Prerequisites for tissue specific and position independent expression of a gene locus in transgenic mice.

The elucidation of general parameters influencing the transcriptional activation of gene loci at distinct stages of development is an essential prerequisite for a reproducibly successful gene transfer in both gene therapy protocols and biotechnology. Up to now research has focused mostly on the identification and characterization of individual cis-regulatory elements by transient transfection and in vitro assays. However, the most relevant assay system to test gene constructs designed for gene therapy protocols is the transgenic animal. In such an experimental system exogenous genes are usually integrated randomly in the chromatin. For gene constructs not fulfilling the requirements for correct gene locus activation this can lead to genomic position effects on gene expression. The consequences are highly variable expression levels and a disturbance of temporal and spatial expression patterns. Hence it is important to examine how cis-elements function in a chromatin context, and how they cooperate during the developmentally controlled activation of an entire gene locus. One among a few gene loci which are sufficiently characterized to enable such investigations is the chicken lysozyme locus. This review summarizes recent results aimed at identifying the necessary prerequisites for a reproducibly correct expression of the lysozyme locus in transgenic mice and the implications of our findings for gene transfer. The complete lysozyme locus is expressed independent of the chromosomal position and at a high level in macrophages of transgenic mice. Correct transgene regulation requires the cooperation of all cis-regulatory elements. Chromatin of the lysozymes locus in both the active and the inactive state is highly structured. Each cis-regulatory element on the chicken lysozyme locus is organized in its own unique chromatin environment, with nucleosomes specifically placed on specific sequences. The transcriptional activation of the lysozyme locus is accompanied by extensive rearrangements of its chromatin structure, which are disturbed when the transgenes are subjects to genomic position effects. Based on these results, we propose that a complete locus is resistant to genomic position effects, and that a distinct chromatin architecture of a gene locus is required for its correct activation.

Different macrophage populations develop from embryonic/fetal and adult hematopoietic tissues.

Immunohistochemical and ultrastructural studies have indicated the existence of a distinct "fetal macrophage" type, differing from monocyte-derived macrophages. In order to characterize macrophages of different ontogenetic origins on the molecular level, we examined their surface-marker and marker-gene expression patterns. We found that macrophages derived from chicken embryos express the lysozyme gene at significantly lower levels than macrophages derived from adult chicken. The same was observed when expression of the chicken lysozyme gene was analyzed in transgenic mice. In three independent mouse lines, mature macrophages derived from embryonic or fetal hematopoietic tissues expressed the transgene at drastically lower levels than macrophages derived from the bone marrow, spleen, or peritoneal cavity of adult mice. Macrophages obtained by in vitro differentiation of mouse embryonic stem cells (a process resembling early embryonic hematopoiesis) displayed the embryo-specific low transgene expression level. Experiments determining the developmental potential of myeloid precursors in culture and immunophenotypic analyses revealed differences between embryo-derived and adult myeloid progenitor populations. In summary, our results provide further evidence for the existence of dissimilar embryonic/fetal and adult macrophage types and describe the first molecular marker for their distinction.

The -3.9 kb DNaseI hypersensitive site of the chicken lysozyme locus harbours an enhancer with unusual chromatin reorganizing activity.

Tissue specific regulation of the chicken lysozyme locus is achieved by a combination of positive and negative cis-regulatory elements. Here we describe the molecular characterization of a newly discovered enhancer element located -3.9kb upstream of the transcription start. The -3.9kb enhancer is activated early in macrophage differentiation, as indicated by chromatin reorganization in macrophage precursor cells. Interestingly, enhancer activation leads to nucleosome phasing. Tissue specificity of expression is achieved by a combination of 5'-sequences with ubiquitous enhancer activity and 3'-flanking sequences. The 5'-half contains binding sites for members of the nuclear factor I transcription family and a yet unknown protein. We could show by in vivo footprinting that the ubiquitously expressed factors occupy their binding sites only in lysozyme expressing cells. We conclude that a specific chromatin architecture may be responsible for the differential activity of the enhancer.

Regulation of the chicken lysozyme locus in transgenic mice.

The chicken lysozyme locus is transcriptionally activated during macrophage differentiation. Each cis-regulatory element has its unique activation stage during cell differentiation, whereby maximal transcriptional activity of the gene is only observed when all cis-elements are active. The complete chicken lysozyme locus is expressed position independently and at a high level in macrophages of transgenic mice. For correct transgene regulation, the cooperation of all cis-regulatory elements is required. These cis-regulatory elements specify the mode of regulation and we observe the same expression pattern of the transgene in the mouse and the endogenous gene in chicken macrophages. This indicates that the transcription factors responsible for chicken lysozyme regulation are highly conserved in evolution. The endogenous mouse lysozyme gene is regulated differently. The chromatin of the lysozyme locus is highly structured in the transcriptionally active, as well as in the inactive state. The transcriptional activation of the lysozyme locus is accompanied by extensive chromatin rearrangements, which are disturbed when one essential cis-regulatory element is deleted and the transgenes are subjects to genomic position effects. Based on these results, we propose that a distinct chromatin architecture of a gene locus is required for its correct activation.

Identification of cis-acting elements as DNase I hypersensitive sites in lysozyme gene chromatin.

DNase I hypersensitive sites in chromatin of eukaryotic cells mark the positions of multifactorial cis-acting elements. Mapping DH sites by indirect end labeling is a convenient procedure used for identifying regulatory elements within extensive regions of chromatin and for gaining information about their functional specificity as well as their fine structure.

Dynamic changes in the chromatin of the chicken lysozyme gene domain during differentiation of multipotent progenitors to macrophages.

The chicken lysozyme locus is regulated in oviduct and macrophages by a complex set of well-characterized cis-regulatory DNA elements. We determined the DNase I hypersensitive chromatin site pattern of the chromatin of the lysozyme locus in retrovirally transformed cell lines representing different stages of myelomonocytic cell differentiation. In the transformed multipotent progenitor stage and in erythroblasts, only a DNase I hypersensitive chromatin site at a silencer element located -2.4 kb upstream of the transcriptional start site is present. At the myeloblast stage DNase I hypersensitive chromatin sites are formed both at the distal enhancer located at -6.1 kb and at the promoter. Later in differentiation, at the monocytic stage, a second DNase I hypersensitive chromatin site appears at the medial enhancer located at -2.7 kb. Parallel with DNase I hypersensitive chromatin site formation at the medial enhancer, the DNase I hypersensitive chromatin site at the silencer element disappears. These chromatin rearrangements correlate with the mRNA expression of the gene that is undetectable in multipotent progenitors and maximal in a lipopolysaccharide-stimulated monocyte cell line. Our results show that the chromatin structure and the transcriptional activity of the gene are tightly coupled during commitment and maturation of the myelomonocytic lineage.

Effect of Copper Sulfate and Lead Acetate on Infection of Pines with Bursaphelenchus xylophilus.

Treatment of 3-year-old Scots, white, and Austrian pine seedlings with copper sulfate or lead acetate significantly affected energy homeostasis and oleoresin production in the seedlings but did not induce wilting of the seedlings. Inoculation of copper sulfate-treated or lead acetate-treated white, Scots, and Austrian pine seedlings with the white pine specific pathotype of Bursaphelenchus xylophilus, VPSt-1, caused a significant increase in oleoresin production, stressed energy homeostasis, and induced rapid wilting of the seedlings. Scots pine lost tolerance and Austrian pine lost resistance to VPSt-1 after the seedlings were treated with either copper sulfate or lead acetate. These results suggest that environmental pollution may significantly affect susceptibility of pines to B. xylophilus and may have a role in establishment of this nematode in uninfested areas.

Stigmatic performance of an EUV spectrograph with a single toroidal grating.

The stigmatic performance of conventionally ruled toroidal gratings used near normal incidence is explored in detail. A single-grating spectrograph, particularly suitable for observing the dynamics of the solar corona in the 520-630-A wavelength range, is described. The arrangement uses a 3600-line/mm grating with a horizontal radius of curvature of 2 m that generates images with a blur of less than 20 microm over an area 2.6 mm high x 80 mm wide, while over an area 6 mm x 80 mm the blur does not exceed 40 microm. In terms of wavelength intervals these blurs correspond to 28 mA and 56 mA, and the width of the area covers a spectral range of 110 A. If the spectrograph is equipped with a 20-microm wide entrance slit, and if this slit is placed in the focal plane of a telescope with 4-m focal length, spatial resolution elements of 1 x 1 (sec of arc)(2) and 1 x 2 (sec of arc)(2) over a slit height of 2 min of arc and 5 min of arc, respectively, result.

Extreme uv spectroheliometer on the Apollo Telescope Mount.

The Harvard College Observatory instrument on the Skylab was a seven-channel spectrometer-spectroheliometer operating in the 280-1340-A wavelength range and capable of a variety of observing modes encompassing both spatial and spectral scans of a wide range of solar features. The instrument design and operational performance are described in detail.

Toroidal grating obtained on an elastic substrate.

The performance of a grating that is elastically deformed from a sphere into a toroid is that expected from ray traces assuming an ideal toroidal surface. Thus, it has become possible to select the wavelength range, in which a toroidal grating spectrograph is near stigmatic, by adjusting the forces acting on the elastic grating substrate. The possibility of producing toroidal gratings by replication of an elastically deformed blank is pointed out.

The developmental activation of the chicken lysozyme locus in transgenic mice requires the interaction of a subset of enhancer elements with the promoter.

The complete chicken lysozyme locus is expressed in a position independent fashion in macrophages of transgenic mice and forms the identical chromatin structure as observed with the endogenous gene in chicken cells. Individual lysozyme cis -regulatory elements reorganize their chromatin structure at different developmental stages. Accordingly, their activities are developmentally regulated, indicating a differential role of these elements in locus activation. We have shown previously that a subset of enhancer elements and the promoter are sufficient to activate transcription of the chicken lysozyme gene at the correct developmental stage. Here, we analyzed to which grade the developmentally controlled chromatin reorganizing capacity of cis -regulatory elements in the 5'-region of the chicken lysozyme locus is dependent on promoter elements, and we examined whether the lysozyme locus carries a dominant chromatin reorganizing element. To this end we generated transgenic mouse lines carrying constructs with a deletion of the lysozyme promoter. Expression of the transgene in macrophages is abolished, however, the chromatin reorganizing ability of the cis -regulatory elements is differentially impaired. Some cis -elements require the interaction with the promoter to stabilize transcription factor complexes detectable as DNase I hypersensitive sites in chromatin, whereas other elements reorganize their chromatin structure autonomously.

Genomic position effects lead to an inefficient reorganization of nucleosomes in the 5'-regulatory region of the chicken lysozyme locus in transgenic mice.

The chicken lysozyme locus is gradually activated during macrophage development exhibiting a specific chromatin structure with each differentiation state. Its small size and the extensive characterization of its cis-regulatory elements allows us to study even subtle changes in chromatin structure of the entire gene locus during transcriptional activation. Tissue-specific and position independent expression of the lysozyme locus in transgenic mice requires the cooperation of all cis-regulatory elements. In order to elucidate further the molecular basis of locus activation, we have determined nucleosome positions within the complete 5'-regulatory region of the chicken lysozyme locus in chicken myeloid cell lines and transgenic mice. Each cis-regulatory element develops its unique nucleosomal structure and each one remodels chromatin differently. The nucleosomal organization of the endogenous gene in chicken cell lines and the transgene in the mouse turned out to be identical, enabling us to study the influence of cis-regulatory deletions on the development of an active chromatin structure in transgenic mice. Transgenes with a deletion of an important cis-regulatory element show an impediment in nucleosome reorganization as compared with the complete lysozyme locus. We demonstrate that multicopy transgene-clusters in position dependently expressing mouse lines exhibit a heterogeneous chromatin organization.

[Retrospective study of the reasons for admission and the principal causes of mortality in the pediatric service of the Port-Gentil General Hospital of (Gabon), June 1985-May 1986]. / Etude rétrospective des motifs d'admission et des principales causes de mortalité au service de pédiatrie de l'Hopital Général de Port-Gentil (Gabon), juin 1985-mai 1986.

A retrospective study of 534 bed-rests permitted us to specify the reasons of admissions and mortality in the hospital service of pediatrics, Port-Gentil, Gabon, from June 1985 to May 1986: a high mortality rate (7.5%) with, specially, diarrheas (46%) and proteinocaloric malnutrition (19%). The other reasons of mortality are constituted by infections and neonatal diseases. Priority in public health seems to be child welfare (vaccinations, primary health care in the whole province of Ogoue, health certificates...).

Photometric calibration of the EUV spectroheliometer on ATM.

This paper describes the derivation of the preflight photometric calibration of the uv spectrometer on Skylab. The calibration of the orbiting instrument through cross-comparison with two rocket instruments is discussed in assessing the observed changes in response to quiet solar regions during the mission. Formulas are presented for the determination of the instrument sensitivity, and an uncertainty of +/-35% is assigned over most of the 296-1340-A wavelength range.

Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNase I hypersensitive site formation.

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.

Solar-blind photoelectric detection systems for satellite applications.

The characteristics of open Magnetic Electron Multipliers (MEM) with continuous dynode and field strips are discussed in view of their use in satellite-borne detection systems. Special emphasis is placed on selection, thermal stabilization, calibration, and long-time performance of the extreme ultraviolet detectors Practical aspects of the design of satellite-borne detection systems, such as monitoring capabilities, immunity to charged particles, and electrical breakdown, are also described. Laboratory studies with fast electronics showed that the useful linear range of an MEM is restricted because of afterpulses. Most characteristics of MEM's reported in the literature appear to be due to the use of relatively slow electronics that do not resolve the afterpulses. Statistical studies of afterpulses are reported, and a tentative explanation for the generation of afterpulses is outlined.

Instrumentation for Combined Dispersion and Absorption Measurements in the VUV.

When the hook method that measures anomalous dispersion is combined with photoelectric photometry, a particularly powerful tool results. An apparatus that combines these techniques over a wavelength range extending into the vacuum ultraviolet has been constructed and used chiefly on the iron-group elements. It consists of hydrogen-discharge light source, a Mach-Zehnder interferormeter, a high temperature furnace, a stigmatic spectrograph, and a photoelectric photometer.

Imaging extreme ultraviolet spectrometer employing a single toroidal diffraction grating: the initial evaluation.

A high-efficiency extreme ultraviolet (EUV) imaging spectrometer has been constructed and tested. The spectrometer employs a concave toroidal grating illuminated at normal incidence in a Rowland circle mounting and has only one reflecting surface. The toroidal grating has been fabricated by a new technique employing an elastically deformable submaster grating which is replicated in a spherical form and then mechanically distorted to produce the desired aspect ratio of the toroidal surface for stigmatic imaging over the selected wavelength range. The fixed toroidal grating used in the spectrometer is then replicated from this surface. Photographic tests and initial photoelectric tests with a 2-D pulse-counting detector system have verified the image quality of the toroidal grating at wavelengths near 600 A. The results of these initial tests are described in detail, and the basic designs of two instruments which could employ the imaging spectrometer for astrophysical investigations in space are briefly described, namely, a high-resolution EUV spectroheliometer for studies of the solar chromosphere, transition region, and corona and an EUV spectroscopic telescope for studies of nonsolar objects.

Intercalibration of SUMER and CDS on SOHO. I. SUMER detector A and CDS NIS.

The results of an intercalibration between the extreme ultraviolet spectrometers Coronal Diagnostic Spectrometer (CDS) and Solar Ultraviolet Measurements of Emitted Radiation (SUMER) onboard the Solar and Heliospheric Observatory (SOHO) are presented. During the joint observing program Intercal_01, CDS and SUMER were pointed at the same locations in quiet Sun areas and observed in the same wavelength bands located around the spectral lines He i 584 A, Mg x 609 A, and Mg x 624 A. The data sets analyzed here consist of raster images recorded by the CDS normal-incidence spectrometer and SUMER detector A and span the time from March 1996 to August 1996. Effects of the different spatial and spectral resolutions of both instruments have been investigated and taken into account in the analysis. We find that CDS measures generally a 30% higher intensity than SUMER in the He i 584-A line, while it measures 9% and 17% higher intensities in Mg x 609 A and Mg x 624 A, respectively. Both instruments show very good temporal correlation and stability, indicating that solar variations dominate over changes in instrumental sensitivity. Our analysis also provides in-flight estimates of the CDS spatial point-spread functions.