Chromosome-based vectors for gene therapy.

Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. There is increasing agreement that the ideal vector for gene therapy should be completely based on chromosomal elements and behave as an independent functional unit after integration into the genome or when retained as an episome. In this review we will first discuss the chromosomal elements, such as enhancers, locus control regions, boundary elements, insulators and scaffold- or matrix-attachment regions, involved in the hierarchic regulation of mammalian gene expression and replication. These elements have been used to design vectors that behave as artificial domains when integrating into the genome. We then discuss recent progress in the use of mammalian artificial chromosomes and small circular non-viral vectors for their use as expression systems in mammalian cells.

In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei.

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3'-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T(4)G(4)), forms a 16-nucleotide 3'-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of K(d) = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (K(d) = 3--5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.

Exploiting chromosomal and viral strategies: the design of safe and efficient non-viral gene transfer systems.

The development of gene transfer systems for therapeutic applications depends, to a large part, on an understanding of chromosomal elements mediating controlled gene expression, and DNA synthesis and maintenance. The integration of transgenes into chromosomes assures their faithful replication and segregation due to the natural functions of the host chromosome, but their expression is susceptible to inactivation by the host-cell apparatus, and they may also cause unwanted mutagenic effects. While episomal vectors are free from these shortcomings, progress in this field suffers from the lack of an in-depth understanding of the accessory functions, although a number of first-generation prototypes have been constructed in the past years. As an immediate solution, small non-viral circular episomal vectors are emerging which not only permit the study of the relevant components in a minimal gene-transfer system, but for which a considerable potential for therapeutic applications can be anticipated.

Association of the telomere-telomere-binding protein complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication.

Telomeric interactions with the nuclear matrix have been described in a variety of eukaryotic cells and seem to be essential for specific nuclear localization. Macronuclear DNA of hypotrichous ciliates occurs in small gene-sized DNA molecules, each being terminated by telomeres. Each macronucleus contains over 10(8 )individual DNA molecules. Owing to the high number of telomeres present in this nucleus it provides an excellent model to study telomere behaviour throughout the cell cycle. In this study we provide experimental evidence that the telomere-telomere-binding protein (TEBP) complex specifically interacts with components of the nuclear matrix in vivo. In the course of replication the specific interaction of the TEBP with components of the nuclear matrix is resolved and an attachment of the telomeres to the matrix no longer occurs.

Both subtelomeric regions are required and sufficient for specific DNA fragmentation during macronuclear development in Stylonychia lemnae.

BACKGROUND: Programmed DNA-reorganization and DNA-elimination events take place frequently during cellular differentiation. An extreme form of such processes, involving DNA reorganization, DNA elimination and DNA fragmentation, is found during macronuclear differentiation in hypotrichous ciliates. Ciliated protozoa can therefore serve as a model system to analyze the molecular basis of these processes during cellular differentiation in eukaryotic cells. RESULTS: Using a biological approach to identify cis-acting sequences involved in DNA fragmentation, we show that in the hypotrichous ciliate Stylonychia lemnae sequences required for specific DNA processing are localized in the 3'- and the 5'-subtelomeric regions of the macronuclear precursor sequence. They can be present at various positions in the two subtelomeric regions, and an interaction between the two regions seems to occur. Sequence comparison revealed a consensus inverted repeat in both subtelomeric regions that is almost identical to the putative Euplotes chromosome breakage sequence (E-Cbs), also identified by sequence comparison. When this sequence was mutagenized, a processed product could no longer be detected, demonstrating that the sequence plays a crucial role in DNA processing. By injecting a construct into the developing macronucleus, which exclusively contains the subtelomeric regions of the Stylonychia alphal-tubulin gene, we show that subtelomeric regions are not only required but are also sufficient for DNA processing in Stylonychia. CONCLUSIONS: Our results indicate that an inverted repeat with the core sequence 5'-TGAA present in both subtelomeric regions acts as a Cbs in Stylonychia. The results allow us to propose a mechanistic model for DNA processing in this ciliate.

A nuclear protein involved in apoptotic-like DNA degradation in Stylonychia: implications for similar mechanisms in differentiating and starved cells.

Ciliates are unicellular eukaryotic organisms containing two types of nuclei: macronuclei and micronuclei. After the sexual pathway takes place, a new macronucleus is formed from a zygote nucleus, whereas the old macronucleus is degraded and resorbed. In the course of macronuclear differentiation, polytene chromosomes are synthesized that become degraded again after some hours. Most of the DNA is eliminated, and the remaining DNA is fragmented into small DNA molecules that are amplified to a high copy number in the new macronucleus. The protein Pdd1p (programmed DNA degradation protein 1) from Tetrahymena has been shown to be present in macronuclear anlagen in the DNA degradation stage and also in the old macronuclei, which are resorbed during the formation of the new macronucleus. In this study the identification and localization of a Pdd1p homologous protein in Stylonychia (Spdd1p) is described. Spdd1p is localized in the precursor nuclei in the DNA elimination stage and in the old macronuclei during their degradation, but also in macronuclei and micronuclei of starved cells. In all of these nuclei, apoptotic-like DNA breakdown was detected. These data suggest that Spdd1p is a general factor involved in programmed DNA degradation in Stylonychia.

A subtelomeric DNA sequence is required for correct processing of the macronuclear DNA sequences during macronuclear development in the hypotrichous ciliate Stylonychia lemnae.

During macronuclear differentiation in ciliated protozoa a series of programed DNA reorganization processes occur. These include the elimination of micronuclear-specific DNA sequences, the specific fragmentation of the genome into small gene-sized DNA molecules, the de novo addition of telomeric sequences to these DNA molecules and the specific amplification of the remaining DNA molecules. Recently we constructed a vector containing the modified micronuclear version of macronuclear destined DNA sequences that was correctly fragmented and telomeres were added de novo after injection into the developing macronucleus. It therefore must contain all the cis- acting sequences required for these processes. We made a series of vectors deleting different sequences from the original vector. It could be shown that at least in the case studied here no micronuclear-specific sequences are required for specific fragmentation of the genome and telomere addition. However, a short subtelomeric sequence at the 3[prime]-end is essential for these processes, whereas no specific cut seems to occur at the 5[prime]-end. In addition, we can show that the processing activity is restricted to a short period of time during macronuclear differentiation and that a preceding transcription is required for correct processing of macronuclear-destined DNA sequences. Possible mechanisms of these processes will be discussed.

Separation of micronuclear DNA of Stylonychia lemnae by pulsed-field electrophoresis and identification of a DNA molecule with a high copy number.

DNA from the hypotrichous ciliatae Stylonychia lemnae was separated by PFGE. In addition to the separation of the macronuclear DNA molecules with a size up to approximately 40 kb, we were able to separate the micronuclear DNA with a size between approximately 90 kb and 2 Mb. One very prominent 90-kb DNA band appeared on the pulsed-field gels. We propose that this 90-kb DNA fragment represents a linear plasmid residing in the micronucleus in a very high copy number. About 10% of the micronuclear DNA consists of the 90-kb DNA molecule. It appears in the micronucleus as well as in the macronuclear anlagen during macronuclear development but not in the mature macronucleus. Thus, the multicopy DNA is eliminated during fragmentation of the macronuclear anlagen DNA in the course of macronuclear development. Therefore, this 90-kb DNA molecule might serve as an excellent tool to study the recognition and elimination of DNA during nuclear differentiation of hypotrichous ciliates.

A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells.

We have developed an episomal replicating expression vector in which the SV40 gene coding for the large T-antigen was replaced by chromosomal scaffold/matrix attached regions. Southern analysis as well as vector rescue experiments in CHO cells and in Escherichia coli demonstrate that the vector replicates episomally in CHO cells. It occurs in a very low copy number in the cells and is stably maintained over more than 100 generations without selection pressure.

Telomeric interactions result in the formation of intramolecular circles behaving as topologically constrained.

The gene-sized macronuclear DNA molecules of hypotrichous ciliates carry telomeric sequences of homogenous length. Incubation of these molecules at low concentrations in the presence of monovalent cations (K+, Na+, Cs+) leads to the formation of intramolecular circles. They can be visualized on one or two-dimensional agarose gels only when KCl is present in the gel. From their electrophoretic behavior on agarose gels as well as on density gradients it can be concluded that they are topologically constrained. Digestion of macronuclear DNA with S1 as well as various exonucleases indicate that both the 3' overhang and the 5' C-rich strand of the telomeric repeat is involved in these interactions. Several models of these interactions are discussed.

The formation of new nucleoli during macronuclear development of the hypotrichous ciliate Stylonychia lemnae visualized by in situ hybridization.

After sexual reproduction in ciliates, the old macronucleus degenerates and a new macronucleus is formed from a micronuclear derivative. Macronuclear development in hypotrichous ciliates includes the formation of polytene chromosomes, degradation of these chromosomes and elimination of DNA, specific fragmentation of macronuclear DNA in short gene-sized DNA molecules and specific amplification of these molecules. After fusion of the two haplid micronuclei, the zygote nucleus divides mitotically; one of the daughter nuclei develops into a new micronucleus and the other into a new macronucleus. A first DNA synthesis phase in the developing macronucleus (macronuclear anlage) leads to the formation of polytene chromosomes. These polytene chromosomes become degraded and up to over 90% of the DNA is eliminated, leading to a DNA-poor stage. During a second DNA synthesis phase, replication bands become visible, and finally the new vegetative macronucleus is formed (for a review see Ammermann et al. 1974, Prescott 1994, Lippe & Eder 1996). As in most other ciliates analysed so far (for a review see Prescott 1994), rDNA occurs as a single-copy gene in the micronucleus but is highly amplified in the vegetative macronucleus (Steinbrück 1990). This amplification of rDNA is accompanied by the formation of many new nucleoli in the course of macronuclear development. In the light microscope, nucleoli become first visible at the beginning of the second DNA synthesis phases and multiply in subsequent rounds of replication (Ammermann et al. 1974). In this report, we describe the amplification of rDNA and the formation of new nucleoli during macronuclear differentiation by in situ hybridization of rDNA to different stages of the developing macronucleus.

Characterization of a family of repetitive sequences that is eliminated late during macronuclear development of the hypotrichous ciliate Stylonychia lemnae.

A repetitive element from the hypotrichous ciliate Stylonychia lemnae was characterized by restriction and hybridization analysis. This repetitive element is present in about 5,000-7,000 copies per haploid genome in the micronucleus and the macronuclear anlagen. Its DNA sequence is very conserved, but the length of the repetitive sequence blocs is variable. In some cases, it is associated with telomeric sequences and macronucleus-homologous sequences. Restriction analysis of genomic micronuclear and macronuclear anlagen DNA and in situ hybridization showed that the repetitive sequences are amplified during the formation of polytene chromosomes. They are localized in many bands of the polytene chromosomes and are eliminated during the degradation of the polytene chromosomes. Possible functions of the repetitive sequences during macronuclear differentiation are discussed.

Mammalian artificial chromosomes--vectors for somatic gene therapy.

Mammalian artificial chromosomes might prove to be useful vectors for somatic gene therapy. The functional elements of such an artificial chromosome are telomeres, a centromere and a replication origin. Recent progress in the characterization of these functional elements of the eukaryotic chromosome will be described. Attempts to construct artificial chromosomes for mammalian cells and their use for somatic gene therapy are discussed.

Sequential excision of internal eliminated DNA sequences in the differentiating macronucleus of the hypotrichous ciliate Stylonychia lemnae.

Elimination of internal eliminated sequences (IES) during macronuclear development of the hypotrichous ciliate Stylonychia lemnae was analyzed in one cluster of macronuclear precursor DNA sequences. The results indicate that IES elimination is a highly ordered process, it starts very early during macronuclear development and has only finished immediately before DNA fragmentation takes place. It occurs in distinct steps and the IES are eliminated in a specific order, where a defined IES is only removed after complete elimination of other IES. Transfection experiments clearly demonstrate that the structure of the IES itself is not sufficient for its correct excision but other cis-acting sequences or additional structural requirements are needed for IES elimination.

Macronucleus structure and macronucleus development in hypotrichous ciliates.

In the course of macronuclear development of the hypotrichous ciliates all genetic information not required for normal growth of the cell is removed from the new macronucleus. This differentiation process involves DNA-splicing, excision of transposons, DNA-fragmentation, selective gene amplification and telomere addition. Since many of the processes observed during macronuclear development, such as DNA-transposition, DNA-rearrangement or selective DNA-amplification, may occur in differentiating cells of higher organisms, this biological system provides an unusual opportunity to study the ways in which DNA-sequences can be manipulated in a differentiating cell.

The processing of macronuclear-destined DNA sequences microinjected into the macronuclear anlagen of the hypotrichous ciliate Stylonchia lemnae.

We describe the construction of a vector carrying the micronuclear versions of two macronuclear DNA molecules, one of which was modified by the insertion of a polylinker sequence. This vector was injected into the polytene chromosomes of the developing macronucleus of Stylonychia and its processing during further macronuclear development and its fate in the mature macronucleus were analyzed. In up to 30% of injected cells the modified macronuclear DNA sequence could be detected. While the internal eliminated sequences (IES) present in the macronuclear precursor DNA sequence are still retained in the mature macronucleus, the modified macronuclear DNA sequence is correctly cut out from the vector, telomeres are added de novo and it is stably retained in the macronucleus during vegetative growth of the cells. This vector system represents an experimental system that allows the identification of DNA sequences involved in the processing of macronuclear DNA sequences during macronuclear development.

A gene from the hypotrichous ciliate Stylonychia lemnae coding for a protein with homology to cyclin B.

A nucleotide (nt) sequence of a DNA molecule from Stylonychia lemnae with an open reading frame encoding a protein showing homology to cyclin B has been determined. The DNA molecule is 3791-nt long and the deduced 444-amino-acid (aa) sequence shares about 30% identity with the sequences of two yeast cyclin-B homologs over a length of about 210 aa.