Hybrid gene regulatory region operable in E. coli

This invention is directed to a novel hybrid regulatory region for directing and regulating transcription and translation of a gene sequence positioned downstream from the hybrid regulatory region. This hybrid regulatory region includes the promoter sequence of the phage lambda P.sub.R promoter-operator region fused to the operator sequence of the phage lambda P.sub.L promoter-operator region.

TECHNICAL FIELD 
This invention relates to the construction of a regulatory sequence which 
provides a new operator region to regulate transcription initiation while 
leaving the promoter intact. This regulatory sequence can be used for the 
regulated transcription and translation of prokaryotic or eukaryotic 
genes. 
BACKGROUND OF THE INVENTION 
The expression of a gene in both prokaryotic and eukaryotic organisms 
involves first the synthesis of RNA from a DNA template followed by 
protein synthesis from the RNA. 
Transcription, the synthesis of RNA from a DNA template and the first step 
in the expression of a gene, is controlled by certain signals present on 
the DNA. These signals are nucleotide sequences which initiate 
transcription and control the amount of transcription taking place at a 
given time. The control signals generally consist of promoter and operator 
regions. The promoter region is a site that is specific for the binding of 
RNA polymerase and is the initiation point for transcription. Operators 
function in conjunction with a repressor to control the amount of 
transcription. 
Transcription of a DNA segment is effected by the enzyme RNA polymerase. 
After RNA polymerase binds to the promoter at the -35 and -10 recognition 
regions (M. Rosenberg and D. Court, Ann. Rev. Genet. 13:319-353, 1979), it 
transcribes nucleotides which encode a ribosome binding site and 
translation initiation signal and then transcribes the nucleotides which 
encode the actual structural gene until it reaches so-called stop signals 
at the end of the structural gene. The RNA polymerase acts by moving along 
the DNA segment and synthesizing single-stranded messenger RNA (mRNA) 
complementary to the DNA. As the mRNA is produced, it is bound by 
ribosomes at the ribosome binding site (also called the Shine-Dalgarno 
region). The ribosomes translate the mRNA, beginning at the translation 
initiation signal and ending at the stop signals, to produce a polypeptide 
having the amino acid sequence encoded by the DNA. 
Through the use of genetic engineering techniques genes from one organism 
can be removed from that organism and spliced into the genetic information 
of a second organism and the polypeptide encoded by that gene expressed by 
the second organism. It is desirous to maximize the expression of the 
foreign gene and thus obtain high yields of the resultant polypeptide. It 
has been realized that one way in which gene expression can be regulated 
is through selection and manipulation of the control signals discussed 
above. 
There is variation among different promoters in their strength and their 
ability to be repressed efficiently. A promoter which cannot be repressed 
easily is of only limited use with genes whose protein product in small 
amounts is toxic to the cell or inhibits maintenance of the plasmid. In 
such situations, maximal repression of the genes is needed to assure that 
the host cell and/or plasmid can grow normally until derepression is 
desired. 
Some promoters also suffer a disadvantage when they are present on 
multi-copy plasmids in that they cannot be repressed efficiently unless a 
suitable repressor also is located on that plasmid and thus present in 
multiple copies. 
Such promoters are in contrast to others which can be repressed fully by 
the amount of repressor made from a single chromosomal gene copy. These 
promoters, however, may have other drawbacks. They may not, for example, 
be as strong as other promoters. 
Various efforts have been made to manipulate different promoter/operator 
systems so as to enhance promoter strength or increase efficiency of 
repression. European Patent Application 067,540 (see also De Boer et al. 
in "Promoters: Structure and Function," ed. R.L. Rodriguez, M.J. 
Chamberlin, Praeger, 1982, pp. 462-481), for example, describes and claims 
a hybrid promoter/operator. This hybrid is constructed by ligating the -10 
region of one promoter/operator sequence, capable of being derepressed by 
induction, downstream from a DNA fragment which comprises the -35 region 
and 5' flanking region of a second promoter which has a stronger signal 
sequence than the first promoter/operator sequence. The two DNA fragments 
are linked at a position between about the -35 and -10 recognition 
sequences for binding of RNA polymerase to the promoter/operator sequence. 
The fusion results in an entirely new promoter sequence. 
Although such a hybrid promoter/operator can be used advantageously in 
certain situations, it still may prove to be unsatisfactory in others. For 
example, although the transcription efficiency of the promoter 
contributing the -10 region may be enhanced, the promoter may not be 
regulated as tightly as desired under certain circumstances. 
There thus remains a need for a regulatory sequence that has a strong 
promoter which can be repressed highly efficiently. Accordingly, it is an 
object of this invention to construct a novel regulatory region having 
these characteristics. It also is an object of this invention to construct 
such a regulatory region that can be ligated conveniently to a variety of 
prokaryotic and eukaryotic genes. 
SUMMARY OF THE INVENTION 
In accordance with the present invention there is disclosed a hybrid 
regulatory region which comprises the intact promoter sequence of a first 
promoter/operator region fused to the operator sequence of a second 
promoter/operator region wherein the operator sequence of said second 
promoter-operator region can regulate the promoter from the first region 
more efficiently than can its native operator sequence.

DETAILED DISCLOSURE OF THE INVENTION 
The present invention relates to a hybrid promoter/operator region capable 
of directing and regulating transcription of a gene sequence positioned 
downstream therefrom which provides an intact strong promoter ligated to a 
new operator region which can regulate transcription initiation more 
efficiently than the promoter's own natural operator(s). The hybrid 
regulatory region of this invention is constructed from two 
promoter/operator regions, a first region which contains a strong promoter 
and a second region which contains an efficient operator. These regions 
are cleaved and fragments taken from them are fused together such that the 
resultant hybrid region comprises the complete promoter sequence of the 
first region and the efficient operator of the second. 
To make this novel hybrid region the first region generally is cleaved at a 
restriction enzyme recognition site located upstream from the complete 
promoter sequence and the second region is cleaved at a site downstream 
from its operator sequence. The appropriate fragments from each of these 
two regions then are fused together in accordance with conventional 
methods so as to form the novel hybrid regulatory region of this 
invention. Alternatively, the first region may be cleaved at a restriction 
enzyme recognition site that is within the nucleotide sequence of the 
promoter provided that when the resulting fragment containing the partial 
sequence of the promoter is fused to the operator sequence of the second 
region, the nucleotide sequence at the 3' end of the operator region is 
such that the complete nucleotide sequence for the promoter will be 
restored. 
The two regions may be cut at a naturally occurring common or complementary 
restriction enzyme recognition site or at a common or complementary site 
which has been introduced into one or both of the regions by in vitro 
mutagenesis. Alternatively, if the DNA fragments taken from the two 
regions have noncomplementary ends, a synthetic DNA segment which matches 
the restriction sites of the fragments can be prepared and used to link 
the two fragments. 
The details of this invention will be set forth below in terms of a 
particular embodiment of this invention. It is to be understood, however, 
that this is done for illustrative purposes only and is not to be 
construed as limiting. 
In one embodiment of this invention the hybrid regulatory region is 
constructed from two phage .lambda. promoter/operator regions. These two 
promoters of phage .lambda., which function early in .lambda. infection, 
are known as P.sub.R and P.sub.L (Eisen, H. and M. Ptashne, The 
Bacteriophage Lambda, A.D. Hershey, ed., Cold Spring Harbor Lab, N.Y., 
1971, pp. 239-270). The P.sub.R sequence provides a strong promoter, but 
the promoter cannot be repressed as efficiently (i.e., to as low a level) 
as .lambda. promoter P.sub.L (Queen, C.J., Mol. Appl. Genet. 2: 1-10 
(1983)). A second disadvantage of the P.sub.R promoter is that when it is 
present on multi-copy plasmids it can be repressed efficiently only when a 
.lambda. repressor also is located on the plasmid and thus in multiple 
copies. When, however, the .lambda. repressor is also present on the 
plasmid, complete derepression of .lambda.P.sub.R cannot be achieved 
efficiently unless the temperature is raised to 42.degree. C. In contrast, 
the P.sub.L promoter can be repressed fully by the amount of repressor 
made from a single chromosomal gene copy, and derepression is effective at 
37.degree.-38.degree. C. The lower induction temperature is useful for 
proteins which may be rendered less active by heating at 42.degree. C. 
The structure of two segments of the .lambda. genome containing promoters 
P.sub.R and P.sub.L is diagrammed in FIG. 1. RNA polymerase binds to each 
promoter at the -35 and -10 regions (Rosenberg, M. et al., Ann. Rev. 
Genet. 31: 319-353 (1979); Hawley, D.K. et al., Nucl Acids Res. 11: 
2237-2255 (1983)). The ability of RNA polymerase to bind each promoter is 
antagonized by the .lambda. repressor (cI protein) which binds at operator 
sites O.sub.L 1, 2 and 3 and O.sub.R 1, 2 and 3 (Ptashne, M. et al., Cell 
19 :1-11 (1980)). 
As shown in FIG. 1, the P.sub.L and P.sub.R regions have a naturally 
occurring common HincII site. The regions are cut with endonuclease 
HincII, then a fragment from each region is fused together, such that the 
sequence upstream from the HincII site (to the left of HincII in FIG. I) 
is the P.sub.L fragment and the sequence downstream from the HincII site 
(to the right of HincII in FIG. I) is the P.sub.R fragment. The hybrid 
region has been designated O.sub.L /P.sub.R. 
The HincII site in P.sub.L and P.sub.R is located within the -35 region of 
each promoter. When the P.sub.L and P.sub.R segments are fused at the 
HincII site, the new regulatory region recreates the exact and complete 
sequence of P.sub.R, for the bases upstream of the HincII cut site are 
identical in P.sub.L and P.sub.R (Rosenberg et al., supra; Hawley et al; 
supra). 
##STR1## 
Similarly, the fusion at the HincII site recreates O.sub.L 2, a portion of 
which is shown above, because the G residue in O.sub.L 2 to the right of 
HincII is also found in O.sub.R 2. The O.sub.L /P.sub.R hybrid has the 
repressor binding characteristics of P.sub.L. The primary repressor 
binding sites O.sub.R 1 and O.sub.L 1 do not have identical DNA sequences 
(Pirrotta, V. Nature 254:114 (1975); Humayun, et al., J. Molec. Biol. 112: 
267 (1977)); thus, the differences between P.sub.R and P.sub.L in their 
ability to be repressed apparently resides in the differences between the 
remaining repressor sites. The O.sub.L /P.sub.R hybrid made in accordance 
with the above-discussed procedure contains the O.sub.L 2 and O.sub.L 3 
repressor sites and the repressor binding characteristics of P.sub.L. The 
O.sub.L /P.sub.R hybrid thus can be repressed to the low basal levels of 
O.sub.L. Furthermore, the O.sub.L /P.sub.R regulatory region can be 
repressed efficiently when the .lambda. repressor gene (cI) is located on 
the chromosome of the bacterial host and derepressed efficiently at 
temperatures less than 42.degree. C. 
In a specific embodiment of the invention, the P.sub.L fragment is derived 
from the plasmid pGW7 (provided by Geoffrey Wilson) which contains a 
segment of the .lambda. genome. The P.sub.R segment is derived from 
plasmid pCQV2 (Queen, C., J. Mol. Appl. Genet. 2:1-10, 1983). pCQV2 
contains an alteration in a segment of the .lambda. DNA sequence such that 
a BamHI site overlaps the ATG of the cro gene, the first gene downstream 
from P.sub.R. When the BamHI site is cleaved and the resulting single 
stranded region removed, an ATG codon is present at the blunt end of the 
hybrid promoter/operator region. The resulting O.sub.L /P.sub.R hybrid 
regulator has been cloned into a plasmid designated pGX2606 (see FIG. 2). 
An E.coli cell culture transformed with this plasmid has been designated 
GX3123 and deposited with the Northern Regional Research Laboratory, 
Peoria, Illinois, as NRRL No. B-15551. 
In this example, the promoter can be repressed by maintaining the plasmid 
in an E.coli cell which carries the gene for wild type .lambda. repressor 
on the chromosome. Alternatively, if the plasmid carrying the O.sub.L 
/P.sub.R region is introduced into a cell which has the gene specifying 
the temperature-sensitive .lambda. repressor mutant, cI857, repression is 
maintained at 30.degree. C. Induction of the cI857 lysogen is obtained by 
raising the temperature to 37.degree.-42.degree. C. expression at a 
desired time (Campbell, A., The Bacteriophage Lambda, ed A.D. Hershey, 
Cold Spring Harbor Lab, N.Y., 1971, pp. 13-44). Nonregulated expression of 
the gene of interest linked to O.sub.L /P.sub.R also can be obtained by 
putting the plasmid into a non-lysogen. With this variation, gene 
expression is constitutive, and the temperature can be maintained at 
37.degree. C. which is the optimal growth temperature for E.coli. 
The hybrid regulatory region of this invention provides a translation 
initiation region derived from the region between the promoter and the 
first gene downstream from the promoter in the plasmid from which it was 
derived, which can be joined to a gene sequence to provide all needed 
translation initiation signals for E.coli. This includes the ribosome 
binding site, known as the Shine-Dalgarno region (Shine, J. and L. 
Dalgarno, Proc. Natl. Acad. Sci. USA, 71:1342-46, 1974) and the ATG. As 
discussed above, for example, the end of the O.sub.L /P.sub.R region 
proximal to the P.sub.R promoter can be digested so as to provide a blunt 
end with an ATG (translation initiation codon) at the terminus. The region 
then can be fused to a gene lacking an ATG. 
Alternatively, the region proximal to the 3' end promoter in this hybrid 
can be altered such that the promoter region no longer carries an ATG 
codon for translation initiation and so can be fused to genes which carry 
their own initiation codon. An example of this using the O.sub.L /P.sub.R 
hybrid is shown by converting the BamHI site to a ClaI site by site 
directed mutagenesis in vitro (Zoller, M.J., et al. in Methods in 
Enzymology 154:329-350 (1987)). 
##STR2## 
In a third embodiment of this invention, a single base change made with in 
vitro mutagenesis can be used to create a restriction site downstream from 
the -10 RNA polymerase recognition site of the hybrid regulatory region. 
Such a cut separates the hybrid promoter/operator from the Shine/Dalgarno 
region (Shine, J. and L. Dalgarno, supra, preceding the first downstream 
gene, thus allowing the insertion of any other natural or synthetic 
Shine/Dalgarno sequence. These substitutions provide additional 
possibilities for high expression. One example shows the insertion of an 
SphI site in the O.sub.L /P.sub.R at such a position by site directed 
mutagenesis (see above). 
##STR3## 
The hybrid promoter/operator regulatory region can be used for 
transcription and translation of various prokaryotic or eukaryotic genes 
either in a regulated or an unregulated form. The efficient repression 
which can be obtained with such a hybrid makes it especially useful for 
fusion to genes whose protein products are toxic to the cell in small 
amounts or inhibit plasmid maintenance. Maximal repression of the 
expression of such genes enables the cells to grow normally and to retain 
the plasmid until derepression is desired. Expression of the genes then 
can be induced when cell viability no longer is important. 
The following examples are intended to further illustrate this invention 
and are not to be construed as limiting. 
I. Cloning of .lambda.P.sub.L and .lambda.P.sub.R Fragments Into 
Intermediate Vectors 
A. Cloning of P.sub.L from PGW7 into pUC8 (FIG. 3) 
Plasmid of pGW7 (8007 base pairs, obtained from Geoffrey Wilson) contains a 
3987 base pair segment of bacteriophage .lambda. DNA from nucleotides 
34498 to 39173 (excluding bases 38104 to 38754 which were deleted). The 
numbering of the residues in .lambda. DNA is from Sanger, F. et al., J. 
Mol. Biol., 162, 729-773 (1982). This region contains the early .lambda. 
promoter P.sub.L from which was isolated a fragment from endonuclease 
sites Bg1II to HindII (HincII) (bases 35615 to 35711). 
Plasmid pGW7 DNA (10.mu.g) was digested with 11.2 units endonuclease Bg1II 
(New England Biolabs, Inc.) for 3 hours at 37.degree. C. in "medium salt" 
restriction buffer (50mM NaCl, 10mM Tris, pH 7.4, 10mM MgSO.sub.4, 1mM 
dithiothreital). The 5566 base pair fragment was isolated after 
electrophoresis in a gel of 1% low melting agarose (Bethesda Research 
Laboratores, Inc.) in E buffer (50 mM Tris, pH 7.5, 30mM sodium acetate, 
3mM EDTA) and extracted from the agarose with butanol as described by 
Langridge et al., Anal. Biochem. 103, 264-271 (1980). The DNA was 
precipitated by addition of 2.5 volumes ethanol and pelleted in an SW40 
Beckman ultracentrifuge rotor at 4.degree. C. and 35,000 rpm for 1 hr. The 
pellet was dried in vacuo and suspended in 200.mu.l H.sub.2 O. 
The isolated 5566 base pair fragment (10.mu..lambda.) was digested with 8 
units endonuclease HindII (Boehringer Mannheim, Gmbh) in medium salt 
buffer for 20.5 hrs. at 37.degree. C. The digest was extracted with phenol 
and ether and subjected to electrophoresis on a 6% polyacrylamide gel 
(acrylamide:bisacrylamide--30:1) in TBE buffer (90 mM Tris, pH 8.3, 90 mM 
boric acid, 4 mM EDTA). After staining the gel with ethidium bromide, the 
desired 110 base pair fragment was cut out and removed from the gel by 
electroelution in 400 .mu.l 0.1X TBE. One ml 0.2M NaCl, 20 mM Tris, pH 
7.4, 1mM EDTA was added and the DNA was purified by passage over an Elutip 
(Schleicher and Schnell, Inc., Keene, N.H.) as suggested by the 
manufacturer. The DNA was precipitated with ethanol as above and pelleted 
in a Beckman SW28 ultracentrifuge rotor at 25000 rpm for 1 hr at 4.degree. 
C. The pellet was dried in vacuo and suspended in 20 .mu.l H.sub.2 O. 
Plasmid pUC8 (Vieira J. and J. Messing. Gene, 19 259-268, 1982), 10 .mu.g, 
was digested with 9.1 units endonuclease Hind II (Boehringer Mannheim, 
GmbH) for 60 min. at 37.degree. C., then another 9.1 units of enzyme was 
added and incubated another 15 hrs. at 37.degree. C. The DNA was 
precipitated in 0.3M sodium acetate, pH 5.5, with 2.5 volume ethanol. The 
dried pellet was suspended in 16 ml H.sub.2 O, to which was added medium 
salt buffer and 20 units endonuclease BamHI in a total reaction volume of 
20 .mu.l. The reaction was incubated for 2 hours at 35.degree. C. and then 
extracted with phenol, precipitated with ethanol, and resuspended in 10 
.mu.l H.sub.2 O. 
For ligation of the P.sub.L fragment to pUC8, approximately 5 ng fragment 
was joined to approximately 30 ng pUC8 in a 20 .mu.l reaction containing 
200 units T4 DNA ligase (New England Biolabs, Inc.), 10 .mu.g/ml bovine 
serum albumin (Bethesda Research Laboratories, Inc.) 0.5mM ATP, 50mM Tris, 
pH 7.8, 10mM MgCl.sub.2, 20 mM dithiothreital. The reaction was carried 
out for 23 hours at 12.degree. C. 
E.coli K12 JM103: F' traD36 proA.sup.+ B.sup.+ lacI.sup.9 
lacZ.DELTA.M15/.DELTA.(lac pro) supE thi rpsL4 sbcB15 endA) was grown in 
YT broth (5g yeast extract, 8g trypstone, 5g NaCl per liter H.sub.2 O) and 
made competent for transformation by CaCl.sub.2 treatment (Cohen, S.N. et 
al., Proc. Natl. Acad. Sci USA, 69, 2110-2114, 1972). Two 200.mu.l samples 
of competent cells (approx. 2.times.10.sup.9 /ml) were each added to 8 
.mu.l ligation mix and kept on ice 40 min. The mix was heat shocked at 
42.degree. C. 2 min., diluted 15-fold in YT broth, incubated at 37.degree. 
C. 1 hr., and plated on selective medium (YT broth with 1.5% Difco agar, 
2.mu.g/ml ampicillin, 2ml/l 0.1 M isopropylthio-.beta.-D-galactoside 
[IPTG], 2ml/l 5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside [Xgal]. 
Ligations which produced plasmids containing the insert were indicated by 
a color change in the colony in the medium. This method for detecting 
inserts is described in more detail by Vieira, J. and J. Messing Gene 19, 
259-269, 1982. 
After 15/hrs incubation at 37.degree. C., 85 colonies were obtained. 
Miniprep DNA was prepared from white colonies by the method of D.S. Holmes 
and M. Quigley Anal. Biochem. 114:193-197 (1981). 
To verify that a 96 bp fragment had been inserted into pUC8, miniprep DNA 
was digested with two endonucleases whose sites border the insert on each 
side. 0.5 .mu.g DNA in a total volume of 20 .mu.l was incubated with 8 
units endonuclease HindIII (Boehringer Mannheim GmbH) in medium salt 
buffer for 1 hr. at 37.degree. C., then for another 4 hrs at 37.degree. C. 
with an additional 8 units HindIII. The reaction was stopped by heating 
for 5 minutes at 65.degree. C. It was brought to 50mMTris, pH 7.4, 100 mM 
NaCl in a volume of 35 .mu.l and digested further with 20 units 
endonuclease EcoRI (New England Biolabs, Inc.) for 15 minutes at 
37.degree. C. A 5 .mu.l sample was analyzed by electrophoresis on a 5% 
polyacrylamide gel in TBE buffer. By digesting with EcoRI and HindIII a 
118 base pair fragment should be obtained if the correct 96 base pair 
.lambda.P.sub.L fragment has been inserted between them. The correct 
isolate was identified as having a fragment which comigrated with a 119 
base pair marker. The identity of the insert was confirmed by DNA 
sequencing (Maxam, A. M. and W. Gilbert Methods in Enzymology, ed. L. 
Grossman, K. Moldave, Academic Press, N.Y. vol. 65, pp. 499-559 (1980)), 
from DNA which had been extracted from cells by a method similar to the 
detergent lysis procedure (Molecular Cloning, ed. T. Maniatis, E. F. 
Fritsch, J. Sambrook, Cold Spring Harbor Laboratory, N.Y. p. 92, 1982). 
The DNA was purified on two CsCl-ethidium bromide gradients by established 
procedures and passed over a column of Biogel A-50 (BioRad Laboratories). 
B. Cloning of P.sub.R from pCQV2 into pUC9 (FIG. 4) 
These procedures were carried out in a manner analogous to the procedures 
described in section A; therefore, only specific changes will be noted 
here. All other details can be assumed to be the same as in section A. 
Plasmid pCQV2 (Queen, C. J. Mol. Appl. Genet. 2, 1-10, 1983) contains 
.lambda.DNA from base numbers 37169 to 38043 and it was modified to 
contain an endonuclease BamHI site overlapping the ATG of the .lambda. cro 
gene. From pCQV2 was isolated a HindIII to BamHI fragment which contains 
most of P.sub.R and the Shine-Dalgarno region (Shine and Dalgarno, supra) 
preceding the .lambda. cro gene. 
pCQV2 (50 .mu.g) was digested with 50 units endonuclease BamHI (Bethesda 
Research Laboratories) in medium salt buffer at 37.degree. C. for 1 hr. 
Endonuclease HindII (Boehringer Mannheim, GmbH) then was added (80 units) 
and digestion was continued 20.5 hrs. at 37.degree. C. The digest was 
extracted with phenol and ether and subjected to electrophoresis on a 
preparative 6% polyacrylamide gel. The 50 base pair BamHI to HindII 
fragment was removed from the gel by electroelution, passed over a 
Schleicher and Schuell Elutip and precipitated with ethanol. 
The vector pUC9 is similar to pUC8 except that the cloning sites from EcoRI 
to HindIII are in the opposite orientation (Vieira, J. and J. Messing Gene 
19, 259-269, 1982) pUC9 (10.mu.g) was digested with endonuclease BamHI and 
HindII as described before. Approximately 15 ng digested pUC9 was joined 
to 0.2 ng P.sub.R fragment in a reaction with 200 units T4 DNA ligase for 
23 hrs. at 12.degree. C. in a reaction volume of 20.mu.l. 
Competent E.coli K12 JM103 cells were transformed with 8.mu.l of the 
ligation and plated on YT agar plates + IPIG + X-gal + ampicillin at 
37.degree. C. After 15 hrs. incubation, there were 326 white colonies. 
Miniprep DNA was prepared from some of these, and it was digested with 
EcoRI and HindIII sites on either side of the insert. The insert (50 base 
pairs) was removed in this way to give a 72 base pair diagnostic fragment. 
DNA from an isolate with the correct size insert was purified and 
sequenced by the Maxam-Gilbert technique to confirm its identity. 
The cloning of the P.sub.L and P.sub.R fragments into pUC8 and pUC9 
resulted in orienting the fragments in the same direction and in placing 
useful endonuclease sites on either side of the inserts. pUC8 containing 
P.sub.L is hereafter referred to as pGX2602 and pUC9 containing P.sub.R as 
pGX2603. 
C. Joining of the P.sub.L and P.sub.R Fragments and Cloning of the Joined 
Piece (FIG. 5) 
Purified DNA (25.mu.g each) of pGX2602 and pGX2603 was digested with 24 
units of endonuclease HincII (same as HindII, New England Biolabs, Inc.) 
in medium salt buffer 2 hrs at 37.degree. C.; another 24 units of enzyme 
were added and incubation continued at 37.degree. C. for 1 hour (pGX2602) 
or 4 hours (pGX2603). The digested DNAs were precipitated with ethanol and 
resuspended in medium salt buffer. pGX2602 was then incubated with 56 
units endonuclease HindIII (New England Biolabs, Inc.) and pGX2603 was 
incubated with 25 units endonuclease PstI (Takara Inc., Japan) at 
37.degree. C. for 2 hrs. The two DNA samples were then mixed, extracted 
with phenol, and precipitated with ethanol. The digestion of both DNAs 
with two different enzymes allowed fewer possible combinations when they 
were joined in the next step. The desired junction was of P.sub.L to 
P.sub.R at the HincII site. 
For joining of the linearized plasmid, the DNA (50.mu.g) was treated with 
2000 units T4 polynucleotide ligase (New England Biolabs, Inc.) in a 
volume of 100 .mu.l for 15 hr. at 16.degree. C. Another 2000 units of 
ligase was added and incubation was continued for another 48 hrs. 
An EcoRI to BamHI fragment which was thought to contain the left operator 
fused to P.sub.R was removed from the joined linear DNA fragments and 
cloned into another plasmid. This was done by first digesting the DNA with 
100 units endonuclease EcoRI (New England Biolabs, Inc.) at 37.degree. C. 
for 2 hrs. and precipitating it with ethanol. The pellet was suspended in 
96 .mu.l 100mM Tris, pH 8.0 and digested with 944 units (4 .mu.l) 
bacterial alkaline phosphatase for 40 min. at 65.degree. C. to remove 5' 
phosphate groups. After three extractions with phenol and an ethanol 
precipitation, the free ends were labeled with .lambda..sup.32 P-ATP by 
incubating in 50 mM Tris, pH 7.4, 10mM MgCl.sub.2, 5mM dithiothreital with 
10 units T4 polynucleotide kinase (P.L. Biochemicals Inc.) and 100 .mu.Ci 
.sup.32 P-ATP (Amersham, Inc. 6300 Ci/m mol) at 37.degree. C. for 35 min. 
Unlabeled ATP was added to lmM and incubated for 10 min at 37.degree. C. 
The mixture was extracted with phenol, and the DNA was precipitated with 
ethanol. The DNA was then digested with 80 units endonuclease BamHI in 
medium salt buffer for 2 hrs. at 37.degree. C., extracted with phenol and 
precipitated with ethanol. The pellet was suspended in 45 .mu.lTBE + dyes 
(80% glycerol, 0.5% bromphenol blue, 0.5% xylene cyanol) and loaded onto a 
3 mm thick 6% polyacrylamide preparatory gel. The gel was made from 11.2 
ml acrylamide (40%; 30:1 acrylamide: bis-acrylamide), 56 ml H.sub.2 O, 7.5 
ml 10X TBE, 0.5ml 10% ammonium persulfate and 55 .mu.l TEMED (BioRad 
Laboratories, Inc.). After electrophoresis at 250V for 1 hr., the gel was 
stained with ethidium bromide, and the 150 base pair EcoRI to BamHI 
fragment was excised, removed from the gel by electroelution, passed over 
a Schleicher and Schuell Elutip and precipitated with ethanol. The amount 
of material at this point was barely detectable by ethidium bromide 
staining, therefore, the fragment was hereafter detected on gels by 
autoadiography since it was end-labeled with .sup.32 P. 
The plasmid pGX1025 was used as the vector for cloning of the O.sub.L 
/P.sub.R fragment. It was digested with endonucleases EcoRI and BamHI 
under conditions described previously, and then it was treated with 
bacterial alkaline phosphatase to remove 5' phosphates and thereby to 
permit recircularization of the plasmid only when it was joined to the 
O.sub.L /P.sub.R fragment. 
Conditions for ligation of the O.sub.L /P.sub.R fragment to the vector were 
as follows: 200 units T4 DNA ligase (New England Biolabs), 500 ng pGX1025 
prepared as described above and the entire recovered O.sub.L /P.sub.R 
fragment (amount unknown) under standard reaction conditions and a 20 
.mu.l total volume. Incubation was at 16.degree. C. for 18 hrs. 
The host for transformation of the ligated DNA was E.coli K12 
JM101(.lambda.) F'traD36 proA.sup.+ B.sup.+ lac19 
lacZ.DELTA.M15/.DELTA.(lac pro) supE thi. Cells (200 .mu.l) were made 
competent and transformed by 8 .mu.l ligation mixture as described for 
JM103(.lambda.). The transformed cell suspension was divided into 200 
.mu.l aliquots and plated on LB agar (1.0% tryptone, 0.5% yeast extract, 
1.5% agar, all from Difco Laboratories, 0.5% NaCl) + 100 .mu.g/ml 
ampicillin at 37.degree. C. for 15 hrs. Approximately 6000 transformed 
colonies were obtained. 
Miniprep DNA was prepared (Holmes and Quigley, supra) from 64 colonies 
grown to saturation in 10ml LB (broth minus agar). The plasmid DNA was 
extracted twice with phenol, precipitated with ethanol, and suspended in 
100 .mu.l 10mM Tris, 1mM EDTA, pH 8.0. A sample of each miniprep DNA, 5 
.mu.l in a total volume of 20 .mu.l, was digested with 12 units 
endonuclease HincII (New England Biolabs, Inc.) in medium salt buffer for 
2 hrs at 37.degree. C. Two isolates had a diagnostic piece of 50-60 base 
pairs when the digest was analyzed by electrophoresis on a 5% 
polyacrylamide minigel. This HincII fragment originated from the HincII 
site internal to the O.sub.L /P.sub.R fragment and from a HincII site just 
3' to the insert in the vector plasmid. Another diagnostic test was to 
digest 5 .mu.l miniprep DNA with 16 units endonuclease BamHI (New England 
Biolabs, Inc.) in medium salt buffer for 2 hrs. at 37.degree. C. The 
completion of the BamHI digestion was confirmed by electrophoresis of a 
small portion of the digest on a 1% agarose minigel. The digest was then 
brought to 100mM NaCl, 50 mM Tris, pH 7.4 and digested with 20 units 
endonuclease EcoRI for 2 hrs at 37.degree. C. The mixture was analyzed by 
electrophoresis on a 5% polyacrylamide gel. The BamHI and EcoRI sites 
flank the O.sub.L /P.sub.R insert; therefore, this digestion should yield 
a fragment of 164bp. The two isolates which had the correct HincII 
fragment also had the correct BamHI to EcoRI fragment. 
In order to confirm the identity of the O.sub.L /P.sub.R insert, DNA was 
purified from one isolate which had the correct restriction pattern and 
subjected to DNA sequencing by the technique of Maxam and Gilbert. The 
sequence was identical to that of the corresponding segments from phage 
.lambda. (Sanger, F., et al. supra). 
The plasmid containing O.sub.L /P.sub.R has been designated pGX2606. An 
E.coli culture transformed with this plasmid has been designated GX3123 
and Deposited with the Northern Regional Laboratory as NRRL No. B-15551. 
EXAMPLE II 
Expression of Human Serum Albumin Gene Under the Control of the O.sub.L 
/P.sub.R Regulatory Region Insertion of an XhoI Cleavage Site Preceding 
the Sequence Coding Mature Human Serum Albumin (HSA) 
The O.sub.L /P.sub.R hybrid region was used to regulate expression of a 
human serum albumin (HSA) gene. In this procedure, the O.sub.L /P.sub.R 
regulatory region supplied the promoter, Shine-Dalgarno region, and ATG 
codon for translation initiation. The O.sub.L /P.sub.R region was ligated 
to a mature HSA coding sequence which contained no ATG codon at its 5' 
end. This form of HSA was created by introducing a restriction site (XhoI) 
which overlapped the codon for the first amino acid of HSA. 
Oligonucleotide-directed mutagenesis was used to modify the wild type 
sequence coding for preproHSA in order to place an XhoI restriction 
endonuclease cleavage site overlapping the 5' end of the mature HSA coding 
sequence. The strategy for this mutagensis and for expression of metHSA in 
E.coli from this modified sequence is outlined in the following diagram 
and described below. 
##STR4## 
The mutagenesis was accomplished in the following steps, adapted from 
Zoller, M. and M. Smith (supra). 
1. A portion of a human serum albumin gene was subcloned into the 
bacteriophage M13mp8, as shown in FIG. 6. Purified DNA from plasmid 
pGX401, containing a full length HSA clone with pre-pro sequences 
(designated hsa-1) was digested with HincII and the 1.35 kb fragment 
comprised of hsa-1 sequences from nucleotides -22 to 1328 was purified by 
electroelution from an agarose gel. M13mp8 was digested with HincII and 
treated exhaustively with bacterial alkaline phosphatase (BAP) to remove 
5' phosphates. BAP-treated M13mp8 DNA was incubated with the purified 
hsa-1 HincII fragment in the presence of T4 DNA ligase at 12.degree. C. 
(1.35:1 molar ratio of vector to insert). The ligation mix was used to 
transfect E.coli strain JM103. The hsa-1 sequence could be inserted into 
M13mp8 in either clockwise or counterclockwise orientation such that the 
single-stranded viral DNA from the recombinants would contain either the 
sense or nonsense strand of hsa-1. To determine the orientation of the 
insert, plaques were screened by hybridization with oligomer probes 
complementary to a portion of the sense or nonsense strands of hsa-1 (as 
described in detail below). An isolate in which the hsa-1 fragment had 
been inserted in the desired orientation was confirmed by restriction 
endonuclease mapping and by DNA sequencing from the 3' HincII site toward 
the XbaI site. The phage containing the cloned hsa-1 fragment was 
designated MGX-2. 
2. The desired mutant differed from the wild type sequence by a single 
nucleotide. A 17 base oligonucleotide was synthesized which was 
complementary to the wild type sequence except for a single base mismatch 
at the position of the desired base change (G.fwdarw.C). 
3. The mutagenic oligonucleotide was used as a primer for DNA synthesis 
with DNA polymerase I. After treatment with DNA ligase the product 
heteroduplex closed circular DNA molecules were purified by alkaline 
sucrose gradient centrifugation, pooled, dialyzed, and used to transfect 
competent E.coli. 
4. The plaques obtained were screened by hybridization of phage DNA to the 
mutagenic oligonucleotide. The principle behind this procedure is that the 
oligonucleotide used to direct the mutagenesis will form a duplex of 
higher thermal stability with mutant DNA, to which it is perfectly matched 
(17 of 17 base it is imperfectly matched (16 of 17 base pairs). Therefore 
the mutant phage can be differentiated from wild type phage in a 
hybridization experiment under conditions which discriminate between 
perfectly matched oligomers and mismatched oligomers (R.B. Wallace, M.J. 
Johnson, T. Hirose, T. Miyake, E.H. Kawashima, and K. Itakura, Nucl. 
Acids. Res. 9:879, 1981). Phage stocks were prepared from individual 
plagues. 20 .mu.l of each phage supernatant was spotted onto 
nitrocellulose filter paper using an S & S Minifold.TM. device (96 well 
capacity) to concentrate the 20 .mu.l onto a small area of the filter. 
Samples were applied in duplicate to make identical 4.times.12 arrays. 
The filter was air dried and baked in vacuo at 80.degree. C. for 2 hours. 
This filter was prehybridized and then hybridized with 5' end labeled 
oligomer (10 pmol in 4 ml) as described in Zoller and Smith, supra. After 
one hour of hybridization at 25.degree. C., the filter was removed from 
the probe solution and rinsed for 2 minutes in 50 ml 6XSSC at 25.degree. 
C. The filter was cut horizontally to separate the identical arrays. The 
top half of the filter was washed at 48.degree. C. for 10 minutes (2X25 ml 
6XSSC) and the bottom half at 52.degree. C. for 10 minutes (2X25 ml 
6XSSC). Filters were air dried and exposed to X-ray film for 12 hours at 
room temperature. It was determined that hsa-1 DNA (MGX2) formed 
mismatched hybrids with the mutagenic oligonucleotide in 1 M salt at 
25.degree. C. which were stable during washes at 48.degree. C. but 
unstable at 52.degree. C. Therefore, duplicate DNA samples from plaques 
obtained after mutagenesis were hybridized at 25.degree. C. and then were 
washed at 48.degree. C. and 52.degree. C. 
5. Double-stranded replicative form DNA was prepared from two 
hybridization-positive (A7,D7) and two hybridization-negative (A8,D8) 
clones. Each DNA was tested for the presence of an XhoI cleavage site. DNA 
from phages A7 and D7 was cleaved by XhoI; DNA from phages A8 and D8 was 
not. The correct location of the XhoI site in the DNA from phages A7 and 
D7 was confirmed by digestion with various other restriction enzymes. DNA 
sequence analysis confirmed the desired base change had occurred. This 
variant of hsa is called hsa-3, and the M13 clone bearing it is called 
MGX4. MGX4 has a restriction site which will cleave precisely at the 5' 
end of the mature HSA coding sequence. 
Reconstruction of hsa-3 in a Plasmid Vector 
The hsa-3 gene was constructed in a plasmid vector suitable for the 
addition of expression signals. Plasmid pGX1031 contains all of the hsa-1 
clone from pGX401, except a small section of the prepro region (3 codons). 
It was used to provide the 3' end of the gene and other necessary vector 
components. FIG. 7 outlines the procedure used to fuse the 5' portion of 
the hsa-3 gene from MGX4 to the 3' end of the hsa-1 gene in pGX1031 in 
order to make pGX1042 containing hsa-3 with the XhoI site. pGX1031 (FIG. 
7) was cut with EcoRI and XbaI, and the fragment shown was purified. This 
fragment was mixed with vector MGX4 DNA cut with the same enzymes, and the 
mixture was incubated with DNA ligase. After transformation of E.coli 
JM101 with the ligation mixture, 1200 ampicillin resistant transformants 
were obtained. Plasmid DNA from 45 of these which were randomly chosen was 
characterized by digestion with several restriction endonucleases, 
including XhoI. The plasmid designated pGX1042 was determined to have the 
desired construction. 
Construction of pGX1043 Containing the OL/PR Regulator Linked to hsa-3 at 
the XhoI Site 
The outline for the fusion of O.sub.L /P.sub.R to hsa-3 is shown in FIG. 8. 
The bacterial host for the transformation was JM101(.lambda.). The O.sub.L 
/P.sub.R promoter should be repressed in this strain. As fragments for 
this construction were not purified, the steps described below were 
performed for reducing the number of parental molecules and one type of 
recombinant plasmid which otherwise would have been recovered. It thus was 
expected that the desired transformant would be highly enriched among the 
colonies recovered. 
The following outline illustrates how the junction between the promoter and 
hsa-3 was made. 
##STR5## 
Plasmid pGX2606 DNA was prepared by digestion with BamHI (Rice, R.H. and 
G.E. Means, J. Biol. Chem. 246:831-832 (1971)). The 5' single-stranded 
ends were removed by mung bean nuclease, and the plasmid was cut again 
with BglI. In order to prevent recircularization of pGX2606 in the 
subsequent ligation, the DNA was treated with bacterial alkaline 
phosphatase. Plasmid pGX1042 DNA was cut with XhoI, treated with mung bean 
nuclease to remove the 5' single-stranded ends, and cut with BglI. 
Approximately 250 ng of each plasmid DNA was mixed and incubated with T4 
DNA ligase at 16.degree. C. for 18 hours. The ligation mixture was cut 
with BamHI to linearize any pGX1042 plasmid which had recircularized and 
to linearize one of the possible recombinant types. 
Approximately 75 ng of ligated DNA was used to transform competent JM101 
(.lambda.). The transformation mixture was plated on medium containing 
ampicillin and incubated at 37.degree. C. 430 transformants were obtained. 
The final plasmid pGX1043 was expected to have the sequence listed (at the 
bottom of the figure above) at the junction between promoter and hsa-3. 
The sequence to the left of the arrow including the ATG and the 
Shine-Dalgarno region (underlined) came from the O.sub.L /P.sub.R segment. 
The sequence to the right of the arrow came from hsa-3. 
The 430 transformants obtained were tested in several ways. 
A. Colony hybridization (M. Grunstein and D.S. Hogness Proc. Natl. Acad. 
Sci U.S.A. 72:3961, 1975). A .sup.32 P-labeled probe from the 5' end of 
hsa was used to detect colonies which carry hsa. The transformants were 
grown in LB medium plus 100 .mu.g/ml ampicillin in 96 well microtiter 
plates at 37.degree. C. Aliquots were transferred with a replicator to 
nitrocellulose filters on LB+ampicillin plates where they were incubated a 
further 5 hr at 37.degree. C. The conditions processing the filters and 
doing the hybridization are described in the above reference. The .sup.32 
P-labeled DNA probe was prepared from a plasmid containing the sequence 
for the 5' end of mature HSA. A 178 base pair fragment from the 5' end was 
labeled with .lambda.-.sup.32 P-ATP using T4 polynucleotide kinase, and 
purifying the desired hsa fragment on a 5% polyacrylamide gel. Known 
positive and negative controls gave the expected results. 39% of the 
transformants had at least this segment of hsa. 
B. Southern blot (E.M. Southern, J. Mol. Biol. 98: 503, 1975). Since the 
host cells were lysogenic for .lambda., the transformants could not be 
tested directly for the .lambda. O.sub.L /P.sub.R sequence by colony 
hybridization. Instead, DNA from 45 transformants which did have hsa 
sequences (identified in step A above) was prepared, plasmid DNA was 
separated from chromosomal DNA on an agarose gel, and a Southern blot was 
prepared from this gel. The correct plasmid DNAs were identified by 
hybridization to a .sup.32 P-labeled O.sub.L /P.sub.R fragment, made by 
end labeling the 164 base pair EcoRI to BamHI fragment from pGX2606. 
Hybridization was carried out as in A. 44 isolates had the O.sub.L 
/P.sub.R sequences. 
C. Identification of correctly-constructed plasmid. Plasmid DNAs from each 
of the 45 transformants tested in step B were analyzed by restriction 
endonuclease digestion. Two clones appeared to have the proper 
construction according to: 1) analysis of the size of the undigested 
plasmids by agarose gel electrophoresis, 2) lack of a BamHI site (the 
pGX1042 parent has a BamHI site but the desired recombinant does not) and 
3) presence of restriction fragments diagnostic for the presence of the 
O.sub.L /P.sub.R regulator. 
D. DNA sequencing. Two of the plasmid DNAs which had all the expected 
characteristics described above were subject to sequencing in phage M13. 
M13 subclones of the O.sub.L /P.sub.R -hsa-3 fusion from pGX1043 were 
constructed by cloning the O.sub.L /P.sub.R -hsa-3 segment (EcoRI to 
HindIII) from pGX1043 into M13mp9 (EcoRI to HindIII). Dideoxy DNA 
sequencing was performed by the method of Sanger, F. et al., Proc. Natl. 
Acad. Sci. USA 74:5463 (1977). An isolate which had the predicted sequence 
was termed pGX1043. 
Expression of metHSA 
In order to test for expression of HSA, plasmid pGX1043 was transferred to 
strain GX1864 which carries the temperature inducible, defective prophage 
.lambda..DELTA.Hl.DELTA.Bam cI857. Transcription was then induced from the 
O.sub.L /P.sub.R promoter by raising the temperature to 42.degree. C., 
and samples taken at different times were analyzed. The samples were 
subjected to electrophoresis in SDS-polyacrylamide gels (U. Laemmli Nature 
227:6880, 1970) followed by the Western blot procedure (H. Towbin et al. 
Proc. Natl. Acad. Sci. U.S. 76:4350, 1979, W. Burnette Anal. Biochem. 
112:195, 1981.) HSA was assayed using anti-HSA antibody followed by goat 
anti-rabbit antibody coupled to horseradish peroxidase. A color 
development procedure was used to visualize the antigen bands. Controls of 
the host strain as well as uninduced cells containing pGX1043 showed no 
stainable bands. Induced pGX1043 DNA gave rise to a major band with a 
mobility corresponding to a molecular weight of 68 kilodaltons (kd). There 
were also minor bands with higher mobilities corresponding to lower 
molecular weights. These minor bands could arise from proteolytic 
degradation of HSA or from abnormal transcription or translation starts 
and stops in the hsa gene. 
By comparing the intensity of the stained 68kd band from pGx1043 with known 
amounts of pure HSA (Sigma Chemical Co.), it was estimated that 0.2% of 
the total protein in extracts of induced pGX1043 was HSA after 2 hours 
induction. This amount of expression was confirmed by performing 
immunoprecipitation from extracts labeled with H-leucine during induction 
as before. Known amounts of HSA (fraction V Sigma Chemical Co.) labeled 
with .sup.14 C-formaldehyde were used as an internal standard (Rice, R.H. 
and G.E. Means). The standard was added to cell extracts which were then 
immunoprecipitated by the method of S.W. Kessler (J. Immunol. 
115:1617-1624, 1975) with minor modifications. The immunoprecipitate was 
subjected to electrophoresis on a 7.5% polyacrylamide gel and the HSA band 
was cut out and ozidized in a Packard sample oxidizer. The .sup.14 C 
O.sub.2 and .sup.3 H.sub.2 O products were separately quantitated by 
liquid scintillation spectrometry. The yield of .sup.3 H-HSA was 
determined by direct comparison to the yield of added known amounts of 
HSA-.sup.14 C standard. The amount of .sup.3 H-HSA was then calculated as 
a percentage of the total .sup.3 H leucine incorporated into bacterial 
protein. The maximum yield of HSA was 0.2% of the total protein. 
An E.coli culture transformed with this plasmid has been designated GX1864 
(pGX1043) and deposited with the Northern Regional Research Labortory, 
Peoria, Ilinois, as NRRL No. B-15613.