Establishment of SSEA-1- and Oct-4-expressing rat embryonic stem-like cell lines and effects of cytokines of the IL-6 family on clonal growth.

Here, we demonstrate long-term cultivation of alkaline phosphatase-positive rat embryonic stem-like (RES) cell lines. RES cells were characterized by their typical growth in highly compacted cell clusters, which were found to be sensitive against enzymatic dissociation. RES cells expressed stage-specific embryonic antigen-1 (SSEA-1) and transcription factor Oct-4, but Oct-4 mRNA was detected at lower levels compared to mouse ES cells. Once established to tissue culture, RES cells were able to grow in the absence of feeder cells under clonal conditions. Cytokines of the interleukin-6 family known to maintain the undifferentiated state of mouse ES cells were comparatively analyzed for their capacity to maintain the undifferentiated growth of two cell lines, RES-1 and RES-15, in a clonal assay. Rat ciliary neurotrophic factor (rCNTF), human oncostatin M (hOSM), and interleukin-6 and soluble interleukin-6 receptor (IL-6/sIL-6R) were found to support clonal growth of RES cells, but the cytokines did not reach the efficiency of the colony forming ability of leukemia inhibitory factor (LIF). When RES-1 and RES-15 cells were cultivated without feeder cells, SSEA-1 expression was maintained after clonal growth in the presence of LIF and LIF + rCNTF, respectively. Oct-4 mRNA was significantly detected in RES-15 cells when cultivated in the absence of feeder cells in media substituted by LIF and/or IL-6/sIL-6R, as well as without cytokines. In summary, rat embryonic stem-like cell lines could be established from rat blastocysts and were able to proliferate as undifferentiated alkaline phosphatase-positive cells. Embryonal stem cell properties, such as SSEA-1 and Oct-4 expression, were maintained by members of the IL-6 family of cytokines, but most significantly by LIF.

Terminal heterochromatin and alternative telomeric sequences in Allium cepa.

The chromosome termini of the onion (Allium cepa) apparently lack Arabidopsis-type telomeric repeats. The terminal Giemsa bands of A. cepa chromosomes contain a 375-bp satellite and (short arm of chromosome 8)/ or (short arm of chromosome 6) rDNA repeats. By means of fluorescence in situ hybridization (FISH) on metaphase chromosomes and on DNA fibres with probes specific for Ty1-copia retroelements and a En/Spm-transposable element-like sequence, respectively, it was demonstrated that the former are rarely and the latter frequently associated with satellite repeats within the terminal heterochromatin. Polymerase chain reaction of polyC-tailed nuclear DNA with anchor primers and single satellite-specific primers yielded amplification products that, after cloning and sequencing, revealed satellite sequences. This supports the idea that satellite repeats represent one class of terminal sequences in A. cepa.

Formation of postsynaptic-like membranes during differentiation of embryonic stem cells in vitro.

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, alpha 1 subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the gamma- and epsilon-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR epsilon-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiation in vitro neuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by "loss of function" analysis in vitro.

Formation and repair of O6-methylguanine in recombination hot spots of plant chromosomes.

Mutagen-induced chromatid aberrations are not randomly distributed along the metaphase chromosomes. In the field bean (Vicia faba), defined late-replicating and transcriptionally inactive heterochromatic regions are preferentially involved. After exposure to the alkylating agent N-methyl-N-nitrosourea (MNU) (10(-3) M, 1 hour), 70% of all aberrations are clustered within 6 segments containing tandemly repeated FokI elements of 59 bp, which comprise approximately 10% of the genome. Using immuno-slot-blot analyses, we have studied the frequency of O6-methylguanine (O6-MeG), a mutagenic lesion important for aberration induction, in total genomic DNA as well as in FokI sequences of the field bean after exposure to MNU. In either case, similar numbers of adducts per nucleotide were found immediately after treatment as well as after 18 hours of recovery, when most adducts were removed and significant amounts of chromatid aberrations were detectable. Peculiarities of long FokI element arrays (e.g., formation of specific tertiary structures), resulting in error-prone recombination repair, rather than preferential formation or delayed repair of O6-MeG are apparently responsible for aberration clustering in these hot spot regions.

The Ty1-copia group retrotransposons of Allium cepa are distributed throughout the chromosomes but are enriched in the terminal heterochromatin.

The genomic organization and diversity of the Ty1-copia group retrotransposons has been investigated in a monocotyledonous plant, Allium cepa. We used the polymerase chain reaction (PCR) to generate sequences corresponding to a conserved domain of the reverse transcriptase gene of Ty1-copia retrotransposons in this plant. Sequence analysis of 27 of these PCR products shows that they are a highly heterogeneous population, a feature which is common in plants but not in yeast and Drosophila. Slot-blot analysis shows there are 100,000-200,000 copies of Ty1-copia group retrotransposons within the A. cepa genome (2C = 31.7 pg), indicating that they are a significant component of the genome of this plant. In situ hybridization to metaphase chromosomes reveals that Ty1-copia retrotransposons are distributed throughout the euchromatin of all chromosomes of A. cepa but are enriched in the terminal heterochromatic regions, which contain tandem arrays of satellite sequences. This is the first clear evidence for the presence of Ty1-copia retrotransposons in the terminal heterochromatin of plants and contrasts with the distribution of these elements in other plant species.

How do Alliaceae stabilize their chromosome ends in the absence of TTTAGGG sequences?

The Arabidopsis-type telomeric repeats (5'-TTTAGGG-3) are highly conserved. In most families of different plant phyla they represent the basic sequence of telomeres that stabilize and protect the chromosome termini. The results presented here show that Alliaceae and some related liliaceous species have no tandemly repeated TTTAGGG sequences. Instead, their chromosomes reveal highly repetitive satellite and/or rDNA sequences at the very ends. These apparently substitute the original plant telomeric sequences in Alliaceae. Both sequence types are very active in homologous recombination and may contribute to the stabilization of chromosome termini via compensation of replication-mediated shortening.

Molecular-cytogenetic characterization of a higher plant centromere/kinetochore complex.

The centromeric region of a telocentric field bean chromosome that resulted from centric fission of the metacentric satellite chromosome was microdissected. The DNA of this region was amplified and biotinylated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)/linker-adapter PCR. After fluorescence in situ hybridization (FISH) the entire chromosome complement of Vicia faba was labelled by these probes except for the nucleolus organizing region (NOR) and the interstitial heterochromatin, the chromosomes of V. sativa and V. narbonensis were only slightly labelled by the same probes. Dense uniform labelling was also observed when a probe amplified from a clearly delimited microdissected centromeric region of a mutant of Tradescantia paludosa was hybridized to T. paludosa chromosomes. Even after six cycles of subtractive hybridization between DNA fragments amplified from centromeric and acentric regions no sequences specifically located at the field bean centromeres were found among the remaining DNA. A mouse antiserum was produced which detected nuclear proteins of 33 kDa and 68 kDa; these were predominantly located at V. faba kinetochores during mitotic metaphase. DNA amplified from the chromatin fraction adsorbed by this serum out of the sonicated total mitotic chromatin also did not cause specific labelling of primary constrictions. From these results we conclude: (1) either centromere-specific DNA sequences are not very conserved among higher plants and are - at least in species with large genomes - intermingled with complex dispersed repetitive sequences that prevent the purification of the former, or (2) (some of) the dispersed repeats themselves specify the primary constrictions by stereophysical parameters rather than by their base sequence.

Primer-induced labeling of pea and field bean chromosomes in situ and in suspension.

A protocol for primed in situ DNA labeling (PRINS) was optimized for pea (Pisum sativum L.) and field bean (Vicia faba L.) chromosomes attached to coverslips. Cloned DNA or synthetic oligonucleotides were used as probes for repetitive DNA sequences (rDNA, Fok-element) and different reaction conditions were tested to achieve the highest specific signal-to-background ratio. A procedure based on direct labeling by fluorescein-dUTP was compared with an indirect one using digoxigenin detected by fluorescently labeled antibody. Under optimal conditions, strong and specific signals were obtained exclusively on chromosome regions known to contain respective DNA sequences. Compared to the direct labeling, significantly stronger signals were obtained when the indirect procedure was used. Both types of labeling were successfully applied to chromosomes in suspension and were shown to produce signals comparable to that obtained with chromosomes attached to coverslips. It is expected that primed in situ DNA labeling en suspension (PRINSES) will provide a basis for flow-cytometric discrimination and sorting of otherwise indistinguishable chromosomes according to their specific fluorescent labeling.

The nodule-specific VfENOD-GRP3 gene encoding a glycine-rich early nodulin is located on chromosome I of Vicia faba L. and is predominantly expressed in the interzone II-III of root nodules.

A nodule-specific cDNA was isolated from a Vicia faba L. nodule cDNA library. Since time course experiments revealed an early expression of this transcript in the nodule, this cDNA coded for an early nodulin and was designated VfENOD-GRP3. Based on tissue print hybridizations, we found a predominant expression of VfENOD-GRP3 transcripts in the interzone II-III region of broad bean root nodules. The encoded early nodulin ENOD-GRP3 was characterized by an N-terminal signal peptide and a C-terminal domain displaying a glycine content of 31%. Sequence analysis of a genomic VfENOD-GRP3 clone revealed that the signal peptide and the glycine-rich domain were specified by two separate exons. Primer extension experiments identified two adjacent transcription start sites for VfENOD-GRP3 transcripts. The common nodulin sequences 'AAAGAT' and 'CTCTT' were present five and three times on both DNA strands of the putative VfENOD-GRP3 promoter, respectively. Additionally, three sequence motifs resembling organ-specific elements of the soybean lbc3 gene promoter and a sequence similar to the binding site 1 for the nodule trans-acting factor Nat2 were identified. From Southern blot data and from sequence analysis of genomic PCR fragments, the presence of a VfENOD-GRP3 gene family was inferred. By PCR experiments using sequence-specific primers and DNA of microisolated chromosomes as a template, this family was located on the long arm of chromosome I.

Immunostaining and interphase arrangement of field bean kinetochores.

More than 100 sera from patients with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) were tested in order to detect antigenic nuclear components of the field bean Vicia faba (2n = 12). Kinetochores of mitotic chromosomes and prekinetochores of interphase cells from root-tip meristems were specifically labelled via an indirect immunofluorescence procedure by antibodies of one of these sera. In 44% of interphase nuclei in which centromeres could be identified, only half (6) of the number of expected prekinetochores (12) was detected, circumstantially indicating at least transient association of homologous centromeres. Some nuclei showed clustering of centromeres at one pole (Rabl configuration). In metaphase chromosomes, each sister kinetochore contained a fluorescent spot. Western blotting of field bean nuclear proteins revealed four antigenic proteins of 28, 30, 64 and 68 kDa.

Sequence organization and the mechanism of interstitial deletion clustering in a plant genome (Vicia faba).

Intercalary deletions and duplication-deletions are types of chromatid aberrations typical of the aberration spectrum observed in the first mitosis of plant cells after mutagen treatment. They are the results of error-prone recombination repair and arise when reunion is not prevented by inhibition of DNA synthesis. Both types of aberrations are nearly exclusively located in chromosome regions composed of tandemly arranged highly repetitive DNA sequences (e.g. FokI elements). The data discussed in the present paper make it possible to arrive at a simple mechanistic interpretation of the origin of these aberration types.

Polymerase chain reaction mediated localization of RFLP clones to microisolated translocation chromosomes of barley.

A new strategy has been devised and used for the physical localization of genetically mapped restriction fragment length polymorphism (RFLP) clones to barley chromosomes. Morphologically distinct translocation chromosomes from synchronized root-tip meristems were microisolated and their DNA was used as a template for polymerase chain reaction with sequence-specific primers. Four RFLP clones were assigned to cytologically defined segments of chromosome 5. This related approximately one-third of the map length of linkage group 5 to approximately one-fifth of the mitotic metaphase length of chromosome 5. The technique may substantially contribute to the connection of the RFLP-based genetic linkage maps with cytological markers of the barley chromosomes.

Utility of DNA amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) from the total genome and defined chromosomal regions of field bean.

Degenerate oligonucleotide primed (DOP)-PCR has emerged as a simple and rapid method for representative amplification of highly complex genomic DNA from humans, mice and Drosophila. The present paper describes the adaptation of this method for use on a plant species, Vicia faba, with a large genome (2C = 30 pg). Specific low-copy-number sequences as well as highly repeated sequences were detectable among DOP-PCR products obtained from small samples of purified genomic DNA (100 pg), DNA from 10 prophase nuclei, 10 flow-sorted chromosomes or 15 microdissected chromosome segments (satellites) following reamplification with sequence-specific primers and/or Southern hybridization. Biotinylated chromosome-specific DOP-PCR products were used for fluorescent in situ hybridization. All chromosomes showed hybridization signals, with the exception of regions containing Fok elements which are not present in the chromosomal DNA targeted by DOP-PCR.

Differentiation of field bean heterochromatin by in situ hybridization with a repeated FokI sequence.

The chromosomes of a field bean line with a reconstructed karyotype (ACB) were hybridized in situ with biotinylated probes of a repetitive Fok I sequence, of DOP-PCR (degenerate oligonucleotide primed polymerase chain reaction) amplified DNA from a chromosome that does not contain this sequence, and with probes containing dispersed repetitive sequences. The results were compared with Giemsa banding, DNA late replication and Fok I in situ digestion patterns. This allowed further differentiation between the chromatin types of this species. Centromeric and NOR-associated heterochromatin as well as euchromatin were shown to be free of Fok I sequence repeats. Among the interstitial late replicating Giemsa bands, subdivided into 'marker' and 'additional' bands, most of the marker bands located at mid-arm positions were composed mainly or exclusively of tandemly arranged Fok I repeats. Some of the marker bands and nearly all of the additional bands located in the vicinity of centromeres were free of FokI sequence repeats, of Fok I recognition sites, and possibly also of dispersed repetitive sequences. They are probably composed of specific, not yet defined, repetitive sequences.

Localization of seed protein genes on flow-sorted field bean chromosomes.

Chromosomes from reconstructed field bean (Vicia faba L.) karyotypes were flow-sorted and the DNA was used for the physical localization of seed storage and nonstorage (USP) protein genes using PCR with sequence specific primers. The data were confirmed and refined by using DNA of microisolated chromosomes of other karyotypes as the target for PCR. The specificity of the PCR products was proved by restrictase digestion into fragments of predicted length or by reamplification using 'nested' primers. The genes are located within defined regions of chromosome I (USP = unknown seed protein genes), II (vicilin genes, legumin B3 genes), III (legumin B4 genes), IV (pseudogenes psi 1) and V (legumin A genes and pseudogenes psi 1). Except for the pseudogene derived from the sequence of legumin B4 gene, all members of each gene family are located in one chromosome region exclusively. This approach proved to be useful for localizing genes that cannot be mapped genetically (due to the lack of allelic variants) and might be applied to integrate physical and genetic maps.

Localization of vicilin genes via polymerase chain reaction on microisolated field bean chromosomes.

A new technique is reported for the physical mapping of low copy DNA sequences on plant chromosomes. Individual chromosomes were microisolated and their DNA used as the target for the polymerase chain reaction in order to identify the chromosome carrying a specific gene sequence. The use of defined translocation chromosomes further refined the resolution of the method to a subchromosomal level. To demonstrate the applicability of the procedure genes have been localized coding for vicilin seed storage proteins on the field bean Vicia faba L. in a region which includes the centromere and the proximal parts of the short and the long arms of chromosome II.

Genome organization of mitochondrial DNA from the non-saccharomycete yeast Arxula adeninivorans LS3.

Mitochondrial (mt) DNA of the asexual ascomycetous yeast Arxula adeninivorans LS3 was isolated and characterized. The mtDNA has a GC content of 30.3 mol%. It is circular and its size, as estimated by restriction analysis performed with nine endonucleases, was 35.5 kbp. Using mt gene-probes from Saccharomyces cerevisiae six structural genes (cob, cox1, cox2, oli1, oli2, and 21S rRNA) were located on the mitochondrial genome of A. adeninivorans. The comparison between the mt genomes of A. adeninivorans and other yeasts showed differences in genome organization.