Comparative analysis of the gene-dense ACHE/TFR2 region on human chromosome 7q22 with the orthologous region on mouse chromosome 5.

Chromosome 7q22 has been the focus of many cytogenetic and molecular studies aimed at delineating regions commonly deleted in myeloid leukemias and myelodysplastic syndromes. We have compared a gene-dense, GC-rich sub-region of 7q22 with the orthologous region on mouse chromosome 5. A physical map of 640 kb of genomic DNA from mouse chromosome 5 was derived from a series of overlapping bacterial artificial chromosomes. A 296 kb segment from the physical map, spanning ACHE: to Tfr2, was compared with 267 kb of human sequence. We identified a conserved linkage of 12 genes including an open reading frame flanked by ACHE: and Asr2, a novel cation-chloride cotransporter interacting protein Cip1, Ephb4, Zan and Perq1. While some of these genes have been previously described, in each case we present new data derived from our comparative sequence analysis. Adjacent unfinished sequence data from the mouse contains an orthologous block of 10 additional genes including three novel cDNA sequences that we subsequently mapped to human 7q22. Methods for displaying comparative genomic information, including unfinished sequence data, are becoming increasingly important. We supplement our printed comparative analysis with a new, Web-based program called Laj (local alignments with java). Laj provides interactive access to archived pairwise sequence alignments via the WWW. It displays synchronized views of a dot-plot, a percent identity plot, a nucleotide-level local alignment and a variety of relevant annotations. Our mouse-human comparison can be viewed at http://web.uvic.ca/~bioweb/laj.html. Laj is available at http://bio.cse.psu.edu/, along with online documentation and additional examples of annotated genomic regions.

Cloning and characterization of three novel genes, ALS2CR1, ALS2CR2, and ALS2CR3, in the juvenile amyotrophic lateral sclerosis (ALS2) critical region at chromosome 2q33-q34: candidate genes for ALS2.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that manifests as selective upper and lower motor neuron degeneration. The autosomal recessive form of juvenile amyotrophic lateral sclerosis (ALS2) has previously been mapped to the 1.7-cM interval flanked by D2S116 and D2S2237 on human chromosome 2q33-q34. We identified three novel full-length transcripts encoded by three distinct genes (HGMW-approved symbols ALS2CR1, ALS2CR2, and ALS2CR3) within the ALS2 critical region. The intron-exon organizations of these genes as well as those of CFLAR, CASP10, and CASP8, which were previously mapped to this region, were defined. These genes were evaluated for mutations in ALS2 patients, and no disease-associated sequence alterations in either exons or intron-exon boundaries were observed. Sequence analysis of overlapping RT-PCR products covering the whole coding sequence for each transcript revealed no aberrant mRNA sequences. These data strongly indicate that ALS2CR1, ALS2CR2, ALS2CR3, CFLAR, CASP10, and CASP8 are not causative genes for ALS2.

Comparative genomic sequence analysis of the Williams syndrome region (LIMK1-RFC2) of human chromosome 7q11.23.

Williams syndrome (WS) is a complex neurodevelopmental disorder arising from a microdeletion at Chr band 7q11.23, which results in a hemizygous condition for a number of genes. Within this region we have completely characterized 200 kb containing the genes LIMK1, WBSCR1, and RFC2. Evidence was also found for WBSCR5 in this region, but not the previously proposed genes WSCR2 and WSCR6. The syntenic region in mouse was also sequenced (115 kb) and characterized, and a comparative sequence analysis with a percent identity plot (PIP) easily allowed us to identify coding exons. This genomic region is GC rich (50.1% human, 49.9% mouse) and contains an unusually high abundance of repetitive elements consisting primarily of Alu (45.4%, one of the highest levels identified to date) in human, and the B family of SINES (30.6% of the total sequence) in mouse. WBSCR1 corresponds to eukaryotic initiation factor 4H, identified in rabbit, and is herein found to be constitutively expressed in both human and mouse, with two RNA and protein products formed (exon 5 is alternatively spliced). The transcription pattern of WBSCR5 was also examined and discussed along with its putative amino acid sequence.

A 39-kb sequence around a blackbird Mhc class II gene: ghost of selection past and songbird genome architecture.

To gain an understanding of the evolution and genomic context of avian major histocompatibility complex (Mhc) genes, we sequenced a 38.8-kb Mhc-bearing cosmid insert from a red-winged blackbird (Agelaius phoeniceus). The DNA sequence, the longest yet retrieved from a bird other than a chicken, provides a detailed view of the process of gene duplication, divergence, and degeneration ("birth and death") in the avian Mhc, as well as a glimpse into major noncoding features of a songbird genome. The peptide-binding region (PBR) of the single Mhc class II B gene in this region, Agph-DAB2, is almost devoid of polymorphism, and a still-segregating single-base-pair deletion and other features suggest that it is nonfunctional. Agph-DAB2 is estimated to have diverged about 40 MYA from a previously characterized and highly polymorphic blackbird Mhc gene, Aph-DAB1, and is therefore younger than most mammalian Mhc paralogs and arose relatively late in avian evolution. Despite its nonfunctionality, Agph-DAB2 shows very high levels of nonsynonymous divergence from Agph-DAB1 and from reconstructed ancestral sequences in antigen-binding PBR codons-a strong indication of a period of adaptive divergence preceding loss of function. We also found that the region sequenced contains very few other unambiguous genes, a partial Mhc- class II gene fragment, and a paucity of simple-sequence and other repeats. Thus, this sequence exhibits some of the genomic streamlining expected for avian as compared with mammalian genomes, but is not as densely packed with functional genes as is the chicken Mhc.

Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.

Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) levels. Here we have identified two Tangier disease (TD) families, confirmed 9q31 linkage and refined the disease locus to a limited genomic region containing the gene encoding the ATP-binding cassette transporter (ABC1). Familial HDL deficiency (FHA) is a more frequent cause of low HDL levels. On the basis of independent linkage and meiotic recombinants, we localized the FHA locus to the same genomic region as the TD locus. Mutations in ABC1 were detected in both TD and FHA, indicating that TD and FHA are allelic. This indicates that the protein encoded by ABC1 is a key gatekeeper influencing intracellular cholesterol transport, hence we have named it cholesterol efflux regulatory protein (CERP).

A yeast artificial chromosome-based physical map of the juvenile amyotrophic lateral sclerosis (ALS2) critical region on human chromosome 2q33-q34.

The autosomal recessive form of juvenile amyotrophic lateral sclerosis (ALS2; RFALS Type 3) has previously been mapped to the 8-cM interval flanked by D2S115 and D2S155 on human chromosome 2q33-q34. We have established a yeast artificial chromosome (YAC) contig spanning an approximately 8-Mb region of the ALS2 candidate region and mapped 52 transcribed DNA sequences including 13 known genes and 39 expressed sequenced tags within this YAC contig. The establishment of a YAC contig and transcript map that spans the region containing the ALS2 mutation is an essential step in the identification of the ALS2 gene.

The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate.

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumours (exostoses) that develop from the growth plate of endochondral bone. This condition can lead to skeletal abnormalities, short stature and malignant transformation of exostoses to chondrosarcomas or osteosarcomas. Linkage analyses have identified three different genes for HME, EXT1 on 8q24.1, EXT2 on 11p11-13 and EXT3 on 19p (refs 6-9). Most HME cases have been attributed to missense or frameshift mutations in these tumour-supressor genes, whose functions have remained obscure. Here, we show that EXT1 is an ER-resident type II transmembrane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). Two EXT1 variants containing aetiologic missense mutations failed to alter cell-surface glycosaminoglycans, despite retaining their ER-localization.

Length of huntingtin and its polyglutamine tract influences localization and frequency of intracellular aggregates.

It is unclear how polyglutamine expansion is associated with the pathogenesis of Huntington disease (HD). Here, we provide evidence that polyglutamine expansion leads to the formation of large intracellular aggregates in vitro and in vivo. In vitro these huntingtin-containing aggregates disrupt normal cellular architecture and increase in frequency with polyglutamine length. Huntingtin truncated at nucleotide 1955, close to the caspase-3 cleavage site, forms perinuclear aggregates more readily than full-length huntingtin and increases the susceptibility of cells to death following apoptotic stimuli. Further truncation of huntingtin to nucleotide 436 results in both intranuclear and perinuclear aggregates. For a given protein size, increasing polyglutamine length is associated with increased cellular toxicity. Asymptomatic transgenic mice expressing full-length huntingtin with 138 polyglutamines form exclusively perinuclear aggregates in neurons. These data support the hypothesis that proteolytic cleavage of mutant huntingtin leads to the development of aggregates which compromise cell viability, and that their localization is influenced by protein length.

Cell death attenuation by 'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex.

Apoptotic cell suicide initiated by ligation of CD95 (Fas/APO-1) occurs through recruitment, oligomerization and autocatalytic activation of the cysteine protease, caspase-8 (MACH, FLICE, Mch5). An endogenous mammalian regulator of this process, named Usurpin, has been identified (aliases for Usurpin include CASH, Casper, CLARP, FLAME-1, FLIP, I-FLICE and MRIT). This protein is ubiquitously expressed and exists as at least three isoforms arising by alternative mRNA splicing. The Usurpin gene is comprised of 13 exons and is clustered within approximately 200 Kb with the caspase-8 and -10 genes on human chromosome 2q33-34. The Usurpin polypeptide has features in common with pro-caspase-8 and -10, including tandem 'death effector domains' on the N-terminus of a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity, including the His and Cys which form the catalytic substrates diad, and residues that stabilize the P1 aspartic acid in substrates. Retro-mutation of these residues to functional caspase counterparts failed to restore proteolytic activity, indicating that other determinants also ensure the absence of catalytic potential. Usurpin heterodimerized with pro-caspase-8 in vitro and precluded pro-caspase-8 recruitment by the FADD/MORT1 adapter protein. Cell death induced by CD95 (Fas/APO-1) ligation was attenuated in cells transfected with Usurpin. In vivo, a Usurpin deficit was found in cardiac infarcts where TUNEL-positive myocytes and active caspase-3 expression were prominent following ischemia/reperfusion injury. In contrast, abundant Usurpin expression (and a caspase-3 deficit) occurred in surrounding unaffected cardiac tissue, suggesting reciprocal regulation of these pro- and anti-apoptotic molecules in vivo. Usurpin thus appears to be an endogenous modulator of apoptosis sensitivity in mammalian cells, including the susceptibility of cardiac myocytes to apoptotic death following ischemia/ reperfusion injury.

Evolution and selection of primate T cell antigen receptor BV8 gene subfamily.

The set of potential T cell receptor specificities is highly diverse. The relative contributions of T cell receptor (TCR) V beta gene segment polymorphisms, duplications, deletions, and gene conversions to this final T cell receptor protein diversity are unknown. To study these mechanisms, we sequenced and compared closely related primate TCR gene segments from BV8S1, S2, and S5. Interspecies comparisons show that these gene segments have sustained multiple duplication, gene conversion, and deletion events during the last 35 million years of anthropoid primate evolution. BV8 coding sequences are generally conserved with respect to their flanking noncoding sequences, but we find no evidence for positive or negative selection in sequences coding for the first two putative complementarity-determining (ligand-binding) regions. Sequences of TCRBV8 gene segments from unrelated humans demonstrate no nonsynonymous substitutions in nonleader regions of either the BV8S1 or S2 gene segments. We conclude that gene duplication, deletion, and conversion mechanism contribute in a substantial way to the overall diversity of the TCRBV8 gene segment repertoire in primate evolution and that germline substitutions and consequent polymorphisms in CDRs 1 and 2 of these gene segments probably do not play an active role in generating TCR beta chain protein variation.

Dextran sulfate can act as an artificial receptor to mediate a type-specific herpes simplex virus infection via glycoprotein B.

Herpes simplex virus (HSV) adsorption to host cells is mediated, at least in part, by the interaction of viral glycoproteins with cell surface glycosaminoglycans such as heparan sulfate and chondroitin sulfate. To investigate the contribution of various cell surface components in the infection pathway, we isolated a mutant cell line, sog9, which is unable to synthesize glycosaminoglycans (B. W. Banfield, Y. Leduc, L. Esford, K. Schubert, and F. Tufaro, J. Virol. 69:3290-3298, 1995). Although HSV-1 and HSV-2 infection of sog9 cells is diminished, the cells are still infected at about 0.5% efficiency, which suggests that these cells normally express at least one nonglycosaminoglycan receptor. In this report, we used sog9 cells to test whether glycosaminoglycan analogs, such as dextran sulfate (DS), could functionally substitute for cellular glycosaminoglycans to initiate HSV infection. We show that high-molecular-weight DS added either prior to or during inoculation stimulated HSV-1 but not HSV-2 infection by up to 35-fold; DS added after viral adsorption had no effect on infection efficiency. Moreover, DS stimulated HSV-1 infection at 4 degrees C, indicating that this compound impinged on an early, energy-independent step in infection. Using radiolabeled virus, we showed that HSV-1 is more efficient than HSV-2 in adsorbing to DS immobilized on microtiter wells. This raised the possibility that only HSV-1 could engage additional receptors to initiate infection in the presence of DS. To determine which viral component(s) facilitated DS stimulation, a panel of intertypic recombinants and deletion mutant viruses was investigated. These assays showed that DS stimulation of infection is mediated primarily by gB-1. Thus, this study provides direct evidence that a principal role for cell surface glycosaminoglycans in HSV infection is to provide an efficient matrix for virus adsorption. Moreover, by using DS as an alternative adsorption matrix (a trans receptor), we uncovered a functional, type-specific interaction of HSV-1 with a cell surface receptor.

Identification of Sonic hedgehog as a candidate gene responsible for holoprosencephaly.

Holoprosencephaly (HPE) is a genetically and phenotypically heterogenous disorder involving the development of forebrain and midface, with an incidence of 1:16,000 live born and 1:250 induced abortions. This disorder is associated with several distinct facies and phenotypic variability: in the most extreme cases, anophthalmia or cyclopia is evident along with a congenital absence of the mature nose. The less severe form features facial dysmorphia characterized by ocular hypertelorism, defects of the upper lip and/or nose, and absence of the olfactory nerves or corpus callosum. Several intermediate phenotypes involving both the brain and face have been described. One of the gene loci, HPE3, maps to the terminal band of chromosome 7. We have performed extensive physical mapping studies and established a critical interval for HPE3, and subsequently identified the sonic hedgehog (SHH) gene as the prime candidate for the disorder. SHH lies within 15-250 kilobases (kb) of chromosomal rearrangements associated with HPE, suggesting that a 'position effect' has an important role in the aetiology of HPE. As detailed in the accompanying report, this role for SHH is confirmed by the detection of point mutations in hereditary HPE patients.

Identification of genes from a 500-kb region at 7q11.23 that is commonly deleted in Williams syndrome patients.

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 delta and epsilon. LIMK1 and WSCR1 may be particularly relevant when explaining cognitive defects observed in WS patients.

Retroviral-type zinc fingers and glycine-rich repeats in a protein encoded by cnjB, a Tetrahymena gene active during meiosis.

We have determined the nucleotide sequence of the cnjB gene from the ciliate Tetrahymena thermophila. This gene is transcriptionally active only during early conjugation, peaking in meiotic prophase. It contains 13 introns, four transcription start points and codes for a putative polypeptide (CnjB) of 1748 amino acids with a calculated molecular weight of 200 kilodaltons and a pl of 7.9. The coding region of cnjB has a low GC content (32% GC) and unusual codon usage. The C-terminal one-third of CnjB consists of three repetitive domains. Introns were absent in this region of cnjB. One of the repetitive domains consists of seven CCHC or retroviral-type zinc fingers, a motif found in one or two copies in retroviral nucleocapsid proteins. This motif has also been found recently in seven copies in the human nucleic-acid binding protein CNBP, in an apparent CNBP homologue in Schizosaccharomyces pombe and in one copy in a Xenopus gene active in early embryos. The other two domains are on either side of the zinc finger domain and contain a repeated glycine-rich motif seen in the heterogeneous nuclear ribonuclear proteins A1 and A2/B1 as well as other proteins. Both CCHC zinc fingers and glycine-rich repeats have been found in proteins with single-stranded nucleic acid-binding activity as well as strand-annealing activity. CnjB is, to our knowledge, the first protein found to contain both types of motifs.