A randomized community intervention trial to increase awareness and knowledge of the role of periconceptional folate in women of child-bearing age.

OBJECTIVES: To determine the effect of a consumer-directed information campaign to increase knowledge of folate for the prevention of neural tube defects among women of child-bearing age, and to measure women's recall of sources of information and knowledge about folate. DESIGN: A community randomized trial. SETTING: Three matched pairs of geographically distinct Local Government Areas in the state of Victoria, Australia.

Identification of the copper chaperone SAH in Ovis aries: expression analysis and in vitro interaction of SAH with ATP7B.

A clone encoding the putative copper chaperone protein Sheep Atx1 Homologue (SAH) was isolated from a sheep liver cDNA library. The 466-bp cDNA encoded a predicted protein of 68 amino acids, with 44 and 81% amino acid identity to the yeast Atx1 and human Atox1 copper chaperone proteins, respectively. The characteristic MTCxxC and KTGK motifs were conserved in SAH.

Cloning, mapping and expression analysis of the sheep Wilson disease gene homologue.

Copper homeostasis in mammals is maintained by the balance of dietary intake and copper excretion via the bile. Sheep have a variant copper phenotype and do not efficiently excrete copper by this mechanism, often resulting in excessive copper accumulation in the liver. The Wilson disease protein (ATP7B) is a copper transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile.

The correlation of clinical phenotype in Friedreich ataxia with the site of point mutations in the FRDA gene.

Most cases of Friedreich ataxia (FRDA) are due to expansions of a GAA trinucleotide repeat sequence in the FRDA gene coding for frataxin, a protein of poorly understood function which may regulate mitochondrial iron transport. However, between 1% and 5% of mutations are single base changes in the sequence of the FRDA gene, causing missense, nonsense, or splicing mutations. We describe three new mutations, IVS4nt2 (T to G), R165C, and L182F, which occur in patients in association with GAA expansions.

Late diagnosis of maternal PKU in a family segregating an arylsulfatase [corrected] E mutation causing symmetrical chondrodysplasia punctata.

Mutations in the arylsulfatase E gene, located on the X chromosome, have been shown to cause chondrodysplasia punctata (CDP). A substitution of arginine with serine at amino acid 12 (R12S) was identified in a patient with typical features of mild symmetrical CDP including mild mental retardation. The proband was institutionalized and was found to have seven full and half siblings all of whom were microcephalic.

Identifying genes predisposing to atopic eczema.

Background: Genetic and environmental factors are known to play a role in the development of atopic diseases, such as asthma, eczema, and rhinitis. However, the atopy gene (or genes) has yet to be defined. Studies of familial asthma have identified several regions that may contain genes predisposing to atopy, but the data for candidate regions do not show agreement, which may be due to heterogeneity, ascertainment bias, or stochastic factors.

Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system.

Gene therapy studies require techniques that allow alteration of human genomic DNA sequences. Bacterial artificial chromosome cloning systems (BACs/PACs) bridge the gap between vectors with small inserts and yeast artificial chromosomes (YACs). We report the use of a second generation BAC vector, pEBAC, containing eukaryotic selectable markers and combining some of the best features of the BAC, PAC and HAEC systems, into which a 185 kb sequence containing the human beta-globin gene cluster was retrofitted.

A series of mutations in the dihydropteridine reductase gene resulting in either abnormal RNA splicing or DHPR protein defects. Mutations in brief no. 244. Online.

Five novel mutations are described which result in the rare hyperphenylalaninemia DHPR-deficiency. Three of these are located at different intron/exon boundaries within the DHPR gene, and disrupt the maturation of the DHPR transcript such that little full-length mRNA can be detected by RT-PCR. Each mutation alters a conserved nucleotide within the splice site consensus sequence, and results in the skipping of an exon and, in one case, the activation of an inappropriate splicing signal.

The role of GMXCXXC metal binding sites in the copper-induced redistribution of the Menkes protein.

The Menkes protein (MNK or ATP7A) is a transmembrane, copper-transporting CPX-type ATPase, a subgroup of the extensive family of P-type ATPases. A striking feature of the protein is the presence of six metal binding sites (MBSs) in the N-terminal region with the highly conserved consensus sequence GMXCXXC. MNK is normally located in the trans-Golgi network (TGN) but has been shown to relocalize to the plasma membrane when cells are cultured in media containing high concentrations of copper.

Novel Mitochondrial DNA Variant That May Give a False Positive Diagnosis for the T8993C Mutation.

Background: Two of the most common mutations in the mitochondrial DNA (mtDNA) of children occur at nucleotide 8993 (nt8993). The base substitutions of T to G (T8993G) and T to C (T8993C) are known to cause neurologic disorders and are routinely screened for in patients suspected of having a mitochondrial disorder. Methods and Results: Both mutations at nt8993 create a novel HpaII restriction endonuclease site and are usually detected by polymerase chain reaction (PCR) amplification of a section of the mtDNA containing nt8993, followed by HpaII digestion.

Segregation of mutations in arylsulphatase E and correlation with the clinical presentation of chondrodysplasia punctata.

Sixteen males and two females with symmetrical (mild) type of chondrodysplasia punctata were tested for mutations in the X chromosome located arylsulphatase D and E genes. We identified one nonsense and two missense mutations in the arylsulphatase E gene in three males. No mutations were detected in the arylsulphatase D gene.

Functional analysis and intracellular localization of the human menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1).

The Menkes protein (MNK or ATP7A) is an important component of the mammalian copper transport pathway and is defective in Menkes disease, a fatal X-linked disorder of copper transport. To study the structure and function of this protein and to elucidate its role in cellular copper homeostasis, a cDNA construct encoding the full-length MNK protein was cloned into a mammalian expression vector under the control of the CMV promoter. Transfection of this plasmid construct into CHO-K1 cells yielded clones that expressed MNK at varying levels.

Centromere protein B null mice are mitotically and meiotically normal but have lower body and testis weights.

CENP-B is a constitutive centromere DNA-binding protein that is conserved in a number of mammalian species and in yeast. Despite this conservation, earlier cytological and indirect experimental studies have provided conflicting evidence concerning the role of this protein in mitosis. The requirement of this protein in meiosis has also not previously been described.

Eukaryotic expression vectors that replicate to low copy number in bacteria: transient expression of the Menkes protein.

A set of low copy number plasmid vectors for mammalian gene expression has been constructed. These vectors are derived from the previously described bacterial low copy number expression vectors, pWSK29 and pWKS30, which are present at six to eight copies per cell. The new plasmids also have the following useful properties: (1) they contain antibiotic resistance markers for the selection of stable mammalian cell lines; (2) they have either constitutive or inducible promoters; (3) a chimeric intron, for enhancing gene expression, is present; (4) they contain unique cloning sites; (5) they have an SV40 polyadenylation signal, and a subset of the vectors have an SV40 origin of replication for episomal replication and transient gene expression.

Direct cloning of human 10q25 neocentromere DNA using transformation-associated recombination (TAR) in yeast.

The transformation-associated recombination (TAR) procedure allows rapid, site-directed cloning of specific human chromosomal regions as yeast artificial chromosomes (YACs). The procedure requires knowledge of only a single, relatively small genomic sequence that resides adjacent to the chromosomal region of interest. We applied this approach to the cloning of the neocentromere DNA of a marker chromosome that we have previously shown to have originated through the activation of a latent centromere at human chromosome 10q25.

Targeted disruption of mouse centromere protein C gene leads to mitotic disarray and early embryo death.

Centromere protein C (CENPC) is a key protein that has been localized to the inner kinetochore plate of active mammalian centromeres. Using gene targeting techniques, we have disrupted the mouse Cenpc gene and shown that the gene is essential for normal mouse embryonic development. Heterozygous mice carrying one functional copy of the gene are healthy and fertile, whereas homozygous embryos fail to thrive.

Gene structure and sequence analysis of mouse centromere proteins A and C.

We have determined the genomic structure and organization of the mouse Cenpa and Cenpc genes. CENPA is a member of the histone H3-like proteins and is thought to replace histone H3 in centromeric nucleosomes. CENPC is a DNA-binding protein that is located at the inner kinetochore plate of active mammalian centromeres.

Isolation and characterization of chondroitin sulfate proteoglycans from embryonic quail that influence neural crest cell behavior.

The movement of neural crest cells is controlled in part by extracellular matrix. Aggrecan, the chondroitin sulfate proteoglycan from adult cartilage, curtails the ability of neural crest cells to adhere, spread, and move across otherwise favorable matrix substrates in vitro. Our aim was to isolate, characterize, and compare the structure and effect on neural crest cells of aggrecan and proteoglycans purified from the tissues through which neural crest cells migrate.

A functional neo-centromere formed through activation of a latent human centromere and consisting of non-alpha-satellite DNA.

We recently described a human marker chromosome containing a functional neo-centromere that binds anti-centromere antibodies, but is devoid of centromeric alpha-satellite repeats and derived from a hitherto non-centromeric region of chromosome 10q25. Chromosome walking using cloned single-copy DNA from this region enabled us to identify the antibody-binding domain of this centromere. Extensive restriction mapping indicates that this domain has an identical genomic organization to the corresponding normal chromosomal region, suggesting a mechanism for the origin of this centromere through the activation of a latent centromere that exists within 10q25.

Detecting fetal abnormalities.

Three per cent of infants suffer from birth defects, (mostly genetic) including single gene diseases, such as cystic fibrosis or thalassaemia; chromosomal aneuploidies such as Down syndrome; or multifactorial conditions such as spina bifida and congenital heart defects. Perhaps the most important reason for focusing attention on genetics in 1997 is that the field has changed dramatically due to advances in technology and in our understanding of the human genome. New opportunities for prevention combined with more effective treatment, represent the new standard of care that the community has the right to expect for genetic disease.

Chromosomal localization of mouse Cenpa gene.

Using a previously isolated mouse centromere protein A (Cenpa) probe, we have localized the gene to the proximal region of mouse Chromosome 5, between the known Il6 and Yes1 loci near [Adra2C-D5H4S43-Hdh]. Comparison of this localization with that of human CENPA, which maps to chromosome 2, is consistent with the presence of a new region of conserved synteny between the two species.

Migration of enteric neural crest cells in relation to growth of the gut in avian embryos.

Neural crest cell migration in the gut and the growth of the mid- and hindgut of avian embryos was investigated by a combination of whole-mount immunofluorescence of the HNK-1 neural crest marker epitope, chorioallantoic membrane grafting and morphometry. HNK-1-labelled cells advanced rostrocaudally in the gut of quail embryos (to the duodenum by stage HH 21, to the umbilicus by HH 25, to the ceca by HH 27, to the cloaca by HH 33). The timetable in chick embryos appeared to be slightly slower, but neural cells were obscured by background fluorescence in this species.