A novel olfactory receptor gene family in teleost fish

While for two of three mammalian olfactory receptor families (OR and V2R) ortholog teleost families have been identified, the third family (V1R) has been thought to be represented by a single, closely linked gene pair. We identified four further V1R-like genes in every teleost species analyzed (Danio rerio, Gasterosteus aculeatus, Oryzias latipes, Tetraodon nigroviridis, Takifugu rubripes). In the phylogenetic analysis these ora genes (olfactory receptor class A-related) form a single clade, which includes the entire mammalian V1R superfamily. Homologies are much lower in paralogs than in orthologs, indicating that all six family members are evolutionarily much older than the speciation events in the teleost lineage analyzed here. These ora genes are under strong negative selection, as evidenced by very small d(N)/d(S) values in comparisons between orthologs. A pairwise configuration in the phylogenetic tree suggests the existence of three ancestral Ora subclades, one of which has been lost in amphibia, and a further one in mammals. Unexpectedly, two ora genes exhibit a highly conserved multi-exonic structure and four ora genes are organized in closely linked gene pairs across all fish species studied. All ora genes are expressed specifically in the olfactory epithelium of zebrafish, in sparse cells within the sensory surface, consistent with the expectation for olfactory receptors. The ora gene repertoire is highly conserved across teleosts, in striking contrast to the frequent species-specific expansions observed in tetrapod, especially mammalian V1Rs, possibly reflecting a major shift in gene regulation as well as gene function upon the transition to tetrapods.     

A gene recently inactivated in human defines a new olfactory receptor family in mammals

The olfactory receptor (OR) gene family constitutes one of the largest multigene families and is distributed among many chromosomal sites in the human genome. Four OR families have been defined in mammals. We previously demonstrated that a high fraction of human OR sequences have incurred deleterious mutations, thus reducing the repertoire of functional OR genes. In this study, we have characterized a new OR gene, 912-93, in primates. This gene is unique and it defines a new OR family. It localizes to human chromosome 11q11-12 and at syntenical sites in other hominoids. The sequence marks a previously unrecognized rearrangement of pericentromeric material from chromosome 11 to the centromeric region of gibbon chromosome 5. The human gene contains a nonsense point mutation in the region corresponding to the extracellular N-terminus of the receptor. This mutation is present in humans of various ethnic groups, but is absent in apes, suggesting that it probably appeared during the divergence of humans from other apes, <4 000 000-5 000 000 years ago. A second mutation, a frameshift at a different location, has occurred in the gorilla copy of this gene. These observations suggest that OR 912-93 has been recently silenced in human and gorilla, adding to a pool of OR pseudogenes whose growth may parallel a reduction in the sense of smell in primates.      

Large multi-chromosomal duplications encompass many members of the olfactory receptor gene family in the human genome

The human genome contains thousands of genes that encode a diverse repertoire of odorant receptors (ORs). We report here on the identification and chromosomal localization of 74 OR-containing genomic clones. Using fluorescence in situ hybridization (FISH), we demonstrate a striking homology among a set of approximately 20 OR locations, illustrating a history of duplications that have distributed OR sequences across the genome. Half of the OR-containing BACs cloned from total genomic DNA and 86% of cosmids derived from chromosome 3 cross- hybridize to a subset of these locations, many to 17 of them. These paralogous regions are distributed on 13 chromosomes, and eight lie in terminal bands. By analyzing clones from an approximately 250 kb clone- walk across one of these sites (3p13), we show that the homology among these sites is extensive (>150 kb) and encompasses both OR genes and intergenic genomic sequences. The FISH signals appear significantly larger at some sites than at the native location, indicating that portions of some duplicons have undergone local amplification/attrition. More restricted duplications involving pairs of other genomic locations are detected with 12% of the OR-BACs. Only a small subset of OR locations is sufficiently diverged from the others that clones derived from them behave as single-copy FISH probes. We estimate that duplications encompassing members of the OR gene family account for >0.1% of the human genome. A comparison of FISH signals at orthologous locations in other primates indicates that a portion of this OR 'subgenome' has been in flux during the divergence of primates, possibly as a mechanism for evolving the repertoire of olfactory receptors.      

A comparison of the human and chimpanzee olfactory receptor gene repertoires

Olfactory receptor (OR) genes constitute the basis of the sense of smell and are encoded by the largest mammalian gene superfamily, with >1000 members. In humans, but not in mice or dogs, the majority of OR genes have become pseudogenes, suggesting that OR genes in humans evolve under different selection pressures than in other mammals. To explore this further, we compare the OR gene repertoire of human with its closest living evolutionary relative, by taking advantage of the recently sequenced genome of the chimpanzee. In agreement with previous reports based on a small number of ORs, we find that humans have a significantly higher proportion of OR pseudogenes than chimpanzees. Moreover, we can reject the possibility that humans have been accumulating OR pseudogenes at a constant neutral rate since the divergence of human and chimpanzee. The comparison of the two repertoires reveals two chimpanzee-specific OR subfamily expansions and three expansions specific to humans. It also suggests that a subset of OR genes are under positive selection in either the human or the chimpanzee lineage. Thus, although overall there is relaxed constraint on human olfaction relative to chimpanzee, species-specific sensory requirements appear to have shaped the evolution of the functional OR gene repertoires in both species.     

Localization of the human membrane-type 2 matrix metalloproteinase gene (MMP15) to 16q12.1 near DNA elements that are part of centromeric and non-centromeric heterochromatin of 11 human chromosomes

We have localized a second gene for membrane-type matrix metalloproteinases, MT2-MMP, to chromosome 16q12 by in situ hybridization. FISH experiments using a genomic PAC clone containing the MT2-MMP gene resulted in an unusual hybridization pattern detecting centromeric and non-centromeric heterochromatin regions or its flanking sequences in 11 human chromosomes in addition to the MT2-MMP locus on chromosome 16q12. The detailed analysis of this hybridization pattern using molecular cytogenetic methods together with the specific hybridization of the MT2-MMP cDNA allowed a refined mapping of the gene to 16q12.1, directly adjacent to the 16q heterochromatin. Our findings may give some insights into the evolution of the MMP gene family.     

Population differences in haplotype structure within a human olfactory receptor gene cluster

We investigated the population differences in patterns of single nucleotide polymorphisms (SNPs) for a 400 kb olfactory receptor (OR) gene cluster on human chromosome 17p13.3. Samples were drawn from 35 individuals, of four different ethnogeographical origins: Pygmies, Bedouins, Yemenite Jews and Ashkenazi Jews. Of the 74 SNPs identified, two segregated between pseudogenized and intact ORs, while a third involved a change in a highly conserved motif proposed to mediate ligand-induced signal transduction. Linkage disequilibrium (LD) was computed based on phase inference across the cluster using Clark's haplotype subtraction algorithm. We also calculated LD directly from the genotypes using the expectation-maximization (EM) algorithm. Both methods yielded very similar results. Our analyses revealed substantial differences in nucleotide diversity, haplotype distribution and LD patterns among the different human populations. In particular, the two Jewish populations had low haplotype diversity and negligible decay of LD across the entire genomic region. Intriguingly, the three functional SNPs segregated at different frequencies in the different ethnogeographical groups, with the Pygmies having higher frequencies of the intact OR genes. Our data suggests that OR genes may have evolved to create different functional repertoires in distinct human populations.     

Spontaneous reiterations of DNA sequences near the ends of adenovirus type 3 genomes

Repeated passage of adenovirus type 3 in HeLa cells has led to a novel stock of variant genomes. Most of the DNA molecules in this stock are characterized by deletions and substitutions of DNA sequences near the left end of the adenovirus type 3 genome map, as reported earlier (C.C. Robinson and C. Tibbetts (1984) Virology 137, 276-286). In this report the characterization of the variant genomes is extended and reveals elongated DNA molecules bearing tandem repetitions of viral DNA sequences near the left and right ends of the viral DNA. Evidence is also presented supporting the cellular DNA origin of short insert sequences found in substitution variants. The elongated variants are of interest because of their novel repeated DNA structures. The locations of these aberrant sequences raise questions about their potential impact on viral gene expression.     

The olfactory receptor gene superfamily of the mouse.

Olfactory receptor (OR) genes are the largest gene superfamily in vertebrates. We have identified the mouse OR genes from the nearly complete Celera mouse genome by a comprehensive data mining strategy. We found 1,296 mouse OR genes (including 20% pseudogenes), which can be classified into 228 families. OR genes are distributed in 27 clusters on all mouse chromosomes except 12 and Y. One OR gene cluster matches a known locus mediating a specific anosmia, indicating the anosmia may be due directly to the loss of receptors. A large number of apparently functional 'fish-like' Class I OR genes in the mouse genome may have important roles in mammalian olfaction. Human ORs cover a similar 'receptor space' as the mouse ORs, suggesting that the human olfactory system has retained the ability to recognize a broad spectrum of chemicals even though humans have lost nearly two-thirds of the OR genes as compared to mice.     

Frequent mutations in the neurotrophic tyrosine receptor kinase gene family in large cell neuroendocrine carcinoma of the lung

The neurotrophic tyrosine receptor kinase (NTRK) family is potentially implicated in tumorigenesis and progression of several neoplastic diseases, including lung cancer. We investigated a large number of pulmonary neuroendocrine tumors (PNETs) and non-small cell lung carcinomas (NSCLCs) without morphological evidence of neuroendocrine differentiation for mutations in the NTRK gene family. A total of 538 primary lung carcinomas, including 17 typical carcinoids (TCs), 10 atypical carcinoids (ACs), 39 small cell lung carcinomas (SCLCs), 29 large cell neuroendocrine carcinomas (LCNECs), and 443 NSCLCs were evaluated by single-strand conformation polymorphism (SSCP) and sequencing of the tyrosine kinase domain (TKD) of NTRK1, NTRK2, and NTRK3. The NTRK1 gene was never found to be mutated. A total of 10 somatic mutations were detected in NTRK2 and NTRK3, mostly located in the activating and catalytic loops. NTRK mutations were seen in 9 (10%) out of 95 PNETs but in 0 out of 443 NSCLCs investigated. No mutations were observed in TCs, ACs, and SCLCs. Interestingly, all the mutations were restricted to the LCNEC histotype, in which they accounted for 31% of cases. A mutational analysis, performed after microdissection of LCNECs combined with adenocarcinoma (ADC), showed that only neuroendocrine areas were positive, suggesting that NTRK mutations are involved in the genesis of the neuroendocrine component of combined LCNECs. Our data indicate that somatic mutations in the TKD of NTRK genes are frequent in LCNECs. Such mutational events could represent an important step in the cancerogenesis of these tumors and may have potential implications for the selection of patients for targeted therapy.     

Tandemly repeated sequences are present at the ends of the DNA of raccoonpox virus

The DNA of raccoonpox virus (RCN) has been characterized by restriction enzyme analysis. DNA hybridization studies showed that all HindIII fragments of the 215-kbp RCN DNA share some nucleotide sequence similarity with fragments of the DNA of cowpox virus (CPV). This information was used to construct a HindIII restriction map of the RCN DNA. The nucleotide sequence of the 2.2-kbp Sal 1 end fragment of the RCN DNA has been determined from a cloned copy of the HindIII O fragment. Of this 2.2-kb region 75% consists of short, tandemly repeated sequences. It does not contain any open reading frames capable of encoding polypeptide chains of more than 62 amino acids. There are six related types of repeated sequence, and these are arranged into two separate sets, each flanked by nonrepeated sequences. The nucleotide sequences of both repeated and nonrepeated sequences within this Sal 1 fragment are extremely similar to those of the Sal 1-generated end fragments of the DNas of CPV and vaccinia virus. The arrangements of the repeated and nonrepeated sequences are also similar in the DNAs of these three viruses. In contrast, the remainder of the RCN DNA is markedly different from the DNAs of other orthopoxviruses. The high degree of similarity between the ends of the RCN DNA and the ends of the other orthopoxvirus DNAs suggest that the complex arrays of repeated and nonrepeated sequences have been conserved because they have a role in virus multiplication.     

Marker chromosomes in direct preparations of human large bowel tumors

A survey of 257 marker chromosomes in 48 primary human large bowel adenocarcinomas showed that 44% were markers with recognized patterns. Chromosomes #1, #3, #5, #8, #9, #13, and #17 were involved most frequently. Markers related to chromosomes #7 and X were not seen in any recognizable form. The unidentified chromosomes were classified as markers with abnormal banding regions. In correlating tumor location and stage of invasion with markers, there were fewer markers in tumors from the right side. However, there was little difference in the number of markers seen in the left-sided tumors, irrespective of histopathologic stage, suggesting that function and microenvironmental conditions between various parts of the colon may be related to these differences. The most striking observation is that 21% of all tumors analyzed were without any obvious markers.     

Variation within the human killer cell immunoglobulin-like receptor (KIR) gene family

The killer cell immunoglobulin-like receptors (KIR) form a family of highly homologous immune receptors that regulate the response of natural killer (NK) cells and some T cells. The genetics of the human KIR family is reviewed in this article. In human populations, diversity in KIR genotype arises from variations in gene content and allelic polymorphism. Comparisons of 81 human KIR sequences reveal past events ofgene duplication and recombination, and indicate that individual KIR genes have diversified from the influence of natural selection. Comparison and compilation of population studies reveal extensive KIR genotype variability within human populations and among them. Genomic analysis shows the KIR genes to be close to each other and separated by homologous sequences that promote haplotype diversification through assymetric recombination. In contrast, homologous recombination appears favored at a unique sequence in the center of the KIR locus, and much haplotypic diversity can be explained by recombination between a limited number of gene-content motifs in the centromeric and telomeric halves of the locus. The importance of NK cells for early defenses against infection suggests that human KIR genotype diversity is the accumulated consequence of a history of numerous and successive selective episodes by different pathogens on human NK-cell responses.     

Fluorescence analysis of late DNA replication in human metaphase chromosomes

Thymidine incorporated as a terminal pulse into chromosomes otherwise substituted with 5-bromodeoxyuridine can be detected by associated bright 33258 Hoechst fluorescence. The location of metaphase chromosome regions identified by this method as last to complete DNA synthesis is consistent with the results of autoradiographic analyses with tritiated thymidine. The very late-replicating regions correspond to a subset of those which appear as bands after chromosomes are stained by quinacrine or modified Giemsa techniques. The high resolution of the 33258 Hoechst fluorescence pattern within individual cells is especially useful for revealing variations in the order of terminal replication. Both homolog asynchrony and fluctuations in the distribution of bright 33258 Hoechst fluorescence within chromosomes from different cells are apparent and localized to individual bands. The results are consistent with the possibility that these bands constitute units of chromosome replication as well as structure.     

A chloroplast DNA mutational hotspot and gene conversion in a noncoding region near rbcL in the grass family (Poaceae)

The noncoding DNA region of the chloroplast genome, flanked by the genes rbcL and psaI (ORF36), has been sequenced for seven species of the grass family (Poaceae). This region had previously been observed as a hotspot area for length mutations. Sequence comparison reveals that short duplications, probably resulting from slipped-strand mispairing, account for many small length differences between sequences but that major mutational hotspots are localized in three small areas, two of which show potential secondary structure. Mutation in one of these hotspots appears to be a result of more complex recombination events. All seven species contain a pseudogene for rpl23 and evidence is presented that this pseudogene is being maintained by gene conversion with the functional gene. Different transition/transversion biases and AT contents between the pseudogene and the surrounding noncoding sequences are noted. In the subfamily Panicoideae there is a deletion in which almost 1 kb of ancestral sequence, including the 3 end of the rpl23 pseudogene, has been replaced by a non-homologous 60-base sequence of unknown origin. Two other deletions of almost the same region have occurred in the grass family. The deleted noncoding region has mutational and compositional properties similar to the rbcL coding sequence and the rpl23 pseudogene. The three independent deletions, as well as the pattern of mutation in the localized hotspots, indicate that such noncoding DNA may be misleading for studies of phylogenetic inference.     

DNA linkage analysis and studies of the androgen receptor gene in a large kindred with complete androgen insensitivity

DNA linkage analysis of the X chromosome and studies with cDNA probes specific for the androgen receptor gene were performed on the largest known kindred with the syndrome of complete androgen insensitivity. The affected subjects (XY) have absent binding of dihydrotestosterone to the androgen receptor (the receptor negative form of androgen insensitivity). In this kindred there was maternal transmission of the gene, with all affected males expressing complete genital feminization. Linkage analysis studies were conducted with two DNA probes, DXS1 and PGK1, localized to the Xq11-Xq13 region of the long arm of the X chromosome near the centromere. The results demonstrate linkage to the markers in the order of DXS1-(AR; PGK1), thus localizing the AR gene to an area between Xq11 and Xq13. Three cDNA probes that span various parts of the androgen receptor gene, including the DNA and steroid binding domain, were used to evaluate the androgen receptor gene in normal individuals, carrier mothers, and affected subjects. Identical restriction fragment patterns were found in all three groups studied. Thus the androgen receptor gene was present in affected subjects without detectable DNA polymorphism at the androgen binding domain. Therefore, despite complete absence of binding to the androgen receptor, the defect in the androgen receptor gene in this kindred is not the result of a gene deletion. The results point to a mutation or a small insertion/deletion as the probable cause of the syndrome.