The flamingo-related mouse Celsr family (Celsr1-3) genes exhibit distinct patterns of expression during embryonic development.

We have isolated cDNAs for three members of a family of seven-pass transmembrane cadherins in mouse (Celsr1, 2 and 3). These three genes represent vertebrate homologues of flamingo/starry night, recently identified as an essential component of the Drosophila planar cell polarity pathway and for the correct formation of dendritic fields within the Drosophila peripheral nervous system. In this study, we show that each member of the mouse Celsr family exhibit distinct patterns of expression within a range of different tissues within the developing embryo. Celsr1 and Celsr2 expression is observed during gastrulation and within the developing nervous system. Celsr3 transcripts, however, are found only at sites of active neurogenesis.

Disruption of a novel imprinted zinc-finger gene, ZNF215, in Beckwith-Wiedemann syndrome.

The genetics of Beckwith-Wiedemann syndrome (BWS) is complex and is thought to involve multiple genes. It is known that three regions on chromosome 11p15 (BWSCR1, BWSCR2, and BWSCR3) may play a role in the development of BWS. BWSCR2 is defined by two BWS breakpoints. Here we describe the cloning and sequence analysis of 73 kb containing BWSCR2. Within this region, we detected a novel zinc-finger gene, ZNF215. We show that two of its five alternatively spliced transcripts are disrupted by both BWSCR2 breakpoints. Parts of the 3' end of these splice forms are transcribed from the antisense strand of a second zinc-finger gene, ZNF214. We show that ZNF215 is imprinted in a tissue-specific manner.

A 7.5 Mb sequence-ready PAC contig and gene expression map of human chromosome 11p13-p14.1.

The region p13 of the short arm of human chromosome 11 has been studied intensely during the search for genes involved in the etiology of the Wilms' tumor, aniridia, genitourinary abnormalities, mental retardation (WAGR) syndrome, and related conditions. The gene map for this region is far from being complete, however, strengthening the need for additional gene identification efforts. We describe the extension of an existing contig map with P1-derived artificial chromosomes (PACs) to cover 7.5 Mb of 11p13-14.1. The extended sequence-ready contig was established by end probe walking and fingerprinting and consists of 201 PAC clones. Utilizing bins defined by overlapping PACs, we generated a detailed gene map containing 20 genes as well as 22 anonymous ESTs which have been identified by searching the RH databases. RH maps and our established gene map show global correlation, but the limits of resolution of the current RH panels are evident at this scale. Initial expression studies on the novel genes have been performed by Northern blot analyses. To extend these expression profiles, corresponding mouse cDNA clones were identified by database search and employed for Northern blot analyses and RNA in situ hybridizations to mouse embryo sections. Genomic sequencing of clones along a minimal tiling path through the contig is currently under way and will facilitate these expression studies by in silico gene identification approaches.

mCelsr1 is an evolutionarily conserved seven-pass transmembrane receptor and is expressed during mouse embryonic development.

Mcelsr1 encodes a protein of 3034 amino acids predicted to contain seven membrane spanning domains having homology to a group of peptide hormone binding G-protein coupled receptors. Its extracellular domain comprises epidermal growth factor-like repeats, laminin A G-domains and cadherin repeats. Homologous genes have been identified in C. elegans and D. melanogaster suggesting that the Celsr gene family is ancient. mCelsr1 mRNA expression precedes gastrulation, is subsequently restricted primarily to ectodermal derivatives and is tightly regulated in the developing central nervous system (CNS). We observe segmentally-restricted gene expression in the developing hindbrain and in the spinal cord dynamic dorso-ventrally restricted 'stripes' of expression.

Generation of a transcription map distal to HLA-F.

We have constructed a transcription map covering a 2 Mb region beginning approximately 1 Mb distal to HLA-F. Cosmids isolated from a chromsome 6 library were positioned by YAC hybridisation, STS and fingerprint analysis. Using direct cDNA selection, exon trapping, and direct genomic sequence analysis, we identified 42 potential exonic fragments in this region. Six fragments corresponded to previously characterised genes, four previously broadly mapped to this region. Five fragments were similar to known genes, eight fragments matched ESTs and 10 of the remaining 23 novel fragments, gave a positive signal on northern analysis. All cDNA fragments were mapped to the YAC and cosmid contig covering the region and with respect to other known genes and STS in this area. The distribution of the cDNA fragments indicated their organisation in three clusters around CpG islands.

A sequence-ready 3-Mb PAC contig covering 16 breakpoints of the Wilms tumor/anirida region of human chromosome 11p13.

A large body of evidence that links alterations of chromosome 11p13 to tumor formation and various developmental disorders has been accumulated. To address the underlying genetic events it would be helpful to have a comprehensive gene map of the region, and this is most readily achieved by generating the complete genomic sequence. Building upon previous mapping and YAC contig analysis we have established a 3-Mb sequence-ready PAC contig. It was constructed by chromosome walking and independently verified by fingerprint analysis of individual clones. The contig starts from the catalase gene on the centromeric side and reaches beyond the PAX6 gene at the 11p13/p14.1 boundary. Additional smaller contigs on either side were identified, but still have to be linked up. The 3-Mb contig spans the central region of deletions encompassing 16 chromosomal breakpoints in patients with WAGR syndrome (Wilms tumor, aniridia, genitourinary malformation, mental retardation), and its construction is an important step in facilitating functional analysis of these genes.

Characterization of a KRAB family zinc finger gene, ZNF195, mapping to chromosome band 11p15.5.

We report the cDNA sequence of the zinc finger gene, ZNF195, which maps to chromosome 11p15.5. ZNF195 contains an N-terminal KRAB domain and 14 tandemly repeated Krüppel type zinc finger motifs at its C-terminus. Northern analysis shows expression of ZNF195 in adult heart, brain, placenta, skeletal muscle, and pancreas with a predominant transcript size of 4.3 kb. There is little expression in adult lung, liver, and kidney. In fetal lung, liver, kidney, and brain, the predominant transcript is 3.5 kb. Fetal brain also expresses a 4.3-kb transcript. RT-PCR analysis shows that two exons, 4a, which contains an inverted Alu sequence, and 4b, are differentially spliced and absent from the major transcript.

Celsr1, a neural-specific gene encoding an unusual seven-pass transmembrane receptor, maps to mouse chromosome 15 and human chromosome 22qter.

We have identified Celsr1, a gene that encodes a developmentally regulated vertebrate seven-pass transmembrane protein. The extracellular domain of Celsr1 contains two regions each with homology to distinct classes of well-characterized motifs found in the extra-cellular domains of many cell surface molecules. The most N-terminal region contains a block of contiguous cadherin repeats, and C-terminal to this is a region containing seven epidermal growth factor-like repeats interrupted by two laminin A G-type repeats. Celsr1 is unique in that it contains this combination of repeats coupled to a seven-pass transmembrane domain. As part of the characterization of the Celsr1 gene, we have determined its chromosomal map location in both mouse and human. The European Collaborative Interspecific Backcross (EUCIB) and BXD recombinant inbred strains were used for mapping Celsr1 cDNA clones in the mouse, and fluorescence in situ hybridization was used to map human Celsr1 cosmid clones on metaphase chromosomes. We report that Celsr1 maps to proximal mouse Chromosome 15 and human chromosome 22qter, a region of conserved synteny. Reverse transcriptase-polymerase chain reaction analysis and in situ hybridization were used to determine the spatial restriction of Celsr1 transcripts in adult and embryonic mice. The results presented here extend our previous finding of expression of the Celsr1 receptor in the embryo and show that expression continues into adult life when expression in the brain is localized principally in the ependymal cell layer, choroid plexus, and the area postrema.

Genomic organization and chromosomal localization of a member of the MAP kinase phosphatase gene family to human chromosome 11p15.5 and a pseudogene to 10q11.2.

Mitogen-activated protein kinase phosphatases (MKPs) play a central role in a variety of signaling pathways. We recently described a novel murine MKP, M3/6, which is uniquely specific for c-Jun N-terminal kinase/stress-activated protein kinase and p38 kinase. Here we report the localization of the human orthologue of this gene, HB5, to within 150 kb of H19 on human chromosome 11p15.5. The gene consists of six exons. Two of the introns in HB5 are not found in other genes of this family, suggesting an evolutionary split between MKPs displaying specificity toward different MAP kinases. An intronless pseudogene is present on chromosome 10q11.2. Although 11p15.5 is an imprinted region, HB5 is almost entirely unmethylated on both alleles in lymphocytes. Chromosome 11p15 has been implicated in the development of a number of tumor types, including lung, a tissue known to express this gene. Loss of heterozygosity was found in one of eight informative lung tumors studied.

Isolation and characterisation of cosmids to intervals within a 4.5Mb region at 6p21.3.

The gene responsible for hereditary haemochromatosis (HH) has recently been identified. One mutation in this gene, termed HFE, has been found in all Australian HH patients. We previously identified a predominant HH ancestral haplotype covering 4.5Mb at 6p21.3, and showed that patients with two copies of this haplotype express a more severe form of the disorder. One key question to now be resolved is why haplotype related variation in phenotypic expression of HH is present if all patients tested have the same HFE mutation. A cosmid resource covering the 4.5Mb HH ancestral haplo type region was obtained. These cosmids provide the material for the completion of a transcript map of this region, and will assist the identification of candidate modifiers of HFE expression.

Physical analysis of the region deleted in the tw18 allele of the mouse tcl-4 complementation group.

We have generated a YAC contig of at least 3.3 Mb from the proximal region of In(17)4 of mouse chromosome 17. This region corresponds to DNA lost in the gastrulation mutant tw18, which belongs to the tcl-4 complementation group. Our most proximal and distal probes lie within the deletion-3.3 Mb apart-indicating that we have not cloned the entire region. The deleted region is contained in a genetic interval of less than 1 cM, suggesting that some suppression of recombination must occur.

Mapping the human Y chromosome by fingerprinting cosmid clones.

We have used Y-specific cosmid clones in a random fingerprinting approach to build contigs on the human Y chromosome. Clones derived from two libraries have been analyzed. The construction of one library is described here, the second was the Y chromosome-specific library LLOYNCO3 "M" (Lawrence Livermore National Laboratory). To date, we have fingerprinted 4430 cosmids: 377 contigs have been constructed containing from 2 to 39 clones. Along with the singletons, we estimate that we have covered 72.5% of the euchomatic portion of the Y chromosome with fingerprinted clones. Sequence tagged sites are being used to anchor cosmids and contigs onto the YAC framework.

Human nicotinamide N-methyltransferase gene: molecular cloning, structural characterization and chromosomal localization.

Genomic DNA clones for nicotinamide N-methyltransferase (NNMT), an enzyme that catalyzes drug and xenobiotic metabolism, were isolated from a human chromosome 11-specific DNA library. Study of one of those clones, when combined with PCR-based experiments performed with human genomic DNA, made it possible to determine the structure of the human NNMT gene. The gene was approximately 16.5 kb in length and consisted of 3 exons and 2 introns. Transcription initiation for the NNMT gene occurred 105-109 nucleotides 5'-upstream from the cDNA translation initiation codon on the basis of the results of both primer extension and 5'-rapid amplification of cDNA ends. NNMT mapped to chromosome band 11q23.1 by fluorescence in situ hybridization.

Seven genes on the short arm of human chromosome 3 map to two regions on Macropus eugenii (tammar wallaby) chromosome 2.

Seven genes were mapped by in situ hybridization to metaphase chromosomes of the marsupial species Macropus eugenii, using a series of human-derived cloned probes (six cosmids and one cDNA). The genes were located in two widely separated clusters on the long arm of M. eugenii chromosome 2, in contrast to their location in a single cluster on the distal half of the short arm of human chromosome 3. Multiple rearrangements had to be involved in the evolutionary divergence of these chromosome segments from the unknown arrangement in the common ancestor.

Tumor cell growth arrest caused by subchromosomal transferable DNA fragments from chromosome 11.

A fundamental problem in the identification and isolation of tumor suppressor and other growth-inhibiting genes is the loss of power of genetic complementation at the subchromosomal level. A direct genetic strategy was developed to isolate subchromosomal transferable fragments (STFs) from any chromosome, each containing a selectable marker within the human DNA, that could be transferred to any mammalian cell. As a test of the method, several overlapping STFs from 11p15 were shown to cause in vitro growth arrest of rhabdomyosarcoma cells. This activity mapped between the beta-globin and insulin genes.

Isolation and expression of linked zinc finger gene clusters on human chromosome 11q.

Proteins that share conserved "zinc finger" motifs represent a class of DNA-binding proteins that have been shown to play a fundamental role in regulating gene expression and to be involved in a number of human hereditary and malignant disease states. We have isolated, characterized, and mapped zinc finger-encoding genes specific to human chromosome 11q to investigate their possible association in the molecular pathogenesis of several disease loci mapped to this chromosome. An arrayed chromosome 11q cosmid library was screened using a degenerate oligonucleotide corresponding to the H/C link consensus sequence of the Drosophila Kruppel zinc finger gene, resulting in the isolation of six putative zinc finger genes. Three of the genes (ZNF123, ZNF125, and ZNF126) were analyzed and shown to contain tandemly repeated zinc finger motifs of the C2-H2 class. All three novel genes were found to be expressed in normal adult human tissues, although the tissue-specific pattern of expression differs markedly. Isolated zinc finger genes were regionally mapped on chromosome 11 using fluorescence in situ suppression hybridization and demonstrated clustering of the genes at 11q13.3-11q13.4 and 11q23.1-11q23.2. Analysis of in situ hybridization to interphase nuclei demonstrated a maximum distance of 1 Mb separating distinct finger genes. This analysis defines two linked multigene families of zinc finger genes to chromosome bands associated with a high frequency of specific translocations associated with malignancies.

High-resolution localization of 69 potential human zinc finger protein genes: a number are clustered.

In this study, we describe the identification and partial characterization of 101 potential human zinc finger protein genes (ZnFPs). These sequences were isolated by hybridization of cosmids, obtained from mouse-human cell lines enriched for chromosome 11p, with an oligonucleotide specific for the "link" sequence between contiguous zinc fingers. Sixty-nine of these cosmids were regionally localized to human prometaphase chromosomes by in situ hybridization. The localization of these cosmids suggests that a number of finger protein genes occur in linked clusters. Their assignment to chromosomes 3p, 11p, 19p, 19qter, 20p, and 21q makes them valuable as markers or "candidate" genes for diseases associated with these chromosome regions.