François-Xavier Wilhelm

A 5′–3′ long‐range interaction in Ty1 RNA controls its reverse transcription and retrotransposition

LTR‐retrotransposons are abundant components of all eukaryotic genomes and appear to be key players in their evolution. They share with retroviruses a reverse transcription step during their replication cycle. To better understand the replication of retrotransposons as well as their similarities to and differences from retroviruses, we set up an in vitro model system to examine minus‐strand cDNA synthesis of the yeast Ty1 LTR‐retrotransposon. Results show that the 5′ and 3′ ends of Ty1 genomic RNA interact through 14 nucleotide 5′–3′ complementary sequences (CYC sequences). This 5′–3′ base pairing results in an efficient initiation of reverse transcription in vitro. Transposition of a marked Ty1 element and Ty1 cDNA synthesis in yeast rely on the ability of the CYC sequences to base pair. This 5′–3′ interaction is also supported by phylogenic analysis of all full‐length Ty1 and Ty2 elements present in the Saccharomyces cerevisiae genome. These novel findings lead us to propose that circularization of the Ty1 genomic RNA controls initiation of reverse transcription and may limit reverse transcription of defective retroelements.

Transfer RNA binding protein in the nucleus of Saccharomyces cerevisiae

A yeast nuclear protein that binds to tRNA was identified using a RNA mobility shift assay. Northwestern blotting and N‐terminal sequencing experiments indicate that this tRNA‐binding protein is identical to zuotin which has previously been shown to bind to Z‐DNA [(1992) EMBO J. 11, 3787–37961. Labeled TRNA and poly(dG‐m5dC) stabilized in the Z‐DNA form identify the same protein on a Northwestern blot. In a gel retardation assay poly(dG‐m5dC) in the Z‐form strongly diminishes the binding of tRNA to zuotin. These studies establish that zuotin is able to bind to both tRNA and Z‐DNA. Zuotin may be transiently associated with tRNA in the nucleus of yeast cells and play a role in its processing or transport to the cytoplasm.

A transposon-like DNA fragment interrupts a Physarum polycephalum histone H4 gene

A recombinant DNA library was screened for histone H4 genes using a sea urchin probe. One recombinant was analysed by restriction enzyme mapping and Southern blotting. The complete DNA sequence of the H4 histone locus was determined. An 86 base pair interrupting sequence was found within the histone H4 coding sequence. The inserted DNA fragment has some characteristics of a transposable element.

Comparison of the DNA repeat length in H1- and H5-containing chromatin (Mature hen erythrocytes, immature chick erythroid cells and hen liver)

The variability of the DNA repeat length in chromatin of eukaryotes is now well documented [ 1-11 ]. The size of the repeating unit ranges from 154 base pairs in Aspergillus nidulans [2] to 241 base pairs in the chromatin of sea urchin sperm [7]. As yet the origin of this phenomenon has not been explained but a few possible causes of the observed variability have been excluded [7-9]; indeed it has been shown that the rate of cell division, the stage in the cell cycle, the genomic activity, the phosphorylation of H1 and the acetylation of histone Ha and H4 could not be correlated to the variation of the repeat length of chromatin. Noll [1 ] and Morris [2] have suggested that there may be a relationship between the structure of historic H1 (as expressed by the content of basic amino acids) and the length of the DNA repeat in chromatin. As a test of this hypothesis Morris [3] has compared the structure of hen liver and hen erythrocyte chromatin in which a large fraction of the histone H1 is replaced by the lysine rich histone Hs; the finding that the repeat length of hen erythrocyte chromatin was longer than that of hen liver chromatin has suggested that there may be a correlation between the increased length of the repeat of hen erythrocyte chromatin and the presence of Hs. In the present work we have compared the DNA repeat length from mature erythroid cells of hen, immature erythroid cells of three-day old chicks [I 2] and hen liver chromatin. We show that the immature chick erythroid cell chromatin has the same repeat length as the hen liver chromatin, both being smaller than that of adult hen erythrocyte chromatin. Since the erythroid cells of chick contain the histone Hs whereas the hen liver cells do not, our results suggest that there is no direct relationship between the presence of Hs and the increased length of the repeating unit in the hen erythrocytes.

The central PPT of the yeast retrotransposon Ty1 is not essential for transposition.

The yeast retrotransposon Ty1 has structural and functional similarities to retroviruses. We report here that, as in retroviruses, the plus-strand DNA of Ty1 is synthesized as two segments. A central DNA flap is formed during reverse transcription consecutive to elongation (with strand displacement) of the upstream segment beyond the central polypurine tract (cPPT) until the replication machinery is stopped at the central termination sequence. Comparison of wild-type and cPPT-mutant Ty1 elements shows that the mutant element lacking the central DNA flap is only twofold defective in transposition.

The structure of Sipunculus nudus erythrocyte chromatin

Abstract The structure of the Sipunculus erythrocyte chromatin has been characterized by electron microscopy and nuclease digestion (staphylococcal nuclease and pancreatic nuclease). Contrary to previous results [1], we were able to isolate and characterize a histone H2B in sipunculid nuclei. Though the histones H2A and H2B were markedly different from their vertebrate homologues, the subunit structure of the chromatin is the same. But the length of the repeat unit of DNA in the chromatin, is 177 ± 5 bp for the sipunculid erythrocyte nuclei, close to that reported for the chromatin of some lower eukaryotes.

Nucleosome core particles can be reconstituted using mixtures of histones from two eukaryotic kingdoms

It is now well known that nucleosome core particles can be reconstituted by mixing the four histones H2a, H2b, H3, H4 and the DNA under appropriate conditions (reviewed [ 11). The most commonly used reconstitution procedure involves mixing of the histones and DNA in high salt (2 M NaCl) followed by a stepwise decrease of the ionic strength down to G 0.25 M NaCl. The nucleosome core particles reconstituted in this way have properties very similar to the native ones as judged by electron microscopy [Z], sedimentation velocity [3], nuclease digestion [4] and the ability to impose a constraint to circular DNA [2]. There is much evidence that the histones H3, H4 and H2a, H2b play distinct roles in the assembly and stability of the nucleosome [2,5-81. Histones H3, H4 have a central role in the formation of the nucleosome and are able to generate particles with properties similar to that of the nucleosome, whereas histones H2a, H2b are necessary to complete and stabilize the nucleosome. The most conserved histones are H3 and H4 and this may be related to their very precise role in chromatin structure. In contrast H2a and H2b have not been as well conserved during evolution. Moreover, variants of H2a and H2b appear sometimes during embryogenesis [9]. This suggests that H2a and H2b do not play a static structural role but that they may play different roles according to the functional requirement of the cell. It has also been suggested that the difference in nucleosomal DNA content observed for the various

Sequence Comparison of the Ty1 and Ty2 Elements of the Yeast Genome Supports the Structural Model of the tRNAiMet–Ty1 RNA Reverse Transcription Initiation Complex

In the reverse transcription initiation complex of the yeast Ty1 retrotransposon, interaction between the template RNA and primer tRNAiMet is not limited to base pairing of the primer binding site (PBS) with ten nucleotides at the 3′ end of tRNAiMet but three regions named boxes 0, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Sequence comparison of 33 Tyl elements and 13 closely related Ty2 elements found in the yeast genome shows that the nucleotide sequence of all elements is highly conserved in the region spanning the PBS and the three boxes. Since this domain of the template RNA encodes a portion of protein TyA, we have calculated its amino acid profile and its nucleotide profile to evaluate the role played by nucleotide sequence conservation in the selection for TyA function and in the maintenance of base pairing interactions for the priming function of Tyl RNA. Our results show that the nucleotide sequence conservation of Ty1 RNA is constrained not only by selection for Ty1 function but also by maintenance of a given nucleotide sequence able to base pair with the tRNAiMet in the primer–template initiation complex.