Fung Chan

NUCLEOPHOSMIN/B23 (NPM) OLIGOMER IS A MAJOR AND STABLE ENTITY IN HELA CELLS

HeLa cell extract was separated by 7% polyacrylamide gel electrophoresis without SDS and subsequently stained with anti-nucleophosmin/B23 (NPM) antibody in a Western blot analysis. Two immunobands were obtained. The major band with a slower electromobility (RF = 0.23) is the NPM oligomer, while the fast-moving minor band is the monomer (RF = 0.56). The oligomer constitutes about 95% of total NPM. The oligomer sedimented faster (10 s) than the monomer in sucrose density gradient centrifugation. Three oligomer bands were identified. NPM oligomer is not affected by treatments with DNase. RNase, 10 mM EDTA, 1 M NaCl, and lyophilization. However, 3 M urea causes reversible dissociation of NPM oligomer into monomer. The level of NPM oligomer remains unchanged in HeLa cells after treatment with the cytotoxic agents, actinomycin D, toyocamycin and camptothecin. These results indicate that NPM oligomer is the major and stable NPM entity in HeLa cells.

Long CTG Tracts from the Myotonic Dystrophy Gene Induce Deletions and Rearrangements during Recombination at the APRT Locus in CHO Cells

ABSTRACT Expansion of CTG triplet repeats in the 3′ untranslated region of the DMPK gene causes the autosomal dominant disorder myotonic dystrophy. Instability of CTG repeats is thought to arise from their capacity to form hairpin DNA structures. How these structures interact with various aspects of DNA metabolism has been studied intensely for Escherichia coli and Saccharomyces cerevisiae but is relatively uncharacterized in mammalian cells. To examine the stability of (CTG)17, (CTG)98, and (CTG)183 repeats during homologous recombination, we placed them in the second intron of one copy of a tandemly duplicated pair of APRT genes. Cells selected for homologous recombination between the two copies of the APRT gene displayed distinctive patterns of change. Among recombinants from cells with (CTG)98 and (CTG)183, 5% had lost large numbers of repeats and 10% had suffered rearrangements, a frequency more than 50-fold above normal levels. Analysis of individual rearrangements confirmed the involvement of the CTG repeats. Similar changes were not observed in proliferating (CTG)98 and (CTG)183 cells that were not recombinant at APRT. Instead, they displayed high frequencies of small changes in repeat number. The (CTG)17 repeats were stable in all assays. These studies indicate that homologous recombination strongly destabilizes long tracts of CTG repeats.

A study of correlation between NPM-translocation and apoptosis in cells induced by daunomycin

Human leukemia K562 and HeLa cells were treated with daunomycin (DA) for 1-4 hr. With the indirect immunofluorescence technique, we observed that the nucleolar protein nucleophosmin/B23 (NPM) shifted its location from the nucleolus to the nucleoplasm (NPM-translocation). The degree of NPM-translocation was determined by the relative immunofluorescent intensity in the nucleoli vs the nucleoplasm (defined as localization index, LI). We found that NPM-translocation, as determined by the decrease of LI, correlates with cytotoxicity. The degrees of NPM-translocation, chromatin condensation, and DNA fragmentation in HeLa cells were determined after treatment with 0.1, 0.5 and 1 microg/mL DA for 1 hr. We found that NPM-translocation (LI < 2.5) was observed in cells during the treatment with 0.5 and 1 but not with 0.1 microg/mL DA. Also, cells treated with 1 microg/mL remained in an NPM-translocated state for a longer time (5-6 hr) than those cells treated with 0.5 microg/mL (1-2 hr). Cells treated with 0.5 and 1 microg/mL DA showed increased levels of chromatin condensation beginning at 5 hr after the drug treatment. The number of cells with condensed chromatin increased with both time and drug concentration. No cells with condensed chromatin were observed in samples treated with 0.1 microg/mL DA, which also showed no significant NPM-translocation. Similar results were observed for induction of DNA fragmentation. We found that the drug concentration required for induction of DNA fragmentation and chromatin condensation coincided with the drug concentration required for NPM-translocation. Taken together, these results indicate that NPM-translocation correlates with apoptosis induced by daunomycin.

Rhodopsin Gene Expression Determines Rod Outer Segment Size and Rod Cell Resistance to a Dominant-Negative Neurodegeneration Mutant

Two outstanding unknowns in the biology of photoreceptors are the molecular determinants of cell size, which is remarkably uniform among mammalian species, and the mechanisms of rod cell death associated with inherited neurodegenerative blinding diseases such as retinitis pigmentosa. We have addressed both questions by performing an in vivo titration with rhodopsin gene copies in genetically engineered mice that express only normal rhodopsin or an autosomal dominant allele, encoding rhodopsin with a disease-causing P23H substitution. The results reveal that the volume of the rod outer segment is proportional to rhodopsin gene expression; that P23H-rhodopsin, the most common rhodopsin gene disease allele, causes cell death via a dominant-negative mechanism; and that long term survival of rod cells carrying P23H-rhodopsin can be achieved by increasing the levels of wild type rhodopsin. These results point to promising directions in gene therapy for autosomal dominant neurodegenerative diseases caused by dominant-negative mutations.

Efficient mutagenesis of the rhodopsin gene in rod photoreceptor neurons in mice

Dominant mutations in the rhodopsin gene, which is expressed in rod photoreceptor cells, are a major cause of the hereditary-blinding disease, autosomal dominant retinitis pigmentosa. Therapeutic strategies designed to edit such mutations will likely depend on the introduction of double-strand breaks and their subsequent repair by homologous recombination or non-homologous end joining. At present, the break repair capabilities of mature neurons, in general, and rod cells, in particular, are undefined. To detect break repair, we generated mice that carry a modified human rhodopsin-GFP fusion gene at the normal mouse rhodopsin locus. The rhodopsin-GFP gene carries tandem copies of exon 2, with an ISceI recognition site situated between them. An ISceI-induced break can be repaired either by non-homologous end joining or by recombination between the duplicated segments, generating a functional rhodopsin-GFP gene. We introduced breaks using recombinant adeno-associated virus to transduce the gene encoding ISceI nuclease. We found that virtually 100% of transduced rod cells were mutated at the ISceI site, with ∼85% of the genomes altered by end joining and ∼15% by the single-strand annealing pathway of homologous recombination. These studies establish that the genomes of terminally differentiated rod cells can be efficiently edited in living organisms.

Defective development of photoreceptor membranes in a mouse model of recessive retinal degeneration

Retinal neurodegeneration occurs in several inherited diseases. Some of the most severe disease alleles involve mutations at the C-terminus of rhodopsin, but in no case is the pathogenic mechanism leading to cell death well understood. We have examined a mouse model of recessive retinal degeneration caused by a knock-in of a human rhodopsin-EGFP fusion gene (hrhoG/hrhoG) at the rhodopsin locus. Whereas heterozygous mutant mice were indistinguishable from control mice, homozygous mutant mice had retinal degeneration. We hypothesized that degeneration might be due to aberrant rhodopsin signaling; however, inhibiting signaling by rearing mice in total darkness had no effect on the rate of degeneration. Using confocal and electron microscopy, we identified the fundamental defect as failed biogenesis of disk membranes, which is observed at the earliest stages of outer segment development. These results reveal that in addition to its role in transport and sorting of rhodopsin to disk membranes, rhodopsin is also essential for formation of disks.

Rhodopsin-EGFP knock-ins for imaging quantal gene alterations.

We have developed an imaging approach to monitor changes in gene structure in photoreceptors. We review here, the strategy and recent progress. Knock-in mice bearing a human rhodopsin-EGFP fusion gene potentially allow detection of a single molecular event: correction of a single copy of a gene within an entire retina. These mice can also be used for imaging rhodopsin distribution, membrane structure, and trafficking in normal mice or in disease states, using confocal or multiphoton fluorescence imaging techniques. They represent tools for studying molecular triggers of photoreceptor development, for following stem cell populations, and for evaluating retinal transplantation experiments.

Abrupt onset of mutations in a developmentally regulated gene during terminal differentiation of post-mitotic photoreceptor neurons in mice

For sensitive detection of rare gene repair events in terminally differentiated photoreceptors, we generated a knockin mouse model by replacing one mouse rhodopsin allele with a form of the human rhodopsin gene that causes a severe, early-onset form of retinitis pigmentosa. The human gene contains a premature stop codon at position 344 (Q344X), cDNA encoding the enhanced green fluorescent protein (EGFP) at its 3′ end, and a modified 5′ untranslated region to reduce translation rate so that the mutant protein does not induce retinal degeneration. Mutations that eliminate the stop codon express a human rhodopsin-EGFP fusion protein (hRho-GFP), which can be readily detected by fluorescence microscopy. Spontaneous mutations were observed at a frequency of about one per retina; in every case, they gave rise to single fluorescent rod cells, indicating that each mutation occurred during or after the last mitotic division. Additionally, the number of fluorescent rods did not increase with age, suggesting that the rhodopsin gene in mature rod cells is less sensitive to mutation than it is in developing rods. Thus, there is a brief developmental window, coinciding with the transcriptional activation of the rhodopsin locus, in which somatic mutations of the rhodopsin gene abruptly begin to appear.