Phage-cosmid hybrid vector, open cos DNA fragments, their method of use, and process of production

A hybrid vector produced from a linearized cosmid and arms from .lambda. phage vector DNA has been developed. The hybrid vector can be used to generate fragments which are useful as vectors in a helper phage-mediated transformation system, permitting large fragments of foreign DNA to be introduced into a host on an industrial scale.

FIELD OF THE INVENTION 
The present invention is directed to a hybrid vector produced from .lambda. 
phage vector DNA and cosmid DNA, open cos DNA fragments, their use in a 
helper phage-mediated transformation system, and a method of producing 
same. 
BACKGROUND OF THE INVENTION 
In 1960, Kaiser and Hogness found that a lysogenic E. coli strain of 
.lambda. phage could uptake .lambda. phage DNA encoding a different 
repressor gene. This was done by infecting the same type of .lambda. phage 
as helper phage in advance at high multiplicity of infection (m.o.i.), and 
the cells would allow the incorporated DNA to replicate and the cells to 
lyse (15) (the preceding and following numbers in parentheses are in 
reference to the numbered list of references contained at the end of the 
specification). This is known as helper phage mediated transformation 
(HPMT). Since 1960, HPMT has been improved and used to demonstrate the 
colinearity of the vegetative genetic map of .lambda. with the physical 
sequence of genes along the DNA (16, 17, 18). The repressor is expressed 
in the lysogen and its protein inhibits the growth of the helper phage. 
However, once the external phage DNA, which encodes a different repressor, 
is incorporated and replicated, the cells lyse. Only .lambda. phage DNA 
can be incorporated when .lambda. phage is used as the helper phage. 
Subsequently, it was discovered that the lambdoid phages and P2 phage had 
the same effect, but the ends of the DNA to be incorporated must coincide 
with the helper phage, suggesting that the structure of the ends of the 
phage DNA ("cohesive" ends) are related to this phenomenon (19). 
In 1981, Miller and Feiss demonstrated how cells infected with the helper 
phage could incorporate the additional phage DNA (20). The authors 
designed the experiments to prove this function using cos-open cosmid DNA 
which was cut by the ter reaction in vitro. They could recover one type of 
hybrid DNA in circular form which consisted of full helper phage and 
cosmid DNA ligated at each cohesive ends. They made three models to 
explain this observation as follows: 
1. Helper phage DNA molecules and free DNA molecules enter the cell without 
interacting and cyclize at random. 
2. Cohesive end annealing between the helper phage and the free DNA is 
necessary for entry into the cell. 
3. A high probability of cohesive end annealing between the free DNA and 
the helper phage might exist as a consequence of the proximity of the 
helper cohesive end(s) to the entry of the free DNA. 
The first model is not likely because the formation of multimers by 
cohesive end annealing is rare, and the plasmids resulting from the 
experiments almost always contain the helper phage chromosome. The second 
model fits their results. However, the possibility of the third model 
cannot be excluded. They also pointed to two types of plasmid instability; 
defective segregation and plasmid breakdown. The former may simply be due 
to a low copy number of the recombinant, and the latter may occur by a 
recA-independent recombination. 
.lambda. DNA is linear when it is in a capsid, but once the phage infects 
E. coli cells, packaged DNA is injected into the cells uncovering the 
termini (i.e., cohesive ends) to be circularized for DNA replication, 
transcription, and translation. The cohesive ends of linear .lambda. phage 
DNA are self-complementary, extruding 12 bases (or nucleotides) and 
facilitating circularization after infection. The E. coli host strain, 
which is a .lambda. lysogen, can adsorb .lambda. phage at a high 
multiplicity of infection (super-infection, m.o.i.=10) without lysis 
because the lysogen produces phage-specific repressor proteins (cI 
repressor) which repress the expression of phage operons. Usually, 10 to 
20 molecules of repressor protein are produced in a lysogenic strain. 
.lambda. phage needs .lambda. receptors on the membrane to adsorb to E. 
coli cells. This phenomenon determines the host specificity, and the 
receptor is encoded on the E. coli chromosome (lamB). Adsorption can be 
attained in a few minutes even at 0.degree. C., and it can be maintained 
without injecting DNA into the cell when the cells are incubated at 
0.degree. C. Adsorption is a reversible reaction, and follows a Poisson 
distribution because only spontaneous molecular collisions between 
.lambda. phage tails and receptor proteins determine efficiency. With a 
short incubation above 15.degree. C., but at permissive temperatures, 
simultaneous DNA injection from the phage heads into the cells takes place 
and the DNAs remain temporally between the outer and inner membranes 
(periplasmic space). This state can be maintained for at least 5 hrs at 
0.degree. C. Flip-flop of injected linear phage DNA occurs on the outer 
membrane while in this state, resulting in the cohesive end of phage DNA 
extruding frequently through the outer membrane, thereby trapping any 
external DNA having cohesive ends (e.g., a foreign DNA segment desired to 
be introduced into the host) by annealing. A temperature shift from 
0.degree. C. to 30.degree. C. or 37.degree. C. allows the DNA to be drawn 
into the cytoplasm. The authors (20) designed an experiment to prove this 
function using cos-open cosmid DNA which was cut by the ter reaction in 
vitro. The mechanism of introducing the injected phage DNA is not known, 
but the host mutant (pel-) does not allow DNA injection from phage 
particles (21). Host factors may play an important role in the process. 
The overall process is illustrated in FIG. 1. 
The ter reaction was constructed by Becker and Gold isolating the key 
enzyme, pA (terminase) (22), and the in vitro system is commercially 
available as a kit (LAMBDA TERMINASE.TM., Gibco BRL). However, since the 
reaction requires unknown E. coli factors, the reaction is not as 
efficient as other single enzyme reactions, which makes duplication 
difficult. Since this ter reaction was the only way to produce the open 
cos arms from the cosmid, application of this method for general cloning 
purposes was not indicated by the authors and, in fact, was actually 
impossible at that time. 
More recently, cloning of long DNA segments has become important in the 
construction of gene libraries and analysis of very long genes, especially 
those of the eukaryotic genomes. It has become clear that many genes are 
too long to be clones as single fragments in standard vectors. The 
bithorax and antennapedia of Drosophila, for example, contain 
transcription units of 75 and 100 kb (1,2,3,4), respectively. The human 
factor VIII is 180 kb (5), and dystrophin's transcription unit is the 
longest found to date (1,800 kb) (6). The ultrabithorax (Ubx) gene is 
regulated by sequences located upstream (50 kb) and within the introns 
(7). And although a gene such as the human .beta.-globulin gene has a 
transcription unit of only few kb, sequences as far as 50 kb upstream 
appear necessary to obtain full expression in transgenic mice (8). Thus, 
the ability to clone large DNA segments containing relatively small genes 
is important. 
These large functional units of DNA need to be manipulated and cloned 
efficiently. In most cases, to examine the functions of cloned segments, 
DNAs can be delivered into the eukaryotic cells with or without the 
vector. Transfection of cultured mammalian cells requires no special 
vector but marker genes are usually needed, e.g., the thymidine kinase 
gene for work with mammalian cells, suppressor tRNA genes for 
transformation of nematodes, and G418 for P-element mediated transfection 
into Drosophila cells (9,10). Most of these genes were isolated from 
libraries constructed in phage vectors which can clone inserts up to 24 kb 
in size, or in cosmid vectors which are useful up to 45 kb in length (11). 
Large regions of the genome are usually cloned in a series of overlapping 
recombinants isolated by chromosome walking. A segment of non-repetitive 
DNA isolated from one end of a recombinant is used as a probe to screen 
recombinant clones containing adjacent sequences. This process is repeated 
until the entire region has been covered. This method requires an 
extraordinary amount of time and labor. Recently, a P1 phage vector and 
its in vitro packaging system were developed and the technique could reach 
a cloning range of 100 kb in E. coli (12). However, undesirable size 
restrictions are associated with this system. 
The YAC system was developed based on the creation of extra chromosomes 
introducing telomeres in yeast (13). In addition, the vector has two small 
units that function as a centromere and as a replication origin (ARS 
element). This technique allows the cloning of much larger segments of 
DNA, up to several hundred kb, but the yeast chromosomes are more 
difficult to handle than bacterial plasmids due to their linear 
configuration, which can be sheared during manipulation. Screening clones 
from yeast colonies is more difficult than from E. coli, and isolating the 
target DNAs from the positive clones requires special techniques, 
necessitating more restrictions during manipulation than when E. coli is 
used. 
Shizuya et al. (4) introduced a new E. coli cloning system based on an 
F-factor vector (BAC). They achieved cloning of DNA up to 300 kb, 
demonstrating the stability of cloned DNA and ease of its manipulation 
because existing passenger DNA is the form of a supercoiled circular 
plasmid. However, since this method depends on electroporation, long DNA 
cannot be incorporated into cells as easily. 
Therefore, a system for E. coli cells is still needed for the cloning of 
large DNA segments at high efficiency and which is easy to use. The 
present inventors have discovered such a system as hereinafter described.

DETAILED DESCRIPTION OF THE INVENTION 
One embodiment of the present invention is a hybrid vector comprising a 
linearized cosmid having a single unique restriction enzyme site between 2 
marker genes, wherein the linearized cosmid is ligated between left and 
right arms produced from digestion of .lambda. phage DNA. The vector is 
useful for producing arms having open cos ends that can be used to 
transform a bacterial host with large segments of foreign DNA on an 
industrial scale. A preferred hybrid vector is .lambda.KAD93.4, which is a 
hybrid phage prepared from .lambda.gtll and pHC79 cosmid DNA. 
A second embodiment of the present invention is a method of transforming a 
bacterial host with a foreign DNA sequence comprising the steps of: 
digesting .lambda. phage vector DNA with a restriction enzyme to generate a 
left arm and a right arm, 
linearizing a cosmid vector with the restriction enzyme to produce 
linearized cosmid DNA, 
ligating the linearized cosmid DNA between the left and right arms to 
produce a hybrid phage-cosmid vector, 
packaging the hybrid phage-cosmid vector into phage heads in vitro, 
infecting a bacterial cell with the phage particles to propagate the phage 
particles thereby forming phage plaques, 
transferring the plaques onto a membrane, 
screening the hybrid phage-cosmid vector from the membrane by hybridizing 
with a DNA probe, 
preparing a phage lysate by infecting the hybrid phage-cosmid vector to the 
host bacterial cell, 
precipitating the cell debris from the lysate, 
removing the bacterial cell's DNA and RNA by nuclease treatment, 
precipitating the propagated phage particles, 
extracting the packaged DNA from the precipitated phage particles, 
digesting the extracted DNA with the restriction enzyme to generate a 
mixture of DNA fragments, 
isolating the shortest two fragments from the mixture of fragments which 
are DNA fragments bearing an open cos site on one end, 
ligating a foreign DNA sequence between the two open cos site fragments, 
and 
transforming a host bacterial cell with the ligated foreign DNA sequence 
having the two open cos sites using a helper phage-mediated transformation 
system. 
The restriction enzyme used in the above method is selected according to 
the type of vector used. A preferred restriction enzyme is EcoRI when 
.lambda.gtll is used. 
The marker genes can be any conventionally used marker gene known to those 
of ordinary skill in the art. Preferred marker genes are antibiotic 
resistance genes. Particular preferred marker genes are amp.sup.r and 
tet.sup.r, encoding ampicillin and tetracycline resistance, respectively. 
Still another embodiment is a bacterial host transformed with a desired 
segment of foreign DNA ligated between the open-cos arms produced from the 
hybrid vector, wherein transformation is effected using the helper 
phage-mediated transformation system. 
Still another embodiment is a purified DNA fragment comprising an open cos 
site on one end and a restriction enzyme site on the other end. 
Preferably, the fragment is up to about 3.3 kb in length. A preferred cos 
site has the sequence 5' GGGCGGCGACCT 3' (SEQ ID NO:1). A preferred 
restriction enzyme site is EcoR1. Preferably, the purified DNA fragment 
further comprises a marker gene to facilitate identification and 
maintenance. 
The foreign DNA sequence desired to be introduced into the bacterial host 
cell can be any DNA sequence up to about 5 megabases (Mb) in length, more 
preferably, up to about 1 Mb in length. For example, suitable foreign DNA 
sequences may be produced from genomic DNA that has been digested with a 
suitable restriction enzyme or exonucleases and fractionated. Any known 
method for preparing DNA fragments can be used to produce a foreign DNA 
sequence that may be introduced into the bacterial host cell using the 
method of the present invention. 
Any vector of .lambda. phage permitting the introduction of a piece of DNA 
may be used as the source of DNA for the hybrid vector. A preferred strain 
of .lambda. phage is .lambda.gtll whose unique cloning site is for EcoRI. 
Any cosmid vector may be used which is autoreplicable and contains a single 
unique restriction enzyme site, which is compatible with that of a 
.lambda. phage vector, between 2 selective markers. Preferably, the cosmid 
vector comprises a set of unique restriction enzyme sites between 2 
antibiotic resistance genes. Preferably, the cosmid contains short cosmid 
arms having a .lambda. cos site at almost the same distance from the 
cloning site for easy fractionation. A short arms region derived from 
.lambda.DNA containing cos is preferred because they reduce the likelihood 
of homologous recombination and they are needed for in vivo phage 
packaging. Preferably, the cosmid is also a low copy and not a run-away 
type plasmid, which will increase the likelihood of homologous 
recombination. A preferred cosmid is pHC79. 
As used herein, the term "cos site" means a sequence of the cohesive end of 
lambda and "cos end" or "open cos site" means a 5'-end overhang of 
.lambda. DNA containing a 12 base sequence: 5' GGGCGGCGACCT 3' (SEQ ID 
NO:1). The cos site is created by the ter reaction including pA and pNu-1. 
In the step of isolating the hybrid phage-cosmid vector from the membrane, 
any suitable DNA probe can be used which will bind to the DNA of the 
hybrid phage-cosmid vector. A preferred probe is a pHC79 DNA fragment. 
Other screening techniques known in the art can be used. 
The host bacterial strain should be a stable lysogen, and the lysogenized 
phage should be blocked to prevent induction and repress expression of the 
super-infected helper phage. A shortage of repressor may induce the 
expression of the super-infected helper phage and cell lysis. Also, the 
host bacterial strain preferably has rec mutations or related mutations 
which minimize recombination. Also, the host bacterial strain should have 
a set of marker genes for easy maintenance and identification. 
A preferred bacterial strain which can be used to construct a host strain 
for HPMT by infecting .lambda.ind- is the SURE.TM. strain (available from 
Stratagene, La Jolla, Calif.) having two rec mutations: recB and J. 
Another preferred host bacterial strain is E. coli strain NS428 
(.lambda.red) and NS433 (.lambda.red), which possesses .lambda.red and the 
recA mutation. Since the latter strain can be lysed at temperatures around 
42.degree. C., it should be grown at 30.degree. C., which ensures slow 
growth and may reduce the level of recombination. In addition, any other 
lysogenic strain with .lambda. phage and the rec mutations can be used. 
Other preferred host bacterial strains include E. coli KD9302 and KD9402. 
The former strain has both recB and J mutations to repress recombination, 
F' with tet.sup.r as a genetic marker, and .lambda.ind- in the genome, and 
the latter is the same as the former except F'. When the former strain is 
cultured in a medium with tetracycline, it is rarely lost due to 
segregation of F'. In addition, both strains are believed to have tandemly 
repeated .lambda.ind- on their genomes, which is advantageous because it 
ensures the production of a sufficient amount of repressor. 
As shown in FIG. 1, helper-phage mediated transformation involves 
lysogenizing E. coli cells with .lambda. phage followed by infection with 
excess .lambda. phage ("helper" phage) at mid log phase. When infection is 
carried out at 0.degree. C., phage can adsorb on the cell surface 
reversibly. Then, a short incubation of the cells at a suitable 
temperature (around 30.degree. to 37.degree. C. depending on the type of 
host strain) allows DNA injection into the periplasmic space of the cells. 
This state can be held for several hours if the cells are kept on ice 
after phage adsorption. Alternatively, in a suitable medium, cells can be 
kept in the freezer for several months without loss of activity. 
The injected helper phage DNA protrudes its ends bearing cos ends 
occasionally outside the surface of the outer membrane. This allows 
foreign DNA ligated between cosmid arms to be annealed to the cos ends of 
the helper phage by mixing with the cells at 0.degree. C. In such a 
manner, the cos end of the helper phage can trap the complementary cos 
site from the foreign DNA. Subsequent incubation at a suitable temperature 
facilitates incorporation of the phage DNA into the cytoplasm with the 
annealed vector-plus-foreign DNA at the cos site. Although the mechanism 
is still not fully understood, proteins produced by E. coli are known to 
play an important role in this stage. This incorporation occurs 
independently of DNA size. 
Once the phage DNA or foreign DNA hybrid has been incorporated into the 
cytoplasm, the hybrid can be circularized by annealing at both cos ends, 
and then ligated to start replication. Since the .lambda. phage 
replication is blocked by the repressor produced by the lysogenic .lambda. 
phage in the host strain, the attached replication origin of the cosmid 
allows replication of the hybrid DNA. The replication can be regulated by 
the ColE1/ori on one of the cosmid arms, while the copy number of the 
hybrid DNA can be regulated as well. The hybrid DNA can be amplified by 
addition of chloramphenicol prior to the DNA extraction in the same manner 
as other relaxed type plasmids. Preferably, a low copy number cosmid DNA 
is selected, which does not interfere with the chromosomal DNA of E. coli 
replication. 
The invention is more clearly illustrated by, though in no way limited to, 
the following examples. 
Construction of a Lysogenic Strain 
To prepare the infective phage as the helper phage in this method, the 
titer must be as high as possible. The method utilizes multiplicity of 
infection (m.o.i. )=10 to prepare competent cells. Since a high 
concentration of the recipient cells is desirable to obtain a high 
transformation rate, the titer of the helper phage should exceed 10.sup.10 
pfu/ml ("pfu" stands for plaque forming unit). Infection in broth cannot 
attain this value. Therefore, a new lysogen strain of E. coli was 
constructed as follows. 
E. coli strain LE392 (e14.sup.- (mcrA) hsdR514 supE44 supF58 lacY1 or 
.DELTA.(laclZY)6 galK2 galT22 metB1 trpR55) and .lambda..sub.cl857sam7. A 
fresh overnight culture of LE392 was poured onto a .lambda. agar plate 
with phage (.lambda..sub.cl857sam7) to make a confluent plate. The plate 
under these conditions is usually clear. But, if propagation is 
established by superinfection, some of the colonies appear on the plate. 
In such a way, 22 colonies were obtained, and after purification these 
were examined by infecting phage. All were resistant to the same phage, 
and the integrated phage was inducible at non-permissive temperature 
(42.degree. C.). Since the repressor of the phage is temperature sensitive 
(CI.sup.857), the lysogen must be grown at permissive temperature 
(30.degree. C.). Then, the culture is shifted to non-permissive 
temperature to inactivate the repressor. After this treatment, cell lysis 
begins because the amber mutation of protein S (Sam7) is suppressed by two 
sup mutations (supE and supF). To get the highest titer of the phage, the 
culture is concentrated before induction rather than at the endpoint of 
induction as with other strains. The latter case sometimes lyses cells 
from inside because of high concentration of phage. This may lead to the 
loss of phage before cell lysis with chloroform. The titer showed 
1.times.10.sup.10 to 10.sup.11 pfu/ml. 
Construction of a Host Strain 
The strain NS428, which is a stable lysogen of .lambda.red, was used. As 
this strain is temperature sensitive, it is necessary to grow the cells at 
30.degree. C. which extended the doubling time. Especially, on the plate, 
the transformants grew extraordinarily slowly. To improve the cell growth, 
a new strain was constructed introducing better mutations. NS428 has a 
recA mutation to prevent recombination, but recA alone is not sufficient 
for the highly repetitive sequences which are common in the eukaryotic 
genomes. SURE cells (Stratagene) are well designed to maintain even Z-form 
DNA by introducing two rec mutations (recB and J). This strain was used to 
construct the lysogen. .lambda.ind- was infected into SURE cells as 
described in the preceding section (Construction of a Lysogenic Strain), 
and the lysogens were isolated. Out of 12 clones, there were typically two 
groups, one of which was more sensitive to superinfection of the helper 
phage, and the other of which was quite stable for superinfection. Since 
.lambda.ind- can be integrated in the genome tandemly, the latter group, 
which is believed to be a tandem .lambda.ind- prophage, was selected. 
Curing of KD9302 Strain 
A new strain of E. coli, KD9302, was constructed and its ability for helper 
phage mediated transformation was tested. Transformation efficiency was 
the same as with NS428 discussed above. For small plasmid DNA, the 
presence of F' may not affect recovery of the target plasmid DNA. Since 
the helper phage mediated transformation aims at cloning extremely long 
DNA fragments in E. coli cells, the size of the cosmid DNA in the cells 
may compete with F' whose size range is from 70-150 kb. The method of 
cosmid extraction may also cause F' DNA contamination which would make 
identification of the target DNA difficult. Therefore, the curing of F' 
from the strain, KD9302, was carried out with three commonly used 
reagents: acridine orange, ethidium bromide and SDS. 
Fifty .mu.L of an overnight culture of KD9302 in MMB (27) was inoculated in 
fresh 5 mL of MMB containing 30 .mu.g/ml acridine orange, 0.5 .mu.g/ml 
ethidium bromide, and 10% SDS, respectively. Then the cultures were 
incubated overnight. Cultures containing acridine orange and ethidium 
bromide grew well, but SDS culture showed very poor growth. Each one was 
streaked on LB agar plate and incubated. Even though the growth of the 
cells in 10% SDS medium was poor, the plate gave enough colonies to be 
examined. From each plate, 50 colonies were transferred on both plain LB 
and LB agar plate containing tetracycline to examine tetracycline 
resistance. If F' is cured, tetracycline resistance is removed as well, 
because its gene is located on the F'. These plates were incubated 
overnight. From SDS treated cells, 3 colonies were isolated showing 
tetracycline sensitivity. From acridine orange treated cells, 1 colony was 
isolated. No colonies were formed on the ethidium bromide plate. These 
colonies were then inoculated in MMB and incubated overnight. The next 
day, cultures were streaked on Davis' minimal medium agar plates, one 
included proline and the other did not. Since proline is linked with 
tetracycline on the F' and the entire proline gene was deleted on the host 
strain, the cured strain should show proline dependence. All isolated 
strains required proline, proving that all strains were cured. Another 
advantage of these strains was that the cells could have two antibiotic 
resistance marker genes to make strain maintenance easier. The new strain 
was named as KD9402. 
Production of Cosmid Arms from Hybrid Vector 
A .lambda. phage vector, .lambda.gtll, and cosmid DNA, pHC79 (available 
from Gibco BRL), were both digested with EcoRI. The hybrid vector was 
constructed with two cos sites, one on .lambda.gtll and the other on the 
cosmid. In the next step shown in FIG. 2, the linearized pHC79 DNA is 
ligated in between the right arm of .lambda.gtll and the left arm of 
.lambda.gtll, after which the hybrid DNA is packaged in vitro into phage 
heads (a kit for which is available from Gibco BRL). Because the packaged 
fraction is not homogeneous, self-ligated lambda phage is dominant in the 
fraction. This produces recombinants by a reaction in vitro. 
When the host strain is infected with the packaged phage particles, two 
types of hybrid DNA are made in vivo as shown in FIG. 2. The hybrid DNA 
vector can be packaged in two different ways using cos sites derived from 
either .lambda.gtll or pHC79. As shown in FIG. 2, the resulting population 
of the hybrid phage includes both forms, denoted A and B in FIG. 2. 
To package the .lambda. phage DNA in vivo, .gamma. protein encoded by the 
.lambda. phage plays an important role. After injecting the .lambda. phage 
DNA into the cytoplasm of a host cell, cos ends annealed and the .lambda. 
DNA was circularized. At this point, so-called "Cairn's type" replication 
takes place. Once a sufficient amount of DNA is accumulated in the cell's 
cytoplasm, .gamma. protein converted the replication from Cairn's to 
"rolling circle" replication for production of concatamers by inhibiting 
the activity of recBC protein. Then cos to cos packaging begins. Protein 
A, Nu-1 and other factors permits uptake of concatamers into .lambda. 
phage heads and causes cleavage at the cos site. As a result, one molecule 
of .lambda. DNA having open-cos sites at both ends is packaged into each 
.lambda. phage head. 
As explained above, .lambda.gtll and pHC79 are preferred for construction 
of the recombinant hybrid vector because: 
1. Total size of cosmid should be as small as possible. 
2. The location of cos site in cosmid vector should be almost the same 
distance from the cloning site (EcoRI site). 
3. Each arm, when produced by cleaving at the cos site, should have a 
marker (e.g., an antibiotic resistance gene). 
4. The cos region should be minimized to minimize the homologous 
recombination. 
5. The origin of replication should be from ColE1 to be a relaxed type 
plasmid whose copy number can be controlled by chloramphenicol. 
Extraction and EcoRI digestion of the hybrid DNA (i.e., forms A and B of 
FIG. 2) produce six fragments, the right arm of .lambda.gtll, the left arm 
of .lambda.gtll, intact .lambda.gtll DNA, linearized pHC79, the right arm 
of pHC79, and the left arm of pHC79. The fragments can be isolated by 
either gel electrophoresis, gel filtration, HPLC, or sucrose density 
gradient ultracentrifugation. In a preferred embodiment, the cos ends are 
treated with alkaline phosphatase, and then the .lambda. DNA is digested 
with EcoRI. The digests are fractionated with either sucrose density 
gradient ultra-centrifugation or gel electrophoresis. The smallest two 
fragments are collected and purified. Only the cosR and cosL fragments 
resulting from the cosmid DNA (shown in fragment B of FIG. 2) are used in 
the present invention. 
Use of Hybrid Vector Cos Arms to Transform Host With Foreign DNA 
There are two main steps involved in this process, one is to prepare 
competent cells, and the other is to transform the cells with a desired 
foreign DNA segment using the cosmid arms produced from the 
above-described hybrid vector. These two main steps can be further broken 
down into the following specific steps. 
Step 1: Preparation of competent cells 
The host strain is inoculated in 10 mL of P medium (as shown in Kaiser et 
al. (15)) and then incubated at 37.degree. C. until the OD.sub.600 
(optical density at 600 nanometers) reaches 0.5. The cells are chilled on 
ice, centrifuged, suspended in 5 ml of Medium 1 (also as shown in Kaiser 
et al. (15)), then the helper phage (.lambda.cI857Sam7) is infected at 
m.o.i.=10. The cells are first incubated on ice for 10 min., then at 
37.degree. C. for 5 min., and then again on ice for 5 min. 
To remove excess non-adsorbed phage, the cells are centrifuged, rinsed with 
5 ml of ice-cold TMC (Tris-Mg.sup.2+ -Ca.sup.2+ buffer, pH 7.0, 
containing 10 mM Tris HCl, 10 mM MgCl.sub.2, and 10 mM CaCl.sub.2), TM 
(Tris-Mg.sup.2+ buffer) or a storage buffer, aliquoted in microfuge tubes 
and frozen in liquid nitrogen (when storage as frozen cells is desired). 
Step 2: Preparation of Foreign DNA to be Transformed 
Any known method of DNA preparation can be applied to prepare the foreign 
DNA that is desired to be transformed using the helper phage-mediated 
transformation method. There are several steps by which transformation 
efficiency can be optimized: 
A. DNA extraction from the source; 
B. DNA sizing (e.g., sonic treatment, partial digestion with restriction 
enzymes, exonuclease treatment, etc.); 
C. DNA fractionation (sucrose density gradient ultracentrifugation, HPLC, 
gel electrophoresis, gel filtration); 
D. end treatment with an exonuclease (e.g., ExoVII) when sonic treatment, 
or exonuclease treatment; 
E. adaptor attachment: if the technique described in step D is used, 
adaptors which are complementary to the cloning site of the vector arms 
are ligated to the insert DNA. The short fragment of the adaptor DNA is 
phosphorylated but the longer fragment is not, thus avoiding concatamer 
formation of the adaptor DNA on the target DNA. 
F. removal of non-ligated adaptor DNA: excess free adaptor DNA should be 
removed efficiently if the technique described in step E is used. If 
adaptor remains in the subsequent steps, it will inhibit efficient 
reactions. Gel filtration, gel electrophoresis, HPLC, or sucrose density 
gradient ultracentrifugation can be used for this purpose. 
Step 3: Ligation of Foreign DNA Between Cosmid Arms 
The foreign DNA described in step 2 is ligated between the cosmid arms 
described above in the section entitled "Production of Cosmid Arms from 
Hybrid Vector". 
Step 4: Transformation 
Competent cells (described above in step 1) which have been stored at 
-80.degree. C. (when frozen cells are used) are thawed at 37.degree. C. 
for 1.5 min. with gentle shaking. Ice-cold 10 mM Tris-HCl (pH 7.1) is 
added to the stock cells to dilute them five-fold, and the cells are 
chilled on ice. After precipitation, the cells are suspended in TM(C) 
(2.times.10.sup.9 ml) and kept on ice for 45 min. The ligated DNA (100 
microliters) is mixed with 200 microliters of the competent cells on ice, 
then the mixture is incubated at 30.degree. C. for 1 h to allow complete 
injection into the cytoplasm of the cell. The cells are spread onto a 
selective plate containing ampicillin and tetracycline. Roughly one 
transformation can be achieved with 10.sup.3 DNA molecules (26). 
Example 1 
Preparation of the Helper Phage 
E. coli K-12 LE392 was infected with (helper) .lambda. phage 
.lambda.cl857Sam7. LE392 was streaked on .lambda. broth and incubated at 
37.degree. C. overnight. Five colonies were selected and cultured in 
.lambda. broth at 37.degree. C. overnight with aeration. With a loop, the 
stock of .lambda.cl857Sam7 phage was streaked onto a .lambda. agar plate, 
and each culture was cross-streaked onto the same plate with a loop. After 
overnight incubation at 37.degree. C., the cultures which had proper 
infectivity formed plaques. One E. coli strain was chosen, inoculated in 
.lambda. broth, and cultured at 37.degree. C. overnight. 500 microliters 
of the LE392 bacterial strain was infected with 1 microliter of the stock 
.lambda. phage (10.sup.10 pfu/ml) at room temperature for 15 min., and 
then transferred to 5 ml of fresh .lambda. broth. The infected strain was 
grown at 37.degree. C. with shaking (275 rpm) until the culture became 
transparent by lysis. This took about 6 hours. Chloroform (100 
microliters) was added to the culture and centrifuged for 10 min. at 
4.degree. C. to collect the supernatant (phage stock). This stock phage 
showed a titer ranging from 1.times.10.sup.8 to 1.times.10.sup.9 pfu/ml 
typically. 
Phage-lysogenized E. coli cells were also prepared in the following way. 
The E. coli freshly prepared overnight culture (LE392, 200 microliters) 
was poured onto a .lambda. agar plate with 10 microliters of phage 
(.lambda.cl857Sam7, diluted to 10.sup.4 /ml) in 4 ml of .lambda. soft 
agar, and incubated at 30.degree. C. overnight. The plates under this 
condition usually become confluent while the E. coli strain lysogenized 
with .lambda. phage will make colonies. The plate showed 22 colonies. 
The lysogen (KD9301) was selected and cultured in 5 ml of .lambda. broth at 
30.degree. C. overnight. 200 microliters of the overnight culture was 
inoculated in 5 ml of fresh .lambda. broth, and cultured at 30.degree. C. 
for about 4 hours until the OD.sub.600 reached 0.5. The culture was 
transferred to a 42.degree. C. water bath to inactivate the cI repressor 
which is a temperature sensitive repressor. Then, the culture was shaken 
at 42.degree. C. for another 5 h. The culture was centrifuged to 
precipitate the cells, and suspended in 500 microliters of TM. Chloroform 
(100 microliters) was added to the culture to lyse the cells, which were 
then centrifuged to remove the cell debris. The supernatant showed a titer 
ranging from 1.times.10.sup.10 to 1.times.10.sup.11 pfu/ml typically. 
Isolation of the Foreign DNA to be Transformed 
For the foreign DNA to be transformed, DNA of .lambda.KAD93.4 was selected. 
.lambda.KAD93.4 DNA was extracted from a phage lysate and isolated at a 
concentration of 0.2 micrograms/ml. 
Transformation of Host Bacterial Strain 
The E. coli strain NS428 was grown overnight at 30.degree. C. to prepare a 
fresh culture, The overnight culture (50 microliters) was inoculated in 5 
ml of broth, and shaken at 30.degree. C. until OD.sub.600 reached 0.8 
after about 4 hours. The cells were harvested by centrifugation, suspended 
in 2 ml of ice cold TM buffer, and stored on ice. The cells were mixed 
with the helper phage at m.o.i=10 on ice, and incubated on ice for 10 min. 
The mixture was incubated at 30.degree. C. for another 10 min. to allow 
phage DNA injection, and ice cooled for one min. 
Ice-cold TM (500 microliters; 10 mM Tris-HCl, pH 7.0-10 mM MgCl.sub.2) was 
added to the mixture, and centrifuged at 4.degree. C. for 1 min. to remove 
the non-infected phage. The cell pellet was then suspended in 200 
microliters of ice-cold TMC (10 mM Tris-HCl, pH 7.0, 10 mM MgCl.sub.2, 10 
mM CaCl.sub.2), and stored on ice until used. To these competent cells 
(200 microliters), 0.1 microgram of .lambda.KAD93.4 DNA in TE (Tris-EDTA 
buffer: 10 mM Tris-HCl at pH 8.0 plus 1 mM EDTA at pH 8.0) was added on 
ice after incubation at 70.degree. C. for 10 min. to dissociate the cos 
ends from self-annealing. The mixture was then incubated on ice for 10 
min. to allow the cos ends to anneal. The mixture was incubated at 
30.degree. C. for 30 min. with gentle swirling, and 1 microliter of 10 
mg/ml DNase I was added to digest the rest of the DNA for transformation. 
One ml of LB broth (Luria-Bertani broth) was added to the mixture. The 
culture was incubated with shaking at 30.degree. C. for 1.5 h, then spread 
(100 microliters) onto an LB agar plate with ampicillin (100 
micrograms/ml) and tetracycline (25 .mu.g/mL). The plates were incubated 
at 30.degree. C. overnight. The colonies on the plates were counted and 6 
randomly picked colonies were cultured in 5 ml of MMB (J. T. Baker Inc., 
Phillipsburg, N.J.) at 30.degree. C. overnight. The transformation 
efficiency was about 5.times.10.sup.6 cells/microgram DNA. 
The resulting cultures were treated to extract the plasmid DNA with the 
SCREEN MAX MINI PLASMID extraction kit (available from J. T. Baker Inc.) 
or the PLASMID FAST kit (available from Amresco, Inc.). 
Example 2 
Construction of Hybrid Phage 
The procedure shown in FIG. 2 was followed as discussed above and Fragment 
B (the open-cos cosmid arms) was isolated. The fragment can be isolated 
either by gel electrophoresis or sucrose density gradient 
ultracentrifugation. 
To fractionate the open-cos arms, the two arms should be almost the same 
size. If they are not substantially the same size, the longer arm will 
migrate or sediment closely with the linear cosmid DNA on the gel or the 
sucrose density gradient, rendering recovery of the two arms difficult. In 
particular, the linear cosmid DNA which is a co-product from the hybrid 
phage DNA will contaminate the arms, because each fragment distributes 
according to the binomial distribution either on the gel or the sucrose 
density gradient. Therefore, the cos site of the cosmid should divide the 
EcoRI digested cosmid evenly to minimize contamination of the linear 
cosmid DNA. Currently, pHC79 is preferred for fulfilling this condition. 
Furthermore, since pHC79 is a pBR322 derivative, it is classified as 
relaxed type plasmid whose replication is independent of the host system 
resulting in stable plasmid maintenance due to its low copy number (up to 
20 copies/cell) and easy amplification with either chloramphenicol or 
spectinomycin. Because plasmid segregation or rearrangement is likely when 
there is a long insert DNA and/or a high copy number, the plasmid should 
have a low copy number when it is maintained, but can be amplified when it 
is to be extracted. This is an advantage of the present invention over the 
BAC system. In addition, since two antibiotic resistance genes locate 
separately on each arm, unexpected rearrangements can be eliminated when 
the clones are grown on a medium with both antibiotics. Rearrangement may 
occur between the segments near the cos site locating apart after the 
passenger DNA is inserted, and homologous recombination may take place. 
Ampicillin and tetracycline resistance genes located on each arm of pHC79 
can segregate the deleted plasmids which have lost one of the antibiotic 
resistance genes as well as the passenger DNA. 
A lambda phage vector, .lambda.gtll (24), and cosmid DNA, pHC79 (25), were 
digested with EcoRI, purified and ligated. The resulting DNA was then 
packaged in vitro into lambda heads (26), and infected into the proper 
host strain to allow in vivo packaging. 
Generated phage were transferred onto a nylon membrane, and screened with a 
probe (pHC79). Positive phage were cultured independently and DNA 
extracted. Five clones were identified as a hybrid phage by restriction 
enzyme analysis, and one clone (.lambda.KAD93.4) was selected as a 
vector-arms producing phage, because the other four were hard to isolate 
DNA from due to their poor growth rate. Since the intensity of each band 
was almost proportional to its size, in vivo packaging occurred at almost 
equal rates for both possible patterns as shown in FIG. 2. 
Those arms were then isolated from agarose gel, and tested for the ability 
to anneal to each other. At a 42.degree. C. incubation in the presence of 
20 mM MgCl.sub.2, the two arms created a new large fragment (6.4 kb) which 
migrated the same as the linearized pHC79 DNA on an agarose gel. Then, 
this fraction was ligated and transformed into E. coli cells (SURE 
competent cells from Stratagene). All plasmid DNA examined (from 24 
colonies) showed the EcoRI sites were intact by EcoRI digestion indicating 
that the recovered arms from the hybrid phage had the expected structure 
for HPMT. 
Conditions for HPMT 
Conditions for HPMT were optimized using the .lambda.KAD93.4 DNA fraction 
as a transforming DNA, .lambda.cI857Sam as a helper phage and NS428 
(N205recA1)(.lambda.cI857 Aam11 Sam7 b2 red3) as a host strain. Since this 
DNA fraction includes a cosmid inside or outside the DNA with open cos 
sites, the total DNA fraction includes a cosmid inside or outside the DNA 
with open cos sites, the total DNA fraction is sufficient to test its 
ability or to optimize conditions for HPMT. It is likely that vast 
recombination will occur to the resulting transformants due to the 
repeated structure of lambda DNA. 
At first, cell growth of the NS428 strain was examined to determine the 
relationship between optical density at 600 nm (OD.sub.600) and viable 
cell number (FIG. 4). Based on the results, a 2.5 h cell culture in LB was 
used for subsequent tests, because the culture was estimated to be 
5.times.10.sup.7 cells/mL when the OD.sub.600 was 0.5. Then, the cells 
were treated as in Ref. (18). 
Multiplicity of infection (m.o.i.) was then optimized as shown in FIG. 5. 
The competent cells were mixed with .lambda.KAD93.4 DNA, transformed, and 
the colonies scored. The best m.o.i. was around 8. 
To determine the linearity of transformation with increasing DNA 
concentration, HPMT was carried out changing the DNA concentration from 
0.5 to 11 .mu.g at m.o.i.=8. The results are shown in the following Table 
1: 
TABLE 1 
______________________________________ 
DNA Efficiency 
colonies (.mu.g) 
(colonies/.mu.g) 
______________________________________ 
37.degree. C. 
4260 0.272 1.57 .times. 10.sup.4 
5820 0.544 1.07 .times. 10.sup.4 
5720 1.088 5.26 .times. 10.sup.3 
8490 2.72 3.12 .times. 10.sup.3 
11150 5.44 2.05 .times. 10.sup.3 
30.degree. C. 
7480 0.272 2.75 .times. 10.sup.4 
9950 0.544 1.83 .times. 10.sup.4 
6670 1.088 6.22 .times. 10.sup.3 
7250 2.72 2.67 .times. 10.sup.3 
11850 5.44 2.18 .times. 10.sup.3 
______________________________________ 
These results show a maximum transformation efficiency of 1.57 
.times.10.sup.4 colonies/.mu.g DNA, which is almost two orders of 
magnitude higher than Ref. (20) (5.15.times.10.sup.2 colonies/.mu.g DNA). 
This may result from the number of open-cos sites in the presently 
invention. The authors of Ref. (20) did not confirm the ter reaction 
during transformation, further showing the higher efficiency of the 
present invention. As well as the regular conditions at 30.degree. C., a 
non-permissive temperature (37.degree. C.) was tested. It is noted that 
more colonies appeared when less DNA was used. When transforming 
efficiencies are plotted with the DNA amount, the curves match a typical 
rectangular hyperbola as shown in FIG. 6. This indicates that annealing 
between the helper phage DNA and the .lambda.KAD93.4 DNA occurred quite 
efficiently and annealing may contribute more than the chance of collision 
between cos sites. At the non-permissive temperature that may cause cell 
lysis, the efficiency was lower than at the permissive temperature. 
All transformants obtained above showed vast levels of recombination. This 
is because the DNA used had the same segments from lambda phage and the 
expected recombinant included the helper phage DNA, and recombination was 
induced, probably at the initial stage of transformation, because the 
structures were stable once it established. This suggests that 
recombination is independent from the recA related homologous 
recombination system. This recombination may be minimized by shortening 
the cos region in pHC79 or improving the host strain using different rec 
mutations. The following table lists some of the strain names and 
corresponding genotypes for the strains referred to in the above 
description: 
______________________________________ 
strain name genotype 
______________________________________ 
KD9302 mcrA.DELTA.(mcrCB-hsdSMR-mrr)171, 
sbcC, rec J, rec B, uvrC, 
umuC::Tn5(kan')supE44, lac, 
gyrA96, relA1, thi-1, endA1, F' 
proAB lacl.sup.q Z.DELTA.M15Tn10(tet.sup.r)!, 
.lambda.ind- lysogen 
KD9402 same as KD9301 except F' minus 
LE392 mcrA, hsdR514, supE44, supF58, 
lacY1, .DELTA.(laclZY)6, galK2, 
galT22, metB1, trpR55 
.lambda..sub.c1857Sam78 
Repressor is temperature 
sensitive. Lysozyme subunit is 
amber mutated. 
.lambda.ind- Induction minus mutant. 
.lambda.KAD93.4 
A hybrid phage of cosmid (pHC79) 
and bacteriophage .lambda.gt11. 
KD9301 LE392(.lambda.cI857Sam7) 
______________________________________ 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it will be apparent to one skilled in the 
art that various changes and modifications can be made therein without 
departing from the spirit and scope of the invention. 
LIST OF REFERENCES CITED 
1. Laughon et al., Mol. Cell Biol., 6(1986):4676. 
2. Strocher et al., id. at 4667. 
3. O'Connor et al., EMBO J., 7(1988):435. 
4. Kornfeld et al., Genes Dev., 3(1989):243. 
5. Robertson, Nature, 327(1987):372. 
6. Gitschier et al., Nature, 312(1984):326. 
7. Peifer et al., Genes Dev., 1(1987):891. 
8. Grosveld et al., Cell, 5(1987):975. 
9. Spradling, Drosophila: A Practical Approach, (IRL Press, Oxford 1986), 
pp. 175-197. 
10. Fire, EMBO J., 5(1986):2673. 
11. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold 
Spring Harbor Laboratory Press, New York (1989). 
12. Sternberg, Proc. Natl. Acad. Sci., USA, 87(1990):103. 
13. Burke et al., Science, 236(1987):806. 
14. Shizuya et al., Proc. Natl. Acad. Sci., USA, 89(1992):8794. 
15. Kaiser et al., J. Mol. Biol., 2(1960):392. 
16. Kaiser, id., 4(1962):275. 
17. Radding et al., id., 7(1963):225. 
18. Kaiser et al., id., 13(1965):78. 
19. Mandel, Mol. Gen. Genet., 99(1967):88. 
20. Miller et al., Virology, 109 (1981):379. 
21. Elliott et al., Mol. Gen. Genet., 161(1978):1. 
22. Freifelder et al., Virology, 74(1976):169. 
23. Becker et al., Proc. Natl. Acad. Sci., USA, 75(1978):4199. 
24. Young et al., Proc. Natl. Acad. Sci., USA, 80(1983):1194. 
25. Hohn et al., Gene, 11(1980):291. 
26. Sternberg et al., id., 1(1977):255. 
27. Kadokami et al., BioTechniques, 17(1994):580. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 1 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GGGCGGCGACCT12 
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