Molecular cytogenetics of the california condor: evolutionary and conservation implications.

Evolutionary cytogenetic comparisons involved 5 species of birds (California condor, chicken, zebra finch, collared flycatcher and black stork) belonging to divergent taxonomic orders. Seventy-four clones from a condor BAC library containing 80 genes were mapped to condor chromosomes using FISH, and 15 clones containing 16 genes were mapped to the stork Z chromosome. Maps for chicken and finch were derived from genome sequence databases, and that for flycatcher from the published literature. Gene content and gene order were highly conserved when individual condor, chicken, and zebra finch autosomes were compared, confirming that these species largely retain karyotypes close to the ancestral condition for neognathous birds. However, several differences were noted: zebra finch chromosomes 1 and 1A are homologous to condor and chicken chromosomes 1, the CHUNK1 gene appears to have transposed on condor chromosome 1, condor chromosomes 4 and 9 and zebra finch chromosomes 4 and 4A are homologous to chicken chromosome arms 4q and 4p, and novel inversions on chromosomes 4, 12 and 13 were found. Condor and stork Z chromosome gene orders are collinear and differentiated by a series of inversions/transpositions when compared to chicken, zebra finch, or flycatcher; phylogenetic analyses suggest independent rearrangement along the chicken, finch, and flycatcher lineages.

Further evidence that the KIAA0319 gene confers susceptibility to developmental dyslexia.

The DYX2 locus on chromosome 6p22.2 is the most replicated region of linkage to developmental dyslexia (DD). Two candidate genes within this region have recently been implicated in the disorder: KIAA0319 and DCDC2. Variants within DCDC2 have shown association with DD in a US and a German sample. However, when we genotyped these specific variants in two large, independent UK samples, we obtained only weak, inconsistent evidence for their involvement in DD. Having previously found evidence that variation in the KIAA0319 gene confers susceptibility to DD, we sought to refine this genetic association by genotyping 36 additional SNPs in the gene. Nine SNPs, predominantly clustered around the first exon, showed the most significant association with DD in one or both UK samples, including rs3212236 in the 5' flanking region (P = 0.00003) and rs761100 in intron 1 (P = 0.0004). We have thus refined the region of association with developmental dyslexia to putative regulatory sequences around the first exon of the KIAA0319 gene, supporting the presence of functional mutations that could affect gene expression. Our data also suggests a possible interaction between KIAA0319 and DCDC2, which requires further testing.

Use of scanning electron microscopy to confirm the identity of lice infesting communally grazed goat herds.

Lice have been described on goats in commercial farming systems in South Africa but not from flocks on communal grazing. During a longitudinal survey on the causes of goat kid mortality, conducted in Jericho district, North West Province, lice were collected from communally grazed indigenous goats. These lice were prepared for and viewed by scanning electron microscopy, and micro-morphological taxonomic details are described. Three species of lice were found in the study area and identified as Bovicola caprae, Bovicola limbatus and Linognathus africanus. Sucking and biting lice were found in ten of the 12 herds of goats examined. Lice were found on both mature goats and kids. Bovicola caprae and L. africanus were the most common biting and sucking lice respectively in all herds examined. Scanning electron microscopy revealed additional features which aided in the identification of the louse species. Photomicrographs were more accurate aids to identification than the line drawings in the literature and facilitated identification using dissecting microscope.

Candidate gene isolation and comparative analysis of a commonly deleted segment of 7q22 implicated in myeloid malignancies.

Monosomy 7 and deletion of 7q are recurring abnormalities in malignant myeloid diseases. Here we extensively characterize an approximately 2-Mb commonly deleted segment (CDS) of 7q22 bounded by D7S1503 and D7S1841. Approximately 1.8 Mb of sequence have been generated from this interval, facilitating the construction of a transcript map that includes large numbers of genes and ESTs. The intron/exon organization of seven genes and expression patterns of three genes were determined, and leukemia samples were screened for mutations in five genes. We have used polymorphic markers from this region to examine leukemia cells for allelic loss within 7q22. Finally, we isolated mouse genomic clones orthologous to several of the characterized human genes. Fluorescence in situ hybridization studies using these clones indicate that a region of orthologous synteny lies on proximal mouse chromosome 5. These resources should greatly accelerate the pace of candidate gene discovery in this region.

The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants.

The pregnane X receptor (PXR)/steroid and xenobiotic receptor (SXR) transcriptionally activates cytochrome P4503A4 (CYP3A4) when ligand activated by endobiotics and xenobiotics. We cloned the human PXR gene and analysed the sequence in DNAs of individuals whose CYP3A phenotype was known. The PXR gene spans 35 kb, contains nine exons, and mapped to chromosome 13q11-13. Thirty-eight single nucleotide polymorphisms (SNPs) were identified including six SNPs in the coding region. Three of the coding SNPs are non-synonymous creating new PXR alleles [PXR*2, P27S (79C to T); PXR*3, G36R (106G to A); and PXR*4, R122Q (4321G to A)]. The frequency of PXR*2 was 0.20 in African Americans and was never found in Caucasians. Hepatic expression of CYP3A4 protein was not significantly different between African Americans homozygous for PXR*1 compared to those with one PXR*2 allele. PXR*4 was a rare variant found in only one Caucasian person. Homology modelling suggested that R122Q, (PXR*4) is a direct DNA contact site variation in the third alpha-helix in the DNA binding domain. Compared with PXR*1, and variants PXR*2 and PXR*3, only the variant PXR*4 protein had significantly decreased affinity for the PXR binding sequence in electromobility shift assays and attenuated ligand activation of the CYP3A4 reporter plasmids in transient transfection assays. However, the person heterozygous for PXR*4 is normal for CYP3A4 metabolism phenotype. The relevance of each of the 38 PXR SNPs identified in DNA of individuals whose CYP3A basal and rifampin-inducible CYP3A4 expression was determined in vivo and/or in vitro was demonstrated by univariate statistical analysis. Because ligand activation of PXR and upregulation of a system of drug detoxification genes are major determinants of drug interactions, it will now be useful to extend this work to determine the association of these common PXR SNPs to human variation in induction of other drug detoxification gene targets.

Strategies for the systematic sequencing of complex genomes.

Recent spectacular advances in the technologies and strategies for DNA sequencing have profoundly accelerated the detailed analysis of genomes from myriad organisms. The past few years alone have seen the publication of near-complete or draft versions of the genome sequence of several well-studied, multicellular organisms - most notably, the human. As well as providing data of fundamental biological significance, these landmark accomplishments have yielded important strategic insights that are guiding current and future genome-sequencing projects.

Comparative physical mapping of targeted regions of the rat genome.

The comparative mapping and sequencing of vertebrate genomes is now a key priority for the Human Genome Project. In addition to finishing the human genome sequence and generating a 'working draft' of the mouse genome sequence, significant attention is rapidly turning to the analysis of other model organisms, such as the laboratory rat (Rattus norvegicus). As a complement to genome-wide mapping and sequencing efforts, it is often important to generate detailed maps and sequence data for specific regions of interest. Using an adaptation of our previously described approach for constructing mouse comparative and physical maps, we have established a general strategy for targeted mapping of the rat genome. Specifically, we constructed a framework comparative map of human Chromosome (Chr) 7 and the orthologous regions of the rat genome, as well as two large (>1-Mb) P1-derived artificial chromosome (PAC)-based physical maps. Generation of these physical maps involved the use of mouse-derived probes that cross-hybridized with rat PAC clones. The first PAC map encompasses the cystic fibrosis transmembrane conductance regulator gene (Cftr), while the second map allows a three-species comparison of a genomic region containing intra- and inter-chromosomal evolutionary rearrangements. The studies reported here further demonstrate that cross-species hybridization between related animals, such as rat and mouse, can be readily used for the targeted construction of clone-based physical maps, thereby accelerating the analysis of biologically interesting regions of vertebrate genomes.

Refinement within single yeast artificial chromosome clones of a minimal region commonly deleted on the short arm of chromosome 7 in Wilms tumours.

Cytogenetic and molecular data indicate an involvement of genes mapped to the proximal portion of the short arm of chromosome 7 (7p) in Wilms tumours (WTs). We have analysed 38 WTs using a panel of eight microsatellite markers mapped to proximal 7p. Loss of heterozygosity (LOH) in tumour, compared with matched constitutional DNA, was identified in eight cases. To define better the minimal region commonly deleted in these tumours, they were analysed with nine additional markers, mapped within the region of interest. One tumour (case 30) showed LOH for only one marker (D7S510), while maintaining heterozygosity for the two immediately flanking loci (D7S555 and D7S668). This result was confirmed by fluorescence in situ hybridisation analysis, which showed that in the majority (65%) of nuclei from tumour 30 hybridising with a bacterial artificial chromosome clone containing the D7S510 locus, only one signal was visible. Noticeably, both markers defining the limits of the observed deleted region are simultaneously present within two distinct overlapping yeast artificial chromosome (YAC) clones mapped to chromosome bands 7p13-p14. This suggests that the maximum length of the missing DNA fragment was approximately 1.3 Mb, corresponding to the length of the smaller of the two YAC clones. In all other cases that showed LOH, the deletion encompassed the 7p13-p14 region. For this reason, we speculate that the identified interval contains a gene whose inactivation is important for the development of at least a fraction of WTs.

Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion.

Pendrin is an anion transporter encoded by the PDS/Pds gene. In humans, mutations in PDS cause the genetic disorder Pendred syndrome, which is associated with deafness and goiter. Previous studies have shown that this gene has a relatively restricted pattern of expression, with PDS/Pds mRNA detected only in the thyroid, inner ear, and kidney. The present study examined the distribution and function of pendrin in the mammalian kidney. Immunolocalization studies were performed using anti-pendrin polyclonal and monoclonal antibodies. Labeling was detected on the apical surface of a subpopulation of cells within the cortical collecting ducts (CCDs) that also express the H(+)-ATPase but not aquaporin-2, indicating that pendrin is present in intercalated cells of the CCD. Furthermore, pendrin was detected exclusively within the subpopulation of intercalated cells that express the H(+)-ATPase but not the anion exchanger 1 (AE1) and that are thought to mediate bicarbonate secretion. The same distribution of pendrin was observed in mouse, rat, and human kidney. However, pendrin was not detected in kidneys from a Pds-knockout mouse. Perfused CCD tubules isolated from alkali-loaded wild-type mice secreted bicarbonate, whereas tubules from alkali-loaded Pds-knockout mice failed to secrete bicarbonate. Together, these studies indicate that pendrin is an apical anion transporter in intercalated cells of CCDs and has an essential role in renal bicarbonate secretion.

A physical map of the human genome.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

Mutational and functional analyses reveal that ST7 is a highly conserved tumor-suppressor gene on human chromosome 7q31.

Loss of heterozygosity (LOH) of markers on human chromosome 7q31 is frequently encountered in a variety of human neoplasias, indicating the presence of a tumor-suppressor gene (TSG). By a combination of microcell-fusion and deletion-mapping studies, we previously established that this TSG resides within a critical region flanked by the genetic markers D7S522 and D7S677. Using a positional cloning strategy and aided by the availability of near-complete sequence of this genomic interval, we have identified a TSG within 7q31, named ST7 (for suppression of tumorigenicity 7; this same gene was recently reported in another context and called RAY1). ST7 is ubiquitously expressed in human tissues. Analysis of a series of cell lines derived from breast tumors and primary colon carcinomas revealed the presence of mutations in ST7. Introduction of the ST7 cDNA into the prostate-cancer-derived cell line PC3 had no effect on the in vitro proliferation of the cells, but abrogated their in vivo tumorigenicity. Our data indicate that ST7 is a TSG within chromosome 7q31 and may have an important role in the development of some types of human cancer.

Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene.

A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5' end of the KRIT1 cDNA sequence. RT-PCR and 5'RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.

Effects of thyroglobulin and pendrin on iodide flux through the thyrocyte.

Iodide transport by thyrocytes involves porters on the apical and basal surfaces of the cell facing the follicular lumen and bloodstream, respectively. Recent work identifies pendrin as an apical porter and shows that follicular thyroglobulin is a transcriptional regulator of the gene encoding pendrin and other thyroid-restricted genes. For example, whereas follicular thyroglobulin suppresses the gene expression and activity of the sodium iodide symporter (NIS), it increases pendrin gene expression. A potential new dynamic for iodide flux and thyroid hormone formation in thyrocytes has thus emerged and is supported by in vivo data.

Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome.

Following the positional cloning of PDS, the gene mutated in the deafness/goitre disorder Pendred syndrome (PS), numerous studies have focused on defining the role of PDS in deafness and PS as well as elucidating the function of the PDS-encoded protein (pendrin). To facilitate these efforts and to provide a system for more detailed study of the inner-ear defects that occur in the absence of pendrin, we have generated a Pds-knockout mouse. Pds(-/-) mice are completely deaf and also display signs of vestibular dysfunction. The inner ears of these mice appear to develop normally until embryonic day 15, after which time severe endolymphatic dilatation occurs, reminiscent of that seen radiologically in deaf individuals with PDS mutations. Additionally, in the second postnatal week, severe degeneration of sensory cells and malformation of otoconia and otoconial membranes occur, as revealed by scanning electron and fluorescence confocal microscopy. The ultrastructural defects seen in the Pds(-/-) mice provide important clues about the mechanisms responsible for the inner-ear pathology associated with PDS mutations.

A balanced reciprocal translocation t(5;7)(q14;q32) associated with autistic disorder: molecular analysis of the chromosome 7 breakpoint.

Autism is a neuropsychiatric disorder characterized by impairments in social interaction, restricted and stereotypic pattern of interest with onset by 3 years of age. The results of genetic linkage studied for autistic disorder (AD) have suggested a susceptibility locus for the disease on the long arm of chromosome 7. We report a girl with AD and a balanced reciprocal translocation t(5;7)(q14;q32). The mother carries the translocation but do not express the disease. Fluorescent in situ hybridization (FISH) analysis with chromosome 7-specific YAC clones showed that the breakpoint coincides with the candidate region for AD. We identified a PAC clone that spans the translocation breakpoint and the breakpoint was mapped to a 2 kb region. Mutation screening of the genes SSBP and T2R3 located just centromeric to the breakpoint was performed in a set of 29 unrelated autistic sibling pairs who shared at least one chromosome 7 haplotype. We found no sequence variations, which predict amino acid alterations. Two single nucleotide polymorphisms were identified in the T2R3 gene, and associations between allele variants and AD in our population were not found. The methylation pattern of different chromosome 7 regions in the patient's genomic DNA appears normal. Here we report the clinical presentation of the patient with AD and the characterization of the genomic organization across the breakpoint at 7q32. The precise localization of the breakpoint on 7q32 may be relevant for further linkage studies and molecular analysis of AD in this region.

Divergent human and mouse orthologs of a novel gene (WBSCR15/Wbscr15) reside within the genomic interval commonly deleted in Williams syndrome.

Williams syndrome (WS) is a contiguous gene deletion disorder resulting in complex and intriguing clinical features. Detailed molecular characterization studies of the genomic segment on human chromosome 7q11.23 commonly deleted in WS have uncovered numerous genes, each of which is being actively studied for its possible role in the etiology of the syndrome. Our efforts have focused on the comparative mapping and sequencing of the WS region in human and mouse. In previous studies, we uncovered important differences in the long-range organization of these human and mouse genomic regions; in particular, the notable absence of large duplicated blocks of DNA in mouse that are present in human. Aided by available genomic sequence data, we have used a combination of gene-prediction programs and cDNA isolation to identify the human and mouse orthologs of a novel gene (WBSCR15 and Wbscr15, respectively) residing within the genomic segment commonly deleted in WS. Unlike the flanking genes, which are closely related in human and mouse, WBSCR15 and Wbscr15 are strikingly different with respect to their cDNA and corresponding protein sequences as well as tissue-expression pattern. Neither the WBSCR15- nor Wbscr15-encoded amino acid sequence shows a statistically significant similarity to any characterized protein. These findings reveal another interesting evolutionary difference between the human and mouse WS regions and provide an additional candidate gene to evaluate with respect to its possible role in the pathogenesis of WS.