Mitosis in vertebrate somatic cells with two spindles: Implications for the metaphase/anaphase transition checkpoint and cleavage

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 μm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles x̄ = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase x̄ = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a “start anaphase” reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.

Identification of transforming growth factor-β- regulated genes in Caenorhabditis elegans by differential hybridization of arrayed cDNAs

Members of the transforming growth factor-β family play critical roles in body patterning, in both vertebrates and invertebrates. One transforming growth factor-β-related gene, dbl-1, has been shown to regulate body length and male ray patterning in Caenorhabditis elegans. We screened arrayed cDNAs to identify downstream target genes for the DBL-1 signaling by using differential hybridization. C. elegans cDNAs representing 7,584 independent genes were arrayed on a nylon membrane at high density and hybridized with 33P-labeled DNA probes synthesized from the mRNAs of wild-type, dbl-1, sma-2, and lon-2 worms. Signals for all the spots representing hybridized DNA were quantified and compared among strains. The screening identified 22 and 2 clones, which were positively and negatively regulated, respectively, by the DBL-1 signal. Northern hybridization confirmed the expression profiles of most of the clones, indicating good reliability of the differential hybridization using arrayed cDNAs. In situ hybridization analysis revealed the spatial and temporal expression patterns of each clone and showed that at least four genes, including the gene for the type I receptor for DBL-1, sma-6, were transcriptionally regulated by the DBL-1 signal.

Spatio-temporally controlled site-specific somatic mutagenesis in the mouse

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type will facilitate studies of gene function and the generation of animal models for human diseases. We have shown previously that conditional recombination–excision between two loxP sites can be achieved in mice by using the Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (Cre-ERT), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ERT under the control of the cytomegalovirus promoter. However, the efficiency of excision varied between tissues, and the highest level (≈40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ERT in a given cell type, we have now crossed Cre-ERT-expressing mice with reporter mice in which expression of Escherichia coli β-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. We show that site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ERT. These results indicate that cell-specific expression of Cre-ERT in transgenic mice can be used for efficient tamoxifen-dependent, Cre-mediated recombination at loci containing loxP sites to generate site-specific somatic mutations in a spatio-temporally controlled manner.

Somatic Cell Mutants Resistant to Retrovirus Replication: Intracellular Blocks during the Early Stages of Infection

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription.

tRNA Nuclear Export in Saccharomyces cerevisiae: In Situ Hybridization Analysis

To understand the factors specifically affecting tRNA nuclear export, we adapted in situ hybridization procedures to locate endogenous levels of individual tRNA families in wild-type and mutant yeast cells. Our studies of tRNAs encoded by genes lacking introns show that nucleoporin Nup116p affects both poly(A) RNA and tRNA export, whereas Nup159p affects only poly(A) RNA export. Los1p is similar to exportin-t, which facilitates vertebrate tRNA export. A los1 deletion mutation affects tRNA but not poly(A) RNA export. The data support the notion that Los1p and exportin-t are functional homologues. Because LOS1 is nonessential, tRNA export in vertebrate and yeast cells likely involves factors in addition to exportin-t. Mutation of RNA1, which encodes RanGAP, causes nuclear accumulation of tRNAs and poly(A) RNA. Many yeast mutants, including those with the rna1-1 mutation, affect both pre-tRNA splicing and RNA export. Our studies of the location of intron-containing pre-tRNAs in the rna1-1 mutant rule out the possibility that this results from tRNA export occurring before splicing. Our results also argue against inappropriate subnuclear compartmentalization causing defects in pre-tRNA splicing. Rather, the data support “feedback” of nucleus/cytosol exchange to the pre-tRNA splicing machinery.

Somatic polyploidization and cellular proliferation drive body size evolution in nematodes

Most of the hypodermis of a rhabditid nematode such as Caenorhabditis elegans is a single syncytium. The size of this syncytium (as measured by body size) has evolved repeatedly in the rhabditid nematodes. Two cellular mechanisms are important in the evolution of body size: changes in the numbers of cells that fuse with the syncytium, and the extent of its acellular growth. Thus nematodes differ from mammals and other invertebrates in which body size evolution is caused by changes in cell number alone. The evolution of acellular syncytial growth in nematodes is also associated with changes in the ploidy of hypodermal nuclei. These nuclei are polyploid as a consequence of iterative rounds of endoreduplication, and this endocycle has evolved repeatedly. The association between acellular growth and endoreduplication is also seen in C. elegans mutations that interrupt transforming growth factor-β signaling and that result in dwarfism and deficiencies in hypodermal ploidy. The transforming growth factor-β pathway is a candidate for being involved in nematode body size evolution.

Inverse radiation dose-rate effects on somatic and germ-line mutations and DNA damage rates

The mutagenic effect of low linear energy transfer ionizing radiation is reduced for a given dose as the dose rate (DR) is reduced to a low level, a phenomenon known as the direct DR effect. Our reanalysis of published data shows that for both somatic and germ-line mutations there is an opposite, inverse DR effect, with reduction from low to very low DR, the overall dependence of induced mutations being parabolically related to DR, with a minimum in the range of 0.1 to 1.0 cGy/min (rule 1). This general pattern can be attributed to an optimal induction of error-free DNA repair in a DR region of minimal mutability (MMDR region). The diminished activation of repair at very low DRs may reflect a low ratio of induced (“signal”) to spontaneous background DNA damage (“noise”). Because two common DNA lesions, 8-oxoguanine and thymine glycol, were already known to activate repair in irradiated mammalian cells, we estimated how their rates of production are altered upon radiation exposure in the MMDR region. For these and other abundant lesions (abasic sites and single-strand breaks), the DNA damage rate increment in the MMDR region is in the range of 10% to 100% (rule 2). These estimates suggest a genetically programmed optimatization of response to radiation in the MMDR region.

Peptide nucleic acid pre-gel hybridization: An alternative to Southern hybridization

We have found that it is possible to use labeled peptide nucleic acid (PNA)-oligomers as probes in pre-gel hybridization experiments, as an alternative for Southern hybridization. In this technique, the PNA probe is hybridized to a denatured DNA sample at low ionic strength and the mixture is loaded directly on to an electrophoresis system for size separation. Ensuing gel electrophoresis separates the single-stranded DNA fragments by length. The neutral backbone of PNA allows for hybridization at low ionic strength and imparts very low mobility to excess PNA. Detection of the bound PNA is possible by direct fluorescence detection with capillary electrophoresis, or the DNA/PNA hybrids can be blotted onto a membrane and detected with standard chemiluminescent techniques. Efficient single bp discrimination was achieved routinely using both capillary and slab-gel electrophoresis.

Somatic and germ cell parasitism in a colonial ascidian: Possible role for a highly polymorphic allorecognition system

A colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction in the wild that results alternatively in colony fusion (chimera formation) or inflammatory rejection. A single, highly polymorphic histocompatibility locus (called Fu/HC) is responsible for rejection versus fusion. Gonads are seeded and gametogenesis can occur in colonies well after fusion, and involves circulating germ-line progenitors. Buss proposed that colonial organisms might develop self/non-self histocompatibility systems to limit the possibility of interindividual germ cell “parasitism” (GCP) to histocompatible kin [Buss, L. W. (1982) Proc. Natl. Acad. Sci. USA 79, 5337–5341 and Buss, L. W. (1987) The Evolution of Individuality (Princeton Univ. Press, Princeton]. Here we demonstrate in laboratory and field experiments that both somatic cell and (more importantly) germ-line parasitism are a common occurrence in fused chimeras. These experiments support the tenet in Buss’s hypothesis that germ cell and somatic cell parasitism can occur in fused chimeras and that a somatic appearance may mask the winner of a gametic war. They also provide an interesting challenge to develop formulas that describe the inheritance of competing germ lines rather than competing individuals. The fact that fused B. schlosseri have higher rates of GCP than unfused colonies additionally provides a rational explanation for the generation and maintenance of a high degree of Fu/HC polymorphism, largely limiting GCP to sibling offspring.

Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine

Somatic mutation accumulation has been implicated as a major cause of cancer and aging. By using a transgenic mouse model with a chromosomally integrated lacZ reporter gene, mutational spectra were characterized at young and old age in two organs greatly differing in proliferative activity, i.e., the heart and small intestine. At young age the spectra were nearly identical, mainly consisting of G·C to A·T transitions and 1-bp deletions. At old age, however, distinct patterns of mutations had developed. In small intestine, only point mutations were found to accumulate, including G·C to T·A, G·C to C·G, and A·T to C·G transversions and G·C to A·T transitions. In contrast, in heart about half of the accumulated mutations appeared to be large genome rearrangements, involving up to 34 centimorgans of chromosomal DNA. Virtually all other mutations accumulating in the heart appeared to be G·C to A·T transitions at CpG sites. These results suggest that distinct mechanisms lead to organ-specific genome deterioration and dysfunction at old age.

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 × 10−7 cm2/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (≤1 × 10−7 cm2/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 × 10−7 cm2/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (VH) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T2-biotin·dT-T2 loop. The third base was a biotinylated uracil (UB) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3′ dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3′ end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5′-FITC, or radiolabeled with [γ-33P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45°C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin–target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.

Fluorescent RNA cytochemistry: tracking gene transcripts in living cells

The advent of jellyfish green fluorescent protein and its spectral variants, together with promising new fluorescent proteins from other classes of the Cnidarian phylum (coral and anemones), has greatly enhanced and promises to further boost the detection and localization of proteins in cell biology. It has been less widely appreciated that highly sensitive methods have also recently been developed for detecting the movement and localization in living cells of the very molecules that precede proteins in the gene expression pathway, i.e. RNAs. These approaches include the microinjection of fluorescent RNAs into living cells, the in vivo hybridization of fluorescent oligonucleotides to endogenous RNAs and the expression in cells of fluorescent RNA-binding proteins. This new field of ‘fluorescent RNA cytochemistry’ is summarized in this article, with emphasis on the biological insights it has already provided. These new techniques are likely to soon collaborate with other emerging approaches to advance the investigation of RNA birth, RNA–protein assembly and ribonucleoprotein particle transport in systems such as oocytes, embryos, neurons and other somatic cells, and may even permit the observation of viral replication and transcription pathways as they proceed in living cells, ushering in a new era of nucleic acids research in vivo.

Electrostatic surface plasmon resonance: Direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches

We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surface-immobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum.