Excess folate during adolescence suppresses thyroid function with permanent deficits in motivation and spatial memory.

Cognitive and memory deficits can be caused or exacerbated by dietary folate deficiency, which has been combatted by the addition of folate to grains and dietary supplements. The recommended dose of the B9 vitamin folate is 400 µg/day for adolescents and non-pregnant adults, and consumption above the recommended daily allowance is not considered to be detrimental. However, the effects of excess folate have not been tested in adolescence when neuro and endocrine development suggest possible vulnerability to long-term cognitive effects. We administered folate-supplemented (8.0 mg folic acid/kg diet) or control lab chow (2.7 mg folic acid/kg diet) to rats ad libitum from 30 to 60 days of age, and subsequently tested their motivation and learning and memory in the Morris water maze. We found that folate-supplemented animals had deficits in motivation and spatial memory, but they showed no changes of the learning- and memory-related molecules growth-associated protein-43 or Gs-α subunit protein in the hippocampus. They had decreased levels of thyroxine (T4) and triiodothyronine (T3) in the periphery and decreased protein levels of thyroid receptor-α1 and -α2 (TRα1 and TRα2) in the hippocampus. The latter may have been due to an observed increase of cytosine-phosphate-guanosine island methylation within the putative thyroid hormone receptor-α promoter, which we have mapped for the first time in the rat. Overall, folate supplementation in adolescence led to motivational and spatial memory deficits that may have been mediated by suppressed thyroid hormone function in the periphery and hippocampus.

The human aminophospholipid-transporting ATPase gene ATP10C maps adjacent to UBE3A and exhibits similar imprinted expression.

Maternal duplications of the imprinted 15q11-13 domain result in an estimated 1%-2% of autism-spectrum disorders, and linkage to autism has been identified within 15q12-13. UBE3A, the Angelman syndrome gene, has, to date, been the only maternally expressed, imprinted gene identified within this region, but mutations have not been found in autistic patients. Here we describe the characterization of ATP10C, a new human imprinted gene, which encodes a putative protein homologous to the mouse aminophospholipid-transporting ATPase Atp10c. ATP10C maps within 200 kb distal to UBE3A and, like UBE3A, also demonstrates imprinted, preferential maternal expression in human brain. The location and imprinted expression of ATP10C thus make it a candidate for chromosome 15-associated autism and suggest that it may contribute to the Angelman syndrome phenotype.

Xist has properties of the X-chromosome inactivation centre.

X-chromosome inactivation is the process by which female mammals (with two X chromosomes) achieve expression of X-chromosomal genes equivalent to that of males (one X and one Y chromosome). This results in the transcriptional silencing of virtually all genes on one of the X chromosomes in female somatic cells. X-chromosome inactivation has been shown to act in cis and to initiate and spread from a single site on the X chromosome known as the X-inactivation centre (Xic). The Xic has been localized to a 450-kilobase region of the mouse X chromosome. The Xist gene also maps to this region and is expressed exclusively from the inactive X chromosome. Xist is unusual in that it appears not to code for a protein but produces a nuclear RNA which colocalizes with the inactive X chromosome. The creation of a null allele of Xist in embryonic stem cells has demonstrated that this gene is required for X inactivation to occur in cis. Here we show that Xist, introduced onto an autosome, is sufficient by itself for inactivation in cis and that Xist RNA becomes localized close to the autosome into which the gene is integrated. In addition, the presence of autosomal Xist copies leads to activation of the endogeneous Xist gene in some cells, suggesting that elements required for some aspects of chromosome counting are contained within the construct. Thus the Xist gene exhibits properties of the X-inactivation centre.

Novel lacZ-based recombination vectors for mammalian cells.

We have constructed two sets of Escherichia coli lacZ-based vectors for use in studies of general mitotic recombination, both in somatic mammalian cells grown in culture and in transgenic animals. The vectors use two mutant copies of the E. coli lacZ gene as their recombination substrates and contain a neo gene for selection of stable transformants. In one vector, pLrec, an SV40 promoter drives lacZ, while the other vector, pArec, utilizes a human non-muscle beta-actin promoter for lacZ expression. Gene conversions, unequal sister chromatid exchanges and reciprocal exchanges between the two lacZ genes result in expression of beta-galactosidase, which can be detected in situ by histochemical staining. These vectors yield rates and frequencies of mitotic intrachromosomal recombination in human and rodent cell lines which are similar to rates reported using conventional recombination vectors. Molecular analysis of recombinational events involving the lacZ-based vectors can be carried out on genomic DNA isolated from clonally expanded populations and individual LacZ+ cells and cell clusters can be analyzed using PCR amplification. These reporter gene-based vectors may facilitate the study of recombination in cells with limited proliferative capacities, allow for analysis of both products of an unequal sister chromatid exchange, and permit in situ detection of recombination in the tissues of transgenic animals.

Expression cloning of multiple human cDNAs that complement the phenotypic defects of ataxia-telangiectasia group D fibroblasts.

Ataxia-telangiectasia (A-T) is an inherited human disease of unknown etiology associated with neurologic degeneration, immune dysfunction, cancer risk, and genetic instability. A-T cells are sensitive to ionizing radiation and radiomimetic drugs, offering the possibility of cloning A-T genes by phenotypic complementation. We have used this sensitivity to isolate the first human cDNAs reported to complement A-T cells in culture. Complementation group D A-T fibroblasts were transfected with an episomal vector-based human cDNA library, approximately 610,000 resultant transformants were treated with the radiomimetic drug streptonigrin-resistant, and nine unrelated cDNAs were recovered from 29 surviving streptonigrin-resistant clones. Five cDNAs were mapped, but none localized to 11q23, the site of A-T complementation group A and C loci. Four of the mapped cDNAs conferred mutagen resistance to A-T D fibroblasts on secondary transfection. One cDNA was identified as a fragment of dek, a gene involved in acute myeloid leukemia. The dek cDNA fragment and pCAT4.5, a 4.5-kb cDNA that mapped to 17p11, independently complemented three different phenotypic abnormalities of A-T D fibroblasts (mutagen sensitivity, hyper-recombination, and radio-resistant DNA synthesis). The pCAT4.5 cDNA did not complement the mutagen sensitivity of an A-T group C fibroblast line, suggesting that it represents a candidate disease gene for group D A-T. Our results indicate that phenotypic complementation alone is insufficient evidence to prove that a candidate cDNA is an A-T disease gene. The complementing cDNAs may represent previously uncharacterized genes that function in the same pathway as does the A-T gene product(s) in the regulation of cellular responses to DNA damage.