Phase-stepped fringe projection by rotation about the camera's perspective center.

A technique to produce phase steps in a fringe projection system for shape measurement is presented. Phase steps are produced by introducing relative rotation between the object and the fringe projection probe (comprising a projector and camera) about the camera's perspective center. Relative motion of the object in the camera image can be compensated, because it is independent of the distance of the object from the camera, whilst the phase of the projected fringes is stepped due to the motion of the projector with respect to the object. The technique was validated with a static fringe projection system by moving an object on a coordinate measuring machine (CMM). The alternative approach, of rotating a lightweight and robust CMM-mounted fringe projection probe, is discussed. An experimental accuracy of approximately 1.5% of the projected fringe pitch was achieved, limited by the standard phase-stepping algorithms used rather than by the accuracy of the phase steps produced by the new technique.

Magnetic resonance imaging in the diagnosis of sacral stress fracture.

Low back and buttock pain in athletes can be a source of frustration for the athlete and a diagnostic dilemma for the doctor. Sacral stress fractures have been increasingly recognised as a potential cause of these symptoms. As plain radiographs are often normal and the radiation load of an isotope bone scan is substantial, the alternative use of magnetic resonance imaging in the diagnosis of a sacral stress fracture is highlighted in this case report.

Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress.

Chronic exposure (>200 days) of HA1 fibroblasts to increasing concentrations of H2O2 or O2 results in the development of a stable oxidative stress-resistant phenotype characterized by increased cellular antioxidant levels, particularly catalase (D. R. Spitz et al, Arch. Biochem. Biophys., 279: 249-260, 1990; D. R. Spitz et al., Arch. Biochem. Biophys., 292: 221-227, 1992; S. J. Sullivan et al., Am. J. Physiol. (Lung Cell. Mol. Physiol.), 262: L748-L756, 1992). Acutely stressed cells failed to develop a stably resistant phenotype or increased catalase activity, suggesting that chronic exposure is required for the development of this phenotype. This study investigates the mechanism underlying increased catalase activity in the H2O2- and O2-resistant cell lines. In H2O2- and O2-resistant cells, catalase activity was found to be 20-30-fold higher than that in the parental HA1 cells and correlated with increased immunoreactive catalase protein and steady-state catalase mRNA levels. Resistant cell lines also demonstrated a 4-6-fold increase in catalase gene copy number by Southern blot analysis, which is indicative of gene amplification. Chromosome banding and in situ hybridization studies identified a single amplified catalase gene site located on a rearranged chromosome with banding similarities to Z-4 in the hamster fibroblast karyotype. Simultaneous in situ hybridization with a Z-4-specific adenine phosphoribosyltransferase (APRT) gene revealed that the amplified catalase genes were located proximate to APRT on the same chromosome in all resistant cells. In contrast, HA1 cells contained only single copies of the catalase gene that were not located on APRT-containing chromosomes, indicating that amplification is associated with a chromosomal rearrangement possibly involving Z-4. The fact that chronic exposure of HA1 cells to either HO2 or 95% O2 resulted in gene amplification suggests that gene amplification represents a generalized response to oxidative stress, contributing to the development of resistant phenotypes. These results support the hypothesis that chronic exposure to endogenous metabolic or exogenous environmental oxidative stress represents an important factor contributing to gene amplification and genomic instability.

Construction of a pilot human YAC library in a recombination-defective yeast strain.

Using high-molecular-weight DNA fragments from a human lymphoblastoid cell line, a pilot collection of 2500 YACs was constructed in YKK115, a recombination-deficient strain of Saccharomyces cerevisiae carrying mutations in both the rad51 and rad52 genes. Analysis of 520 clones from the current library by pulsed-field gel electrophoresis revealed more than 97% single YACs with an insert size averaging 340 kb. Fluorescent in situ hybridization (FISH) performed with 37 clones on metaphase chromosomes suggested a high proportion mapping at centromeric (7) or telomeric (4) locations. The results are consistent with the stabilization of YACs in strains disarmed in recombination functions [Kohno, K., Oshiro, T., Kishine, H., Wada, M., Takeda, H., Ihara, N., Imamoto, F., Kano, Y. and Schlessinger, D. (1997) Human YACs unstable in a rad52 single mutant strain become stable in rad51rad52 double mutant. Gene, 000, 000-000 (GENE 10429)], and further suggest that the YACs may include regions that have been difficult to clone in other strains.

Linkage and physical mapping of X-linked lissencephaly/SBH (XLIS): a gene causing neuronal migration defects in human brain.

While disorders of neuronal migration are associated with as much as 25% of recurrent childhood seizures, few of the genes required to establish neuronal position in cerebral cortex are known. Subcortical band heterotopia (SBH) and lissencephaly (LIS), two distinct neuronal migration disorders producing epilepsy and variable cognitive impairment, can be inherited alone or together in a single pedigree. Here we report a new genetic locus, XLIS, mapped by linkage analysis of five families and physical mapping of a balanced X;2 translocation in a girl with LIS. Linkage places the critical region in Xq21-q24, containing the breakpoint that maps to Xq22.3-q23 by high-resolution chromosome analysis. Markers used for somatic cell hybrid and fluorescence in situ hybridization analyses place the XLIS region within a 1 cM interval. These data suggest that SBH and X-linked lissencephaly are caused by mutation of a single gene, XLIS, that the milder SBH phenotype in females results from random X-inactivation (Lyonization), and that cloning of genes from the breakpoint region on X will yield XLIS.

X chromosome map at 75-kb STS resolution, revealing extremes of recombination and GC content.

A YAC/STS map of the X chromosome has reached an inter-STS resolution of 75 kb. The map density is sufficient to provide YACs or other large-insert clones that are cross-validated as sequencing substrates across the chromosome. Marker density also permits estimates of regional gene content and a detailed comparison of genetic and physical map distances. Five regions are detected with relatively high G + C, correlated with gene richness; and a 17-Mb region with very low recombination is revealed between the Xq13.3 [XIST] and Xq21.3 XY homology loci.

4.5-Mb YAC STS contig at 50-kb resolution, spanning Xq25 deletions in two patients with lymphoproliferative syndrome.

Sequence-tagged site (STS) content mapping in yeast artificial chromosomes (YACs) was used to cover the region deleted in two patients affected with X-linked lymphoproliferative disorder. The order of markers includes, centromere to telomere, DXS8009-DXS1206-DXS8078-DXS8044-DXS982- DXS6811-DXS8093-AFM240xblO- DXS75-DXS737-DXS100-DXS6-DXS1046-DXS803 8. The order of six major markers is confirmed by fluorescent in situ hybridization, and all the markers assigned by linkage mapping fall within a 1.6-cM interval. The contig comprises 90 clones containing 89 STSs, yielding a resolution of 50 kb; DNA in a gap just telomeric to DXS8044 has not been found in > 20 equivalents of YACs or bacterial clones. The two deletions were found to have centromeric breakpoints that lie close to DXS1206 and may be identical; the telomeric breakpoints are -150 kb apart, one falling between DXS737 and DXS100, the other between DXS100 and DXS1046. Several STSs near the breakpoints show weak amplification from more than one site; one gives products from three groups of YACs, and lie, respectively, within 50 kb of the centromeric and the two telomeric deletion borders. Such partially duplicated segments of DNA are candidates for involvement in the formation of the deletions.

Amplification and altered expression of the hsc70/U14 snoRNA gene in a heat resistant Chinese hamster cell line.

We have recently demonstrated that the heat resistant phenotype of the HR-1 variant isolated from HA-1 Chinese hamster fibroblasts after a series of heat shocks is associated with the increased expression of Hsc70, the constitutive form of Hsp70 (Laszlo and Li 1985). Here, we report the cloning and characterization of the Chinese hamster hsc70 gene and its organization and expression in wild type HA-1 and permanently heat resistant HR-1 cells. DNA sequencing revealed that the structure and nucleotide sequence of the hamster hsc70 gene is highly homologous to the human and rat genes coding for Hsc70. Three of the eight introns of the hamster hsc70 gene encode U14 small nucleolar RNAs, as has been demonstrated in other species. Although putative transcriptional elements, including a TATA box, two inverted CAT boxes, and two sets of heat shock elements (HSEs) are completely conserved in the human and hamster hsc70 genes, the regulation of expression of the hamster hsc70 gene is different from that reported for its human counterpart in that the mRNA coding for Hsc70 increases at least 10-fold after a mild heat shock in Chinese hamster cells while no induction of Hsc70 by heat shock has been reported in human cell lines. In situ hybridization revealed a complex chromosomal rearrangement in HR-1 cells which results in the 4- to 5-fold amplification of the hsc70 gene as indicated by genomic Southern blots. In association with this amplification of the hsc70 gene, the levels of Hsc70 mRNA and U14 snoRNA are increased in the HR-1 cells under both normal growing conditions and after heat shock. Thus, the elevated expression of both Hsc70 and U14 snoRNA might play a role in the heat resistant phenotype of the HR-1 cells. This is the first report of the amplification of a heat shock gene and the possible induction of gene amplification by heat shock.

MRI diagnosis of accessory soleus muscle strain.

An accessory soleus muscle is a rare anatomical variant. Clinical awareness of this entity and the appropriate use of magnetic resonance imaging (MRI) can yield a definitive diagnosis in those patients with symptomatic accessory muscles. A case is presented in which the unique sensitivity of MRI in the evaluation of exertional muscle injuries proved useful in the detection of an accessory soleus muscle strain.

A human calcium-activated potassium channel gene expressed in vascular smooth muscle.

Large-conductance Ca(2+)-activated K+ (BK) channels are widespread and functionally heterogeneous. In other classes of K+ channels, functional heterogeneity derives from large gene families, alternative splicing, heterologous subunit composition, and functional modulation. The molecular basis of mammalian BK channel heterogeneity is unknown, since only a single gene (mSlo) has been identified. BK channels in native vascular smooth muscle have an apparent Ca2+ sensitivity approximately 10-fold greater than native brain or skeletal muscle channels, or cloned mSlo channels. Using mSlo as a low-stringency probe, we screened human arterial smooth muscle and genomic libraries extensively in search of genes or splice variants with novel properties. We isolated the human homologue of mSlo, including two novel splice variant forms, but found no other related genes. Electrophysiological characterization of the hSlo clones in Xenopus oocytes and Chinese hamster ovary cells gave BK currents that were not measurably different from mSlo currents. However, coexpression of hSlo with a recently cloned beta-subunit derived from smooth muscle dramatically increased apparent Ca2+ sensitivity. Thus alpha-subunits alone may not determine Ca2+ sensitivity of vascular smooth muscle BK channels. hSlo was mapped to human chromosome 10q23.1, and the genomic structure was analyzed. Immediately after the amino terminal, two unusual regions of trinucleotide repeating sequences are present. The first of these regions encodes polyglycine, and the second encodes polyserine. Both regions of repeated sequence are conserved between the mouse and human genome.

YAC contigs mapping the human COL4A5 and COL4A6 genes and DXS118 within Xq21.3-q22.

Sequence-tagged sites (STSs) were developed for three loci of uncertain X chromosomal localization (DXS122, DXS137, and DXS174) and were used to seed YAC contigs. Two contigs now total about 3.3 Mb formatted with 34 STSs. One contains DXS122 and DXS174 within 250 kb on single YACs; it is placed in Xq21.3-q22.1 by FISH analysis, which is consistent with somatic cell hybrid panel analyses and with the inclusion of a probe that detects polymorphism at the DXS118 locus already assigned to that general region. The other contig, which contains DXS137, is in Xq22.2 by FISH, consistent with cell hybrid analyses and with the finding that it covers the human COL4A5 and COL4A6 genes known to be in that vicinity. In addition to extending the cloned coverage of this portion of the X chromosome, these materials should aid, for example, in the further analysis of Alport syndrome.

Positional cloning of cDNAs from the human chromosome 3p21-22 region identifies a clustered organization of zinc-finger genes.

The human 3p21-22 region is frequently involved in karyotype rearrangements associated with malignancies. The high frequency of allelic loss in this region has been associated with virtually all small cell lung carcinomas and many renal carcinomas. These findings suggest that at least one tumor-suppressor gene might be located in 3p21-22. We have recently reported the isolation of a 750-kb yeast artificial chromosome (YAC) contig from 3p21-22. Here, we describe three new genes isolated from the 3p YAC contig by using a cDNA hybridization selection. Remarkably, the three new genes encode zinc-finger proteins, indicating the presence of a cluster of zinc-finger genes in human chromosome 3p21.

Exclusion of chromosomal mosaicism in amniotic fluid cultures: determination of number of colonies needed for accurate analysis.

Most laboratories use the in situ or flask culture method and a two-stage approach to mosaicism detection. Determination of the optimum number of metaphases to be counted depends largely on whether or not the colonies that grow from the cells in the amniotic fluid can be considered independent. Previous statistical analysis of data from mixed male and female amniocyte cultures indicated that for mosaicism detection these colonies can be considered independent (Cheung et al., 1990). This analysis was repeated with a set of mosaic cases from two independent prenatal diagnosis programmes. The same degree of colony independence was found with this set of data. This result was used to calculate the level of mosaicism that is excluded at a particular confidence level when set numbers of colonies are analysed at each stage. The tables generated apply directly to the in situ method and with modification they can be used with the flask method. The conclusions are (1) analysis of cells from multiple colonies enhances the likelihood of excluding true mosaicism; (2) analysis of more than one metaphase per colony offers little advantage in excluding mosaicism; and (3) the two-stage approach is the most efficient. These conclusions should be used together with the expected clinical outcome of the actual cytogenetic abnormality, as discussed by Hsu et al. (1992).

Cloning and chromosomal localization of the gene coding for human protein kinase CK1.

A cDNA clone coding for human protein kinase CK1 (casein kinase 1) has been isolated and sequenced demonstrating that it corresponds to a homolog of the CK1 alpha form found in bovine brain. The derived amino acid sequence of the human CK1 alpha is identical to the bovine counterpart except that it contains 12 extra amino acids at the carboxyl end. Using this cDNA sequence and PCR amplification, YAC genomic clones that contain this human CK1 alpha sequence have been isolated. These YACs have been used for fluorescent in situ hybridization in order to localize the human CK1 alpha gene to chromosome 13q13.

Magnetic resonance imaging in sports medicine--an overview.

As more Britons become health conscious and are involved in a variety of sporting events, sports-related injuries are a daily occurrence. Although evaluation of bony abnormalities resulting from acute and chronic sports injury by conventional radiography and bone scintigraphy has been satisfactory, the assessment of soft tissue and tendinous injury is more difficult and imprecise. Due to its superior contrast sensitivity and multiplanar imaging capability, magnetic resonance imaging has already shown great promise in delineating soft-tissue and tendinous abnormalities. In addition, marrow pathology is exquisitely displayed.

Epidermoid tumour of the cerebellopontine angle: diagnosis by MRI.

Epidermoids are the third most common cerebellopontine angle tumour, and their diagnosis in the last two decades has been largely obtained using computed tomography (CT). More recently magnetic resonance imaging (MRI) has become a valuable diagnostic tool, and the signal characteristics and enhancement pattern of an epidermoid following intravenous gadolinium with MRI is presented and the literature reviewed.