ABSTRACT
There are two types of 1-day field tests available for young Swedish Warmblood sport horses; one test for 3-year olds and one more advanced test for 4-year olds. Conformation, gaits and jumping ability are evaluated at both tests. Studies on various genetic parameters were based on about 20 000 tested horses. The data for 4-year olds consisted of 30 years of testing. The aims of the study were to estimate genetic parameters for results from different time periods, and to estimate heritabilities for, and genetic correlations between, traits scored in the two tests. The judgement of traits was shown to have been changed during the 30 years of testing, resulting in changes in higher heritabilities in, and stronger genetic correlations between, later time periods. In the first time period, records showed higher residual and lower genetic variances than in the subsequent time periods. Genetic correlations between traits recorded in the first and last time period deviated considerably from unity. Further studies are needed to investigate how to treat data from the early period in genetic evaluations. Heritabilities were moderate to high for conformation traits (0.24 to 0.58) at both types of tests, except for correctness of legs (0.08). The heritabilities for gait traits were also moderate to high (0.37 to 0.53). For jumping traits, the heritabilities ranged between 0.17 and 0.33. The highly positive genetic correlations (0.82 to 0.99) between corresponding traits tested at the simpler test for 3-year olds and at the ridden test of 4-year olds implied that it would be desirable to include the test results of 3-year olds into the genetic evaluation as breeding values for Swedish Warmbloods for many years has only been based on results from 4-year olds.
ABSTRACT
Inpp5b is an ubiquitously expressed type II inositol polyphosphate 5-phosphatase. We have disrupted the Inpp5b gene in mice and found that homozygous mutant males are infertile. Here we examine the causes for the infertility in detail. We demonstrate that sperm from Inpp5b(-/-) males have reduced motility and reduced ability to fertilize eggs, although capacitation and acrosome exocytosis appear to be normal. In addition, fertilin beta, a sperm surface protein involved in sperm-egg membrane interactions that is normally proteolytically processed during sperm transit through the epididymis, showed reduced levels of processing in the Inpp5b(-/-) animals. Inpp5b was expressed in the Sertoli cells and epididymis and at low levels in the developing germ cells; however, mice lacking Inpp5b in spermatids and not in other cell types generated by conditional gene targeting, were fully fertile. The abnormalities in mutant sperm function and maturation appear to arise from defects in the functioning of Sertoli and epididymal epithelial cells. Our results directly demonstrate a previously unknown role for phosphoinositides in normal sperm maturation beyond their previously characterized involvement in the acrosome reaction. Inpp5b(-/-) mice provide an excellent model to study the role of Sertoli and epididymal epithelial cells in the differentiation and maturation of sperm.
Subject(s)
Membrane Glycoproteins/metabolism , Metalloendopeptidases/metabolism , Phosphoric Monoester Hydrolases/deficiency , Phosphoric Monoester Hydrolases/genetics , Spermatozoa/enzymology , Spermatozoa/physiology , ADAM Proteins , Animals , Female , Fertilins , Fertilization in Vitro , Gene Targeting , Infertility, Male/enzymology , Infertility, Male/genetics , Infertility, Male/pathology , Infertility, Male/physiopathology , Inositol Polyphosphate 5-Phosphatases , Male , Mice , Mice, Knockout , Phosphoric Monoester Hydrolases/physiology , Protein Processing, Post-Translational , Sperm Count , Sperm Motility/genetics , Sperm Motility/physiology , Spermatogenesis/genetics , Spermatogenesis/physiology , Spermatozoa/growth & developmentABSTRACT
Palmitoyl protein thioesterase (PPT) is the defective enzyme in infantile neuronal ceroid lipofuscinosis (INCL), which is a recessively inherited, progressive neurodegenerative disorder. We present here the cloning, chromosomal mapping, genomic structure, and the expression of the cDNA of mouse PPT. The mouse PPT gene spans >21 kb of genomic DNA and contains nine exons with a coding sequence of 918 bp. Fluorescence in situ hybridization to metaphase chromosomes localized the mouse PPT gene to the chromosome 4 conserved syntenic region with human chromosome 1p32 where the human PPT is located. PPT is expressed widely in a variety of mouse tissues. The mouse PPT cDNA is conserved highly with the human and rat PPT both at the nucleotide and amino acid sequence level. Transient expression of mouse PPT in COS-1 cells yielded a 38/36-kD differentially glycosylated polypeptide that was also secreted into culture media. Immunofluorescence analysis of transiently transfected HeLa cells indicated lysosomal localization of mouse PPT. Based on the high conservation of the gene and polypeptide structure as well as similar processing and intracellular localization, the function of PPT in mouse and human are likely to be very similar.
Subject(s)
DNA, Complementary/biosynthesis , Gene Expression Regulation , Genes , Thiolester Hydrolases/genetics , Amino Acid Sequence , Amino Acids/analysis , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , DNA, Complementary/analysis , HeLa Cells , Humans , Intracellular Fluid/enzymology , Male , Mice , Molecular Sequence Data , Thiolester Hydrolases/biosynthesisABSTRACT
Neuronal ceroid lipofuscinoses (NCL) represent a group of common progressive encephalopathies of children with a global incidence of 1 in 12,500. NCL are divided into three autosomal recessive subtypes, all assigned to different chromosomal loci. The infantile subtype of NCL (INCL) is characterized by early visual loss and mental deterioration, and leads to a vegetative state of the patients by 3 years of age. We pursued the identification of the gene defective in INCL, enriched in the Finnish population by a positional cloning approach and identified mutations in the palmitoyl-protein thioesterase (PPT) gene in INCL patients. We have further shown that PPT represents a novel lysosomal enzyme and is routed to the lysosomes via the mannose 6-phosphate receptor-mediated pathway. The worldwide most common mutation in the PPT gene, INCLFin, results in the deficient routing of the mutant PPT to lysosomes and undetectable enzyme activity in the brain tissue of patients. Our results suggest that INCL can be classified as a new member of lysosomal enzyme deficiencies.
Subject(s)
Chromosome Mapping , Cloning, Molecular , Neuronal Ceroid-Lipofuscinoses/genetics , Adolescent , Child , Child, Preschool , Chromosome Aberrations/genetics , Chromosome Disorders , Chromosomes, Human, Pair 1 , DNA Mutational Analysis , Finland , Genes, Recessive/genetics , Humans , Infant , Neuronal Ceroid-Lipofuscinoses/classification , Neuronal Ceroid-Lipofuscinoses/diagnosis , Palmitoyl-CoA Hydrolase/geneticsABSTRACT
Palmitoyl protein thioesterase (PPT) is an enzyme that removes palmitate residues from various S-acylated proteins in vitro. We recently identified mutations in the human PPT gene in patients suffering from a neurodegenerative disease in childhood, infantile neuronal ceroid lipofuscinosis (INCL), with dramatic manifestations limited to the neurons of neocortical origin. Here we have expressed the human PPT cDNA in COS-1 cells and demonstrate the lysosomal targeting of the enzyme via the mannose 6-phosphate receptor-mediated pathway. The enzyme was also secreted into the growth medium and could be endocytosed by recipient cells. We further demonstrate the disturbed intracellular routing of PPT carrying the worldwide most common INCL mutation, Arg122Trp, to lysosomes. The results provide evidence that INCL represents a novel lysosomal enzyme deficiency. Further, the defect in the PPT gene causing a neurodegenerative disorder suggests that depalmitoylation of the still uncharacterized substrate(s) for PPT is critical for postnatal development or maintenance of cortical neurons.
Subject(s)
Lysosomes/enzymology , Neuronal Ceroid-Lipofuscinoses/enzymology , Thiolester Hydrolases/metabolism , Amidohydrolases , Animals , Biological Transport , COS Cells , Endocytosis , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/metabolism , Glycosylation , Hexosaminidases , Humans , Lysosomal Storage Diseases/enzymology , Mannose/analysis , Mannose/metabolism , Molecular Weight , Mutation , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Peptides/metabolism , Phosphorylation , Receptor, IGF Type 2/physiology , Thiolester Hydrolases/analysis , Thiolester Hydrolases/chemistry , Thiolester Hydrolases/geneticsSubject(s)
Chromosome Aberrations/genetics , Chromosomes, Human, Pair 13 , Chromosomes, Human, Pair 16 , Neuronal Ceroid-Lipofuscinoses/genetics , Neuronal Ceroid-Lipofuscinoses/therapy , Adolescent , Adult , Child , Child, Preschool , Gene Expression , Humans , In Vitro Techniques , Infant , Infant, Newborn , Neuronal Ceroid-Lipofuscinoses/diagnosis , PrognosisABSTRACT
We have previously characterized intrachromosomal rearrangements at 1p32 fusing the first exon of the RLF gene with L-myc. Here we present the full-length cDNA sequence of the 6251 bp RLF mRNA. The predicted 1914 amino acid Rlf protein contains sixteen widely spaced zinc finger motifs, and is related to the Zn-15 transcription factor. RLF is widely expressed in fetal and adult tissues, suggesting that it has a general role in transcriptional regulation. The zinc fingers are not contained in the 79 amino acid N-terminal region of RLF involved in the RLF-L-myc fusions, and the transforming ability of the RLF-L-myc and the normal L-myc proteins is indistinguishable. These findings suggest that the role of the rearrangements fusing RLF and L-myc is to deregulate the tightly controlled expression of the L-myc gene.
Subject(s)
DNA-Binding Proteins/genetics , Gene Rearrangement , Genes, myc , Trans-Activators/genetics , Transcription Factors/genetics , Zinc Fingers , Adult , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Complementary/chemistry , DNA-Binding Proteins/physiology , Guanine Nucleotide Exchange Factors , Humans , Molecular Sequence Data , RNA, Messenger/analysis , Trans-Activators/physiologyABSTRACT
FISH techniques have opened new possibilities for high-resolution genome mapping. Effective utilization of these techniques for the rapid orientation and ordering of adjacent and overlapping probes as well as for the characterization of long-range genomic contigs would facilitate physical mapping and positional cloning efforts. Here, we have evaluated our recently developed improved fiber-FISH technique for the physical mapping of a 500-kb region at 1p32 as well as for the detection of genomic rearrangement affecting this region. Our fiber-FISH technique is based on the hybridization of probes to unfixed linearized DNA fibers on a microscope slide. Preparation of the target DNA from cells embedded in pulsed-field gel electrophoresis (PFGE) blocks makes it possible to obtain long intact DNA fibers that give an excellent signal-to-noise ratio in FISH. The linear range of the method reached from 2 to 500 kb with a measuring accuracy approaching that of PFGE. Fiber-FISH was used to establish the order, orientation, and distances for several probes for this region, including six large insert phage, cosmid, and P1 clones and seven genomic subclones. This has significantly facilitated our efforts to develop a genomic contig for this region, recently discovered to contain the gene for inherited neuronal ceroid lipofuscinosis (INCL). Finally, we also demonstrated how rearrangements affecting the L-myc gene at this locus in small-cell lung cancer can be visualized with fiber-FISH. In conclusion, fiber-FISH is very useful for high-resolution physical mapping and contig evaluation as well as for detecting genetic rearrangements.
Subject(s)
Chromosome Mapping/methods , Collagen/genetics , Fiber Optic Technology , Genes, myc/genetics , In Situ Hybridization, Fluorescence/methods , Transcription Factors/genetics , Carcinoma, Small Cell/genetics , Carcinoma, Small Cell/pathology , Chromosomes, Human, Pair 1 , DNA/chemistry , DNA Probes , Electrophoresis, Gel, Pulsed-Field , Gene Rearrangement , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Tumor Cells, CulturedABSTRACT
When used with metaphase chromosomes, fluorescence in situ hybridization (FISH) makes it possible to localize probes to individual chromosome bands and to establish the order of probes separated by > or = 2-3 Mb in dual-color hybridizations. We evaluated the use of mechanically stretched chromosomes as hybridization targets for increased mapping resolution. Mapping resolution was tested by pair-wise hybridizations with probes from the 1p32-p33 region, spanning distances from 20 to approximately 1500 kb. Probes separated by > or = 170 kb could be ordered relative to one another and to the centromere-telomere axis of the chromosome. The advantages of the technique are the simple procedure for preparing the slides, the straightforward interpretation of the results, and the ability to score the predominant order from < 10 stretched chromosomes. However, because of the variability of stretching from one sample to another, the calculation of actual physical distances between probes is not possible. To illustrate the utility of this method, we showed that the gene for receptor tyrosine kinase TIE lies centromeric to COL9A2, RLF, and L-MYC genes at 1p32. The use of mechanically stretched chromosomes provides < or = 10-fold increased mapping resolution as compared with conventional metaphase FISH. Thus, the technique effectively bridges the gap between metaphase mapping and ultra-high-resolution mapping (1-300 kb) techniques, such as the DNA fiber FISH.
Subject(s)
Chromosome Mapping/methods , Chromosomes, Human/ultrastructure , In Situ Hybridization, Fluorescence/methods , Adult , Centrifugation , Chromosome Mapping/instrumentation , Chromosomes, Human/drug effects , Chromosomes, Human/genetics , Chromosomes, Human, Pair 1/drug effects , Chromosomes, Human, Pair 1/genetics , Chromosomes, Human, Pair 1/ultrastructure , Cosmids/genetics , Cosmids/ultrastructure , Humans , Hypotonic Solutions/pharmacology , Metaphase , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cell Surface/genetics , Receptors, TIE , Sensitivity and Specificity , Stress, MechanicalABSTRACT
Neuronal ceroid lipofuscinoses (NCL) represent a group of common progressive encephalopathies of children which have a global incidence of 1 in 12,500. These severe brain diseases are divided into three autosomal recessive subtypes, assigned to different chromosomal loci. The infantile subtype of NCL (INCL), linked to chromosome 1p32, is characterized by early visual loss and rapidly progressing mental deterioration, resulting in a flat electroencephalogram by 3 years of age; death occurs at 8 to 11 years, and characteristic storage bodies are found in brain and other tissues at autopsy. The molecular pathogenesis underlying the selective loss of neurons of neocortical origin has remained unknown. Here we report the identification, by positional candidate methods, of defects in the palmitoyl-protein thioesterase gene in all 42 Finnish INCL patients and several non-Finnish patients. The most common mutation results in intracellular accumulation of the polypeptide and undetectable enzyme activity in the brain of patients.
Subject(s)
Mutation , Neuronal Ceroid-Lipofuscinoses/genetics , Palmitoyl-CoA Hydrolase/genetics , Amino Acid Sequence , Base Sequence , Brain/enzymology , Chromosomes, Human, Pair 1 , DNA Primers , Humans , Lymphocytes/enzymology , Molecular Sequence Data , Neuronal Ceroid-Lipofuscinoses/enzymology , Restriction MappingABSTRACT
Infantile neuronal ceroid lipofuscinosis (INCL, CLN1) is a neurodegenerative disorder in which the biochemical defect is unknown. We earlier assigned the disease locus to chromosome 1p32 in the immediate vicinity of the highly informative HY-TM1 marker by linkage and linkage disequilibrium analysis. Here we report the construction of PFGE maps on the CLN1 region covering a total of 4 Mb of this relatively poorly mapped chromosomal region. We established the order of loci at 1p32 as tel-D1S57-L-myc-HY-TM1-rlf-COL9A2-D1S193-D1S6 2-D1S211-cen by combining data obtained from analysis of a chromosome 1 somatic cell hybrid panel, PFGE, and interphase FISH. We isolated YACs and constructed two separate YAC contigs, the loci L-myc, HY-TM1, rlf, and COL9A2 being present on a 1000-kb contig and the markers D1S193, D1S62, and D1S211 on a YAC contig spanning a maximum of 860 kb. Within the 1000-kb contig we were able to identify five CpG islands in addition to those associated with the earlier cloned genes. The YAC contigs as well as the physical map provide us with tools for the identification of the INCL gene.
Subject(s)
Chromosomes, Artificial, Yeast , Chromosomes, Human, Pair 1 , Neuronal Ceroid-Lipofuscinoses/genetics , Base Sequence , Chromosome Mapping , Electrophoresis, Gel, Pulsed-Field , Genetic Markers , Humans , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
Fluorescence in situ hybridization (FISH) and pulse field gel electrophoresis (PFGE) are essential techniques in physical mapping and in positional cloning. We present a technique that utilizes agarose-embedded high molecular weight DNA prepared for PFGE as a target for FISH. The agarose blocks are melted, and the DNA is extended on a poly-L-lysine-coated microscope slide. The resulting DNA fibers appear on the slide as long straight strands and are a suitable target for high resolution FISH mapping as demonstrated here with cosmid and plasmid hybridizations.
Subject(s)
Chromosome Mapping/methods , DNA/metabolism , In Situ Hybridization, Fluorescence , Sepharose , Cosmids , Electrophoresis, Gel, Pulsed-Field , Humans , Lymphocytes/chemistry , Microscopy, Fluorescence , Plasmids , Polylysine , Restriction MappingABSTRACT
In the positional cloning of a disease gene with an unknown protein defect a spectrum of molecular biological methods is applied after linkage has been established. It is reasonable to analyze in detail any relevant candidate gene mapping to the particular chromosomal region. We report here the refined chromosomal assignment of SCPx/SCP2, a gene coding for the protein that is believed to have an important role in lipid metabolism by transporting many kinds of lipid molecules between organelles. This gene represents an excellent candidate gene for infantile neuronal ceroid lipofuscinosis, earlier assigned to 1p32 by us, since lipid metabolism in the patient's brain appears to be significantly disturbed. Expression and structural analyses by Northern, Southern and Western blotting as well as SSCP and direct sequencing did not detect any differences between the cDNAs of patients and controls. Consequently, it is unlikely that a mutation in SCPx/SCP2 is the underlying cause of this severe neurodegenerative disease of childhood.
Subject(s)
Acetyl-CoA C-Acetyltransferase , Carrier Proteins/genetics , Chromosomes, Human, Pair 1 , Genes , Neuronal Ceroid-Lipofuscinoses/genetics , Plant Proteins , Chromosome Mapping , Humans , In Situ Hybridization, Fluorescence , Polymorphism, GeneticABSTRACT
The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in neurons and other cell types. Inheritance is autosomal recessive. Three main childhood subtypes are recognized: infantile (Haltia-Santavuori disease; MIM 256743), late infantile (Jansky-Bielschowsky disease; MIM 204500), and juvenile (Spielmeyer-Sjögren-Vogt, or Batten, disease; MIM 204200). The gene loci for the juvenile (CLN3) and infantile (CLN1) types have been mapped to human chromosomes 16p and 1p, respectively, by linkage analysis. Linkage analysis of 25 families segregating for late-infantile NCL has excluded these regions as the site of this disease locus (CLN2). The three childhood subtypes of NCL therefore arise from mutations at distinct loci.
Subject(s)
Alleles , Neuronal Ceroid-Lipofuscinoses/genetics , Child, Preschool , Chromosomes, Human, Pair 1 , Chromosomes, Human, Pair 16 , Female , Genetic Linkage , Humans , Infant , Male , Neuronal Ceroid-Lipofuscinoses/classification , Tripeptidyl-Peptidase 1ABSTRACT
Infantile neuronal ceroid lipofuscinosis, INCL, CLN1, is an autosomally inherited progressive neurogenerative disorder. The disease results in the massive death of cortical neurons, suggesting an essential role for the CLN1 gene product in the normal neuronal maturation during the first years of life. Identification of new multiallelic markers has now made possible the construction of a refined genetic map encompassing the CLN1 locus at 1p32. Strong allelic association was detected with a new, highly polymorphic HY-TM1 marker. We incorporated this observed linkage disequilibrium into multipoint linkage analysis, which significantly increased the informativeness of the limited family material and facilitated refined assignment of the CLN1 locus.
Subject(s)
Chromosomes, Human, Pair 1 , Neuronal Ceroid-Lipofuscinoses/genetics , Base Sequence , DNA , Genetic Markers , Humans , Linkage Disequilibrium , Molecular Sequence DataABSTRACT
The infantile form of neuronal ceroid lipofuscinosis (INCL) is a progressive encephalopathy in children < 2 years old. The disease is one of the Finnish diseases, enriched in this genetically isolated population. The gene responsible for INCL has been recently assigned to the short arm of human chromosome 1. Here we describe DNA-based prenatal and carrier diagnostics using a highly polymorphic marker (HY-TM1) which demonstrates a strong allelic association to the disease locus. 88% of Finnish INCL patients were observed to have the same affected genotype, suggesting that one major CLN1 mutation is enriched in this population. In contrast, all the non-Finnish INCL patients had different allele combinations.
Subject(s)
Carrier State/diagnosis , Neuronal Ceroid-Lipofuscinoses/genetics , Prenatal Diagnosis , Alleles , Base Sequence , Female , Finland , Genetic Markers , Humans , Linkage Disequilibrium , Male , Molecular Sequence Data , Neuronal Ceroid-Lipofuscinoses/diagnosis , Polymerase Chain Reaction , Polymorphism, Genetic , Polymorphism, Restriction Fragment LengthABSTRACT
This overview describes recent advances in molecular biology of neuronal ceroid lipofuscinoses (CLN). Despite intensive research during last 20 years, the basic defects of these autosomal recessive-progressive encephalopathies of childhood remain unknown. Consequently, no specific cure is available. Methods of positional cloning (reverse genetics) starting from random linkage approach have been applied to search for gene defects in the infantile and juvenile forms of the disease. The results of this random search for disease loci have for the first time revealed molecular heterogeneity of CLN diseases. The gene defect causing the infantile form has been assigned to 1p32 in the Finnish family material, whereas the disease locus of the juvenile form has been localized to 16p12 in European and Canadian families. Finally, the gene defect causing the late infantile form has been excluded from both 1p32 and 16p12 chromosomal regions, referring to a third, still unknown locus causing CLN disease. Consequently, reliable prenatal and carrier diagnostics have now become possible in families with the infantile and juvenile forms of the disease, and DNA-based prenatal diagnostics have been successfully applied in the infantile form. Most importantly, the assignment of gene loci has brought these fatal brain diseases within the reach of molecular cloning strategies that eventually will result in revealing both the infantile and juvenile CLN genes and in identifying corresponding gene products.
Subject(s)
Neuronal Ceroid-Lipofuscinoses/genetics , Child , Child, Preschool , Chromosome Mapping , Chromosomes, Human, Pair 1 , Chromosomes, Human, Pair 16 , Diagnosis, Differential , Female , Genetic Carrier Screening , Humans , Infant , Molecular Biology , Neuronal Ceroid-Lipofuscinoses/classification , Neuronal Ceroid-Lipofuscinoses/diagnosis , Pregnancy , Prenatal DiagnosisABSTRACT
In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin beta and profilin-actin gamma. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg2+, and can be crystallized. From the isolated profilin-actin complexes the beta- and gamma-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin.