Erika Csonka

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.

Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species.

A 60-Mb murine chromosome consisting of murine pericentric satellite DNA and two bands of integrated marker and reporter genes has been generated de novo in a rodent/human hybrid cell line (mM2C1). This prototype mammalian artificial chromosome platform carries a normal centromere, and the expression of its β-galactosidase reporter gene has remained stable under selection for over 25 months. The novel chromosome was transferred by a modified microcell fusion method to mouse [L-M(TK−)], bovine (P46) and human (EJ30) cell lines. In all cases, the chromosome remained structurally and functionally intact under selection for periods exceeding 3 months from the time of transfer into the new host. In addition, the chromosome was retained in three first- generation tumours when L-M(TK−) cells containing the chromosome were xenografted in severe combined immunodeficiency mice. These data support that a murine satellite DNA-based artificial chromosome can be used as a functional mammalian artificial chromosome and can be maintained in vivo and in cells of heterologous species in vitro.

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.

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.

Comparative Analysis of Nuclear Transfer Embryo-Derived Mouse Embryonic Stem Cells. Part I: Cellular Characterization

Embryonic stem cells derived from nuclear transfer embryos (ntESCs) are particularly valuable for regenerative medicine, as they are a patient-specific and histocompatible cell source for the treatment of varying diseases. However, currently, little is known about their cellular and molecular profile. In the present study, in a mouse model different donor cell-derived ntESCs from various genetic backgrounds were compared with reference ESCs and analyzed comprehensively at the cellular level. A number of pluripotency marker genes were compared by flow cytometry and immunocytochemistry analysis. Significant differences at the protein level were observed for POU5F1, SOX2, FGF4, NANOG, and SSEA-1. However, such differences had no effect on in vitro cell differentiation and cell fate: derivatives of the three germ layers were detected in all ntESC lines. The neural and cardiac in vitro differentiation revealed minor differences between the cell lines, both at the mRNA and protein level. Karyotype analyses and cell growth studies did not reveal any significant variations. Despite some differences observed, the present study revealed that ntESC lines had similar differentiation competences compared to other ESCs. The results indicate that the observed differences may be related to the genotype rather than to the nuclear transfer technology.

Cloning, characterization and localization of Chinese hamster HP1 isoforms.

The Chinese hamster is one of the few mammalian species that are characterized by relatively poor heterochromatin content. It was intriguing to test whether or not the lack of large blocks of heterochromatin in the hamster chromosomes could be correlated with the absence or species-specific differences of the HP1 proteins, the main structural components of heterochromatin. To address this, we attempted to clone HP1 from the Chinese hamster. It is shown here that all three isoforms of HP1 known in mammals are present in hamster, and the amino acid sequences deduced from the cDNAs of the isoforms are 97–100% identical to those of the known mammalian homologues. All three isoforms are localized mainly in heterochromatic regions in the native chromosomes and nuclei. The hamster HP1α gene was cloned, sequenced and mapped to the short arm of hamster chromosome 2.These data indicate that the Chinese hamster has all the HP1 components necessary for the establishment of heterochromatin. The limited amount of heterochromatin in hamster cells may probably be attributed to the unusual satellite DNA content of the hamster genome.

Chromosomal Aneuploidy in Azoospermic Men

Abstract The aim of this study was to estimate the prevalence of chromosome abnormalities in patients with azoospermia, in our material. Preoperative evaluation included routine andrological investigations with 2 semen analysis, ultrasound, hormonal and genetic examinations. In the last three years, 73 biopsies were performed for testicular sperm extraction in 71 patients. Non-obstructive azoospermia was diagnosed in 53 patients (79%). Karyotyping was performed on lymphocyte preparations of 36 men with diagnosis of infertility. In order to obtain exact diagnosis, cytogenetic methods (QFQ-, GTG-, CBG-band and FISH analysis) were complemented with molecular genetic techniques. Patients were included in the assisted reproduction programme on the base of their genetic results. The most characteristic cases were sex numerical deviations, such as XXY (3 cases), XYY (2 cases) and mosaic X0 syndromes (1 case).

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.