Ovule-specific MADS-box proteins have conserved protein-protein interactions in monocot and dicot plants

OsMADS13 is a rice MADS-box gene that is specifically expressed in developing ovules. The amino acid sequence of OsMADS13 shows 74% similarity to those of FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, the products of two MADS-box genes that are necessary and sufficient to determine ovule identity in Petunia. To assess whether OsMADS13, the putative rice ortholog of FBP7 and FBP11, has an equivalent function, several analyses were performed. Ectopic expression of FBP7 and FBP11 in Petunia results in ectopic ovule formation on sepals and petals. Here we show that ectopic expression of OsMADS13 in rice and Arabidopsis does not result in the formation of such structures. Furthermore, ectopic expression of FBP7 and FBP11 in Arabidopsis also fails to induce ectopic ovule formation. To determine whether protein-protein interactions involving putative class D MADS-box proteins have been conserved, yeast two-hybrid assays were performed. These experiments resulted in the identification of three putative partners of OsMADS13, all of them encoded by AGL2-like genes. Interestingly the Petunia FBP7 protein also interacts with AGL2-like proteins. The evolutionary conservation of the MADS-box protein partners of these ovule-specific factors was confirmed by exchange experiments which showed that the protein partners of OsMADS13 interact with FBP7 and vice versa.     

Autistic disorder and chromosome 15q11-q13: construction and analysis of a BAC/PAC contig

Autistic disorder (AD) is a neurodevelopmental disorder that affects approximately 2-10/10,000 individuals. Chromosome 15q11-q13 has been implicated in the genetic etiology of AD based on (1) cytogenetic abnormalities; (2) increased recombination frequency in this region in AD versus non-AD families; (3) suggested linkage with markers D15S156, D15S219, and D15S217; and (4) evidence for significant association with polymorphisms in the gamma-aminobutyric acid receptor subunit B3 gene (GABRB3). To isolate the putative 15q11-q13 candidate AD gene, a genomic contig and physical map of the approximately 1.2-Mb region from the GABA receptor gene cluster to the OCA2 locus was generated. Twenty-one bacterial artificial chromosome (BAC) clones, 32 P1-derived artificial chromosome (PAC) clones, and 2 P1 clones have been isolated using the markers D15S540, GABRB3, GABRA5, GABRG3, D15S822, and D15S217, as well as 34 novel markers developed from the end sequences of BAC/PAC clones. In contrast to previous findings, the markers D15S822 and D15S975 have been localized within the GABRG3 gene, which we have shown to be approximately 250 kb in size. NotI and numerous EagI restriction enzyme cut sites were identified in this region. The BAC/PAC genomic contig can be utilized for the study of genomic structure and the identification and characterization of genes and their methylation status in this autism candidate gene region on human chromosome 15q11-q13.     

Molecular analysis of 24 Alagille syndrome families identifies a single submicroscopic deletion and further localizes the Alagille region within 20p12.

Alagille syndrome (AGS) is a clinically defined disorder characterized by cholestatic liver disease with bile duct paucity, peculiar facies, structural heart defects, vertebral anomalies, and ocular abnormalities. Multiple patients with various cytogenetic abnormalities involving 20p12 have been identified, allowing the assignment of AGS to this region. The presence of interstitial deletions of varying size led to the hypothesis that AGS is a contiguous gene deletion syndrome. This molecular analysis of cytogenetically normal AGS patients was performed in order to test this hypothesis and to refine the localization of the known AGS region. Investigation of inheritance of simple tandem repeat polymorphism alleles in 67 members of 24 cytogenetically normal Alagille families led to the identification of a single submicroscopic deletion. The deletion included loci D20S61, D20S41, D20S186, and D20S188 and presumably intervening uninformative loci D20S189 and D20S27. The six deleted loci are contained in a single YAC of 1.9 Mb. The additional finding of multiple unrelated probands who are heterozygous at each locus demonstrates that microdeletions at known loci within the AGS region are rare in cytogenetically normal patients with this disorder. This suggests that the majority of cases of AGS may be the result of a single gene defect rather than a contiguous gene deletion syndrome.     

Autistic Disorder and Chromosome 15q11–q13: Construction and Analysis of a BAC/PAC Contig

Autistic disorder (AD) is a neurodevelopmental disorder that affects approximately 2–10/10,000 individuals. Chromosome 15q11–q13 has been implicated in the genetic etiology of AD based on (1) cytogenetic abnormalities; (2) increased recombination frequency in this region in AD versus non-AD families; (3) suggested linkage with markers D15S156, D15S219, and D15S217; and (4) evidence for significant association with polymorphisms in the γ-aminobutyric acid receptor subunit B3 gene (GABRB3). To isolate the putative 15q11–q13 candidate AD gene, a genomic contig and physical map of the approximately 1.2-Mb region from the GABA receptor gene cluster to the OCA2 locus was generated. Twenty-one bacterial artificial chromosome (BAC) clones, 32 P1-derived artificial chromosome (PAC) clones, and 2 P1 clones have been isolated using the markers D15S540, GABRB3, GABRA5, GABRG3, D15S822, and D15S217, as well as 34 novel markers developed from the end sequences of BAC/PAC clones. In contrast to previous findings, the markers D15S822 and D15S975 have been localized within the GABRG3 gene, which we have shown to be approximately 250 kb in size. NotI and numerous EagI restriction enzyme cut sites were identified in this region. The BAC/PAC genomic contig can be utilized for the study of genomic structure and the identification and characterization of genes and their methylation status in this autism candidate gene region on human chromosome 15q11–q13.     

Homozygosity mapping, to chromosome 11p, of the gene for familial persistent hyperinsulinemic hypoglycemia of infancy.

Familial persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a rare, autosomal recessive disease of unregulated insulin secretion, defined by elevations in serum insulin despite severe hypoglycemia. We used the homozygosity gene-mapping strategy to localize this disorder to the region of chromosome 11p between markers D11S1334 and D11S899 (maximum LOD score 5.02 [theta = 0] at marker D11S926) in five consanguineous families of Saudi Arabian origin. These results extend those of a recent report that also placed PHHI on chromosome 11p, between markers D11S926 and D11S928. Comparison of the boundaries of these two overlapping regions allows the PHHI locus to be assigned to the 4-cM region between the markers D11S926 and D11S899. Identification of this gene may allow a better understanding of other disorders of glucose homeostasis, by providing insight into the regulation of insulin release.     

Autosomal recessive familial exudative vitreoretinopathy is associated with mutations in LRP5

Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disorder that affects both the retina and vitreous body. Autosomal recessive FEVR was diagnosed in multiple individuals from three consanguineous families of European descent. A candidate-locus-directed genome scan shows linkage to the region on chromosome 11q flanked by markers D11S905 and D11S1314. The maximum LOD score of 3.6 at theta =0 is obtained with marker D11S987. Haplotype analysis confirms that the critical region is the 22-cM (311-Mb) interval flanked by markers D11S905 and D11S1314. This region contains LRP5 but not FZD4; mutations in both of these genes cause autosomal dominant FEVR. Sequencing of LRP5 shows, in all three families, homozygous mutations R570Q, R752G, and E1367K. This suggests that mutations in this gene can cause autosomal recessive as well as autosomal dominant FEVR.     

Refinement of the Multiple Exostoses Locus (EXT2) to a 3-cM Interval on Chromosome 11

Hereditary multiple exostoses (EXT) is an autosomal dominant skeletal disorder characterized by the formation of multiple exostoses on the long bones. EXT is genetically heterogeneous, with at least three loci involved: one (EXT1) in the Langer-Giedion region on 8q23-q24, a second (EXT2) in the pericentromeric region of chromosome 11, and a third (EXT3) on chromosome 19p. In this study, linkage analysis in seven extended EXT families, all linked to the EXT2 locus, refined the localization of the EXT2 gene to a 3-cM region flanked by D11S1355 and D11S1361/D11S554. This implies that the EXT2 gene is located at the short arm of chromosome 11, in band 11p11-p12. The refined localization of EXT2 excludes a number of putative candidate genes located in the pericentromeric region of chromosome 11 and facilitates the process of isolating the EXT2 gene.     

Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3'-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200,000 U/mg), but was of lower Mr (23,000) than human erythropoietin produced in COS cells (30,000) or purified from urine (30,000 to 38,000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosaccharides from this Mr 23,000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18,000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.     

Temporal and Spatial Expression of MADS Box Genes, FBP7 and FBP11, During Initiation and Early Development of Ovules in Wild Type and Mutant Petunia hybrida

Abstract: The temporal and spatial distribution of the Petunia Floral Binding Proteins 7 and 11 (FBP7/11) were determined immunocytochemically during ovule initiation and development. In wild type plants, FBP7/11 were first detected in the placenta before ovule primordia were formed. At ovule primordium stage, FBP7/11 levels increased in the placenta and appeared in ovule primordia at the sites where integument primordia developed. At the megagametogenesis stage, FBP7/11 were present at high levels in the placenta, funicle and integument, but not in the nucellus or gametophyte. Transgenics with cosuppression of FBP7/11 formed normal ovule primordia on the placenta from which both normal ovules and carpel-like structures developed. The amount of FBP7/11 was low in the ovules and undetectable in the carpel-like structures. Plants with ectopic expression of FBP7/11 developed normal ovules on the placenta and, in addition, ovule- and carpel-like structures on sepals. Placental and sepal ovules showed the same labeling pattern as observed in wild type ovules. FBP7/11 levels were, however, low or undetectable in the carpel-like structures. The results indicate that FBP7/11 only have indirect roles in ovule primordium initiation. However, at least small quantities are needed for proper ovule differentiation. Thus, the amount of FBP7/11 is related to the type of development after primordium formation, i.e., towards the formation of real ovules or carpel-like structures.     

Linkage disequilibrium at the Angelman syndrome gene UBE3A in autism families.

Autistic disorder is a neurodevelopmental disorder with a complex genetic etiology. Observations of maternal duplications affecting chromosome 15q11-q13 in patients with autism and evidence for linkage and linkage disequilibrium to markers in this region in chromosomally normal autism families indicate the existence of a susceptibility locus. We have screened the families of the Collaborative Linkage Study of Autism for several markers spanning a candidate region covering approximately 2 Mb and including the Angelman syndrome gene (UBE3A) and a cluster of gamma-aminobutyric acid (GABA(A)) receptor subunit genes (GABRB3, GABRA5, and GABRG3). We found significant evidence for linkage disequilibrium at marker D15S122, located at the 5' end of UBE3A. This is the first report, to our knowledge, of linkage disequilibrium at UBE3A in autism families. Characterization of null alleles detected at D15S822 in the course of genetic studies of this region showed a small (approximately 5-kb) genomic deletion, which was present at somewhat higher frequencies in autism families than in controls.     

A major locus for myoclonus-dystonia maps to chromosome 7q in eight families

Myoclonus-dystonia (M-D) is an autosomal dominant disorder characterized by myoclonic and dystonic muscle contractions that are often responsive to alcohol. The dopamine D2 receptor gene (DRD2) on chromosome 11q has been implicated in one family with this syndrome, and linkage to a 28-cM region on 7q has been reported in another. We performed genetic studies, using eight additional families with M-D, to assess these two loci. No evidence for linkage was found for 11q markers. However, all eight of these families showed linkage to chromosome 7 markers, with a combined multipoint LOD score of 11.71. Recombination events in the families define the disease gene within a 14-cM interval flanked by D7S2212 and D7S821. These data provide evidence for a major locus for M-D on chromosome 7q21.     

Linkage of the gene for an autosomal dominant form of juvenile amyotrophic lateral sclerosis to chromosome 9q34.

We performed genetic mapping studies of an 11-generation pedigree with an autosomal dominant, juvenile-onset motor-systems disease. The disorder is characterized by slow progression, distal limb amyotrophy, and pyramidal tract signs associated with severe loss of motor neurons in the brain stem and spinal cord. The gene for this disorder, classified as a form of juvenile amyotrophic lateral sclerosis (ALS), is designated "ALS4." We performed a genomewide search and detected strong evidence for linkage of the ALS4 locus to markers from chromosome 9q34. The highest LOD score (Z) was obtained with D9S1847 (Z=18.8, recombination fraction of .00). An analysis of recombinant events identified D9S1831 and D9S164 as flanking markers, on chromosome 9q34, that define an approximately 5-cM interval that harbors the ALS4 gene. These results extend the degree of heterogeneity within familial ALS syndromes, and they implicate a gene on chromosome 9q34 as critical for motor-neuron function.     

A gene for autosomal dominant hypohidrotic ectodermal dysplasia (EDA3) maps to chromosome 2q11-q13.

Autosomal dominant hypohidrotic ectodermal dysplasia (ADHED) is a disorder characterized by fine, slow-growing scalp and body hair, sparse eyebrows and eyelashes, decreased sweating, hypodontia, and nail anomalies. By genetic linkage analysis of a large ADHED kindred, we have mapped a gene for ADHED (EDA3) to the proximal long arm of chromosome 2 (q11-q13). Obligate recombinations localize EDA3 to an approximately 9-cM interval between D2S1321 and D2S308, with no apparent recombinations with markers D2S1343, D2S436, D2S293, D2S1894, D2S1784, D2S1890, D2S274, and CHLC.GAAT11C03.     

The isolation and properties of multiple forms of folate binding protein in cultured KB cells

The folate binding proteins (FBPs) of KB cells which were cultured in normal (N) and folate-deficient (D) medium have been characterized. The 200,000 g supernate of lysed cells contained two FBPs which could be separated by DEAE-Bio-Gel A chromatography, indicating that they differ in ionic charge although they could not be separated by gel filtration through Sephadex G-100 (apparent Mr approximately 40,000). Two species of FBP, a major form of apparent Mr approximately 160,000 and a minor form of apparent Mr approximately 40,000, were identified by gel filtration through Sephadex G-150 in the membrane component of the cells after solubilization with Triton X-100. An additional FBP was isolated and purified by affinity chromatography from the medium in which these cells were cultured. By gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent Mr of this FBP was approximately 44,000. The association constants for pteroylglutamic acid of the FBPs in the 200,000 g cell lysate supernate, culture medium, and Triton-solubilized membrane were similar and the relative affinity of folate analogs for the FBP, vis-à-vis pteroylglutamic acid, was similar for all species. An antiserum raised to the purified FBP from the culture medium precipitated the FBPs in the 200,000 g cell lysate supernate, Triton-solubilized membrane, and culture medium, indicating antigenic homology among these FBPs. There was no unsaturated FBP in the 200,000 g cell lysate supernate or medium when KB cells were cultured in N medium. However, when cells were cultured in D medium, the unsaturated FBP of the 200,000 g cell supernate and culture medium was substantial (9.2 and 14.1 pmol/mg protein, respectively). Unsaturated FBP was detected in the membrane of normal cells but this also increased when these cells were cultured in D medium (4.5 to 756 pmol/mg protein), indicating that the FBPs of these cellular compartments are normally saturated by folate. After 16 weeks of culture in D medium, the total folate binding capacity of the membrane-associated FBP was twofold greater than that of normal KB cells, indicating the induction of FBP.     

Osteoporosis-pseudoglioma syndrome, a disorder affecting skeletal strength and vision, is assigned to chromosome region 11q12-13.

Osteoporosis-pseudoglioma syndrome (OPS) is an autosomal recessive disorder characterized by severe juvenile-onset osteoporosis and congenital or juvenile-onset blindness. The pathogenic mechanism is not known. Clinical, biochemical, and microscopic analyses suggest that OPS may be a disorder of matrix homeostasis rather than a disorder of matrix structure. Consequently, identification of the OPS gene and its protein product could provide insights regarding common osteoporotic conditions, such as postmenopausal and senile osteoporosis. As a first step toward determining the cause of OPS, we utilized a combination of traditional linkage analysis and homozygosity mapping to assign the OPS locus to chromosome region 11q12-13. Mapping was accomplished by analyzing 16 DNA samples (seven affected individuals) from three different consanguineous kindreds. Studies in 10 additional families narrowed the candidate region, supported locus homogeneity, and did not detect founder effects. The OPS locus maps to a 13-cM interval between D11S1298 and D11S971 and most likely lies in a 3-cM region between GSTP1 and D11S1296. At present, no strong candidate genes colocalize with OPS.     

Role of Ser-65 in the activity of alpha-galactosidase A: characterization of a point mutation (S65T) detected in a patient with Fabry disease

Fabry disease is a genetic disorder caused by deficient activity of alpha-galactosidase A (alpha-Gal A). Recent gene analysis of a Fabry patient revealed a point mutation (S65T) resulting in a significant decrease of enzyme activity (Chen, C.-H., et al. (1998) Hum. Mutat. 11, 328-330). In order to evaluate the role of Ser-65 in the alpha-Gal A activity and the molecular mechanism of its deficient enzyme activity in mammalian cells, we prepared gene products of S65T, S65A, and E66D mutations of alpha-Gal A by using an expression system with baculovirus/insect cells and characterized the kinetic and physical properties of those purified enzymes. The Km values of mutant enzymes were 3.5 (S65T), 3.4 (S65A), and 2.3 mM (E66D), using 4-methylumbelliferyl alpha-D-galactoside as a substrate, and the Vmax values were 2.7 x 10(6) (S65T), 3.4 x 10(6) (S65A), and 2.5 x 10(6) units/mg (E66D), respectively, which were similar to those of the normal enzyme (Km, 2.3 mM; Vmax, 2.3 x 10(6) units/mg). The in vitro stability of mutant enzymes at neutral pH was significantly reduced (S65T, 4% of normal; S65A, 29%; E66D, 54%). The intracellular alpha-Gal A activities of S65T, S65A, and E66D in COS1 cells transfected with corresponding plasmid DNAs were markedly lower than the normal enzyme activity (9, 26, and 68% of normal, respectively). However, intracellular enzyme activities were enhanced to 34% (S65T), 44% (S65A), and 80% (E66D) of normal, respectively, by cultivation of the cells with 20 microM 1-deoxygalactonojirimycin (a potent inhibitor of alpha-Gal A) for 24 h. These results suggest that Ser-65 is responsible for the stability of alpha-Gal A but not for the enzyme function.     

The human cationic amino acid transporter (ATRC1): physical and genetic mapping to 13q12-q14.

The product of the mouse Rec-1 locus is an integral membrane protein that determines susceptibility to infection by murine ecotropic retroviruses. Recently it has been determined that its role in normal cell metabolism is transport of the cationic amino acids, arginine, lysine, and ornithine across the plasma membrane. Southern blot analysis of genomic DNA from a panel of 48 mouse-human somatic cell hybrids assigned the human version of this gene, ATRC1, to chromosome 13. Chromosomal in situ hybridization localized the gene to 13q12-q14. A restriction fragment length polymorphism (RFLP) was detected with TaqI. There were two alleles with frequencies of 0.29 and 0.71. Pairwise linkage analysis established linkage between ATRC1 and ATP1AL1, D13S1, D13S6, D13S10, D13S11, D13S21, D13S22, D13S33, D13S36, and D13S37. Multilocus linkage analysis of five of the loci indicated that the most likely order of loci in this region was D13S11-ATP1AL1-ATRC1-D13S6-D13S33.     

Autosomal dominant nanophthalmos (NNO1) with high hyperopia and angle-closure glaucoma maps to chromosome 11.

Nanophthalmos is an uncommon developmental ocular disorder characterized by a small eye, as indicated by short axial length, high hyperopia (severe farsightedness), high lens/eye volume ratio, and a high incidence of angle-closure glaucoma. We performed clinical and genetic evaluations of members of a large family in which nanophthalmos is transmitted in an autosomal dominant manner. Ocular examinations of 22 affected family members revealed high hyperopia (range +7.25-+13.00 diopters; mean +9.88 diopters) and short axial length (range 17.55-19.28 mm; mean 18.13 mm). Twelve affected family members had angle-closure glaucoma or occludable anterior-chamber angles. Linkage analysis of a genome scan demonstrated highly significant evidence that nanophthalmos in this family is the result of a defect in a previously unidentified locus (NNO1) on chromosome 11. The gene was localized to a 14.7-cM interval between D11S905 and D11S987, with a maximum LOD score of 5. 92 at a recombination fraction of .00 for marker D11S903 and a multipoint maximum LOD score of 6.31 for marker D11S1313. NNO1 is the first human locus associated with nanophthalmos or with an angle-closure glaucoma phenotype, and the identification of the NNO1 locus is the first step toward the cloning of the gene. A cloned copy of the gene will enable examination of the relationship, if any, between nanophthalmos and less severe forms of hyperopia and between nanophthalmos and other conditions in which angle-closure glaucoma is a feature.