Strontium chromium(II) diphosphate, SrCrP2O7
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Journal Article↗
Biomedical subjects
K Maass
Publications and source records attributed to K Maass.
Retargeting of retroviral integration sites for the predictable expression of transgenes and the analysis of cis-acting sequences.
The transcriptional activity of transgenes in eukaryotic host cells critically depends on the sites of their integration where it is modulated by interactions between the promoter and surrounding chromatin structures. Retroviruses integrate their genome into chromosomal loci favoring expression from one long-terminal repeat (LTR). We have therefore developed a strategy in which retroviral vectors are provided with "tags", that is, targets for a site-specific recombinase (Flp). Presence of two 48 bp Flp recognition target (FRT) sequences permits the excision of a selection marker whereby the reading frame of a reporter gene (lacZ) is restored and beta-galactosidase activity can be monitored to characterize the integration site regarding the level and stability of expression. The location of the remaining FRT tag permits the subsequent Flp-mediated insertion of a truncated selection marker which is then expressed from the LTR. This step represents an authentic site-specific recombination event which can be demonstrated by a number of criteria, among these its reversibility in the presence of Flp activity. Thereby the "expression trap" principle permits the efficient recovery of stable insertion events, and if a gene of interest is linked to the truncated marker, the established properties of a given genomic site can be utilized for transcription studies or for the generation of highly expressing clones, even for biotechnological purposes.
3T3 Cells↗
A thymocyte factor SATB1 suppresses transcription of stably integrated matrix-attachment region-linked reporter genes.
SATB1 specifically recognizes and binds to specialized genomic regions with an ATC sequence context with high base-unpairing propensity. Such base-unpairing regions (BURs) are typically identified within nuclear scaffold- or matrix-attachment regions (S/MARs). SATB1 is a homeodomain protein and is predominantly expressed in thymocytes. We obtained BHK cell lines expressing low levels of SATB1 by stable transfection and investigated its effect on stably integrated MAR-linked SV40 enhancer/promoter-driven luciferase reporter genes. For this study, both naturally occurring and synthetic MARs, as well as an AT-rich non-MAR control, were tested. Previous studies demonstrated that MAR sequences augment transcription of the linked reporter luciferase gene. Here, we show that SATB1 dramatically reduces the high levels of MAR-linked luciferase gene transcription. Transcription was virtually abolished for a reporter gene surrounded by two MARs at the 5' and 3' ends of the gene, which otherwise confer the highest level of transcriptional augmentation. On the other hand, SATB1 did not affect expression of an AT-rich non-MAR-linked luciferase gene or of endogenous housekeeping genes. This study shows that SATB1 acts as a strong transcriptional suppressor on a reporter gene linked to MARs when it is stably integrated into chromatin.
Animals↗
Scaffold/matrix-attached regions act upon transcription in a context-dependent manner.
Scaffold/matrix-attached regions (S/MARs) are cis-acting elements with a function outside transcribed regions and in introns. Although they usually augment transcriptional rates, their action is highly context-dependent. We cloned an 800 bp S/MAR element from the upstream border of the human interferon-beta domain at various positions within a transcribed region of 4.3 kb. By use of retroviral gene transfer, the vector could be integrated into target cells as a single copy enabling a rigorous definition of the distance between the S/MAR and the transcriptional start site. At a distance of about 4 kb, the S/MAR supported transcriptional initiation, whereas at distances below 2.5 kb, transcription was essentially shut off. Controls proved the functionally of all constructs in the transient expression phase and ruled out any influence of S/MAR position on transcript stability. Moreover, no pausing or premature termination was observed within these elements. We suggest that the protein binding partners of S/MARs change according to the topological status, explaining these divergent S/MAR effects.
3T3 Cells↗
Anatomy of highly expressing chromosomal sites targeted by retroviral vectors.
The eukaryotic genome contains chromosomal loci with a high transcription-promoting potential. For their identification in cultured cells, transfer of a reporter gene has to be performed by a technique that grants the integration of individual copies. We have applied retroviral vectors in conjunction with inverse polymerase chain reaction techniques to reconstruct a number of these sites for a further characterization. Remarkably, all examples conform to the same design in that the process of retroviral infection selected a scaffold- or matrix-attached region (S/MAR) that was flanked by DNA with high bending potential. The S/MARs are of an unusual type in that they show a high incidence of certain dinucleotide repeats and the potential to act as topological sinks. The anatomy of retroviral integration sites reveals principles that can be exploited for the development of predictable transgenic systems on the basis of expression and targeting vectors.
3T3 Cells↗
Permeability of the blood-retinal barrier and the blood-aqueous barrier in type I diabetes without diabetic retinopathy: simultaneous evaluation with fluorophotometry.
For the evaluation of a possible malfunction of the blood-retinal barrier (BRB) and the blood-aqueous barrier (BAB) in type I diabetes without manifest angiopathy after i.v. injection of sodium fluorescein, the permeability of the BRB (P) and the permeability coefficient of the BAB [P(a)] were simultaneously determined by fluorophotometry in 34 eyes of 34 type-I diabetics [hemoglobin (Hb)A1c = 6.6% +/- 0.9%] without retinopathy whose age ranged from 19 to 38 years (mean, 30.5 +/- 5 years); the diabetes duration was between 5 and 18 years. Fluorescein angiography was performed to exclude nonperfused areas. In all, 34 eyes of 34 healthy volunteers whose age ranged between 23 and 34 years (mean, 28.5 +/- 3.3 years) served as controls; in this group, fluorophotometry was performed twice to evaluate reproducibility. The mean BAB permeability coefficient in diabetics [P(a) = 5.3 +/- 1.8 x 10(-4)/min] was significantly increased (P = 0.00003) as compared with control values [P(a) = 3.7 +/- 0.7 x 10(-4)/min]; BRB permeability in diabetics (P = 3.2 +/- 1.4 x 10(-7) cm/s) was raised, with this elevation being of lower significance (P = 0.019; controls, P = 2.6 +/- 0.7 x 10(-7) cm/s). We found a decrease in BRB permeability depending on diabetes duration (r = -0.15; P = 0.007) that was not significant in the BAB (r = -0.1; P = 0.24). No correlation was found to exist between permeability and hemoglobin (Hb)A1c values either in the BAB or in the BRB. The reproducibility in controls was 9% in BRB determinations and 12% in BAB measurements.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Scaffold-attached regions (SAR elements) mediate transcriptional effects due to butyrate.
The expression of certain genes has been reported to respond positively to sodium butyrate. This study demonstrates the same feature for two marker genes under the control of five different promoters. In all examples, the stimulatory effect is largest if one or especially two scaffold/matrix-attached regions (SAR/MAR elements) are present adjacent to the gene, and in one case, the stimulation depends entirely upon this situation. These results are observed with several SAR sequences including those obtained by oligomerizing short stretches of DNA surrounding a core motif. It is suggested that butyrate exerts important actions at the level of the chromatin structure.
Animals↗
Scaffold-attached regions from the human interferon beta domain can be used to enhance the stable expression of genes under the control of various promoters.
We have transfected DNA corresponding to the complete chromatin domain of human interferon beta (huIFN-beta) gene into mouse L cells. In this construct, which is flanked by scaffold-attached regions (SARs), the gene's transcription was enhanced 20-30-fold with respect to DNAs containing only the immediate regulatory elements. To elucidate the role of SAR elements in the transcriptional enhancement, their position was varied relative to several artificial promoter-gene combinations. It was found that SARs enhance general promoter functions in an orientation- and partially distance-independent manner; their effect is restricted to the integrated state of transfected templates. During the phase of transient expression, SAR elements were generally found to have an antagonizing effect.
Animals↗
[Comparative methodologic studies of closing volume and increase in airway resistance volumes].
The early recognition of obstructive lung diseases plays an important role in the subsequent therapy; the closing volume, determined by means of single-breath oxygen test has been established as a suitable parameter. This technique, however, has not succeeded as a routine method due to prohibitive costs. In a clinical study the closing volume and the easier estimatable so-called flow-resistance elevation volume (Ros-volume-curve parameter obtained from oscilloresistometry/volumetry) were compared and a correlation sought. It could be concluded, that for characterization of small airways diseases the closing volume is a more suitable parameter than the flow-resistance elevation volume.
Adolescent↗
Chromatin domain surrounding the human interferon-beta gene as defined by scaffold-attached regions.
Regions attached to the nuclear scaffold have been traced after transfecting a 36-kilobase (kb) piece of DNA, surrounding the human interferon-beta gene, into mouse L-cells. An extended attached region starts 1.7 kb upstream from the gene and a moderate binding site immediately downstream. These findings could be confirmed by reconstitution experiments in vitro which predict another scaffold-attached region (SAR) starting 12 kb downstream from the gene. Since no other transcripts originate from DNA between the major SARs, these elements could be involved in interferon gene regulation.
Animals↗
Chromatin structure and induction-dependent conformational changes of human interferon-beta genes in a mouse host cell.
Multiple copies of a human interferon-beta gene introduced into a mouse host cell line can be activated by induction with double-stranded RNA. Several induction-dependent changes of the chromatin structure could be traced by mapping techniques using four different agents [DNase I, micrococcus nuclease, bromoacetaldehyde and methidiumpropyl-EDTA X iron(II)]. Our data show that all copies of the interferon gene have adopted a very similar conformation in the host cell and respond to the inducing stimulus in a highly synchronous fashion. Detailed induction-specific changes were observed best with the chemical reagents which disclose a specific hypersensitive site within a sequence that has been shown to be required for the induction process (around position -80) and three other regions which, in addition to the transcribed region itself, gain single-strand character by an auxiliary process which can be mimicked by the addition of butyrate to the medium and may therefore involve histone hyperacetylation. Six discrete 'phased' nucleosomes are present upstream from the gene and are modulated by induction. At least four nucleosomes are located downstream. The interferon genes are largely protected from micrococcus nuclease in the inactive state. Gene activation increases access to micrococcus nuclease and DNase I indicating gross conformational changes on a higher level of chromatin structure.
Animals↗
Links between effects of butyrate on histone hyperacetylation and regulation of interferon synthesis in Namalva and FS-4 cell lines.
The human Namalva lymphoma cell line being an established producer, predominantly of alpha interferon, has been reported to enhance interferon synthesis after a preincubation in butyrate containing media. We have performed the corresponding experiments with FS-4 fibroblast cells and show that the synthesis of human beta interferon is adversely affected by this treatment. Both cell types are hyperacetylated by the fatty acid to a comparable extent. However, after the withdrawal of butyrate, the persistence of highly acetylated forms of histone H4 is insufficient in the case of FS-4 to endure the interferon induction period. Concerning fibroblasts, deacetylation proceeds to a hypoacetylated state which is reversed only slowly. With lymphoid cells on the other hand, acetylated H4 specimens are much more stable and persist for more than eight hours in the absence of butyrate. Moreover, the acetylation reactions are supported by other Friend cell stimulators which by themselves are no inhibitors of deacetylase activities.
Acetamides↗