Bing-Ling Huang

Human ARX gene: genomic characterization and expression.

Arx is a homeobox-containing gene with a high degree of sequence similarity between mouse and zebrafish. Arx is expressed in the forebrain and floor plate of the developing central nervous systems of these vertebrates and in the presumptive cortex of fetal mice. Our goal was to identify genes in Xp22.1-p21.3 involved in human neuronal development. Our in silico search for candidate genes noted that annotation of a human Xp22 PAC (RPCI1-258N20) sequence (GenBank Accession No. AC002504) identified putative exons consistent with an Arx homologue in Xp22. Northern blot analysis showed that a 3.3kb human ARX transcript was expressed at high levels in fetal brain. A 5.9kb transcript was expressed in adult heart, skeletal muscle, and liver with very faint expression in other adult tissues, including brain. In situ hybridization of ARX in human fetal brain sections at various developmental stages showed the highest expression in neuronal precursors in the germinal matrix of the ganglionic eminence and in the ventricular zone of the telencephalon. Expression was also observed in the hippocampus, cingulate, subventricular zone, cortical plate, caudate nucleus, and putamen. The expression pattern suggests that ARX is involved in the differentiation and maintenance of specific neuronal cell types in the human central nervous system. We also mapped the murine Arx gene to the mouse genome using a mouse/hamster radiation hybrid panel and showed that Arx and ARX are orthologues. Therefore, investigations in model vertebrates may provide insight into the role of ARX in development. The recent identification of ARX mutations in patients with various forms of mental retardation make such studies in model organisms even more compelling.

AluY insertion (IVS4‐52ins316alu) in the glycerol kinase gene from an individual with benign glycerol kinase deficiency

Glycerol kinase deficiency has three distinct forms: an isolated form which may be benign or symptomatic, and a complex form which is symptomatic and part of an Xp21 contiguous gene syndrome. Here we report the case of a male with benign isolated glycerol kinase deficiency who was incidentally identified after observation of pseudohypertriglyceridemia. DNA sequencing of this subjects glycerol kinase gene showed the insertion of an AluY sequence in intron 4 of the glycerol kinase gene. Although Alu insertions have been implicated in other diseases, and a closely related AluY element is found as an insert in the C1 inhibitor gene in patients with hereditary angioedema, this is the first case of glycerol kinase deficiency caused by an Alu insertion. Hum Mutat 15:316–323, 2000.

Ahch, the mouse homologue of DAX1: cloning, characterization and synteny with GyK, the glycerol kinase locus.

We cloned the murine full-length cDNA encoding Ahch, the mouse homologue of DAX1 (DSS-AHC Region on Human X Chromosome, Gene1) which is the gene responsible for human X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Sequence analysis revealed that the murine and human cDNAs have 65% aa identity and 75% aa similarity overall. The cysteine residues in the putative DNA binding domain, which may interact with Zn2+ ions to form zinc fingers, are 100% conserved between the two species, indicating that the novel zinc-finger structures in DAX1 may be functional. In addition, mouse interspecific backcrosses show that the Ahch gene is closely linked to the glycerol kinase locus, GyK, on the mouse X chromosome, indicating that the order of the loci is conserved in this syntenic region between mouse and human.

Primate DAX1, SRY, and SOX9: evolutionary stratification of sex-determination pathway.

The molecular evolution of DAX1, SRY, and SOX9, genes involved in mammalian sex determination, was examined in six primate species. DAX1 and SRY have been added to the X and Y chromosomes, respectively, during mammalian evolution, whereas SOX9 remains autosomal. We determined the genomic sequences of DAX1, SRY, and SOX9 in all six species, and calculated K(a), the number of nonsynonymous substitutions per nonsynonymous site, and compared this with the K(s), the number of synonymous substitutions per synonymous site. Phylogenetic trees were constructed by means of the DAX1, SRY, and SOX9 coding sequences, and phylogenetic analysis was performed using maximum likelihood. Overall measures of gene and protein similarity were closer for DAX1 and SOX9, but DAX1 exhibited nonsynonymous amino acid substitutions at an accelerated frequency relative to synonymous changes, similar to SRY and significantly higher than SOX9. We conclude that, at the protein level, DAX1 and SRY are under less selective pressure to remain conserved than SOX9, and, therefore, diverge more across species than does SOX9. These results are consistent with evolutionary stratification of the mammalian sex determination pathway, analogous to that for sex chromosomes.

Glycerol homeostasis and metabolism in glycerol kinase carrier mice

To examine glycerol homeostasis and metabolism is essential for understanding of pathogenesis and evaluation of treatment efficacy in disorders of glycerol metabolism. In this study, we designed the intraperitoneal glycerol tolerance test (IPGlyTT) and studied glycerol tolerance in vivo using glycerol kinase (Gyk) carrier (C) and wild type (WT) mice. Serum glycerol concentrations in WT almost normalized at 90 min after injection, whereas Gyk C mice retained high serum glycerol concentrations at least until 180 min after injection. These results showed that glycerol tolerance was impaired in Gyk C mice compared to WT mice. The IPGlyTT is useful in accessing glycerol homeostasis and metabolism in animal models such as Gyk C mice and will be valuable in assessing therapeutic interventions in Gyk KO mice.

Glycerol Kinase (GK) Point Mutations in Patients with Isolated GK Deficiency: Possible Genotype-Phenotype Relationships

Glycerol Kinase (GK) Point Mutations in Patients with Isolated GK Deficiency: Possible Genotype-Phenotype Relationships

Adrenal Hypoplasia Congenita and Male Pseudohermaphroditism: New Syndromic Association Association Additional Sex Determining Gene(s)

Adrenal Hypoplasia Congenita and Male Pseudohermaphroditism: New Syndromic Association Association Additional Sex Determining Gene(s)

Glycerol Kinase Missense Mutations Provide Structure-Function But Not Genotype-Phenotype Insights. † 641

Glycerol Kinase Missense Mutations Provide Structure-Function But Not Genotype-Phenotype Insights. † 641

BACTERIAL SPECIES IDENTIFICATION AFTER AMPLIFICATION WITH A UNIVERSAL PRIMER PAIR. |[dagger]| 1059

Our group and others have shown that a single, universal primer pair designed to anneal to highly conserved regions within the bacterial 16S ribosomal RNA coding sequence (16S rDNA) can amplify genomic DNA from a wide variety of bacterial organisms. Since the highly conserved regions of the 16S rDNA flank regions of the gene which vary between species, we proposed to determine whether products amplified with a universal primer pair could then be used for identification of the species with specific hybridization probes. We examined a variety of bacterial 16S rDNA sequences from GenEMBL, a non-overlapping nucleotide sequence database, using FastA (GCG, Madison, WI). These sequences were aligned using PileUp (GCG, Madison, WI) and 19-21 bp sequences were identified with apparent species specificity. PCR amplification was performed with the universal primer pair, p806R and p8FPL, and amplification products were evaluated by manual and automated methods. Dot blots were prepared with the PCR products from both Gram positive and Gram negative organisms and hybridized with end-labeled species specific probes. Using this approach we were able to differentiate between PCR products generated from different bacterial DNA templates. We conclude, that individual species can be identified by species specific sequence variability within amplification products generated following PCR with a universal bacterial primer pair. We speculate that the application of molecular genetic technology for the diagnosis of bacterial infections will reduce the cost and the time for identification of pathogens, will allow for the molecular triage of sepsis, and will improve the quality of care for patients with infections.(E.R.B. McCabe is an Academic Associate for Corning-Nichols Institute.)