Cloning method for trapping human origins of replication

A method of identifying a DNA sequence containing a human origin of replication (hORI) is disclosed. The method includes the steps of (A) providing fragments of human genomic DNA suitable for cloning into a bacterial plasmid, (B) ligating the fragments into a bacterial plasmid comprising (i) a bacterial origin of replication, (ii) a bacterial selection marker, and (iii) a mammalian selection marker, (C) transforming bacterial host cells with the plasmid, (D) selecting transformed bacterial host cells using the bacterial selection marker, (E) isolating plasmid DNA from the transformed bacterial host cells, (F) transfecting human cells with the isolated plasmid DNA, (G) selecting transfected human cells using the mammalian selection marker, (H) isolating extrachromosomal DNA from selected human cells, (I) digesting a suspension of the extrachromosomal DNA with a restriction endonuclease capable of cleaving methylated, but not unmethylated DNA, and (J) purifying undigested extrachromosomal DNA from the suspension.

FIELD OF THE INVENTION 
The present invention relates to a method of cloning human origins of 
replication (ORIs). 
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BACKGROUND OF THE INVENTION 
A great deal of our knowledge on origins of replication (ORIs) comes from 
the study of ORIs in animal viruses. The SV40 and polyomavirus origins of 
replication occur in highly accessible, nucleosome-free regions of about 
450 base pairs (bp) which are activated by T antigen protein encoded by 
the viral genome. This nucleosome-free gap contains both transcription and 
replication controlling elements. Large T antigen alone (90 kDa) initiates 
each round of viral DNA synthesis. However, several transcription factors 
are known to bind to the 21 bp repeats and the 72 bp enhancers of the SV40 
ORI; these transcription factors could interact with large T to enhance 
initiation of replication. 
Three rather extensive regions within SV40 ORI, termed sites I, II and III, 
bind large T (Hay 1982). The SV40 core ORI consists of three domains of a 
total length of 64 bp: (i) a sequence with an imperfect inverted repeat; 
(ii) a palindrome with four GAGGC pentanucleotide repeats recognized by T 
antigen; and (iii) a 17-bp A-Trich segment (Deb, et al., 1986a; Parsons, 
et al., 1990). T antigen contacts and melts bases in the imperfect 
inverted repeat, structurally distorts the GAGGC pentanucleotide domain 
the bends and untwists the AT-rich domain (Deb, et al., 1986b; Borowiec 
and Hurwitz, 1988; Parsons, et al., 1990). All three domains of core ORI 
are protected by the T antigen toward digestion by DNase I (Deb and 
Tegtmeyer, 1987). 
In the presence of ATP twelve molecules of large T antigen are assembled in 
the form of two hexamers on the SV40 core ORI (Mastrangelo, et al., 1989; 
Tsurimoto, et al., 1989a, 1989b). Assembly of a hexamer first occurs on 
the early half core ORI and then on the late half; the formation of these 
hexamers melts the early ORIs and untwists the late half core ORIS; 
melting and untwisting releases large T antigen molecules from the GAGGC 
pentanucleotides to act as helicases at flanking DNA regions (Parsons, et 
al., 1991). 
The B subunit of DNA polymerase a (68 kDa) mediates the assembly of the 180 
kDa subunit with T antigen acting as a molecular tether linking the two 
proteins (Collins, et al., 1993). Thus, T antigen enhances the ability of 
DNA polymerase .alpha. to prime the synthesis of new DNA chains and to 
extend pre-existing DNA chains (Erdile, et al., 1991; Collins, et al., 
1993). In the absence of T antigen DNA polymerase a synthesizes about 7-13 
nt per binding event and then dissociates from DNA (Copeland and Wang, 
1991); the ability of T antigen to translocate in the 3' to 5' direction 
along the DNA in an ATP-consuming process and the linkage of T antigen to 
the 180 kDa catalytic subunit of DNA polymerase .alpha. via the 70 kDa B 
subunit would be expected to hold the polymerase and make single-stranded 
template available, thus increasing enzyme processivity (Collins, et al., 
1993). 
Unlike E. coli, which uses a single start point to replicate its DNA, 
eukaryotic cells use multiple replication origins (Huberman and Riggs, 
1968; Linskens and Huberman, 1990). The existence of multiple 
replicons--chromosomal segments that are replicated from a single origin 
and whose size, number and temporal order of replication is cell type- and 
developmental stage-specific (Edenberg and Huberman, 1975; Hand, 
1978)--has promoted the idea of chromatin compartmentalization into 
domains. The 300 kb locus comprising the murine immunoglobulin heavy chain 
gene segments is a single replicon (Brown, et al., 1987). 
Both RNA and DNA viruses, using either prokaryotic or eukaryotic cells for 
their proliferation, usually possess a unique, and in some cases (i.e., 
HSV), two or three origins of replication. For example, both the DNA of 
SV40, a virus causing cancer in monkeys (5 kb), and the genome of E. coli 
(3.times.10).sup.6 bp) are replicated from a single origin. However, 
eukaryotes, due to their vast content in DNA, require multiple origins in 
replication. For example, the genome of the fruit fly Drosophila 
melanogaster (.about.10.sup.8 bp) and the DNA in haploid human nuclei 
(.about.3.times.10.sup.9 bp) use about 5,000 and 60,000 start points of 
DNA replication, respectively. 
Of the 60,000 or so ORIs from human cells, five specific ORIs have 
apparently been identified as of June 1996. The known human ORIs are: 
(i) That of the .beta.-globin gene complex (Kitsberg, et al., 1993; 
Boulikas, 1993). Earlier studies on the replication of the .beta.-globin 
multigene cluster showed temporal directionality and led to the 
identification of potential initiation sites for the replication of the 
.beta.-globin gene complex (Dhar, et al., 1988); 
(ii) that of the c-myc gene (Iguchi-Ariga, et al., 1988; Ariga, et al., 
1989; Vassilev and Johnson, 1990); 
(iii) the ORI in the 18S/28S ribosomal DNA 44 kb repeating unit (Little, et 
al., 1993); 
(iv) the ORI of the human HSP70 gene (Taira, et al., 1994); and 
(v) the ORI of the CHAT gene (Boulikas, et al., 1996). 
These ORIs are presumably activated by transcription factors (TFs) and 
replication initiator proteins which may include ssDNA-binding proteins 
(Bergemann, et al., 1992) and cruciform DNA-binding proteins (Pearson, et 
al., 1995). One TF involved in replication initiation may be the 
oncoprotein c-myc which promotes cellular DNA replication by binding to a 
cloned human putative ORI sequence (Iguchi-Ariga, et al., 1987a), c-myc 
can substitute for SV40 large T-antigen in an in vitro SV40 replication 
system (Iguchi-Ariga, et al., 1987b; Classon, et al., 1987). A region 
approximately 2 kb upstream of the transcription start site of the human 
c-myc gene contains a putative ORI of 210 bp (Boulikas, 1996, herein 
incorporated by reference) which is also a transcription enhancer 
containing c-myc binding sites (Iguchi-Ariga, et al., 1988a; Umekawa, et 
al., 1988). This fragment contains the 22-nucleotide binding site 
determined by DNase footprinting and mobility shift assays; this 
interaction is involved in both upregulating transcription as well as 
replication of the c-myc gene domain (Ariga, et al., 1989). 
DNA sequences enriched in origins of replication termed ors have been 
isolated by extrusion of single-stranded newly synthesized DNA at the 
replication fork from actively replicating monkey cells in culture 
(Zannis-Hadjopoulos et al., 1985). pBR322 plasmid harboring several cloned 
ors sequences have been shown to be autonomously and extrachromosomally 
replicating after their transfection into HeLa cells (Frappier and 
Zannis-Hadjopoulos, 1987; Rao et al, 1990; Landry and Zannis-Hadjopoulos, 
1991). 
Two chromosomal origins of replication within the DHFR amplicon (240 kb) 
mapped by two different approaches (Anachkova and Hamlin, 1988; Leu and 
Hamlin, 1989) are located at a distance of about 20 kb from one another. 
One of these origins had been localized in a 4.3 kb fragment (Burhans, et 
al., 1986) and was narrowed down to a 450 bp fragment by mapping the site 
where the strand specificity of the Okazaki fragments switches (Burhans, 
et al., 1990). The presence of two independent origins for the amplified 
DHFR locus was confirmed by Handeli, et al. (1989) using a novel mapping 
procedure. Multiple initiation sites are apparently used for the 
replication of this gene repeat lying within a 28 kb region (Vaughn, et 
al., 1990). These data do not contradict the model that precise DNA 
sequences are used for the initiation of DNA replication in mammalian 
cells since .sup..about. 1000 copies of the DHFR gene that may include DNA 
elements that function in replication initiation are present within an 
amplicon. 
Plasmids carrying the cad gene and flanking regulatory sequences were able 
to function as autonomously replicating episomes in mammalian cells 
(Carroll, et al., 1987). A bidirectional origin in the native locus and in 
episomally amplified murine adenosine deaminase loci has been found 
(Carroll, et al., 1993). The region of DNA replication in the murine 
immunoglobulin heavy chain gene has been identified and the octamer motif 
has been suggested as a putative DNA replication origin in mammalian cells 
(Iguchi-Ariga, et al., 1993). Similar studies are consistent with the 
presence of a replication origin in the mdr-1 gene (Ruiz, et al., 1989). 
The chicken has one H5 gene displaying a polarity with respect to its 
replication in expressing and non-expressing cell types; these data are 
compatible with an origin in the 5' flanking region used for the 
replication of the avian .beta.-globin gene in erythroid cells and from an 
origin in the 3' flanking region in nonerythroid cell types (Trempe, et 
al., 1988). Shot-gun cloning experiment aimed at identifying mammalian 
genomic sequences with ARS activity in yeast has identified autonomously 
replicating sequences (Roth, et al., 1983; Montiel, et al., 1984; Ariga, 
et al., 1985). 
SUMMARY OF THE INVENTION 
In one aspect, the invention includes a method of identifying a DNA 
sequence containing a human origin of replication (hORI) is disclosed. The 
method includes the steps of (A) providing fragments of human genomic DNA 
suitable for cloning into a bacterial plasmid, (B) ligating the fragments 
into a bacterial plasmid comprising (i) a bacterial origin of replication, 
(ii) a bacterial selection marker, and (iii) a mammalian selection marker, 
(C) transforming bacterial host cells with the plasmid, (D) selecting 
transformed bacterial host cells using the bacterial selection marker, (E) 
isolating plasmid DNA from the transformed bacterial host cells, (F) 
transfecting human cells with the isolated plasmid DNA, (G) selecting 
transfected human cells using the mammalian selection marker, (H) 
isolating extrachromosomal DNA from selected human cells, (I) in a 
suspension containing said extrachromosomal DNA, cleaving 
bacterially-synthesized DNA. The uncleaved extrachromosomal DNA in the 
suspension comprises a DNA sequence containing a human origin of 
replication. Such uncleaved extrachromosomal DNA may be purified from the 
suspension and/or used to transform host cells for amplification of the 
plasmid. 
In one general embodiment, the fragments of human genomic DNA are generated 
by digestion of human genomic DNA with a suitable restriction endonuclease 
(e.g., EcoRI). The fragments may also be produced by other DNA 
fragmentation methods, including sonication and shearing by passage 
through a narrow-bore syringe needle. 
The fragments typically have an average size of more than about 100 base 
pairs (bp) in length and less than about 10 kbp in length. Preferably, the 
average size is between about 2 kbp and about 6 kbp in length (e.g., 4 
kbp). 
The plasmid may be any plasmid suitable for transforming bacterial cells, 
though it is typically a pUC-derived or pBR322-derived plasmid (see, e.g., 
Sambrook, et al., or Ausubel, et al., for a description of bacterial 
plasmids). The bacterial origin of replication (ORI) may be, for example, 
a pUC or pBR322 ORI. The bacterial host cells are preferably E. coli 
cells. 
The bacterial selection marker can be any of a variety of known bacterial 
selectable markers, such a gene conferring resistance to a selected 
antibiotic. Examples of such antibiotics (and corresponding genes) include 
ampicillin (.beta.-lactamases), tetracycline, chloramphenicol 
(chloramphenicol acetyltransferases), and kanamycin (kanamycin 
phosphotransferases). 
Competent bacterial cells may be transformed with the plasmid using any 
standard bacterial cell transformation method, including calcium chloride 
transformation or electroporation. See, for example, Ausubel, et al., 
Chapter 1, for methods of making competent cells and methods for 
introducing plasmids into bacterial cells. 
Similarly, the mammalian selection marker can be selected from any of a 
variety of mammalian selectable markers. Examples of such markers include 
adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, APH) , 
dihydrofolate reductase (DHFR), hygromycin-b-phosphotransferase (HPH), 
thymidine kinase (TK), and xanthine-guanine phosphoribosyltransferase 
(XGPRT, gpt). Selection conditions for the above markers can be found, 
e.g., in Ausubel, et al., Chapter 9. A preferred mammalian selection 
marker is aminoglycoside phosphotransferase, a neomycin-resistance gene. 
Cells possessing plasmids containing this gene are preferably selected 
using neomycin or G418 (geneticin). 
Any of a variety of different human cells or cell lines may be used in the 
transfection step. Exemplary human cells suited for such transfection are 
K562 human erythroleukemia cells. As above, the transfection may be 
carried out using any known method of mammalian cell transfection. Such 
methods include calcium phosphate transfection, transfection using 
DEAE-dextran, liposome-mediated transfection, and electroporation. 
Preferred transfection methods are liposome-mediated transfection using 
cationic liposomes and electroporation. 
Extrachromosomal DNA may be isolated from transfected human cells using, 
for example, the Hirt extraction method. 
The suspension of extrachromosomal DNA is typically cleaved by digestion 
with a restriction endonuclease capable of cleaving methylated (typically 
at adenine residues), but not unmethylated DNA. An exemplary restriction 
endonuclease capable of cleaving methylated, but not unmethylated DNA, is 
DpnI. 
As stated above, the uncleaved extrachromosomal DNA comprises a DNA 
sequence containing a human origin of replication. In one general 
embodiment of the invention, this uncleaved extrachromosomal DNA is used 
for a second round of transfection and selection according to steps 
(F)-(J), above. 
These and other objects and features of the invention will become more 
fully apparent when the following detailed description is read in 
conjunction with the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION 
I. Multiple ORIs in Higher Eukaryotes 
According to the present invention, the observations summarized above can 
be reconciled by the following model: higher eukaryotes, due to the higher 
sequence complexity of replication origins, have developed a more 
sophisticated mechanism for firing specific sets of origins than the more 
simple genomes of viruses, bacteria, and mitochondria; replication 
enhancers, proposed to coincide with transcriptional enhancers, act 
synergistically with a core origin to enhance the efficiency of initiation 
of DNA replication (Boulikas, et al., 1995a, 1995b, herein incorporated by 
reference). Like transcription, requiring more than one enhancer (e.g., 
three enhancers and one promoter in the case of the histone H5 gene; 
Rousseu, et al., 1993), origin in higher eukaryotes may similarly require 
more than one enhancer occasionally located at a large distance from the 
core ORI. Thus, the longer the fragment of DNA dissected from the human 
genome in the range of 10-20 kb, the higher the probability of finding 
enhancer elements cooperating with the core origin. 
This model reconciles the studies of Calos and coworkers (Heinzel, et al., 
1991; Krysan and Calos, 1991; Krysan, et al., 1989) finding that large 
fragments from the human genome can confer autonomous replication with 
studies showing that small defined genomic fragments in the size range of 
500 bp from primates can drive the episomal replication of bacterial 
plasmids in humans cells (Zannis-Hadjopoulos, et al., 1995; Frappier and 
Zannis-Hadjopoulos, 1987; Iguchi-Ariga, et al., 1988; Ariga, et al., 1989; 
Rao, et al. , 1990; Landry, et al, 1991). In these studies the particular 
plasmid used (defective EBV, pBR322 Blue-script, etc.) and the transfected 
cell type may have a role in autonomous replication. A special case in 
such discrepancies are amplified genes like the chorion genes in fruit 
flies (Delidakis and Kafatos, 1989) and the DHFR amplicon in CHO cells 
expected to possess multiple origins or truncated origins and although 
initiating their replication at various sites along the genomic DNA 
nevertheless may use the same DNA sequence characteristics. 
About 50,000 origins of replication are thought to be present in each 
nucleus in mammalian cells during all developmental stages; some are used 
at early and others at later stages of development (Spradling and 
Orr-Weaver, 1987). In addition, a fraction of ORIs, linked with 
housekeeping genes, is presumably active at all stages of development. 
Thus, a net decrease in the number of active ORIs takes place during 
development (Callan, 1974). Origins of transcriptionally active genes are 
"fired" during early S-phase; this is in contrast to the heterochromatin 
and transcriptionally inactive genes which are replicated in late S-phase 
(Holmquist, 1987; Dhar, et al., 1988). The activation of replication 
origins during the early versus mid and late- S-phase of the cell cycle is 
governed by some unknown mechanism which seems to be associated with the 
transcription status of a gene and its attachment to the nuclear matrix. 
II. Matrix-Attached Regions (MARs) 
Matrix-attached regions (MARs) are responsible for the structuring of 
genomes into chromatin domains. Specific interactions between DNA sequence 
motifs on MARs and matrix proteins are responsible for the formation of 
the boundaries of chromatin units. Specifically, MARs (together with 
matrix proteins) constrain polynuceosomes into loops or domains, thereby 
insulating them from the effects of chromatin structure and torsional 
strain from flanking domains. MAR sequences have an average size of 500 
bp, are spaced about every 30 kb, and are believed to harbor or lie next 
to the origins of replications (ORIs) of the eukaryotic genome. 
Matrix proteins comprise one type of MAR-associating proteins. The 
MAR-associating proteins include (i) the classical matrix proteins, not 
displaying a strict DNA sequence specificity but rather a preference for 
AT-rich motifs (lamins A, B, topoisomerase II, histone Hi, Nuc2.sup.+, 
SAF-A, SATB1, nucleoli, matrins, calmodulin); (ii) a number of 
transcription factors (NF-1, ATF, Sp1, AP-1, C/EBP, RFP, T antigen, 
steroid hormone receptors, Tat of HIV, E7 of HPV-16); (iii) 
transcriptional adaptors such as retinoblastoma; (iv) a number of enzymes 
(histone deacetylases, casein kinase II, DNA polymerase .alpha. and 
.beta.); (v) intermediate filaments; (vi) hnRNA protein components; and 
(vii) the Ser-Arg family of protein splicing factors (see Table II in 
Boulikas, 1995, herein incorporated by reference). 
The presence of this complex assortment of structural and regulatory 
molecules in the matrix, some of which play an important role in DNA 
replication and transcription, as well as overwhelming evidence from 
analysis of MAR structures and in situ localization of DNA replication and 
transcription complexes together indicate that the nuclear matrix plays a 
fundamental, unique role in nuclear processes and that the structuring of 
genomes into domains has a functional significance. 
III. Activation and Silencing of Origins of Replication 
A program of specific silencing of origins of replication may drive the 
differential gene expression and the establishment of cell memory during 
development. The early Xenopus embryo, for example, increases the number 
of replication origins in order to replicate its genome within 10 to 15 
minutes (Callan, 1974). A similar process has been found in Drosophila 
embryos (Blumenthal et al., 1974). During subsequent stages of development 
different origins of replication may be suppressed in different cell 
types. This resembles the differential gene expression as well as the 
pattern of DNA methylation, starting from fully unmethylated DNA in the 
fertilized egg. 
The mammalian genome contains more ORIs than actually needed for its 
replication at a particular stage of development; the number of active 
origins decreases with pattern and cell type determination during 
development (Spradling, et al. 1987). As development proceeds, different 
replication origins, which are supposed to be anchored to the nuclear 
matrix, might be activated with a concomitant decrease in the total number 
of active origins. Thus, the multiplicity of origins may drive the 
differential gene expression and cell type formation during embryogenesis 
(Taylor, 1977, 1984; Brown et al., 1987; Hatton et al., 1988). 
IV. Replicons 
Replicons may physically coincide with large chromatin domains formed by 
the permanent class of MARs. According to the present invention, large 
domains may be subdivided into smaller subdomains by the facultative class 
of MARS. In line with this proposal, the entire 300 kb region encompassing 
the murine immunoglobulin heavy chain gene region was found to be a single 
replicon (Brown et al., 1987). Yet, this region was determined to contain 
multiple matrix anchorage sites spaced every 20 to 70 kb (Cockerill, 
1990). 
Martin and Oppenheim (1977) found that the average fork-to-fork distances 
in SV40-transformed Chinese hamster lung cells were shorter, or that there 
were more forks per unit length of DNA in transformed compared to 
nontransformed cells of the same type: The mean replicon size of the 
transformed cells was about 31 mm as compared with .about.44 mm for the 
nontransformed cells. Since 10 bp of DNA have a length of 3.4 nm, then the 
3.times.10.sup.9 bp of the Chinese hamster genome have a length of 1.02 m. 
If the length of the DNA (1.02.times.10.sup.6 mm) is divided by the size 
of the replicon (31 or 44 mm), we obtain .about.33,000 replicons per 
haploid genome for transformed and .about.23,200 replicons per haploid 
genome for normal cells. It was thought that the T antigen, coded by SV40 
and acting as an initiator of host DNA synthesis, was responsible for the 
increase in the number of replication initiation sites during 
transformation of cells by SV40. 
Replication enhancers are defined as DNA elements that interact with the 
core origin to enhance manyfold replication efficiency. This interaction 
is via proteins bound to the enhancer region with proteins bound to the 
core ORI and looping out of DNA. Core ORI by itself functions as a 
transcriptional enhancer for the gene(s) in the same chromatin loop. One 
core ORI may require more than one transcription enhancers for its 
activation. Replication specificity is conferred by a class of replication 
initiator proteins, comprising cruciform-binding proteins, that need to 
interact with transcription factors at the core ORI for origin firing; 
transcription specificity is provided by the interaction of the 
transcription/replication enhancer with the immediate 5' flanking promoter 
region. Core ORIs in this model possess a great number of transcription 
factor binding sites in addition to the initiator protein sites; both 
initiator proteins and transcription factors cooperate in origin firing. 
The developmental programs leading to inactivation of specific genes 
during cell type formation are proposed to be tightly linked with programs 
leading to the inactivation of ORIS; thus active ORIs are also active 
enhancers whereas inactive ORIs are linked with transcriptionally-inactive 
genes and are identical to enhancers inactivated during development or 
differentiation. Tumor cells are expected to be able to assemble a larger 
number of active enhancers because of activation of early embryo- or fetal 
stage-specific transcription factors, resulting in the activation of early 
developmental stage-specific ORIS. 
The capacity of papillomaviruses for autonomous extrachromosomal 
replication into the appropriate host (DiMaio, et al., 1982; 
Mitrani-Rosenbaum, et al., 1983; Giri, et al., 1983; Meneguzzi, et al., 
1984) has prompted the identification of DNA sequence elements in their 
genome able to maintain this function for human and animal gene therapy or 
cell culture transfection studies. A fragment of 69% of the BPV1 genome, 
after deletion of the L1 and L2 sequences coding for major structural 
proteins of the virus, has been used to drive the episomal expression of 
IL-2 cDNA under control of the murine metallothinein promoter in human 
SCLC cells in ex vivo studies; the 69% fragments contains the E1 to E8 
early genes and regulatory regions including its origin of replication 
(see Cassileth, et al., 1995). 
Several cloning vectors able to episomal replication have been constructed 
which use the SV40 or the related polyoma virus ORI and the T-antigen cDNA 
to express the T antigen protein in human or animal cells and effect the 
episomal replication of the plasmid containing the SV40 ORI (Zhu, et al., 
1993; Thierry, et al., 1995). However, T antigen exerts profound effects 
on host gene regulation leading to deregulation of cell growth, known as 
transformation (although, on the other hand, the polyoma large T antigen 
has not been shown to cause oncogenic transformation but to facilitate the 
establishment of embryo fibroblasts as permanent cell lines 
(Rassoulzadegan, et al., 1983) and to cooperate with other oncogenes in 
the tumorigenic transformation of primary cells (Ruley, 1983). 
It is desirable for human gene therapy applications to use natural human 
ORIs in order to drive the episomal replication of therapeutically 
important genes. To-date, due to our incomplete knowledge of human 
sequences able to act as ORIs no vectors with human ORIs have been used in 
gene therapy. 
Experiments performed in support of the present invention on the human CHAT 
gene ORI indicate that this ORI can be used to construct episomal plasmids 
that might find application in human gene therapy. The use of human ORIs 
is desirable over viral ORIs used in gene transfer to somatic cells (Zhu, 
et al., 1993; Thierry, et al., 1995) that seem to require the presence of 
a viral replication initiator protein cDNA in the same vector for the 
episomal retention of the plasmid. The rather small size of this human ORI 
(513 bp or 1435 bp if fragments C,D, and E are combined together) makes it 
attractive for use with retroviral or adenoviral and AAV vectors because 
of their limited payload capacity. 
V. Method for Isolating Human ORIs 
A methodology is described for isolating human sequences with strong origin 
of replication (ORI) activity. Human genomic DNA is isolated and digested 
with restriction endonucleases into fragments of an average size of about 
4 kb. Alternatively, the human genomic DNA used is 1% of total DNA 
attached to the nuclear matrix, known to be enriched in human ORIs. 
The DNA fragments are then ligated into a bacterial plasmid which cannot 
replicate in human cells, containing neomycin and ampicillin resistance 
genes. The ligation mixture is used to transform competent E. coli cells 
(such as strain DH5.alpha.) and clones are grown on ampicillin/neomycin 
plates. Resistant clones are isolated and used to make a plasmid DNA prep, 
which contains a mixture of several hundred thousand different recombinant 
plasmid molecules. 
Human sequences with ORI function are selected by transfection of the 
mixture of the recombinant plasmids into human cells in culture, in the 
presence or absence of 1 mg/ml of G418 (geneticin) for selecting 
transfected cells expressing the plasmid. Episomal and extrachromosomal 
plasmids are extracted from the human cells at 3 days and 30 days 
posttransfection. The plasmids are treated with DpnI, which destroys the 
bacterially-replicated plasmids to eliminate input plasmid DNA. The 
DpnI-resistant plasmids are used to transform E. coli cells and to isolate 
individual colony plasmids. 
The total plasmid isolate may be subjected into a second cycle of 
transfection into human cells in culture and selection of the 
extrachromosomally-replicating plasmids. Individual clones selected in 
this way can drive the autonomous replication of selected heterologous 
genes (e.g., therapeutically important genes human cells in vivo and in 
vitro, and are useful in human gene therapies. 
The isolated ORIs can further be used in footprinting assays of 
transcription/replication factors to identify the regions of interaction 
with MAR-associating proteins. Further, oligonucleotides from these 
regions, immobilized on Sepharose beads, can be used to react with a 
mixture of transcription/replication factors isolated from human cells to 
identify their corresponding proteins in binding assays. This approach may 
be used to isolate novel nuclear proteins involved in the activation of 
human ORIs. 
The following example is intended to illustrate but in no way limit the 
invention. 
Materials and Methods 
Reagents such as restriction enzymes, modifying enzymes, and the like may 
be purchased from a variety of commercial suppliers, including New England 
Biolabs (Beverly, Mass.), United States Biochemical (USB; Cleveland, 
Ohio), Boehringer Mannheim (Indianapolis, Ind.), and Strategene (La Jolla, 
Calif.). 
A. Isolation and Nuclear Matrix Preparation 
Nuclei are isolated using the procedure of Boulikas (1988). According to 
this method, cultured cells are lysed in 1% Triton X-100 and nuclei are 
collected by centrifugation through a 60% glycerol cushion at 
13,000.times.g for 4 minutes in "EPPENDORF" tubes (Boulikas, 1988). Nuclei 
isolated from 2.5.times.10.sup.7 cells are digested with 1,000 units 
micrococcal nuclease (MNase) at 37.degree. C. for 10 minutes in 0.5 ml 50 
mM Tris.HCl, pH 7.5, 25 mM KCl, 1 mM CaCl.sub.2, 4 mM MgCl.sub.2. 
Nuclear matrices are isolated by an adaptation of the methods of Cockerill 
and Garrard (1986) and Boulikas (1986). All procedures are at 0.degree. C. 
in the continuous presence of 0.1 mM N-p-tosyl-L-lysine chloromethyl 
ketone to minimize proteolysis and in "EPPENDORF" tubes using short-time 
spins to avoid nuclear matrix disaggregation. According to this method, 
digested nuclei are centrifuged in "EPPENDORF" tubes for 2 minutes in a 
microfuge (13,000.times.g) to yield a supernatant fraction S1, enriched in 
active chromatin (Rose and Garrard, 1984). The pellet (P1), containing 
digested nuclei, is then lysed in 0.5 ml 2 mM EDTA, 3 mM Tris, pH 7.5, and 
the lysed nuclei are centrifuged at 13,000.times.g for 3 minutes. 
The supernatant fraction (S2) of the lysed nuclei centrifugation contains 
the bulk of nucleosomes mainly as mononucleosome particles due to the 
extensive treatment with MNase. The pellet fraction, P2, containing the 
residual of the chromatin loops and the nuclear matrix, is resuspended in 
0.5 ml 2M NaCl, 2 mM EDTA, 10 mM Tris, pH 7.5, and immediately centrifuged 
(3 minutes) to separate the residual histones from the nuclear matrix (NM) 
fraction. The DNA content of the nuclear matrix fraction that can be 
monitored by the extent of digestion of nuclei by MNase (Boulikas, 1985) 
typically represents 1-2 % of total DNA and typically has fragments 
ranging in size from 0.1-5.0 kb (Boulikas and Kong 1993a,b). The nuclear 
matrix fraction is resuspended in 0.2 ml 5M urea. Then 0.2 ml 2% SDS is 
added and the nuclear matrix proteins are removed with proteinase K (0.1 
mg/ml), at 37.degree. C. for 16 hours. DNA is extracted twice with 
phenol/chloroform and precipitated with 70% ethanol. NM DNA preparations 
are size-fractionated on agarose gels and purified using Qiagen columns 
for cloning. 
B. Agarose Gel Electrophoresis 
The various nuclear fractions are adjusted to 1% SDS, 10% glycerol, 0.05% 
bromophenol blue in TBE buffer (89 mM Tris, 89 mM boric acid, pH 8.5, 1 mM 
EDTA) and loaded directly onto 1% agarose gels containing 10 .mu.g/ml 
ethidium bromide, 0.1% SDS in TBE buffer. The electrophoresis buffer is 
TBE buffer containing 0.1% SDS. 
C. Cloning of the Nuclear Matrix DNA in pBlueScript 
Pieces from the low-melting agarose gel containing 100-700 bp or 0.7-5.0 
kbDNA fragments and about 3 .mu.g of nuclear matrix DNA are excised under 
low-energy UV light. Exposure to UV is minimized to avoid formation of 
cyclobutane dimers and (6-4) photoproducts on the DNA that cause 
C.fwdarw.T transitions during replication (see Boulikas, 1992). 
One volume of TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA) is added to the gel 
piece, melted at 65.degree. C., extracted once with phenol, once with 
phenol/chloroform, once with chloroform, and precipitated by the addition 
of 2 vol. ethanol and 0.1 vol. of 3M sodium acetate at -70.degree. C. 
overnight in Eppendorf tubes. 
DNA is collected by centrifugation at 0.degree. C. for 20 minutes 
(13,000.times.g, microfuge), washed in 70% ethanol and resuspended in 20 
.mu.l TE. In some experiments the DNA fragments are phosphorylated at 
their 5' ends using T4 polynucleotide kinase (GIBCO/BRL, Grand Island, 
N.Y.) and ATP as described (Wu, et al., 1987). The 3' recessed ends 
generated by MNase (Sollner-Webb, et al., 1978) are filled in with 1 .mu.l 
(4 units) Klenow fragment of DNA polymerase in TE buffer, extracted with 1 
vol. phenol, once with phenol/chloroform and ethanol-precipitated. 
The DNA pellet is dried in a Speedvac for 5 min, resuspended in 7 .mu.l 
H.sub.2 O, 1 .mu.l 10.times. ligation buffer, 1 .mu.l "BLUESCRIPT" vector 
(Stratagene, La Jolla, Calif.) cut with EcoRV, and 1 .mu.l T4 ligase (USB) 
and incubated at 14.degree. C. for 16 hours. DNA is directly transformed 
in JM109 or DH5.alpha. competent E. coli cells and plated on LB/agar with 
ampicillin (100 .mu.g/ml), X-gal and IPTG. Plates are incubated at 
37.degree. C. overnight and screened for white colonies. Individual 
colonies are picked and grown in 3 ml LB + ampicillin (100 .mu.g/ml). 
Plasmid minipreps digested with EcoRI + HindIII are analyzed for the size 
of the insert on 1% agarose gels. 
A family of new vectors is constructed containing the neomycin resistance 
gene, the lacZ gene of E. coli encoding .beta.-galactosidase, and a 
polylinker whereupon cloning of a MAR/ORI insert would cause disruption of 
the b-galactosisdase gene and loss of the blue color in colonies, and the 
kanamycin-resistance gene. Cloning in such vectors is performed using 
standard methods (e.g., Maniatis, et al., 1982; Sambrook, et al., Ausubel, 
et al.). 
E. DNA Sequencing 
DNA sequencing is performed using the dideoxy method and the Sequenase II 
kit (USB). For this purpose, double-stranded DNA plasmid is denatured, 
mixed with universal or reverse primers (USB), and DNA strand synthesis is 
performed according to the manufacturer's instructions and the method of 
Mierendorf and Pfeffer (1987). The sequencing reaction DNA is analyzed on 
gels (4% acrylamide, 50% w/v urea, 60 cm long, 0.4 mm thick) by running at 
about 65 Watts for 2.5-3 hours. The gels are dried on Whatman 3 MM filter 
paper and exposed for autoradiography for about 24 hours at room 
temperature using X-Omat film. 
F. DpnI Assays and Bromodeoxyuridine Incorporation 
Fragments of DNA isolated using the methods of the invention can be tested 
for their efficacy in driving the autonomous replication of a bacterial 
plasmid after its introduction into cells in culture in transient 
transfection experiments. A preferred method for testing replication of a 
DNA fragment in higher eukaryotes is the DpnI-resistance assay on Hirt 
extracts (Hirt, 1967) at 48-72 post-transfection. According to this 
method, cells are lysed with SDS in the presence of about 0.5 M NaCl; the 
lysate is left at 0.degree. C. for several hours. Under these conditions 
high molecular weight genomic DNA forms a large complex with the SDS-NaCl 
complex which can be removed following centrifugation at 13,000 to 
25,000.times.g for about 30 minutes-1 hour, leaving the low size plasmid 
or viral DNA in the supernatant (Hirt, 1967). The plasmid DNA in the 
supernatant is extracted, digested with DpnI, the fragments are separated 
by electrophoresis on agarose gels, and blotted on nylon or nitrocellulose 
filters using the bacterial plasmid as a probe. DpnI-resistant plasmids 
are those replicated in eukaryotic cells and lacking methylation on 
adenine residues characteristic of molecules replicated in E. coli. Only 
bacterially-made DNA that carries methylated A that is cleaved by DpnI 
(Frappier and Zannis-Hadjopoulos, 1987). 
Although identification of a genomic sequence that confers autonomous 
replication to a plasmid does not guarantee that the sequence will 
function as an origin of replication in vivo., most sequences capable of 
driving the extrachromosomal replication have sequence motifs 
characteristic of ORIs and could be used as origins at a certain 
developmental stage or cell type. 
Another method for determining autonomous replication is from the 
incorporation of the heavy bromodeoxyuridine in place of thymidine in the 
newly synthesized DNA followed by isolation of low molecular weight DNA 
(Hirt, 1967) and separation of the heavy and light extrachromosomal DNA by 
CsCl density gradient centrifugation (Frappier and Zannis-Hadjopoulos, 
1987). 
G. Two-Dimensional Mapping Techniques 
The development of two-dimensional agarose gel electrophoresis on 
neutral/neutral (Brewer and Fangman, 1987) or neutral/alkaline (Huberman, 
et al., 1987) gels has prompted the identification of origins of 
replication. Replication intermediates (forks, bubbles, q-shaped or Cairns 
structures) from the yeast 2 mm plasmid terminate in multiply interlocked 
catenanes and are subsequently resolved by the cell machinery to monomer 
plasmids; restriction fragments derived from the Cairns structures contain 
replication forks and bubbles that are separated from one another and from 
nonreplicated linear DNA by low voltage agarose gel electrophoresis which 
resolves DNA primarily by mass, followed by a second dimensional gel 
electrophoresis in a higher percentage gel and at a higher voltage, 
separating DNA by shape as well as by mass (Brewer and Fangman, 1987). 
According to the procedure of Huberman, et al. (1987), replication 
intermediates from 2 mm plasmid of yeast are separated on neutral agarose 
gels which are further separated by size and into nascent and parental 
strands on the second dimensional alkaline agarose gels. Closed circular, 
nicked circular, full-length linear, as well as oligomers of these forms 
give discrete spots on 2D gels. Transfer of the DNA fragments from the 2D 
gels onto nylon membrane filters and hybridization with short probes from 
different areas of the suspected replication origin reveals the direction 
of fork movement (Huberman, et al., 1987). If a particular genome-derived 
restriction fragment does not contain an origin of replication, the 
replication intermediates of this fragment will be fork-shaped, because a 
replication fork will proceed through the fragment from one end to the 
other; genomic probes from the origin of replication will reveal the 
presence of bubble-shaped replication intermediates arising from two forks 
proceeding bidirectionally from the origin. Fork-shaped versus 
bubble-shaped intermediates give rise to different arc patterns on 2D gel 
blots (Brewer and Fangman, 1987, 1988; Rivier and Rine, 1992). Since 
replicating DNA represents a small fraction of total DNA, especially in 
asynchronous cell cultures, these analyses can be facilitated by selective 
adsorption of the partially single-stranded DNA of replication 
intermediates on benzoylated naphthoylated DEAE-cellulose (Huberman, et 
al., 1987). 
The 2D agarose gel technique does not have sufficient resolution to 
determine whether initiation occurs at a single site or from multiple 
dispersed or closely spaced ORIs present over a small genomic region in 
the range of 500 bp. 
EXAMPLE 1 
Isolation of Human ORI sequences 
Human genomic DNA is isolated and digested with restriction endonucleases 
into fragments of an average size of approximately 4 kb; the fragments are 
inserted into cloning vectors which contain: (i) a bacterial plasmid ORI 
permitting replication in E. coli such as pUC or pBR322 ORI (ii) the 
ampicillin-resistance gene allowing selection of the plasmids in E. coli 
cells; (iii) a neomycin-resistance gene near the cloning site to be driven 
by the cloned human sequence; (iv) the firefly luciferase gene in the 
opposite orientation from the neomycin-resistance gene and on the other 
side of the cloning site to be driven by the same human DNA sequence 
element; (v) the LacZ gene containing the cloning site and the Sp6/T7 
promoters at the flanks of the cloning site allowing white/blue colony 
selection after treatment of bacteria with IPTG and X-Gal in order to 
identify the plasmids with human sequence inserts forming white colonies 
(FIG. 1). 
The ligation mixture is used to transform competent E. coli cells (such as 
strain DH5.alpha.), the total transformed population of cells is cultured, 
and plasmids are isolated. The resulting plasmid prep is a mixture of 
several hundred thousand different plasmid molecules which, upon 
electrophoresis on agarose gels, give a smear with size range higher than 
that of the control plasmid without insert. At this stage, it is possible 
to size-fractionate the plasmids from the gel and pursue the cloning of 
large, medium, and small inserts separately. 
The plasmid preparation is purified using Qfagen plasmid purification 
columns, complexed with cationic liposomes (e.g., the commercially 
available lipofectin or transfectam), and is used to transfect K562 human 
erythroleukemia cells. Selection of the proper cell line is important 
since different cell types use a subset of different ORIs for the 
replication of their genome (Boulikas 1995a). 
The plasmid mixture is introduced into the cells, the cell culture is split 
into two, and the cells are cultured in the presence or absence of G418 (1 
mg/ml). This antibiotic is destroyed by those human cells which express 
the neomycin-resistance gene. Such cells are able to expand during 
culturing. Thus, a selection of the human DNA sequences able to drive the 
neomycin-R gene is made at this stage. 
A further selection is performed for plasmids capable episomal 
(extrachromosomal) replication in human cells in view of the function of 
the insert as an ORI. Episomally replicated plasmids are isolated from the 
human cultured cells using, e.g., the Hirt extraction method (1967), and 
treated with DpnI, which digests only the plasmids which have been 
replicated in E. coli cells (DpnI recognizes the GATC tetranucleotide only 
when the A is methylated). Only bacteria (not human or other eukaryotic) 
cells can methylate A in this sequence. This tetranucleotide is recognized 
by dam methyltransferase which methylates the adenine residue at the 
N.sup.6 position. 
The plasmids that have been replicated in human cells and that survive DpnI 
digestion contain human sequences able to function as ORIs in the host 
mammalian cells. Such a plasmid isolate is used to transform E. coli; 
total plasmid DNA is isolated, and can optionally be subjected to a second 
round of transfection of human cells and selection. Individual clones are 
isolated as white colonies from IPTG/X-Gal smeared agar plates. Bacteria 
are grown from individual colonies, the plasmid is isolated, the insert is 
sequenced using the Sp6/T7 primers, and its ORI function is tested using 
three different approaches (i) the ability to sustain the episomal 
replication in human cells after transfection of the plasmid; (ii) using 
the PCR method of Vassilev and Johnson (1989); and (iii) using the 
two-dimensional mapping approach (Huberman, et al., 1987) Clones isolated 
using the above protocol are expected to represent or contain human ORIs. 
Such sequences may be used, for example, in plasmid vectors for driving 
the episomal replication of therapeutically important genes in animal 
tissues after in vivo injection and in human tissues in clinical trials.