Human and mouse RAD17 genes: identification, localization, genomic structure and histological expression pattern in normal testis and seminoma
ABSTRACT Recently, the human orthologue to the cell cycle checkpoint genes rad17 (Schizosaccharomyces pombe) and RAD24 (Saccharomyces cerevisiae), called HRAD17, has been isolated and localized to chromosome 4. Independently, we have isolated the HRAD17 transcript and mapped it to chromosome 5q13 between the CCNB1 and BTF2p44cen genes. Furthermore, we have identified the complete exon-intron structure of HRAD17. The gene is organized into 14 exons, the translation initiation site lies within exon 2, and the stop codon within exon 14. Two further HRAD17 pseudogenes, HRAD17P1 and HRAD17P2, were identified on chromosomes 7p21 and 13q14.3, respectively, encompassing exons 3-14 and bearing 84% and 93% homology, respectively. Additionally, we have isolated the coding region of the mouse orthologue, Mrad17, and mapped it on chromosome 13 between Ccnb1 and Btf2p44, the same two genes between which it maps in human. The predicted Mrad17 polypeptide encompasses 687 amino acids and shows 89% similarity to HRAD17. Both genes are most highly expressed in testis compared to all other tissues, as shown by Northern blot hybridization. Histological studies, based on in situ hybridization with radioactively labeled antisense HRAD17 riboprobes, showed a strong expression within the germinal epithelium of the seminiferous tubuli in normal testis whereas in testicular tumors (seminomas) only weak, diffuse signals were seen. In light of the known function of the yeast orthologue at meiotic and mitotic checkpoints, as well as the strong expression in testis and weak expression in seminomas, we suggest a putative involvement of HRAD 17 in testicular tumorigenesis.
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ABSTRACT: During every S-phase cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. It is a tremendous task, given the large sizes of mammalian genomes and the required precision of DNA replication. A major threat to the accuracy and efficiency of DNA synthesis is the presence of damaged DNA, e.g. abasic sites, single stranded DNA breaks, DNA crosslinks and adducts. This damage can be caused by exogenous agents, e.g. UV light, ionizing radiation, or environmental carcinogens, but is also an inevitable consequence of normal cellular metabolism. Replicative DNA polymerases, which carry out the bulk of DNA synthesis, evolved to do their job extremely precisely and effficiently. However, they are unable to use damaged DNA as templates, and, consequently, are stopped at most DNA lesions. Failure to restart such stalled forks can result in major chromosomal aberrations and lead to cell dysfunction or death. Therefore, a well-coordinated response to replication perturbation is essential for cell survival and wellbeing. It involves adjusting cell cycle progression to the emergency situation, and the use of specialized pathways promoting replication recovery. The aim of this thesis was to contribute to our understanding of the mechanisms the cell employs to deal with replication problems.
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ABSTRACT: Besides the well-known applications of bacterial artificial chromosomes (BACs) in classical molecular genetics, BACs are also used for molecular cytogenetic studies. BACs, as well as other locus-specific probes like cDNA, plasmids, cosmids, fosmids, P1-clones or yeast artificial chromosomes (YACs), can be labeled with fluorochromes and applied in FISH experiments. Various applications are possible, like gene mapping, FISH banding, determination of chromosomal breakpoints, characterization of derivative chromosomes, studies of interphase architecture, and karyotypic evolution studies. Here the basic principle of hybridizing BACs in situ on chromosome preparations is outlined. Moreover, an overview of possible issues that can be studied using BACs as FISH probes is provided. Finally, a shortened and more efficient FISH protocol using microwave treatment which yields results that can be evaluated within a few hours is presented.Fluorescence in situ Hybridization (FISH) – Application Guide, Edited by T Liehr, 12/2008: pages 53-60; Springer.
Mammalian Genome 01/2001; 12(4):326-328. DOI:10.1007/s003350010276 · 2.88 Impact Factor