In vitro method for concerted integration of donor DNA molecules using retroviral integrase proteins

A method of analysis of concerted integration in which viral integrase enzyme is first incubated with donor DNA molecules followed by incubation with target DNA molecules. The donor DNA molecule having at least one unique restriction site for analysis of concerted integration product.

BACKGROUND OF THE INVENTION 
Upon infection of cells by retroviruses, a large-size viral nucleoprotein 
complex is observed in the cytoplasm. These complexes vary in size from 
160 S for murine leukemia virus (MLV) to 80 S for human immunodeficiency 
virus (HIV) type-1. The viral nucleoprotein complexes contain newly 
synthesized blunt-ended linear viral DNA that is subsequently trimmed by 
two nucleotides at its 3' OH termini by the viral integrase(IN). IN can 
catalyze the concerted integration of the recessed viral DNA termini into 
exogenous DNA targets (full-site reaction) mimicking the in vivo reaction 
. Both the trimming and integration of the viral DNA by IN in the 
nucleoprotein complexes requires the metal cofactor Mg.sup.2+. 
Similar trimming and integration reactions can also be catalyzed by 
purified IN in vitro. IN derived from bacterial expression systems or 
purified from avian myeloblastosis virus (AMV) can trim two nucleotides 
from the termini of oligonucleotides or DNA restriction fragments 
containing viral long terminal repeat (LTR) sequences . The recessed DNA 
substrates can then be integrated into other DNA targets by IN. The 
majority of the observed integration events with these substrates involve 
the insertion of a single LTR terminus into one strand of the target DNA 
(half-site reaction). Expressed IN requires Mn.sup.2+ for efficient 
catalysis of either the trimming or the strand transfer reactions while 
AMV IN can effectively use Mg.sup.2+ or Mn.sup.2+ for these reactions. To 
date, expressed IN is not capable of efficiently performing the concerted 
insertion of viral-like DNA substrates into target DNA using either 
divalent cation. 
DESCRIPTION OF THE INVENTION 
The present invention relates to a method and a kit for efficient 
integration of DNA donor molecules into DNA target molecules using 
retrovirus integrase , hereinafter referred to as IN. The present 
invention also relates to a method for studying integrase such as 
screening of HIV-1 or HIV-2 integrase inhibitors, production of transgenic 
animals and gene transfer. 
According to the present invention, IN purified from virus or IN in virus 
particles and specifically designed donor substrates, hereinafter referred 
to as donor DNA, are used to mimic the integration of retrovirus DNA in 
vivo. This does not exclude the possibility that suitable expressed IN 
could perform concerted integration. The reaction assay conditions and 
donor DNA molecules being such that they induce a high cyclization 
probability for the donor DNA molecules in the reaction solution. 
Concerted integration of a single donor DNA requires the transient 
formation of a circle molecule prior to this event. The donor DNA 
molecules could also have regions capable of readily bending in solution 
and located appropriately to enhance cyclization of the donor DNA 
molecules in reaction solution. It should be understood that cyclization 
of donor DNA is necessary for the concerted integration of the two donor 
termini, in which each termini has a dimer of IN. Furthermore, the 
condition inducing cyclization may also be influenced by temperature, 
concentration of reactants, order of addition of reactants, and divalent 
metal cation. 
The donor DNA molecules should have at least one unique restriction site 
for analysis of successful and efficient concerted integration. This 
analysis may be readily performed on agarose gels. Preferably, the donor 
DNA molecules have at least one genetic marker for isolation or 
characterization of concerted integration products. 
Preferably, the donor DNA molecules can be labeled at their 5' ends, using 
radioactive probes but not excluding other detection probes. 
For efficient concerted integration of a donor DNA molecule into a target 
molecule, IN and the donor DNA molecule are incubated in reaction buffer 
which allows formation of transient circles. Preferably, the reaction 
buffer contains 20 mM MgCl.sub.2, 10% dimethyl sulfoxide, 0.05% Nonidet 
P-40, 5% polyethylene glycol, and 200 mM NaCl. After formation of 
transient circles of donor DNA with IN, the target molecule is introduced 
into the reaction and the integration reaction is allowed to proceed. 
After the integration reaction is complete, the reaction products may be 
analyzed for the unique restriction site and the genetic marker on the 
donor DNA molecule. 
Preferably, the donor molecules should be of a optimum size for the 
cyclization of the DNA to occur in solution. The size can vary with 
certain DNAs but should be in the 700 bp range, with several hundred base 
pairs on either size. It should be understood that small size DNA 
molecules are too rigid for cyclization in assay solution while large size 
DNA molecules are too flexible for stable cyclization. DNA sequences that 
readily bend in solution may be introduced in the donor DNA thereby 
increasing the size of the donor that can be used because its cyclization 
frequency should be higher. 
Preferably, the donor molecule should lack internal DNA sequences, such as 
A/T rich regions, that IN can readily bind to thereby lowering the 
concentration of IN in solution. Also, non-specific binding of IN to 
regions other then the donor termini would allow formation of DNA loop 
structures that inhibit the formation of transient circles by IN. 
Preferably, the target DNA molecule for in vitro analysis should be a 
circle whose size could be easily varied. The circular target DNA and 
unique restriction site on the donor DNA molecules makes analysis of the 
concerted integration reaction easy by the use of gel electrophoresis. 
This invention could be readily applied to the study and identification of 
HIV-1 or HIV-2 IN inhibitors. HIV-1 virions can readily perform the 
concerted integration of the appropriate DNA donor that matches the size 
criteria stated above into a circular target. 
Preferably, for the production of transgenics and for the practice of gene 
transfer, the donor molecule would be complexed with IN prior to their 
transfer into the animals or cells. As noted below, this formation of 
IN/donor preintegration complexes is the most efficient method to produce 
concerted integration recombinants with target DNA. In transgenics and 
gene transfer the host genome acts as the target DNA.

PREFERRED EMBODIMENT OF THE INVENTION 
The invention is further explained by referring to preferred embodiment and 
the following and examples. 
This invention relates to the conditions necessary to reconstitute a viral 
nucleoprotein complex capable of efficiently performing the concerted 
integration reaction. IN purified from AMV is capable of the full-site 
integration reaction using a linear 3.4 Kbp plasmid-based virus-like 
substrate as donor and gtwes as target. The reaction required Mg.sup.2+ 
and approximately 0.25% of the donor substrate was inserted into the 
target in a concerted fashion. The data demonstrated that AMV IN is 
capable of catalyzing the full-site reaction but conditions for formation 
of preintegration complexes capable of efficient concerted integration 
were not optimal. 
To optimize conditions to investigate IN/donor preintegration complexes 
capable of concerted integration, the 3.4 Kbp donor molecule and the 
target were changed. The size of the donor was reduced to 528 bp, referred 
to as termed M-2 (FIG. 1A) to optimize the cyclization probability (j 
factor)(Ref.1) of the molecule. A transient circle is an apparent 
necessity for concerted integration as well as for trimming of the viral 
genome by IN in vivo. The formation of DNA looped structures by AMV IN in 
the 3.4 Kbp donor molecule map to A/T rich regions (Ref.2). The ability of 
IN to form DNA looped structures may hinder the formation of 
preintegration complexes capable of the full-site reaction. M-2 lacks A/T 
rich regions to overcome this problem. The target was changed to a small 
circular DNA (pGEM-3, 2,867 bp) to provide an easier analysis of IN/donor 
complexes and its interactions with target DNA. 
The full-site integration reaction could occur via two pathways using the 
above substrates (FIG. 1). First, the termini of a single M-2 molecule can 
be inserted by IN into circular pGEM (FIG. 1B). Second, the termini of two 
individual M-2 molecules can be inserted in a concerted fashion at the 
same site into pGEM (FIG. 1D). The insertion of a single M-2 terminus by 
IN into pGEM producing a half-site integration reaction can also occur 
(FIG. 1C). M-2 contains a unique restriction site not found in the target 
molecule thereby permitting isolation and analysis of half-site and 
full-site integration recombinants by agarose gel electrophoresis (FIG. 
1). M-2 also contained the supF gene that was used for genetic isolation 
of individual full-site recombinants to characterize the target site. 
Bacteria and Plasmids. 
The plasmid R35 (Ref.2 ) was the source of the avian 65-bp LTR insert and 
the supF gene (469 bp). Both DNA fragments were used to reconstruct the 
M-2 donor fragment into pUCl9 (New England BioLabs). pGEM-3 was from 
Promega. Escherichia coli (strain CA244), that had amber mutations in the 
lacZ gene and for tryphotan biosynthesis, was obtain from the E.coli 
Genetic Stock Center at Yale University. CA244 cells transformed by 
M-2/pGEM recombinants or R-35 containing the supF gene were grown at 
37.degree. C. in M9 medium that contained autoclaved casamino acids (2 
g/L), lactose (0.2%), 0.5 mM IPTG, 40 .mu.g/ml X-Gal, and ampicillin (30 
.mu.g/ml). 
Construction of donor M-2, SEQ ID. NO;1 
The 65 bp LTR circle junction was recovered from R35 by EcoRI digestion. 
The isolated fragment was ligated and then digested by NdeI. The DNA was 
cloned into the NdeI site of pBR322 lacking its EcoRI site. A plasmid was 
selected that contained a single U3/U5 insert having NdeI termini and an 
internalized EcoRI site. The amplified DNA was digested with EcoRI and the 
supF gene was cloned into this site by blunt-end ligation. Digestion of 
this plasmid by NdeI yielded a 528 bp restriction fragment that contained 
U3 and U5 LTR termini and the internalized supF gene. This restriction 
fragment was termed M-2 (FIG. 1) and served as a donor substrate. M-2 also 
contained the polylinker site derived from piAN7 that has unique BglII and 
XbaI sites. The XbaI site located near the U3 terminus of M-2 was used to 
create a donor molecule that lacked its U3 terminus but was similar in 
size to M-2. The XbaI/M-2 donor was use to help identify and characterize 
half-site M-2/pGEM recombinants. M-2 was subsequently cloned into the NdeI 
site of the high copy-number pUC19 for large scale isolation of the 
restriction fragment. 
Labeling of M-2. 
The supercoiled forms of pUC19 containing M-2 and pGEM were isolated by 
velocity sedimentation on sucrose gradients to remove any small size DNA 
or RNA fragments. M-2 was released from pUC19 by NdeI digestion and was 
isolated by low-m elt agarose gel electrophoresis. Following purification, 
M-2 was dephosphorlyated and 5' end labeled using .gamma.-32P ATP and T4 
polynucleotide kinase. The specific activity of M-2 was generally 5,000 to 
10,000 cpm per ng of DNA. The pUC19 plasmid containing M-2 was also 
digested by Xbal and Ndel releasing M-2 lacking its U3 terminus (FIG. 1A). 
This fragment (490 bp) was labeled at both of its 5' ends as described 
above. Xbal digestion of M-2 removed the first two nucleotides of the 
adjacen t Bglll site. 
Assay Conditions. 
The standard reaction mixture contained 20 mm Tris-hydrochloride (pH 7.5), 
5 mM dithiothreitol, 5 mM MgCl.sub.2, 10% dimethyl sulfoxide, 0.05% 
Nonidet P-40, 5% polyethylene glycol, and 200 mM NaCl.The preincubation 
step included incubation of M-2 with IN at 0.degree. C. for 10 min in 20 
.mu.l aliquots. The molar ratio of dimeric IN (33 ng) to M-2 (15 ng) was 
usually set at 12 unless otherwise indicated. The catalytic reaction was 
initiated by the addition of supercoiled pGEM followed by the immediate 
incubation at 37.degree. C. The standard concentrations of labeled M-2 and 
pGEM were 15 ng and 100 ng, respectively. The molar ratio of M-2 to pGEM 
was 1. Scale-up reactions maintained the same molar ratios of enzyme to 
DNA substrates for isolation of various M-2/pGEM recombin ants. Some 
reaction conditions were modified as indicated in the text. 
Analysis of M-2/pGEM recombinants. 
After the integration reaction, each reaction was stop with sodium dodecyl 
sulfate and proteinase K at final concentrations of 1% and 1 mg/ml, 
respectively. The samples were further incubated at 37.degree. C. for 2 hr 
and then subjected to phenol-chloroform (1:1) and ether extractions. The 
DNA was precipitated by ethanol. Aliquots of each sample were subjected to 
electrophoresis on 1 or 1.5% agarose gels which were dried and the 
radioactive products were quantitated by a Molecular Dynamics 
PhosphorImager. The dried gels were also exposed to X-ray films with or 
without an intensifying screen. For BglII restriction analysis of the 
M-2/pGEM recombinants, each 20 .mu.l reaction mixture was digested by 12 
units of Bglll for 2 hr to ensure complete digestion. The samples were 
subjected to electrophoresis on 1.5% agarose gels in a Tris-borate-EDTA 
buffer with 0.5 .mu.g/ml of ethidium bromide for 13 hr at 100 volts. The 
gels were dried and analyzed as described above. Linear DNA fragments 
(Boehringer Mannheim), pGEM, and R35(Ref.2) were used as unlabeled 
molecular weight markers. Linear R35 (3.4 Kbp) was 5' end labeled with 
.gamma.-32P ATP and was used to identify the same size BglII linerarized 
M-2/pGEM recombinants which resulted from the concerted insertion of M-2 
into pGEM. 
Characterization of M-2/pGEM recombinants resulting from concerted 
integration events. 
The 3.4 kbp Bglll linearized DNA obtained from digestion of all of the 
M-2/pGEM recombinants was isolated from scale-up integration reactions. 
The Bglll digested recombinants were subjected to electrophoresis on 1.5% 
agarose gels and the wet gel was exposed to X-ray film. The desired 
fragment was excised and electroeluted from the gel slice. The labeled DNA 
was purified by a Wizard PCR Prep column (Promega) and stored at 
-20.degree. C. The purified DNA was ligated and analyzed by agarose gel 
electrophoresis or transformed into E coli HB101,Epicuran Coli Sure 
(Stratagene) or CA244 cells. Colonies were screened for plasmids that were 
analyzed by size, restriction enzymes, and DNA sequencing. Primers for 
sequencing were located within M-2 near both the U3 and U5 termini and 
were used to sequence the donor/target junctions. Sequencing was 
accomplished by the didexoxy method. The purified DNA was also examined by 
electron microscopy to determine the size and structure of the linearized 
DNA and other DNA structures as previously described (Ref.2 ). Individual 
M-2/pGEM recombinants 13 (&gt;3.4 Kbp circular DNA, 3.4 Kbp circular DNA , 
and linear 3.92 Kbp ) were also isolated and analyzed as described above. 
Purification of integrase. 
AMV IN was purified to near homogeneity as previously described (Ref.2 ). 
Parameters for production and physical quantitation of concerted 
integration events. 
I wanted to devise a scheme which would permit the use of a simple approach 
to investigate concerted integration of a donor molecule into a DNA 
target. This scheme should allow an easy indepth examination of reaction 
conditions which promote full-site reactions. Such an approach is 
cartooned in FIG. 1. The linear donor molecule (M-2) has recessed U3 and 
U5 termini, is 528 bp in length which is optimal for forming transient 
circles , and has a unique Bglll restriction site (FIG. 1A). Concerted 
integration of a single linear M-2 molecule into circular pGEM-3 (2,867 
bp) by AMV IN would result in the formation of a 3.4 Kbp circle, which 
when digested by Bglll, produces a 3.4 Kbp linear molecule (FIG. 1B). 
Half-site integration of M-2 into pGEM would result in a stick and ball 
structure also with a mass of 3.4 Kbp (FIG. 1C); Bglll digestion of this 
half-site recombinant would result in the formation of two different stick 
and ball structures ( "A" or "B"), depending on whether the U3 or U5 
terminus was inserted. Prior XbaI digestion of M-2 (FIG. 1A) would produce 
a molecule capable of only producing U5 half-site recombinants (stick and 
ball structure "A") whose mass is 3.36 Kbp (FIG. 1C). Lastly, concerted 
integration could also result from a single insertion event involving two 
separate M-2 molecules (or 2 XbaI/M-2 molecules) into circular pGEM at one 
site (FIG. 1D). The integration of 5' end labeled M-2 or Xbal digested M-2 
into circular pGEM can be easily followed by restriction enzyme digestion 
and agarose gel electrophoresis. 
IN/M-2/pGEM preintegration complexes. 
I wanted to test the above protocol (FIG. 1) for the production of 
concerted integration recombinants. I first tested reaction conditions 
which were conducive for formation of complexes capable of performing the 
concerted integration reaction employing a linear 3.4 Kbp donor and 
.lambda.gtWES. IN, M-2, and pGEM were preincubated together on ice for 10 
min with Mg2+ prior to incubation at 37.smallcircle. (FIG. 2). The donor 
to target molar ratio was 16 and the IN dimer to M-2 molar ratio was 12. 
The reactions were stopped at the indicated times and were analyzed by gel 
electrophoresis on 1.5% agarose (FIG. 2, lanes 2 and 3). Only two major 
labeled M-2/pGEM products were formed, one which comigated with a nicked 
circular 3.4 Kbp marker and the other group migrating slightly slower. 
Electron microscopy of both DNA species confirmed that the majority 
(.about.97%) of the 3.4 Kbp M-2/pGEM recombinants were of the ball and 
single stick model ("A", FIG. 1C) and the slower migrating group of 
recombinants were circular pGEM with two or more independent M-2 inserts 
(data not shown). These structural data were confirmed using XbaI digested 
M-2, which lacks the U3 terminus (FIG. 1). Only single insertion events of 
U5 ends of XbaI/M-2 into pGEM migrating with a mass of 3.36 Kbp were 
observed by gel electrophoresis (FIG. 2, lanes 9 and 10) or by electron 
microscopy (data not shown). 
Digestion of the M-2/pGEM recombinants containing circles by Bglll should 
result in the cleavage of all Bglll sites regardless of whether they are 
half-site or full-site recombinants. With all half-site reactions, 
one-half of radioactivity will be lost upon Bglll digestion while no 
radioactivity would be lost by Bglll digestion of full-site reactions 
because no 5' -end labeled termini are lost(FIGS. 1B and 1C). Two major 
products were produced by Bglll digestion of M-2/pGEM recombinants formed 
under these above conditions (FIG. 2, lanes 4 and 5). The slowest moving 
DNA migrated with a mass of 3.4 Kbp ("A" product) and the fastest moving 
DNA ("B" product) nearly comigrated with nicked circular pGEM (2.86 Kbp). 
The "A" and "B" products derived by Bglll digestion of M-2/pGEM 
recombinants would be the result of half-site U5 and U3 insertions, 
respectively. As expected, digestion of Xbal/M-2 recombinants with Bglll 
(FIG. 2, lanes 7 and 8) did not alter the migration of these recombinants 
nor identified any recombinants containing U3 insertions. 
The digestion of M-2/pGEM recombinants produced under these reaction 
conditions did not reveal a significant quantity of linear 3.4 Kbp product 
suggesting that few full-site M-2/pGEM recombinants were formed (FIG. 2, 
lanes 4 and 5). The 3.4 Kbp donor molecule served as an identical 
molecular weight marker for the expected Bglll linearized M-2/pGEM 
recombinant (FIG. 2, lane 6). Prolonged exposure of this gel did result in 
a minor product comigrating at 3.4 Kbp (FIG. 2, lanes 4 and 5). 
IN/M-2 preintegration complexes. 
To increase the efficiency of the full-site integration reaction, several 
reactants and procedures were modified. The formation of IN/M-2 
preintegrations complexes were allowed to occur on ice prior to the 
addition of target. The molar ratio of M-2 to PGEM was decreased to 1 from 
16. The IN to M-2 molar ratio was held constant at 12. Preincubation of 
IN/M-2 together prior to addition of target and subsequent incubation at 
37.degree. C. for 30 min resulted in the formation of three major M-2/pGEM 
recombinant species (FIG. 3,lanes 3). The two slowest migrating M-2/pGEM 
recombinants in these lanes of FIG. 3 were previously described (FIG. 2, 
lanes 2 and 3) and the fastest moving M-2/pGEM recombinant is presumed to 
be 3.92 Kbp linear DNA (FIG. 1D) . I will address this linear 3.92 Kbp 
M-2/pGEM recombinant later. Bglll digestion of the reaction products 
observed in lane 3 of FIG. 3 resulted in the formation of a labeled 
product (lane 4) which comigrated with the 3.4 Kbp linear marker (lane 5). 
Quantitation using PhosphoImager analysis showed that the linearized 
M-2/pGEM recombinant (FIG. 3, lane 4) represented 22% of the radioactively 
associated with the non-digested Bglll 3.4 Kbp and 3.92 Kbp M-2/pGEM 
recombinants(lane 3). 
Bglll digestion of M-2/pGEM recombinants also reaffirmed early data on the 
preference of U3 over U5 termini for both the trimming and strand transfer 
reactions by AMV IN by a 2 to 1 margin. This same U3 over U5 preference 
was observed by Bglll digestion of either M-2/pGEM recombinants when 
either circles (FIG. 3, lane 4, products "A" and "B"). 
Physical and genetic analysis of concerted M-2/pGEM recombinants. 
I needed to confirm that the linearized 3.4 Kbp M-2/pGEM recombinants were 
the result of a full-site integration reaction. Several 30 min scale-up 
reactions were performed and the samples were subjected to Bglll 
digestion. The linearized 3.4 Kbp DNA was identified on wet 1.5% agarose 
gels by autoradiography and was eluted and purified. The 3.4 Kbp 
recombinant was ligated and then analyzed again by 1.5% agarose gel 
electrophoresis (FIG. 4, lanes 2 and 3). The ligated DNA comigated with 
the original circular 3.4 Kbp molecule (FIG. 4,lane 1). In three separate 
experiments, the maximum amount of the circular 3.4 Kbp product produced 
by ligation was 75% suggesting that some of the DNA ends were damaged upon 
elution and purification of the linearized DNA. Digestion of the ligated 
3.4 Kbp circle by Bgll, which only cuts pGEM and not M-2, again resulted 
in the production of only linear 3.4 Kbp DNA (data not shown). 
To further establish that only linear recombinant DNA is present in the 
Bglll linearized 3.4 Kbp band, the purified DNA was analyzed by electron 
microscopy. Counting 200 molecules, 70% of the molecules were linear 
structures 3.4 Kbp +150 bp in length, 1% were branched structures of the 
same size,15% were linear molecules of various sizes and 15% were circles 
of 2.86 Kbp in length. The various smaller size linear molecules probably 
represent degraded 3.4 Kbp DNA. The 2.86 Kbp circular DNA is unused pGEM 
(&gt;96% of the target DNA is not used in the 30 min reactions as observed by 
ethidium bromide staining of DNA). The contaminating pGEM represents a 
very minor population of topological forms of PGEM induced by the presence 
of ethidium bromide in the 1.5% agarose gels that comigrates with the 
labeled 3.4 Kbp linearized DNA. 
Does the linearized 3.4 Kbp recombinant DNA represent true concerted 
integration events? The only definite way to establish that each 
recombinant contains the correct host duplicated site that is observed 
upon concerted integration in vivo is by the genetic selection of 
individual recombinants. The purified 3.4 Kbp Bglll linearized DNA (FIG. 
4) was ligated and transformed into E. coli (HB101, Epicuran Coli Sure 
cells, or CA244). Screening of individual colonies demonstrated that 5 to 
10% of the plasmids were 3.4 Kbp in length when either HB101 or Sure cells 
were used as hosts. Also, fill-in and repair of the ligated 3.4 Kbp DNA by 
E coli DNA polymerase I before transformation did not increase the 
percentage of rescued 3.4 Kbp plasmids.The rest of the plasmids were 2.86 
Kbp in length. Restriction enzyme analysis of all the rescued plasmids 
demonstrated that the 3.4 Kbp DNA were concerted integration products and 
the 2.86 Kbp DNA was pGEM. DNA sequence analysis of 20 recombinants of 3.4 
kbp in length demonstrated that all of them had the correct avian 
duplication host size (18 six bp; 1 five bp; and 1 seven bp). These above 
data demonstrated that the 3.4 Kbp M-2/pGEM recombinants were the products 
of full-site integration events. The observation that only 5 to 10% of the 
rescued plasmid were the correct recombinants suggests that the ligation 
of the 3.4 Kbp was incomplete (FIG. 4) and unused pGEM target was present 
as demonstrate by electron microscopy. 
To decrease the pGEM background in the genetic assay, we transformed the 
ligated 3.4 Kbp DNA into CA244 cells which have amber mutations in the 
lacZ gene and for tryptophan biosynthesis. M-2 contains the supF gene 
which when inserted into pGEM in a concerted matter (FIG. 1B) would permit 
the replication of this recombinant plasmid but not pGEM in CA244 cells. 
Transformation of CA244 resulted in the production of only blue colonies 
(FIG. 5) and all of the rescued plasmids were 3.4 Kbp in length. DNA 
sequence of eight plasmids verified that they were all the result of 
concerted integration events. 
Characterization of the IN/M-2 complexes capable of concerted integration. 
I wanted to compare the synthesis rate of the concerted integration events 
with respect to all other half-site reactions and the utilization of the 
input M-2 substrate (FIG. 6). Standard preincubation of IN/M-2 in the 
present of Mg2+ prior to the synaptic reaction with pGEM was employed 
(FIG. 3). Aliquots were removed at the indicated times and the DNA samples 
were analyzed by 1% agarose gel electrophoresis to quantitate 
disappearance of input substrate and formation of all recombinants (See 
FIG. 8 as an example). An equal aliquot of each sample was also digested 
or not digested with Bglll to quantitate the concerted integration 
reaction (FIG. 6A). As shown in FIG. 6B, most of the catalysis occurs in 
the first 40 min of incubation at 37.degree. C. Eighty percent of the 
input M-2 substrate is used (data not shown) and both the synthesis of the 
3.4 Kbp circular and linear DNAs are leveling off. Maximal insertion of 
M-2 into other M-2 molecules occurs earlier at 20 min followed by their 
disappearance with further incubation. These presumed half-site M-2/M-2 
recombinants were probably subjected to the disintegration reaction which 
AMV IN in the presence of Mg2+ is capable of performing using appropriate 
disintegration substrates composed of oligonucleotides (data not shown). 
Further analysis of all the M-2/pGEM recombinants by Bglll digestion and 
1.5% gel electrophoresis demonstrated that the concerted integration 
events (linearized 3.4 Kbp DNA) occurred at a linear rate for 
approximately 40 min but does not level off entirely (FIG. 6A and 6B). 
After 2h of incubation, the 3.4 Kbp linearized DNA represents 80% of the 
3.4 Kbp circle population. The linearized 3.4 Kbp DNA derived from the 
reaction at these latter times has the same properties as shown previously 
for linearized 3.4 Kbp produced at 30 min. 
Several of the major components in a standard reaction mixture were 
separately removed to determine whether individual components affected the 
full-site integration reaction. The reactions were for 30 min at 370 
.degree. C. with the standard 10 min preincubation of IN/M-2 on ice. The 
samples were subjected to Bglll digestions , gel electrophoresis, and 
PhosphorImager analysis. The individual removal of Nonidet P-40 appear to 
had little affect on the full-site and half-site reactions into pGEM. Both 
of these catalytic reactions were influenced equally by DMSO with maximum 
catalysis occurring between 10 and 15% DMSO. There was an approximate 3 to 
4 fold increase in all M-2/pGEM recombinants, including full-site 
recombinants, above those observed with no DMSO present. PEG also was 
necessary for maximum stimulation. The addition of glycerol(up to 10%) to 
the standard reaction mixture did not affect the integration reaction. 
While the present invention has been described by reference to preferred 
embodiment, it should be understood that modifications and variations of 
the invention may be derived without departing from the spirit of the 
invention. 
References: 
1. Hochschild, A. 1991. Detecting cooperative protein-DNA interactions and 
DNA loop formation by footprinting. Methods of Enzymology 208:343-361. 
2. Grandgenett, D. P., R. Inman, A. Vora, and M. Fitzgerald. 1993. 
Comparison of DNA binding and integration half-site selection by avian 
myeloblastosis virus integrase. J. Virology 67: 2628-26 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 7 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 491 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY:linear 
(ii) MOLECULE TYPE: other nucleic acid 
(iii) HYPOTHETICAL: no 
(iv) ANTI-SENSE: no 
(vi) ORIGINAL SOURCE: Combination of avian or HIV-1 retrovirus 
DNA and piAN7 plasmid. 
(vii) IMMEDIATE SOURCE: Same as in 2,vi. 
(ix) FEATURE: 
(D) OTHER INFORMATION: A linear double-standed DNA,termed M-2 
in the original application. Contains amber supressor 
sequences and restriction enzyme sites. Termini contain 
retrovirus long terminal repeat sequences that the viral 
integrase uses for concerted integration. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TATGAAGCCTTCTGCTTCATGCAGGTGCTCGTAGTCGAATTAGCTTGCGT50 
TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAAT100 
CGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCA150 
GGCATTACCCGTCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT200 
CTACGGGGTCTGAACGGATCTCAATTCTTTCGGACTTTTGAAAGTGATGG250 
TGGTGGGGGAAGGATTCGAACCTTCGAAGTCGATGACGGCAGATTTAGAG300 
TCTGCTCCCTTTGGCCGCTCGGGAACCCCACCACAGGTAATGCTTTTACT350 
GGCCTGCTCCTTATCGGGAAGCGGGGCGCATCATATCAAATGACGCGCCG400 
CTGTAAAGTGTTACGTTGAGAAAGAATTCCCGGGGATCCGTCGACCTGCA450 
GATCTCTAGAAGCTAATTCAAGAGTATTGCATAAGACTACA491 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY:linear 
(ii) MOLECULE TYPE: other nucleic acid 
(iii) HYPOTHETICAL: no 
(iv) ANTI-SENSE: no 
(vi) ORIGINAL SOURCE: Combination of avian or HIV-1 retrovirus 
DNA, piAN7 plasmid and pGEM plasmid. 
(vii) IMMEDIATE SOURCE: Same as in 2,vi. 
(ix) FEATURE: 
(D) OTHER INFORMATION: The sequence is the bottom strand of 
M-2 U5 and the pGEM target of the top clone shown in 
Figure 14 of original application. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CTTCATACTGGGCTG15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY:linear 
(ii) MOLECULE TYPE: other nucleic acid 
(iii) HYPOTHETICAL: no 
(iv) ANTI-SENSE: no 
(vi) ORIGINAL SOURCE: Combination of avian or HIV-1 retrovirus 
DNA, piAN7 plasmid and pGEM plasmid. 
(vii) IMMEDIATE SOURCE: Same as in 2,vi. 
(ix) FEATURE: 
(D) OTHER INFORMATION: The sequence is the top strand of 
M-2 U3 and the pGEM target of the top clone shown in 
Figure 14 of original application. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CTACACCAGTAGTAG15 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY:linear 
(ii) MOLECULE TYPE: other nucleic acid 
(iii) HYPOTHETICAL: no 
(iv) ANTI-SENSE: no 
(vi) ORIGINAL SOURCE: Combination of avian or HIV-1 retrovirus 
DNA. piAN7 plasmid and pGEM plasmid. 
(vii) IMMEDIATE SOURCE: Same as in 2,vi. 
(ix) FEATURE: 
(D) OTHER INFORMATION: The sequence is the bottom strand of 
M-2 U5 and the pGEM target of the middle clone shown in 
Figure 14 of original application. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
CTTCAATATGAGTAA15 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY:linear 
(ii) MOLECULE TYPE: other nucleic acid 
(iii) HYPOTHETICAL: no 
(iv) ANTI-SENSE: no 
(vi) ORIGINAL SOURCE: Combination of avian or HIV-1 retrovirus 
DNA, piAN7 plasmid and pGEM plasmid. 
(vii) IMMEDIATE SOURCE: Same as in 2,vi. 
(ix) FEATURE: 
(D) OTHER INFORMATION: The sequence is the top strand of 
M-2 U3 and the pGEM target of the middle clone shown in 
Figure 14 of original application. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CTACACTCATATATA15 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY:linear 
(ii) MOLECULE TYPE: other nucleic acid 
(iii) HYPOTHETICAL: no 
(iv) ANTI-SENSE: no 
(vi) ORIGINAL SOURCE: Combination of avian or HIV-1 retrovirus 
DNA. piAN7 plasmid and pGEM plasmid. 
(vii) IMMEDIATE SOURCE: Same as in 2,vi. 
(ix) FEATURE: 
(D) OTHER INFORMATION: The sequence is the bottom strand of 
M-2 U5 and the pGEM target of the bottom clone shown in 
Figure 14 of original application. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CTTCAACCATTCCTT15 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 bases 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY:linear 
(ii) MOLECULE TYPE: other nucleic acid 
(iii) HYPOTHETICAL: no 
(iv) ANTI-SENSE: no 
(vi) ORIGINAL SOURCE: Combination of avian or HIV-1 retrovirus 
DNA, piAN7 plasmid and pGEM plasmid. 
(vii) IMMEDIATE SOURCE: Same as in 2,vi. 
(ix) FEATURE: 
(D) OTHER INFORMATION: The sequence is the top strand of 
M-2 U3 and the pGEM target of the bottom clone shown in 
Figure 14 of original application. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CTACAAATGGTGCAA15 
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