Kazuhiro Kagotani

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.

Replication Timing Properties within the Mouse Distal Chromosome 7 Imprinting Cluster

Genomic imprinting is characterized by allele-specific expression of genes within chromosomal domains. Here we show, using fluorescence in situ hybridization (FISH) analysis, that the large chromosomal domain of the mouse distal chromosome 7 imprinting cluster, approximately 1 Mb in length between p57Kip2 and H19 genes, replicates asynchronously between the two alleles during S-phase. At the telomeric side of this domain, we found a transition from asynchronous replication at the imprinted p57Kip2 gene to synchronous replication at the Nap2 gene. Two-color FISH suggested that the paternal allele of this whole domain replicates earlier than its maternal allele. Treatment of the cells with a histone deacetylase inhibitor abolished this allele-specific feature accompanied with accelerated replication of the later-replicating allele at a domain level. Allele-specific asynchronous replication was observed even in ES cells. These results suggest that this imprinting cluster consists of a large replication domain which is already found at the early stage in development.

Cloning, sequencing, and chromosomal localization of two tandemly arranged human pseudogenes for the proliferating cell nuclear antigen (PCNA).

We have characterized a human genomic clone carrying two pseudogenes for the proliferating cell nuclear antigen (PCNA), which were tandemly arranged on human Chromosome (Chr) 4. One is a processed pseudogene that showed a 73% nucleotide homology to the human PCNA cDNA and possessed none of the introns existing in the functional PCNA gene. This pseudogene presumably arose by reverse transcription of a PCNA mRNA followed by integration of the cDNA into the genome. The other is a 5’ and 3’ truncated pseudogene that showed a nucleotide homology to a 3’ region of the exon 4 and to a 5’ region of the exon 5 of the PCNA gene and did not have the intronic sequence between the exons 4 and 5. Both pseudogenes had the same nucleotide deletion as compared with the human functional PCNA gene. A phylogenetic analysis of PCNA gene family, including the functional PCNA gene and another PCNA pseudogene located on a different chromosome, revealed that the truncated pseudogene exhibits the closest evolutionary relationship with the processed pseudogene, suggesting, that the truncated pseudogene was generated by duplication of the processed pseudogene after translocation to Chr 4. Furthermore, fluorescence in situ hybridization revealed that these pseudogenes are located on the long arm of Chr 4, 4q24.

Characterization of a Processed Pseudogene of Human ΨHSP40 on Chromosome 2q32

A pseudogene for the human Hsp40 gene has been characterized (ΨHSP40). The pseudogene sequence shows 90% similarity to the human Hsp40 mRNA at the nucleotide level. No introns were found in the region corresponding to the human Hsp40 cDNA, and two direct repeats flank this same region. Because of these features, the pseudogene can be classified as a processed pseudogene. ΨHSP40 was assigned to chromosome 2q32 by in situ hybridization. This is the first report of a pseudogene for a member of the DnaJ (Hsp40) family protein gene.