Generation and characterization of an ordered lambda clone array for the 460-kb region surrounding the murine Xist sequence

The Xist sequence has several characteristics that make it a potential candidate for the X-inactivation center. To investigate the role of Xist and adjacent sequences lying within the X-inactivation center candidate region, a 460-kb region surrounding the murine Xist sequence has been arrayed in lambda contigs with a combination of IRS-PCR-based hybridization and YAC fragmentation. The orientation of the Xist sequence in relation to the telomere and centromere of the X Chromosome (Chr) has been established with this contig and shown to be inverted compared to that in human.     

Comparative mapping on the mouse X Chromosome defines a myotubular myopathy equivalent region

The gene for X-linked myotubular myopathy (MTM1) has been localized to a 300-kb critical region in human Xq28 between IDS and GABRA3. As part of an effort to clone this gene, we developed a YAC contig on the mouse X Chromosome (Chr) which includes loci homologous to those within the human MTM1 critical region. The murine contig consists of 18 YACs and spans 2.5–3.0 Mb. We have aligned the human and murine physical maps by isolating conserved mouse genomic fragments, including CpG islands and trapped exons. We believe that the simultaneous isolation of genes from both mouse and human and continued comparative mapping will prove helpful in the eventual identification of MTM1 and other genes in the region. Received: 9 February 1996 / Accepted: 30 March 1996     

Genetic and Physical Mapping of a Gene Encoding a Methyl CpG Binding Protein, Mecp2, to the Mouse X Chromosome

The methyl CpG binding proteins (MeCP1 and MeCP2) are a class of proteins that bind to templates containing symmetrically methylated CpGs. Using an interspecific backcross segregating a number of X-linked markers, we have localized the Mecp2 gene in mouse to the X chromosome close to the microsatellite marker DXMit1. Detailed physical mapping utilizing an available YAC contig encompassing the DXMit1 locus has localized the Mecp2 gene to a 40-kb region between the L1cam and the Rsvp loci, indicating the probable position of a homologue on the human X chromosome.     

A targeted deletion upstream of <Emphasis Type="Italic">Snrpn</Emphasis> does not result in an imprinting defect

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the disturbance of imprinted gene expression within human chromosome 15q11–q13. Some cases of PWS and AS are caused by microdeletions near the SNRPN gene that disrupt a regulatory element termed the imprinting center (IC). The IC has two functional components; an element at the promoter of SNRPN involved in PWS (PWS-IC) and an element 35 kilobases (kb) upstream of SNRPN involved in AS (AS-IC). To further understand the function of the IC, we sought to create a mouse model for AS-IC mutations. We have generated two deletions at a location analogous to that of the human AS-IC. Neither deletion produced an imprinting defect as indicated by DNA methylation and gene expression analyses. These results indicate that no elements critical for AS-IC function in mouse reside within the 12.8-kb deleted region and suggest that the specific location of the AS-IC is not conserved between human and mouse. Camilynn I. Brannan was Deceased     

Sub-milliMorgan map of the proximal part of mouse chromosome 17 including the hybrid sterility 1 gene

We have generated a high-resolution genetic map, 0.071 cM per backcross animal, of the 13 cM T–H2 region of the mouse Chromosome (Chr) 17. The map contains two phenotypic loci, T and Hst1, 12 RFLP markers, and 24 microsatellite loci. The Hst1 gene was mapped to a chromosomal interval contained within a single 580-kb YAC clone. The FFEH11 YAC is 0.44 cM long and carries, besides the Hst1 gene, five polymorphic DNA markers and recombination breakpoints of six backcross animals. Two candidate genes for Hst1 were identified based on their location and testicular expression. These are Tbp and D17Ph4e. The sub-milliMorgan map of the T–H2 region revealed significant clustering of (CA)_n loci. The clustering, if shown to be a common feature in the mouse genome, may cause gaps in the physical map of the mouse genome. Received: 11 September 1995 / Accepted: 9 October 1995     

Physical Mapping of 2000 kb of the Mouse X Chromosome in the Vicinity of the Xist Locus

A physical map encompassing approximately 2.0 megabases (Mb) in the region of the mouse X-inactivation center has been constructed. The map extends from the Gjb-1 locus to the Xist locus and demonstrates the order of probes inseparable by genetic analysis. The deduced locus order is as follows: Gjb-1, Ccg-1, DXCrc171, Rps4, Phka, DXCrc177, DXCrc318, Xist . Detailed physical mapping in the region between the Phka and Xist loci indicates the position of CpG-rich islands associated with the 5′ end of genes. The DXCrc177 and DXCrc318 loci, both defined by probes derived from linking clones, are associated with CpG-rich islands. The map provides a framework for the isolation of underlying sequences in the mouse X-inactivation center region.     

Bisulfite Genomic Sequencing-Derived Methylation Profile of theXistGene throughout Early Mouse Development

Differential epigenetic modification by methylation of CpG dinucleotides is a candidate mechanism that may identify the alleles of imprinted genes and result in monoallelic expression of either the maternal or the paternal allele. Determination of the allelic methylation status of imprinted genes in the gametes and during early development is constrained by the limiting quantities of genomic DNA available from these early developmental stages. To circumvent this problem we have used bisulfite genomic sequencing to determine the allelic methylation status of the minimal promoter and a 1-kb region within theXistgene during preimplantation development. We find that the parentalXistalleles are not differentially methylated in these regions. Our findings are discussed in the context of previous conflicting data obtained using methylation-sensitive restriction enzyme digestion followed by PCR amplification to assay for methylation.     

Cloning, Mapping, and Expression of Two Novel Actin Genes, Actin-like-7A (ACTL7A) and Actin-like-7B (ACTL7B), from the Familial Dysautonomia Candidate Region on 9q31

Two novel human actin-like genes, ACTL7A and ACTL7B, were identified by cDNA selection and direct genomic sequencing from the familial dysautonomia candidate region on 9q31. ACTL7A encodes a 435-amino-acid protein (predicted molecular mass 48.6 kDa) and ACTL7B encodes a 415-amino-acid protein (predicted molecular mass 45.2 kDa) that show greater than 65% amino acid identity to each other. Genomic analysis revealed ACTL7A and ACTL7B to be intronless genes contained on a common 8-kb HindIII fragment in a “head-to-head” orientation. The murine homologues were cloned and mapped by linkage analysis to mouse chromosome 4 in a region of gene order conserved with human chromosome 9q31. No recombinants were observed between the two genes, indicating a close physical proximity in mouse. ACTL7A is expressed in a wide variety of adult tissues, while the ACTL7B message was detected only in the testis and, to a lesser extent, in the prostate. No coding sequence mutations, genomic rearrangements, or differences in expression were detected for either gene in familial dysautonomia patients.     

DNA loop domains in a 1.4-Mb region around the human hprt gene mapped by cleavage mediated by nuclear matrix-associated topoisomerase II

We have mapped the positions in a ∼1.4-Mb region of genomic DNA around the human hprt gene which are accessible in vivo to cleavage by topoisomerase II associated with the nuclear matrix. These positions, which are interpreted as the boundaries of DNA loop domains, were mapped in K562 cells by examining the truncation of rare-cutter restriction fragments separated by pulsed field gel electrophoresis after topoisomerase II-mediated cleavage, using seven linked markers mapped in this region as probes for indirect end-labeling. Eleven cleavage positions were detected and were interpreted as defining ten loop domains of lengths between 70 and 210 kb (average ∼135 kb); the hprt gene resides in a 150-kb loop domain. Loop domain boundaries coincided with three of the fifteen deletion breakpoints mapped in a 600-kb sector of this region in human lymphocytes, within the limits of resolution of pulsed field gel electrophoresis; this correlation was not statistically significant. Received: 14 June 1998 / Accepted: 4 September 1998