A polymorphic dinucleotide repeat in the rat nucleolin gene forms Z-DNA and inhibits promoter activity.

Many sequences in eukaryotic genomes have the potential to adopt a left-handed Z-DNA conformation. We used a previously described assay based on the binding of a mAb to Z-DNA to inquire whether Z-DNA is formed in the rat nucleolin (Ncl) gene in metabolically active, permeabilized nuclei. Using real-time PCR to measure Z-DNA formation, the potential Z-DNA sequence element Z1 [(CA)(10)(CG)(8)] in the promoter region was found to be enriched 571- to 4,040-fold in different cell lines, whereas Z2 [AC(GC)(5)CCGT(CG)(2)] in the first intron was enriched 12- to 34-fold. Ncl promoter activity was 1.5- to 16-fold stronger than that of the simian virus 40 promoter and enhancer. This activity was further increased 36-54% when Z1 was deleted. The inhibitory effect of Z1 on Ncl promoter activity was independent of location and orientation. The Ncl Z1 element is identical to the genetic marker D9Arb5. Five allelic variants of Z1 were identified by sequence analysis of genomic DNA from various rats. The two most common alleles differed significantly (up to 27%) in their capacity to inhibit Ncl promoter activity. This finding suggests that differences in Z-DNA formation by polymorphic dinucleotide repeats may be one of the factors contributing to genetic variation.

DNA methylation contributes to tissue- and allele-specific expression of the T-cell differentiation marker RT6.

We investigated the role of DNA methylation in gene regulation of the rat T-cell differentiation marker RT6. Analysis of the methylation status of various tissues revealed that the RT6 promoter was hypomethylated in RT6-expressing tissues, and methylated in nonexpressing ones. Remarkably, among RT6-nonexpressing tissues, the extent of methylated regions varied greatly between lymphatic tissues, where regions larger than 23 kb were methylated, and nonlymphatic tissues, where methylation was restricted to a 3- to 4-kb region surrounding the promoter. We have previously shown that cis-regulatory elements determine differential expression of the two RT6 alleles in a subpopulation of T cells. We now show that the RT6 alleles in these cells differed in their methylation status. The promoter region of the silent allele was methylated, while that of the transcribed allele was not. Upon treatment of RT6-nonexpressing thymoma cells with the methyltransferase inhibitor 5-azacytidine, RT6 expression was induced. In RT6 heterozygous hybridoma cells, expressing only one RT6 allele, induction of the silent, methylated RT6 allele was observed. Sensitivity of the RT6 promoter to DNA methylation was demonstrated by promoter-specific in vitro methylation, which inhibited RT6 promoter activity, while that of the SV40 promoter was not influenced. Our findings indicate that DNA methylation plays an important role in the control of monoallelic and tissue-specific RT6 expression.

DNA methylation and Z-DNA formation as mediators of quantitative differences in the expression of alleles.

With draft copies of several model genomes available in the near future, attention is turning towards the genetic mechanisms that determine differences between individuals. While mutations in protein coding regions affect the structure of gene products, polymorphisms outside such regions may cause quantitative differences in gene expression. Here we summarize observations indicating that such differences may be mediated by allele-specific alterations in the modification or structure of DNA. Mono-allelic expression of the rat T-cell differentiation marker RT6 in a subpopulation of cells is associated with allele-specific differences in DNA methylation in the RT6 promoter. In contrast to previously described examples of mono-allelic expression, these are determined neither stochastically nor by parental origin, but by cis-acting elements within the alleles. An attractive candidate is a rodent identifier (ID) element exclusively present in the RT6a allele. In the case of the rat nucleolin gene, a polymorphic dinucleotide repeat in the 5' region modulates promoter strength and forms left-handed Z-DNA in vivo. Models explaining putative effects of Z-DNA formation on transcription are presented. These observations suggest novel mechanisms by which repetitive DNA, an abundant source of polymorphism in the mammalian genome, may exert quantitative effects on gene expression.

Telerehabilitation services using web-based telecommunication.

A World Wide Wide-based telerehabilitation platform has been demonstrated in a laboratory environment. This platform allows a rehabilitation provider to thoroughly evaluate the progress of a patient remotely with the same care and measurement precision that would be possible if the provider and the patient were in the same room. The platform was designed to be Web-based so that the service could be offered at the same price without regard to long distance telecommunication facility charges. The Web-based implementation allows enough bandwidth for a simultaneous video teleconference and a precision data acquisition mode even when the Web connection is a low cost analog modem computer interface at both ends of the connection.

Expression of the RT6 mono(ADP-ribosyl)transferases is regulated by two promoter regions.

The structure of the RT6 mono(ADP-ribosyl)transferase gene was studied. Analysis of cDNA clones revealed eight exons and suggested two independent transcriptional start sites. The existence of the downstream initiation site was confirmed by S1-nuclease protection and localized to position +29 of exon 2. The corresponding 5' flanking regions were found to contain typical promoter structures such as TATA- and CCAAT-boxes. Comparison with sequences deposited in the TRANSFAC database of transcription factor binding sites revealed few putative regulatory elements in the region associated with exon 1 (promoter 1). In contrast, several elements contained in the regulatory regions of other T cell-specific genes, such as ets, lyf-1 and ikaros were found in in promoter 2. Analysis of RT6-transcripts showed this region to be the most active promoter in spleen cells of adult rats. Finally, transient transfection assays with reporter gene constructs showed promoter 2 to mediate T-cell specific transcription.