Preferential S-phase pairing of the imprinted region on distal mouse chromosome 7.

Fluorescence in situ hybridization (FISH) has been used to visualize the spatial orientation of homologous chromosome regions in interphase nuclei of exponentially growing mouse fibroblasts. Simultaneous labeling of replicating DNA with the halogenated base analog 5-bromodeoxyuridine (BrdU) shows preferential somatic pairing of the imprinted region on distal mouse chromosome 7 during the S phase of the cell cycle. Trans-interactions between oppositely imprinted chromosome regions may be important for the maintenance of imprinting.     

Identification of the mouse paternally expressed imprinted gene Zdbf2 on chromosome 1 and its imprinted human homolog ZDBF2 on chromosome 2

In mammals, both the maternal and paternal genomes are necessary for normal embryogenesis due to parent-specific epigenetic modification of the genome during gametogenesis, which leads to non-equivalent expression of imprinted genes from the maternal and paternal alleles. In this study, we identified a paternally expressed imprinted gene, Zdbf2, by microarray-based screening using parthenogenetic and normal embryos. Expression analyses showed that Zdbf2 was paternally expressed in various embryonic and adult tissues, except for the placenta and adult testis, which showed biallelic expression of the gene. We also identified a differentially methylated region (DMR) at 10 kb upstream of exon 1 of the Zdbf2 gene and this differential methylation was derived from the germline. Furthermore, we also identified that the human homolog (ZDBF2) of the mouse Zdbf2 gene showed paternal allele-specific expression in human lymphocytes but not in the human placenta. Thus, our findings defined mouse chromosome 1 and human chromosome 2 as the loci for imprinted genes.     

Assignment of a gene encoding ornithine decarboxylase to the proximal region of chromosome 12 in the mouse

Ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthetic pathway, is encoded by at least one member of a multi-gene family in the mouse. Analysis of a polymorphism in ODC structure in recombinant inbred strains has enabled assigning a functional ODC structural gene (Odc) to the proximal region of mouse chromosome 12 between Apob and Es25. Linkage of Odc to Apob and Ah is conserved in the mouse and human genomes.     

Composition and structure of the centromeric region of rice chromosome 8

Understanding the organization of eukaryotic centromeres has both fundamental and applied importance because of their roles in chromosome segregation, karyotypic stability, and artificial chromosome-based cloning and expression vectors. Using clone-by-clone sequencing methodology, we obtained the complete genomic sequence of the centromeric region of rice (Oryza sativa) chromosome 8. Analysis of 1.97 Mb of contiguous nucleotide sequence revealed three large clusters of CentO satellite repeats (68.5 kb of 155-bp repeats) and >220 transposable element (TE)-related sequences; together, these account for approximately 60% of this centromeric region. The 155-bp repeats were tandemly arrayed head to tail within the clusters, which had different orientations and were interrupted by TE-related sequences. The individual 155-bp CentO satellite repeats showed frequent transitions and transversions at eight nucleotide positions. The 40 TE elements with highly conserved sequences were mostly gypsy-type retrotransposons. Furthermore, 48 genes, showing high BLAST homology to known proteins or to rice full-length cDNAs, were predicted within the region; some were close to the CentO clusters. We then performed a genome-wide survey of the sequences and organization of CentO and RIRE7 families. Our study provides the complete sequence of a centromeric region from either plants or animals and likely will provide insight into the evolutionary and functional analysis of plant centromeres.     

Appropriate expression of imprinted genes on mouse chromosome 12 extends development of bi-maternal embryos to term

Recently, we reported that the restored regulation of imprinted gene expression from two regions -H19 differentially methylated region (H19-DMR) and intergenic germline-derived DMR (IG-DMR) - is sufficient for accomplishing full-term development in mice. In the present study, we determined the developmental ability of the bi-maternal embryos (BMEs) containing the non-growing oocyte genome with the IG-DMR deletion (ng(Deltach12)) and fully-grown (fg) oocyte genome. Foetuses derived from ng(Deltach12)/fg BMEs were alive at E19.5 but could not survive further. Comparison with BMEs derived from Igf2+/- ng/fg genomes suggests that bi-allelic H19 expression might be involved in foetal development.     

Vrn-D4 is a vernalization gene located on the centromeric region of chromosome 5D in hexaploid wheat

Natural variation in wheat requirement of long exposures to cold temperatures to accelerate flowering (vernalization) is mainly controlled by the Vrn-1, Vrn-2, Vrn-3, and Vrn-4 loci. The first three loci have been well characterized, but limited information is available for Vrn-4. So far, natural variation for Vrn-4 has been detected only in the D genome (Vrn-D4), and genetic stocks for this gene are available in Triple Dirk (TDF, hereafter). We detected heterogeneity in the Vrn-1 alleles present in different TDF stocks, which may explain inconsistencies among previous studies. A correct TDF seed stock from Japan carrying recessive vrn-A1, vrn-B1, and vrn-D1 alleles was crossed with three different winter cultivars to generate F₂ mapping populations. Most of the variation in flowering time in these three populations was controlled by a single locus, Vrn-D4, which was mapped within a 1.8 cM interval flanked by markers Xcfd78 and Xbarc205 in the centromeric region of chromosome 5D. A factorial ANOVA for heading time using Vrn-D4 alleles and vernalization as factors showed a significant interaction (P < 0.0001), which confirmed that the Vrn-D4 effect on flowering time is modulated by vernalization. Comparison of the different Triple Dirk stocks revealed that Vrn-B1, Vrn-D1, and Vrn-D4 all have a small residual response to vernalization, but Vrn-D4 differs from the other two in its response to short vernalization periods. The precise mapping and characterization of Vrn-D4 presented here represent a first step toward the positional cloning of this gene.     

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.     

Recurrent HSR in the centromeric region of chromosome 8 in breast cancer

In a sample of 44 human breast cancers, the presence of HSRs was detected in 15 cases (36%). The occurrence of HSRs seems correlated poorly with tumor progression, but the number of HSRs per cell certainly does. Chromosome 8 was the most frequently involved, and the HSRs were always located near its centromere, suggesting that an amplification of gene(s) located in this region has occurred. Among these, LHRH is a candidate, but further studies are needed to confirm or reject this hypothesis.     

Microfluorometric detection of asymmetry in the centromeric region of mouse chromosomes

A lateral asymmetry in the centromeric region of mouse chromosomes is revealed in studies involving the BUdR quenching of 33258 Hoechst fluorescence. This cytologically detected asymmetry may reflect the unequal distribution of thymidine between the two chains of mouse satellite DNA.     

The size and sequence organization of the centromeric region of arabidopsis thaliana chromosome 5.

We have determined the size of the centromeric region of Arabidopsis thaliana chromosome 5, which corresponds to the genetically defined centromere by Copenhaver et al. (Science, 286, 2468-2474, 1999) on the basis of restriction analysis. As a large clone gap was present in the previously constructed contig map of the centromeric region, the restriction map of this region was constructed using Asc I, Not I, Apa I and Pme I and genomic DNA from a hypomethylated strain. The size of the centromeric region finally estimated by combination with the sequence data of cloned regions at both sides was 4.35 megabases (Mb). This value is over 2 Mb longer than those estimated in our previous work and also by Copenhaver et al. Combing this centromeric region with the physical map previously constructed, the entire length of chromosome 5 becomes 31 Mb. Although the internal moiety of the centromeric region has not been sequenced yet because of extremely high repetition, the result of sequence analysis from both sides toward the inside strongly suggests that the centromeric region is composed of the central 2.9-Mb domain consisting of mainly 180-bp repeats and Athila retrotransposons and flanking regions containing various types of transposons. On the basis of these observations, a structural model for the centromeric region is discussed.     

The size and sequence organization of the centromeric region of Arabidopsis thaliana chromosome 4.

We have determined the genome structure of the centromeric region of Arabidopsis thaliana chromosome 4 by sequence analysis of BAC clones obtained by genome walking, followed by construction of a physical map using DNA of a hypomethylated strain. The total size of the centromeric region, corresponding to the recombinant inbred (RI) markers between mi87 and mi167, was approximately 5.3 megabases (Mb). This value is over 3 Mb longer than that previously estimated by the Arabidopsis Genome Initiative (Nature, 408, 796-815, 2000). Although we could not cover the entire centromeric region by BAC clones because of the presence of highly repetitive sequences in the middle (2.7 Mb), the cloned regions spanning approximately 1 Mb at both sides of the gap were newly sequenced. These results together with the reported sequences in the adjacent regions suggest that the centromeric region is principally composed of a central domain of 2.7 Mb, consisting of mainly 180-bp repeats and Athila elements, and upper and lower flanking regions of 1.55 Mb and 1 Mb, respectively. The flanking regions were predominantly composed of various types of transposable elements, except for the upper end moiety in which a large 5S rDNA array (0.65 Mb) and central domain-like sequence are present. Such an organization is essentially identical to the centromeric region of chromosome 5 reported previously.     

Peg5/Neuronatin is an imprinted gene located on sub-distal chromosome 2 in the mouse.

We have established a systematic screen for imprinted genes using a subtraction-hybridization method with day 8.5 fertilized and parthenogenetic embryos. Two novel imprinted genes, Peg1/Mest and Peg3, were identified previously by this method, along with the two known imprinted genes, Igf2 and Snrpn. Recently three additional candidate imprinted genes, Peg5-7 , were detected and Peg5 is analyzed further in this study. The cDNA sequence of Peg5 is identical to Neuronatin, a gene recently reported to be expressed mainly in the brain. Two novel spliced forms were detected with some additional sequence in the middle of the known Neuronatin sequences. All alternatively spliced forms of Peg5 were expressed only from the paternal allele, confirmed using DNA polymorphism in a subinterspecific cross. Peg5/Neuronatin maps to sub-distal Chr 2, proximal to the previously established imprinted region where imprinted genes cause abnormal shape and behavior in neonates.     

Paternal expression of a novel imprinted gene, Peg12/Frat3, in the mouse 7C region homologous to the Prader-Willi syndrome region.

Paternally expressed imprinted genes (Pegs) were systematically screened by comparing gene expression profiles of parthenogenetic and normal fertilized embryos using an oligonucleotide array. A novel imprinted gene, Peg12/Frat3, was identified along with 10 previously known Pegs. Peg12/Frat3 is expressed primarily in embryonic stages and might be a positive regulator of the Wnt signaling pathway. It locates next to the Zfp127 imprinted gene in the mouse 7C region, which has syntenic homology to the human Prader-Willi syndrome region on chromosome 15q11-q13, indicating that this imprinted region extends to the telomeric side in the mouse.     

An alpha satellite DNA polymorphism specific for the centromeric region of chromosome 13

Alpha satellite DNA is composed of variants of a short consensus sequence that are repeated in tandem arrays in the centromeric heterochromatin of each human chromosome. To define centromeric markers for linkage studies, we screened human genomic DNA for restriction fragment length polymorphisms using a probe detecting alphoid sequences on chromosomes 13 and 21. We describe one such DNA polymorphism. Analysis of linkage of this DNA marker to other polymorphic markers in the CEPH pedigrees demonstrates linkage to markers on the proximal long arm of chromosome 13 and defines the centromeric end of the linkage map of this chromosome.