Expression of Xist in mouse germ cells correlates with X-chromosome inactivation.

Mammals compensate for different doses of X-chromosome-linked genes in male (XY) and female (XX) somatic cells by terminally inactivating all but one X chromosome in each cell. A transiently inactive X chromosome is also found in germ cells, specifically in premeiotic oogenic cells and in meiotic and postmeiotic spermatogenic cells. Here we show that the Xist gene, which is a expressed predominantly from the inactive X-chromosome in female somatic cells, is also expressed in germ cells of both sexes, but only at those stages when an inactive X chromosome is present. This suggests support for the putative role of Xist as a regulator of X-chromosome inactivation and suggest a common mechanism for the initiation and/or maintenance of X-chromosome inactivation in all cell types.

Mutation of the CDKN2A 5' UTR creates an aberrant initiation codon and predisposes to melanoma.

Approximately 8-12% of melanoma is inherited in an autosomal dominant fashion with variable penetrance. A chromosome 9p21 locus has been linked to this disease in 50-80% of affected families. CDKN2A (also known as P16, INK4, p16INK4A and MTS1) is allelic to this locus and encodes a cdk4/cdk6 kinase inhibitor that constrains cells from progressing through the G1 restriction point. Although germline CDKN2A coding mutations cosegregate with melanoma in 25-60% of families predisposed to the disease, there remains a number of mutation-negative families that demonstrate linkage of inherited melanoma to 9p21 markers. We show here that a subset of these kindreds possess a G-->T transversion at base -34 of CDKN2A, designated G-34T. This mutation gives rise to a novel AUG translation initiation codon that decreases translation from the wild-type AUG. The G-34T mutation is not seen in controls, segregates with melanoma in families and, on the basis of haplotyping studies, probably arose from a common founder in the United Kingdom. Characterization of this and other CDKN2A non-coding mutations should have an impact on current efforts to identify susceptible melanoma-prone families and individuals.

Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex.

Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and export-bound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran--GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion of NUP60 renders Nup2p nucleoplasmic and compromises Nup2p-mediated recycling of Kap60p/Srp1p. Depletion of Ran--GTP by metabolic poisoning, disruption of the Ran cycle, or in vitro by cell lysis, results in a shift of Nup2p from the nucleoplasm to the cytoplasmic face of the NPC. This mobility of Nup2p was also detected using heterokaryons where, unlike nucleoporins, Nup2p was observed to move from one nucleus to the other. Together, our data support a model in which Nup2p movement facilitates the transition between the import and export phases of nucleocytoplasmic transport.

Intersection of the Kap123p-mediated nuclear import and ribosome export pathways.

Kap123p is a yeast beta-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a DeltaRAI1-induced decrease in the overall biogenesis efficiency.

Plasmodium brasilianum antigen for use in the indirect hemagglutination test.

Antigen was prepared from Plasmodium brasilianum harvested from an infected spider monkey. This antigen was attached to aldehyde-fixed, human type O cells, and was tested against sera from human cases of P. malaria, P. vivax, P. falciparum, and P. ovale infection, and sera from noninfected persons. At dilutions of 1:16 or greater the antigen failed to react in sera from noninfected persons. It reacted at titers of 16 or above with sera from 85% of the persons with P. malariae infection, 83% with P. vivax infection, 70% with P. falciparum infection, and 70% with P. ovale infection.

Synthetic-aperture chirp confocal imaging.

An imaging system that combines synthetic-aperture imaging, holography, and an optical chirp with confocal imaging is described and analyzed. Comparisons are made with synthetic-aperture radar systems. Adaptation of several synthetic-aperture radar techniques to the optical counterparts is suggested.

Pseudo-second-derivative matrix and its application to automatic lens design.

A method of approximating the homogeneous second derivatives of a merit function with respect to a set of design variables is presented. If these derivatives are used in an automatic lens design program, the familiar problem of stagnation may usually be avoided.

Fast MTF Calculation in the Presence of Diffraction.

Calculating the MTF of a lens from the lens specification is very time consuming when done in a straightforward manner. One method is to compute the Fourier transform of the line spread function. Even when done on a computer, this is a lengthy calculation. Our programs instead construct a mathematical model of the image. This model has the property that its Fourier transform is known almost immediately when the model is completed.

Lensless imaging by spatial Fourier synthesis holography.

The technique of Fourier synthesis holography is extended to the spatial domain. A spatially extended source is decomposed into its Fourier components, and a hologram of an object distribution is formed at each spatial frequency and stored in a computer. Upon synthesis in the computer a clear image can be formed of the object without the use of lenses.

Improved imagery through scattering materials by quasi-Fourier-synthesis holography.

An improvement on the technique of Fourier-synthesis holography is proposed and demonstrated. Artifacts produced during the process of sampling are eliminated when the laser is swept over a continuous bandwidth between samples. The advantages of Fourier-synthesis holography, such as the ability to select the gating time delay and to shape the autocorrelation function after data acquisition, are retained.

Noise analysis for the holographic first-arriving-light technique.

A noise analysis is performed on an electronic holography first-arriving-light system. Analytical expressions for the signal-to-noise ratio caused by the dominant noise terms are derived. The effect of various system parameters on the signal-to-noise ratio is explored; numerical and experimental examples are given.

Motion-resolved imaging of moving objects embedded within scattering media by the use of time-gated speckle analysis.

Spectral analysis of a time-evolving speckle pattern is used to provide motion-resolved detection of moving objects embedded within scattering media. Results show that the ability to detect small nonstationary scattering objects and to discriminate between objects moving at different rates is greatly enhanced.

Analysis of Fourier synthesis holography for imaging through scattering materials.

The technique of Fourier synthesis holography to image through scattering materials is analyzed in detail. A broad spectral source is decomposed into its Fourier components, and a hologram is formed at each wavelength and stored in the computer. Upon synthesis in the computer, a clear image can be formed of the obscured object. Post-data-acquisition processing such as selection of the gating time delay and autocorrelation shaping are also demonstrated.

Ensemble-averaged imaging through highly scattering media.

A method for imaging through highly scattering media is described that consists of forming a multiplicity of holograms and performing an extensive averaging process. This process produces an estimate of the phase distribution across the exiting surface of the medium. This information is combined with the available magnitude data to form an ensemble-averaged wave front that can be backprojected to form an image of absorbers within or behind the scattering medium.

Use of Fourier synthesis holography to image through inhomogeneities.

A method for image formation through inhomogeneities is demonstrated. A broad spectral source is decomposed into its Fourier components, and a hologram is recorded at each wavelength through a diffusing medium. When the holograms are synthesized in a computer, a clear image can be formed of the obscured object.

Semiquantitative analysis of X-linked gene expression during spermatogenesis in the mouse: ethidium-bromide staining of RT-PCR products.

We have used analysis of ethidium-bromide-stained reverse transcriptase-polymerase chain reaction (RT-PCR) products to assess the effects of X-chromosome inactivation during spermatogenesis in the mouse. RT-PCR was performed on total RNA from eight different spermatogenic cell types, including premeiotic spermatogonia, meiotic spermatocytes, and postmeiotic spermatids, to detect transcripts from five different X-linked structural genes (Pgk-1, Zfx, Pdha-1, Hprt, and Phka) and two autosomal genes (Pgk-2 and beta-actin). Relative intensities of ethidium-bromide-stained RT-PCR products representing transcripts from each gene in each cell type were analyzed by densitometry using the Image program (version 1.4, NIH), and normalized against beta-actin values. These results suggest a coordinate inactivation of the X-linked loci at the onset of meiosis, followed by variable rates of decline of corresponding transcript levels reflecting differential mRNA stabilities and/or leaky expression after inactivation. Technically, these results indicate that analysis of ethidium-bromide-stained RT-PCR products can be used to provide a "semiquantitative" indication of relative levels of specific transcripts in a developing cell lineage without using radioactive probes to quantitate these products.

Three-dimensional confocal imaging of objects embedded within thick diffusing media.

Confocal scanning methods are modified to allow 3-D imaging of objects embedded within thick diffusing media. A method called exfoliative deconvolution is used to sharpen a volume image in which the blur is depth variant. Experimental results are presented.

Resolution-enhanced detection of moving objects embedded within scattering media using time-gated speckle methods.

The holographic first-arriving-light method in combination with the speckle differencing method is used to provide resolution-enhanced detection of moving objects embedded in scattering media. Results show that the first-arriving-light technique provides significant resolution improvements over standard speckle differencing.

Detection of moving objects embedded within scattering media by use of speckle methods.

Speckle-pattern subtraction methods are used for the detection of moving objects embedded in scattering media. Results show that the ability to detect small nonstationary objects is greatly enhanced.

Imaging absorbing structures within thick diffusing media.

A linear and space invariant model for imaging absorbing structures within thick diffusing structures illuminated by coherent light is developed. Experimental, theoretical, and computer simulation results compare ordinary broad beam images with confocal and deconvolved-confocal images; the resolution is improved by factors of ~2 and 3, respectively.