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PubMed2003

The phylogenetic history of the MHC class I gene families in pig, including a fossil gene predating mammalian radiation.

More than 990 kb of the 1200 kb in the SLA class I region of the pig major histocompatibility complex (MHC) have been sequenced. The present study was designed to establish the evolution of this region which was best understood by distinguishing three periods. The most recent period, which extended from 40 to 15 mya, probably corresponded to five rounds of duplication of a basic unit. This unit consisted of a single class I gene linked to widely dispersed repeats, and one SLA-specific repeat motif. The duplications gave rise to six SLA classical class I genes. The second evolutionary period corresponded to the emergence of the SLA nonclassical class I genes, i.e. after the suidae separated from the other artiodactyl species about 65 mya. The third period appeared to correspond to a much more remote age when the ancestor of the gene SLA-11 existed. Comparative studies of the human and pig sequences of the class I-containing segments indeed revealed the presence within the human HSR1-ZNF segment of relics of a human class I fossil gene which appeared to be orthologous to the 5' moiety of the SLA-11 pseudogene. This was the first evidence that a class I gene existed in this location at least 110-120 mya in the MHC class I region of the precursor of the mammalian species. Human/pig sequence comparison also revealed that the presumably functional pig MIC2 gene was probably orthologous to the human functional MICA or MICB genes.

Animals↗
PubMed2004

Structure analysis of two Toxoplasma gondii and Neospora caninum satellite DNA families and evolution of their common monomeric sequence.

A family of repetitive DNA elements of approximately 350 bp-Sat350-that are members of Toxoplasma gondii satellite DNA was further analyzed. Sequence analysis identified at least three distinct repeat types within this family, called types A, B, and C. B repeats were divided into the subtypes B1 and B2. A search for internal repetitions within this family permitted the identification of conserved regions and the design of PCR primers that amplify almost all these repetitive elements. These primers amplified the expected 350-bp repeats and a novel 680-bp repetitive element (Sat680) related to this family. Two additional tandemly repeated high-order structures corresponding to this satellite DNA family were found by searching the Toxoplasma genome database with these sequences. These studies were confirmed by sequence analysis and identified: (1). an arrangement of AB1CB2 350-bp repeats and (2). an arrangement of two 350-bp-like repeats, resulting in a 680-bp monomer. Sequence comparison and phylogenetic analysis indicated that both high-order structures may have originated from the same ancestral 350-bp repeat. PCR amplification, sequence analysis and Southern blot showed that similar high-order structures were also found in the Toxoplasma-sister taxon Neospora caninum. The Toxoplasma genome database (http://ToxoDB.org ) permitted the assembly of a contig harboring Sat350 elements at one end and a long nonrepetitive DNA sequence flanking this satellite DNA. The region bordering the Sat350 repeats contained two differentially expressed sequence-related regions and interstitial telomeric sequences.

Animals↗
PubMed2003

Nucleotide sequencing analysis of the swine 433-kb genomic segment located between the non-classical and classical SLA class I gene clusters.

Genome analysis of the swine leukocyte antigen ( SLA) region is needed to obtain information on the MHC genomic sequence similarities and differences between the swine and human, given the possible use of swine organs for xenotransplantation. Here, the genomic sequences of a 433-kb segment located between the non-classical and classical SLA class I gene clusters were determined and analyzed for gene organization and contents of repetitive sequences. The genomic organization and diversity of this swine non-class I gene region was compared with the orthologous region of the human leukocyte antigen ( HLA) complex. The length of the fully sequenced SLA genomic segment was 433 kb compared with 595 kb in the corresponding HLA class I region. This 162-kb difference in size between the swine and human genomic segments can be explained by indel activity, and the greater variety and density of repetitive sequences within the human MHC. Twenty-one swine genes with strong sequence similarity to the corresponding human genes were identified, with the gene order from the centromere to telomere of HCR - SPR1 - SEEK1 - CDSN - STG - DPCR1 - KIAA1885 - TFIIH - DDR - IER3 - FLOT1 - TUBB - KIAA0170 - NRM - KIAA1949 - DDX16 - FLJ13158 - MRPS18B - FB19 - ABCFI - CAT56. The human SEEK1 and DPCR1 genes are pseudogenes in swine. We conclude that the swine non-class I gene region that we have sequenced is highly conserved and therefore homologous to the corresponding region located between the HLA-C and HLA-E genes in the human.

Animals↗
PubMed2005

Genomic sequence analysis of the 238-kb swine segment with a cluster of TRIM and olfactory receptor genes located, but with no class I genes, at the distal end of the SLA class I region.

Continuous genomic sequence has been previously determined for the swine leukocyte antigen (SLA) class I region from the TNF gene cluster at the border between the major histocompatibility complex (MHC) class III and class I regions to the UBD gene at the telomeric end of the classical class I gene cluster (SLA-1 to SLA-5, SLA-9, SLA-11). To complete the genomic sequence of the entire SLA class I genomic region, we have analyzed the genomic sequences of two BAC clones carrying a continuous 237,633-bp-long segment spanning from the TRIM15 gene to the UBD gene located on the telomeric side of the classical SLA class I gene cluster. Fifteen non-class I genes, including the zinc finger and the tripartite motif (TRIM) ring-finger-related family genes and olfactory receptor genes, were identified in the 238-kilobase (kb) segment, and their location in the segment was similar to their apparent human homologs. In contrast, a human segment (alpha block) spanning about 375 kb from the gene ETF1P1 and from the HLA-J to HLA-F genes was absent from the 238-kb swine segment. We conclude that the gene organization of the MHC non-class I genes located in the telomeric side of the classical SLA class I gene cluster is remarkably similar between the swine and the human segments, although the swine lacks a 375-kb segment corresponding to the human alpha block.

Amino Acids↗
PubMed1999

Gene organization of the quail major histocompatibility complex (MhcCoja) class I gene region.

Class I genomic clones of the quail (Coturnix japonica) major histocompatibility complex (MhcCoja) were isolated and characterized. Two clusters spanning the 90.8 kilobase (kb) and 78.2 kb class I gene regions were defined by overlapping cosmid clones and found to contain at least twelve class I loci. However, unlike in the chicken Mhc, no evidence for the existence of any Coja class II gene was obtained in these two clusters. Based on comparative analysis of the genomic sequences with those of the cDNA clones, Coja-A, Coja-B, Coja-C, and Coja-D (Shiina et al. 1999), these twelve loci were assigned to represent one Coja-A gene, two Coja-B genes (Coja-B1 and -B2), four Coja-C genes (Coja-C1-C4), four Coja-D genes (Coja-D1-D4), and one new Coja-E gene. A class I gene-rich segment of 24.6 kb in which five of these genes (Coja-B1, -B2, -D1, -D2 and -E) are densely packed were sequenced by the shotgun strategy. All of these five class I genes are very compact in size [2089 base pairs (bp)-2732 bp] and contain no apparent genetic defect for functional expression. A transporter associated with the antigen processing (TAP) gene was identified in this class I gene-rich segment. These results suggest that the quail class I region is physically separated from the class II region and characterized by a large number of the expressible class I loci (at least seven) in contrast to the chicken Mhc, where the class I and class II regions are not clearly differentiated and only at most three expressed class I loci so far have been recognized.

ATP-Binding Cassette Transporters↗
PubMed2001

Analysis of a 1-Mb BAC contig overlapping the mouse Nkrp1 cluster of genes: cloning of three new Nkrp1 members, Nkrp1d, Nkrp1e, and Nkrp1f.

The murine Nkrp1 gene family encodes three previously identified activation and inhibitory receptors expressed on natural killer (NK) cells. This family includes the gene for NKR-P1C (NK1.1), the most specific serologic marker on C57BL/6-derived NK cells and is localized in a gene cluster in the NK gene complex (NKC). To further analyze the Nkrp1 family, we constructed and analyzed a bacterial artificial chromosome contig. A genomic organization of the Nkrp1 family was obtained and three new Nkrp1 genes were isolated from interleukin-2-activated NK cells. Thus, the Nkrp1 family adds to the repertoire of receptors expressed by NK cells.

Amino Acid Sequence↗
PubMed2006

The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies.

Seventeen non-allelic laccase genes and one gene footprint are present in the genome of Coprinopsis cinerea. Two gene subfamilies were defined by intron positions and similarity of deduced gene products, one with 15 members (lcc1-lcc15) and one with 2 members (lcc16, lcc17). The first subfamily divides in the phylogenetic tree of deduced proteins into smaller clusters that probably reflect recent gene duplication events. Different laccase genes diverged from each other both by frequent synonymous and non-synonymous codon changes. Mainly synonymous codon changes accumulate in alleles, with up to 12% total codon differences between given pairs of alleles. Overexpression of the 17 laccase genes under the control of a constitutive promoter identified nine active enzymes from subfamily 1. All of these showed laccase activities with DMP (2,6-dimethoxy phenol) as substrate but only eight of them also with ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)]. Lcc16 and Lcc17 share certain sequence features with ferroxidases but enzyme assays failed to show such activity. Lcc15 is expected to be non-functional in laccase activity due to an internal deletion of about 150 amino acids. Transcripts were obtained from all genes but splice junctions for three genes were not congruent with translation into a functional protein.

Alleles↗
PubMed2001

A major deletion in the cucumber mitochondrial genome sorts with the MSC phenotype.

The cucumber mitochondrial genome is unique because of its large size, paternal transmission, and the existence of a paternally transmitted mosaic (MSC) phenotype spontaneously appearing after independent tissue culture experiments. Transmission studies eliminated paternal imprinting as the genetic basis for the MSC phenotype. We identified a 13 kb region (JLV5-DEL) in the wild-type mitochondrial genome that was absent from three MSC lines. This deleted region was paternally transmitted with the MSC phenotype through the F3 and test-cross generations. Southern hybridizations and PCR amplifications using primers within the JLV5 region revealed that rare wild-type sorters possessed the wild-type region. MSC plants possess the wild-type region at levels (approximately 0.002) below detection by standard PCR reactions and Southern hybridizations, using genomic DNA. Sequence analysis of the wild-type contig revealed no homologies to mitochondrial genes and no open reading frames within JLV5-DEL. PCR amplifications across JLV5-DEL using MSC mtDNA revealed that the loss of this region was not a simple deletion and may be associated with a rearrangement. Because no genic regions were identified within the wild-type JLV5 region, the specific lesion conditioning the MSC phenotype may be associated with the putative rearrangement or another mutation that occurred during tissue culture or exists substoichiometrically in the parental line and is transmitted together with JLV5 within the same mitochondrion.

Contig Mapping↗
PubMed2002

A large duplication associated with dominant white color in pigs originated by homologous recombination between LINE elements flanking KIT.

The Dominant White (I/KIT) locus is one of the major coat color loci in the pig. Previous studies showed that the Dominant White (I) and Patch (IP) alleles are both associated with a duplication including the entire KIT coding sequence. We have now constructed a BAC contig spanning the three closely linked tyrosine kinase receptor genes PDGFRA-KIT-KDR. The size of the duplication was estimated at about 450 kb and includes KIT, but not PDGFRA and KDR. Sequence analysis revealed that the duplication arose by unequal homologous recombination between two LINE elements flanking KIT. The same unique duplication breakpoint was identified in animals carrying the I and IP alleles across breeds, implying that Dominant White and Patch alleles are descendants of a single duplication event. An unexpected finding was that Piétrain pigs carry the KIT duplication, since this breed was previously assumed to be wild type at this locus. Comparative sequence analysis indicated that the distinct phenotypic effect of the duplication occurs because the duplicated copy lacks some regulatory elements located more than 150 kb upstream of KIT exon 1 and necessary for normal KIT expression.

Alleles↗
PubMed2004

Genomic and cytological analysis of the Y chromosome of Drosophila melanogaster: telomere-derived sequences at internal regions.

The genomic analysis of heterochromatin is essential for studying chromosome behavior as well as for understanding chromosome evolution. The Y chromosome of Drosophila melanogaster is entirely heterochromatic and the under-representation of this chromosome in genomic libraries together with the difficulty of assembling its sequence has made its study very difficult. Here, we present the construction of bacterial artificial chromosome (BAC) contigs from regions h14, h16 and the centromeric region h18. The analysis of these contigs shows that telomere-derived sequences are present at internal regions. In addition, immunostaining of prometaphase chromosomes with an antibody to the kinetochore-specific protein BubR1 has revealed the presence of this protein in some Y chromosome regions rich in telomere-related sequences. Collectively, our data provide further evidence for the hypothesis that the Drosophila Y chromosomes might have evolved from supernumerary chromosomes.

Animals↗
PubMed2005

Detection of putative secreted proteins in the plant-parasitic nematode Heterodera schachtii.

The beet cyst nematode Heterodera schachtii is an important pathogen worldwide, but its molecular characterization has been limited to studying individual genes of interest. We undertook a high-throughput genomic approach and drastically increased the number of available sequences for this parasite. A total of 2,662 expressed sequence tags were grouped into 1,212 clusters representing a nonredundant catalog of H. schachtii genes. Implementing a bioinformatic workflow, we identified 50 sequences coding for candidate secreted proteins. All of these contain a putative signal peptide required for entry into the secretory pathway and lack any transmembrane domain. Included are previously postulated cell-wall-degrading enzymes and other parasitism-related genes. Moreover, we provide the first report of an arabinogalactan endo-1,4-beta-galactosidase enzyme (EC 3.2.1.89) in animals. As sequence data increase at a rapid rate, developing high-throughput genomic screening is a necessity. The in silico approach described here is an effective way to identify putative secreted proteins and prioritize candidates for further studies.

Animals↗
PubMed2004

The complete nucleotide sequence and multipartite organization of the tobacco mitochondrial genome: comparative analysis of mitochondrial genomes in higher plants.

Tobacco is a valuable model system for investigating the origin of mitochondrial DNA (mtDNA) in amphidiploid plants and studying the genetic interaction between mitochondria and chloroplasts in the various functions of the plant cell. As a first step, we have determined the complete mtDNA sequence of Nicotiana tabacum. The mtDNA of N. tabacum can be assumed to be a master circle (MC) of 430,597 bp. Sequence comparison of a large number of clones revealed that there are four classes of boundaries derived from homologous recombination, which leads to a multipartite organization with two MCs and six subgenomic circles. The mtDNA of N. tabacum contains 36 protein-coding genes, three ribosomal RNA genes and 21 tRNA genes. Among the first class, we identified the genes rps1 and psirps14, which had previously been thought to be absent in tobacco mtDNA on the basis of Southern analysis. Tobacco mtDNA was compared with those of Arabidopsis thaliana, Beta vulgaris, Oryza sativa and Brassica napus. Since repeated sequences show no homology to each other among the five angiosperms, it can be supposed that these were independently acquired by each species during the evolution of angiosperms. The gene order and the sequences of intergenic spacers in mtDNA also differ widely among the five angiosperms, indicating multiple reorganizations of genome structure during the evolution of higher plants. Among the conserved genes, the same potential conserved nonanucleotide-motif-type promoter could only be postulated for rrn18-rrn5 in four of the dicotyledonous plants, suggesting that a coding sequence does not necessarily move with the promoter upon reorganization of the mitochondrial genome.

Base Sequence↗
PubMed2000

Spectrum of color gene deletions and phenotype in patients with blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked ocular disease characterized by poor visual acuity, nystagmus, and photodysphoria in males with severely reduced color discrimination. Deletions, rearrangements and point mutations in the red and green pigment genes have been implicated in causing BCM. We assessed the spectrum of genetic alterations in ten families with BCM by Southern blot, polymerase chain reaction, and sequencing analysis, and the phenotype was characterized by ophthalmoscopy, fluorescein angiography, and a battery of tests to assess color vision in addition to routine ophthalmological examination. All families showed clinical features associated with BCM. Acuities were reduced in all affected males, and photopic b-wave was reduced by more than 90% in seven families. In three families, however, the photopic b-wave response showed uncharacteristic relative preservation of 30-80% (of the clinical low-normal value). The color vision was unusually preserved in two affected males, but this was not correlated with photopic electroretinography retention. Progressive macular atrophy was observed in affected members of two BCM families while the rest of the families presented with normal fundus. In nine families deletions were identified in the gene encoding the red-sensitive photopigment and/or in the region up to 17.8 kb upstream of the red gene which contains the locus control region and other regulatory sequences. In the same nine families the red pigment gene showed a range of deletions from the loss of a single exon to loss of the complete red gene. In one family no mutation was found in the exons of the red gene or the locus control region but showed loss of the complete green gene. No association was observed between the phenotypes and genotypes in these families.

Adult↗
PubMed2005

Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region.

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.

Chromosomes, Artificial, Bacterial↗