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Two-dimensional target characterization using inverse profiling approaches with total-field phaseless data is discussed. Two different inversion schemes are compared. In the first one, the intensity-only data are exploited in a minimization scheme, thanks to a proper definition of the cost functional. Specific normalization and starting guess are introduced to avoid the need for global optimization methods. In the second scheme [J. Opt. Soc. Am. A21, 622 (2004)], one exploits the field properties and the theoretical results on the inversion of quadratic operators to derive a two-step solution strategy, wherein the (complex) scattered fields embedded in the available data are retrieved first and then a traditional inverse scattering problem is solved. In both cases, the analytical properties of the fields allow one to properly fix the measurement setup and identify the more convenient strategy to adopt. Also, indications on the number and types of sources and receivers to be used are given. Results from experimental data show the efficiency of these approaches and the tools introduced.

A new solution approach to inverse scattering from aspect-limited phaseless measurements of the total field is introduced and discussed. In analogy with the case of measurements on closed curves [J. Opt. Soc. Am. A21, 622 (2004)], the procedure splits the problem into two different steps. In the first step, amplitude and phase of the scattered field are estimated from only amplitude information of the total field. By properly extending the concept of reduced radiated field to the case of scattered fields (as a function of both illumination and measurement variables) and taking advantage of the properties of the square amplitude distribution of the total field, criteria are given for an optimal choice of the measurement setup and a successful retrieval. Then the complex permittivity profile is reconstructed in the second step, starting from the scattered fields estimated in the previous step. Numerical examples are provided to assess the effectiveness of the whole chain in the presence of noise-corrupted data and the relevance of the representation introduced for the scattered fields.

A new approach for quantitative electromagnetic imaging of scatterers located in free space from phaseless data is proposed and discussed. The procedure splits the problem into two steps. In the first one, we solve a phase-retrieval problem for the total field, thus estimating the amplitude and phase of the scattered field. Careful analysis of properties and possible representations of both scattered and incident fields allow us to introduce a criterion for an optimal choice of the measurement setup and a successful retrieval. Then the complex permittivity profile is reconstructed in the second step by use of the estimated scattered field. Numerical examples are provided to check the whole chain in the presence of noise-corrupted data.

X-linked nonspecific mental retardation (MRX) accounts for ~25% of mental retardation in males. A number of MRX loci have been mapped on the X chromosome, reflecting the complexity of gene action in central nervous system (CNS) specification and function. Eleven MRX genes have been identified, but many other causative loci remain to be refined to the single gene level. In 21 MRX families, the causative gene is located in the pericentromeric region; and we report here the identification by linkage analysis of a further such locus, MRX81. The new MRX locus was identified by two- and multi-point parametric analysis carried out on a large Italian family. Tight linkage of MRX81 to DNA markers ALAS2, DXS991, and DXS7132 was observed with a maximum LOD score of 3.43. Haplotype construction delineates an MRX81 critical region of 8 cM, the smallest MRX pericentromeric interval so far described, between DXS1039 and DXS1216, and placing it in Xp11.2-Xq12. So far, automated sequencing of two candidates in the region, the MRX gene oligophrenin (OPHN1) and the brain-specific ephrinB1 (EFNB1) gene, in DNA from affected males excluded their candidacy for MRX81, suggesting a novel disease gene. © 2003 Wiley-Liss, Inc.

No abstract.

The region Xq21.3/Yp11.1 represents the largest segment of homology between the sex chromosomes in humans, though no recombination occurs in male meiosis. It presumably arose as a transposition from the X to the Y chromosome; the present-day organization in the latter chromosome indicates a paracentric inversion that disrupted its continuity. Moreover, an X-specific block (defined by the marker DXS214) is embedded in the region. Previously, no hypotheses about the length, origin, or evolution of this X-specific segment have been proposed. Here we report on the refinement of the size and the sequence of the distal boundary of the X-specific block. Furthermore, we have tracked by FISH experiments the evolution of this region in primates. This further clarifies the multistep mechanism of origin for the XY homology region, by demonstrating that the X-specific block was deleted from the Y chromosome after the initial transfer from the X chromosome.

A full-length mouse glucose-6-phosphate dehydrogenase (G6PD) cDNA has been isolated and sequenced, and the evolutionary conservation of many portions of the sequence has been verified by comparison with that of human and other sources.

A contig of 20 yeast artificial clones (YACs) has been assembled across 1.5 Mb of Xq28 and formatted with nine previously reported probes and nine STSs developed from the sequence of probes and end fragments of YACs. YAC end fragments were obtained by subcloning, Alu-vector PCR, or primer-ligation PCR methods. Eighteen of the YACs were recovered from a library specific for Xq24-q28; two that fill a gap were obtained from a second library made from total human DNA. One region, containing probes pX78c and 2A1.1, was unstable in YACs, but it was possible to generate a self-consistent map of DNA over the entire contig. Overlaps were confirmed by Southern blot analyses of YAC DNAs, and pulsed-field gel electrophoresis confirmed the extent of the contig and identified at least four CpG islands in the region. Copyright 1993, 1999 Academic Press

The a-galactosidase A activity from fibroblasts of five Fabry patients and five controls has been separated from a-galactosidase B through small DEAE-cellulose columns and in some experiments by treatment of the fibroblast extracts with Sepharose coupled to anti-a-galactosidase B antibodies. By these independent methods, it has been shown that there is a residual a-galactosidase A in Fabry's disease, which is immunologically similar to the a-galactosidase A from the controls. The a-galactosidase A from all of the patients and controls has the same apparent Km value for the synthetic substrate 4-methylumbelliferyl-a-galactoside. Four out of five patients have a thermostable a-galactosidase A, while the fifth has a thermolabile enzyme like that from the controls. The amount of immunologically active a-galactosidase A seems to be decreased in the patients tested.

One hundred nineteen YACs were assembled into 6 contigs spanning about 7.1 Mb of Xq28. The contigs were formatted with 65 STSs and 136 hybridization probes and were extensive enough to be aligned and oriented by published genetic linkage and somatic cell hybrid panel data. Selected YACs from the entire region were mapped with five rare-cutter restriction enzymes to infer the position of putative CpG islands indicative of gene locations; 48 such sites were identified by the near-coincidence of at least three rare-cutter sites. The analysis defined three subregions of Xq28: 4 Mb of moderate GC and CpG island content from the Xq27 border through the GABRA locus; 1.5 to 2 Mb, extending to the G6PD gene, that is variably and poorly cloned, but contains a high concentration of CpG islands and GC; and about 1.5 Mb between G6PD and the telomere, which is generally low in CpG and GC levels, including a subtelomeric DNA region that shows extensive homology to Yq DNA. Copyright 1994, 1999 Academic Press

The human Xp11.23–p11.22 interval has been implicated in several inherited diseases including Wiskott–Aldrich syndrome; three forms of X-linked hypercalciuric nephrolithiaisis; and the eye disorders retinitis pigmentosa 2, congenital stationary night blindness, and Åland Island eye disease. In constructing YAC contigs spanning Xp11.23–p11.22, we have previously shown that the region around the synaptophysin (SYP) gene is refractory to cloning in YACs, but highly stable in cosmids. Preliminary analysis of the latter suggested that this might reflect a high density of coding sequences and we therefore undertook the complete sequencing of a SYP-containing cosmid. Sequence data were extensively analyzed using computer programs such as CENSOR (to mask repeats), BLAST (for homology searches), and GRAIL and GENE-ID (to predict exons). This revealed the presence of 29 putative exons, organized into three genes, in addition to the 7 exons of the complete SYP coding region, all mapping within a 44-kb interval. Two genes are novel, one (CACNA1F) showing high homology to α1 subunits of calcium channels, the other (LMO6) encoding a product with significant similarity to LIM-domain proteins. RT-PCR and Northern blot studies confirmed that these loci are indeed transcribed. The third locus is the previously described, but not previously localized, A4 differentiation-dependent gene. Given that the intron–exon boundaries predicted by the analysis are consistent with previous information where available, we have been able to suggest the genomic organization of the novel genes with some confidence. The region has an elevated GC content (>53%), and we identified CpG islands associated with the 5′ ends of SYP, A4, and LMO6. The order of loci was Xpter–A4–LMO6–SYP–CACNA1F–Xcen, with intergenic distances ranging from ∼300 bp to ∼5 kb. The density of transcribed sequences in this area (>80%) is comparable to that found in the highly gene-rich chromosomal band Xq28. Further studies may aid our understanding of the long-range organization surrounding such gene-enriched regions.

The contents of Alu- and L1-containing TaqI restriction fragments were assessed by Southern blot analyses across YAC contigs already assembled by other means and localized within Xq24-q28. Fingerprinting patterns of YACs in contigs were concordant, and using software based on that of M. V. Olson et al. (1986, Proc. Natl. Acad. Sci. USA 83: 7826) to analyze digitized data on fragment sizes, fingerprinting itself could establish matches among about 40% of a test group of 435 YACs. At 100-kb resolution, both repetitive elements were found throughout the region, with no apparent enrichment of Alu or L1 in DNA of G compared to that found in R bands. However, consistent with a random overall distribution, delimited regions of up to 100 kb contained clusters of repetitive elements. The local concentrations may help to account for the reported differential hybridization of Alu and L1 probes to segments of metaphase chromosomes. Copyright 1993, 1999 Academic Press

A yeast artificial chromosome sequence-tagged site-based (YAC/STS) physical map of 22.5 Mb of the Xq24–q26 cytogenetic band region of the human X chromosome has been assembled. DNA coverage includes 857 large-insert clones formatted with 405 STSs to provide ninefold depth of DNA. At five points, no bridging clones have been recovered from 20 X-chromosome equivalents of human DNA in YACs or bacterial clones, but the placement of 25 (“CA”)npolymorphic markers permits the ordering of contigs by comparison with the genetic linkage map and radiation hybrid data. The map localizes the X3000 translocation breakpoint and six genes (ANT2, NDUFA1, LAMP2, OCRL, IGSF1, and HDGF) at better than 100-kb resolution. The relatively gene-poor nature of the region is consistent with relatively low uniform 34–42% GC content in STSs across nearly all of the region.

A patient with primary thrombocythemia, who was heterozygous for glucose-6-phosphate dehydrogenase deficiency (GdB/GdMed), was investigated to test for the clonal origin of this myeloproliferative disorder. In order to assess somatic cell mosaicism in various tissues, we have made use of the different rate of utilization of 2-deoxyglu-cose-6-phosphate, an analog of glucose-6-phosphate, by normal glucose-6-phosphate dehydrogenase and by the Mediterranean variant: the results demonstrate that essential thrombocythemia is a clonal disease involving the erythrocytic, granulocytic, and megakaryocytic series, without affecting monocytes, T lymphocytes, and non-T lymphocytes.

Human-hamster somatic cell hybrids have been obtained by fusion of a CHO line (NA31) doubly deficient in hypoxanthine guanine phosphoribosyltransferase and glucose 6-phosphate dehydrogenase (G6PD) with normal G6PD(+) human fibroblasts. Analysis of NA31 extracts has revealed that, although G6PD activity is nearly absent, significant activity can be detected with 2-deoxyglucose 6-phosphate as substrate, so that the mutant and normal forms of the enzyme can both be easily detected. The cell hybrids obtained express human G6PD. The human G6PD subunits are distributed in homodimeric molecules as well as in human-hamster heterodimeric molecules. However, whereas the amount of hamster G6PD subunits present in the hybrid is similar to that in the hamster parental cells, the amount of human G6PD subunits is decreased by 3- to 10-fold when compared to the human parental cell. These results indicate that either the expression of the G6PD gene or the stability of the gene product is altered in the hybrid. By mutagenesis and selection in diamide (a substance that oxidizes intracellular glutathione), we have isolated a clone with a 3- to 5-fold increase in human G6PD activity. This derivative may have an increased rate of expression of the human G6PD structural gene.

Most genes on the X chromosome undergo “inactivation,” being transcribed from only one copy in female somatic cells, but several human genes have been shown to be expressed from both the active and the otherwise inactivated homologue. To assess further the fraction and location of genes that escape inactivation, we have analyzed the inactivation status of a set of 73 expressed sequence tags that were derived from the sequencing of cDNA collections and mapped to the X chromosome. Of 33 that were expressed in cultured cells, as assessed by reverse transcription and PCR, 4 (about 12%) were transcribed from both the active and the inactive X chromosome. Two, RPS4 and PCTAIRE1, are already known to escape inactivation; the other 2, of unknown function, include a short cDNA with a full open reading frame and a transcript with no detectable open reading frame. They map, respectively, to Xp11.3–p11.4 and Xp22.2; both regions were previously reported to encode sequences transcribed from the inactive X. Neither transcript has a corresponding sequence on the Y. Thus, they exhibit double dosage in females compared to males, and inactivation status may be inconsequential for these transcribed sequences.

A cosmid containing 36.4 kb of high GC human DNA centromeric to the G6PD gene has been analyzed. The sequence was 99.9% precise, based on the comparison of 4.3 kb that overlaps an earlier analysis of 20.1 kb containing G6PD. Properties of the entire 52 kb region that may be characteristic of high GC portions of the genome include a very high density of sixty-two half or full Alu sequences, or 1,2/kb, and an absence of L1 sequences. Other highly repetitive sequences include 11 MER sequences, one of them interrupted at two positions by groups of 3 Alu elements. In segments of unique sequence, computer-aided analysis predicted three possible genes, one of which has thus far been confirmed by the recovery of a corresponding cDNA, both by a direct hybridization method and by a PCR-based method based on a primer pair inferred from the genomic sequence. The cDNA has been sequenced, and is completely concordant with counterpart genomic sequence; it has no resemblance to any previously described gene.

The X and Y chromosomes that maintain human dimorphism are thought to have descended from a single progenitor, with the Y chromosome becoming largely depleted of genes1. A number of genes, however, retain copies on both X and Y chromosomes and escape the inactivation that affects most X-linked genes in somatic cells2. Many of those genes are present in two pseudoautosomal regions (PARs) at the termini of the short (p)3and long (q)4,5arms of the sex chromosomes. For both PARs, pairing facilitates the .exchange of information, ensuring the homogeni-sation of X and Y chromosomal material in these regions6–10. We report here a strikingly different regulation of expression of a gene in Xq PAR. Unlike all Xp PAR genes studied so far, a synaptobrevin-like gene, tentatively named SYBL1, undergoes X inactivation. In addition, it is also inactive on the Y chromosome, thereby maintaining dosage compensation in an unprecedented way.

Actin-binding protein-280 (ABP-280) is a dimeric actin filament crosslinking protein that promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. We have mapped the ABP-280 filamin gene (FLN) to Xq28 by Southern blot analysis of somatic cell hybrid lines, by fluorescence in situhybridization, and through identification of portions of the FLN gene within cosmids and YACs mapped to Xq28. The FLN gene is found within a 200-kb region centromeric to the G6PD locus and telomeric to DSX52 and the color vision locus.

Sequences located several kilobases both 5′ and 3′ of the stably transcribed portion of several genes hybridize to radio-labeled pure fragments of the alternating sequence poly (dG-dT)(dC-dA) [poly(GT)]. The genes include the ribosornal DNA of mouse, rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300 bp of adjoining DNA show perfect runs of poly(GT) (>30bp) in all but the human 5′ region of rDNA, which shows a somewhat different alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of the locations of tracts of alternating purlne:pyrimidine between evolutionarily diverse species is consistent with a possible functional role for these sequences.

Glucose-6-phosphate dehydrogenase (G6SPD) is an ubiquitous enzyme which by determinig the NADPH level has a crucial role in NADPH-mediated reductive processes in all cells (1). The structural gene for G6PD, Gd, is X-linked in mammals and on the basis of its expression in many tissues, it can be regarded as a typical “housekeeping” gene (2). Over 300 variants of the protein are known, many of which have deficient enzyme activity. Nearly 100 of these variants are polymorphic in various populations (3). The mammalian enzyme is a homodimer or a homotetramer with a subunit moleculat weight of {small tilde} 54000 daltons (4). Here we report the isolation of cDNA clones from HeLa cells, SV40-transformed human fibroblasts, human placenta and human teratocarcinoma cell lines. These clones have enabled us to sequence the entire coding region of Gd . Thus, the entire amino acid sequence of human G6PD is provided for the first time. This work is the first step for structural analysis of G6PD variants and for an understanding of the biological features of this enzyme at the molecular level .

Starting from sequences of at least 60 bp, PCR-based screening has been developed to recover cDNAs from libraries without the necessity for hybridization or extensive DNA extraction steps. The method maintains the indefinite availability of even scarce cDNA libraries and provides an estimate of the relative abundance of the mRNA species. Isolation of a cDNA clone can be done in less than a week. cDNAs were isolated that were cognate for fragments of expressed sequences and for an exon predicted from genomic sequence.