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K Scherrer

Publications and source records attributed to K Scherrer.

At least 91 records · Page 5Linked to original sources
PubMed1984

Altered globin gene transcription pattern and the presence of a 7-8 kb alpha A globin gene transcript in avian erythroblastosis virus-transformed cells.

Immature chick erythroid cells transformed by avian erythroblastosis virus (AEV) display an altered pattern of globin gene transcription leading to the abortive phenotypic expression of such transcripts. Detectable adult globin gene-specific RNA sequences, confined exclusively to the nucleus, are uniquely of the alpha A type. The alpha A globin-specific sequences occur in transcripts 7-8 kb long from which the 5' contiguous alpha D gene product is absent, and also in fragments smaller than 9S globin mRNA. The implication of this observation for schemes of post-transcriptional regulation of gene expression and viral transformation are discussed.

Alpharetrovirus↗
PubMed1983

The translation of the messenger for the poly(A)-binding protein-associated with translated mRNA is suppressed. A case of cytoplasmic repression in duck erythroblasts.

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the Mr 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.

Animals↗
PubMed1983

Messenger RNA for the 73,000-dalton poly(A)-binding protein occurs as translationally repressed mRNP in duck reticulocytes.

Poly(A)-containing mRNA has been prepared from the polyribosomes and post-polyribosomal mRNP fraction of duck reticulocytes. The coding capacity of the respective mRNA populations has been examined by translation in vitro followed by two-dimensional electrophoresis of the 35S-labeled polypeptides. A detailed analysis of these results is given elsewhere (Imaizumi-Scherrer, M.-T., Maundrell, K., Civelli, O., and Scherrer, K. (1982) Dev. Biol. 93, 126-138). Here, we focus on one of these translation products which migrates as a slightly basic protein of 73,000 molecular weight. By two-dimensional electrophoretic analysis and partial peptide mapping, we show that this protein is indistinguishable from the poly(A)-binding protein. We conclude that the majority of the coding sequences for this protein are translationally repressed in the reticulocyte cytoplasm.

Animals↗
PubMed1983

Differential repression of specific mRNA in erythroblast cytoplasm: a possible role for free mRNP proteins.

Two types of in vivo untranslated 'free' mRNA-protein particles (mRNP) were isolated from duck erythroblast cytoplasm and characterised. Both types, namely the highly purified globin mRNA-specific '20S' mRNP and the '35S' mRNP containing a heterogenous non-globin mRNA population, are not translatable in rabbit reticulocyte lysates, but yield active mRNA upon deproteinisation. In vivo, 90% of globin mRNA is translated, but the majority of mRNA types are found in the inactive mRNP fraction, including fully repressed mRNA species. Searching for the factors controlling differential mRNA repression, we characterised and compared the protein composition of globin and '35S' mRNP using two dimensional gel electrophoresis, in vivo labelling with [35S]methionine and in vivo phosphorylation. The major proteins ubiquitously bound to globin or any other mRNA in the polyribosomes (e.g., the 73 K mol. wt. poly(A) binding protein) were not detected in purified inactive mRNP. In the latter some polypeptides appear to be associated with only one of the two inactive mRNA types while some others are common to both mRNPs. Furthermore, different rates of synthesis and phosphorylation characterize the protein populations of the two types of repressed mRNP. The specificity in composition and metabolism of the populations of polypeptides associated with different subpopulations of inactive cytoplasmic mRNA, as shown here, argues in favour of a role of mRNP proteins in mRNA recognition and selective translational repression, possibly in association with the ScRNA previously found as components of the free mRNP and able to inhibit protein synthesis.

Animals↗
PubMed1983

Determination of the primary sequence of the duck alpha D globin mRNA and comparison of all adult duck and chick globin mRNA sequences.

The nucleotide sequence of the duck alpha D globin mRNA was determined. Its main feature is an exceptionally short 3' non-coding segment of only 46 nucleotides, placed after the coding sequence of 141 codons. The last of the 6 adult globin mRNA of duck and chicken being thus sequenced, a comparison of all their features has become possible. Comparing the duck alpha D mRNA to the related sequence in the chicken, we found greater homology than comparing it to the linked alpha A globin sequence in the same species. Extensive homology can be found for a same globin chain alpha A, alpha D or beta in between different avian species including also the goose and the ostrich; the avian alpha globin chains show a lower degree of sequence conservation in between species than the beta chains. In contrast, within one species the three globin sequences have further diverged. The divergence between the alpha A and alpha D globin within a same species point to individual functional specificity and hence independent evolution and suggest that a mechanism of 'gene conversion' did not operate in between the avian alpha globin genes. Two segments of the amino acid sequence which we named 'A alpha' and 'B alpha' remain homologous in all avian alpha globins; two other regions 'A beta' and 'B beta' are identical in between the beta globins. Segment A is placed at the 5' end of exon II, and segment B at the 3' end of the same exon; some amino acids in those segments are involved in the Heme binding site. Being almost identical in all know mammalian and avian globins of the alpha respectively the beta type, regions A and B seem to represent the best conserved sequences in adult globin mRNA maintained during the divergence of species.

Animals↗
PubMed1982

In vitro transformation by avian erythroblastosis virus recruits cells of erythroid lineage with varying degree of differentiation.

We have previously shown that AEV infection of leukosis-free chickens provokes an erythroleukemia in the infected birds, and that the target cell for virus transformation is most likely an early pro-erythroblast comparable to the BFU-E. The virus infection correlates with a block in differentiation which was studied at a specific cellular gene level, namely that of the globin genes. In live birds, the viral transformation has been found to affect the transcriptional activity to two out of three adult globin genes and the phenotypic expression of the third. Indeed, the alpha A gene that is transcribed is silent as well, since its transcription product is fully eliminated in the nucleus following an "abortive processing". Chick bone marrow cells can also be transformed in vitro by AEV in which case the virus recruits, as targets, cells of the erythroid lineage belonging to a wider spectrum and hence in varying degrees of differentiation. In contrast to the results obtained with in vivo transformed cells, molecular hybridization with globin cDNA probes showed that in the erythroblasts transformed in vitro under tissue culture conditions all the three adult globin genes are transcriptionally active. In fact, low but detectable amounts of globin mRNA sequences are present in both the nuclear and cytoplasmic compartment of the cell prior to exposure to any chemical inducers of red cell differentiation. Judging from our present results, we may propose that AEV transformation in vitro does not shut off out only modulates the already functioning erythroid cell differentiation program.

Alpharetrovirus↗
PubMed1981

Isolation of globin pre-messenger RNA on thiol-agarose by terminally mercurated complementary DNA.

A mercurated DNA complementary to globin mRNA was prepared by the addition of mercurated poly(dC) tails to the 3'-end of the molecule using the enzyme terminal deoxynucleotidyl transferase. The mercurated complementary DNA was retained efficiently on thiol-agarose from which it was eluted by 2-mercaptoethanol. Hybridization of the mercurated probe to globin mRNA led to a specific selection of the latter from a mixed population of RNA through hybrid retention on thiol-agarose. In some pilot experiments this technique was applied for the isolation of globin gene-specific pre-mRNA. Pulse-labeled RNA up to 3 X 10(6) MW was thus isolated with prominent peaks of 1.5 kb ('15 S'), 4.0 kb ('28 S') and up to about 10 kb. Electron microscopical analysis revealed pre-mRNA molecules of up to 1.2 mu (about 4.5 kb) in length isolated by hybridization to (Hg)cDNA; in control experiments, hybridization of this high MW RNA was competed out by highly purified globin mRNA. These data provide another indication for the existence of globin gene transcripts in the 10 kb range, i.e. transcripts larger than the about 1 500 nucleotides long '15 S' pre-mRNA, the substrate to final splicing. Such 'giant' transcripts can be interpreted as either the obligatory primary pre-mRNA or only facultative transcripts of the globin genes.

Animals↗
PubMed1981

Modification of the methylation pattern in the vicinity of the chicken globin genes in avian erythroblastosis virus transformed cells.

Having previously found a reduced transcription of globin genes and an abortive processing of the already transcribed globin pre-mRNA in Avian Erythroblastosis Virus (AEV) transformed cells (1), we compared the genomic DNA of these cells with that of normal chicken erythroblasts, using 32-P-labelled cDNA probes specific for the beta, alpha A and alpha D globin sequences. Restriction endonuclease digestion, electrophoresis of digests in agarose gels, Southern blotting and hybridization were carried out. Our results show that the overall genome organization is not disturbed in the immediate neighbourhood of the adult globin genes; the observed restriction fragments are identical for both DNAs after EcoRI, HindIII, BamHI and XbaI digestion, using the beta, alpha A and alpha D globin cDNA probes. However, we observe specific modifications at some methylation sites in the beta, beta-like and alpha D regions: after HpaII or MspI digestion in the alpha D region and after HhaI digestion in the beta and beta-like region, heavier bands appear in the transformed cell DNA in addition to the ones observed in normal DNA. This implies that, at some specific sites, the transformed cell DNA is more methylated than the normal erythroblast DNA. The possible significance of this observation is discussed.

Alpharetrovirus↗
PubMed1981

Systematic punctuation of eukaryotic DNA by A+T-rich sequences.

Isodenaturation of avian and mammalian DNA of Mr greater than 2.5 X 10(8) in 85% (vol/vol) formamide led to the observation by electron microscopy of A+T-rich zones ("AT-rich linkers") of 300-3000 base pairs which are distributed over the entire genome in a characteristic pattern. Linkers of mean length 800 base pairs are found either isolated or in clusters of about four to six linkers of more heterogeneous size separated on average by 2500 base pairs (in duck DNA). In between clusters, single linkers segment the DNA at distances of 10 to more than 100 kilobase pairs, with a majority in the range of 10-30 kilobase pairs. An analogous organization of linkers is found in rat and mouse DNA. The internal organization of the clusters varies, however, in a fashion that might be related to the large amount of light satellite DNA in the mouse and its apparent absence in rat and avian DNA. It is possible to fragment the DNA under appropriate conditions by the single-strand-specific nuclease S1 at the site of these A+T-rich zones and to obtain, on alkaline sucrose gradients, a bimodal pattern of DNA fragments of the size corresponding to the pattern observed by electron microscopy. The implications of this observation for DNA organization, chromatin structure, units of transcription and replication, and possible targets of A+T-specific drugs are discussed.

Adenine↗