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Biomedical subjects

M P Calos

Publications and source records attributed to M P Calos.

At least 19 recordsLinked to original sources
PubMed2001

Directed evolution of a recombinase for improved genomic integration at a native human sequence.

We previously established that a unidirectional site-specific recombinase, the phage phiC31 integrase, can mediate integration into mammalian chromosomes. The enzyme directs integration of plasmids bearing the phage attB recognition site into pseudo attP sites, a set of native sequences related to the phage attP recognition site. Here we use two cycles of DNA shuffling and screening in Escherichia coli to obtain evolved integrases that possess significant improvements in integration frequency and sequence specificity at a pseudo attP sequence located on human chromosome 8, when measured in the native genomic environment of living human cells. Such integrases represent custom integration tools that will be useful for modifying the genomes of higher eukaryotic cells.

Base Sequence↗
PubMed2001

Phage R4 integrase mediates site-specific integration in human cells.

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.

Attachment Sites, Microbiological↗
PubMed2001

Epstein-Barr virus/human vector provides high-level, long-term expression of alpha1-antitrypsin in mice.

We have constructed plasmid DNA vectors that contain Epstein-Barr virus (EBV) sequences and the human gene (SERPINA1) encoding alpha1-Antitrypsin (AAT). We demonstrate that a plasmid carrying the full SERPINA1 on a 19-kb genomic fragment and the EBV gene EBNA1 and its family of repeats binding sites undergoes efficient extrachromosomal replication in dividing mammalian tissue culture cells. Therefore, use of a whole genomic therapeutic gene to provide both replication and gene expression may be an effective gene therapy vector design, if the target cells are dividing. The efficacy of this same vector for expression of AAT in vivo in the nondividing cells of mouse liver was determined by hydrodynamic injection of naked plasmid DNA by means of the tail vein. A single injection of an EBV/genomic SERPINA1 vector provided >300 microg/ml of AAT, which approached normal plasma levels and persisted for the >9-month duration of the experiment. These data exceed most previously reported values, probably due to sequences in the genomic DNA that resist silencing of gene expression, possibly in combination with favorable effects on expression provided by the EBV sequences. These results demonstrate that plasmid DNA with the correct cis-acting sequences can provide in vivo long-term expression of protein at high levels that are therapeutically relevant for gene therapy.

Animals↗
PubMed2001

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase.

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

3T3 Cells↗
PubMed2000

An extrachromosomal tetracycline-regulatable system for mammalian cells.

We have modified the tetracycline-regulatable system so that all components are present on a stable extrachromosomal vector that can replicate in a wide variety of mammalian cells. An EBV/human ori vector is used to carry the system, overcoming the species specificity of conventional Epstein-Barr virus vectors. By placing the transcriptional transactivator gene under autoregulation, better induction characteristics are obtained. This system offers greater speed and sensitivity than previously reported methods. It can be applied within 3-4 weeks and produces an induction range of several hundred-fold with a low background.

Animals↗
PubMed2000

A phage integrase directs efficient site-specific integration in human cells.

The integrase from the Streptomyces phage phiC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the phiC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.

Bacteriophages↗
PubMed2000

Mammalian genomes contain active recombinase recognition sites.

Recombinases derived from microorganisms mediate efficient site-specific recombination. For example, the Cre recombinase from bacteriophage P1 efficiently carries out recombination at its loxP target sites. While this enzyme can function in mammalian cells, the 34bp loxP site is expected to be absent from mammalian genomes. We have discovered that sequences from the human and mouse genomes surprisingly divergent from loxP can support Cre-mediated recombination at up to 100% of the efficiency of the native loxP site in bacterial assays. Transient assays in human cells demonstrate that such pseudo-lox sites also support Cre-mediated integration and excision in the human cell environment. Pseudo sites for Cre and other recombinases may be useful for site-specific insertion of exogenous genes into mammalian genomes during gene therapy and other genetic engineering processes.

Animals↗
PubMed1999

Epstein-Barr virus plasmid model system for analyzing recombination in human cells.

Homologous recombination stimulated by a double-strand break at a desired target site offers a method to achieve site-specific integration useful for gene therapy and other genetic engineering. To test parameters needed for this strategy, we developed an Epstein-Barr virus shuttle vector model system as a genetic tool. This extrachromosomal plasmid assay system has several advantages over a chromosomal assay. The system detects all classes of recombination events without selection and allows rapid analysis of the frequency and nature of recombination events. We found that a double-strand break at the target site stimulated a large increase in recombination frequency. The resulting recombinants included one-sided insertion events, as well as two-sided or gene conversion events. A circular donor substrate was more effective in recombination than linearized donor DNA.

Base Sequence↗
PubMed1999

Effects of homology length and donor vector arrangement on the efficiency of double-strand break-mediated recombination in human cells.

We use an Epstein-Barr virus (EBV) plasmid model chromosome system to study how different donor plasmid constructs affect recombination stimulated by an I-SceI-induced double-strand break in the target sequence in human cells. The entire 3.5 kb lacZ gene was efficiently recombined into a target EBV vector lacking lacZ sequences, but having limited homology to the donor plasmid. A donor plasmid with lacZ flanked by sequence homologous to the target consistently generated gene conversion events and was more effective than a donor carrying lacZ outside the same sequence homology. Reducing the length of homology between the target and donor from 5.5 kb to 1 kb caused only a 3-fold drop in recombination frequency, contrasting with the exponential dependence on homology length seen when no DSB is present in the target. These results document a DSB-induced 175-fold increase in recombination of a heterologous gene into a target, requiring only limited flanking homology.

Cells, Cultured↗
PubMed1998

Telomeric sequences from human herpesvirus 6 do not mediate nuclear retention of episomal DNA in human cells.

Telomeric repeat sequences (TRS) have been identified close to, but not at, the genome termini of several lymphotropic herpesviruses, including human herpesviruses 6 and 7 (HHV-6, HHV-7). The functional significance of these motifs remains uncertain. Since telomeric sequences can mediate stable retention of episomal DNA in yeast, we have tested whether the TRS motifs from HHV-6 might mediate a similar function in human cells. Several candidate sequences were assessed for their ability to provide nuclear retention to an autonomously replicating vector in rapidly dividing human tissue culture cells, including HHV-6 TRS DNA, as well as telomeric DNA from human cells and sequences from Epstein-Barr virus (EBV). However, only a vector carrying the EBV-derived retention mechanism showed a significant level of nuclear retention. Neither the HHV-6 TRS motifs, nor human telomeric sequences, mediated nuclear retention of episomal DNA in human cells.

Cell Line, Transformed↗
PubMed1998

Epstein-Barr virus vectors for gene expression and transfer.

Vectors based on components of Epstein-Barr virus (EBV) have found increasingly wide applications in biotechnology. Three areas of recent advancement comprise the use of EBV vectors to improve the convenience of gene expression systems, the development of EBV vectors for gene transfer in gene therapy, and the use of EBV components to generate large vectors carrying sizable regions of genomic DNA.

Animals↗
PubMed1996

The potential of extrachromosomal replicating vectors for gene therapy.

Persistence of DNA vectors in target cells is advantageous in most applications of gene therapy. Particularly when target cells are undergoing proliferation, vector longevity will depend on either the integration of the vector into the chromosomes or the ability of the vector to replicate and be retained extrachromosomally. Vectors that efficiently integrate in a nonrandom fashion are currently unavailable, and those that can replicate extrachromosomally provide a major alternative strategy. Several classes of such vectors are under development, carrying mechanisms for prolonging DNA retention in mammalian nuclei that extend vector lifetime in non-proliferating cells as well. The vectors utilize either chromosomal or viral elements to mediate replication and retention, and have a large size capacity for insertion of genes of interest. I discuss the state of the art for these vectors, including the assets and limitations of their future use in gene therapy.

Animals↗
PubMed1996

Long-term gene expression from autonomously replicating vectors in mammalian cells.

We tested the longevity of gene expression provided by autonomously replicating vectors. The vectors contain segments of human genomic DNA that provide efficient replication initiation and sequences derived from Epstein-Barr virus that provide nuclear retention. In order to monitor gene expression, the vectors also carry an expression cassette containing the chloramphenicol acetyl transferase gene. Replicating and control vectors were transfected into rapidly dividing human 293 cells, and gene expression and DNA retention were monitored. Gene expression decreased rapidly from a control vector lacking replication and retention functions, reaching near background levels by 10 days. Vectors having both replication and retention showed greatly prolonged gene expression, with vector DNA still detectable after 2 months. Autonomously replicating vectors also prolonged gene expression in rodent cells, in human lung epithelial cells, and in slowly dividing cell cultures. These results demonstrate the utility of this autonomous replication system for long-term retention and expression of introduced genes in mammalian cells. Such vectors could be useful for gene therapy, in combination with any of several methods for introduction of DNA.

Animals↗
PubMed1995

Autonomous replication in Drosophila melanogaster tissue culture cells.

This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and test ed them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA were able to replicate autonomously, as were random human and Escherichia coli genomic DNA fragments. Most of the plasmids were detectable 18 days after transfection in the absence of selection, suggesting that transfected DNA is maintained in Drosophila cells without rapid loss or degradation. The finding that all plasmids containing Drosophila, human or bacterial DNA replicate autonomously in Drosophila cells suggests that the signals that direct autonomous replication in Drosophila contain a low degree of sequence specificity. A two-dimensional gel analysis of initiation on one of the plasmids was consistent with many dispersed initiation sites. Low sequence specificity and dispersed initiation sites also characterize autonomous replication in human cells and Xenopus eggs and may be general properties of autonomous replication in animal cells.

Animals↗
PubMed1995

Replication of centromere II of Schizosaccharomyces pombe.

The centromeric DNAs of Schizosaccharomyces pombe chromosomes resemble those of higher eukaryotes in being large and composed predominantly of repeated sequences. To begin a detailed analysis of the mode of replication of a complex centromere, we examined whether any sequences within S. pombe centromere II (cen2) have the ability to mediate autonomous replication. We found a high density of segments with such activity, including at least eight different regions comprising most of the repeated and unique centromeric DNA elements. A physical mapping analysis using two-dimensional gels showed that autonomous replication initiated within the S. pombe sequences in each plasmid. A two-dimensional gel analysis of replication on the chromosomes revealed that the K and L repeat elements, which occur in multiple copies at all three centromeres and comprise approximately 70% of total centromeric DNA mass in S. pombe, are both sites of replication initiation. In contrast, the unique cen2 central core, which contains multiple segments that can support autonomous replication, appears to be repressed for initiation on the chromosome. We discuss the implications of these findings for our understanding of DNA replication and centromere function.

Base Sequence↗
PubMed1994

Physical mapping of origins of replication in the fission yeast Schizosaccharomyces pombe.

We isolated four fragments from the Schizosaccharomyces pombe genome that mediate autonomous replication. A two-dimensional gel analysis revealed that in each case initiation could be mapped to within the S. pombe sequences. In three of the fragments, initiation could be mapped to one discrete location. In the fourth fragment, subcloning and two-dimensional gel analysis suggested that two discrete origins of replication were located within 3 kb of each other. When in proximity, usually only one of these origins fired, suggesting origin interference. Two-dimensional gel analysis of the four origin fragments at their genomic locations demonstrated that each is used in the chromosomes, but in only a subset of cells or cell divisions. The S. pombe genome appears to contain many discrete origins, not all of which fire in any given cell and some of which are closely spaced. Not I/Sfi I mapping of the five origins from this and a previous study indicates that they are randomly distributed throughout the genome and appear to be representative of chromosomal origins of replication in this organism. We compare the features of S. pombe replication origins with those of S. cerevisiae and animal cells.

Chromosome Mapping↗
PubMed1994

Specific initiation at an origin of replication from Schizosaccharomyces pombe.

Using a genetic assay for efficient autonomous replication, we have isolated from Schizosaccharomyces pombe a 6.2-kb fragment which shows the properties expected of an origin of DNA replication in S. pombe. A 2.8-kb subclone of the fragment has the same replication properties. Two-dimensional gel analysis of replication intermediates throughout plasmids carrying the 6.2- or 2.8-kb fragments shows that replication initiates only in a specific region, which can be localized to within several hundred base pairs, in the fragments. This region is also a site of replication initiation in the S. pombe chromosome where the fragments normally reside. These results provide strong evidence that initiation of replication in S. pombe is localized and mediated by specific DNA sequence signals.

Cloning, Molecular↗