The human RAD18 gene product interacts with HHR6A and HHR6B

During DNA replication, lesion bypass is an important cellular response to unrepaired damage in the genome. In the yeast Saccharomyces cerevisiae, Rad6 and Rad18 are required for both the error-free and error-prone lesion bypass mechanisms. Furthermore, Rad6–Rad18 interaction is thought to be critical at an early step during lesion bypass in yeast. Two closely related human homologs of yeast Rad6 have been identified as HHR6A and HHR6B. Here, we report a full-length cDNA coding for the human homolog of yeast Rad18. The human RAD18 gene codes for a protein of 484 amino acid residues with a calculated molecular weight of 54 804 Da, and the gene is localized to chromosome 3 between reference intervals D3S3591 and D3S1283. Human RAD18 protein (hRAD18) was found to interact with HHR6A and HHR6B. When co-expressed in yeast cells, stable hRAD18–HHR6A and hRAD18–HHR6B protein complexes were identified and purified to near homogeneity. Thus, through interaction and complex formation with HHR6A and HHR6B, RAD18 protein may play an important role in lesion bypass mech­anisms in humans. Consistent with its role as a fundamental lesion bypass protein, the RAD18 gene is ubiquitously expressed in various human tissues.     

Localization of the gene encoding R kappa B (NFRKB), a tissue-specific DNA binding protein, to chromosome 11q24-q25.

Although NF (nuclear factor)-kappa B binds in vitro to several of the kappa B regulatory elements found in cellular and viral genes, another DNA binding protein, R kappa B, also binds to a related variant of the kappa B site that regulates interleukin-2 receptor alpha-chain gene expression, a critical event in T cell activation. Southern blot analysis of a human-mouse somatic cell hybrid panel and in situ hybridization using a fluorescent genomic R kappa B probe have allowed assignment of the R kappa B gene (NFRKB) to 11q24-q25. The NFRKB locus is in close proximity to the chromosomal breakpoint implicated in Ewing sarcoma, but it does not appear to span this region. Nonetheless, NFRKB may be particularly useful as the most telomeric marker thus far assigned to 11q.     

Localization of the nucleotide excision repair gene ERCC6 to human chromosome 10q11-q21.

We have cloned the human DNA excision repair gene ERCC6 by virtue of its ability to correct the uv sensitivity of Chinese hamster overy cell mutant UV61. This mutant is a member of complementation group 6 of the nucleotide excision repair-deficient rodent mutants. By means of in situ hybridization and Southern blot analysis of mouse x human somatic cell hybrids, the gene was localized to human chromosome 10q11-q21. An RFLP detected within the ERCC6 locus can be helpful in linkage analysis.     

Localization of the active type I DNA topoisomerase gene on human chromosome 20q11.2-13.1, and two pseudogenes on chromosomes 1q23-24 and 22q11.2-13.1

Summary Different subfragments of a cDNA coding for DNA topoisomerase I were used as probes to determine the chromosomal localization of topoisomerase I sequences in human cells. Southern blotting of restricted DNA from a panel of rodent-human somatic cell hybrids revealed the localization of the complete gene on chromosome 20 and the presence of two truncated topoisomerase I pseudogene sequences on chromosomes 1 and 22. In situ chromosome hybridzation experiments confirmed these results showing the location of the complete gene on band q11.2–13.1 of chromosome 20, and the location of the pseudogene sequences on band q23–24 of chromosome 1 and q11.2–13.1 of chromosome 22.     

Regional mapping of genes encoding human steroidogenic enzymes: P450scc to 15q23-q24, adrenodoxin to 11q22; adrenodoxin reductase to 17q24-q25; and P450c17 to 10q24-q25

Steroid hormones are synthesized by a complex array of 10 enzymes. The genes for each of these have now been cloned, and previous work has determined the regional chromosomal assignments of six of these. We used in situ hybridization to determine the regional chromosomal assignments of the four remaining enzymes. The CYP11A1 gene encodes mitochondrial P450scc, which converts cholesterol to pregnenolone, and is located on 15q23-q24. The gene for adrenodoxin, which receives electrons from adrenodoxin reductase and transfers them to P450scc, is on 11q22 while its pseudogenes are on 20q11-q12. The gene for adrenodoxin reductase is on 17q24-q25. The CYP17 gene encodes P450c17, which has both 17 alpha-hydroxylase and 17,20-lyase activities, and is located on 10q24-q25. None of the 10 genes involved in human steroidogenesis is closely linked to another gene for a steroidogenic enzyme.     

Assignment of the GDH loci to human chromosomes 10q23 and Xq24 by in situ hybridization

More precise localisations of the glutamate dehydrogenase gene (GLUD) to chromosome 10q23 and of the pseudogene GLUDP1 to X q24 are proposed.     

The human cytochrome b5 gene and two of its pseudogenes are located on chromosomes 18q23, 14q31-32.1 and 20p11.2, respectively

Using very high stringency hybridization conditions for the Southern blot hybridization analysis of hamster-human cell hybrid DNA, we were able to map the human cytochrome b₅ gene and two of its pseudogenes (psgb₅1 and psgb₅2) unambiguously to chromosomes 18, 14, and 20. These localizations were confirmed and extended to 18q23, 14q31-32.1, and 20p11.2 by using a combination of nonisotopic in situ hybridization of chromosomal spreads and the polymerase chain reaction analysis of DNA samples isolated from somatic cell hybrids retaining deletions or translocations of chromosome 18.     

HHR23B, a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro.

A protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces cerevisiae repair gene product Rad23 (Masutani et al., EMBO J. 13:1831-1843, 1994). To elucidate the roles of these proteins in "genome-overall" repair, we expressed the XPC protein in a baculovirus system and purified it to near homogeneity. The recombinant human XPC (rhXPC) protein exhibited a high level of affinity for single-stranded DNA and corrected the repair defect in XP-C whole-cell extracts without extra addition of recombinant HHR23B (rHHR23B) protein. However, Western blot (immunoblot) experiments revealed that XP-C cell extracts contained excess endogenous HHR23B protein, which might be able to form a complex upon addition of the rhXPC protein. To investigate the role of HHR23B, we fractionated the XP-C cell extracts and constructed a reconstituted system in which neither endogenous XPC nor HHR23B proteins were present. In this assay system, rhXPC alone weakly corrected the repair defect, while significant enhancement of the correcting activity was observed upon coaddition of rHHR23B protein. Stimulation of XPC by HHR23B was found with simian virus 40 minichromosomes as well as with naked plasmid DNA and with UV- as well as N-acetoxy-2- acetylfluorene-induced DNA lesions, indicating a general role of HHR23B in XPC functioning in the genome-overall nucleotide excision repair subpathway.     

Localization of the gene encoding insulin-degrading enzyme to human chromosome 10, bands q23----q25.

Insulin-degrading enzyme (IDE) is a cytosolic proteinase involved in the cellular processing of insulin. Using somatic cell hybrid analysis and in situ chromosomal hybridization, we have localized the gene encoding IDE to human chromosome 10, bands q23----q25. The murine Ide gene was previously mapped to Chromosome 19; together, these results suggest that the IDE gene is a member of a conserved syntenic group on human chromosome 10, bands q23----q25 and mouse Chromosome 19.     

Mapping of the human gene for the human immunodeficiency virus type 1 enhancer binding protein HIV-EP2 to chromosome 6q23-q24.

The human gene encoding the human immunodeficiency virus type 1 enhancer binding protein HIV-EP2 has been isolated. Using Southern analysis of human-rodent somatic cell hybrid DNA with a human HIV-EP2-specific cDNA probe, the HIV-EP2 gene was assigned to chromosome 6. The gene was further localized to the region 6q23-24 by fluorescence in situ hybridization.     

Familial eosinophilia maps to the cytokine gene cluster on human chromosomal region 5q31-q33.

Familial eosinophilia (FE) is an autosomal dominant disorder characterized by peripheral hypereosinophilia of unidentifiable cause with or without other organ involvement. To localize the gene for FE, we performed a genomewide search in a large U.S. kindred, using 312 different polymorphic markers. Seventeen affected subjects, 28 unaffected bloodline relatives, and 8 spouses were genotyped. The initial linkage results from the genome scan provided evidence for linkage on chromosome 5q31-q33. Additional genotyping of genetic markers located in this specific region demonstrated significant evidence that the FE locus is situated between the chromosome 5q markers D5S642 and D5S816 (multipoint LOD score of 6.49). Notably, this region contains the cytokine gene cluster, which includes three genes-namely, those for interleukin (IL)-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF)-whose products play important roles in the development and proliferation of eosinophils. These three cytokine genes were screened for potential disease-specific mutations by resequencing of a subgroup of individuals from the present kindred. No functional sequence polymorphisms were found within the promoter, the exons, or the introns of any of these genes or within the IL-3/GM-CSF enhancer, suggesting that the primary defect in FE is not caused by a mutation in any one of these genes but, rather, is caused by another gene in the area.     

Long-range mapping of the gene for the human alpha 5(IV) collagen chain at Xq22-q23.

The X-linked kidney disorder known as Alport syndrome (AS) has been shown to be due to mutations in the gene for an alpha 5 chain of type IV collagen that maps to Xq22-23. Using overlapping cDNA clones that represent approximately 90% of this gene and pulsed-field gel electrophoresis, we have constructed a 2.4-Mb long-range restriction map around the locus. All of the cDNA clones lie within a 360-kb segment of DNA bounded by CpG islands that contain sites for the rare-cutting enzymes BssHII, MluI, NotI, NruI, SalI, and SfiI. High-resolution PFGE mapping with XhoI shows that the gene is at least 110 kb in size and is one of the largest collagen genes characterized to date. This map will prove useful in the characterization of mutations in individuals affected with AS and will also provide information as to the location of other genes in the region.