Novel Nanotube Multiquantum Dot Devices.

Addressable quantum states well isolated from the environment are of considerable interest for quantum information science and technology. Carbon nanotubes are an appealing system, since a perfect crystal can be grown without any missing atoms and its cylindrical structure prevents ill-defined atomic arrangement at the surface. Here, we develop a reliable process to fabricate compact multielectrode circuits that can sustain the harsh conditions of the nanotube growth. Nanotubes are suspended over multiple gate electrodes, which are themselves structured over narrow dielectric ridges to reduce the effect of the charge fluctuators of the substrate. We measure high-quality double- and triple-quantum dot charge stability diagrams. Transport measurements through the triple-quantum dot indicate long-range tunneling of single electrons between the left and right quantum dots. This work paves the way to the realization of a new generation of condensed-matter devices in an ultraclean environment, including spin qubits, mechanical qubits, and quantum simulators.

Mutation analysis of Wilson disease in the Spanish population -- identification of a prevalent substitution and eight novel mutations in the ATP7B gene.

Wilson disease (WD) is a copper metabolism disorder characterized by hepatic and/or neurological damage. More than 200 mutations in the ATP7B gene causing this autosomal recessive defect have been reported. In certain populations, a high prevalence of particular mutations allows rapid screening and diagnosis of the disease. We identified the ATP7B alterations in Spanish patients with WD. Mutations in the ATP7B gene were analysed in a total of 64 individuals from 40 different WD families by PCR amplification, single-strand conformation polymorphism (SSCP) analysis and sequencing. Twenty-one different ATP7B gene mutations were identified, eight of which were novel. 74% of the disease alleles were characterized among the 40 unrelated probands. We identified a prevalent mutation in our population (Met645Arg), present in 55% of this 40 patients. The frequency of the remaining ATP7B alterations was low. In addition, 17 different polymorphic variants were found. There is remarkable allele heterogeneity in WD in the Spanish population. Nevertheless, SSCP screening for the most frequent mutations in our population is feasible and leads to the detection of about 74% of the mutated chromosomes. Molecular diagnosis of WD is very useful in clinical practice to confirm or support clinical suspicion.

Smoldering multiple myeloma: natural history and recognition of an evolving type.

Patients with smoldering multiple myeloma (SMM) meet the diagnostic criteria of multiple myeloma (MM) but are asymptomatic. Between January 1978 and July 2001, 53 patients (median age 63 years) were diagnosed with SMM. The median serum M-protein and proportion of bone marrow plasma cells were 36 g/l and 27% respectively. Two subsets of SMM were identified: (i) evolving SMM (n = 22), characterized by a progressive increase in serum M-protein, a previously recognized monoclonal gammopathy of undetermined significance (MGUS) and a significant higher proportion of IgA type and (ii) non-evolving SMM (n = 26) with stable M-protein that abruptly increases when symptomatic MM develops. Thirty-four patients developed symptomatic MM. The median time to progression in the overall series was 3.2 years and the only feature associated with a shorter time to progression was the evolving versus non-evolving type (1.3 vs. 3.9 years respectively, P = 0.007). The pattern of progression consisted of anaemia, lytic bone lesions or both, without renal failure, hypercalcaemia or extramedullary plasmacytomas. Fifty-seven per cent of patients that required chemotherapy showed no or minimal response. The median survival from diagnosis and from progression was 8.2 and 3.5 years respectively.

A novel mutation (V89L) in the presenilin 1 gene in a family with early onset Alzheimer's disease and marked behavioural disturbances.

A novel mutation (V89L) in the presenilin 1 (PSEN1) gene is described in a family with pathologically confirmed Alzheimer's disease. The mutation was identified in two affected members with early onset Alzheimer's disease characterised by early and marked behavioural disturbances. The mutation is located on the same side of the helix as other described mutations in the first transmembrane domain and its relation to other mutations in this helix suggests that they share a common pathogenic mechanism.

A novel presenilin 2 gene mutation (D439A) in a patient with early-onset Alzheimer's disease.

The authors describe a novel missense mutation in the presenilin 2 (PSEN2) gene at residue 439 that predicts an aspartate-to-alanine substitution (D439A). This mutation was found in a 58-year old patient who displayed a progressive dementia at the age of 52. The mutation was absent in his cognitively normal relatives. Haplotype analysis indicated that his affected mother was the most probable mutation carrier. The D439A mutation is located near the C-terminal end of the PS2 protein, a region critical for endoproteolytic processing.

Detection of the presenilin 1 gene mutation (M139T) in early-onset familial Alzheimer disease in Spain.

In a family with early-onset Alzheimer disease (EOAD) from Spain we found a mutation in the presenilin 1 (PS1) gene that predicts a methionine-to-threonine change at the PS1 residue 139 (M139T). This mutation was previously reported in a independent French family. The age of onset of the disease was similar in the affected members from both families, suggesting a specific age of expression (range 47-50 years). The detection of the M139T mutation in an independent EOAD family strongly supports the pathogenicity of this mutation in familial Alzheimer disease (AD).

Paternal isodisomy 13 in a normal newborn infant after trisomy rescue evidenced by prenatal diagnosis.

Maternal and paternal uniparental disomy of chromosome 13 have been associated with normal phenotypes. We report on a new case of paternal isodisomy 13 in a phenotypically normal girl. Prenatal diagnosis had shown a 46,XX,-13,der(13;13) karyotype in chorionic villi and a 45,XX,der(13;13) karyotype in amniocytes and fetal blood. Molecular studies demonstrated that the de novo der(13;13) was an isochromosome 13 of paternal origin. This observation supports the lack of imprinting effects on chromosome 13 and trisomy rescue as a mechanism leading to uniparental disomy in cases involving isochromosomes.

Analysis of the polymorphic (GT)(n) repeat at the dopamine beta-hydroxylase gene in Spanish patients affected by schizophrenia.

The presence of a polymorphic (GT)(n) repeat, a microsatellite repeat, at the human dopamine beta-hydroxylase (DBH) gene had been previously investigated in healthy people and in schizophrenic patients. The different DBH genotypes had been found to be associated to different DBH biochemical function, but no differences were found in the allelic and genotype frequencies between schizophrenic and control groups. To further clarify the potential involvement of the variation at the DBH gene in schizophrenia we have studied the DBH (GT)(n) repeat in a sample of 47 Spanish schizophrenic patients, in their healthy relatives (n = 72), and in a control population (n = 74). We have been able to identify five different variants of the DBH gene (A1, A2, A3, A4, A5) in the different groups. Subsequent statistical analysis revealed that the genotypes as well as the allele frequencies did not differ significantly among schizophrenic patients and the control population. Interestingly, the allelic variant A2 and the genotype A4/A2 were significantly more frequent in schizophrenic patients as compared with their healthy relatives. However, the association of the A2 allele with schizophrenia was not supported by the haplotype relative risk analysis of transmitted versus nontransmitted alleles. Therefore, although it will be important to extend the present analysis in a larger sample of schizophrenic patients and controls, our results suggest that the (GT)(n) does not seem to play a major role in the genetics of schizophrenia at least in this group of Spanish schizophrenic patients. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:88-92, 2000.

Trisomy/tetrasomy 21 mosaicism in CVS: interpretation of cytogenetic discrepancies between placental and fetal chromosome complements.

Trisomy/tetrasomy 21 mosaicism was found in chorionic villi (semidirect preparation) obtained from a 40 year old pregnant woman. Since both cell lines were abnormal, the couple elected for pregnancy termination. Placenta and fetal tissue samples were obtained for cytogenetic study. Long term cultured villi showed a non-mosaic trisomy 21 karyotype, while other tissues showed either a normal karyotype or normal/trisomy21 mosaicism. These discrepancies could be explained by a modified "bottle neck" embryogenic model with a trisomic zygote and a non-disjunction event taking place in one of the first divisions. Our case emphasises the need for confirmatory studies in other tissues when mosaicism is encountered in chorionic villi, even if all cell lines are abnormal.

Parental origin and meiotic stage of non-disjunction in 139 cases of trisomy 21.

The parental origin and the meiotic stage of non-disjunction have been determined in 139 Down syndrome patients with regular trisomy 21 and in their parents through the analysis of DNA polymorphism. The meiotic error is maternal in 91.60% cases and paternal in 8.39% of cases. Of the maternal cases, 72.41% were due to meiosis I errors (MMI) and 27.58% were due to meiosis II errors (MMII). Of the paternal cases, 45.45% were due to meiosis I (PMI) and 54.54% were due to meiosis II (PMII). The mean maternal ages were 31.6 +/- 5.3 (+/- SD) years in errors from MMI, 32.3 +/- 6.4 years in errors from MMII, 31.4 +/- 4.6 years in errors from PMI and 29.5 +/- 2.7 years in errors from PMII. No significant statistical differences were observed between maternal and paternal errors, further supporting the presence of a constant chromosome 21 non-disjunction error type.

Apolipoprotein E epsilon 4 alleles and meiotic origin of non-disjunction in Down syndrome children and in their corresponding fathers and mothers.

An increased risk of Alzheimer disease (AD) has been reported in young mothers of Down syndrome (DS) probands. Subsequently, an increased frequency of the apolipoprotein E (apoE) allele epsilon 4 has been found in mothers (< or = 32 years) of DS children due to meiosis II (MII) errors providing a potential explanation for the increased risk of AD in DS mothers. In the present study we genotyped apoE and determined the origin of non-disjunction of 132 mothers and the corresponding fathers and DS children from Spain. Unexpectedly no epsilon 4 alleles have been detected in MII mothers of < or = 32 years of age (P = 0.02). Thus our study not only fails to find the effect previously reported, but it detects an opposite correlation. An increase in the epsilon 4 frequency (0.227) is detected in MI mothers <28 as compared to the epsilon 4 frequency present in MI mothers >28 years of age (0.089), although the differences are not significant if correction for multiple comparisons is applied. The simplest overall interpretation of the previously reported and present findings is that the detected associations are due to random statistical variation rather than to some real effect of the epsilon 4 allele. However the important potential implications of alternative explanations imply that this issue deserves further clarification in independent studies in other populations.

Cloning, sequencing and characterization of the rat hereditary hemochromatosis promoter: comparison of the human, mouse and rat HFE promoter regions.

We have cloned and sequenced 1398bp of the rat HFE gene promoter region. The alignment of the rat promoter HFE sequence with the HFE promoter sequence from human and mouse detected several highly conserved sequences present at orthologous or heterologous positions in the three species. Subsequent analysis of the conserved promoter sequences identified the presence of 10 novel transcription elements present in the promoter regions of the human, mouse and rat HFE genes (GATA, NF-IL6, AP1, AP2, CREB, PEA3, gamma-IRE, GFI1, HNF-3beta, HFH2). Different gel retardation analyses performed with rat-liver nuclear extracts have confirmed the presence of factors binding to some of these transcription elements. This represents the first data concerning the identification of potential transcriptional elements of the HFE promoter in these three species. The expression pattern of the transcription factors corresponding to the novel elements identified in the HFE promoter is consistent with the potential role of the HFE promoter in the transcription regulation and function of the HFE gene. Knowledge of the identified conserved elements in the HFE promoter from human, mouse and rat provides the basis for subsequent in-vitro or in-vivo studies leading to identification of the detailed mechanisms involved in the regulation of the iron metabolism and the design of potential future alternative therapies.

Evolution of protamine P1 genes in mammals.

Protamine P1 genes have been sequenced following PCR amplification from 11 mammals representing five major mammalian orders: Rodentia (rat and guinea pig), Carnivora (cat and bear), Proboscidea (elephant), Perissodactyla (horse), and Artiodactyla (camel, deer, elk, moose, and gazelle). The predicted amino acid sequence for these genes together with previously reported sequences results in a data set of 25 different P1 genes and 30 different P1 amino acid sequences. The alignment of all these sequences reveals that protamines are amongst the most rapidly diverging proteins studied. In spite of the large number of differences there are conserved motifs that are also common to birds such as the N-terminal ARYR followed by the triple alternating SRSRSR phosphorylation site. The central region contains 3 arginine clusters consisting of 5-6 arginines each. The C-terminus appears to be the most variable region of the protamines. Overall the molecular evolution of P1 genes is in agreement with the expected species evolution supporting that these genes have evolved vertically.

Demonstration of trans-acting factors binding to the promoter region of the testis-specific rat protamine P1 gene.

Potential regulatory DNA elements within the rat protamine P1 promoter region have been identified using gel retardation assays and DNase I footprinting analysis with rat nuclear extracts obtained from different tissues. Distinctive gel shift bands are generated by rat nuclear extracts from mature testis, kidney, brain, spleen and liver, which bind to the cis-acting SRE (Serum response element) and Prot1C (Protamine 1 Consensus) oligonucleotide sequences [Queralt R. and Oliva R. Gene. 133 (1993) 197-204]. In vitro DNase I footprinting analysis demonstrates protection of the SRE region at positions -124 to -115. In addition, we have detected protection in two new regions adjacent to the SRE that we called SAP (SRE Adjoining Protection; nt -153 to -141) and SEP (SRE Extended Protection; nt-114 to -100), respectively. These sequences, SAP and SEP, show no apparent consensus homology with cis-acting elements of other known transcription factors. Two additional DNase I protected regions have also been found at positions +27 to +46 and +61 to +88, the first of which contains the sequence +42 TCGNNNNNGCCAA +30 recognized by nuclear factor I (NFI).

Direct sequencing of the human protamine P1 gene and application in forensic medicine.

Protamines are among the most variable nuclear proteins known in eukaryotes. In order to learn more about their evolution and function in humans and to explore the possibility of potential applications in forensic medicine we have developed a rapid method to amplify and directly sequence the protamine P1 gene simultaneously in many different samples. The method takes only 3.5 h from genomic DNA to the sequencing reactions. Despite the high variability of these genes only one polymorphic site was detected at the coding region level in different individuals. This polymorphic variation does not create a change in the amino-acid sequence of the protamine. Because all the protamine genes sequenced from different species are markedly different among them as well as to the human sequence, amplification and direct sequencing of this gene can be used to unequivocally identify the human or animal origin of biological specimens. Furthermore, the single polymorphic site detected in the human P1 gene could be useful in conjunction with other markers in identification studies in humans.

Identification of conserved potential regulatory sequences of the protamine-encoding P1 genes from ten different mammals.

In order to detect regulatory conserved DNA elements within the protamine 1-encoding gene (P1) promoter, we have sequenced this region from the rat, guinea pig, gorilla, orangutan, anubis baboon and red monkey P1 genes and compared it to the homologous human, bull, boar and mouse nucleotide (nt) sequences. We demonstrate the presence of a consensus sequence, HSMCYTCAYAAT (Prot1C: protamine 1 consensus), from nt position -64 to position -53 in all P1 genes whose promoter sequences are now known. We also show that sequences similar to Prot1C are found in the promoter region of other testis-specific genes, such as the transition protein 1-encoding gene promoter which is thought to have derived from the P1 genes. The relevance of this conserved element in the expression of P1 genes is strongly supported by the recent demonstration of a mouse testis trans-acting factor [Tet-1; Tamura et al., J. Biol. Chem. 267 (1992) 4327-4332] which binds and matches in the mouse the first 11 bp of the corresponding consensus Prot1C sequence reported here. Another highly conserved element (TGTGAGG) has been identified 20 +/- 3 nt upstream from Prot1C. This sequence forms a perfect palindrome with the central 7 nt of Prot1C and is absent in the homologous region of other genes. Further upstream, at positions -113 to -132, a third highly conserved region is present (MATGCCCATATWTGGRCAYG) which is similar to the c-fos SRE (serum-response element) and contains the central core common to all SREs. This element has not been found in the homologous region of other sperm-specific genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Evolution of protamine P1 genes in primates.

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine P1s indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10-13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24-29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39-44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest-neighbor matrices and trees based on maximum parsimony indicates that P1 genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine P1s are expressed in germ line rather than somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)