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1.
The entire chicken lysozyme gene locus including all known cis-regulatory sequences and the 5' and 3' matrix attachment sites defining the borders of the DNase I sensitive chromatin domain, is expressed at a high level and independent of its chromosomal position in macrophages of transgenic mice. It was concluded that the lysozyme gene locus carries a locus control function. We analysed several cis-regulatory deletion mutants to investigate their influence on tissue specificity and level of expression. Position independent expression of the gene is lost whenever one of the upstream tissue specific enhancer regions is deleted, although tissue specific expression is usually retained. Deletion of the domain border fragments has no influence on copy number dependency of expression. However, without these regions an increased incidence of ectopic expression is observed. This suggests that the domain border fragments may help to suppress transgene expression in inappropriate tissues. We conclude, that position independent expression of the lysozyme gene is not controlled by a single specific region of the locus but is the result of the concerted action of several tissue specific upstream regulatory DNA elements with the promoter.  相似文献   

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We have examined in some detail the chromatin structure of a 6.2 kilobase pair (kbp) chromosomal region containing the chicken beta-globin gene. The chromatin structure was probed with three nucleases, DNase I, micrococcal nuclease, and DNase II, and the rate of digestion of specific subfragments of the region was compared with the rate of bulk DNA digestion. We have characterized the rate of digestion of each fragment in terms of a sensitivity factor which measures the sensitivity of a fragment to a particular nuclease relative to bulk DNA. The sensitivity factors were determined by a least squares curve fitting method based on target analysis. In nuclei isolated from 14-day-old chicken embryo red blood cells, the entire 6.2-kbp region shows approximately a 10- to 20-fold increase in sensitivity to DNase I, a 3-fold increased sensitivity to micrococcal nuclease, and a 6-fold increased sensitivity to DNase II. In addition to the adult beta-globin gene, this region contains 5' and 3' flanking sequences, the 5' half of the inactive, embryonic globin gene, epsilon, and some repeated sequences. There is no obvious correlation between these genetic elements and the overall chromatin structure as measured by the nuclease sensitivity. This same region shows little or no special sensitivity in nuclei isolated from 14-day-old chicken embryo brain. Furthermore, fragments of the inactive ovalbumin gene show little or no sensitivity in either red blood cells or brain. These results support the conclusion that the entire 6.2-kbp region is largely packaged as active chromatin in 14-day-old chicken embryo red blood cells.  相似文献   

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The mitochondrial uncoupling protein gene is rapidly induced in mouse brown fat following cold exposure. To identify cis-regulatory elements, approximately 50 kb of chromatin surrounding the uncoupling protein gene was examined for its hypersensitivity to DNase I. Seven DNase I-hypersensitive sites were identified in the 5'-flanking DNA, and one site was identified in the 3'-flanking DNA. Transgenic mice with an uncoupling protein minigene were generated by microinjection of fertilized eggs with a transgene containing 3 kb of 5'-flanking DNA and 0.3 kb of 3'-flanking DNA. Expression of the transgene is restricted to brown fat and is cold inducible. Four additional transgenic lines were generated with a second transgene containing a 1.8-kb deletion in the 5'-flanking DNA, and expression of this minigene is absent in all tissues analyzed. A DNase I-hypersensitive site located in the 1.8-kb deletion contains a cyclic AMP response element that binds a brown fat tumor enriched nuclear factor. On the basis of these observations, we propose that a cis-acting regulatory sequence between -3 and -1.2 kb of the 5'-flanking region, possibly at a DNase I-hypersensitive site, is required for controlling uncoupling protein expression in vivo.  相似文献   

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M C Alevy  M J Tsai  B W O'Malley 《Biochemistry》1984,23(10):2309-2314
We have cloned a 36-kilobase segment of chicken DNA containing the gene coding for glyceraldehyde-3-phosphate dehydrogenase [GAPDH (EC 1.2.1.12)], a glycolytic enzyme which is expressed constitutively in all cell types. Using defined segments of this cloned DNA as probes, we have determined the DNase I sensitive domain of the GAPDH natural gene in the hen oviduct. When nuclei isolated from hen oviduct were treated with DNase I under conditions known to preferentially degrade actively transcribed genes (i.e., 15-20% of the DNA rendered perchloric acid soluble), a region of approximately 12 kilobase(s) (kb) containing the GAPDH coding sequences and flanking DNA was found to be highly susceptible to digestion by DNase I. Approximately 4 kb downstream from the end of the coding sequences, there was an abrupt transition from the DNase I sensitive or "open" configuration to the resistant or "closed" configuration. The chromatin then remained in a closed conformation for at least 10 kb further downstream. On the 5' side of the gene, the transition from a sensitive to a resistant configuration was located about 4 kb upstream from the gene. In addition, we have localized two repeated sequences in the area of DNA that was cloned. One of these is of the CR1 family of middle repetitive elements. It is located about 18 kb 3' to the gene and as such lies well outside of the DNase I sensitive region which encompasses GAPDH. The other repetitive element is of an uncharacterized family. It is located upstream from the gene and appears to be within a region of transition from the DNase I sensitive to resistant states.  相似文献   

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The three CD3 genes on human chromosome 11q23 encode proteins (gamma, delta and epsilon) which form part of the antigen receptor on T lymphocytes. All three genes are clustered within 50 kb and are activated approximately contemporaneously during the early stages of T cell ontogeny. In order to pinpoint potential regulatory sequences important for locus activation and tissue-specific gene expression, the chromatin structure of almost 90 kb of this region has been probed in five cell lines using the endonuclease pancreatic DNase I. A set of DNase I hypersensitive (HS) sites has been defined in T cell chromatin, five of which were strong and not found in non-T cells, with the exception of the erythroleukaemia cell line K562, in which three sites were weakly expressed, correlating with a low level of delta mRNA. The subset of five HS sites map close to the CD3 genes and lie in regions which contain elements of defined function: the gamma promoter; the delta promoter and its 3' enhancer; and the epsilon promoter and its 3' enhancer. Since no further major T cell-restricted HS sites lie within the 90kb of the CD3 locus analysed, these five regions may contain all the sequences important for CD3 gene expression.  相似文献   

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The complete chicken lysozyme gene locus is expressed copy number dependently and at a high level in macrophages of transgenic mice. Gene expression independent of genomic position can only be achieved by the concerted action of all cis regulatory elements located on the lysozyme gene domain. Position independency of expression is lost if one essential cis regulatory region is deleted. Here we compared the DNase I hypersensitive site (DHS) pattern formed on the chromatin of position independently and position dependently expressed transgenes in order to assess the influence of deletions within the gene domain on active chromatin formation. We demonstrate, that in position independently expressed transgene all DHSs are formed with the authentic relative frequency on all genes. This is not the case for position dependently expressed transgenes. Our results show that the formation of a DHS during cellular differentiation does not occur autonomously. In case essential regulatory elements of the chicken lysozyme gene domain are lacking, the efficiency of DHS formation on remaining cis regulatory elements during myeloid differentiation is reduced and influenced by the chromosomal position. Hence, no individual regulatory element on the lysozyme domain is capable of organizing the chromatin structure of the whole locus in a dominant fashion.  相似文献   

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We have cloned an 11.3-kb rat genomic DNA fragment encompassing the muscle regulatory factor 4 (MRF4) protein-coding sequence, 8.5 kb of 5'-flanking sequence, and 1.0 kb of 3'-flanking sequence. In order to study MRF4 gene expression, the rat myogenic cell line, L6J1-C, which expresses the endogenous MRF4 gene only in differentiated myofibers, was transfected stably with the full-length genomic clone and various 5' deletions. RNase protection assays demonstrated that MRF4 genes containing as little as 430 bp of 5'-flanking sequence exhibited an increase in expression as the cells differentiated into myofibers, indicating that elements responsible for fiber-specific expression are contained within this cloned DNA fragment. Similar up-regulation was observed with genes containing 1.5 kb of 5'-flanking sequence. Interestingly, MRF4 genes containing 5.0 kb and 8.5 kb of 5'-flanking sequence were up-regulated to even higher levels, suggesting that additional myofiber-specific regulatory elements located between 1.5 and 5.0 kb upstream from the coding region play a role in regulating the expression of this muscle-specific gene.  相似文献   

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We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.  相似文献   

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《The Journal of cell biology》1996,134(5):1333-1344
We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse pro alpha 2 (I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse pro alpha 2 (I) collagen gene, cloned upstream of either the Escherichia coli beta- galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal pro alpha 2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse pro alpha 1(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro alpha 2(I) collagen gene.  相似文献   

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The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19. Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells. A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.  相似文献   

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