Assignment of fibulin-5 (FBLN5) to human chromosome 14q31 by in situ hybridization and radiation hybrid mapping.
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Calcium-Binding Proteins↗
Biomedical subjects
R A Schultz
Publications and source records attributed to R A Schultz.
Neurotoxicity in calves induced by the plant, Nierembergia hippomanica Miers var. violacea Millán in South Africa.
The plant Nierembergia hippomanica var. violacea has been incriminated in field outbreaks of neurotoxicity in calves in the Free State Province. Hepatotoxicity and electrocardiogram (ECG) deviations were induced in a sheep dosed with 5 g/kg dried plant material on four consecutive days. A calf dosed with 2.5 g/kg dried plant material, on two consecutive days, did not show overt clinical changes. Voluntary ingestion of approximately 30 g/kg fresh flowering plants by a second calf resulted in nervous signs characterized by chewing motions, protrusion of the tongue, dysphagia, hypermetria, ataxia, paresis and lateral recumbency. Salivation, dehydration and cardiac irregularities completed the clinical picture. Clinical chemistry changes revealed muscle damage and increased serum urea and creatinine concentrations indicative of kidney involvement. This is the first confirmed outbreak of Nierembergia hippomanica var. violacea intoxication of stock in South Africa.
Animals↗
Differential expression of mitochondrial DNA replication factors in mammalian tissues.
Mitochondrial biogenesis and mitochondrial DNA (mtDNA) replication are regulated during development and in response to physiological stresses, but the regulatory events that control the abundance of mtDNA in cells of higher eukaryotes have not been defined at a molecular level. In this study, we observed that expression of the catalytic subunit of DNA polymerase gamma (POLgammaCAT) mRNA varies little among different tissues and is not increased by continuous neural activation of skeletal muscle, a potent stimulus to mitochondrial biogenesis. Increased copy number for the POLgamma locus in a human cell line bearing a partial duplication of chromosome 15 increased the abundance of POLgammaCAT mRNA without up-regulation of mtDNA. In contrast, expression of mitochondrial single-stranded DNA-binding (mtSSB) mRNA is regulated coordinately with variations in the abundance of mtDNA among tissues of mammalian organisms and is up-regulated in association with the enhanced mitochondrial biogenesis that characterizes early postnatal development of the heart and the adaptive response of skeletal myofibers to motor nerve stimulation. In addition, we noted that expression of mtSSB is concentrated within perinuclear mitochondria that constitute active sites of mtDNA replication. We conclude that constitutive expression of the gene encoding the catalytic subunit of mitochondrial DNA polymerase is sufficient to support physiological variations in mtDNA replication among specialized cell types, whereas expression of the mtSSB gene is controlled by molecular mechanisms acting to regulate mtDNA replication or stability in mammalian cells.
Animals↗
Krimpsiekte in a sheep following a single dose of Tylecodon ventricosus (Burm. f.) Toelken and the isolation of tyledoside D from this plant species.
Tylecodon ventricosus induced severe respiratory distress in two penned sheep without any electrocardiographic abnormalities being recorded. Based on the results it appears as if T. ventricosus predominantly induces the neuromuscular syndrome referred to as krimpsiekte. A single, relatively large intraruminal dose of 10.0 g/kg induced krimpsiekte in one sheep. Treatment with 5.0 g/kg activated charcoal on two consecutive days did not prevent the development of krimpsiekte. A bufadienolide, tyledoside D, was isolated from semi-dried plant material.
Animals↗
Structure and localization of the human gene encoding SR-BI/CLA-1. Evidence for transcriptional control by steroidogenic factor 1.
The scavenger receptor, class B, type 1 receptor (SR-BI) mediates the selective transport of lipids from high density lipoprotein to cells. We describe the structure and subchromosomal location of human SR-BI and provide evidence that it is regulated by the transcription factor, steroidogenic factor 1 (SF-1). SR-BI resides on chromosome 12q24.2-qter, spans approximately 75 kilobase pairs, and contains 13 exons. RNA blot analysis of human tissues reveals an expression pattern similar to that described previously for rodents with the highest levels of mRNA in the adrenal gland, ovary, and liver. Unlike rodents, human SR-BI was expressed at high levels in the placenta. The transcription start site for SR-BI was mapped, and DNA sequence analysis revealed a binding site for SF-1 in the proximal 5'-flanking sequence. SF-1, an orphan member of the nuclear hormone receptor gene family, plays a key role in the regulation of steroidogenesis and is expressed at high levels in steroidogenic tissues. SF-1 binds to the SR-BI promoter in a sequence-specific manner, and efficient transcription from this promoter in adrenocortical Y1 cells is dependent on an intact SF-1 site. These data extend our understanding of SF-1 function within steroidogenic tissues and suggest that SR-BI, which serves to supply selected tissues with lipoprotein-derived lipids, is part of the repertoire of SF-1-responsive genes involved in steroidogenesis.
Base Sequence↗
Genomic organization, chromosomal localization, and expression of the murine thromboxane synthase gene.
Thromboxane synthase (TS) is a membrane-bound cytochrome P450 enzyme catalyzing the synthesis of TxA2, a potent modulator of vascular smooth muscle contraction and platelet aggregation. TS plays an important role in hemostasis and may be intimately involved in the etiology of cardiovascular, renal, and immune diseases. Restriction enzyme mapping, subcloning, and DNA sequencing analysis of recombinant phage lambda and P1 clones revealed that exons encoding the 1.9-kb mouse TS mRNA are dispersed over >150 kb genomic DNA. Determination of the intron-exon splicing junctions established that the mouse TS gene (Tbxas1) is encoded by 13 exons ranging in size from 53 (exon III) to 315 bp (exon IX). Genomic Southern analysis and fluorescence in situ hybridization suggested that the gene is a single-copy gene, located on chromosome 6 near the midpoint between the centromere and the Igkappa gene. An alternatively spliced variant of the Tbxas1 transcript, lacking the exon XII-encoded sequence, has been detected in normal mouse tissues. Ribonuclease protection and 5'-RACE assays identified at least five major transcription start sites clustered within 31 bp of the Tbxas1 promoter. The 5'-most start site is not preceded by a TATA box, suggesting transcription can be initiated in a TATA-independent manner. Transfection analyses indicated that the expression of Tbxas1 is controlled by a short (70-bp) positive regulatory sequence and several upstream repressive elements. Mutational studies further demonstrated that NF-E2/AP-1 and Sp1 exerted activating and repressive, respectively, effects on the promoter. These studies provide the genetic tools and information for TS research in mice, which should expedite understanding of the genetic contribution of TS in normal physiology as well as in disease states.
Alternative Splicing↗
Construction and screening of a cosmid library generated from a somatic cell hybrid bearing human chromosome 15.
A cosmid library has been constructed with DNA isolated from a mouse/human hybrid cell line designated A15, which was previously characterized and shown to retain chromosome 15 as the only human material. The library was generated and stored as 34 independent pools of primary colonies at 8-10,000 colonies per pool. Screening colonies representing pools of this library by hybridization with a human-specific repetitive probe has facilitated the identification of random clones bearing human inserts. To data, 43 unique clones have been isolated and the inserts mapped by fluorescence in situ hybridization (FISH) to chromosome 15. An STS was generated for each of these clones by end sequencing of the inserts. Two of these clones, c36 and MR23, were mapped by FISH to 15q26.1, and end-sequence data revealed homology to different regions of the FES protooncogene. Sequence generated from the other end of the c36 insert was found to match the previously identified and unmapped coding sequence EST00075. In addition to the identification of such random clones, establishment of the library as pools was expected to facilitate the PCR-based identification of unique clones representing specific regions of chromosome 15. Screening of the library pools with primers specific to the FES region led to the recovery of five independent clones facilitating the development of a cosmid contig encompassing the FES gene. One of the cosmids isolated by PCR-based analysis was derived from the same pool as M23 and was subsequently shown to be identical to M23. The data offer the first report of a chromosome 15 cosmid library and demonstrate the value of utilizing pools to evaluate libraries generated from complex sources like somatic cell hybrids.
Animals↗
Confirmation of homozygosity for a single nucleotide substitution mutation in a Cockayne syndrome patient using monoallelic mutation analysis in somatic cell hybrids.
The identification of individuals homozygous for a specific mutation offers advantages for the elucidation of molecular mechanisms of hereditary disease states. Cockayne syndrome is a rare autosomal recessive disorder, the molecular basis of which is complicated by significant genetic and clinical heterogeneity. The genes associated with both genetic complementation groups, CSA and CS-B, have been identified. We have previously identified a number of CSA mutations, including a single base substitution that introduces a stop codon (322Tyr-->Stop) mutation in the C-terminal region for at least one allele of the CSA gene in a severely affected patient. We now present data confirming the existence of homozygosity in this patient using a strategy with general applicability. Somatic cell hybrids were established by fusing patient cells with mouse A9 cells. Screening with chromosome 5 specific polymorphic markers facilitated identification of hybrid clones bearing only one of the distinct CSA alleles. Sequencing of a portion of the human CSA gene in a subset of these hybrids permitted monoallelic mutation analysis and confirmed the presence of the 322Tyr-->Stop mutation in both alleles.
Alleles↗
Correction of the Bloom syndrome cellular phenotypes.
Bloom syndrome (BLM) is a genetic disorder associated with predisposition to cancer and chromosome instability. However, the most readily recognized clinical feature of the syndrome is growth retardation. Introduction of the previously cloned BLM gene into BLM cells yielded correction of the chromosome instability and slow growth phenotypes. Additionally, asynchronous cultures of complemented clones revealed a lower percentage of cells in S-phase than uncomplemented BLM cells. These results support the notion that BLM is a defect in which short stature, chromosome instability and cancer predisposition are all associated with an error in DNA replication.
Adenosine Triphosphatases↗
Krimpsiekte, associated with thalamic lesions, induced by the neurotoxic cardiac glycoside, cotyledoside, isolated from Tylecodon wallichii (Harv.) Toelken subsp. wallichii.
The specific neurotoxic principle of Tylecodon wallichii (Harv.) Toelken subsp. wallichii, the cause of krimpsiekte in small stock, was isolated and identified as the previously described cumulative bufadienolide, cotyledoside. Krimpsiekte was experimentally induced in two sheep by the repeated intravenous administration of cotyledoside at the rate of 0.01-0.015 mg/kg body mass. On day 9, both animals developed clinical signs typical of krimpsiekte, which is characterized by tremors, paresis and recumbency. Both sheep had difficulty in controlling their hindquarters when attempting to lie down. No significant electrocardiograph abnormalities were detected during the experiment which confirms that cotyledoside at low doses does not overtly affect the electrical activity of the heart. No gross lesions were observed in the sheep. The most significant microscopic lesions comprised mild brain oedema and pronounced vacuolation of the white matter of thalamic nuclei. These lesions might explain some of the motor function deficiencies clinically observed in this syndrome. The previously held contention that these neurotoxic cardiac glycosides are indeed the cause of krimpsiekte is, therefore, confirmed.
Animals↗
Robertsonian (15q;15q) translocation in a child with Angelman syndrome: evidence of uniparental disomy.
A balanced Robertsonian translocation 45,XY,t(15q15q) was detected in a patient with mental retardation, microcephaly, and hypertonia. Deletion of the 15q11q13 region was unlikely based on fluorescence in situ hybridization studies that revealed hybridization of appropriate DNA probes to both arms of the Robertsonian chromosome. Inheritance of alleles from 13 highly polymorphic DNA markers on chromosome 15 showed paternal uniparental isodisomy. The clinical, cytogenetic, and molecular results are consistent with a diagnosis of Angelman syndrome.
Angelman Syndrome↗
Identification and molecular confirmation of a small chromosome 10q duplication [dir dup(10)(q24.2-->q24.3)] inherited from a mother mosaic for the abnormality.
We describe a family in which two siblings exhibited developmental delay, reduced muscle tone and mild muscle weakness. Cytogenetic evaluation demonstrated that both children had a tandem duplication of a small portion of the long arm of chromosome 10 [46,XX or XY,dir dup(10)(q24.2-->q24.3)], inherited from their clinically normal mother, who was found to be mosaic for the duplicated chromosome 10. Fluorescence in situ hybridization approaches, including total chromosome painting and the use of regional specific cosmid probes, were used to confirm the chromosome 10q origin of the duplicated material. This is the smallest confirmed duplication of this portion of chromosome 10 reported to date.
Child↗
The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH.
The hereditary disease Cockayne syndrome (CS) is characterized by a complex clinical phenotype. CS cells are abnormally sensitive to ultraviolet radiation and are defective in the repair of transcriptionally active genes. The cloned CSB gene encodes a member of a protein family that includes the yeast Snf2 protein, a component of the transcriptional regulator Swi/Snf. We report the cloning of the CSA cDNA, which can encode a WD repeat protein. Mutations in the cDNA have been identified in CS-A cell lines. CSA protein interacts with CSB protein and with p44 protein, a subunit of the human RNA polymerase II transcription factor IIH. These observations suggest that the products of the CSA and CSB genes are involved in transcription.
Amino Acid Sequence↗
MHC class I gene organization in > 1.5-Mb YAC contigs from the H2-M region.
Sixteen yeast artificial chromosome (YAC) clones have been mapped to the H2-M region at the distal end of the mouse major histocompatibility complex (MHC) on chromosome 17. Analysis of the YACs with single- and multicopy probes yielded a proximal contig spanning a minimum of 800 kb and a distal contig of 700 kb. A probe for the conserved fourth exon of MHC class I genes detected 19 restriction fragments, including 6 of the 8 previously characterized H2-M class I genes, in the proximal contig. This contig spans the gap from the M to the T region and includes the T1 gene. By contrast, only two class I genes, M2 and M3, were found in the distal contig. These two genes, which are both expressed, may mark the end of the MHC. The order among nine class I genes and seven other markers was determined in the cloned DNA from the centromere as T1, Tu32A, (M1-M7-M8), Tu32B, B30, M6, M4, M5, Mog, Tu42A parallel M2, Leh525, M3, Tu42B, where the orientation with respect to the centromere is unknown for M1-M7-M8.
Animals↗
Human liver mitochondrial carnitine palmitoyltransferase I: characterization of its cDNA and chromosomal localization and partial analysis of the gene.
Using the cDNA for rat liver mitochondrial carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) as a probe, we isolated its counterpart as three overlapping clones from a human liver cDNA library. Both the nucleotide sequence of the human cDNA and the predicted primary structure of the protein (773 aa) proved to be very similar to those of the rat enzyme (82% and 88% identity, respectively). The CPT I mRNA size was also found to be the same (approximately 4.7 kb) in both species. Screening of a human genomic library with the newly obtained cDNA yielded a positive clone of approximately 6.5 kb which, upon partial analysis, was found to contain at least two complete exons linked by a 2.3-kb intron. Oligonucleotide primers specific to upstream and downstream regions of one of the exon/intron junctions were tested in PCRs with DNA from a panel of somatic cell hybrids, each containing a single human chromosome. The results allowed unambiguous assignment of the human liver CPT I gene to the q (long) arm of chromosome 11. Additional experiments established that liver and fibroblasts express the same isoform of mitochondrial CPT I, legitimizing the use of fibroblast assays in the differential diagnosis of the "muscle" and "hepatic" forms of CPT deficiency. The data provide insights into the structure of a human CPT I isoform and its corresponding gene and establish unequivocally that CPT I and CPT II are distinct gene products. Availability of the human CPT I cDNA should open the way to an understanding of the genetic basis of inherited CPT I deficiency syndromes, how the liver CPT I gene is regulated, and which tissues other than liver express this particular variant of the enzyme.
Amino Acid Sequence↗
Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination.
Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating spermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replication. In contrast, elevated levels of DNA ligase III mRNA were observed in primary spermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells.
Amino Acid Sequence↗
Isolation of the toxin responsible for gousiekte, a plant-induced cardiomyopathy of ruminants in southern Africa.
Gousiekte was induced in sheep and goats by the oral and intravenous (i/v) administration of methanolic and ethanolic precipitates of aqueous extracts of Pavetta harborii. Further purification of the toxic principle was carried out by dialysis and ion-exchange chromatography. The toxin was eluted from the cation-exchange resin with 1 mol/l of ammonium acetate and finally purified on Sephadex G-10 and silica gel. Spots on thin-layer chromatographic plates (TLC) were visualized with ninhydrin. Gousiekte was induced with the pure toxin after i/v administration in goats. The yield of pure toxin was about 10 mg/kg of dried leaves.
Africa, Southern↗
In vivo effects of a novel calcium antagonist (R56865) against induced epoxyscillirosidin and tulp poisoning in sheep.
Two anaesthetized sheep were intoxicated with epoxyscillirosidin, the main cardio-active bufadienolide, extracted from Homeria pallida (Natal yellow tulp). The epoxyscillirosidin was injected intravenously as a bolus of 50 micrograms/kg, followed 30 min later by a continuous infusion in a normal saline drip (0.9% NaCl) at 25 micrograms/kg/h. In addition, another two conscious sheep were poisoned by intraruminal dosing of 1,25 g/kg of dried H. pallida plant material. Electrocardiograms, heart and respiratory rates and venous-acid-base levels were recorded prior to and at approximately 30-60 min intervals during the course of the experiment. Additional recordings were made when animals showed signs of intoxication. R56865 (Janssen Pharmaceutica, Pty Ltd), a novel Ca++ antagonist, was administered at the first distinct signs of cardiac disturbances in the sheep given epoxyscillirosidin and after development of tachycardia and dyspnoea in those that received plant material. Activated charcoal was drenched at 3 g/kg to both sheep that received H. pallida about 1 h after the initial administration of R56865. All H. pallida sheep and one of the epoxyscillirosidin sheep survived. The signs of intoxication with H. pallida, namely groaning and tachypnoea, abated within minutes of treatment with R56865, but returned c. 30 min later in both animals. The treatment apparently had little effect on heart rate and EKG changes. One of the epoxyscillirosidin sheep was treated while exhibiting paroxysmal ventricular tachycardia. Although a transient improvement in conduction disturbance was recorded, the animal died soon afterwards. The results of this study indicate that the in vivo response of R56865 against induced bufadienolide cardiac disturbance in sheep is not as evident as that observed with R56865 against similar cardiac disturbance in vitro. The potential use of R56865 together with activated charcoal is discussed.
Animals↗