Linker histone H1 plays an important role in chromatin folding in vitro. To study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. H1 depletion caused dramatic chromatin structure changes, including decreased global nucleosome spacing, reduced local chromatin compaction, and decreases in certain core histone modifications. Surprisingly, however, microarray analysis revealed that expression of only a small number of genes is affected. Many of the affected genes are imprinted or are on the X chromosome and are therefore normally regulated by DNA methylation. Although global DNA methylation is not changed, methylation of specific CpGs within the regulatory regions of some of the H1 regulated genes is reduced. These results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns.
Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.
Sanfilippo syndrome type III A (Mucopolysaccharidosis (MPS) III
N-Acetylglucosaminyltransferase III (GlcNAc-TIII), the product of the Mgat3 gene, transfers the bisecting GlcNAc to the core mannose of complex N-glycans. The addition of this residue is regulated during development and has functional consequences for receptor signaling, cell adhesion, and tumor progression. Mice homozygous for a null mutation at the Mgat3 locus (Mgat3 ⌬ ) or for a targeted mutation in the Mgat3 gene (previously called Mgat3 neo , but herein renamed Mgat3 T37 because the allele generates inactive GlcNAc-TIII of ϳ37 kDa) were found to exhibit retarded progression of liver tumors. Matrix-assisted laser desorption/ionization time-offlight mass spectrometry of neutral N-glycans from kidneys revealed no significant differences, and both mutants showed the expected lack of N-glycan species with an additional GlcNAc. However, the two mutants differed in several biological traits. Mgat3 T37/T37 homozygotes in a mixed or 129SvJ background were retarded in growth rate and exhibited an altered leg clasp reflex, an altered gait, and defective nursing behavior. Pups abandoned by Mgat3 T37/T37 mothers were rescued by wildtype foster mothers. None of these Mgat3 T37/T37 traits were exhibited by Mgat3 ⌬/⌬ mice or by heterozygous mice carrying the Mgat3 T37 mutation. Similarly, no dominant-negative effect was observed in Chinese hamster ovary cells expressing truncated GlcNAc-TIII in the presence of wild-type GlcNAc-TIII. However, compound heterozygotes carrying both the Mgat3 T37 and Mgat3 ⌬ mutations exhibited a marked leg clasp reflex, indicating that in the absence of wild-type GlcNAc-TIII, truncated GlcNAc-TIII causes this phenotype. The Mgat3 gene was expressed in brain at embryonic day 10.5 and thereafter and in neurons of adult cerebellum. The mutant Mgat3 gene was also highly expressed in Mgat3 T37/T37 brain. This may be the basis of the unexpected neurological phenotype induced by truncated, inactive GlcNAc-TIII in the mouse.The N-glycans of mammalian glycoproteins vary widely in structure, but the biological significance of this variation is largely unknown. A well studied example is the bisecting GlcNAc. This residue is transferred to the -linked Man of the core of N-glycans by the glycosyltransferase termed N-acetylglucosaminyltransferase III (GlcNAc-TIII 1 ; EC 2.4.1.144) (1), the product of the Mgat3 gene (2). The presence of the bisecting GlcNAc alters the lectin binding properties of a cell, a fact initially revealed by the gain-of-function Chinese hamster ovary (CHO) glycosylation mutant LEC10, which expresses GlcNAc-TIII (3). LEC10 cells are ϳ15-fold more resistant to ricin and ϳ10-fold more sensitive to the toxicity of the erythroagglutinin from Phaseolus vulgaris (E-PHA) compared with wild-type CHO cells, reflecting dramatic changes in binding of these lectins to N-linked Gal residues of cell-surface glycoproteins. Similarly, the ectopic expression of an Mgat3 cDNA reduces the expression of terminal ␣3-Gal residues, a key determinant in xenotransplantation (4, 5). The regulated expression o...
We have compared the sequences of a major class of kinetoplast DNA (kDNA) minicircle (pLURkE3) of Leishmania strain UR6 with other minicircle sequences from different Leishmania species. Alignment of these sequences allowed the selection of a pair of oligonucleotides suitable as primers in polymerase chain reaction (PCR) which is specific for Leishmania parasites. PCR with this genus-specific primer set is capable of detecting 1 femtogram of kDNA. These primers have been tested with kDNAs from both old world and new world Leishmania species. The results indicate that the primers may be suitable for detection of any kind of leishmaniasis.
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