Masahiro Nogami

Cloning, expression and chromosome locations of the human DNMT3 gene family

DNA methylation plays an important role in animal development and gene regulation. In mammals, several genes encoding DNA cytosine methyltransferases have been identified. DNMT1 is constitutively expressed and is required for the maintenance of global methylation after DNA replication. In contrast, the murine Dnmt3 family genes appear to be developmentally regulated and behave like de novo DNA methyltransferases in vitro. In this study, we have cloned human DNMT3A and DNMT3B that encode full-length DNMT3A and DNMT3B proteins with 98% and 94% amino acid sequence identity to their murine homologues. The DNMT3A and DNMT3B show high homology in the carboxy terminal catalytic domain and contain a conserved cysteine-rich region, which shares homology with the X-linked ATRX gene of the SNF2/SWI family. We have mapped human DNMT3A and DNMT3B to chromosomes 2p23 and 20q11.2 respectively, and determined the DNMT3B genomic structure. We further show that DNMT3A expression is ubiquitous and can be readily detected in most adult tissues, whereas DNMT3B is expressed at very low levels in most tissues except testis, thyroid and bone marrow. Significantly, both DNMT3A and DNMT3B expression is elevated in several tumor cell lines to levels comparable to DNMT1. The cloning of the human DNMT3 genes will facilitate further biochemical and genetic studies of their functions in establishment of DNA methylation patterns, regulation of gene expression and tumorigenesis.

Inactivating Mutations in the 25-Hydroxyvitamin D3 1α-Hydroxylase Gene in Patients with Pseudovitamin D–Deficiency Rickets

BACKGROUND Pseudovitamin D-deficiency rickets is characterized by the early onset of rickets with hypocalcemia and is thought to be caused by a deficit in renal 25-hydroxyvitamin D3 1alpha-hydroxylase, the key enzyme for the synthesis of 1alpha,25-dihydroxyvitamin D3. METHODS We cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase complementary DNA (cDNA) using a mouse 1alpha-hydroxylase cDNA fragment as a probe. Its genomic structure was determined, and its chromosomal location was mapped by fluorescence in situ hybridization. We then identified mutations in the 1alpha-hydroxylase gene in four unrelated patients with pseudovitamin D-deficiency rickets by DNA-sequence analysis. Both the normal and the mutant 1alpha-hydroxylase proteins were expressed in COS-1 cells and were assayed for 1alpha-hydroxylase activity. RESULTS The gene for 25-hydroxyvitamin D3 1alpha-hydroxylase was mapped to chromosome 12q13.3, which had previously been reported to be the locus for pseudovitamin D-deficiency rickets by linkage analysis. Four different homozygous missense mutations were detected in this gene in the four patients with pseudovitamin D-deficiency rickets. The unaffected parents and one sibling tested were heterozygous for the mutations. Functional analysis of the mutant 1alpha-hydroxylase protein revealed that all four mutations abolished 1alpha-hydroxylase activity. CONCLUSIONS Inactivating mutations in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene are a cause of pseudovitamin D-deficiency rickets.

Intranuclear arrangement of human chromosome 12 correlates to large-scale replication domains

Abstract.The intranuclear arrangement of human chromosome 12 in G0(G1) nuclei from human myeloid leukemia HL60 cells was analyzed by multicolor fluorescence in situ hybridization (FISH) using band-specific cosmid clones as probes. Pairs of differently colored cosmids were detected on paraformaldehyde-fixed HL60 nuclei, and their relative positions, internal or peripheral, in individual nuclei were scored. Our results suggest that the intranuclear arrangement of human chromosome 12 is not random. Some chromosomal domains, including the centromere, were located in the periphery of the nucleus, while other domains, including the telomeres, were positioned in the internal areas of the nucleus in G0(G1) cells. Based on the replication banding patterns of metaphase spreads, human chromosome 12 was divided roughly into five large domains. Interestingly, the clones in late replicating domains were preferentially localized in the nuclear periphery, whereas clones in early replicating domains were arranged in the internal areas of the nuclei. The DNA replication timing of each cosmid determined by FISH-based assay did not reflect the replication bands, but an overall profile of the replication timing was relatively correlated with these domains on chromosome 12. These results suggest that the intranuclear arrangement of a human chromosome is correlated with the large-scale replication domains, even before DNA replication.

Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2–q24.3, 3q27, 13q12.3–q13, and 7q11.23

Replication factor C is a multimeric primer-recognition protein consisting of five subunits (p145, p40, p38, p37, and p36.5) and is essential for the processive elongation of DNA chains catalyzed by DNA polymerase delta or epsilon in human cells. We have mapped the locations on human chromosomes of the genes coding for the four smaller subunits [p36.5 (RFC5), p37 (RFC4), p38 (RFC3), and p40 (RFC2)] using both PCR amplification from DNAs of a panel of somatic hybrids and fluorescence in situ hybridization to bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23, respectively.

Genomic organization and chromosomal localization of the human casein gene family

Abstract Five yeast artificial chromosome (YAC) clones containing the human casein gene family were isolated and characterized to study the control mechanisms for the expression of these genes. Partial restriction analysis in conjunction with the chromosomal fragmentation method and fluorescence in situ hybridization (FISH) analysis were performed to construct a detailed physical map of the casein gene family and to determine the chromosomal localization of these genes. The isolated YAC clones 748F3, 750D11, 882G11, 886B3 and 960D2 were 1.2 Mb, 860 kb, 800 kb 1.5 Mb and 1.5 Mb in size, respectively. The clones 748F3, 882G11, 886B3 and 960D2 contained the entire casein gene family, while the κ-casein gene was absent in 750D11. The human αS1-, β- and κ-casein genes were found to be closely linked and arranged in the order αS1-β-κ. The distance between αS1 and β, and between αS1 and κ was approximately 10 and 300 kb, respectively. The β-casein gene was oriented in the opposite direction to the αS1- and κ-casein genes. The casein gene family was localized to chromosome 4q21.1 by FISH analysis.

Newly identified repeat sequences, derived from human chromosome 21qter, are also localized in the subtelomeric region of particular chromosomes and 2q13, and are conserved in the chimpanzee genome

Subtelomeric regions have been a target of structural and functional studies of human chromosomes. Markers having a defined structure are especially useful to such studies. Here, we report 93 bp tandem repeat sequences found in the subtelomeric region of human chromosome 21q. They were also detected in the telomeric region of several other chromosomes. Interestingly, the repeat was also found in the 2q13 region which is known to be a position of chromosomal fusion, a major difference between the human and chimpanzee karyotypes. To the best of our knowledge, this repetitive sequence is a new member of human subtelomeric interspersed repeats.