Structures of the genes encoding the alpha and beta subunits of the Neurospora crassa mitochondrial ATP synthase.

We have isolated and sequenced cDNA and genomic clones encoding the alpha and beta subunits of the Neurospora crassa ATP synthase. The genes are not linked to each other: atp-1(alpha) maps to either linkage group I or V, and atp-2(beta) lies on linkage group II. The two genes resemble each other in having a large number of introns, five in atp-1 and seven in atp-2, mostly positioned near their 5' ends and varying in length from 60-332 bp. The coding regions of both genes have a high G+C content (59%) and use a low number of codons, 46 (atp-1) and 44 (atp-2), a feature associated with highly expressed genes. Northern-blot analysis shows both genes are expressed at high levels during mycelial growth. Comparison of the exon-intron structures of the beta-subunit-encoding gene with those from human and tobacco showed a similar number of introns, several closely positioned, but no exact conservation in position, size or sequence of introns.     

A Genetic Analysis of the Werner Syndrome Region on Human Chromosome 8p

Werner syndrome (WRN) is an inherited disorder that produces symptoms of premature aging. This disease is caused by a recessive mutation that has previously been mapped to chromosome 8p. We have now used genetic linkage analysis to map the WRN gene relative to chromosome 6 reference loci, to screen candidate genes, and to identify a novel dinucleotide repeat polymorphic marker closely linked to WRN. The WRN locus was mapped relative to the marker loci, PLAT, ANK1, D8S135, and D8S87 of the comprehensive chromosome 8 linkage map. The heregulin (HRG) and the fibroblast growth factor receptor 1 genes (FGFR1) have been mapped to chromosome 8p and are involved in cellular growth. Recombination events were detected between WRN and the HRG and FGFR1 genes, excluding them as candidates for the WRN gene. A polymorphic marker generated in this study, WT251, is linked to WRN at a recombination fraction of 0.006, with a lod score of 16.5.     

Linkage analysis of the Duffy blood group marker with several chromosome 1 genes in an extended pedigree with Charcot-Marie-Tooth disease

Summary We have recently demonstrated tight linkage of the Duffy blood group marker to the -spectrin gene in an extended pedigree with Charcot-Marie-Tooth neuropathy. To determine a more precise location of the Duffy blood group locus on the chromosome 1 map we have tested several more chromosome 1 genes for linkage with this marker. We found suggestive linkage with the antithrombin III and apolipoprotein A2 genes and conclusive linkage with the gene coding for -nerve growth factor.     

Tyrosine hydroxylase and insulin-like growth factor II but not insulin are adjacent in the teleost species barramundi, Lates calcarifer

In humans, the genes encoding tyrosine hydroxylase (TH), insulin and insulin-like growth factor II (IGF-II) form an extremely tight linkage group on chromosome 11p15. Characterisation of the homologous genomic region of a teleost, the barramundi Lates calcarifer , revealed tight linkage of the TH and IGF-II genes, and the absence of the gene encoding insulin.     

Linkage disequilibrium--understanding the evolutionary past and mapping the medical future

Linkage disequilibrium--the nonrandom association of alleles at different loci--is a sensitive indicator of the population genetic forces that structure a genome. Because of the explosive growth of methods for assessing genetic variation at a fine scale, evolutionary biologists and human geneticists are increasingly exploiting linkage disequilibrium in order to understand past evolutionary and demographic events, to map genes that are associated with quantitative characters and inherited diseases, and to understand the joint evolution of linked sets of genes. This article introduces linkage disequilibrium, reviews the population genetic processes that affect it and describes some of its uses. At present, linkage disequilibrium is used much more extensively in the study of humans than in non-humans, but that is changing as technological advances make extensive genomic studies feasible in other species.     

Meiotic linkage mapping of 52 genes onto the canine map does not identify significant levels of microrearrangement

In an effort to extend our understanding of the evolutionary relationship between the canine and human genomes, we have developed and positioned 52 new gene-associated polymorphic markers on the canine meiotic linkage map. Canine-specific PCR primers were developed from the consensus of published sequences of several mammalian genomes and were designed to span intronic regions, thus optimizing the probability that a polymorphic site was included. The resulting markers were analyzed on a panel of three-generation canine reference families and the data were incorporated into the current meiotic linkage map. The data were compared with those generated by three chromosome paint studies in an effort to understand the distribution and frequency of microrearrangements within the canine genome. Forty-eight of 52 genes map to a chromosomal region predicted to contain genes from the corresponding region of the human genome according to all published reciprocal chromosome paint studies. Meiotic linkage mapping data for three genes can be used to resolve discrepancies between the published reciprocal chromosome paint studies, and for an additional two genes, meiotic mapping data allow evolutionary breakpoints to be more precisely defined. We conclude that microrearrangements of evolutionarily conserved segments between the canine and human genomes are rare, occurring for less than 0.5% of gene data reported to date. In addition, we have found that the placement of genes on the meiotic linkage map is a useful mechanism for resolving discrepancies between existing data sets. Received: 7 February 2001 / Accepted: 9 May 2001     

A long-range restriction map of the interleukin-4 and interleukin-5 linkage group on chromosome 5

The genes for two of the hematopoietic growth factors, interleukin-4 and interleukin-5, are located on a small segment of chromosome 5 (q23-31), which is frequently deleted in myeloid disorders. Using pulsed-field gel electrophoresis, we demonstrate physical linkage of these two genes and present a long-range restriction map of the locus. The two genes are closely linked (maximum separation, 310 kb) and appear to be separated by an HTF island. We were unable to physically link these genes to two other closely related hematopoietic growth factor genes, interleukin-3 and granulocyte/macrophage colony-stimulating factor, which also map to this region of the genome. The clustering of these and other growth-related genes suggests that a higher order of genetic organization exists in this region of the chromosome.     

Cloning and characterization of myogenic regulatory genes in three Ictalurid species

We report sequence, tissue expression and map-position data for myogenin, MYOD1, myostatin and follistatin in three Ictalurid catfish species: channel catfish (Ictalurus punctatus), blue catfish (I. furcatus) and white catfish (Ameiurus catus). These genes are involved in muscle growth and development in mammals and may play similar roles in catfish. Amino acid sequences were highly conserved among the three Ictalurid species (>95% identity), moderately conserved among catfish and zebrafish (approximately 80% identity), and less conserved among catfish and humans (approximately 40-60% identity) for all four genes. Gene structure (number of exons and introns and exon-intron boundaries) was conserved between catfish and other species for all genes. Myogenin and MYOD1 expression was limited to skeletal muscle in juvenile channel catfish, similar to expression patterns for these genes in other fish and mammalian species. Myostatin was expressed in a variety of tissues in juvenile channel catfish, a pattern common in other fish species but contrasting with data from mammals where myostatin is primarily expressed in skeletal muscle. Follistatin was expressed in juvenile catfish heart, testes and spleen. All four genes contained polymorphic microsatellite repeats in non-coding regions and linkage analysis based on inheritance of these microsatellite loci was used to place the genes on the channel catfish linkage map. Information provided in this study will be useful in further studies to determine the role these genes play in muscle growth and development in catfish.     

Assignment of the mulibrey nanism gene to 17q by linkage and linkage-disequilibrium analysis.

Mulibrey nanism (MUL) is an autosomal recessive disorder with unknown basic metabolic defect. It is characterized by growth failure of prenatal onset, characteristic dysmorphic features, constrictive pericardium, hepatomegaly as a consequence of constrictive pericardium, yellowish dots in the ocular fundi, and J-shaped sella turcica. Hypoplasia of various endocrine glands, causing hormone deficiencies, is common. Here we report the assignment of the MUL gene, by linkage analysis in Finnish families, to a 7-cM region flanked by D17S1799 and D17S948 on chromosome 17q. Multipoint linkage analysis gave a maximum LOD score of 5.01 at loci D17S1606-D17S1853 and at D17S1604. The estimate of the critical MUL region was further narrowed to within approximately 250 kb of marker D17S1853 by linkage disequilibrium analysis. Positional candidate genes that belong to the growth hormone and homeobox B gene clusters were excluded. These data confirm the autosomal recessive inheritance of MUL and allow highly focused attempts to clone the gene.     

Testing for genetic linkage in families by a variance-components approach in the presence of genomic imprinting

Some genes that affect development and behavior in mammals are known to be imprinted; and > or = 1% of all mammalian genes are imprinted. Hence, incorporating an imprinting parameter into linkage analysis may increase the power to detect linkage for these traits. Here we propose theoretical justifications for a recently developed model for testing of linkage, in the presence of genetic imprinting, between a quantitative-trait locus and a polymorphic marker; this is achieved in the variance-components framework. We also incorporate sex-specific recombination fractions into this model. We discuss the effects that imprinting and nonimprinting have on the power of the usual variance-components method and on the variance-components method that incorporates an imprinting parameter. We provide noncentrality parameters that can be used to determine the sample size necessary to attain a specified power for a given significance level, which is useful in the planning of a linkage study. Optimal strategies for a genome scan of potentially imprinted traits are discussed.     

Characterization of embryonic globin genes of the zebrafish

Hemoglobin switching is a complex process by which distinct globin chains are produced during stages of development. In an effort to characterize the process of hemoglobin switching in the zebrafish model system, we have isolated and characterized several embryonic globin genes. The embryonic and adult globin genes are found in clusters in a head-to-head configuration. One cluster of embryonic and adult genes is localized to linkage group 3, whereas another embryonic cluster is localized on linkage group 12. Several embryonic globin genes demonstrate an erythroid-specific pattern of expression early during embryogenesis and later are downregulated as definitive hematopoiesis occurs. We utilized electrospray mass spectroscopy to correlate globin genes and protein expression in developing embryonic red cells. The mutation, zinfandel, has a hypochromic microcytic anemia as an embryo, but later recovers in adulthood. The zinfandel gene maps to linkage group 3 near the major globin gene locus, strongly suggesting that zinfandel represents an embryonic globin defect. Our studies are the first to systematically evaluate the embryonic globins in the zebrafish and will ultimately be useful in evaluating zebrafish mutants with defects in hemoglobin production and switching.     

Saturating the genetic map of Arabidopsis thaliana with embryonic mutations

One goal of the Arabidopsis genome project is to identify every gene with an essential function in growth and development. Towards that end, the results are reported here of a mapping project designed to enhance the linkage map of Arabidopsis and establish a valuable resource of mutations in essential genes with known map locations. Embryo-defective (emb) mutations were chosen because they represent the most common heritable defect identified following mutagenesis in Arabidopsis. Multiple marker lines with easily scored phenotypes were constructed to facilitate mapping efforts. Recombination data were obtained for 169 mutants defective in embryo-genesis. The chromosomal locations of 110 emb genes are presented in this report. Twenty-six of these genes are tagged with T-DNA. Nine other mutants isolated following seed transformation appear to contain chromosomal translocations. Another 31 mutant genes in the collectiohave been assigned to a linkage group but not yet placed on the map. Nineteen examples of duplicate alleles have also been found. This is consistent with the estimate that approximately 500 genes readily mutate to give an embryo-defective phenotype in Arabidopsis. With continued progress, it may therefore be possible to approach saturation for this important class of mutations. Molecular cloning of these genes should be facilitated by identifying cDNAs and genomic sequences that map to similar locations.     

Northern hybridization analysis of VH gene expression in murine monoclonal antibodies directed to cancer-associated ganglioside antigens having various sialic acid linkages

The expression of the VH genes in 46 murine hybridoma cells that secrete mAb directed to the cancer-associated carbohydrate Ag, especially acidic glycolipids such as gangliosides and sulfated glycoplipids, was analyzed by Northern hybridization of poly(A)+ RNA of hybridoma with cDNA probes for nine VH gene families. Different hybridomas tended to express VH genes of the same family when the cognate Ag had the same or similar carbohydrate structures; i.e., the VH genes of the J558 family (group 1) were preferentially expressed in the mAb directed to various gangliosides that have NeuAc alpha (or NeuGc alpha) 2-3 and/or 2-8 linkage (71%), the most common linkage of sialic acid residues in the gangliosides of higher animals, and the hybridomas directed to sulfated glycolipids also expressed mainly the VH genes of the J558 family (80%). In contrast, the five mAb directed to various gangliosides with NeuAc alpha 2-6 linkage were exclusively encoded by the VH genes of Q52 family (group 2, 100%), and three antibodies directed to gangliosides with a NeuAc alpha 2-9 linkage all expressed genes of J606 family (group 6, 100%). The VH family usage was largely correlated with the linkage of sialic acid residues in the cognate carbohydrate Ag, but was not correlated at all with the difference in the fine specificities toward the core neutral carbohydrate chain, to which the sialic acid residues were attached. These findings suggest that the VH gene family in these anticarbohydrate antibodies is selected, depending primarily on the linkage of the sialic acid residues in carbohydrate Ag; these residues form the immunodominant sugar residue in the respective antigenic determinant.     

The gene for dominant white color in the pig is closely linked to ALB and PDGRFRA on chromosome 8.

White is a widespread coat color among domestic pig breeds and is controlled by an autosomal dominant gene I. The segregation of this gene was analyzed in a reference pedigree for gene mapping developed by crossing the European wild pig and a Large White domestic breed. The gene for dominant white color was shown to be closely linked to the genes for albumin (ALB) and platelet-derived growth factor receptor alpha (PDGFRA) on chromosome 8. An unexpected phenotype with patches of colored and white coat was observed among the F1 and F2 animals. The segregation data indicated that the phenotype was controlled by a third allele, denoted patch (Ip), most likely transmitted by one of the Large White founder animals. It is shown that the ALB, PDGFRA, I linkage group shares homologies with parts of mouse chromosome 5, human chromosome 4, and horse linkage group II, all of which contain dominant genes for white or white spotting. Candidate genes for the dominant white and patch mutations in the pig are proposed on the basis on these linkage homologies and the recent molecular definition of the dominant white spotting (W) and patch (Ph) mutations in the mouse.     

An amplified fragment length polymorphism map of the silkworm

The silkworm (Bombyx mori L.) is a lepidopteran insect with a long history of significant agricultural value. We have constructed the first amplified fragment length polymorphism (AFLP) genetic linkage map of the silkworm B. mori at a LOD score of 2.5. The mapping AFLP markers were genotyped in 47 progeny from a backcross population of the cross no. 782 x od100. A total of 1248 (60.7%) polymorphic AFLP markers were detected with 35 PstI/TaqI primer combinations. Each of the primer combinations generated an average of 35.7 polymorphic AFLP markers. A total of 545 (44%) polymorphic markers are consistent with the expected segregation ratio of 1:1 at the significance level of P = 0.05. Of the 545 polymorphic markers, 356 were assigned to 30 linkage groups. The number of markers on linkage groups ranged from 4 to 36. There were 21 major linkage groups with 7-36 markers and 9 relatively small linkage groups with 4-6 markers. The 30 linkage groups varied in length from 37.4 to 691.0 cM. The total length of this AFLP linkage map was 6512 cM. Genetic distances between two neighboring markers on the same linkage group ranged from 0.2 to 47 cM with an average of 18.2 cM. The sex-linked gene od was located between the markers P1T3B40 and P3T3B27 at the end of group 3, indicating that AFLP linkage group 3 was the Z (sex) chromosome. This work provides an essential basic map for constructing a denser linkage map and for mapping genes underlying agronomically important traits in the silkworm B. mori L.     

Anchoring of canine linkage groups with chromosome-specific markers

A high-resolution genetic map with polymorphic markers spaced frequently throughout the genome is a key resource for identifying genes that control specific traits or diseases. The lack of rigorous selection against genetic disorders has resulted in many breeds of dog suffering from a very high frequency of genetic diseases, which tend to be breed-specific and usually inherited as autosomal recessive or apparently complex genetic traits. Many of these closely resemble human genetic disorders in their clinical and pathologic features and are likely to be caused by mutations in homologous genes. To identify loci important in canine disease genes, as well as traits associated with morphological and behavioral variation, we are developing a genetic map of the canine genome. Here we report on an updated version of the canine linkage map, which includes 341 mapped markers distributed over the X and 37 autosomal linkage groups. The average distance between markers on the map is 9.0 cM, and the linkage groups provide estimated coverage of over 95% of the genome. Fourteen linkage groups contain either gene-associated or anonymous markers localized to cosmids that have been assigned to specific canine chromosomes by FISH. These 14 linkage groups contain 150 microsatellite markers and allow us to assign 40% of the linkage groups to specific canine chromosomes. This new version of the map is of sufficient density and characterization to initiate mapping of traits of interest. Received: 23 February 1999 / Accepted: 28 April 1999     

A radiation hybrid map of 18 growth factor, growth factor receptor, hormone receptor, or neurotransmitter receptor genes on the distal region of the long arm of chromosome 5.

The distal portion of the long arm of human chromosome 5 contains an impressive number of genes encoding growth factors, growth factor receptors, and hormone/neurotransmitter receptors. The order of and relative distance between 18 of these genes was determined by radiation hybrid mapping. There is only a single gap in a contiguous radiation map from 5q22-5q35. For this set of radiation hybrids, one map unit (centiray) corresponds to 20-50 kb of DNA. Close physical proximity for several pairs of loci was predicted by the map. Two sets of these were found to be contained in single YAC clones. The physical map produced by radiation hybrid mapping should prove useful in efforts to identify four disease genes that have been assigned to distal 5q by linkage studies.     

Walking the interactome for prioritization of candidate disease genes

The identification of genes associated with hereditary disorders has contributed to improving medical care and to a better understanding of gene functions, interactions, and pathways. However, there are well over 1500 Mendelian disorders whose molecular basis remains unknown. At present, methods such as linkage analysis can identify the chromosomal region in which unknown disease genes are located, but the regions could contain up to hundreds of candidate genes. In this work, we present a method for prioritization of candidate genes by use of a global network distance measure, random walk analysis, for definition of similarities in protein-protein interaction networks. We tested our method on 110 disease-gene families with a total of 783 genes and achieved an area under the ROC curve of up to 98% on simulated linkage intervals of 100 genes surrounding the disease gene, significantly outperforming previous methods based on local distance measures. Our results not only provide an improved tool for positional-cloning projects but also add weight to the assumption that phenotypically similar diseases are associated with disturbances of subnetworks within the larger protein interactome that extend beyond the disease proteins themselves.