Katalin Fodor

De novo chromosome formations by large-scale amplification of the centromeric region of mouse chromosomes

Chromosomes formedde novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of ∼30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formedde novo by an H-type amplification revealed that it is composed of a tandem array of 10–12 large (∼30 Mb) amplicons each marked with integrated ‘foreign’ DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of ∼7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are ∼7.5-Mb blocks of satellite DNA flanked by nonsatellite sequences. We suggest that the formationde novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

Evidence for a megareplicon covering megabases of centromeric chromosome segments.

We have analysed the replication of the heterochromatic megachromosome that was formedde novo by a large-scale amplification process initiated in the centromeric region of mouse chromosome 7. The megachromosome is organized into amplicons ∼30 Mb in size, and each amplicon consists of two large inverted repeats delimited by a primary replication initiation site. Our results suggest that these segments represent a higher order replication unit (megareplicon) of the centromeric region of mouse chromosomes. Analysis of the replication of the megareplicons indicates that the pericentric heterochromatin and the centromere of mouse chromosomes begin to replicate early, and that their replication continues through approximately three-quarters of the S-phase. We suggest that a replication-directed mechanism may account for the initiation of large-scale amplification in the centromeric regions of mouse chromosomes, and may also explain the formation of new, stable chromosome segments and chromosomes.

A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe’s disease in the Twitcher mouse

Abstract.Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe’s disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies.

Polyethylene-glycol induced fusion of bacterial protoplasts

SummaryDirect selection for recombinants by supplemented minimal media from polyethylene-glycol (PEG)-induced fusion of protoplasts of polyauxotrophic strains of B. megaterium revealed striking physiological influences on the yield of recombinants. Cytoplasmic state of the protoplasts to be fused, rather than genetic events, determined the number of colonies obtained on the selection media. It is suggested that the physiological effects primarily influenced the ability of the fused protoplasts to revert to bacillary form.

Novel method to load multiple genes onto a mammalian artificial chromosome.

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe’s disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.

The chAB4 and NF1-related long-range multisequence DNA families are contiguous in the centromeric heterochromatin of several human chromosomes

We have investigated the large-scale organization of the human chAB4-related long-range multisequence family, a low copy-number repetitive DNA located in the pericentromeric heterochromatin of several human chromosomes. Analysis of genomic clones revealed large-scale ( approximately 100 kb or more) sequence conservation in the region flanking the prototype chAB4 element. We demonstrated that this low copy-number family is connected to another long-range repeat, the NF1-related (PsiNF1) multisequence. The two DNA types are joined by an approximately 2 kb-long tandem repeat of a 48-bp satellite. Although the chAB4- and NF1-like sequences were known to have essentially the same chromosomal localization, their close association is reported here for the first time. It indicates that they are not two independent long-range DNA families, but are parts of a single element spanning approximately 200 kb or more. This view is consistent both with their similar chromosomal localizations and the high levels of sequence conservation among copies found on different chromosomes. We suggest that the master copy of the linked chAB4-PsiNF1 DNA segment appeared first on the ancestor of human chromosome 17.

Generation of induced pluripotent stem cells by using a mammalian artificial chromosome expression system

Direct reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPS) was achieved recently by overexpression of four transcription factors encoded by retroviral vectors. Most of the virus vectors, however, may cause insertional mutagenesis in the host genome and may also induce tumor formation. Therefore, it is very important to discover novel and safer, non-viral reprogramming methods. Here we describe the reprogramming of somatic cells into iPS cells by a novel protein-based technique. Engineered Oct4, Sox2 and Klf4 transcription factors carrying an N-terminal Flag-tag and a C-terminal polyarginine tail were synthesized by a recently described mammalian artificial chromosome expression system (ACEs). This system is suitable for the high-level production of recombinant proteins in mammalian tissue culture cells. Recombinant proteins produced in this system contain all the post-translational modifications essential for the stability and the authentic function of the proteins. The engineered Oct4, Sox2 and Klf4 proteins efficiently induced the reprogramming of mouse embryonic fibroblasts by means of protein transduction. This novel method allows for the generation of iPS cells, which may be suitable for therapeutic applications in the future.

Inherited Tetrasomy of the 15p-15q11.2 Region Associated with Normal Phenotypes

We analyzed the 15p-15q11.2 region of human chromosome 15 that was tetrasomic in a three-generation family with a chromosome 15-derived small supernumerary marker chromosome (der15, sSMC). This stable, inherited sSMC was present in four family members. The breakpoint that led to the formation of der15 was established by fluorescence in situ hybridization (FISH) using 18 bacterial artificial chromosomes (BACs) mapping to the pericentric region of 15q. The breakpoint was mapped to an 11 kb sequence in the 15q11.2b sub-band, and it resides in a NF1 pseudogene, within the BAC 810K23 clones. FISH mapping verified the localization of the overlapping 18 BAC clones in the 15q11.2 region, and confirmed that large duplications were spanning to 15q13.1 including the PWS/AS critical region. Carriers of the der15 pseudodicentric isochromosome are healthy, and these cases support that inherited tetrasomy of the 19.4 Mb chromosomal segment of the 15p-q11.2b region has no phenotypic consequences.