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The Shwachman–Bodian–Diamond syndrome (SBDS) protein family occurs widely in nature, although its function has not been determined. Comprehensive database searches revealed SBDS homologues from 159 species, including examples from all sequenced archaeal and eukaryotic genomes and all eukaryotic kingdoms. Sequence alignment with ClustalX and MUSCLE algorithms led to the identification of conserved residues that occurred predominantly in the amino-terminal FYSH domain where they appeared to contribute to protein folding or stability. Only SBDS residue Gly91 was invariant in all species. Four distantly related protists were found to have two divergent SBDS genes in their genomes. In each case, phylogenetic analyses and the identification of shared sequence features suggested that one gene was derived from lateral gene transfer. We also identified a shared C-terminal zinc finger domain fusion in flowering plants and chromalveolates that may shed light on the function of the protein family and the evolutionary histories of these kingdoms. To assess the extent of SBDS functional conservation, we carried out complementation studies of SBDS homologues and interspecies chimeras in Saccharomyces cerevisiae. We determined that the FYSH domain was widely interchangeable among eukaryotes, while domain 2 imparted species specificity to protein function. Domain 3 was largely dispensable for function in our yeast complementation assay. Overall, the phylogeny of SBDS was shared with a group of proteins that were markedly enriched for RNA metabolism and/or ribosome-associated functions. These findings link Shwachman–Diamond syndrome to other bone marrow failure syndromes with defects in nucleolus-associated processes, including Diamond–Blackfan anemia, cartilage–hair hypoplasia, and dyskeratosis congenita.

Preaxial polydactyly is a congenital hand malformation that includes duplicated thumbs, various forms of triphalangeal thumbs, and duplications of the index finger. A locus for preaxial polydactyly has been mapped to a region of 1.9 cM on chromosome 7q36 between polymorphic markers D7S550 and D7S2423. We constructed a detailed physical map of the preaxial polydactyly candidate region. With a combination of methods we identified and positioned 11 transcripts within this map. By recombination analysis on families with preaxial polydactyly, using newly developed polymorphic markers, we were able to reduce the candidate region to approximately 450 kb. The homeobox gene HLXB9, a putative receptor C7orf2, and two transcripts of unknown function, C7orf3 and C7orf4, map in the refined candidate region and have been subjected to mutation analysis in individuals with preaxial polydactyly.

The human XRCC2 gene, complementing a hamster cell line (irs1) hypersensitive to DNA-damaging agents, was previously mapped to chromosome 7q36.1. Following radiation reduction of human/hamster hybrids, the gene was found to be associated with the marker D7S483. Yeast artificial chromosomes (YACs) carrying D7S483 were fused to the irs1 cell line to identify a YAC that complemented the sensitivity defect. Transcribed sequences were isolated by direct cDNA selection using the complementing YAC, and these were mapped back to the YAC and hybrids to define a 400-kb region carrying XRCC2. Sequencing of cDNAs led to the identification of both known and novel gene sequences, including a candidate for XRCC2 with homology to the yeast RAD51 gene involved in the recombinational repair of DNA damage. Strong support for the candidacy of this gene was obtained from its refined map position and by the full complementation of irs1 sensitivity with a 40-kb cosmid carrying the gene.

Mutations in the human presenilin genes (PSEN1 and PSEN2) are associated with early onset familial Alzheimer disease. The presenilin genes encode integral membrane proteins with similar structures, which suggests that they may have closely related, but as yet unknown functions. Analysis of the 5' upstream sequence and the structure of the PSEN1 gene reveals that the 5' sequence contains multiple putative transcription regulatory elements including clusters of STAT elements involved in transcriptional activation in response to signal transduction. The first four exons contain untranslated sequences, with Exons 1 and 2 representing alternate initial transcription sites. The function of these alternate initial exons is unclear. Exon 4 bears the first ATG sequence. The last 12 bp of Exon 4 is used as an alternative splice donor site. Exon 9 is alternately spliced in leukocytes, but not in most other tissues. Splicing of Exon 9 is predicted to cause significant structural changes to the protein. The majority of transcripts expressed in most tissues are polyadenylated 1127 bp from the TAG stop codon in Exon 13. A small proportion of transcripts contain the same 5'UTR and ORF but are polyadenylated 4435 bp from the stop codon. The longer polyadenylated transcripts contain three additional palindromes and at least one additional stem-loop structure with stabilities greater than -16 kcal/mol.

Williams syndrome (WS) is a multisystem developmental disorder caused by the deletion of contiguous genes at 7q11.23. Hemizygosity of the elastin (ELN) gene can account for the vascular and connective tissue abnormalities observed in WS patients, but the genes that contribute to features such as infantile hypercalcemia, dysmorphic facies, and mental retardation remain to be identified. In addition, the size of the genomic interval commonly deleted in WS patients has not been established. In this study we report the characterization of a 500-kb region that was determined to be deleted in our collection of WS patients. A detailed physical map consisting of cosmid, P1 artificial chromosomes, and yeast artificial chromosomes was constructed and used for gene isolation experiments. Using the techniques of direct cDNA selection and genomic DNA sequencing, three known genes (ELN, LIMK1, and RFC2), a novel gene (WSCR1) with homology to RNA-binding proteins, a gene with homology to restin, and four other putative transcription units were identified. LIMK1 is a protein kinase with two repeats of the LIM/double zinc finger motif, and it is highly expressed in brain. RFC2 is the 40-kDa ATP-binding subunit of replication factor C, which is known to play a role in the elongation of DNA catalyzed by DNA polymerase δ and ϵ. LIMK1 and WSCR1 may be particularly relevant when explaining cognitive defects observed in WS patients.

A genetic locus (RP3) for X-linked retinitis pigmentosa (XLRP) has been assigned to Xp21 by genetic linkage studies and has been supported by two Xp21 male deletion patients with XLRP. RP3 appears to be the most centromeric of several positioned loci, including chronic granulomatous disease (CGD), McLeod phenotype (XK), and Duchenne muscular dystrophy (DMD). In one patient, BB, the X-chromosome deletion includes RP3 and extends to within the DMD locus. Using a DMD cDNA, the centromeric endpoint of this patient was cloned and used as a starting point for chromosome walking along a normal X chromosome. A single-copy probe, XH1.4, positioned near the centromeric junction but deleted in BB, was used along with a CGD cDNA probe to establish a refined long-range physical map. Both probes recognized a common Sfi I fragment of 205 kb. As the CGD gene covers approximately 30–60 kb, the RP3 locus has been restricted to approximately 150–170 kb. A CpG island, potentially marking a new gene, was identified within the Sfi I fragment at a position approximately 35 kb from the deletion endpoint in BB.

Five different mutations have been identified in the gene causing cystic fibrosis (CF) through sequencing regions encompassing exons 1-8, including the 5' untranslated leader. Two of these apparent mutations are missense mutations, one in exon 3 (Gly to Glu at position 85; G85E) and another in exon 5 (Gly to Arg at 178; G178R), both causing significant changes in the corresponding amino acids in the encoded protein--cystic fibrosis transmembrane conductance regulator (CFTR). Two others affect the highly conserved RNA splice junction flanking the 3' end of exons 4 and 5 (621 + 1G----T, 711 + 1G----T), resulting in a probable splicing defect. The last mutation is a single-basepair deletion in exon 4, causing a frameshift. These five mutations account for the 9 of 31 non-delta F508 CF chromosomes in our Canadian CF family collection and they are not found in any of the normal chromosomes. Three of the mutations, 621 + 1G----T, 711 + 1G----T, and G85E, are found in the French-Canadian population, with 621 + 1G----T being the most abundant (5/7). There are two other sequence variations in the CFTR gene; one of them (129G----C) is located 4 nucleotides upstream of the proposed translation initiation codon and, although present only on CF chromosomes, it is not clear whether it is a disease-causing mutation; the other (R75Q) is most likely a sequence variation within the coding region.

Cytoplasmic dynein is a large multisubunit microtubule-based motor protein, which mediates movement of numerous intracellular organelles. We report here the identification of the human homologue of cytoplasmic dynein intermediate chain 1 gene (DNCI1) located on human chromosome 7q21.3-q22.1. The mouse orthologue (Dnci1) was identified along with another highly related gene, Dnci2, and their RNA in situ expression patterns were examined during mouse embryogenesis. Dnci1 was found to have a highly restricted expression domain in the developing forebrain as well as the peripheral nervous system (PNS), while Dnci2 displayed a broad expression profile throughout the entire central nervous system and most of the PNS. A dynamic expression profile was also found for Dnci2 in the developing mouse limb bud. The data presented here provide a framework for the further analysis of the functional role of Dnci1 and Dnci2 in mouse and DNCI1 in human.

Cloning and characterization of the cystic fibrosis transmembrane conductance regulator (CFTR) gene led to the identification and isolation of cDNA and genomic sequences that cross-hybridized to the first nucleotide binding fold of CFTR. DNA sequence analysis of these clones showed that the cross-hybridizing sequences corresponded to CFTR exon 9 and its flanking introns, juxtapositioned with two segments of LINE1 sequences. The CFTR sequence appeared to have been transcribed from the opposite direction of the gene, reversely transcribed, and co-integrated with the L1 sequences into a chromosome location distinct from that of the CFTR locus. Based on hybridization intensity and complexity of the restriction fragments, it was estimated that there were at least 10 copies of the "amplified" CFTR exon 9 sequences in the human genome. Furthermore, when DNA segments adjacent to the insertion site were used in genomic DNA blot hybridization analysis, multiple copies were also detected. The overall similarity between these CFTR exon 9-related sequences suggested that they were derived from a single retrotransposition event and subsequent sequence amplification. The amplification unit appeared to be greater than 30 kb. Physical mapping studies including in situ hybridization to human metaphase chromosomes showed that multiple copies of these amplified sequences (with and without the CFTR exon 9 insertion) were dispersed throughout the genome. These findings provide insight into the structure and evolution of the human genome.

The region of chromosome 14q24 has been of particular interest as it is known to contain one of the early-onset Alzheimer disease genes (AD3). Other genes of medical interest, such as arrhythmogenic right ventricular cardiomyopathy, have been mapped to this region by linkage analysis or chromosome rearrangements. We have focused on the region of a balanced translocation (2;14)(p25;q24). Members of a family with this translocation all have anterior polar cataracts, suggesting the presence of a gene involved in lens integrity at the vicinity of the breakpoint. The chromosome 14 breakpoint has been defined between the short tandem repeats D14S289 and D14S277, a region of overlap for yeast artificial chromosomes (YACs) 888b2 and 934d4. We have extended the study of the region to 2 Mb on chromosome 14 and present a physical map of this region, including several sequence-tagged sites. New probes were generated using several end clones and inter- Alu PCR fragments from YACs. cDNA selection was used to identify transcribed sequences. Mapping and alignment of 17 nonoverlapping cDNAs completed by sequence and expression pattern analysis suggested that a minimum of eight putative transcription units is present in this region: six of these units correspond to five new genes and one member of a new gene family.

As part of efforts to identify candidate genes for disorders mapped to 21q22.3, we have constructed a 405-kb cosmid contig encompassing five tightly linked markers mapping to this region. A subset of these cosmids was used to identify cDNA fragments by the method of hybrid selection. We present here the cDNA sequence of one such gene (GT335) mapping to this region. The gene is expressed as a 1.7-kb transcript predominantly in heart and skeletal muscle, potentially displays alternate splicing, and is predicted to encode a protein 268 amino acids in length. GT335 spans an estimated 13 kb of genomic DNA and is split into seven exons. Five of the six introns conform to the GT . . . AG consensus for intronic splice junctions; the sixth contains nonconventional (AT . . . AC) intronic junctions. We screened this gene for single-basepair mutations using single-strand conformation polymorphism and sequence analysis of both cDNA and genomic DNA from a number of unrelated individuals and have identified several sequence variations, two of which cause conservative amino acid substitutions. This gene is well conserved evolutionarily, with homologs identified in zebrafish and Escherichia coli, suggesting that it plays an important role in basic cellular metabolism.

An integrated approach involving physical mapping, identification of transcribed sequences, and computational analysis of genomic sequence was used to generate a detailed transcription map of the 1. 0-Mb region containing the breast cancer susceptibility locus BRCA2 on chromosome 13q12-q13. This region is included in the genetic interval bounded by D13S1444 and D13S310. Retrieved sequences from exon amplification or hybrid selection procedures were grouped into physical intervals and subsequently grouped into transcription units by clone overlap. Overlap was established by direct hybridization, cDNA library screening, PCR cDNA linking (island hopping), and/or sequence alignment. Extensive genomic sequencing was performed in an effort to understand transcription unit organization. In total, approximately 500 kb of genomic sequence was completed. The transcription units were further characterized by hybridization to RNA from a series of human tissues. Evidence for seven genes, two putative pseudogenes, and nine additional putative transcription units was obtained. One of the transcription units was recently identified as BRCA2 but all others are novel genes of unknown function as only limited alignment to sequences in public databases was observed. One large gene with a transcript size of 10.7 kb showed significant similarity to a gene predicted by the Caenorhabditis elegans genome and the Saccharomyces cerevisiae genome sequencing efforts, while another contained a motif sequence similar to the human 2',3' cyclic nucleotide 3' phosphodiesterase gene. Several retrieved transcribed sequences were not aligned into transcription units because no corresponding cDNAs were obtained when screening libraries or because of a lack of definitive evidence for splicing signals or putative coding sequence based on computational analysis. However, the presence of additional genes in the BRCA2 interval is suggested as groups of putative exons and hybrid selected clones that were transcribed in consistent orientations could be localized to common physical intervals.

The cDNA for the human alpha-adducin gene has been cloned, and different alternately spliced forms have been identified. We report the complete genomic organization of the human alpha-adducin gene and these alternately spliced forms. The human alpha-adducin gene, spanning approximately 85 kb, consists of 16 exons ranging in size from 34 to 1892 bp. One of the spliced forms of the human alpha-adducin gene results from alternate use of the 5' splice donor site for exon 10, while another results in a truncated protein following insertion of 34 bp comprising exon 15, followed by a premature stop codon. This alternate spliced form of alpha-adducin is predicted to result in an altered carboxyl terminus that would eliminate a protein kinase and calmodulin binding site. Seven nucleotide substitutions and 4 insertion/deletions were also identified. The 5' region of the human alpha-adducin gene contains one Sp1 site, two AP2 sites, and two CAAT boxes. No TATA box was apparent, consistent with features of a housekeeping gene. We have mapped another cDNA within the first intron of the human alpha-adducin gene, suggesting overlapping genes in this 4p16.3 genomic region.

Five G C -containing oligonucleotides that include the recognition sequences of rare-cutting restriction enzymes have been used to isolate almost 100 different genomic segments from chromosome 7 that contain recognition sites for those enzymes. Hybridization and washing at 27°C allow the use of 8-bp radiolabeled oligonucleotides to detect specific G C -containing sequences in less than 1 ng of cloned DNA. This method was used to isolate 9 positive clones from 138 previously isolated single-copy probes from a flow-sorted chromosome 7 library. The specificity of the method was confirmed by showing that clones that gave positive hybridization signals also contained the corresponding restriction site. The oligonucleotides were also used to analyze approximately 12,000 kb of genomic sequence from a newly constructed chromosome 7 cosmid library that yielded 88 positive cosmids from 350 analyzed. The average distances between binding sites ranged from 200 to 690 kb and was independent of the number of CpG residues present in the oligonucleotide. Confirmation that clones containing restriction sites for these rare-cutting enzymes are located near genes was obtained by hybridization to RNA and cross-species DNA blots.

The human homologs of the mouse homeo box-containing genes, En-1 and En-2, which show homology to the Drosophila engrailed gene, have been isolated. The human EN1 gene was mapped to chromosome 2 by analysis of mouse-human somatic cell hybrids. The human EN2 gene was localized to chromosome 7, 7q32-7qter, by analysis of rodent-human somatic cell hybrids and cell lines carrying portions of chromosome 7.