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H P Roest

Publications and source records attributed to H P Roest.

10 recordsLinked to original sources
PubMed2000

Characterization of mRAD18Sc, a mouse homolog of the yeast postreplication repair gene RAD18.

The RAD18 gene of the yeast Saccharomyces cerevisiae encodes a protein with ssDNA binding activity that interacts with the ubiquitin-conjugating enzyme RAD6 and plays an important role in postreplication repair. We identified and characterized the putative mouse homolog of RAD18, designated mRAD18Sc. The mRAD18Sc open reading frame encodes a 509-amino-acid polypeptide that is strongly conserved in size and sequence between yeast and mammals, with specific conservation of the RING-zinc-finger and the classic zinc-finger domain. The degree of sequence conservation between mRAD18Sc, RAD18, and homologous sequences identified in other species (NuvA from Aspergillus nidulans and Uvs-2 from Neurospora crassa) is entirely consistent with the evolutionary relationship of these organisms, strongly arguing that these genes are one another's homologs. Consistent with the presence of a nuclear translocation signal in the amino acid sequence, we observed the nuclear localization of GFP-tagged mRAD18Sc after stable transfection to HeLa cells. mRNA expression of mRAD18Sc in the mouse was observed in thymus, spleen, brain, and ovary, but was most pronounced in testis, with the highest level of expression in pachytene-stage primary spermatocytes, suggesting that mRAD18Sc plays a role in meiosis of spermatogenesis. Finally, we mapped the mRAD18Sc gene on mouse chromosome 6F.

Amino Acid Sequence↗
PubMed1999

The ubiquitin system in gametogenesis.

Ubiquitin is a ubiquitous and highly conserved protein of 76 amino acid residues, that can be covalently attached to cellular acceptor proteins. The attachment of ubiquitin to target proteins is achieved through a multi-step enzymatic pathway, which involves activities of ubiquitin-activating E1 enzymes, ubiquitin-conjugating E2 enzymes, and ligating E3 enzymes. Mono- or poly-ubiquitination of proteins can lead to protein degradation or modification of protein activity. Many components of the complex ubiquitin system show remarkable evolutionary conservation, from yeast to mammalian species. The ubiquitin system is essential to all eukaryotic cells. Among others, several signal transduction cascades show involvement of the ubiquitin system, but there are currently little data supporting a specific role of the ubiquitin system in hormonal control of reproduction. Interestingly, during gametogenesis, many specialized and important aspects of the ubiquitin system become apparent. Components of the ubiquitin system appear to be involved in different steps and processes during gametogenesis, including control of meiosis, and reorganization of chromatin structure.

Animals↗
PubMed1999

Histone ubiquitination and chromatin remodeling in mouse spermatogenesis.

Male infertility in HR6B knockout mice is associated with impairment of spermatogenesis. The HR6B gene is a mammalian, autosomal homolog of the Saccharomyces cerevisiae gene Rad6 encoding a ubiquitin-conjugating enzyme. In addition, X-chromosomal HR6A has been identified, in human and mouse. RAD6 in yeast is required for a variety of cellular functions, including sporulation, DNA repair, and mutagenesis. Since RAD6 and its mammalian homologs can ubiquitinate histones in vitro, we have investigated the pattern of histone ubiquitination in mouse testis. By immunoblot and immunohistochemical analysis of wild-type mouse testis, a high amount of ubiquitinated H2A (uH2A) was detected in pachytene spermatocytes. This signal became undetectable in round spermatids, but then increased again during a relatively short developmental period, in elongating spermatids. No other ubiquitinated histones were observed. In the HR6B knockout mice, we failed to detect an overt defect in the overall pattern of histone ubiquitination. For somatic cell types, it has been shown that histone ubiquitination is associated with destabilization of nucleosomes, in relation to active gene transcription. Unexpectedly, the most intense uH2A signal in pachytene spermatocytes was detected in the sex body, an inactive nuclear structure that contains the heterochromatic X and Y chromosomes. The postmeiotic uH2A immunoexpression in elongating spermatids indicates that nucleosome destabilization induced by histone ubiquitination may play a facilitating role during histone-to-protamine replacement.

Animals↗
PubMed1998

Knockout mouse model and gametogenic failure.

To evaluate the function of a defined gene in gametogenesis, exciting opportunities are offered by the introduction of techniques to generate knockout mice. In this short article, we briefly describe a few gene knockout mouse models, which show a phenotype that involves impairment of gametogenesis and/or fertility. The focus will be on the mHR6B gene knockout mouse, which shows male infertility. The mHR6B gene encodes an ubiquitin-conjugating enzyme, and the data point to an important role of the ubiquitin pathway in gametogenesis.

Animals↗
PubMed1997

A Rhizobium leguminosarum biovar trifolii locus not localized on the sym plasmid hinders effective nodulation on plants of the pea cross-inoculation group.

Introduction of the Sym plasmid pRL1JI into the cured Rhizobium leguminosarum bv. trifolii strain RCR5 resulted in a strain, designated RBL5523, that was expected to nodulate plants of the pea cross-inoculation group. However, effective nodulation occurred only on Vicia sativa plants, not on V. hirsuta or Pisum sativum. After random Tn5 mutagenesis, a derivative of RBL5523 was isolated that effectively nodulated and fixed nitrogen on P. sativum and V. hirsuta. Characterization of the mutant, RBL5787, indicated the cell surface components, extracellular polysaccharides, lipopolysaccharides, and outer membrane proteins, as well as the pattern of Nod metabolites, were indistinguishable from those of the parental strain. To obtain an indication of the function of the mutated locus, the flanking regions were sequenced and used to perform searches in protein and nonredundant nucleotide data-bases. No significant similarity or homology with any known sequence was detected.

Molecular Sequence Data↗
PubMed1996

Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in mice causes male sterility associated with chromatin modification.

The ubiquitin-conjugating yeast enzyme RAD6 and its human homologs hHR6A and hHR6B are implicated in postreplication repair and damage-induced mutagenesis. The yeast protein is also required for sporulation and may modulate chromatin structure via histone ubiquitination. We report the phenotype of the first animal mutant in the ubiquitin pathway: inactivation of the hHR6B-homologous gene in mice causes male infertility. Derailment of spermatogenesis becomes overt during the postmeiotic condensation of chromatin in spermatids. These findings provide a parallel between yeast sporulation and mammalian spermatogenesis and strongly implicate hHR6-dependent ubiquitination in chromatin remodeling. Since heterozygous male mice and even knockout female mice are completely normal and fertile and thus able to transmit the defect, similar hHR6B mutations may cause male infertility in man.

Amino Acid Sequence↗
PubMed1996

Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification.

RAD6, a member of the expanding family of ubiquitin-conjugating (E2) enzymes, functions in the so-called "N-rule" protein breakdown pathway of Saccharomyces cerevisiae. In vitro, the protein can attach one or multiple ubiquitin (Ub) moieties to histones H2A and B and trigger their E3-dependent degradation. Rad6 mutants display a remarkably pleiotropic phenotype, implicating the protein in DNA damage-induced mutagenesis, postreplication repair, repression of retrotransposition, and sporulation. RAD6 transcription is strongly induced upon UV exposure and in meiosis, suggesting that it is part of a damage-induced response pathway and that it is involved in meiotic recombination. It is postulated that the protein exerts its functions by modulating chromatin structure. Previously, we have cloned two human homologs of this gene (designated HHR6A and HHR6B) and demonstrated that they partially complement the yeast defect. Here we present a detailed characterisation of their expression at the transcript and protein levels. Both HHR6 proteins, resolved by 2-dimensional immunoblot analysis, are expressed in all mammalian tissues and cell types examined, indicating that both genes are functional and constitutively expressed. Although the proteins are highly conserved, the UV induction present in yeast is not preserved, pointing to important differences in damage response between yeast and mammals. Absence of alterations in HHR6 transcripts or protein upon heat shock and during the cell cycle suggests that the proteins are not involved in stress response or cell cycle regulation. Elevated levels of HHR6 transcripts and proteins were found in testis. Enhanced HHR6 expression did not coincide with meiotic recombination but with the replacement of histones by transition proteins. Immunohistochemistry demonstrated that the HHR6 proteins are located in the nucleus, consistent with a functional link with chromatin. Electron microscopy combined with immunogold labeling revealed a preferential localisation of HHR6 in euchromatin areas, suggesting that the protein is associated with transcriptionally active regions. Our findings support the idea that both HHR6 genes have overlapping, constitutive functions related to chromatin conformation and that they have a specific role in spermatogenesis, involving Ub-mediated histone degradation.

Animals↗
PubMed1995

Isolation and characterization of ropA homologous genes from Rhizobium leguminosarum biovars viciae and trifolii.

ropA encodes a 36-kDa outer membrane protein of Rhizobium leguminosarum bv. viciae strain 248 which constitutes the low-M(r) part of antigen group III (R.A. de Maagd, I.H.M. Mulders, H.C.J. Canter Cremers, B.J.J. Lugtenberg, J. Bacteriol. 174:214-221, 1992). We observed that genes homologous to ropA are present in strain 248 as well as in other R. leguminosarum strains, and we describe the cloning and characterization of two of these genes. Sequencing of a 2.2-kb Bg/II fragment from R. leguminosarum bv. viciae strain 248 that hybridizes with ropA revealed one large open reading frame of 1,074 bp encoding a mature protein of 38.096 kDa. Homology between this gene and ropA is 91.8% on the DNA level. Homology on the amino acid level is only 69.9% as a result of a frameshift. On the basis of homology and immunochemical characteristics, we conclude that this gene encodes the high-M(r) part of the outer membrane protein antigen group III that is repressed during symbiosis. We named this gene ropA2. The second gene that we cloned was the ropA homologous gene of R. leguminosarum bv. trifolii strain LPR5020. Except for amino acid 43, the N-terminal part of the corresponding protein appeared to be identical to the first 51 amino acids of RopA of strain 248. The transcription start sites of both genes were determined, and the promoter regions were compared with that of ropA of strain 248. No clear consensus sequence could be deduced. The relationship of ropA and ropA2 of R. leguminosarum bv. viciae strain 248 with two similar genes from Brucella abortus is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Isolation of ropB, a gene encoding a 22-kDa Rhizobium leguminosarum outer membrane protein.

As judged from immunochemical detection, the levels of outer membrane antigen groups II and III of Rhizobium leguminosarum bv. viciae strain 248 decrease during bacteroid differentiation (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J. J. Lugtenberg, J. Bacteriol. 171:1136-1142, 1989). Using a newly developed colony blot assay, a cosmid clone expressing the Mab8 epitope of the outer membrane antigen group II of R. l. bv. viciae strain 248 was selected in Rhizobium meliloti LPR2120. From this cosmid the gene encoding this epitope was cloned and characterized. An open reading frame of 636 nucleotides was found and predicted to encode a protein with a calculated molecular mass of 22.5 kDa. After subtraction of the predicted 23 amino acid signal peptide, a M(r) of 20.3 kDa was calculated for the mature protein. This gene, designated ropB, was not active in Escherichia coli under the control of its own promoter. The C-terminal amino acid of the protein is a phenylalanine residue which is required for efficient translocation of outer membrane proteins. Membrane spanning amphipathic beta-sheets are predicted to represent a major part of the secondary structure of the protein. A model of the topology of the protein is presented. We determined the start of transcription in order to analyze the promoter region. No homology was found with other known promoter sequences. The ropB gene appeared to be well-conserved in R. leguminosarum and Agrobacterium tumefaciens strains. An attempt was made to mimic the immunochemical decrease of RopB ex planta. Neither the various growth conditions tested nor the addition of nodule or plant extracts resulted in a reduction of the Mab8 epitope to bacteroid levels.

Amino Acid Sequence↗