Biomedical subjects

K Scherrer

Publications and source records attributed to K Scherrer.

At least 73 records · Page 4Linked to original sources

PubMed1988

The poly(A)-binding protein facilitates in vitro translation of poly(A)-rich mRNA.

To investigate the role of the 73-kDa poly(A)-binding protein in protein synthesis, the effect of the addition of homo-polyribonucleotides on the translation of polyadenylated and non-adenylated mRNA was studied in the rabbit reticulocyte lysate. Poly(A) was found to be the most effective polynucleotide in inhibiting duck-globin mRNA translation, whereas it had no effect on the translation of polyribosomal duck-globin mRNP, or on the endogenous synthesis of the rabbit reticulocyte lysate. The translation of poly(A)-free mRNA was not affected by the addition of poly(A). Furthermore, we found that the inhibiting effect of poly(A) can be reversed by addition of purified poly(A)-binding protein. It is thus likely that the 73-kDa poly(A)-binding protein is an essential factor necessary for poly(A)-rich mRNA translation.

Animals↗

PubMed1988

The duck beta-globin gene cluster contains a single enhancer element.

An erythroid-specific enhancer was previously identified in the 3'-flanking region of the beta adult gene in chicken and duck, by transfection into AEV transformed chicken erythroblasts. Here we show that the duck enhancer is equally active in erythroid human K562 cells, presenting an embryonic/fetal program of globin gene expression. Furthermore, no other enhancer was found within the 20 kb of DNA including four beta-like globin genes as well as a 1.5 kb upstream and a 3 kb downstream sequence.

Animals↗

PubMed1988

Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts.

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A+) mRNA detection by hybridization to [3H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21,000-120,000 range. The Mr 120,000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21,000 protein acceptor is abundant in PMS and a Mr 34,000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.

Animals↗

PubMed1988

Isolation and characterization of stable nuclear matrix preparations and associated DNA from avian erythroblasts.

A novel procedure for isolation of nuclear matrices from chicken erythroblast cells was elaborated. The influence of variations in the isolation procedure on structural integrity and morphology of nuclear matrices as well as on properties of the nuclear matrix-associated DNA fractions was investigated. The incubation of isolated nuclei in the presence of Cu2+ ions provided significant stabilization of the nuclear matrix. Copper treatment of nuclei did not affect the properties of the nuclear skeleton-associated DNA fraction. In both copper-stabilized as well as unstabilized nuclei, nuclear matrix-attached DNA was digested to the same extent with nucleolytic enzymes, and could be totally removed from nuclear matrices by 2 M NaCl-2 M urea treatment. The fine morphology of the nuclear matrix did not change after extraction of nuclear skeleton-associated DNA fragments. In the presence or absence of copper ions, matrix DNA was found to be qualitatively different compared with total DNA, in particular with respect to the representation of specific repetitive sequences of the chicken beta globin gene domain.

Animals↗

PubMed1988

The association of prosomes with some of the intermediate filament networks of the animal cell.

The small RNP complexes of defined morphology and biochemical composition termed prosomes, first isolated from the cytoplasm associated with repressed mRNA (Martins de Sa, C., M.-F. Grossi de Sa, O. Akhayat, F. Broders, and K. Scherrer. J. Mol. Biol. 1986. 187:47-493), were found also in the nucleus (Grossi de Sa, M.-F., C. Martins de Sa, F. Harper, O. Coux, O. Akhayat, P. Gounon, J. K. Pal, Y. Florentin, and K. Scherrer. 1988. J. Cell Sci. 89:151-165). Immunofluorescence, immunoelectron microscopy, and immunochemical studies using mAbs directed against some of the prosomal proteins of duck erythroblasts indicate that in the cytoplasm of HeLa and PtK cells, prosome antigens are associated with the intermediate filament network of the cytokeratin type.

Actin Cytoskeleton↗

PubMed1988

Cytolocalization of prosomes as a function of differentiation.

Prosomes, ubiquitous ribonucleoprotein (RNP) particles of defined biochemical and morphological structure, first isolated as a subcomplex of the repressed globin mRNP in avian and mouse erythroblasts, were also found in the cytoplasm of other vertebrates associated with other mRNAs. Here we show that prosomes are also present in the cell nucleus and, furthermore, that the cytolocalization of specific prosomal peptides is a function of differentiation. Four monoclonal antibodies, raised against the duck prosomal proteins, p27K, p28K, p29K and p31K (K = 10(3) Mr) react to variable degree with prosomes of chicken, mouse, and human cells. Immunocytochemical and biochemical analyses show that all four antigens are present in both the cytoplasm and the nucleus of avian erythroblasts and avian erythroblastosis virus (AEV)-transformed erythroleukaemic cells. Interestingly, the prosomes disappear in the course of the terminal differentiation of erythroblasts to mature erythrocytes. Although all the four prosomal antigens tested are present in both the nuclear and cytoplasmic compartments, slight differences in the immunofluorescent patterns indicate that each antigen may have a particular cytological distribution that varies in the course of differentiation.

Animals↗

PubMed1988

Differential cytolocalization of prosomes in axolotl during oogenesis and meiotic maturation.

The prosomes, a novel type of small RNA-protein complex previously characterized in avian and mammalian cells, were isolated from axolotl (Ambystoma mexicanum) oocytes and identified by sedimentation analysis and protein composition. The prosomal nature of these particles was further ascertained by immunoblot analysis with anti-duck prosome monoclonal antibodies. By in vitro [35S]methionine labelling, de novo synthesis of prosomal proteins could be detected neither during oogenesis nor meiotic maturation. The results obtained by both indirect immunofluorescence and immunoblot analyses demonstrated a dramatic change in the localization of prosomal antigens during oocyte development. They were initially detected in the oocyte cytoplasm, during oocyte growth. At the end of vitellogenesis (stages V-VI), they entered the nucleus (germinal vesicle) and were accumulated there to the highest concentration. During oocyte maturation, after nuclear envelope breakdown, prosomal antigens were found to be localized again in the cytoplasm, until fertilization. No specific localization of prosomal antigens in mature oocytes, unfertilized and fertilized eggs was observed within the oocyte cytoplasm in relation to the cytoplasmic rearrangements leading to grey crescent formation.

Ambystoma mexicanum↗

PubMed1988

Presence and distribution of specific prosome antigens change as a function of embryonic development and tissue-type differentiation in Pleurodeles waltl.

The prosomes, biochemically well characterized small RNA-protein complexes, found associated with mRNA in all eukaryotic cells tested, have been identified as maternal components in sea urchin and chick embryos. In this study, we investigated their presence and cytolocalization in the oocytes and embryos of Pleurodeles waltl by immunoblot analysis and immunofluorescence, using monoclonal antibodies prepared against duck prosome proteins. Of the four antibodies tested, three recognized the corresponding antigens in oocyte total protein extracts. Immunofluorescence analysis, using the three prosomal antibodies, demonstrated a drastic change in the localization of the prosome antigens, which changed from the cytoplasm to the nucleus during oogenesis. In the nucleus, in diplotene stages, prosomal antigens appeared to be associated with the lampbrush chromosomes and the nuclear matrix. During embryogenesis, the subcellular distribution of the prosome antigens was a function of development and differentiation: in the cleavage stages up to the mid-blastula they were localized in the cytoplasm and on the plasma membrane, while in the late blastula, gastrula and neurula they were in the nucleus. Interestingly, one of the prosome antigens, p31K, was found to be in a different location in certain cells in the animal pole of the mid-blastula and was absent in the neural tissue in the neurula. In still later stages, in the free-swimming larva, all three antigens were localized in the cytoplasm, specifically in certain cell types in the epidermal tissues. Furthermore, they were sectorially distributed in the cytoplasm. These data taken together indicate the possible presence of tissue-type-specific prosome antigens in Pleurodeles. Differentiation-dependent subcellular localization of the prosome antigens suggests a cell-compartment-related multiple function of prosomes.

Animals↗

PubMed1987

A new type of prosome-like particle, composed of small cytoplasmic RNA and multimers of a 21-kDa protein, inhibits protein synthesis in vitro.

A large fraction of the translationally repressed non-globin messenger RNA in duck erythroblasts is present in non-polyribosomal free mRNP structures which sediment in the 30-40-S range ('35 S'). In 0.5 M KCl, they form core complexes which show a pronounced peak at about 32 S containing mRNA and a discrete spherical RNP particle with a diameter of about 12 nm and the typical morphology of a prosome [H.-P. Schmid et al. (1984) EMBO J. 3, 29-34]. Buoyant density measurements and chromatography on oligo(dT)-cellulose indicate that this particle is bound to mRNA; it can be released from the mRNA by treatment of the free mRNP fraction with SDS. This prosome-like particle inhibits the translation of mRNA in vitro. It is composed primarily of multimers of a single 21-kDa protein and at least one species of RNA of about 80-100 nucleotides. It is resistant to dissociation by 2 M CS2SO4 and 1% SDS; the 21-kDa protein is not attacked by proteinase K unless the particle is extracted with phenol prior to treatment with the protease. The small RNA moiety of the particle hybridizes to the poly(A)-rich mRNA derived from the free mRNPs, as well as to polyribosomal mRNA. These data indicate that prosomes may serve to regulate mRNA translation; they show furthermore that prosome-like particles (about 600 kDa mass) may be built of up to 25 molecules of a single specific protein, rather than of the entire set of about 20 prosomal proteins previously identified.

Animals↗

PubMed1987

Co-evolution of base composition and codon usage in Xenopus laevis and human globin genes with long-range DNA organization of their genome.

Eucaryotic DNA is punctuated by many A+T-rich segments that we named A+T-rich linkers. Two types of these A+T-rich linkers can be distinguished: (i) isolated A+T-rich linkers, and (ii) A+T-rich linkers crowded in clusters. We have analysed the distribution of A+T-rich linker across the alpha- and beta-globin gene domain in Xenopus laevis and human genomes using isodenaturation and electron microscopy. Comparison of our data with those previously obtained for the avian globin genes leads us to conclude that genes can be harboured indifferently in either domain. A correlation is established between the presence of A+T-rich linker inside introns and flanking regions and the A+T content of the coding sequence. For the coding sequence, a high A+T content is strongly correlated with high A+T content in the codon's third position and weakly in the first position.

Animals↗

PubMed1987

Transcription of the alpha globin gene domain in normal and AEV-transformed chicken erythroblasts: mapping of giant globin-specific RNA including embryonic and adult genes.

The genomic domain of about 20 kbp of the chicken alpha-type globin genes, framed by AT-rich linkers (ATRLs; Moreau et al. 1982) and repetitive sequences (Broders et al. 1986), was cut into 13 fragments and subcloned. The in vitro labelled individual restriction fragments were used to test the extent of the transcribed domain by blot-hybridization of nuclear RNA in large excess from normal adult chicken and Avian Erythroblastosis Virus (AEV)-transformed erythroblasts. In both these types of cells, the AT-rich segments situated 6 kbp upstream of the first gene as well as all the domain including the embryonic pi and the adult alpha D and alpha A genes down to the AT-rich segment placed 3 kbp downstream were found to be transcribed. Electrophoresis of nuclear RNA, Northern blotting and hybridization with most of the nick-translated DNA probes revealed in all cases the presence of heterogeneous globin RNA molecules in the 3-12 kb range, as well as some distinct RNA bands. Single-stranded RNA probes of some genomic segments indicated asymmetrical transcription of the minus strand. A 12 kb globin-specific RNA including the pi and alpha A genes but not the intervening alpha D gene was observed in AEV-transformed cells: it includes sequences located far upstream and downstream from the alpha globin genes and might represent a processing product of a full length transcript spanning the whole domain. Reverse transcription by extension of primers placed in the first exon of each of the three globin genes confirmed the presence of continuous transcripts of the domain including the two adult and the embryonic globin genes.

Alpharetrovirus↗

PubMed1987

A transcriptional enhancer located between adult beta-globin and embryonic epsilon-globin genes in chicken and duck.

We have detected a transcriptional enhancer sequence downstream from the adult beta-globin (beta A-globin) genes of chicken and duck. DNA segments from the beta-globin coding and flanking sequences were cloned into expression vectors containing the SV40 promoter linked to either the T antigen gene or the cat gene. The expression of these genes was measured in a chicken erythroid cell line transfected with the recombinant plasmids. We found that segments located about 400 bp downstream from the poly(A) site of both the chicken and duck beta A-globin genes (and about 1.5 kb upstream from the embryonic epsilon-globin gene) stimulate transcription of the test genes about five-fold. In chicken essentially the same segment was also found by others to act as an erythroid cell-specific enhancer [Hesse et al., Proc. Natl. Acad. Sci. USA 83 (1986) 4312-4316; Choi and Engel, Nature 323 (1986) 731-734]. The sequence containing this enhancer is conserved in evolution. A high degree of homology, reaching 84% in a segment 180 bp in length, was found between chicken and duck despite an evolutionary divergence of 70 myr.

Animals↗

PubMed1986

Replication origins are attached to the nuclear skeleton.

DNA fragments containing replication origins (oriDNA) were isolated from a chicken erythroblast cell line by a modified procedure of Zannis-Hadjopoulos et al. and studied in the renaturation reaction driven by either total or nuclear matrix DNA (nmDNA) from the same cells or from mature erythrocytes. We found that the unique sequences of nmDNA from erythroblasts (5 kb long) represented a specific subset of sequences constituting about a quarter of total DNA unique sequences, while the erythrocyte nmDNA 5 kb fragments constitute only about one tenth of total unique DNA and all are recovered among erythroblast nmDNA. Virtually all oriDNA sequences are present in the fraction of erythrocyte nmDNA. Thereafter, the putative positions of replication origins within the alpha-globine gene domain have been mapped by hybridization experiments. They were found to coincide with the previously established positions of permanent sites of DNA attachment to the nuclear matrix.

Animals↗

PubMed1986

Prosomes. Ubiquity and inter-species structural variation.

The "prosomes", a novel type of ubiquitous ribonucleoprotein particle of extraordinary stability and of defined electron microscopical structure, have been characterized in several cell types and species. Identified as a 19 S sub-component of free mRNA-protein complexes, including globin and other repressed mRNA, in the cytoplasm of duck, mouse and HeLa cells, they were previously found to inhibit protein synthesis in vitro. In all cells studied, electron microscopy shows an identical, seemingly ring-like but rather raspberry-shaped particle of 12 nm diameter, resistant to EDTA and 1% (w/v) Sarkosyl. Two-dimensional electrophoretic analysis of prosomal proteins shows a characteristic pattern in the 19,000 to 35,000 Mr range of pI 4 to 7, with an additional 56,000 Mr component specific to avian species. The prosomes found in globin mRNA-protein complexes contain about 25 protein components, 16 of which have identical molecular weight and pI values in duck and mouse, and which are also found in the prosomes of the heterogeneous free mRNPs of HeLa cells. Seral and monoclonal antibodies raised in mice against the prosomes of duck erythroblasts cross-react with some of the proteins of the mouse and HeLa cell particles. Prosomes isolated from duck and mouse globin mRNP, both contain small cytoplasmic RNAs of 70 to 90 nucleotides, which represent about 15% of the particle mass. The molecular weight and the 3'-terminal oligonucleotide of each one of these small cytoplasmic RNAs are identical in the two animal species; fingerprints of their oligonucleotides generated by RNase T1 show that more than 80% of spots are identical. In contrast, the prosomes of HeLa cells, associated with a large population of repressed mRNA, contain at least 12 small cytoplasmic RNA species. All prosomal RNAs tested so far hybridize to mRNA. The data available indicate that prosomes constitute a novel class of ubiquitous cellular ribonucleoprotein complexes, present in the nucleus and cytoplasm that, in its structural variations shown here, reflects function and species.

Animals↗

PubMed1986

Conservation and variation in the large scale organisation of the globin gene domains of duck and chicken.

The genomic DNA of cloned recombinants containing the duck globin genes was compared to that of the analogous domains of the chicken. A 36 kb insert including the three alpha-type globin genes was isolated from a newly prepared duck genomic library in the cosmid PJB8; another recombinant contained a 45 kb insert with the four beta globin genes. In the alpha globin gene domain, the relative positions of genes, of repetitive sequences, and of the A + T-rich segments (AT-rich linkers, ATRLs) which frame the gene cluster (Moreau et al. 1982), were found to be closely maintained between duck and chicken. Although ATRLs and repetitive sequences also frame the gene cluster in the beta globin domains of duck and chicken, there is more genetic drift in their relative positions than in the alpha domain. It is of interest that several repetitive DNA segments were detected in the chicken beta globin domain which do not exist in corresponding positions in the duck. In view of the strict conservation in both species of genes and their relative positions in the cluster, this observation seems to exclude a simple function of repetitive sequences in the control of individual genes. The data are discussed with regard to the possible significance of repetitive and AT-rich DNA segments in genome organisation and function.

Animals↗

PubMed1986

Correlations of repetitive and AT-rich DNA segments within the chicken globin gene domains.

The repetitive DNA segments were mapped within a 30 Kbp genomic domain including (in 5' to 3' order) the chicken embryonic pi and adult alpha D (minor) and alpha A (major) globin genes. Two repeats map 5 and 8 Kbp upstream from the embryonic pi gene and another 3 Kbp downstream of the adult alpha A gene. These repetitive DNA sequences are placed within, or immediately adjacent to the AT-rich DNA segments framing this domain. Similar correlations exist also within the chicken beta globin gene domain. The positions of these AT-rich and repetitive DNA segments framing the alpha globin gene domain also correlate with other already explored features of long range DNA organisation, as clusters of sites of DNAse I hypersensitivity and differential methylation, sites of Matrix-DNA attachment, and with the beginning and end of the transcribed domain.

Adenine↗

PubMed1985

AT-rich linkers in long range organisation of Drosophila DNA.

AT-rich segments were mapped in 6 different domains of the Drosophila melanogaster genome by partial denaturation of cloned genomic DNA segments and observation in the electron microscope (EM). As found in a previous investigation (Moreau et al., 1982), three types of AT-rich segments could be distinguished: 1) AT-rich linkers with a very high AT content, 600 +/- 200 bp long; 2) clusters of such AT-linkers extending over up to 10 Kbp and 3) AT-rich stretches which are shorter and of lower AT content compared to AT-linkers. Six genes previously localized in these domains were found to lie in relatively GC-rich segments framed by AT-rich elements of the 3 types: the Larval Serum Protein LSP-1 alpha gene is framed by 2 AT-linkers, the LSP-1 beta and LSP-2 genes by two AT-rich stretches, an the LSP-1 gamma gene by a cluster and a stretch. The 55 Kbp genomic segment encompassing the P6 gene contains and AT-cluster of about 15 Kbp and several AT-linkers and AT-rich stretches. The 85 Kbp domain containing the P1 gene includes 3 AT-rich clusters of about 10 Kbp each framing GC-rich domains punctuated by AT-linkers and stretches. This study shows that AT-mapping allows a rapid diagnosis of large genomic DNA domains in relation to the AT-rich segments which, possibly, are of significance with regard to genome organisation and function.

Animals↗

PubMed1984

The prosome: an ubiquitous morphologically distinct RNP particle associated with repressed mRNPs and containing specific ScRNA and a characteristic set of proteins.

A novel ribonucleoprotein (RNP) particle showing a highly compact and characteristic structure in the electron microscope was found associated with globin and other repressed mRNA in the cytoplasm of duck, mouse and HeLa cells. This 19S complex is of extraordinary stability: dissociated by 0.5 M KCl or EDTA from the (still repressed) core globin mRNP, it can be purified on gradients containing 1% Sarkosyl, and resists (unfixed) caesium sulphate-dimethylsulphoxide density centrifugation. Its density of 1.31 g/cm3 indicates an RNP complex with a 15% RNA component. In mouse and duck it contains approximately 10 proteins in the 20 000-30 000 mol. wt. range, a few components of 50 000-70 000 mol. wt., and two specific small cytoplasmic RNAs (ScRNA) of 70-90 nucleotides. Both of these RNAs have identical 3'-terminal oligonucleotides. We propose the name 'prosome' for this ScRNP particle which somehow participates in negative control of mRNA translation, and we believe will prove to be ubiquitous to animal species.

Animals↗