Cbx-rs2 (M31), a mouse homolog of the Drosophila Heterochromatin protein 1 gene, maps to distal chromosome 11 and is nonallelic to Om.
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Alleles↗
Biomedical subjects
R Feil
Publications and source records attributed to R Feil.
Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing.
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5-Methylcytosine↗
Developmental control of allelic methylation in the imprinted mouse Igf2 and H19 genes.
The Insulin-like growth factor 2 (Igf2) and H19 genes are reciprocally imprinted and closely linked. Igf2 encodes a fetal growth-factor and is predominantly expressed from the paternal allele, while H19 is expressed from the maternal allele and encodes a transcript which may downregulate cellular proliferation. One of the epigenetic modifications thought to be involved in parental imprinting is DNA methylation. Here we analyse methylation in two regions of the Igf2 gene, one approx. 3 kb upstream of the gene and one in the 3' part of the gene. Both regions are more methylated on the expressed paternal chromosome. Genomic sequencing of individual chromosomes in the first region shows this parent-specific methylation to be highly mosaic; interestingly, individual sperm chromosomes carry different methylation patterns into the egg. In the more 3' region, which is fully methylated in sperm, the level of methylation on the paternal allele is highly tissue-specific and is correlated with expression of the gene in fetal tissues. Hence, the paternal allele is highly methylated in fetal liver (high expression) but is undermethylated in fetal brain (virtually no expression). Adult choroid plexus, a brain tissue in which Igf2 is expressed from both alleles and H19 is not expressed, represents an apparent loss of imprinting. Here, both Igf2 and H19 adopt a paternal type methylation pattern on both parental chromosomes. Analysis of early-passage androgenetic and parthenogenetic embryonic stem (ES) cells shows that the methylation patterns of Igf2 and H19 on maternal and paternal chromosomes are very similar. Androgenetic and parthenogenetic teratomas derived from these ES cells show the appropriate paternal and maternal patterns, respectively, of allelic methylation in both genes. Our results suggest that allelic methylation patterns in Igf2 and H19 arise early in embryogenesis and change progressively during development. Some of these developmental changes are apparently under tissue-specific control.
Alleles↗
High-level expression of functional cGMP-dependent protein kinase using the baculovirus system.
The understanding of the structure and function of cGMP-dependent protein kinase (cGMP kinase) has been hindered by the difficulty to obtain large quantities of functional enzyme. A recombinant baculovirus encoding bovine cGMP kinase I alpha was constructed and purified. Infected insect cells synthesized large amounts of soluble and biologically active cGMP kinase I alpha representing up to 10% of the total cell extract protein. The recombinant enzyme had an identical apparent molecular mass, cGMP affinity and kinase activity as the native bovine lung enzyme. The high-level expression of functional cGMP kinase I alpha should provide an excellent tool to study further the structure and function of cGMP kinase.
Animals↗
Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters.
Adrenoleukodystrophy (ALD) is an X-linked disease affecting 1/20,000 males either as cerebral ALD in childhood or as adrenomyeloneuropathy (AMN) in adults. Childhood ALD is the more severe form, with onset of neurological symptoms between 5-12 years of age. Central nervous system demyelination progresses rapidly and death occurs within a few years. AMN is a milder form of the disease with onset at 15-30 years of age and a more progressive course. Adrenal insufficiency (Addison's disease) may remain the only clinical manifestation of ALD. The principal biochemical abnormality of ALD is the accumulation of very-long-chain fatty acids (VLCFA) because of impaired beta-oxidation in peroxisomes. The normal oxidation of VLCFA-CoA in patients' fibroblasts suggested that the gene coding for the VLCFA-CoA synthetase could be a candidate gene for ALD. Here we use positional cloning to identify a gene partially deleted in 6 of 85 independent patients with ALD. In familial cases, the deletions segregated with the disease. An identical deletion was detected in two brothers presenting with different clinical ALD phenotypes. Candidate exons were identified by computer analysis of genomic sequences and used to isolate complementary DNAs by exon connection and screening of cDNA libraries. The deduced protein sequence shows significant sequence identity to a peroxisomal membrane protein of M(r) 70K that is involved in peroxisome biogenesis and belongs to the 'ATP-binding cassette' superfamily of transporters.
ATP Binding Cassette Transporter, Subfamily D, Mem↗
Expression of cGMP-dependent protein kinase in Escherichia coli.
Cyclic GMP-dependent protein kinase (cGMP kinase) is involved in the relaxation of smooth muscle. The enzyme has been cloned and expressed in eukaryotic cell lines but so far not in prokaryotic cells. Three vectors were constructed for the expression of I alpha cGMP kinase in Escherichia coli. Transformation with the pET3a/cgk vector which uses the T7 RNA polymerase/promotor system resulted in efficient accumulation of cGMP kinase. Most of the protein was in an insoluble and catalytic inactive form. Various solubilization and refolding conditions did not yield an active enzyme. A small fraction of the cGMP kinase was present in the soluble cell extract. This fraction bound cGMP with high affinity but had no cGMP stimulated kinase activity. To prevent aggregation two additional vectors were constructed. (I) A bacterial leader sequence, which directs the export of proteins into the periplasmic space, was fused to the amino-terminus of the cGMP kinase. (II) A gram/gram+ shuttle vector for expression under the control of the tac promotor was used. Both constructs directed the synthesis of an insoluble and inactive cGMP kinase. These results suggest that large amounts of cGMP kinase can be expressed in E. coli, but mainly in an insoluble and inactive form. In contrast to eukaryotic cells, bacteria may lack systems for correct protein folding and/or posttranslational modification that are crucial for the productive folding and/or activation of cGMP kinase.
Amino Acid Sequence↗
Prenatal diagnosis and management of fetal hydrocephaly and lissencephaly.
Two cases of prenatal diagnosis of lissencephaly are presented in the context of a series of 118 cases of prenatally diagnosed hydrocephalus. Within this series there was one case of Walker-Warburg syndrome and another of Miller-Dieker syndrome. It is stressed that the cases reported here of ventriculomegaly diagnosed in utero show a very different outcome from those in published studies of fetal hydrocephalus which only deal with patients in whom the diagnosis was determined after birth. In those postnatal series there is a considerable selection bias, and the fate of the fetuses reported here was much worse than in postnatal series. Of the 118 fetuses 6 had fetal infections, 6 had chromosomal abnormalities, 26 had associated spina bifida, 64 fetuses had associated other anomalies, and only 28 had isolated hydrocephalus. Although it is difficult to determine the prognosis individually after prenatal diagnosis of ventriculomegaly, the data presented here may be helpful in counseling parents prenatally. The counseling should be performed with the collaboration of obstetricians, pediatricians, surgeons, and geneticists.
Agenesis of Corpus Callosum↗
Purification and characterization of a novel thermostable 4-alpha-glucanotransferase of Thermotoga maritima cloned in Escherichia coli.
Maltodextrin glycosyltransferase (4-alpha-glucanotransferase) of the extremely thermophilic ancestral bacterium Thermotoga maritima has been purified from an Escherichia coli clone expressing the corresponding T. maritima MSB8 chromosomal gene. T. maritima 4-alpha-glucanotransferase, an approximately 53-kDa monomeric enzyme, is the most thermophilic glycosyltransferase described to date. It retained more than 90% of its maximum activity at temperatures from 55 degrees C up to 80 degrees C. The proposed action modus is the transfer of 1,4-alpha-glucanosyl chains, thus resulting in the disproportionation of 1,4-alpha-glucans. It converted soluble starch, amylopectin, and amylose, thereby changing the iodine staining properties of these substrates. The addition of low-molecular-mass malto-oligosaccharides, which act as glucanosyl acceptor molecules, enhanced the reaction and resulted in the formation of a series of linear maltohomologues from two to more than nine glucose units in size. Use of either of the malto-oligosaccharides maltotetraose, maltopentaose, maltohexaose, or maltoheptaose as sole substrate also yielded linear maltohomologues. On the other hand, maltose and maltotriose were not disproportionated by 4-alpha-glucanotransferase, although both were good acceptors for glucanosyl transfer. Glucose did not function as an acceptor in transfer reactions. Glucose also never appeared as a reaction product. The chain length of glucanosyl segments transferred ranged from two to probably far more than six glucose residues. Comparison of the N-terminal amino acid sequence of 4-alpha-glucanotransferase with other published protein sequences revealed significant similarity to sequences near the N-termini of various eucaryotic maltases and bacterial cyclodextrin glycosyltransferases, suggesting its relatedness on the molecular level with other starch- and maltodextrin-converting enzymes.
Amino Acid Sequence↗
Adrenoleukodystrophy: a complex chromosomal rearrangement in the Xq28 red/green-color-pigment gene region indicates two possible gene localizations.
We have characterized a complex chromosomal rearrangement in band Xq28, in an adrenoleukodystrophy patient who also has blue-cone monochromacy. A 130-kb region upstream from the color-vision pigment genes was isolated as yeast artificial chromosome or cosmid clones. Another Xq28 sequence, not included in the above region, was obtained by cloning a deletion breakpoint from the patient. Using probes derived from the cloned sequences, we have shown that the rearrangement affects the color-pigment genes and includes two deletions, most likely separated by a large (greater than 110-kb) inversion. One deletion encompasses part of the pigment gene cluster and 33 kb of upstream sequences and accounts for the patient's blue-cone monochromacy. If this rearrangement also caused ALD, the disease gene would be expected to lie within or close to one of the deletions. However, deletions were not detected in a 50-kb region upstream of the red-color-pigment gene in 81 other ALD patients. Two CpG islands were mapped, at 46 and 115 kb upstream from the pigment genes.
Adrenoleukodystrophy↗
A 195-kb cosmid walk encompassing the human Xq28 color vision pigment genes.
By using cosmid walking, we have cloned a 195-kb region from chromosome band Xq28 that encompasses the red and green color pigment genes and 85 kb of flanking sequences. This has allowed us to confirm that the color pigment genes are within very homologous units arranged in tandem array. Each unit contains two BssHII sites and one NruI site that are frequently methylated in male leukocyte DNA. A NotI and an EagI site are present 6 kb upstream from the red pigment gene promoter; the NotI site was shown to be unmethylated in the active X chromosome in leukocytes and may represent a CpG island for the whole cluster. We have identified another CpG island, 61 kb 3' from the last green pigment gene, that is unmethylated in leukocytes on the active X chromosome, but methylated on the inactive X. This island is flanked by sequences conserved in evolution and may thus correspond to an expressed gene. We also describe an informative three-allele restriction fragment length polymorphism within the pigment gene cluster.
Biological Evolution↗
Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum.
The assimilation of NH(4) (+) causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoenolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH(4) (+) assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.
Journal Article↗
Relationship between NH(4) Assimilation Rate and in Vivo Phosphoenolpyruvate Carboxylase Activity : Regulation of Anaplerotic Carbon Flow in the Green Alga Selenastrum minutum.
The rate of NH(4) (+) assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH(4) (+) assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H(14)CO(-) (3) into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C(3) organism.
Journal Article↗
Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: I. Regulation of Carbon Metabolism and Succinate as a Fermentation Product.
The onset of anaerobiosis in darkened, N-limited cells of the green alga Selenastrum minutum (Naeg.) Collins elicited the following metabolic responses. There was a rapid decrease in energy charge from 0.85 to a stable lower value of 0.6 accompanied by rapid increases in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also a large increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase, can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase activity. These changes resulted in an approximately twofold increase in the rate of starch breakdown indicating a Pasteur effect. The Pasteur effect was accompanied by accumulation of d-lactate, ethanol and succinate as fermentation end-products, but not malate. Accumulation of succinate was facilitated by reductive carbon metabolism by a partial TCA cycle (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). An initial stoichiometric decline in aspartate and increases in succinate and alanine suggests that aspartate catabolism provides an initial source of carbon for reduction to succinate under anoxic conditions. These observations allow us to develop a model for the regulation of anaerobic carbon metabolism and a model for short-term and long-term strategies for succinate accumulation in a green alga.
Journal Article↗
The swine steroid 21-hydroxylase gene (CYP21): cloning and mapping within the swine leucocyte antigen complex.
A swine genomic cosmid library constructed from a genotypically SLA homozygous Large White individual was screened with a murine genomic 21-hydroxylase probe. A clone which contained a pig 21-hydroxylase gene was isolated and after subcloning, the 5' region of the gene was sequenced. The deduced amino acid sequence corresponded almost exactly to the NH2 terminal portion of the steroid 21-hydroxylase from porcine adrenal microsomes. Comparison of the first 99 amino acid residues of both sequences revealed three substitutions comprising two leucine residues in positions 10 and 13, and one arginine residue in position 55 for our sequence, instead of threonine in position 10 and lysine in position 13 and 55 for the isolated enzyme. A swine homologous probe was derived from the isolated 21-hydroxylase gene and used for gene assignment by RFLP studies in two swine leucocyte antigen (SLA) informative families. The results demonstrate that the swine 21-hydroxylase gene is located within or close to the swine MHC. Taken together, the present results suggest the existence of a single 21-hydroxylase gene per haploid genome.
Animals↗
Physical and genetic mapping of polymorphic loci in Xq28 (DXS15, DXS52, and DXS134): analysis of a cosmid clone and a yeast artificial chromosome.
Sequences corresponding to the Xq28 loci DXS15, DXS52, DXS134, and DXS130 were shown to be present in a 140-kb yeast artificial chromosome (YAC XY58, isolated by Little et al.). This YAC clone appears to contain a faithful copy of this genomic region, as shown by comparison with human DNA and with a cosmid clone that contains probes St14c (part of the DXS52 sequences) and cpX67 (DXS134). cpX67 and St14c are contained in 11 kb and detect the same MspI RFLP polymorphism. A comparison of the YAC restriction map and pulsed-field gel electrophoresis data leads us to propose the following order of loci: DXS52(VNTR)-DXS33-DXF22S3-DXS130-DXS134 -DXS52-DXS15-DXS52, this whole cluster being comprised within 575 kb. The physical proximity of the DXS15, DXS52, and DXS134 loci led us to reinvestigate recombination events that had been reported between these loci in families from the Centre d'Etude du Polymorphisme Humain. Our results do not support the assumption that this region shows increased recombination.
Chromosomes, Fungal↗
The red-green visual pigment gene region in adrenoleukodystrophy.
Although recent data established that a specific very-long-chain fatty acyl-CoA synthetase is defective in X-linked adrenoleukodystrophy (ALD), the ALD gene is still unidentified. The ALD locus has been mapped to Xq28, like the red and green color pigment genes. Abnormal color vision has been observed in 12 of 27 patients with adrenomyeloneuropathy (AMN), a milder form of ALD. Furthermore, rearrangements of the color vision gene cluster were found in four of eight ALD kindreds. This led us to propose that a single DNA rearrangement could underlie both ALD and abnormal color vision in these patients. Study of 34 French ALD patients failed to reveal a higher than expected frequency of green/red visual pigment rearrangements 3' to the red/green color vision gene complex. The previous report of such rearrangements was based on small numbers and lack of knowledge that the frequency of "abnormal" color vision arrays on molecular analysis was twice as high as expected on the basis of the frequency of phenotypic color vision defects. The red/green color pigment (R/GCP) region was studied by pulsed-field gel electrophoresis in 14 of these patients, and we did not find any fragment size difference between the patients and normal individuals who have the same number of pigment genes. The R/GCP region was also analyzed in 29 French and seven North American ALD patients by using six genomic DNA probes, isolated from a cosmid walk, that flank the color vision genes. No deletions were found with probes that lie 3' of the green pigment genes. One of the eight previously reported ALD individuals has a long deletion 5' of the red pigment gene, a deletion causing blue cone monochromacy. This finding and the previous findings of a 45% frequency of phenotypic color vision defects in patients with AMN may suggest that the ALD/AMN gene lies 5' to the red pigment gene and that the frequent phenotypic color vision anomalies owe their origin to deleted DNA that includes regulatory genes for color vision. It is possible, however, that phenotypic color vision anomalies in AMN may be phenocopies secondary to retinal or neural involvement by the disease. The single case of blue cone monochromacy may therefore be a fortuitous coincidence of two diseases.
Adrenoleukodystrophy↗
Decreased-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana. Impact on mass-action ratios and fluxes to sucrose and starch, and estimation of Flux Control Coefficients and Elasticity Coefficients.
1. Subcellular-compartment-specific decreased-activity mutants of phosphoglucose isomerase in Clarkia xantiana were used to analyse the control of sucrose and starch synthesis during photosynthesis. Mutants were available in which the plastid phosphoglucose isomerase complement is decreased to 75% or 50% of the wild-type level, and the cytosol complement to 64%, 36% or 18% of the wild-type level. 2. The effects on the [product]/[substrate] ratio and on fluxes to sucrose or starch and the rate of photosynthesis were studied with the use of saturating or limiting light intensity to impose a high or low flux through these pathways. 3. Removal of a small fraction of either phosphoglucose isomerase leads to a significant shift of the [product]/[substrate] ratio away, from equilibrium. We conclude that there is no 'excess' of enzyme over that needed to maintain its reactants reasonably close to equilibrium. 4. Decreased phosphoglucose isomerase activity can also alter the fluxes to starch or sucrose. However, the effect on flux does not correlate with the extent of disequilibrium, and also varies depending on the subcellular compartment and on the conditions. 5. The results were used to estimate Flux Control Coefficients for the chloroplast and cytosolic phosphoglucose isomerases. The chloroplast isoenzyme exerts control on the rate of starch synthesis and on photosynthesis in saturating light intensity and CO2, but not at low light intensity. The cytosolic enzyme only exerts significant control when its complement is decreased 3-5-fold, and differs from the plastid isoenzyme in exerting more control in low light intensity. It has a positive Control Coefficient for sucrose synthesis, and a negative Control Coefficient for starch synthesis. 6. The Elasticity Coefficients in vivo of the cytosolic phosphoglucose isomerase were estimated to lie between 5 and 8 in the wild-type. They decrease in mutants with a lowered complement of cytosolic phosphoglucose isomerase. 7. The implications of these results for regulation and for evolution are discussed.
Chloroplasts↗
[On the demonstration of isomeric phenylethanolamine].
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Chromatography, Gas↗