GB2135678A

GB2135678A - Ribosomal RNA operon and method of incorporating same in plasmids and bacteria

Info

Publication number: GB2135678A
Application number: GB08403900A
Authority: GB
Inventors: Edward Alan Morgan
Original assignee: Research Foundation of the State University of New York
Current assignee: Research Foundation of the State University of New York
Priority date: 1983-02-17
Filing date: 1984-02-14
Publication date: 1984-09-05
Also published as: GB8403900D0; GB2135678B

Abstract

An operon comprising a bacterial ribosomal RNA promoter, a bacterial ribosomal RNA promoter termination sequence and an intermediate sequence between said promoter and said promoter termination sequence, wherein fewer than 2,000 sequential base pairs of said operon sequentially naturally occur, an operon having an rRNA read-through sequence and an operon having an rRNA promoter termination sequence together with an unspecified promoter. Plasmids and bacteria containing the operons are also described.

Description

SPECIFICATION Ribosomal RNA Operon and Method of Incorporating Same in Plasmids and Bacteria Historically, man has manipulated the genetic structure of microorganisms, plants and animals primarily by selection of desirable natural mutants of living organisms or by cross fertilization of organisms followed by selection of a desirable strain. Such methods have given us desirable microorganisms such as the yeasts that are used in baking and that are used in fermentation for the manufacture of beverages such as beer and wine. Other such microorganisms produce antibiotics and others are responsible for production of certain foods such as pickles and sauerkraut. Other selected microorganisms are used in desirable degradation processes such as the microorganisms used in waste disposal.Such genetic manipulation has not been limited to microorganisms and has also resulted in improved species of plants and animals such as hybrid food crops and animals having desirable meat, milk or egg production.

It has recently become technically possible to move genes from one cell type to another (usually from plants and animals to bacteria) by use of techniques developed in the study of molecular biology of prokaryotic cells (cells which are prenucleus) and eukaryotic cells (cells which contain a nucleus which are usually cells of higher organisms). Such a result is exceedingly desirable since man no longer has to rely on the appearance of spontaneous mutants.It is now possible to transfer genes from higher plants or animals (eukaryotic cells) to place them into bacterial cells (prokaryotic cells) by means of a vector. "Genes", as used herein, means a segment of DNA (deoxyribonucleic acid) which carries genetic information. "Vector", as used herein, is any composition or structure which can carry genes into the cell for replication (manufacture of additional similar DNA fragments), and also usually for transcription (manufacture of an RNA segment) and translation (manufacture of a polypeptide, usually a protein, from the information contained in an RNA segment). The vector is usually a phage virus to which the gene has been attached or a plasmid (circular rings of DNA which are relatively small in size when compared with the length of chromosomal DNA).Chromosomal DNA is a long string of DNA which contains most of the genetic information in a cell.

The structure of DNA and RNA (ribonucleic acid) is based upon the arrangement of bases along alternating residues of certain sugars and phosphate. In the case of DNA, the alternating sugar residue is deoxyribose and in the case of RNA, the alternating sugar residue is ribose. The bases in the case of DNA are radicals of the chemicals thymine, cytosine, adenine, or guanine. In RNA, the bases are uracil, cytosine, adenine, or guanine. It is the arrangement of the bases which determines the genetic information. RNA is usually either messenger RNA (mRNA) which carries information from the DNA as an intermediary in the formation of polypeptides or is a transfer RNA (tRNA) which acts between the messenger RNA and amino acids to combine the amino acids in a particular sequence based upon the sequence and information contained in the messenger RNA.The transfer RNA seems to attach to both the messenger RNA and to a particular amino acid thus arranging the amino acids in the proper order.

Each amino acid has its own transfer RNA which recognizes only particular sequences along a messenger RNA thus making certain that the old sequence in the messenger RNA is properly translated into the appropriate amino acid. Sequences of amino acids (polypeptides), usually a protein, may have many different functions depending upon the particular sequence. Such polypeptides may for example act as enzymes which are organic catalysts, hormones which act as regulators, antibodies which are produced in response and defense against foreign materials called antigens, structural and contractile proteins and blood and plasma proteins including albumins, fibrinogen vital in blood clotting and hemoglobin which carries oxygen.

It has been known that bacterial plasmids such as those found in the bacterial genuses salmonella, shigella, proteus, bacillus, pseudomonoas, streptomyces and all gram negative enteric bacteria such as Escherichia coli could be cleaved, new genetic codes (usually for a desired polypeptide) could be then inserted into the plasmid and the plasmid could then be replaced into a bacteria for replication generally followed by transcription and translation to form the desired polypeptide structure. "Cleaved", as used herein, is intended to mean cleaved or broken. "Restricted", is intended to mean cleaved by any means but usually by use of a restriction enzyme.

It has been further recognized that one method for cleaving is by utilizing restriction enzymes followed by insertion of the desired DNA sequence. In particular, such methods are set forth in "Molecular Cloning a Laboratory Manual" by Maniatis et al, published 1 982 by Cold Spring Harbor Laboratory. Table 4.1 beginning on page 100 of the manual lists numercus restriction enzymes and the sequence and location of cleavage.

Although such procedures are well known to those skilled in the art, there remain serious problems with respect to the utilization of inserted plasmids for replication of the plasmid, transcription of the inserted sequence to form the appropriate RNA and translation of messenger RNA to the appropriate polypeptide.

In particular, it is highly desirable to utilize a plasmid which will replicate at a high rate so that multiple copies of the desired DNA sequence will be obtained. Furthermore, it is highly desirable that the plasmid contain a promoter upstream from the desired DNA sequence which will strongly promote transcription of the DNA sequence to the appropriate transfer RNA or messenger RNA sequence. It is further desirable to obtain transcription without premature termination which often occurs especially when eukaryotic DNA sequences are inserted into plasmids which are utilized in prokaryotic bacterial cells. This is true since eukaryotic DNA sequences often contain sequences of bases which do not cause termination in the eukaryotic organism but do cause termination in a prokaryotic organism such as a bacteria.

In the prior art, it was recognized that ribosomal RNA operators were strong operators but there was extreme difficulty in making the operators function outside of their natural location. It has recently been found that an operon containing ribosomal RNA operators could be made to function provided that the parts of both the beginning and end of the operon was utilized, i.e. especially the promoter and promoter termination sequence. The "promoter termination sequence" is the termination sequence properly associated with termination of transcription commencing at the promoter.The promoter termination sequence is in general the sequence which is desired for termination of transcrption commencing at the promoter rather than premature termination which may be caused by undesirable nonsense codons or premature, usually undesirable, sequences which act as terminators located between the promoter and the promoter termination sequence. The promoter termination sequence has also been termed the "transcription termination sequence".

A complete ribosomal RNA operon, including the natural sequence between the promoter and promoter termination sequence, was inserted in the prior art into a plasmid (see Morgan et al "Some rRNA Operons in E. coll have tRNA Genes at their Distal Ends". Cell, Volume 13, pages 335-344 1978). Such a plasmid had little utility since the plasmid was exceedingly large, i.e., about 27,000 base pairs long and had an exceedingly large number of base pairs in the ribosomal RNA operon including operators, promoter, promoter termination sequence, and intermediate sequence (the sequence between the ribosomal RNA promoter and promoter termination sequence). The entire operon had a size of about 5,800 base pairs.

The large size of the plasmid made it unsuitable for genetic engineering purposes since plasmids of such large size are generally rapidly lost from a bacterial cell and since plasmids of such large size replicate slowly. In addition, the long operon made cleavage of the intermediate sequence, followed by insertion of a new desired DNA sequence, impractical. Such impracticality partially results due to the large number of restriction nuclease sites in a such a long intermediate sequence and in such a large plasmid. Furthermore, such sites and their location are difficult to characterize. In addition, the insertion of an additional sequence into such a large plasmid would make the plasmid even more unstable in the bacterial organism.

it has further been recently discovered that large operons having a ribosomal RNA promoter, ribosomal RNA read through sequence and ribosomal RNA promoter termination sequence could be provided with an intermediate sequence in chromosomal DNA (not in a plasmid). These experiments demonstrated that the read through sequence of the ribosomal RNA could in fact permit transcription through a sequence which would have normally been a termination sequence. (See Morgan "Insertions of Tn10 into an E. coliribosomal RNA Operon are incompletely Polar", Volume 21 of Cell, pages 257 265, 1980 and Brewster et al "Tn9 and íS1 Inserts in a Ribosomal Ribonucleic Acid Operon of Escherichia coli are incompletely Polar", Journal of Bacteriology, December 1981, pages 897--903.

These insertions, however, merely provided chloroamphencol resistance and tetracycline resistance to bacteria The insertions had no other utility and since they appeared in chromosomal DNA, replicated very slowly. The only utility therefore was merely for the survival of the microorganisms in the presence of the particular antibiotic. Furthermore since the operons were large, they had all of the problems associated with large operons as previously discussed.

Brief Description of the Invention In accordance with the invention, there is a provided an operon comprising a promoter, a promoter termination sequence and usually an operator and an intermediate sequence between the promoter and promoter termination sequence. The operon contains at least one DNA sequence from a bacterial ribosomal RNA operon selected from a ribosomal RNA promoter, a ribosomal RNA promoter termination sequence or a ribosomal RNA read through sequence. Such an operon is referred to herein as a ribosomal RNA operon. Such a sequence is referred to herein as a "ribosomal RNA functional group". When a ribosomal RNA promoter is present, a ribosomal RNA promoter termination sequence is also usually present.Fewer than 2,000 sequential base pairs, preferably fewer than 1,500 sequential base pairs and most preferably fewer than 1,000 sequential base pairs of the operon naturally sequentially occur either in a plasmid or elsewhere. "Naturally sequentially occur" means to naturally occur in an operon with a ribosomal RNA functional group. "Naturally occurring sequence" means the sequence naturally sequentially occurs in an operon with a ribosomal RNA functional group.

The operons can include an intermediate sequence between the promoter and promoter termination sequence containing a subsequence which does not naturally occur between the promoter and the promoter termination sequence. The intermediate sequence may be very long including a chain in a naturally occurring sequence of up to 2,000 base pairs in addition to even longer sequences which do not naturally occur. The intermediate sequence can also be very short including only a few base pairs. "Does not naturally occur", as used herein, means that the sequence does not naturally appear in the particular order at the particular location. "Naturally sequentially occur", means the occurrence of the base pairs in their natural unaltered order.

The sub-sequence which does not naturally occur in the operon, is usually a sequence which either comprises a eukaryotic DNA sequence which does not naturally occur in bacteria or comprises a prokaryotic DNA sequence which does not naturally occur between the ribosomal RNA promoter and the termination sequence. When the intermediate sequence comprises a prokaryotic DNA sequence, the sequence usually codes for a protein product having a utility other than solely for the survival or selection of a bacteria in the presence of an antibiotic.

In this connection, the read through sequence is often desirable. The read through sequences in certain operons of the present invention can cause transcription of a DNA sequence, intermediate the promoter and promoter termination sequence through a sequence which normally would terminate more of the transcription of the intermediate sequence in a bacteria in the absence of the read through sequence. Such a read through sequence is desirably used in conjunction with a ribosomal RNA promoter but may alternatively be used with other promoters.Since the read through sequence permits transcription through sequences which would otherwise act as terminators in bacteria but either do not act as terminators or act as terminators with reduced strength when the read through sequence is present, the read through sequence is effective to assist transcription of certain eukaryotic sequences which are easily read in higher life forms but which are either not transcribed or transcribed with reduced efficiency in bacteria in the absence of the read through sequence.

Desirably, the operon further contains at least one and preferably a plurality of restriction nuclease sites between the promoter and the promoter termination sequence. The restriction nuclease sites permit easy insertion of desired intermediate sequences for replication and translation.

Due to its strength, the promoter is preferably a ribosomal RNA promoter and the termination sequence is preferably a ribosomal RNA promoter termination sequence. The ribosomal RNA promoter and promoter termination sequences can, however, be used independently in conjunction with other promoters or termination sequences.

One special such operon comprises a bacterial ribosomal RNA promoter, a bacterial ribosomal RNA promoter termination sequence and an intermediate sequence between the promoter and the promoter termination sequence, wherein the operon has a length of fewer than 2,000 base pairs, preferably fewer than 1 ,000 base pairs, and wherein the operon contains at least one restriction nuclease site beween the promoter and the termination sequence. This operon is particularly good for genetic engineering purposes since the small operon coupled with the restriction sites makes stable insertion of other desired sequences into the operon relatively easy.

The invention further comprises plasmids incorporating the operons of the present invention. For the reasons previously discussed, the inclusion of such operons into plasmids gives rise to plasmids which are very desirable for genetic engineering purposes.

A plasmid in accordance with the present invention, containing an operon of the present invention, may be of any size but preferably contains fewer than 25,000 base pairs, more preferably fewer than 1 5,000 base pairs and most preferably fewer than 10,000 base pairs. This is true since smaller plasmids are more stable in the cells, usually replicate faster, and usually are easier to manage when desired sequences are being inserted.

The invention further comprises bacteria containing the novel piasmids of the invention and the methods for making the plasmids.

Detailed Description of the Invention As previously discussed, the ribosomal RNA promoter is a very strong promoter which increases the efficiency of replication and thus increases the efficiency of translation. The promoter can act to transcribe-DNA sequences to the desirable RNA sequence such as transfer RNA (tRNA) or messenger RNA (mRNA). The strong promotion of transcription of DNA sequence to messenger RNA also tends to increase the efficiency of translation to the appropriate polypeptide chain from the messenger RNA.

The strength of ribosomal RNA promoters in systems which have little utility as easy genetic engineering tools has previously been demonstrated. See e.g., Kjeldgaard et al "Regulation of Biosynthesis of Ribosomes, in Nomura, M., Tissieres, A., and Lengyel, P. (Eds.), Ribosomes", Cold Spring Harbor Laboratories, N.Y. pages 369-392.

In E. coli ribosomal RNA transcription units are known and are identified as rrnA through H from seven different bacterial chromosomal locations. None of such ribosomal RNA transcription units naturally occur on a plasmid (although such units are known to occur on an episome which is an extremely large plasmid-like ring approximately one-half the size of a chromosome). For a discussion of such ribosomal RNA genes (for transcription to ribosomal RNA's) see Morgan "Ribosomal RNA Genes in Escherichia coli" The Cell Nucleus, Volume X, copyright 1982 by Academic Press Inc. The preferred ribosomal promoter is an rrnC promoter.The preferred bacterium for utilizing the plasmids and DNA sequences in accordance with the present invention is Escherichia coli. The promoter is preferably a ribosomal RNA promoter and the termination sequence is preferably a ribosomal RNA promoter termination sequence; however, other promoters and termination sequences can be used in any plasmid containing the ribosomal RNA read through sequence in accordance with the present invention.

As previously discussed, the read through sequence can cause transcription of a DNA sequence, intermediate the promoter termination sequence, through a sequence which normally would terminate more of the transcription of the intermediate sequence in a bacterium in the absence of the read through sequence. In other words, the ribosomal RNA read through sequence, causes transcription through DNA sequences which would otherwise stop or reduce transcription of DNA downstream. The effectiveness of the read through sequence has been previously demonstrated in systems which are not suitable for easy use in recombinant DNA procedures.Again, see "Insertions of Tn 10 into an E. coli Ribosomal RNA Operon are incompletely Polar", Morgan, Cell, Volume 21, pages 257-265, August 1980 and "Tn9 and IS Inserts in a Ribosomal Ribonucleic Acid Operon of E. coli are incompletely Polar" Brewster et al, Journal of Bacteriology, December 1981, pages 897-903. These papers demonstrate that the normal termination sequences in Tn 10 and Tn9 have reduced effectiveness when placed downstream from a ribosomal RNA read through sequence.

The read through sequence seems particularly effective in permitting transcription through nonsense codons (DNA sequences which are not recognized for insertions of a nucleic acid) and Rho dependent termination sequences which require Rho, a protein involved in termination of transcription in E. coli, to reduce or abolish the antitranscription (polar) effect that premature nonsense codons or mutations have on expression of downstream genes. The read through sequence similarly seems to have some effect on Rho independent termination sequences such as the sequence contained in Tn 10.

It is understood that Tn 10 and Tn9 are transposons, i.e., a piece of DNA that inserts itself, or a mobile DNA element. The transposon may carry a gene to give a different property. Tn9 is a particular transposon which carries a gene for chloroamphencol resistance and Tn 10 is a particular transposon that carries a gene for tetracycline resistance.

Such read through is particularly important when sequences which are inserted are certain eukaryotic sequences which can be read in higher life forms but which often can not be read in bacteria due to the existence of DNA sequences which are not recognized or transcribed by the bacteria.

Sub-sequences which do not naturally occur in conjunction with ribosomal RNA operons which can be inserted into the intermediate sequence between the promoter and promoter termination sequence can be essentially any DNA sequence which can be transcribed in the bacteria. When a ribosomal RNA read through sequence is present, greater flexibility occurs since more of such subsequences can be transcribed.

Examples of such intermediate sequences are those which code for messenger RNA for structural proteins, contractile proteins, antibodies, enzymes, blood proteins including gamma globulins, albumins, fibrinogens and hemoglobin and for polypeptide hormones. Specific examples of such polypeptide hormones are insulin, human growth hormone and interferon. Other such hormones are ACTH, luteinizing hormones, secretin, gastrin, parathyroid hormone and various hormone releasing hormones. Examples of such enzymes are the digestive enzymes including pepsin, trypsine, chymotrypsin, elastase, carboxypeptidase, peptidase, amylase, maitase, lactase, sucrase, and lipases.

Other such enzymes are those responsible for DNA replication, transcription to RNA and translation from RNA to polypeptides and those enzymes such as restriction enzymes responsible for breaking down or cleaving DNA or RNA. Many other such enzymes could be mentioned since enzymes control essentially all biological chemical functions including chemical synthesis and chemical breakdown.

In accordance with one of the methods of the invention, a plasmid of the invention can be made by treating another small plasm it of the present invention with an appropriate restriction enzyme. The small plasmid shouid have at least one restriction nuclease site between the promoter and promoter termination sequence. After such treatment, the treated plasmid is combined with a desired DNA sequence for replication and transcription, having ends suitable for combination with the restricted sites of the plasmid. The resulting plasmid is then introduced into host bacteria and the bacteria is grown in a suitable medium to produce or replicate the desired plasmid.Such procedures, once the originai plasmid of the present invention is available, are well known to those skilled in the art and are for example, set forth in "Molecular Cloning a Laboratory Manual" by Maniatis et al, published by Cold Spring Harbor Laboratory, 1982.

Such a method for making a certain type of plasmid, in accordance with the present invention, comprises treating a particular plasmid with a restriction enzyme. The particular plasmid contains an operon comprising a bacterial ribosomal RNA promoter, a bacterial ribosomal RNA promoter termination sequence and an intermediate sequence between the promoter and the promoter termination sequence. The operon has a length of fewer than 2,000 base pairs and desirably fewer than 1,000 base pairs. The operon contains at least one restriction nuclease site between.the promoter and the promoter termination sequence within the intermediate sequence. After treatment with the appropriate restriction enzyme, the treated plasmid is combined with a DNA sequence having ends suitable for combination with the restricted sites of the plasmid. The resulting plasmid is then introduced into a host bacteria and the bacteria is grown in a suitable medium to produce the desired plasmid. The same method may be used whether or not the starting particular plasmid contains intermediate sequences such as the read through sequence.

The invention further includes a method for making a plasmid of the present invention containing a ribosomal RNA promoter and a ribosomal RNA promoter termination sequence and a small intermediate sequence, by treating a plasmid of the prior art. Such plasmids of the prior art were too large for easy use in genetic engineering and the intermediate sequence, between the promoter and promoter termination sequence, was too large for practical use for genetic engineering.

In accordance with the present invention, the method comprises treating such a large plasmid with a restriction enzyme, permitting the cleaved plasmids to recombine, introducing resulting plasmids into bacteria, growing colonies of the resulting bacteria, obtaining purified plasmids from such colonies, analyzing the plasmids, selecting the appropriate plasmid and introducing the appropriate plasmid into a host bacteria to replicate the desired plasm id. In the event that the selected plasmid is still too large for practical use, the procedure may be repeated by again treating with a restriction enzyme, followed by recombination, reintroduction, growing of the colonies and obtaining purified plasmids followed by analysis and selection.While the technology to practice the individual steps of the method were well known to those skilled in the art, e.g., as described in "Molecular Cloning a Laboratory Manual" (previously cited) and in "Advanced Bacterial Genetics" by Davis et al, published by Cold Spring Harbor Laboratory, 1 980, the combination of steps and of their use upon the particular starting materials were not previously known and were not practiced.

A method for making a certain type of plasmid in accordance with this method is as follows: a) A plasmid containing a ribosomal RNA operon containing in excess of 2,000 base pairs and containing a ribosomal RNA promoter and a ribosomal RNA termination sequence, is treated with a restriction enzyme. The cleaved plasmids are then permitted to recombine. The recombined plasmids are then introduced into bacteria and colonies of the resulting bacteria are grown. Purified plasmids are then obtained from such colonies and are analyzed.

b) Step a) is repeated as necessary until the appropriate plasmid is found containing the desired ribosomal RNA operon which contains fewer than 2,000 base pairs; and c) The appropriate plasmid is introduced into a host bacteria to reproduce the desired plasm id.

Plasmids of the invention containing an intermediate subsequence which does not naturally occur may similarly be made by first introducing a desired sequence into the prior art plasmid of a size which is impractical for common use.

In accordance with such a method, a desired sequence, which does not naturally occur, is combined with a transposon, the transposon is introduced into a vector such as a phage or another plasmid, and the desired vector is then selected and purified by methods known to those skilled in the art. The vector is then introduced into a bacteria containing a prior art plasmid of large size having a ribosomal RNA promoter and a ribosomal RNA promoter termination sequence. A bacteria containing the resulting plasmid comprising a ribosomal RNA promoter, a ribosomal RNA promoter termination sequence and an intermediate sequence is then selected and the plasmid is purified. If the selected plasmid is too large or contains more than the desired number of base pairs in a natural sequence in the ribosomal RNA operon, the plasmid is further treated with a restriction enzyme as previously described.Again, the technology to practice the individual steps of this method of the present invention are known; however, the combined steps and their use upon the particular starting materials are not known for the purpose of obtaining a plasmid or operon of the present invention.

Plasmids of the present invention can also be made by cloning an operon of the invention onto a plasmid which did not previously contain such an operon. This method will be further discussed in the Example.

Operons of the present invention having fewer than 2,000 sequential base pairs in a naturally occurring sequence can be isolated by restricting a plasmid of the present invention having restriction nuclease sites on each side of said operon with an appropriate restriction enzyme and isolating the restricted operon in accordance with known procedures. Desirably such restriction sites are within about 50 base pairs of the ends of the operon.

Replicating the plasmid, transcribing the plasmid and associated desired sequences into the appropriate RNA's and translating messenger RNA's into polypeptides (including proteins) is simply accomplished by growing a bacterium containing a desired plasmid of the invention in a suitable medium. Such growth techniques are well known to those skilled in the art as are preparation techniques for removing and purifying desired plasmid, RNA and polypeptide products. The invention includes the method for production of RNA which contains its DNA code in a plasmid which comprises growing a bacteria containing a plasmid of the invention in a suitable medium. The invention also includes the method wherein the RNA is messenger RNA and the bacteria produce a polypeptide by translation of the messenger RNA.

In accordance with the present invention, a natural sequential length between a ribosomal RNA promoter and promoter termination sequence in the operon is substantially reduced which permits easy characterization of the reduced sequence and permits stable insertion of the plasmid into a bacterium. Furthermore, such reduction of the natural sequence length between the promoter and promoter termination sequence and reduction of plasmid size permits formation of a stable plasmid with a relatively large insertion of a sequence which does not naturally occur between the promoter and terminator. In addition, the utilization of a ribosomal RNA promoter results in a very strong promotion of sequences intermediate the promoter and promoter termination sequence to the appropriate RNA.

The following example serves to illustrate and not limit the present invention. Unless otherwise indicated, all recombinent DNA procedures were performed as described by Maniatis et al "Molecular Cloning a Laboratory Manual" published by Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982, at pp. 1-545.

EXAMPLE Internal DNA was removed from the complete rrnC operon on the large pLC22-36 plasmid containing a rrnC ribosomal RNA promoter and ribosomal RNA termination sequence by restriction with EcoR1 restriction enzyme. The obtaining of pLC22-36 plasmid is discussed by Morgan et al "Some RNA operons in E. coli have tRNA genes at their distal ends", Cell, vol. 13, pp. 335-344. This deleted restriction nuclease sites that would hinder subsequent replication (cloning) and demonstrated the viability of bacterial strains harboring a modified rrnC operon. (Step 1 in Figure 1). The resulting plasmid, pRS 1, has a deletion of rrn DNA extending from the middle of the 1 6S rRNA gene to seven nucleotides before the tRNAaSP gene.The tRNAtrP gene is eight base pairs (bp) downstream of the tRNAasP gene and is immediately followed by sequences that participate in the base-paired stem of the transcription termination sequence. A Sallfragment of pRS1 was then cloned onto pBR322 plasmid to obtain pRS2 plasmid (Figure 1). More DNA was removed downstream of the rrnC operon (Step 3 of Figure 1) to obtain plasmid pRS3. pRS3 was cut with Bail and Hpal and ligated All resulting plasmids that could be recovered after transformation of bacterial strain EM322 unexpectedly contained less DNA than predicted from circularization of the plasmid at the Ball and Hpal ends.The plasmid with the least DNA deleted, pRS4 (Figure 2), was analysed extensively by restriction nuclease digestion, which revealed that the unexpected deletion in pRS4 included only E. coli chromosomal DNA upstream of the Bcil site in rrnC. To further define the extent of the deletion the Hpa 1-EcoRi fragment from pRS1 containing the rrnC promoters was labeled with 32pi at the EcoR1 end and annealed to pRS4. A 975 base single stranded fragment was protected from S1 nuclease digestion in this hybrid, demonstrating that the deletion terminates in or near the rrnC P1 promoter, but leaves the rrn P2 promoter and downstream DNA sequences intact.This plasmid is important because it expresses very strongíy from a single rrn promoter and may be useful in studies of regulation of rrnC promoters.

Certain plasmids containing both the P1 and the P2 rrnC ribosomal RNA promoters were unstable or lethal when the plasmids are too small; however, it was discovered that the Hpal--Bglllfragment containing the rrnC region of pRS1 could be cloned into Pvull-BamHl cut pBR322 when we used selections for tRNAtrP or nonsense suppressor genes of tRNAtrp, and the resulting plasmids, pLB5, pLB5-Su7, pLB5-Su8 (Figure 3), did not undergo problematical rearrangements during routine manipulations. This was surprising, as a DNA sequence between the Ball and Pvull sites of pBR322 codes for a portion of a protein needed to repress copy number. This region was retained during construction of pRS4 but removed during construction of pLB5.Therefore, the rrnC region is stable on pLB5 even though pLB5 probably has a higher copy number than pRS4. The region in which the Hpa 1Bg 111 fragment is inserted may contribute to plasmid instability or lethality, and that the rrn region itself does not invariably lead to plasmid instability or lethality.

pRS4 (Figure 2) has approximately 1 80 base pairs of leader DNA between the P2 promoter and the start of the 1 6S rRNA gene. It was desirable to retain this DNA because of possible involvement in regulation. Most of the remaining 680 base pairs of the 1 6S rRNA gene were removed by digestion of pRS4 with Bc 11 and Xmal (an isoschizomer of Sma 1), followed by filling in the protruding ends with Klenow fragment of DNA polymerase 1 and blunt end ligation to regenerate a Smal site, giving rise to pRS5 (Figure 2). pRS5 has 20 base pairs of DNA from near the 5' end of the 1 6S rRNA gene and 66 base pairs of DNA from near the middle of the 1 6S rRNA gene.This deletion therefore moves certain restriction nuclease sites nearer to the rrn promoter, reduces the size of the transcript to near the minimum needed to retain all desired features of the rrn operon, and does not interfere with the useful properties of the rrnC tRNA genes. Similar deletions could be introduced into the other plasmids described herein.

Plasmids pRS1, pRS2, pRS3, pRS4, pRS4-Su7, pRS5, pRS5-Su7, pRS5-Su8, pLB5, pLB5- Su7 and pLB5-Su8 are all plasmids in accordance with the present invention which contain a ribosomal RNA rrnC promoter and rrnC promoter termination sequence with read through sequence.

pRS1 has a total size of about 23,000 base pairs and a ribosomal RNA operon of about 1,300 base pairs. pRS2 has a total size of about 11,300 base pairs and a ribosomal RNA operon of about 1,300 base pairs. pRS3 has a total size of about 7,200 base pairs and a ribosomal RNA operon of about 1,300 base pairs. pRS4 has a total size of about 4,700 base pairs and a ribosomal RNA operon of about 1,300 base pairs. pRS5 has a total size of about 4,100 base pairs and a ribosomal RNA operon of about 700 base pairs. pLB5 has a total size of about 4,600 base pairs and a ribosomal RNA operon of about 1300 base pairs. By comparison, starting plasmid pLC22-36 has an overall size of about 27,000 base pairs with a ribosomal RNA operon of about 5,800 base pairs.

Restriction nuclease sites for the pRS4, pRS5, pRS4-Su7, pRS5-Su7, and the pRS5-Su8 plasmids are as shown in Figure 2 and for the pLB5, pLB5-Su7, and pLB 5--Su8 plasmids are as shown in Figure 3. Restriction nuclease sites marked with an asterisk are predicted from the sequences of rrnB, rrnC and pBR322, but have not been tested experimentally. All other restriction nuclease sites have been experimentally confirmed. rrn DNA is represented by the white enclosed area and non-rrn chromosomal DNA by the thin line. pRS1, pRS2, pRS3, pRS4, pRS5, and pLB5 plasmid designations as used herein and in the claims are intended to include the Su7 and Su8 derivatives as well as those other modifications which can be easily derived by those skilled in the art.The plasmids set forth in this example and bacteria containing them are available from the State University of New York at Buffalo, Amherst Campus and have been deposited with Northern Regional Research Laboratories, Peoria, Illinois prior to application for this patent. E. coli bacteria containing pLC22-36, pRS1, pRS2, pRS3, pRS4, pRS5, and pLB5 plasmids have Northern Regional Research Laboratories (deposited on 14th February 1 983) deposit numbers NRRLB-15281, NRRLB-15282, NRRLB-15287, NRRLB-15285, NRRLB-1 5283, NRRLB-1 5284, and NRRLB-15286 respectively.Plasmid pLB6, another plasmid of the present invention containing the ribosomal RNA promoter termination sequence but not the ribosomal RNA promoters was prepared by cloning the EcoR l-Bg lii fragment of pRS 1 into EcoR l-BamH 1 cut pUC8 plasmid described by Vieira et al "pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with universal primers" Gene, Vol. 19, pp. 259-268, 1982.

Table 1 shows the characteristics of the deposited plasmids and bacterial strains containing them.

TABLE 1 Strain Designation Genotype Deposit # 1. pLC22-36/EM22 pLC22-36/ara(amber)galK(amber) NRRLB-1 5281 galE lac(amber) trp(amber) tsxr ivl recA 2. EM315 pRS1/ara(amber) gaiK(amber) galE NRRLB-1 5282 iac(amber) trp(amber) tsxr ilv recA #' Strr trpT-Su7X 3. EM3 14 pRS4/ara(amber) gaiK(amher) galE NRRLB-1 5283 iac(amber) trp(amber) tsxr ilv recA #' Strr trpT-Su7X 4.EM323 pRS5/arn(arnber) galK(amber) galE NRRLB-1 5284 iac(amber) trpramber) tsxt ilv recA #' Strr trpT-Su7X 5. EM329 pRS3/arafamber) gaiK(amber) galE NRRLB-1 5285 iac(amber) trp(amber) tSXr' ilv recA Ar Strr trpT-Su7X 6. EM340 pLB5/ara(amber) gaiK(amber) galE NRRLB-1 5286 lac(amber) trpramberJ tSXr ilv recA Ar Strr trpT-Su7X 7. pRS2/EM2 pRS2/F+ ilvl hls29(amber) pro2 tSXr NRRLB-1 5287 trpR55 trpA 9605(amber) 8. pLB6/EM322 pLB6/ara(amber) gaiK(amber) galE NRRLB-1 5288 lac(amber) trpramber) tSXr ilv recA #' Strr

Claims

1. An operon comprising a bacterial ribosomal RNA promoter, a bacterial ribosomal RNA promoter termination sequence and an intermediate sequence between said promoter and said promoter termination sequence, wherein fewer than 2,000 sequential base pairs of said operon sequentially naturally occur.

2. The operon of claim 1 wherein said intermediate sequence contains at least one restriction nuclease site.

3. The operon of claim 2 wherein the operon has a size of fewer than 1,000 base pairs.

4. The operon of claim 2 wherein said operon includes a read through sequence which causes transcription of the intermediate DNA sequence through a sequence which normally would terminate more of the transcription of the intermediate sequence in a bacteria in the absence of the read through sequence.

5. The operon of claim 3 wherein said operon includes a read through sequence which causes transcription of the intermediate DNA sequence through a sequence which normally would terminate more of the transcription of the intermediate sequence in a bacteria in the absence of the read through sequence.

6. An operon comprising a promoter, a promoter termination sequence and an intermediate DNA sequence between the promoter and the termination sequence, wherein fewer than 2,000 sequential base pairs of said operon naturally sequentially occur and wherein the operon includes a ribosomal RNA read through sequence which can cause transcription of a DNA sequence, intermediate said promoter and said promoter termination sequence, through a sequence which normally would terminate more of the transcription of the intermediate sequence in a bacteria in the absence of the read through sequence.

7. The operon of claim 6 wherein at least one restriction nuclease site is present between the promoter and the promoter termination sequence and after the read through sequence.

8. An operon which comprises a promoter and a bacterial ribosomal RNA promoter termination sequence wherein fewer than 2,000 sequential base pairs of said operon naturally sequentially occur.

9. The operon of claim 2 wherein said ribosomal RNA promoter is an E. coli ribosomal RNA promoter.

10. The operon of claim 9 wherein the ribosomal RNA promoter is a rrnC promoter.

11. The operon of claim 4 wherein said ribosomal RNA promoter is an E. coli ribosomal RNA promoter.

12. The operon of claim 11 wherein the ribosomal RNA promoter is a rrnC promoter.

13. The operon of claim 2 wherein the operon has a plurality of restriction nuclease sites between the promoter and the promoter termination sequence.

14. The operon of claim 3 wherein the operon has a plurality of restriction nuclease sites between the promoter and the promoter termination sequence.

1 5. The operon of claim 4 wherein the operon has a plurality of restriction nuclease sites between the promoter and the promoter termination sequence.

1 6. The operon of claim 2 wherein the operon contains a DNA sequence which reduces the effectiveness of the ribosomal RNA promoter.

1 7. A plasmid containing an operon of claim 1.

1 8. A plasmid containing an operon of claim 2.

19. A plasmid containing an operon of claim 2 wherein the plasmid has a size of fewer than 15,000 base pairs.

20. A plasmid containing an operon of claim 2 wherein the plasmid has a size of fewer than 10,000 base pairs.

21. A plasmid containing an operon of claim 3.

22. A plasmid containing an operon of claim 3 wherein the plasmid has a size of fewer than 10,000 base pairs.

23. A plasmid containing an operon of claim 4.

24. A plasmid containing an operon of claim 4 wherein the plasmid has a size of fewer than 1 5,000 base pairs.

25. A plasmid containing an operon of claim 5.

26. A plasmid containing an operon of claim 6.

27. A plasmid containing an operon of claim 7.

28. A plasmid containing an operon of claim 8.

29. A plasmid containing an operon of claim 9.

30. A plasmid containing an operon of claim 1 0.

31. A plasmid containing an operon of claim 11.

32. A plasmid containing an operon of claim 12.

33. A plasmid containing an operon of claim 13.

34. A plasmid containing an operon of claim 1 4.

35. A plasmid containing an operon of claim 1 5.

36. A plasmid containing an operon of claim 1 6.

37. A plasmid selected from the group consisting of pRS1, pRS2, pRS3, pRS4 and pLB5.

38. A bacterium containing a plasmid of claim 17.

39. A bacterium containing a plasmid of claim 18.

40. A bacterium containing a plasmid of claim 19.

41. A bacterium containing a plasmid of claim 20.

42. A bacterium containing a plasmid of claim 21.

43. A bacterium containing a plasmid of claim 22.

44. A bacterium containing a plasmid of claim 23.

45. A bacterium containing a plasmid of claim 24.

46. A bacterium containing a plasmid of claim 25.

47. A bacterium containing a plasmid of claim 26.

48. A bacterium containing a plasmid of claim 27.

49. A bacterium containing a plasmid of claim 28.

50. A bacterium containing a plasmid of claim 29.

51. A bacterium containing a plasmid of claim 30.

52. A bacterium containing a plasmid of claim 31.

53. A bacterium containing a plasmid of claim 32.

54. A bacterium containing a plasmid of claim 33.

55. A bacterium containing a plasmid of claim 34.

56. A bacterium containing a plasmid of claim 35.

57. A bacterium containing a plasmid of claim 36.

58. A bacterium containing a plasmid of claim 37.

59. The bacterium of claim 38 wherein the bacterium is an E. coli.

60. The bacterium of claim 39 wherein the bacterium is an E. coli.

61. The bacterium of claim 40 wherein the bacterium is an E. coli.

62. The bacterium of claim 41 wherein the bacterium is an E. coli.

63. The bacterium of claim 42 wherein the bacterium is an E. coli.

64. The bacterium of claim 43 wherein the bacterium is an E. coli.

65. The bacterium of claim 44 wherein the bacterium is an E. coli.

66. The bacterium of claim 45 wherein the bacterium is an E. coli.

67. The bacterium of claim 58 wherein the bacterium is an E. coli.

68. A method for making a bacterium which comprises introducing a plasmid of claim 1 7 into an E. coli bacterium.

69. A method for making a bacterium which comprises introducing a plasmid of claim 1 8 into an E. coli bacterium.

70. A method for making a bacterium which comprises introducing a plasmid of claim 1 9 into an E. coil bacterium.

71. A method for making a bacterium which comprises introducing a plasmid of claim 20 into an E. coli bacterium.

72. A method for making a bacterium which comprises introducing a plasmid of claim 21 into an E. coli bacterium.

73. A method for making a bacterium which comprises introducing a plasmid of claim 22 into an E. coil bacterium.

74. A method for making a bacterium which comprises introducing a plasmid of claim 23 into an E. bacterium.

75. A method for making a bacterium which comprises introducing a plasmid of claim 37 into an E. coli bacterium.

76. A method for making the plasmid of claim 1 8 which comprises treating a plasmid; containing an operon comprising a bacterial ribosomal RNA promoter, a bacterial ribosomal RNA promoter termination sequence and an intermediate sequence between said promoter and said promoter termination sequence, wherein said operon has a length of fewer than 2000 base pairs and wherein said intermediate sequence contains at least one restriction nuclease site; with an appropriate restriction enyzme; combining the treated plasmid with a DNA sequence having ends suitable for combination with the restricted sites of the plasmid resulting from treatment with the restriction enzyme; introducing the resulting plasmid into a host bacteria; and growing the bacteria in a suitable medium to produce the desired plasmid.

77. A method for making the plasmid of claim 25 which comprises treating a plasmid; containing an operon comprising a bacterial ribosomal RNA promoter, a bacterial ribosomal RNA promoter termination sequence and an intermediate sequence between said promoter and said promoter termination sequence, wherein said operon has a length of fewer than 1,000 base pairs, wherein said intermediate sequence contains at least one restriction nuclease site and wherein said operon contains a read through sequence which causes transcription of an intermediate DNA sequence through a sequence which normally would terminate more of the transcription of the intermediate sequence in the absence of the read through sequence; with an appropriate restriction enzyme, combining the treated plasmid with a DNA sequence having ends suitable for combination with the restricted sites of the plasmid resulting from treatment with the restriction enyzme; introducing the resulting plasmid into a host bacteria; and growing the bacteria in a suitable medium to produce the desired plasmid.

78. A method for the preparation of the plasmid of claim 1 8 which comprises: a) treating a plasmid; containing a ribosomal RNA operon containing in excess of 2,000 base pairs and containing a ribosomal RNA promoter and a ribosomal RNA termination sequence; with a restriction enzyme, permitting the cleaved plasmids to recombine, introducing resulting plasmids into bacteria, growing colonies of the resulting bacteria, obtaining purified plasmids from such colonies, and analyzing the plasmids; b) repeating step a) as necessary until the appropriate plasmid is found containing the desired ribosomal RNA operon which contains fewer than 2,000 base pairs; and c) introducing the appropriate plasmid into a host bacteria to reproduce the desired plasmid.

79. A method for obtaining an operon having a ribosomal RNA promoter and ribosomal RNA promoter termination sequence and having fewer than 2,000 sequential base pairs in a naturally occurring sequence which comprises treating a plasmid of claim 17, having restriction nuclease sites on each side of said operon, with an appropriate restriction enzyme and isolating the restricted operon.

80. A method for the production of a polypeptide which contains its DNA code for the mRNA for the polypeptide in a plasmid of claim 1 7 which comprises growing a bacteria containing said plasmid followed by isolation of the polypeptide.

81. A method for the production of a polypeptide which contains its DNA code for the mRNA for the polypeptide in a plasmid of claim 1 8 which comprises growing a bacteria containing said plasmid followed by isolation of the polypeptide.

82. A method for the production of a polypeptide which contains its DNA code for the mRNA for the polypeptide in a plasmid of claim 1 9 which comprises growing a bacteria containing said plasmid followed by isolation of the polypeptide.

83. A method for the production of a polypeptide which contains its DNA code for the mRNA for the polypeptide in a plasmid of claim 20 which comprises growing a bacteria containing said plasmid followed by isolation of the polypeptide.

84. A method for the production of a polypeptide which contains its DNA code for the mRNA for the polypeptide in a plasmid of claim 22 which comprises growing a bacteria containing said plasmid followed by isolation of the polypeptide.

85. A. method for the production of a polypeptide which contains its DNA code for the mRNA for the polypeptide in a plasmid of claim 23 which comprises growing a bacteria containing said plasmid followed by isolation of the polypeptide.

86. A method for the production of a polypeptide which contains its DNA code for the mRNA for the polypeptide in a plasmid of claim 24 which comprises growing a bacteria containing said plasmid followed by isolation of the polypeptide.