Showing papers in "Mammalian Genome in 1997"

Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment

Abstract: For an understanding of the aberrant biology seen in mouse mutations and identification of more subtle phenotype variation, there is a need for a full clinical and pathological characterization of the animals. Although there has been some use of sophisticated techniques, the majority of behavioral and functional analyses in mice have been qualitative rather than quantitative in nature. There is, however, no comprehensive routine screening and testing protocol designed to identify and characterize phenotype variation or disorders associated with the mouse genome. We have developed the SHIRPA procedure to characterize the phenotype of mice in three stages. The primary screen utilizes standard methods to provide a behavioral and functional profile by observational assessment. The secondary screen involves a comprehensive behavioral assessment battery and pathological analysis. These protocols provide the framework for a general phenotype assessment that is suitable for a wide range of applications, including the characterization of spontaneous and induced mutants, the analysis of transgenic and gene-targeted phenotypes, and the definition of variation between strains. The tertiary screening stage described is tailored to the assessment of existing or potential models of neurological disease, as well as the assessment of phenotypic variability that may be the result of unknown genetic influences. SHIRPA utilizes standardized protocols for behavioral and functional assessment that provide a sensitive measure for quantifying phenotype expression in the mouse. These paradigms can be refined to test the function of specific neural pathways, which will, in turn, contribute to a greater understanding of neurological disorders.

A medium density genetic linkage map of the bovine genome

Abstract: A cattle genetic linkage map was constructed which covers more than 95 percent of the bovine genome at medium density Seven hundred and forty six DNA polymorphisms were genotyped in cattle families which comprise 347 individuals in full sibling pedigrees Seven hundred and three of the loci are linked to at least one other locus All linkage groups are assigned to chromosomes, and all are orientated with regards to the centromere There is little overall difference in the lengths of the bull and cow linkage maps although there are individual differences between maps of chromosomes One hundred and sixty polymorphisms are in or near genes, and the resultant genome-wide comparative analyses indicate that while there is greater conservation of synteny between cattle and humans compared with mice, the conservation of gene order between cattle and humans is much less than would be expected from the conservation of synteny This map provides a basis for high-resolution mapping of the bovine genome with physical resources such as Yeast and Bacterial Artificial Chromosomes as well as providing the underpinning for the interpolation of information from the Human Genome Project

Genealogy of the 129 inbred strains: 129/SvJ is a contaminated inbred strain

Abstract: The 129 mouse is the most widely used strain in gene targeting experiments. However, numerous substrains exist with demonstrable physiological differences. In this study a set of simple sequence length polymorphisms (SSLPs) was used to determine the relatedness of selected 129 substrains. 129/SvJ was significantly different from the other 129 substrains and is more accurately classified as a recombinant congenic strain (129cX/Sv), being derived from 129/Sv and an unknown strain. This mixed genetic background could complicate gene targeting experiments by reducing homologous recombination efficiency when constructs and ES cells are not derived from the same 129 substrain. Additionally, discrepancies due to different genetic backgrounds may arise when comparing phenotypes of genes targeted in different 129-derived ES cell lines.

A whole-genome radiation hybrid panel for bovine gene mapping

Abstract: Although the idea of irradiation and fusion gene transfer was published more than 20 years ago (Goss and Harris 1975) and employed in an isolated mapping experiment ten years later (Willard et al. 1985), it was not systematically employed as a human gene mapping tool until resurrected by Cox and associates (1990) for constructing a high-resolution map of human Chr 21. Wholegenome radiation hybrid (WG-RH) mapping utilizing irradiated diploid human cells rather than single chromosome hybrids was revived by Walter and colleagues (1994) and has subsequently become a major tool for high-resolution mapping of the human genome. As reviewed by McCarthy (1996), panels of human radiation hybrids (RH) have been effectively utilized to integrate linkage and physical maps, to anchor or order large insert contigs, and to construct expressed sequence tag (EST) maps that already contain more than 12,000 entries and are growing rapidly. RH mapping has not been effectively utilized in constructing maps of other mammalian species, with the exception of the mouse (Schmitt et al. 1996). This situation is destined to change, however, owing to the potential of the technique for integrating linkage and physical maps. It is an especially powerful tool for comparative gene mapping, since chromosomal order can be established for expressed genes that are usually conserved between species but often recalcitrant to linkage mapping for lack of allelic variation. The bovine donor cell l used in constructing this panel were a normal diploid fibroblast culture established from an Angus bull, JEW38. Nearly confluent flasks were trypsinized and suspended in Gibco DMEM without supplements. Approximately 10 7 cells were irradiated at room temperature in 10 ml suspension medium in a T75 flask. A cobalt 60 source delivered 185 rad/min for a total dose of 5000 rad. Attached cells were removed with trypsin, and all cells were suspended in ca/mg-free Hank's balanced salt solution (HBSS), pH 8.0, at 106 cells/ml. One-half ml (5 x 105 cells) was removed as control and 4.5 ml used for fusion. The recipient Chinese hamster TKfibroblast line A23 was kindly provided by David Cox (Stanford University School of Medicine). These cells were also suspended in HBSS at 106/ml, and 0.7 ml of this suspension was removed as control and 9 ml used for fusion. JEW38 (4.5 x 10 6 cells) and A23 (9 x 10 6 cells) were thoroughly mixed, pelleted, and resuspended. One-half ml PEG (Boehringer Mannheim polyethylene glycol 1500 in 50% sterile solution) was slowly added with constant mixing. After 2 min, 10 ml HBSS, pH 8.0, was also added slowly with gentle mixing. Cells were pelleted, then resuspended in 5 ml HBSS, pH 8.0, for 15 min at 37~ Each control line was exposed to PEG by the same process. The fusion suspension was mixed into Gibco DMEM to 10% FBS plus HAT (Sigma) plus 5 x 10 -7 M ouabain to a total volume of 90 ml. Ten ml of this mixture was dispensed to each of nine 100-mm plates (approximately 1.5 x 105 cells/plate). Controls were mixed in the same solution and plated identically. All were incubated at 37~ in 5% CO 2. All JEW38 control cells were dead on day 7, while one A23 colony, apparently a TK revertant, sur-

Two alpha(1,2) fucosyltransferase genes on porcine chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci.

Abstract: The Escherichia coli F18 receptor locus (ECF18R) has been genetically mapped to the halothane linkage group on porcine Chromosome (Chr) 6. In an attempt to obtain candidate genes for this locus, we isolated 5 cosmids containing the alpha (1,2)fucosyltransferase genes FUT1, FUT2, and the pseudogene FUT2P from a porcine genomic library. Mapping by fluorescence in situ hybridization placed all these clones in band q11 of porcine Chr 6 (SSC6q11). Sequence analysis of the cosmids resulted in the characterization of an open reading frame (ORF), 1098 bp in length, that is 82.3% identical to the human FUT1 sequence; a second ORF, 1023 bp in length, 85% identical to the human FUT2 sequence; and a third FUT-like sequence thought to be a pseudogene. The FUT1 and FUT2 loci therefore seem to be the porcine equivalents of the human blood group H and Secretor loci. Direct sequencing of the two ORFs in swine being either susceptible or resistant to adhesion and colonization by F18 fimbriated Escherichia coli (ECF18) revealed two polymorphisms at bp 307 (M307) and bp 857 (M857) of the FUT1 ORF. Analysis of these mutations in 34 Swiss Landrace families with 221 progeny showed close linkage with the locus controlling resistance and susceptibility to E. coli F18 adhesion and colonization in the small intestine (ECF18R), and with the locus of the blood group inhibitor S. A high linkage disequilibrium of M307-ECF18R in Large White pigs makes the M307 mutation a good marker for marker-assisted selection of E. coli F18 adhesion-resistant animals in this breed. Whether the FUT1 or possibly the FUT2 gene products are involved in the synthesis of carbohydrate structures responsible for bacterial adhesion remains to be determined.

Mutations in the Cacnl1a4 calcium channel gene are associated with seizures, cerebellar degeneration, and ataxia in tottering and leaner mutant mice.

Abstract: Tottering and leaner, two mutations of the mouse tottering locus, have been studied extensively as models for human epilepsy. Here we describe the isolation, mapping, and expression analysis of Cacnl1a4, a gene encoding the alpha subunit of a proposed P-type calcium channel, and also report the physical mapping and expression patterns of the orthologous human gene. DNA sequencing and gene expression data demonstrate that Cacnl1a4 mutations are the primary cause of seizures and ataxia in tottering and leaner mutant mice, and suggest that tottering locus mutations and human diseases, episodic ataxia 2 and familial hemiplegic migraine, represent mutations in mouse and human versions of the same channel-encoding gene.

Sucrose consumption in mice : major influence of two genetic loci affecting peripheral sensory responses

Abstract: Individual variability in sucrose consumption is prominent in humans and other species. To investigate the genetic contribution to this complex behavior, we conducted behavioral, electrophysiological, and genetic studies, using male progeny of two inbred mouse strains (C57BL/6ByJ [B6] and 129/J [129]) and their F2 hybrids. Two loci on Chromosome (Chr) 4 were responsible for over 50% of the genetic variability in sucrose intake. These loci apparently modulated intake by altering peripheral neural responses to sucrose. One locus affected the response threshold, whereas the other affected the response magnitude. These findings suggest that the majority of difference in sucrose intake between male B6 and 129 mice is due to polymorphisms of two genes that influence receptor or peripheral nervous system activity.

Characterization, chromosomal localization, and genetic variation of the porcine heart fatty acid-binding protein gene

Abstract: The purpose of this study was to detect genetic variation in the porcine H-FABP gene, a candidate gene for meat quality traits in pigs. Lambda phages containing the porcine H-FABP gene were isolated by plaque hybridization with human H-FABP cDNA. The coding and flanking intronic sequences of the porcine H-FABP gene were determined as well as 1.6 kb of the 5 ′ upstream region. The various potential regulatory sequences in this region are in accordance with the function and expression of the protein in muscle and mammary tissue. Furthermore, comparison with the homolog region of the mouse identified a highly conserved 13-bp element (CTTCCT [A/C] TTTCGG) that may be involved in regulation of expression. The porcine H-FABP gene was localized on Chromosome (Chr) 6 by porcine sequence-specific PCR on DNA from a pig/rodent cell hybrid panel. In addition, part of the H-FABP gene was screened for genetic variation by PCR-RFLP analysis. Three PCR-RFLPs were detected, one in the upstream region (HinfI) and two in the second intron (HaeIII and MspI). In most pig breeds the corresponding alleles have a variable distribution, possibly a consequence of selective breeding. This genetic variation will enable us to investigate the role of the H-FABP locus in porcine production and meat quality traits.

Genetical and physical assignments of equine microsatellites - First integration of anchored markers in horse genome mapping

Abstract: Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one other marker, with a total of 21 markers being involved in these linkages. In parallel, 18 of the microsatellites could be assigned to their chromosomal region by FISH. These assignments involved eight equine autosomes: ECA1, 2, 4, 6, 9, 10, 15, and 16. The genetical and physical mappings revealed by this study represent a significant extension of the current knowledge of the equine genome map.

Myostatin maps to the interval containing the bovine mh locus.

Abstract: Myostatin (GDF-8) is a member of the transforming growth factor-β superfamily and plays a role in muscle growth and development. Mice having targeted disruption of this gene display marked increases in muscle mass, a phenotype similar to the muscular hypertrophy (mh) in several cattle breeds. Physical mapping data developed from YAC clones indicate the bovine myostatin gene lies close to the centromere of bovine Chromosome (Chr) 2 (BTA2) at 2q11, indistinguishable from the cytogenetic location of the mh locus. In addition, a polymorphism in the second intron of the gene was used to show that myostatin maps within the interval previously shown to contain mh. These data suggest myostatin may be the gene causing muscular hypertrophy in cattle.

Mapping of the melatonin receptor 1a (MTNR1A) gene in pigs, sheep, and cattle

Abstract: and Implications Human and sheep Melatonin receptor 1a (MTNR1A) gene information was used to clone a portion of the coding region of this gene in pigs, and to identify polymorphisms of the gene for its assignment to both the genetic linkage and physical maps. MTNR1A maps to pig chromosome 17, establishing a new region of conserved synteny between this chromosome and human chromosome 4. Furthermore, we have assigned MTNR1A to bovine chromosome 27 and sheep chromosome 26. The addition of genes like MTNR1A to livestock genome maps allows questions about evolutionary events and the genetic basis for quantitative traits in livestock to be addressed.

IAP insertion in the murine LamB3 gene results in junctional epidermolysis bullosa.

Abstract: The laminin-5 molecule functions in the attachment of various epithelia to basement membranes. Mutations in the laminin-5-coding genes have been associated with Herlitz junctional epidermolysis bullosa (HJEB), a severe and often lethal blistering disease of humans. Here we report the characterization of a spontaneous mouse mutant with an autosomal recessive blistering disease. These mice exhibit sub-epithelial blisters of the skin and mucosal surfaces and abnormal hemidesmosomes lacking subbasal dense plates. By linkage analysis the genetic defect was localized to a 2-cM region on distal Chromosome (Chr) 1 where a laminin-5 subunit gene, LamB3, was previously localized. LamB3 mRNA and laminin-5 protein were undetectable by Northern blot analysis and immunohistochemical methods, respectively. DNA sequence analysis indicated that the LamB3 genetic defect resulted from disruption of the coding sequence by insertion of an intracisternal-A particle (IAP) at an exon/intron junction. These findings suggest a role for laminin-5 in hemidesmosome formation and indicate that the LamB3IAP mutant is a useful mouse model for HJEB.

A candidate model for angelman syndrome in the mouse

Abstract: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are well-recognized examples of imprinting in humans. They occur most commonly with paternal and maternal 15ql 1-13 deletions, but also with maternal and paternal disomy. Both syndromes have also occurred more rarely in association with smaller deletions seemingly causing abnormal imprinting. A putative mouse model of PWS, occurring with maternal duplication (partial maternal disomy) for the homologous region, has been described in a previous paper but, although a second imprinting effect that could have provided a mouse model of AS was found, it appeared to be associated with a slightly different region of the chromosome. Here, we provide evidence that the same region is in fact involved and further demonstrate that animals with paternal duplication for the region exhibit characteristics of AS patients. A mouse model of AS is, therefore, strongly indicated.

The human DAZ genes, a putative male infertility factor on the Y chromosome, are highly polymorphic in the DAZ repeat regions.

Abstract: The DAZ genes on the human Y Chromosome (Chr) are strong candidates for the azoospermia factor AZF. They are frequently deleted in azoospermic or severely oligospermic males and are expressed exclusively in germ cells. In addition, the DAZ genes share a high degree of similarity with a Drosophila male infertility gene, boule. The predicted DAZ proteins contain an RNA recognition motif (RRM), and multiple copies of a repeat (the DAZ repeat) in tandem array. To understand the DAZ gene family and its expression, the DAZ genomic structure and RNA transcripts in numerous males, as well as several DAZ cDNA clones were analyzed. The results of genomic Southern blot showed that each male contains multiple DAZ genes with varying numbers of DAZ repeats, and that the copy number of the DAZ repeats are polymorphic in the population. The presence of multiple species of DAZ transcripts with different copy number and arrangement of the DAZ repeats in an individual suggests that more than one DAZ gene are transcribed. The existence of multiple functional DAZ genes complicates the analysis of genotype/phenotype correlations among males with varying sperm counts.

Interval-specific congenic strains (ISCS): an experimental design for mapping a QTL into a 1-centimorgan interval.

Abstract: A general experimental design that allows mapping of a quantitative trait locus (QTL) into a 1-cM interval is presented. The design consists of a series of strains, termed “interval-specific congenic strains (ISCS)”. Each ISCS is recombinant at a specific 1-cM sub-interval out of an ordered set of sub-intervals, which together comprise a wider interval, to which a QTL was previously mapped. It is shown that a specific and previously detected QTL of moderate or even small effect can be accurately mapped into a 1-cM interval in a program involving a total of no more than 1000 individuals. Consequently, ISCS can serve as the ultimate genetic mapping procedure before the application of physical mapping tools for positional cloning of a QTL.

Mapping murine loci for seizure response to kainic acid

Abstract: Mature DBA/2J (D2) mice are very sensitive to seizures induced by various chemical and physical stimuli, whereas C57BL/6J (B6) mice are relatively seizure resistant. We have conducted a genome-wide search for quantitative trait loci (QTLs) influencing the differential sensitivity of these strains to kainic acid (KA)-induced seizures by studying an F2 intercross population. Parental, F1, and F2 mice (8–10 weeks of age) were injected subcutaneously with 25 mg/kg of KA and observed for 3 h. Latencies to focal and generalized seizures and status epilepticus were recorded and used to calculate an overall seizure score. Results of seizure testing indicated that the difference in susceptibility to KA-induced seizures between D2 and B6 mice is a polygenic phenomenon with at least 65% of the variance due to genetic factors. First-pass genome screening (10-cM marker intervals) in F2 progeny (n = 257) documented a QTL of moderate effect on Chromosome (Chr) 1 with a peak LOD score of 5.5 (17% of genetic variance explained) localized between D1Mit30 and D1Mit16. Provisional QTLs of small effect were detected on Chr 11 (D11Mit224–D11Mitl4), 15 (D15Mit6–D15Mit46) and 18 (D18Mit9–D18Mitl44). Multiple locus models generally confirmed the Mapmaker/QTL results and also provided evidence for another QTL on Chr 4 (D4Mit9). Multilocus analysis of seizure severity suggested that additional loci on Chrs 5 (D5Mit11), 7 (D7Mit66), and 15 (D15Nds2) might also contribute to KA-induced seizure response. Overall, our results document a complex genetic determinism for KA-induced seizures in these mouse strains with contributions from as many as eight QTLs.

Organization and expression of bovine TSPY.

Abstract: We have isolated genomic sequences as well as transcripts from the bovine homolog of the human testis-specific protein, Y-encoded, TSPY which—in both species—is located on the Y Chromosome (Chr), organized as a gene family with a variable number of members, and expressed exclusively in the testis. 1266 bp of bovine TSPY specific sequence have been isolated from a testis cDNA library, by RT-PCR analyses and by Rapid Amplification of cDNA Ends (RACE). A bovine TSPY gene 4 is organized in seven exons, and transcripts are polyadenylated at various 3′ ends. Consensus polyadenylation signals AA UUAAA are missing. Microheterogeneous sequence variation is found between TSPY family members. In addition, homologies to other Y-located repeated sequence families, BRY, have been discovered; these sequences are presumably derived from ancient members of the TSPY cluster, now forming a separate, probably nonfunctional subfamily. Bovine TSPY is subject to differential splicing. In the adult, it is expressed in early germ-cell stages, and expression could also be detected in fetal testis. Comparison with the human homolog shows the highest degree of similarity in the coding regions of exons 2, 3, and 4, which are also precisely conserved regarding their length.

Variability of canine microsatellites within and between different dog breeds.

Abstract: Polymorphic animal microsatellites have proved valuable genetic markers For this project, the variability of 19 canine microsatellite loci was examined within and between three pure breeds of dog: Greyhounds, Labradors, and German Shepherds The number of alleles, absolute and relative frequencies, and the statistics that express polymorphism within a breed were determined The evolutionary relationships among these closely related dog breeds were estimated by genetic distance measures developed for use with microsatellite loci According to the pairwise genetic distances, Greyhounds and German Shepherds had longer diverse evolutionary histories than Greyhounds and Labradors or Labradors and German Shepherds Although a few breed-specific alleles were observed, the significant differences between breeds are in their relative frequencies and distribution of the alleles across a locus None of the three pure dog breeds corresponds to Hardy-Weinberg equilibrium A considerable reduction in intrapopulation variation was observed within three pure breeds, compared with the population of individuals belonging to 15 dog breeds This reduction was especially pronounced in the Greyhound breed, which expressed the lowest degree of variation Intrapopulation variations of Labradors and German Shepherds did not differ significantly, that of Labradors being only slightly higher The intra-species variation of dogs is lower than in humans, mouse, or rat, but similar to that in domestic animals, probably reflecting similarly high inbreeding coefficients However, some highly informative loci were common to all dog breeds tested so far Such population data are necessary for mapping studies and linkage analysis in dogs

Linkage map and congenic strains to localize blood pressure QTL on rat chromosome 10.

Abstract: Our purposes were to develop a linkage map for rat Chromosome (Chr) 10, using chromosome-sorted DNA, and to construct congenic strains to localize blood pressure quantitative trait loci (QTL) on Chr 10 with the map. The linkage mapping panel consisted of three F2 populations totaling 418 rats. Thirty-two new and 29 known microsatellite markers were placed on the map, which spanned 88.9 centiMorgans (cM). The average distance between markers was 1.46 cM. No markers were separated by more than 6.8 cM. Four congenic strains were constructed by introgressing various segments of Chr 10 from the Milan normo-tensive strain (MNS) onto the background of the Dahl salt-sepsitive (S) strain. A blood pressure QTL with a strong effect on blood pressure (35-42 mm Hg) when expressed on the S background was localized to a 31-cM region between DIOMco6 and DIOMcol. The region does not include the locus for inducible nitric oxide synthase (Nos2), which had been considered to be a candidate locus for the QTL.

Cloning and mapping of a human and mouse gene with homology to ecto-ATPase genes

Abstract: The human CD39-like-l gene (CD39L1) was isolated from a selected cDNA library enriched for transcripts from regions of human Chromosome (Chr) 9q. Database searches with sequences of one group of clones from the selected cDNA library showed strong amino acid homology to the lymphoid cell activation antigen CD39, an ecto-apyrase gene from human and mouse. The full-length sequence for CD39L1 identified a putative 472 amino acid protein with greater than 60% identity with the chicken muscle ecto-ATPase protein, as well as homology to a number of other known ecto-ATPases and ecto-apyrases from rat, garden pea, yeast, and Toxoplasma gondii. A high level of amino acid identity suggests that CD39L1 is closely related to the chicken muscle ecto-ATPase. The presence of the human ABC2 gene on an overlapping cosmid and hybridizations to somatic cell mapping panels suggest that CD39L1 maps to human Chr 9q34. A mouse homolog was isolated (showing greater than 78% nucleotide sequence identity) and mapped by FISH to mouse Chr 2, the syntenic region of human 9q34. The genomic structure of CD39L1 reveals 9 exons covering less than 7 kb.

Linkage mapping of wool keratin and keratin-associated protein genes in sheep

Abstract: The keratins and keratin-associated proteins (KAPs) are a large heterogeneous group of proteins that make up about 90% of the wool fiber. Heteropolymers of the type I and type II keratins (encoded by the KRTI.n and KRT2.n gene families respectively) form the microfibrils of the wool fiber (Powell and Rogers 1994). These microfibrils are embedded in a matrix of KAPs. The KAPs have been divided into three major groupings on the basis of their protein sequences: the high glycine-tyrosine KAPs, the high sulfur KAPs, and the ultra-high sulfur KAPs (Powell and Rogers 1994). The high glycine-tyrosine KAPs are encoded by the KAP6.n multigene family as well as by the single genes, KAP7 and KAP8. The high sulfur family is encoded by three multigene families, KAPI.n, KAP2.n, and KAP3.n, while the ultra-high sulfur family consists of at least two multigene families, the cortex-specific KAP4.n family and the cuticle-specific KAP5.n family (Powell and Rogers 1994). Trichohyalin (THH) is another important wool follicle protein encoded by a single gene, although this protein is also expressed in other epidermal tissues (O'Keefe et al. 1993). The objectives of this project were to identify polymorphism in members of the KRT and KAP gene families and to locate these genes on the sheep genetic linkage map. A genetic linkage map of the sheep genome has been constructed by screening polymorphic markers through full-sib pedigrees of the AgResearch International Mapping Flock (IMF; Crawford et al. 1995). This resource consists of nine three-generation full-sib pedigrees in which four F 1 TexelxCoopworth cross sires were mated with nine F~ (MerinoxRomney) x (PerendalexCoopworth) cross dams to generate between 7 and 17 offspring for each mating (Crawford et al. 1995). PCR products specific for the genes KRT1.2, KRT2.10, KRT2.13, KAPI.1, KAP1.3, KAP2.3, KAP3.2, KAP5.1, KAP6.1, KAP7 and Trichohyalin were obtained (Table 1), and eight new polymorphic markers for KRT and KAP loci were identified including PCR-RFLPs, Southern RFLPs, microsatellites, and SSCPs (Table 1). These new polymorphic markers, in addition to previously published polymorphism at KRT and KAP loci (Rogers et al. 1993, 1994), were screened through the IMF. Nine KRT and KAP genes together with Trichohyalin were mapped onto the sheep genetic linkage map, and six of these represent new assignments (Table 1). The high glycine-tyrosine KAPs (KAP6.1, 7, 8) were mapped to OOV1 (Fig. 1) and were closely linked to one another. RFLP studies have previously demonstrated linkage between KAP6, 7, and 8 (Parsons and Cooper 1995). The mapping of Trichohyalin to OOV1 is a new assignment. The 650-base pair (bp) PCR amplimer obtained for Trichohyalin was smaller than expected (889 bp) from the genomic sequence used to design the primers (O'Keefe et al.

Towards construction of a canine linkage map: establishment of 16 linkage groups.

Abstract: 1Norwegian Kennel Klub and Department of Morphology, Genetics and Aquatic Biology, Section of Genetics, P.O. Box. 8146 Dep., N-0033 Oslo, Norway ZDepartment of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Box 7023, 750 07 Uppsala, Sweden 3Department of Animal Science and Animal Health, Division of Animal Genetics, The Royal Veterinary and Agricultural University, Btilowsvej, 13, DK-1870, Fredriksberg C, Copenhagen, Denmark ~The Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK 5Centre for Preventive Medicine, Animal Health Trust, PO Box 5, Newmarket, Suffolk CB8 7DW, UK 6Medical Technology and Medicine, Michigan State University, B228 Life Science, East Lansing, Michigan 48824-1317, USA 7Institute of Animal Breeding, University of Berne, Bremgartenstrasse 109 a, 3012 Berne, Switzerland

Identification and mapping of a novel human gene, HRMT1L1, homologous to the rat protein arginine N-methyltransferase 1 (PRMT1) gene.

Abstract: Human chromosome (Chr) 21 is the smallest and one of the most intensely studied autosomes. It has served as a paradigm for the Human Genome Project, being the first chromosome for which a detailed genetic and a high-resolution physical map have been produced (6th International Workshop on Chromosome 21, Cold Spring Harbor, 1996). Having accomplished the initial goals of the Genome Project, the next aim is to describe the complete sequence and the full complement of genes residing on the chromosome. Although 700-1000 genes are predicted to map to Chr 21, less that 10% of these have been identified to date (Genome Database, Version 6.0, November 1996). We are enriching the transcription map of 21q by a combination of methods, such as expressed sequence tag (EST) database searching and cDNA selection. As a result of two independent approaches, we have been able to identify various novel transcripts. Here we report the isolation of a human homolog (HRMT1L1) of the rat protein arginine N-methyltransferase 1 gene (PRMT1, Genbank accession number U60882, Lin et al. 1996), its fine mapping on Chr 21, and its expression pattern. We have screened the EST databases with over 1200 singlecopy sequences that have been putatively assigned to human Chr 21 by a variety of methods. To date, we have shown that 158 of these sequences have identity at the nucleotide level with various expressed sequences (Katsanis and Fisher, unpublished). The HRMT1L1 gene was identified by this route when we screened dbEST with three independent sequences: 21ES184 (146bp, L25245), 21ES057 (145bp, L25224), and 21ES074 (306bp, L25503). These three sequences are partial cDNAs selected from a Chr 21 specific cosmid library (Cheng et al. 1994). All three clones identified a collection of 5 ESTs from dbEST, which formed two discrete contigs. Contig 1 (one EST plus 21ES184 and 21ES057) was 354 bp. Contig 2 (four cDNAs plus 21ES074) was 877 bp. Contig 1 contained an open reading frame (ORF) spanning its complete sequence. Contig 2 did not have an ORF but contained a polyA § tail and a putative polyadenylation signal, suggesting it contained a possible 3' UTR. We screened the EST databases with both contigs and identified a total of 56 cDNAs from various human libraries [infant and adult brain, heart, liver, senescent fibroblasts, breast, spleen, lung, melanocyte, multiple sclerosis lesions (Lennon et al. 1996)]. We collected the 5' and the 3' sequences of all clones identified by our two contigs and aligned them. From the IMAGE consortium ID numbers of the 56 cDNAs, we observed that both contigs shared a set of the same clones. Thus the two contigs appear to be derived from different regions of the same locus. To bridge between the two contigs and confirm our finding, we

Organization of the bovine casein gene locus

Abstract: In bovine milk, four types of caseins (O~s~-, cts2-, 13-, and K-casein) have been identified, each encoded by a single copy gene. For each of these, several genetic variants have been described. The casein genes are clustered in a region on Chromosome (Chr) 6 (Ferretti et al. 1990; Threadgill and Womack 1990; Gallagher et al. 1994), and their overall organization is conserved between species (Mercier and Vilotte 1993). The three genes encoding the calcium-sensitive caseins (Otsl, oLs2, and 13) have originated from a common ancestral gene through intraand intergenic duplication and exon shuffling (Groenen et al. 1993) and share common regulatory motifs in the proximal 5' flanking region (Groenen et al. 1992). The K-casein gene is not evolutionarily related to these genes, although its expression pattern is similar and its protein product is essential for micelle formation and stability (Alexander et al. 1988). The proximal 5' flanking region of the K-casein gene is organized differently from that of the other casein genes (Alexander et al. 1988). However, all four casein genes are coordinately expressed at high levels in a tissueand stage-specific fashion. For a good understanding of expression regulation of gene loci, it is essential to gain insight into the nature and location of cisacting control elements and the higher order chromatin structure of these loci. Thus, to unravel the mechanisms that underlie the coordinate expression of all four casein genes, detailed knowledge of the organization of the casein'locus is required. The sequences o~ all four bovine casein genes [oL~l (Koczan et al., 1991); 13 (Bonsing et al. 1988; Gorodetsky et al. 1988); ct~2 (Groenen et al. 1993); K (Alexander et al. 1988)] and their order in the bovine casein gene locus [ctsl-[~--as2-K (Ferretti et al. 1990; Threadgill and Womack 1990)] have been described previously. However, data relating to the distance between these genes, the physical map of the locus, and its size are conflicting [less than 200 kb (Threadgill and Womack 1990); 250-300 kb (Ferretti et al., 1990)]. Moreover, their relative transcriptional orientations remain to be determined. In this study a detailed map of the bovine casein gene locus was established by combining PFGE-based mapping of bovine genomic DNA with the isolation and restriction analysis of cosmid and phage clones comprising various parts of the bovine casein region. The size of the casein locus, the position of the bovine casein genes, and their relative transcriptional orientation have been determined. Agarose blocks of high-molecular-weight DNA for PFGE analysis were prepared from bovine mammary epithelial cells (MAC-T; Huynh et al. 1991), white blood cells, and sperm cells of a Holstein Friesian dairy bull (the same animal used for the cosmid library) according to standard protocols (Birren and Lai 1993). Agarose blocks (80-100 p~l) were prepared at a concentration of l07 cells/ml for MAC-T cells and white blood cells and at 2 x l07 cells/ml for sperm cells (6-10 ixg DNA/block). For restriction enzyme digestion, blocks were added to lx restriction enzyme

Eyes absent: A gene family found in several metazoan phyla

Abstract: Genes related to the Drosophila eyes absent gene were identified in vertebrates (mouse and human), mollusks (squid), and nematodes (C. elegans). Proteins encoded by these genes consist of conserved C-terminal and variable N-terminal domains. In the conserved 271-amino acid C-terminal region, Drosophila and vertebrate proteins are 65-67% identical. A vertebrate homolog of eyes absent, designated Eya2, was mapped to Chromosome (Chr) 2 in the mouse and to Chr 20q13.1 in human. Eya2 shows a dynamic pattern of expression during development. In the mouse, expression of Eya2 was first detected in 8.5-day embryos in the region of head ectoderm fated to become the forebrain. At later stages of development, Eya2 is expressed in the olfactory placode and in a variety of neural crest derivatives. In the eye, expression of Eya2 was first detected after formation of the lens vesicle. At day 17.5, the highest level of Eya2 mRNA was observed in primary lens fibers. Low levels of Eya2 expression was detected in retina, sciera, and cornea. By postnatal day 10, Eya2 was expressed in secondary lens fibers, cornea, and retina. Although Eya2 is expressed relatively late in eye development, it belongs to the growing list of factors that may be essential for eye development across metazoan phyla. Like members of the Pax-6 gene family, eyes absent gene family members were probably first involved in functions not related to vision, with recruitment for visual system formation and function occurring later.

The effect of selective genotyping on QTL mapping accuracy.

Abstract: The detection of quantitative trait loci (QTL) requires large sample sizes to attain reasonable power (Soller et al. 1976). For reduction of the number of individuals needed to be genotyped in markerQTL linkage experiments, a procedure termed \"selective genotyping\" has been proposed for experimental species (Darvasi and Soller 1992; Lander and Botstein 1989; Lebowitz et al. 1987) and has been adapted for humans as well (Risch and Zhang 1995). With selective genotyping, only individuals from the high and low phenotypic extremes are genotyped. It has been shown that the number of individuals genotyped to attain a given power can be decreased significantly, at the expense of a moderate increase in the number of individuals phenotyped (Darvasi and Soller 1992). The major limitation of this approach is that if the experiment is aimed at analyzing a number of traits, then by selecting the extremes of each trait one would select most of the population and thus no reduction in genotyping can be obtained. Selective genotyping is thus most appropriate for the cases where only one trait is being analyzed. This conclusion is valid when selective genotyping is applied to QTL detection. However, after a QTL is detected, its map location will be estimated. In this case, additional markers at chromosomal regions of interest will be used to provide better estimation of QTL map location, since for QTL detection alone, relatively wide marker spacing is adequate (Darvasi and Soller 1994). These additional markers will be at a specific chromosomal region, and thus they will ordinarily concern a single specific QTL and a single specific trait only. Consequently, with respect to these markers, selective genotyping can be applied, even if the initial experimental population was used to map QTL affecting several traits. The proportion of the population selected for genotyping with the additional markers will be determined by the effect of selective genotyping on QTL mapping accuracy. This aspect of selective genotyping is the object of the present study. The model used to investigate the effect of selective genotyping on QTL mapping accuracy consists of a 100-cM chromosome with a single QTL located at its center. A backcross population, originating from crosses between two inbred lines with alternative alleles for all markers and for the QTL, is assumed. The quantitative trait is taken to have a normal distribution with equal variance, cr 2, for all QTL genotypes; and standardized gene effects of 0 and d for the QTL genotypes Qq and QQ respectively. Darvasi and colleagues (1993) have shown that QTL mapping accuracy when using an infinite number of markers is the same as that achieved by interval mapping using moderate marker spacing. Consequently, simulation results obtained on the assumption of an infinite number of markers apply for the actual experimental case where markers spaced at fairly wide intervals are used. On this basis and for simplicity of calculations only, an infinite number of markers, represented in the simulations by a marker every 0.1 cM, is assumed. Monte Carlo simulations were carried out according to the above model, and a maximum likelihood estimate (MLE) was obtained for QTL map location, as detailed in Darvasi and colleagues (1993). A 95% empirical confidence interval (CI) was obtained from 1000 replicated simulations for each parameter combination. Heron, CI is referred to as the length of the 95% confidence interval. The equivalence of various experimental designs (BC, F2, halfsibs) when using selective genotyping has also been presented (Darvasi and Soller 1992). Consequently, the present model serves as a close approximation to a wide range of actual experimental conditions. Previous studies have shown that CI is mainly a function of sample size, N, and gene effect, d (Darvasi et al. 1993). In order to explore the independent effect of N and d, six different parameter combinations of N and d were chosen: N = 500 with d = 2.0, 0.7 and 0.5; and N = 2000 with d = 1.0, 0.35 and 0.25. The values of d were chosen to provide similar CI with the two different sample sizes. Confidence intervals were estimated selecting a total proportion, p, of the population (p/2 at each phenotypic extreme). Figure 1 presents CI as a function of the proportion selected, p, for the six parameter combinations. As expected, CI increases when smaller proportions of the population are selected. It can be seen that population size and gene effect, independently, do not substantially affect the way CI changes as a function of p. That is, the influence of proportion selected on CI will be similar for any particular combination of d and N that determines the same CIs when selecting the entire population. Most importantly, it can be seen that in all cases, selecting more than 40-50% of the population does not reduce the CI.

Quantitative trait loci for estrogen-dependent pituitary tumor growth in the rat

Abstract: Growth control is of fundamental importance to biology in general and of critical importance to cancer research in particular. Tumors develop when control of the normal growth process is lost. The rat pituitary is a model system for control of estrogen-dependent growth. Chronic estrogen treatment induces uncontrolled growth in the pituitaries of Fischer 344 (F344) rats, but not of Brown Norway (BN) rats. We have identified five quantitative trait loci (QTL) for estrogen-dependent pituitary mass (Edpm) in an F2 intercross of F344 and BN. These QTL reside on rat Chromosomes (Chrs) 2, 3, 5, and 9 and explain a total of 55% of the genetic variance in the F2. We have also detected suggestive evidence for a QTL on rat Chr 14. For Edpm2- 1, Edpm2- 2, Edpm3, and Edpm5, the F344 allele corresponds with increased pituitary mass, as expected. Surprisingly, for Edpm9 and the suggested QTL on Chr 14, the BN allele corresponds with increased pituitary mass. We also find evidence for interaction (epistasis) between Edpm3 and Edpm9 and between Edpm5 and the suggested QTL on Chr 14.

Comparative chromosome painting between two marsupials : origins of an XX/XY1Y2 sex chromosome system

Abstract: Cross-species chromosome painting was used to investigate genome rearrangements between tammar wallaby Macropus eugenii (2n = 16) and the swamp wallaby Wallabia bicolor (2n = 10♀/11♂), which diverged about 6 million years ago. The swamp wallaby has an XX female:XY1Y2 male sex chromosome system thought to have resulted from a fusion between an autosome and the small original X, not involving the Y. Thus, the small Y1 should represent the original Y and the large Y2 the original autosome. DNA paints were prepared from flow-sorted and micro-dissected chromosomes from the tammar wallaby. Painting swamp wallaby spreads with each tammar chromosome-specific probe gave extremely strong and clear signals in single-, two-, and three-color FISH. These showed that two tammar wallaby autosomes are represented unchanged in the swamp wallaby, two are represented by different centric fusions, and one by a tandem fusion to make the very long arms of swamp wallaby Chromosome (Chr) 1. The large swamp wallaby X comprises the tammar X as its short arm, and a tandemly fused 7 and 2 as the long arm. The acrocentric swamp wallaby Y2 is a 2/7 fusion, homologous with the long arm of the X. The small swamp wallaby Y1 is confirmed as the original Y by its painting with the tammar Y. However, the presence of sequences shared between the microdissected tammar Xp and Y on the swamp wallaby Y2 implies that the formation of the compound sex chromosomes involved addition of autosome(s) to both the original X and Y. We propose that this involved fusion with an ancient pseudoautosomal region followed by fission proximal to this shared region.

Genetic variation in the porcine myogenin gene locus.

Abstract: The myogenin (MYOG) gene fulfills a key function in muscle differentiation by controlling the onset of myoblast fusion and the establishment of myofibers. In meat-producing animals like pigs and cattle, myofiber numbers have been related to growth capacity. We have characterized the porcine MYOG gene to detect genetic variation at this locus and to relate it to growth characteristics. MYOG gene fragments were isolated by PCR on genomic DNA and by screening a genomic library with a mixture of the four human MyoD cDNA fragments. Both the exons and promoter region were very similar to the human and mouse genes. Southern blot analysis of 105 unrelated pigs revealed three polymorphic MspI sites, located in the promoter region, the second intron, and at the 3' side of the gene. PCR-RFLP tests detecting four MYOG alleles were developed. PCR analysis of a panel of pig-rodent somatic cell hybrids confirmed the genetic localization of MYOG on pig Chromosome (Chr) 9. The PCR-RFLP tests and microsatellite markers on Chr 9 offer the possibility to genotype large numbers of pigs for studies of genetic linkage to meat deposition and growth characteristics.