Anne Bergmann

Fluorescence in Situ hybridization mapping of human chromosome 19: Mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting

Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested).

The fucosyltransferase locus FUT1 maps distal to apolipoprotein loci E and C2 on human chromosome 19

The location of the fucosyltransferase locus FUT1 relative to the apolipoprotein E/C2 loci on human chromosome 19 has remained unclear. We determined by a combination of physical mapping and fluorescence in situ hybridization that this fucosyltransferase gene maps distal to the apolipoprotein loci.

Structure, sequence and location of the UQCRFS1 gene for the human Rieske Fe-S protein.

We have identified and studied the chromosomal location of the human Rieske Fe-S protein-encoding gene UQCRFS1. Mapping by hybridization to a panel of monochromosomal hybrid cell lines indicated that a UQCRFS1 partial cDNA was derived from either chromosome 19 or 22. By screening a human chromosome 19 specific genomic cosmid library with a probe from this cDNA sequence, we identified a corresponding cosmid. Portions of this cosmid were sequenced directly. The exon, exon:intron junction and flanking sequences verified that this cosmid contains the genomic locus. Fluorescent in situ hybridization (FISH) was performed to localize this cosmid to chromosome band 19q12.

Genes encoding the H,K-ATPase α and Na,K-ATPase α3 subunits are linked on mouse Chromosome 7 and human Chromosome 19

We have used linkage analysis and fluorescence in situ hybridization to determine the chromosomal organization and location of the mouse (Atp4a) and human (ATP4A) genes encoding the H,K-ATPase α subunit. Linkage analysis in recombinant inbred (BXD) strains of mice localized Atp4a to mouse Chromosome (Chr) 7. Segregation of restriction fragment length polymorphisms in backcross progeny of Mus musculusxMus spretus mating confirmed this assignment and indicates that Atp4a and Atp1a3 (gene encoding the murine Na,K-ATPase α3 subunit) are linked and separated by a distance of ∼2 cM. Analysis of the segregation of simple sequence repeats suggested the gene order centromere-D7Mit21-D7Mit57/Atpla3-D7Mit72/Atp4a. A human Chr 19-enriched cosmid library was screened with both H,K-ATPase α and Na,K-ATPase α3 subunit cDNA probes to isolate the corresponding human genes (ATP4A and ATP1A3, respectively). Fluorescence in situ hybridization with gene-specific cosmid clones localized ATP4A to the q13.1 region, and proximal to ATP1A3, which maps to the q13.2 region, of Chr 19. These results indicate that ATP4A and ATP1A3 are linked in both the mouse and human genomes.

Interrogating Transcriptional Regulatory Sequences in Tol2-Mediated Xenopus Transgenics

Identifying gene regulatory elements and their target genes in vertebrates remains a significant challenge. It is now recognized that transcriptional regulatory sequences are critical in orchestrating dynamic controls of tissue-specific gene expression during vertebrate development and in adult tissues, and that these elements can be positioned at great distances in relation to the promoters of the genes they control. While significant progress has been made in mapping DNA binding regions by combining chromatin immunoprecipitation and next generation sequencing, functional validation remains a limiting step in improving our ability to correlate in silico predictions with biological function. We recently developed a computational method that synergistically combines genome-wide gene-expression profiling, vertebrate genome comparisons, and transcription factor binding-site analysis to predict tissue-specific enhancers in the human genome. We applied this method to 270 genes highly expressed in skeletal muscle and predicted 190 putative cis-regulatory modules. Furthermore, we optimized Tol2 transgenic constructs in Xenopus laevis to interrogate 20 of these elements for their ability to function as skeletal muscle-specific transcriptional enhancers during embryonic development. We found 45% of these elements expressed only in the fast muscle fibers that are oriented in highly organized chevrons in the Xenopus laevis tadpole. Transcription factor binding site analysis identified >2 Mef2/MyoD sites within ∼200 bp regions in 6 of the validated enhancers, and systematic mutagenesis of these sites revealed that they are critical for the enhancer function. The data described herein introduces a new reporter system suitable for interrogating tissue-specific cis-regulatory elements which allows monitoring of enhancer activity in real time, throughout early stages of embryonic development, in Xenopus.