Fons Cremers

The epidermal growth factor.

Epidermal growth factor (EGF) is a single polypeptide of 53 amino acid residues which is involved in the regulation of cell proliferation. Egf exerts its effects in the target cells by binding to the plasma membrane located EGF receptor. The EGF receptor is a transmembrane protein tyrosine kinase. Binding of EGF to the receptor causes activation of the kinase and subsequently receptor autophosphorylation. The autophosphorylation is essential for the interaction of the receptor with its substrates. These bind to the receptor by the so‐called SH2 domains.

Expression pattern of parathyroid hormone/parathyroid hormone related peptide receptor mRNA in mouse postimplantation embryos indicates involvement in multiple developmental processes

In this paper we describe the cloning of the mouse Parathyroid Hormone/Parathyroid Hormone related Peptide Receptor (PTH/PTHrPR) cDNA and expression of its mRNA during mouse postimplantation development from day 5.5 until day 15.5 post coitum (p.c.). In support of a model from previous studies, in which parietal endoderm differentiation is regulated by the interaction of the PTH/PTHrPR and Parathyroid Hormone related Peptide (PTHrP), high levels of PTH/PTHrPR mRNA levels were detected in developing parietal endoderm from day 5.5 p.c. and onwards. In the embryo proper, PTH/PTHrPR mRNA expression was mainly detected at sites of epithelium/mesenchyme interactions, starting at day 9.5 p.c. in the epithelium of the intestine and later in the mesenchyme of the lung, the epithelium of meso- and metanephric tubuli, the dermis and at all sites where bone formation takes place. The complexity of the PTH/PTHrPR expression pattern suggests tight developmental regulation and indicates multiple roles in embryogenesis for the receptor and its ligands, not only in extraembryonic tissue but also in the formation of various organs.

In vivo import of plastocyanin and a fusion protein into developmentally different plastids of transgenic plants

Transgenic tomato plants that constitutively express a foreign plastocyanin gene were used to study protein transport in different tissues. Normally expression of endogenous plastocyanin genes in plants is restricted to photosynthetic tissues only, whereas this foreign plastocyanin protein is found to be present in all tissues examined. The protein is transported into the local plastids in these tissues and it is processed to the mature size. We conclude that plastids of developmentally different tissues are capable of importing precursor proteins that are normally not found in these tissues. Most likely such plastids, though functionally and morphologically differentiated, have similar or identical protein import mechanisms when compared to the chloroplasts in green tissue.

Ultrastructural localization of active genes in nuclei of A431 cells

We have studied the ultrastructural localization of active genes in nuclei of the human epidermoid carcinoma cell line A431. Nascent RNA was labeled by incorporation of 5‐bromouridine 5′‐triphosphate, followed by pre‐embedment or postembedment immunogold labeling and electron microscopy using ultrasmall gold‐conjugated antibodies and silver enhancement. This combination of techniques allowed a sensitive and high resolution visualization of RNA synthesis in the nucleus. Transcription sites were identified as clusters of 3–20 gold particles and were found throughout the nucleoplasm. The clusters had a diameter of less than 200 nm. The distribution of clusters of gold particles in nuclei is preserved in nuclear matrix preparations. Nascent RNA is associated with fibrillar as well as with granular structures in the matrix. A431 nuclei contained on average about 10,000 clusters of gold particles. This means that each cluster represents transcription of probably one active gene or, at most, a few genes. Our study does not provide evidence for aggregation of active genes. We found transcription sites distributed predominantly on the surface of electron‐dense nuclear material, probably lumps of chromatin. This supports a model of transcription activation preferentially on the boundary between a chromosome domain and the interchromatin space.

Genes encoding ferredoxins from Anabaena sp. PCC 7937 and Synechococcus sp. PCC 7942: structure and regulation

The gene encoding ferredoxin I (petF1) from the filamentous cyanobacterium Anabaena sp. PCC 7937 (Anabaena variabilis ATCC 29413) was cloned by low stringency hybridization with the ferredoxin cDNA from the higher plant Silene pratensis. The petF1 gene from the unicellular cyanobacterium Synechococcus sp. PCC 7942 (Anacystis nidulans R2) was cloned by low stringency hybridization with the petF1 gene from Anabaena sp. PCC 7937. One copy of the petF genes was detected in both organisms, and a single transcript of about 630 b was found for Synechococcus sp. PCC 7942. Both the Synechococcus sp. PCC 7942 and the Anabaena sp. PCC 7937 petF1 genes contain a 297 bp open reading frame coding for a small acidic protein, consisting of 98 amino-acid residues, with a molecular mass of about 10.5 kDa.The ferredoxin content of Synechococcus sp. PCC 7942 is strongly reduced under ironlimited growth conditions. The slight decrease in the amount of ferredoxin transcript found under iron limitation does not account for the more severe reduction in ferredoxin protein observed. The main regulation of the ferredoxin content probably is effected at the level of translation and/or degradation. Although ferredoxin expression can be strongly reduced by iron stress, the ferredoxin function seems to be indispensable, as Synechococcus sp. PCC 7942 appeared refractory to yield mutants lacking the petF1 gene.

Discovery of a new RNA-containing nuclear structure in UVC-induced apoptotic cells by integrated laser electron microscopy

Background information. Treatment of cells with UVC radiation leads to the formation of DNA cross‐links which, if not repaired, can lead to apoptosis. γ‐H2AX and cleaved caspase 3 are proteins formed during UVC‐induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and γ‐H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy).

Localization of nuclear RNA by pre- and post-embedding in situ hybridization using different gold probes.

SummaryPre-embedding and post-embedding in situ hybridization techniques were compared for the localization of RNAs in the nucleus. 28S rRNA and transcripts of the epidermal growth factor receptor (EGF-receptor) were localized with both hybridization methods. PRE-embedding hybridizations were performed on cells permeabilized with Triton X-100, whereas post-embedding hybridizations were carried out on Lowicryl K4M sections. From these studies it was concluded that, for labelling of 28S rRNA, the post-embedding in situ hybridization is preferred, whereas EGF-receptor transcripts were successfully detected only after pre-embedding in situ hybridization. Furthermore, the detection of the hybrids with ultra-small gold particles was compared to the detection with 6 nm gold particles in both pre- and post-embedding in situ hybridization studies. From our results it is concluded that the use of ultra-small gold particles results in higher label efficiency. Therefore, ultra-small gold particles are preferable to 6 nm gold particles for the detection of hybrids in high-resolution in situ hybridization experiments.

Visualization of contact sites between outer and inner envelope membranes in isolated chloroplasts

Abstract Contact sites between outer and inner envelope membranes of isolated chloroplasts have been studied at the ultrastructural level by two different electron microscopical methods, i.e., freeze fracturing and freeze substitution followed by ultrathin sectioning. Both methods demonstrate that approx. 10% of the chloroplast population exhibits blister-like structures. Cross fractures and ultrathin sections of freeze-substituted chloroplasts reveal that the blisters originated from a separation of the outer and inner envelope membranes. Exposure of isolated chloroplasts to hypertonic conditions results in an almost complete separation of the two envelope membranes, except for small regions in which the two membranes are in close contact. These contact sites are clearly recognized in freeze-fracture replicas by ridges of a high density of intramembrane particles. In addition, cross fractures and thin sections of freeze-substituted chloroplasts demonstrate the presence of small vesicles associated with the outer envelope membrane. As indicated by the opacity of the vesicle and demonstrated by immuno-gold labeling, these vesicles, which originate from the inner envelope membrane, contain stroma-derived proteins.

Localisation of EGF‐Receptor mRNA in the nucleus of A431 cells by light microscopy.

We have localized the mRNA of the epidermal growth factor receptor (EGF‐receptor) in nuclei of A431 cells by non‐radioactive in situ hybridization at the light microscopical level using digoxigenin‐labelled DNA probes. Both formaldehyde and glutaraldehyde fixations were tested before the hybridization was performed. Glutaraldehyde, compared with formaldehyde fixation, gives a less diffuse hybridization signal, which is easier to localize. Therefore, glutaraldehyde was used as a fixative in the hybridization experiments. It is demonstrated that the mRNA of the EGF‐receptor is present in restricted domains mainly located around the nucleolus. This location of the EGF‐receptor mRNA was unaltered after extraction of chromatin. Therefore it is concluded that the messenger RNA of the EGF‐receptor is attached to the nuclear matrix. A possible biological role for the location of mRNA of the EGF‐receptor around the nucleolus is discussed.

Silene plastocyanin is fully functional in transgenic tobacco

Abstract Transgenic tobacco plants, expressing the Silene pratensis (white campion) gene for the precursor of plastocyanin, were analysed with respect to the functionality of the gene product. The gene was found to be expressed in all tissues that were examined due to the strong and constitutive cauliflower mosaic virus 35S promoter. In green tissue the Silene protein was transported into chloroplasts and routed to the chloroplasts lumen, where it was found processed to its mature size. In non-photosynthetic tissue the protein is transported into chromoplasts or leucoplasts. In plants grown in tissue culture the amount of endogenous tobacco plastocyanin was found to be reduced significantly, but the Silene plastocyanin was clearly detectable. When plants were dependent on photosynthesis for growth, due to depletion of sucrose from the medium, still only Silene plastocyanin was present. This strongly suggests that the Silene protein can take over photosynthesis in transgenic tobacco when the endogenous plastocyanin is not present. Silene plastocyanin is considered to be fully functional, since both its transport to the chloroplast and its function in photosynthesis were retained in transgenic tobacco plants.