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The chicken lysozyme gene domain is flanked by nuclear matrix attachment regions (MARs) on each side. We have previously shown that bilaterally flanking 5'MARs in stably transfected artificial genetic units enhance expression of a reporter transgene and dampen position effects of the chromatin structure at the site of integration. The 5' MAR was now dissected into smaller fragments that were monitored for effects on transgene expression in mouse 3T3 cells by a similar assay. Fragments, which contain 1.32 and 1.45 kb and represent the upstream and the downstream half, respectively, of the 5' MAR, retained the ability to stimulate transgene expression as well as the ability to reduce the variation in the level of expression. However, a 452 bp subfragment (H1-HaeII), which still exhibits specific binding to nuclear matrices and contains two high-affinity binding sites for the abundant nuclear matrix protein ARBP, lost both of those abilities. A dimerized 177 bp sequence from fragment H1-HaeII, which also binds selectively to nuclear matrices and includes a duplicated ARBP binding site, was also unable to stimulate reporter gene expression. Furthermore, a 0.65 kb subfragment containing an intrinsically bent sequence did not affect an elevated reporter gene expression and its dampening. Our results show that the ability of MAR fragments to bind to nuclear matrices is not sufficient to enhance and insulate transgene expression in stably transfected cells.

Matrix attachment regions (MARs) partition the genome into functional and structural loop-domains. Here, we determined the relative matrix affinity of cloned fragments of the chicken lysozyme 5' MAR. We show that this region contains a non-curved high-affinity binding site, which is 3' followed by a strongly curved DNA sequence that exhibits weak matrix binding. DNA curvature is not a physical property required for strong matrix binding. Possible biological functions of this sequence arrangement, particularly of the strongly curved DNA, are discussed.

The chicken lysozyme gene domain is distinguished by a broad knowledge of how its expression is regulated. Here, we examined the in vivo replication of the lysozyme gene locus using polymerase chain reaction amplification and competitive polymerase chain reaction of size-fractionated, nascent DNA strands. We found that DNA replication initiates at multiple sites within a broad initiation zone spanning at least 20 kilobases, which includes most of the lysozyme gene domain. The 5 border of this zone is probably located downstream of the lysozyme 5 nuclear matrix attachment region. Preferred initiation occurs in a 3-located subzone. The initiation zone at the lysozyme gene locus is also active in nonexpressing liver DU249 cells. Furthermore, examining the timing of DNA replication at the lysozyme gene locus revealed that the gene locus replicates early during S phase in both HD11 and DU249 cells, irrespective of its transcriptional activity.

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The glioma-amplified sequence (GAS) 41 protein has been proposed to be a transcription factor. To investigate its functional role in vivo, we attempted to knock out the GAS41 gene by targeted disruption in the chicken pre-lymphoid cell line DT40. Heterozygous GAS41؉/؊ cell lines generated by the first round of homologous recombination express approximately half the normal level of GAS41 mRNA. However, a homozygous GAS41؊/؊ cell line with both GAS41 alleles disrupted was not obtained following the second round of transfection, indicating that the GAS41 gene is essential for cell viability. Indeed, homozygous GAS41؊/؊ cell lines with two disrupted GAS41 alleles can be generated following substitution of the endogenous gene by stable integration of GAS41 cDNA controlled by a tetracyclineregulated CMV promoter. Inactivation of this promoter by tetracycline withdrawal results in rapid depletion of GAS41, causing a significant decrease in RNA synthesis and subsequently cell death. Thus, our results indicate that GAS41 is required for RNA transcription.The glioma-amplified sequence (GAS) 1 41 gene was identified for the first time as an amplified sequence in the chromosome region 12q13-15, a region known to be involved in gene amplification in human gliomas. The gene was found to be amplified in 23% of glioblastomas and in 80% of grade I astrocytomas, suggesting that gene amplification occurs also in early stages of tumor development (1, 2). GAS41 is a highly conserved protein found in human, Arabidopsis, Drosophila, Caenorhabditis elegans, yeast (2, 3), and chicken (this study).It has been recently shown by immunoprecipitation experiments that GAS41 interacts specifically with the nuclear mitotic apparatus (NuMA) protein in vivo. The C-terminal 50 amino acids are necessary for this binding (3). NuMA is a constituent of the nuclear matrix prepared by DNase I and high salt treatment of interphase nuclei (4), and it binds specifically to matrix attachment regions in vitro (5). In mitotic cells, NuMA is associated with the spindle poles (4). Immunofluorescence microscopic studies revealed a punctate distribution of GAS41 restricted to the nucleus in interphase cells, suggesting that the protein is associated with the nuclear matrix. In mitotic cells, however, GAS41 is found throughout the cell, in apparent contrast to NuMA located at the spindle poles (3).Sequence comparison revealed high homology of GAS41 to AF-9 and ENL in the proline-rich N-terminal region (2, 3). These nuclear proteins containing a transcriptional activation domain at the C-terminal 90 amino acids are believed to represent a new class of transcription factors (6, 7). GAS41 has been predicted to exhibit ␣-helical structures containing a significantly above average percentage (27%) of acidic amino acids in the 60-amino acid C-terminal region (2). Negatively charged ␣-helical structures are present in transcriptional activation domain of several eukaryotic transcription factors (8). In contrast to AF-9 and ENL, GAS41 lacks a typical DNA-binding domain (2...

In this study, we identified a polymorphism in the 5

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