Nonflowering plants possess a unique folate-dependent phenylalanine hydroxylase that is localized in chloroplasts

Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism.     

Cloning and characterization of the ribosomal RNA gene complex from the plant pathogen Verticillium albo-atrum

Abstract The DNA coding for the ribosomal RNA gene complex (rDNA) has been cloned from isolate 621P(PV1) of Verticillium albo-atrum which is pathogenic for hops ( Humulus lupulus ). The rDNA was mapped using a range of restriction enzymes. The functional units of the intergenic spacer (IGS), 18S, 5.8S and 25S regions were located by hybridization to specific gene probes from the rDNA complex of Aspergillus nidulans . The start points of the 18S and 5.8S regions were confirmed by partial sequencing. A genomic restriction enzyme map was found to be identical with the map of the cloned DNA. The rDNA repeat was 7.6 kb in length and this was used as an homologous probe to analyse the size of the repeat in 18 hop isolates of V. albo-atrum strains and in one isolate from alfalfa (Luc2). All of the isolates had a repeat size of 7.6 kb except for Luc2 where the rDNA complex was 8.4 kb.     

Association of Family Environment with Children's Television Viewing and with Low Level of Physical Activity

Objective: This study examined associations between the family environment and children's television (TV) viewing and likelihood of being low‐active. Research Methods and Procedures: In 2001, children were recruited from 19 primary schools in Melbourne, Australia. Parents completed a questionnaire about their child's TV viewing and the family environment. Children also completed a questionnaire and wore an accelerometer for 8 days. Movement counts were used to identify low‐active children (lowest quartile). Data were analyzed in May 2004. Results: The sample consisted of 878 children (mean age = 11.5 ± 0.6 yrs). Multiple logistic regression revealed that socioeconomic status [adjusted odds ratios (AOR) = 0.4 boys], frequency families watched TV together (AOR = 2.0 boys), mothers’ (AOR = 1.8 boys; AOR = 2.5 girls) and fathers’ (AOR = 2.6 boys; AOR = 2.8 girls) TV viewing, and rules prohibiting TV during mealtimes (AOR = 0.6 boys; AOR = 0.6 girls) related to children watching TV ≥2 h/d. Variables associated with low‐level physical activity included self‐reported enjoyment of Internet use (AOR = 1.7 boys) and preference for watching TV (AOR = 2.3 girls), perception that mother uses computer a lot (AOR = 1.9 boys) and likes using the computer (AOR = 0.6 girls), fathers’ reported computer/electronic games use (AOR = 1.7 girls), frequency families used computer together (AOR = 0.4 girls), rules that TV viewing must be supervised (AOR = 1.9 boys; AOR = 0.6 girls), and having pay TV (AOR = 0.6 boys) and electronic games at home (AOR = 2.6 boys). Discussion: These findings suggest that the relationships between the family environment and TV viewing and low‐level activity are complex and that these behaviors are distinct.     

Partial Deletion of the Sulfate Transporter SLC13A1 Is Associated with an Osteochondrodysplasia in the Miniature Poodle Breed

A crippling dwarfism was first described in the Miniature Poodle in Great Britain in 1956. Here, we resolve the genetic basis of this recessively inherited disorder. A case-control analysis (8∶8) of genotype data from 173 k SNPs revealed a single associated locus on CFA14 (P_raw <10^–8). All affected dogs were homozygous for an ancestral haplotype consistent with a founder effect and an identical-by-descent mutation. Systematic failure of nine, nearly contiguous SNPs, was observed solely in affected dogs, suggesting a deletion was the causal mutation. A 130-kb deletion was confirmed both by fluorescence in situ hybridization (FISH) analysis and by cloning the physical breakpoints. The mutation was perfectly associated in all cases and obligate heterozygotes. The deletion ablated all but the first exon of SLC13A1, a sodium/sulfate symporter responsible for regulating serum levels of inorganic sulfate. Our results corroborate earlier findings from an Slc13a1 mouse knockout, which resulted in hyposulfatemia and syndromic defects. Interestingly, the metabolic disorder in Miniature Poodles appears to share more clinical signs with a spectrum of human disorders caused by SLC26A2 than with the mouse Slc13a1 model. SLC26A2 is the primary sodium-independent sulfate transporter in cartilage and bone and is important for the sulfation of proteoglycans such as aggregan. We propose that disruption of SLC13A1 in the dog similarly causes undersulfation of proteoglycans in the extracellular matrix (ECM), which impacts the conversion of cartilage to bone. A co-dominant DNA test of the deletion was developed to enable breeders to avoid producing affected dogs and to selectively eliminate the mutation from the gene pool.