at 400x magnification under transmitted and fluo¬ rescent illumination. Selection of transformants. Transformation was carried out on Ca 2+-treated cells (10) stored for a maximum of 3 weeks in 20% glycerin at -70 C. After addition of Col El plasmid DNA, cells were left on ice for 30 min then put at 30°C for 10 min. Broth at room temperature was added and tubes were returned to 30°C for 90 min. to allow for expression of plasmid-conferred immunity to colicin. Immunity selection was done by spreading 10 cells on an L-agar plate and applying seven 15 1 drops of colicin in successive 3-fold dilutions around the plate. The highest concentration of colicin used was 50 Kill Units/ml. Immune cells survived in the cleared areas created by the drops of from 10 to 1 Kill Units/ml. In some experiments where plasmid pDS1107 (see description below) was used, transformants were also selected with ampicillin (16) . Bacterial Strains. All bacterial strains used in this study were obtained from Graham Walker. Strain Parent Relevant Genotype AB1157 E. coli K12 tlU -l ,__j__li-6 , jςhi-1 r l___.Yl ,gaiK2 , ϋJLa-14 ,2^1-5 ,mtl-l ,£___A2 , his-4 , aj_ E3 , str-31. tsx-33 , sup37 GW1000 AB1157 £__Δ+,_L___≤_ + ,iifiAll (sj_lA) ,_ϋ___is, ____-! (____M41) GW2730 GW1000 l£_iA (Def) (lej_-A71 : : Tn5) . recA441 GW2735 GW2730 l£χA(Def ) ( lexA71: :Tn5 ) , recA+ GW2733 GW2730 i≤ltA (Def) (ieiA71 r : Tn5) , recA56 The sfl mutation is present in all of the strains used. This mutation promotes viability of cells that are deficient in lexA. it is customary to indicate strains that make repressor defective LexA protein by the symbol le_&A(Def) rather than just lexA or lexA " . The l£X 71: :Tn5 mutation used contains a Tn5 insertion in the early region of lexA gene and is believed to be completely lacking in LexA repressor protein. Further information on these strains is reported elsewhere (13) . Plasmid strains. In addition to wild type ColEl a plasmid containing a Tnl insertion in ColEl was used (pDS1107) . This insertion does not interfere with colicin synthesis. It was used in cases where it was convenient to have a drug marker to select for trans¬ formants. The plasmid is further described elsewhere (4). Determinations of plasmid copy number. The relative plasmid copy number of different cultures was determined by isolating plasmid DNA from comparable numbers of cells. The alkaline lysis method was used to isolate plasmid DNA (15) . Relative amounts of plasmid DNA were determined by comparing the intensity of bands after gel electrophoresis and ethidium bromide staining. Usually several independent isolates were compared at different dilutions. For making estimates of the relative amounts of plasmid DNA in different preparations the method is accurate to +30 percent. Results In most of the studies reported here two bacterial strains were used that contain mutations in either the recA • or the lexA genes or both: (1) Strain GW1000 contains a unique mutation in the recA. gene called tif. This strain makes a RecA protein with a temperature activatable protease. (2) Strain GW2730 contains tif and a lexA71 mutation which completely eliminates LexA repressor activity. In selecting for ColEl transform¬ ants of these strains I was concerned about possible lack of viability because of the anticipated high level of cea gene expression. Transformation was done in several ways to make sure that the cells that were transformed did not represent a special subpόpulation of a particular strain. Some comparisons were made with plasmid pDS1107 which carries an ampicillin resistance drug marker in a nonessential region of the plasmid. This permitted transformants to be made by either se¬ lecting for immunity to colicin or by selecting for resistance to ampicillin. The frequency of transform¬ ants obtained was comparable when using either selection technique. Furthermore transformants initially selected for ampicillin resistance also proved to be immune to colicin and vice versa. Comparison between the recA*, lexA* strain AB1175 and strains GW1000 and GW2730 showed that approximately the same number of transformants were obtained with all three strains. Thus all strains bearing colicin plasmids (with ColEl or pDS1107) proved to be equally viable, at least under our conditions of selection. The only qualification to be noted here is that strain GW2730/ColEl grows more slowly as evidenced by the smaller colonies it produced on plates; it also has a cell doubling time of about 40 percent longer than the comparable plasmidless strain in liquid culture. Quantitatively, the intracellular colicin concentration in the total cultures was measured by the numbers of kill units. This unit is a measure of the potency of a crude cell lysate to kill sensitive plasmidless cells. A convenient spot test is described in Methods for determining the relative number of kill units in dif¬ ferent extracts. Use of the spot test showed that the yield of colicin in plasmid-containing cells varied over a range of about 5000-fold from fully repressed to fully induced conditions (table 1) . A recA + .lexA ÷ strain AB1157/ColEl showed a seven fold increase in colicin synthesis on raising the temperature from 30°C to 42°C. The amount of colicin synthesis in AB1157/ColEl cells was greatly augmented by mitomycin C treatment (see table 1) . Host mutations in the recA and/or the lexA gene also lead to a much higher level of colicin synthe¬ sis. The ϋf-containing mutant of recA GWlOOO/ColEl make about 50 times more colicin than the comparable £__.A strain at 30°C. This level was increased about 100-fold on raising the temperature to 42°C. The j_U,le___.A(Def) containing mutant (GW2730/ColEl) made about 500-fold more colicin at 30°C than a comparable recA "1" lexA + strain (AB1157/ColEl) and this level was __________ Colicin activity found in various strains of E. coli grown under different conditions. Growth Colicin Strain Genotype Conditions Activityt recA lejA AB1157 + + 30°C 1 AB1157 + + 42°C + 7.0 AB1157 + + 30°+MTC* 560 GW1000 iif + 30°C 50 GW1000 tif + 42°C + 4500 GW2730 iif - 30°C 500 GW2730 iif - 42°C + 5000 GW2730 iif - 30°+MTC* 300 GW2730 tif - ' 42°+MTC* 5200 *MTC is added at a concentration of 1.0 g/ml for a period of 3 hours before examing the cells. + Cells.grown at 42°C are obtained from cultures grown to mid-expotential phase at 30°C. The temperature upshift is allowed to proceed for 3 hours prior to making the cell extracts. tThe numbers are for relative colicin activity measured as kill units as explained in- Materials and Methods. increased another 10 to 20 fold on raising the tempera¬ ture to 42°C. Mitomycin C treatment did not increase the level of colicin made in (GW2730/ColEl) at either temperature. At very high levels of colicin the amounts of colicin can be directly estimated by gel analyses of crude lysates following standard procedures (30) . Under fully induced conditions we estimate that colicin comprises as much as 25 percent of the soluble protein in a crude extract. The most striking distinction between cells induced to make colicin in tif mutants or tif. lexA(Def) mutants and those induced to make colicin in rec *, lexA* cells by mitomycin C treatment is the greatly increased sui- cide rate in the latter. • tif mutants or tif, lejcA(Def) mutants containing ColEl show a steady increase in the number of viable cells for several hours after a temper- ature upshift to 42°C. Growth gradually slows as it does in control cells because of depletion of the growth medium. By contrast mitomycin C treated cells show a 90 percent loss of viable cells two hours after exposure to the drug and the killing effect is progressive with time. A major goal of the current investigation was to study the colicin synthesis of individual cells. Measurement of colicin synthesis in mass cultures grown in liquid broth provides one with an average value of the colicin content per cell. Likewise measurements on clones on agar plates derived from single cells merely indicate that some members of the clone are making colicin. These types of measurements could be interpreted in terms of two extreme models: all cells produce ap- proximately the same amounts of colicin or some cells produce little or no colicin and the remaining cells produce a high level of colicin. Obviously situations in between these two extremes might be also considered. In order to discriminate between these possibilities it was necessary to adopt procedures for assaying individ¬ ual cells for colicin synthesis and viability. Three types of measurements on single cells from induced cultures were made. The first measurements involved the lacunae assay which gives an estimate of the number of cells making appreciable levels of colicin (see Materi¬ als and Methods) . In order to determine the percentage of cells in an induced culture which can give rise to lacunae it was necessary to make a viable cell count on an aliquot from the same culture. This was done by the standard procedure of plating an aliquot of the colicin producing cells and counting clones after overnight incubation. The lacunae assay was most useful in assessing the situation in cultures that showed a low level of colicin synthesis. For example a culture of the recA+,lex+ strain AB1175/ColEl produced at a very low level of colicin under normal growth conditions at 37°C. The lacunae assay indicated that less than 1 percent of the cells in such a normally growing culture account for most of the colicin synthesis. Thus the cells in such a culture appear to be of two types, the smaller fraction of which are active in colicin synthesis. On maximally induced cultures of GWlOOO/ColEl or GW2730/ColEl the ratios of lacunae producing cells to viable cells varied from 0.3 to 1.0. Such variability limited the usefulness of this method for the purpose at hand which was to determine if colicin producing cells are also viable. Another objection to using this method was that induced cells frequently exist as short chains in a liquid culture. A chain of three cells giving rise to a clone on plating will score as one viable cell on a plate assay even though it may contain from one to three viable cells. Similarly such a cell cluster will give rise to a single clearing in a lacunae assay. This aggregation problem will lead to an overestimate for the ratio of colicin producing cells to replication compe¬ tent cells. In general, maximally induced cells produce larger lacunae suggesting that the colicin synthesizing cells are making more colicin at 42°C than at 30°C. Other than this all the lacunae assay did was to confirm what we knew already which was that an appreciable percentage of the cells were highly active in colicin synthesis in tif or ϋ-f " ,l≤__A(Def) strains. The second type of measurement which was made on single cells involved use of a colicin specific antibody and fluorescent staining technique. Cells from an induced culture were spread on a glass slide, permeabilized by brief exposure to heat and an atmosphere of chloroform, then treated with an immune serum made in rabbit against purified colicin. Finally the preparation was treated with goat anti-rabbit antibody tagged with the fluores¬ cent dye rhodamine. Results of this type showed that at least 90 percent of the cells in an induced culture were high colicin positive as indicated by a halo surrounding those cells presumed to contain large amounts of colicin when viewed using fluorescence microscopy. Mixture of cells from an induced and an uninduced culture allowed estimation of the approximate percentage of fluorescent cells expected for such a mixture. The results of this experiment strongly support the view that the vast majority of cells in an induced culture are highly active in colicin synthesis. Results discussed are for the tif strain (GWlOOO/ColEl) five hours after raising the temperature from 30°C to 42°C. Comparable results have been obtained with GW2730/ColEl cells at 30°C and 42°C. In this case considerably less fluorescence was observed for cells grown at 30°C than at 42°C. This is consistent with the results reported in table 1 that GW2730/ColEl cells make 10 to 20 times more colicin at the higher temperature. The third and final type of measurement that was made on induced single cells consisted of time lapse photomicro¬ graphs of tif cells containing (GWlOOO/ColEl) grown at 42°C and deposited on a thin agar plate containing L broth with 0.1% glucose. Cells were examined immediate¬ ly after plating and- 30, 60 and 90 minutes thereafter. Immediately after plating from liquid broth agar, the field contains single cells or doublets. Careful com- parison of this field with subsequent observations taken at 30 minutes intervals shows that all cells in the culture were growing and dividing. These irregular shapes of the microclones were a surprise. In fact normal wild type cells showed the same behavior at this stage in their growth on agar plates. As they become larger the clones adopt the more rounded appearance when plated at the appropriate dilution. Discussion The level of colicin synthesis was measured in cells with different genetic constitutions under different growth conditions. The primary purpose of these mea¬ surements was to determine the effects of different facts on colicin synthesis and to see how cell viability was influenced by the level of colicin synthesis. Care must be taken in measuring colicin synthesis since after prolonged periods of high levels of synthesis colicin passes across the cell membrane. It is difficult to • quantitatively measure colicin synthesis after transport as it becomes partially inactivated in the liquid growth medium. Therefore most of the measurements reported here have been made on cells grown at 30°C or within three hours of shifting the temperature to 42°C. Under these conditions only a very small percentage of the colicin is lost from the cell. Growth medium -is also a very significant factor affect¬ ing the amounts of colicin synthesis as well as the cell viability. Some growth media result in a high loss of viability in colicin induced cultures; others do ' not (e.g., see reference 26) . Most of the studies were done in L broth where there is little loss of viability of plasmid-containing cells except when mitomycin C is present. The level of colicin synthesis is quite sensi¬ tive to the glucose concentration (11) . It was found that optimal levels of colicin synthesis are obtained at glucose concentrations of 0.1%. Optimum levels of colicin synthesis were obtained in mutant R_ coli cells that are tif or tif, ie∑A(Def) at 3 to 6 hours after a temperature upshift from 30°C to 42°C (table 1) . The levels ' of colicin synthesis found under these conditions were about 5000 fold greater than those obtained with recA , le A + cells at 30°C on a per cell basis. It was estimated that about 25 percent of the cytoplasmic protein is colicin under these conditions. From this it has been calculated that the average cell contains about 250,000 copies of 56 kilodalton colicin protein. Viability of induced cells was examined by using tech¬ niques that permitted detection of the approximate levels of colicin synthesis and viability on single cells. It was concluded that at high levels of induc¬ tion cells are uniformly induced and uniformly viable. The effect of mitomycin C (MTC) on colicin synthesis was measured on both recA*, lexA* cells and tif, lexA(Def) cells." MTC greatly augments colicin levels on the former cells but has no augmentation effect on the tif, lexA(Def) cells at either 30°C or 42°C. This is consis¬ tent with the fact that the main effect of MTC on colicin synthesis is due to the destruction of the LexA protein. Thus when there is no functional LexA protein the level of colicin synthesis is not increased by MTC. Cell viability decreases dramatically in the presence of MTC. Even though MTC induces colicin synthesis it seems unlikely that this is the cause of MTC-induced lethality for two reasons: (1) comparable levels of colicin can be produced in the absence of MTC without loss of via¬ bility and (2) there are indications MTC-induced lethal¬ ity is primarily a function of kil gene expression rather than cea gene expression (see Background) . Since the kil gene is probably in the same operon as the cea gene (5) it is not clear if the MTC-induced lethality is due to a differential effect of the drug on kil gene expression or to the concerted actions of mitomycin and the kil gene product. This question is under investiga- tion. A most interesting finding from these studies was that colicin synthesis was strongly dependent on growth temperature. In some organisms so-called heat shock proteins are produced at high levels for long times after a temperature upshift but in ___ coli the response is transitory (e.g., see reference 27). Examples of prolonged elevated synthesis at high temperatures are unknown for ___ coli proteins. Kennedy (12) was the first to observe a temperature effect on colicin synthesis, but his experiments were done in lexA cells where the levels of colicin synthe¬ sis are never very high because of the repression effect of the LexA protein. Here it was observed a 10 to 20 fold elevation in colicin synthesis in the lexA(Def) strain GW2730/ColEl 3 hours after a temperature upshift. The lexA(Def) mutation in this strain contains a Tn5 insertion in the early region of the lexA gene which should eliminate all functional LexA repressor. Never¬ theless the presence of the temperature inducible RecA protease also present in this strain might in some way be responsible for the big increase in colicin synthesis on the temperature upshift. For this reason two other l£&A(Def) strains were examined that contained different alleles of the recA gene; strain GW2735 was recA + and strain GW2733 contained the recA56 mutant which elimi¬ nates RecA protease activity. Strain GW2733 grows more slowly. Nevertheless both of these strains when carry- ing plasmid showed a comparable enhancement in colicin synthesis after a temperature upshift. It was concluded that the elevated level of colicin synthesis at high temperatures has nothing to do with either the LexA or the RecA proteins. Another possible explanation for the temperature effect on colicin synthesis was ' that the plasmid copy number might be changing. If the number of copies of plasmid rose dramatically at higher temperatures the increased colicin synthesis could be explained by a change in the gene dosage. Copy number studies indicated a slight lowering in copy number on raising temperature. It is concluded that preferential synthesis of colicin at higher temperatures must result from a specific enhancement in cea gene expression; this enhancement could be occurring at either the transcription or trans¬ lation levels. Further experiments are planned to determine the cause of increased colicin synthesis at elevated temperatures. Second Series of Experiments Materials and Methods 1. The Ej. coli K12 strain ' 514 was used in most of the studies reported here (2) . This strain contains wild type recA* and le£.A + genes. The ___ coli K12 strain GW1000 contains a tif mutation of recA gene (26) . The RecA protein in this mutant has a hyperactive protease especially at elevated temperatures. Other properties of the strain are described elsewhere (13) . 2. All of the restriction enzymes used were obtained from International Biotechnologies, Inc. 3. Assay for mitomycin C-induced lethality. Cells were grown in TS broth (Tryptose 2% and NaCl 0.5% at 37°C. In idexponential phase 0.4 g/ml of mitomycin C was added to the cells and growth continued for an addition¬ al period of 2 to 6 hours. Viable cells were determined by plating suitable samples of mitomycin C-free cells on 0.5% agar plates containing TS broth. Colonies arising after overnight growth were taken as an estimate of the number of viable cells in the original culture and the mitomycin C-treated culture. 4. Preparation of plasmid mutants. All plasmid mu- tants were made from ColEl or pDS1107, a ColEl deriva¬ tive containing a Tnl insertion (see figure 2 and ref¬ erence 4) . Three plasmids with deletions pAFl (dele¬ tion of base pairs 5280 to 6549) , pAF2 (deletion of base pairs 1652 to 3432) and pSZl (deletion of base pairs 1682 to 4627) were obtained by exhaustive digestion with a single restriction endonuclease, Pstl, PvuII or Xmnl respectively. The resulting DNA fragments containing the replication origins were recircularized with T4 DNA ligase. Three plasmids with deletions, pSZ2 (deletions of base pairs 4072 to 6646) , pSZ3 (deletion of base pairs 5145 to 6646) and pSZ4 (deletion of base pairs 5280 to 6646) were obtained by exhaustive digestion with combinations of two different restriction enzymes, Ahalll-EcoRl, SacII-EcoRl or Pstl-EcoRl respectively. After digestion these fragments were blunt-ended by treatment with T4 DNA polymerase in the presence of the four deoxynucleoside triphosphates. This enzyme de¬ grades ends containing 3' overhangs and fills in ends containing 5' overhangs. The blunt ended linear frag- ment were then circularized by treatment with T4 DNA ligase. After ligation the plasmids were transfected into strain 514. Transfected cells were selected by the immunity property which permits survival on agar plates containing colicin. Plasmids were reisolated from the transfected cells and their structures were confirmed by restriction enzyme analysis. Two plasmid mutants were made from pDSH07 which con¬ tains a Tnl insertion around base pair 4100 (see figure 2) This plasmid was convenient to use because it con- tains an ampicillin resistance marker that facilitates selection of mutant variants of the plasmid constructed in vitro. A kil mutant (pSZ5) was constructed from pDS1107 by first treating exhaustively with Nrul endonclease. This endonuclease produces a single blunt end cleavage be¬ tween base pairs 478 and 479 which is 40 bases from the start of the kil gene reading frame (base pairs 448 to 583) . The resulting linear fragment was reacted with a 20 fold excess of an 8 base blunt end linker containing an EcoRl site (GGAATTCC) in the presence of T4 DNA ligase. This product was redigested with Nrul to remove plasmid that recircularized without incorporating link- er. After transfection the construction of the reisola- ted plasmid was verified by restriction enzyme digestion and gel electrophoresis. This analysis showed that the plasmid had lost its Nrul site and gained a second EcoRl site (normally pDS1107 like ColEl contains only 1 EcoRl recognition site) . The plasmid pAGl contains a 103 bp duplication (base pairs 5979 to 364) in the region bounding the cea and the imm genes (see figure 3) . The following procedure was used to construct the plasmid. ColEl was digested with EcoRl and Haelll. This treatment led to the pro¬ duction of 12 blunt-ended Haelll fragments and 2 frag¬ ments, 360 bp and 670 bp, which contain one Haelll blunt end and one EcoRl 5' overhang. pDS1107 was digested with EcoRl and calf alkaline phosphatase (CAP) . CAP treatment prevented self-ligation in the subsequent step. The fragments from the EcoRl Haelll digestion were mixed with the linearized pDS1107 and treated with T4 ligase, first under conditions favoring staggered end ligation and then under conditions favoring blunt end ligation. The reaction mixture should contain a vari¬ ety of products. First transfectants were selected for ampicillin resistance. Then they were selected for colicin producing cells. Finally plasmids were isolat¬ ed from different amp , cea* clones and analyzed by restriction enzyme treatment duplication of the Haelll B segment of ColEl. Results ColEl containing cells in liquid culture were treated with 0.4 g/ml MTC in mid-exponential phase. At speci¬ fied time intervals samples were removed from the cul¬ ture, sedimented, resuspended and plated to assay for the number of viable cells. About 90 percent of the cells containing the ColEl plasmid were killed within one hour. The effect was progressive so that after 6 hours exposure to the drug less than 0.1 percent of the cells survived (see figure 4) . Comparable plasmidless cells showed better than 50 percent survival after a six hour exposure to the drug and the effect of prolonged exposure was not progressive. Indeed the population usually seemed to recover after the first two hours of exposure as evidenced by an increased number of survi- vors. Nine mutants of ColEl were examined for their effect on MTC killing. The structures of these plasmids are indicated in figure 2 (more data on the precise loca- tion of the mutations is given in Materials and Meth¬ ods) . In the absence of drug these plasmids did not influence the cell growth rate and their copy number (the number of plasmids per cell) was comparable except for plasmid pAF2 which tends to be present in slightly higher numbers. The relative copy number was visually estimated on an ethidium bromide stained gel after electrophoresis of a plasmid preparation. Whereas more refined methods exist for plasmid copy number estima¬ tion this procedure was considered adequate for the purpose at hand. Two of these mutant plasmids pAF2 and pSZl, contained deletions upstream of the cea gene (figure 2) . Cells with these plasmids showed the usual high sensitivity to MTC (see figure 4A) . Drug hypersensitivity was also observed for cells carrying the pDSH07 plasmid which contains a Tnl insertion about 100bps upstream from the cea gene (figure 4b) . Several mutant plasmids, paAFl, and pSZ3 and pSZ4, carried deletions within the cea gene. These mutants also made their host cells hyper¬ sensitive to MTC (results not shown) . Finally, three mutants, pSZ2, pAGl and pSZ5, were found to be .greatly reduced in their ability to increase drug sensitivity for harboring cells (see figures 4A and 4B) . The drug sensitivity for such cells was just slightly greater than that observed for plasmidless cells. ' The plasmid pSZ2 contains a large deletion which removes most of the cea gene including the promoter and some sequences upstream from the promoter. The pAGl plasmid contains a tandem duplication (1030 bps) of a region bordering the cea and imm genes (see figure 3 for details) . Plasmid pSZ5 contains a mutation situated exclusively in the kil gene. The plasmids pSZ5 and pDS1107 were examined in more detail in a tif strain of JL_. coli (GW1000) in which colicin synthesis is induced by a temperature upshift (30°C to 41°C) in the absence of MTC. Under these conditions of induction cell death is not apparent (see Spangler, R., Zhang, S. and Zubay, G. , Colicin Synthe- sis and Cell Death, J. Bact., submitted). The effect of a temperature upshift was examined 6 hours and 18 hours after a temperature upshift. Intracellular colicin levels were examined by acrylamide gel electrophoresis of whole cell lysates. After six hours both cells contained high levels of colicin as evidenced by the prominence of a 56 kilodalton band characteristic of colicin. After 18 hours at 41°C the cells carrying the pSZ5 Jsϋ " plasmid gave a similar gel profile. In marked contrast the cells carrying the pDS1107 kil* plasmid have lost almost all of their colicin. With neither culture was there any evidence for cell death or lysis. Thus after 18 hours at 41°C both cultures contained about the same number of cells as measured by turbidi- metry or by plating. Examination of 6 clones arising from the 18 hour cultures in .both cases showed that unmodified plasmid was present in the cells of both cultures. The growth medium after 18 hours exposures was also examined by gel analysis. For this purpose the protein present in the cell-free growth medium was precipitated by addition of a 20-fold excess of acetone. The precipitate was collected by centrifugation and the acetone removed by air drying. The sample derived from the pSZ5 containing cells showed almost no protein. The sample derived from pDS1107 containing cells after 18 hours at 41°C showed a substantial concentration of protein dominated by a prominent band with an estimated molecular weight of about 27 kilodaltons but also con¬ taining some 56 kilodalton protein characteristics of colicin. The extracellular fluid from the two cultures were also tested for the presence of colicin by the kill unit bioassay. This assay measures the killing effect of colicin on normal plasmidless ___ coli cells which are sensitive. to colicin as described elsewhere (Spangler, R., Zhang, S. and Zubay, G. , Colicin Synthesis and Cell Death, J. Bact., submitted). By this assay the extra¬ cellular fluid from the GW1000/pDS1107 Jϋ + cells was always 100-1000 times more potent than that from the GW1000/pSZ5 kil " cells. Parallel gel analyses were done on cell extracts and the extracellular fluid from strain 514 cells containing either pSZ5 or pDS1107. The cells were examined 4 hours after exposure to 0.4 g/ml MTC as in figure 4. Cells carrying the pDS1107 ( i + ) plasmid showed no accumula¬ tion of colicin. All of the colicin was in the extra¬ cellular fluid along with a spectrum of other proteins very similar to that found in a whole cell lysate. This suggests th.at all of the colicin has been transported and that a substantial percentage of the " cells (30 percent) have lysed. Cells carrying the pSZ5 (kil " ) plasmid accumulated a significant amount of colicin (about one quarter of that observed in strain GW1000 after six hours). A very small amount ,of colicin was also observed in the extracellular fluid but only minute traces of other proteins. This suggests that even though colicin transport is strongly inhibited there is some indication that it is selectively transported. Discussion The currently favored model for MTC-induced lethality in ColEl containing cells is that lethality results from expression of the kil gene (e.g., see reference 25). This model is supported by findings in this study. Lethality is usually measured by plating efficiency of cells previously exposed to MTC in liquid culture. High levels of MTC (2 g/ml or greater) will kill plasmidless cells but only plasmid-containing cells are extensively killed by low levels (0.4 g/ml) of the drug (compare no plasmid and ColEl in figure 4A) . Deletions in the plasmid outside of the cea-imm-kil gene cluster do not lower the killing effect (see pSZl and pAF2 in figure 4A) . Deletions within the cea gene do not lower the killing effect unless they remove the cea gene promoter. Thus plasmids pAFl, pSZ3 and pSZ4 which contain exten- sive deletions of the coding region of the cea gene (see figure 2) do not lower the killing effect of the plasmid. But pSZ2, which also removes the cea promoter (see figure 2) , greatly lowers drug-induced killing (see figure 4A) . These findings are in agreement with those of Shafferman (24) and Suit (25). Thus far examples of plasmids which are cea + , imm + , kil " or cea + , imm~, Jlil " but none of which are cea + , imm~ f kil + have been considered . In the Introduction it was mentioned that a cea , imm " plasmid isolated by Inselburg grew best in liquid culture when typsin was present. Since the mutation in this plasmid was due to a Tn3 insertion into the imm gene it is very likely that a polar effect would result making the strains effec- tively kil " . This was never tested for but it might help account for the survival of cells containing this plasmid since they would be expected to transport colicin poorly. It would be informative to have plasmid mutants that are altered in immunity expression without being altered in kil gene expression. In fact the plasmid pSZ4 appears to be such a plasmid. Measured by resistance to exogenous colicin this plasmid shows a much higher level of immunity than plasmid pAFl or ColEl (reference 15 and our unpublished observations) . The plasmid pSZ4 has 100 base pairs less than pAFl in the promoter-distal region of the cea gene (figure 2) . The reasons for the large effect of this gene segment on immunity expression not known. The most important point here is that plasmid mutations do exist which have a huge effect on immunity without significantly affecting kil expression. The cea and the imm reading frames abut one another to the extent that their stop codons share two base pairs (see caption to figure 1) . A mutant containing a du¬ plication of this region was constructed (see figure 3) to determine the effect that eliminating the overlap would have on immunity and drug-induced killing. No effect on immunity was seen (results not shown) but drug-induced killing was greatly reduced (see pAGl, figure 4A) . Since this duplication introduces an addi¬ tional Tl transcription termination site between the cea and the kil gene it is possible that this is the cause of the reduced killing. This interpretation is consis- tent with the findings of Shafferman (24) and Suit (25) that transposons inserted in the cea ' gene usually block killing. The most important mutant plasmid generated for these studies was pSZ5 which contains a small DNA insertion in the middle of the kil gene.' This is the only known mutation which is solely located within the kil gene. The properties of this mutant plasmid confirm and ex¬ tend expectations from previous work. Thus MTC sensi- tivity is largely eliminated (see figure 4A) and colicin is synthesized but cannot be found in the extracellular fluid. The striking absence of all but a trace of extracellular colicin in induced cells containing the pSZ5 plasmid indicated that the kil gene is needed for efficient transport of colicin. Whereas it is clear that' extracellular colicin is dras¬ tically reduced in the absence of the Kil protein, this by itself does not explain how the Kil protein functions in colicin release. The first question to resolve is whether the Kil protein stimulates colicin release by a discreet secretion process or whether lysis is required. These results favor the conclusion that colicin release does not require lysis. Thus when strain GW1000 con¬ taining either plasmid is shifted from 30°C to 41°C this results in a high level of colicin synthesis. The turbidity of both cultures rises steadily until it reaches a plateau. Plating of samples from either culture yields approximately the same viable cell count. Indeed the only difference to be found between the two cultures is that the l + plasmid-containing cells lose most of their colicin in addition to minor amounts of some other proteins. Since there is no evidence for cell lysis, this loss of protein probably involves a Kil protein-dependent transport process. This does not out rule the participation of other specific protein factors as suggested by Yamada et al (29) . The spectrum of proteins released upon prolonged incu¬ bation of kil plasmid-containing cells is complex. Some of these proteins are clearly plasmid encoded but some may also be host encoded. Prolonged incubation results in the selective loss or reduction of several intracellular proteins in addition to the 56 kilodalton colicin. Colicin is unstable in the extracellular state (e.g., see reference 1) and this may account for the low intensity of the 56 kilodalton band in the extracellular fluid. From the large amount of the 27 kilodalton protein found in the extracellular fluid it is tempting to suggest that this band represents a major degradation product of the colicin. In this connection it may be relevant that a polypeptide of this size is the second most abundant species synthesized in the ColEl DNA- directed system for protein synthesis (2). It was shown that this polypeptide is encoded by the promoter-proxi- mal region of the cea gene. Other minor proteins or peptide fragments found in the extracellular fluid may be host encoded or plasmid encoded. The most puzzling aspect of cell death associated with colicin production is related to the fact that the conditions of induction are critical. On the one hand when induction is caused by DNA-damaging reagents such as ultraviolet irradiation or MTC, massive cell death accompanied by cell lysis results when the wild type plasmid is present. On the other hand when induction is caused by host mutants that result in a deficiency of the LexA repressor, cell death does not necessarily follow. The critical factor may be the Kil protein concentration which in turn could be related to the extent to which transcripts stop at the Tl and the T2 terminators (see Figure 1) . Only the latter tran¬ scripts would lead to Kil protein synthesis. Possibly the effectiveness of termination at Tl is influenced by metabolic conditions. With the help of the pSZ5 mutant plasmid it should now be possible to systematically investigate the effect of different modes of colicin induction on the types of transcripts that are synthe¬ sized. Attempts are also being made to assay for the Kil protein directly. Third Series of Experiments In an attempt to determine the efficiency of the col promoter in the production of a foreign fusion protein the following experiments were conducted. The gene for a hybrid fusion protein containing the first 41 amino acids of chlora phenicol transacetylase and all except the first 8 amino acids of beta- galactosidase (CMBG) was inserted into two different plasmids (pA03 or pXT5) 130 or 91 nucleotides downstream (respectively) from the transcription start site of the colicin gene (Figure 6) . This location is probably suboptimal for expression, as is the ribosome recognition site in these constructions. Nevertheless, this plasmid when transfected into the tif strain expresses the CMBG protein at a very high level upon temperature induction via a temperature shift from 30°C to 41°C. The presence of this protein was detected by standard acrylamide gel electrophoresis or by the standard orthonitrophenolate assays for beta- galactosidase (Figure 5) . The gel assay indicates that the hybrid CMBG protein is the dominant protein in such cells after a few hours of induced growth. The kinetic data (Figure 5) shows that induction is immediate on temperature upshift and continues at a linear rate for at least 400 min. Results on uninduced cultures show a low level of beta- Gal activity. This low level of activity in uninduced cells probably arises from spontaneous induction of a small percentage of the cells in the culture. The level of plasmid encoded beta-Gal activity in the vast majority of cells is probably very low. This low level of activity of the test gene in the expression vector could be a major asset of this system for proteins that are detrimental to bacterial growth. They can be kept under tight negative control in this system for proteins that are detrimental to bacterial growth. They can be kept under tight negative control in this system until induction is desired. Induction is also very simple and reproducible merely requiring a temperature upshift. The expression system described herein is probably comparable in efficiency to the best available expression systems, and in addition it possesses the advantages described above. Fourth Series of Experiments Several plasmid modifications and constructions were made with the goal of finding conditions under which IFN- A could be expressed at an elevated level. IFN-^A was chosen because it was regarded as protein that might be of some practical importance. As a source of the IFN- αA gene we used pER33(31). In the pER33 plasmid the distance between the ribosome recognition site and the ATG start codon is 6 bases above the optimal. This distance was optimized by removing six of the intervening bases. This construction starts ' from pER33 and ends with pAOl. In this construction pER33 is partially digested with Hindlll. The resulting mixture is digested with T4 DNA polymerase in the presence of dTTp. This is gently treated with Mung bean nuclease and the product is blunt end ligated with T4 ligase. This product is used in transfection. From the amp r transformants a plasmid having the structure of plasmid pAOl was isolated. In subsequent constructions pAOl was used instead of pER33. The goal of the next construction was to move the interferon gene together with its ribosome recognition site as close as possible to the comparable site occupied by the col gene in ColEl. We settled for a location 23 bp further downstream from the translation start site. The relevant plasmid is called pA03. The pA03 plasmid was constructed from pAOl and pXT5 (Figure 7) . We have just explained the construction of pAOl. pXT5 is a derivative of ColEl which contains a large deletion (2948 bp) including part of the rom gene and a large insertion (1427 bp) of the tetracycline resistance region from pBR322. The 2948 bp segment is first removed by Xmnl digestion. Then the tet r gene is released from pBR322 by double digestion with EcoRl and Aval to form a second fragment. Flush ends are formed on this fragment by treatment with mung bean nuclease. The two fragments are then combined and blunt-end ligated. Two plasmids result from this treatment, pXT5, or pXT2 when the fragment .is in the reversed orientation. The pA03 plasmid was constructed from pAOl and pXT5 (Figure 7) . pXT5 was first treated with Ji&IIl and flC_Rl. The larger of two fragments was isolated and treated first with mung bean nuclease and then with calf alkaline phosphatase. Next, pAOl was treated with Hpal and Hindlll. The small fragment (880 bp) if this digestion was isolated and treated with mMung bean nuclease. Two two fragments, one from pXT5 and one from pAOl, were blunt end ligated, as shown, to obtain pA03. The ELISA assay for interferon The pA03 plasmid was transfected into E . coli strain GW1000, to produce strain pAO3/GW1000. Standard conditions of induction were used. Assays for interferon were performed by the ELISA assay. This assay requires the use of monoclonal antibody to interferon. Small wells are coated with the antibody. After proper rinsing monoclonal antibody conjugated to horseradish peroxidase is added. After rinsing, the horseradish peroxidase is assayed colorimetrically. The assay is performed so that the amount of peroxidase activity is directly proportional to the amount of interferon in the interferon-containing extract. Induction of strain pAO3/GW1000 by a temperature upshift (30° to 40°c) for periods of 5 hours consistently gives values of interferon of 100 fold or more over the uninduced levels. References 1. Brey, R.N. 1982. Fragmentation of Colicins A and El by Cell Surface Proteins. J. Bact. 149. 306- 315. 2. Chen, H-Z, and Zubay, G. 1983. Analysis of ColEl Expression in Vitro After Chromsome Fragmentation. J. Bact. ULi. 650-655. 3. Davis, B.D., Dulbecco, R., Eisen, H.N., and Ginsberg, H.S. 1980. Microbiology, pp. 319-321, Harper and Row, New York. 4. Dougan, G. , and Sherratt, D. 1977. The Transposon Tnl as a Probe for Studying ColEl Structure and Function. Molec. Gen. Genet. 151, 151-160. 5. Ebina, Y., Kishi, F., and Nakazawa, A. 1982. Direct Participation of LexA Protein in Repression of Colicin El Synthesis. J. Bact. 150, 1479-1481. 6. Ebina, Y., Takahara, Y. , Kishi, F., Nakazawa, A., and Brent, R. 1983. LexA protein is a repressor of the colicin El gene. J. Biol. Chem 258. 13258- 13261. 7. Ebina, Y. , and Nakazawa, A., 1983. Cyclic AMP- dependent Initiation and -dependent Termination of ColicinEl Gene Transcription. J. Biol. Chem. 258. 7072-7078. 8. Emerick, A.W. 1982. Read-through Transcription from a Derepressed Tn3 Promoter Affects ColEl Functions on a ColEl: :Tn3 Composite Plasmid. Mol. Gen. Genet. 125., 408-417. 9. Hakkart, M.J.J., Veltkamp, E., and Nijkamp, J.J. 1981. Protein H Encoded by Plasmid CloDF13 In¬ volved in Lysis of the Bacterial Host. I. Lo¬ calization of ' the Gene and Identification and Subcellular Localization of the Gene H Product. Mol. Gen. Genet. 121, 318-325. 10. Hakkart, M.J.J., Veltkamp, E., and Nijkamp, H.J.J. 1981. Protein H Encoded by Plasmid CloDF13 In¬ volved in Lysis of the Bacterial Host. II. Func¬ tions and Regulation of Synthesis of the Gene H Product. Mol. Gen. Genet. 183. 326-332. 11. Inselburg, J. 1979. Studies of Colicin Induction with an imm " col + Mutant of the Plasmid. Molec. Gen. Genet. 121, 333-338. 12. Kennedy, C.K. 1971. Induction of colicin produc¬ tion by high temperature or inhibition of protein synthesis. J. Bact. 108. 10-19. 13. Krueger, J.H., Elledge, S.J., and Walker, G.C. 1983. Isolation and Characterization of Tn5 Inser¬ tion Mutations in the lexA Gene of Escherichia Coli. J. Bact. l≤ , 1368-1378. 14. Little, J.W. , and Mount, D.W. 1982. The SOS Regu¬ latory System of Escherichia Coli. Cell ___, 11-22. 15. Maniatis, T. , Fritsch, E.F., and Sambrook, J. 1982. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, Long Island, New York. 16. Nakazawa, A. and Tamada, T. , 1972. Stimulation of Colicin El Synthesis by Cyclic 3*, 5* - Adenosine Monophosphate in Mitomycin C - Induced Escherichia Coli. Biochem. Biophys. Res. Comm. 4JL, 1004- 1009. 17. Ohkubo, H., Shimada, K., and Takaga, Y. 1979. Isolation and Characterization of a mutant ColEl Plasmid That Allows Constitutive Colicin El Synthe¬ sis; J. Bact. H2., 311-315. 18. Oka, A., Nomura, N. , Morita, M., Sugisaki, H., Sugimoto, K., and Takanami, M. , 1979. Nucleotide Sequence of Small ColEl Derivatives: Structure of the Regions Essential for Autonomous Replication and Colicin El Immunity. Mol. Gen. Genet. 171, 151-159. 19. Ozeki, H., Stocker, B.A.D., and Margerie, H. de. 1959. Production of colicine by single bacteria. Nature 1 , 337-339. 20. Patient, R.K. 1979. Characterization of In Vitro Transcription Initiation and Termination Sites in ColEl. Nucleic Acids Research 6_, 2647-2665. 21. Sabik, J.F., Suit, J.L., and Luria, S.E. 1983. cea-kil Operon of the ColEl Plasmid. J. Bact. 153, 1479-1485. 22. Schwartz, S.A. and Helinksi, D.K., 1971. Purifica¬ tion and characterization of colicin El. J. Biol. Chem. 2 , 6318-6327. 23. Shafferman, A., Cohen, S. and Flashner, Y., 1978. A DNA Segment Within. the Colicin El Structural Gene on ColEl Affecting Immunity to Colicin. Molec. Gen. Genet. 1£1, 259-264. 24. Shafferman, A., Flashner, Y., and Cohen, S. 1979. ColEl DNA Sequences Interacting in cis, Essential for Mitomycin C Induced Lethality. Molec. Gen. Genet. 176. 139-146. 25. Suit, J.L., Fan, M-L. J. , Sabik, J.K., LaBarre, R., and Luria, S.E. 1983. Alternative forms of lethal¬ ity in mitomycin C induced bacteria carrying ColEl plasmids. Proc. Natl. Acd. Sci. _\__, 579-583. 26. Tessman, E.S. and Peterson, P.K. 1980. ϋf-depen- dent induction of colicin El, prophage lambda, and filamentation in Escherichia coli K-12. J. Bact. 143. 1307-1317. 27. Tilly, K., McKittrick, N. , Zulicz, M. and Georgopoulos, C. 1983. The dnaK Protein Modulates the Heat-Shock Response of Escherichia coli. Cell 14., 641-646. 28. Yamada, M. , Ebina, Y. , Mujata, T. , Nakazawa, T. , and Nakazawa, A., 1982. Nucleotide Sequence of the Structural Gene for Colicin El and Predicted Struc¬ ture of the Protein. Proc. Natl. Acad. Sci. USA 21, 2827-2831. 29. Yamada, M., Miki, T. and Nakazawa, A., 1982. Translocation of Colicin El Through The Cytoplas- mic Membrane of Escherichia coli. FEBS Letters ]___,, 465-468. 30. Yang, H.L., Ivashkiv, L., Chen, H.Z., Zubay, G. and Cashel, M., 1980. Cell-free coupled transcription translation system for investigation of linear DNA segments. Proc. Natl. Acad. Sci. U.S.A. ___, 7029- 7053. 31. Dworkin-Rastl, E., Swetly, P. and Dworkin, M., 1983. Construction of expression plasmids producing high levels of human leukocyte-type interferon in Escherichia coli. Gene 21,237-248.

Claims: What is claimed is: 1. A double-stranded DNA plasmid adapted for expres¬ sion of a foreign polypeptide in a suitable ___ coli cell carrying a temperature inducible RecA protease gene comprising in 5' to 3' order: a DNA sequence which includes a temperature induc¬ ible promoter, including its associated ribosomal 10 binding site, which promoter is derived from a bacterial plasmid and which promoter normally directs expression of a bacteriocin selected from the group consisting of colicin and the peptide encoded by the clo gene; 15 a DNA sequence which encodes .an ATG initiation codon; a DNA sequence which encodes the foreign polypep- 20 tide; a DNA sequence that is a gene which facilitates high copy number replication of the plasmid; 25 a DNA sequence that is a gene associated with a selectable or identifiable phenotypic trait mani¬ fested when the plasmid is present in the host cell; and 30 a DNA sequence that contains an origin of replica¬ tion from a bacterial plasmid capable of autonomous replication in the host cell. 2. A plasmid of claim 1, wherein the temperature 35. inducible RecA protease gene is a mutant tif gene. 3. A plasmid of claim 1, wherein the promoter is the col promoter from plasmid ColEl. 4. A plasmid of claim 1, wherein the promoter is the col promoter from plasmid CloDF13. 5. A plasmid of claim 3, wherein the ATG initiation codon is derived from the coding sequence of the Cea protein of plasmid ColEl. 6. A plasmid of claim 1, wherein the gene which facil¬ itates high copy number replication is a mutant yorn gene. 7. A plasmid of claim 1, wherein the phenotypic trait is drug resistance. 8. A plasmid of claim 7, wherein the drug resistance is ampicillin resistance. 9. A plasmid of claim 7, wherein the drug resistance is tetracycline resistance. 10. A plasmid of claim 1, wherein the origin of repli- cation is derived from ColEl. 11. A plasmid of claim 1, wherein the foreign polypeptide is chloramphenicol transacetylase. 12. A plasmid of claim 1, wherein the foreign polypeptide is a hybrid polypeptide comprising at least a portion of chloramphenicol transacetylase. 13. A plasmid of claim 1, wherein the foreign polypeptide is an interferon. 14. A plasmid of claim 13, wherein the foreign polypeptide is IFN- A. 15. A suitable ___, coli cell which includes the plasmid of claim 1. 16. A method for producing a foreign polypeptide which comprises growing E__ coli cells of claim 15 under suitable conditions such that the gene which en¬ codes the polypeptide is expressed and the polypeptide produced and recovering the polypeptide so produced. 17. A method of claim 16, wherein the E__ coli cells are grown at a temperature sufficient to induce en¬ hanced expression of the polypeptide. 18. A foreign polypeptide produced according to the method of claim 16. • 19. A hybrid polyeptpide comprising at least a portion of chloramphenicol transacetylase produced according to the method of claim 16. 20. Interferon-α A produced according to the method of claim 16.

Inventors: ZUBAY GEOFFREY L, ZUBAY, GEOFFREY, L.
Applicants: UNIV COLUMBIA, THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF
Application year: 1986
Main classification: C12P21
IPC codes: C07K14, C12N15