Bloom's syndrome. III. Analysis of the chromosome aberration characteristic of this disorder

The following hypothesis is put forward: X chromatin in man condenses around a center which is situated on Xq at a short distance from the centromere. The hypothesis is based on, and explains, two classes of observations. (1) Abnormal X chromosomes that have the assumed center in duplicate form bipartite Barr bodies in part of the cells. The frequency of bipartite bodies and the distance between the two parts seem to be determined by the distance between the postulated centers. (2) A large number of variously abnormal X chromosomes have been described. Almost all of them possess the postulated center and it seems possible that the very few apparent exceptions represent misidentifications of chromosome Xq — as isochromosome i(Xp). According to the hypothesis, chromosomes lacking the center would form no Barr body and therefore presumably would not be inactivated, thus leaving the cell severely unbalanced. Furthermore, absence of the center might interfere with the viability of the chromosome itself.     

Recovery of Colony-Forming Ability and Genetic Marker Activity by UV-Damaged Hemophilus influenzae

The rate of recovery of UV-irradiated Hemophilus influenzae from acriflavine-sensitized loss of colony-forming ability was studied at various acriflavine concentrations, UV doses, and temperatures. This rate (as calculated from an equation based upon certain assumptions) was on the order of 0.07 per minute per cell at 37°C. This did not vary greatly with UV dose or acriflavine concentration, but did with temperature, giving a ΔH‡ of about 16 kcal/mole. In another set of experiments, cells bearing two genetic markers (resistance to 2000 μg/ml streptomycin and to 2.5 μg/ml novobiocin) were irradiated and then incubated without acriflavine. DNA extracts made from samples taken after various periods of incubation time were assayed on antibiotic-sensitive cells using acriflavine to inhibit repair during and following transformation. It was found that both in vivo irradiated markers were reactivated in the donor to approximately the same extent (with a rate constant of 0.04 per minute). This result was in contrast to the results obtained when extracted DNA bearing the same markers was irradiated in vitro and used to transform cells. In this latter case the streptomycin marker was much more sensitive than the novobiocin marker. This difference is interpreted as being due to the mechanics of the transformation system.     

Plant transformation by microinjection techniques

Several techniques have been developed for introducing cloned genes into plant cells. Vectorless delivery systems such as PEG-mediated direct DNA uptake (e.g. Pasz-kowski et al. 1984), electroporation (e.g. Shillito et al. 1985), and fusion of protoplasts with liposomes (Deshayes et al. 1985) are routinely used in many experiments (see several chapters of this issue). A wide range of plant species, dicotyledonous as well as monocotyledonous, has been transformed by these vectorless DNA transfer systems. However, the availability of an efficient protoplast regeneration system is a prerequisite for the application of these techniques. For cells with intact cell walls and tissue explants the biological delivery system of virulent Agrobacterium species has been routinely used (for review see Fraley et al. 1986). However, the host range of Agrobacterium restricts the plant species, which can be transformed using this vector system. In addition, all these methods depend on selection systems for recovery of transformants. Therefore a selection system has to be established first for plant species to be transformed. The microinjection technique is a direct physical approach, and therefore host-range independent, for introducing substances under microscopical control into defined cells without damaging them. These two facts differentiate this technique from other physical approaches, such as biolistic transformation and macroinjection (see chapters in this issue). In these other techniques, damaging of cells and random manipulation of cells without optical control cannot be avoided so far. In recent years microinjection technology found its application in plant sciences, whereas this technique has earlier been well established for transformation of animal tissue culture cells (Capecchi 1980) and the production of transgenic animals (Brin-ster et al. 1981, Rusconi and Schaffner 1981). Furthermore, different parameters affecting the DNA transfer via microinjection, such as the nature of microinjected DNA, and cell cycle stage, etc, have been investigated extensively in animal cells (Folger et al. 1982, Wong and Capecchi 1985), while analogous experiments on plant cells are still lacking.     

The carotenoid zeaxanthin and ‘high-energy-state quenching’ of chlorophyll fluorescence

The possibility that zeaxanthin mediates the dissipation of an excess of excitation energy in the antenna chlorophyll of the photochemical apparatus has been tested through the use of an inhibitor of violaxanthin de-epoxidation, dithiothreitol (DTT), as well as through the comparison of two closely related organisms (green and blue-green algal lichens), one of which (blue-green algal lichen) naturally lacks the xanthophyll cycle. In spinach leaves, DTT inhibited a major component of the rapidly relaxing high-energy-state quenching' of chlorophyll fluorescence, which was associated with a quenching of the level of initial fluorescence (F₀) and exhibited a close correlation with the zeaxanthin content of leaves when fluorescence quenching was expressed as the rate constant for radiationless energy dissipation in the antenna chlorophyll. Green algal lichens, which possess the xanthophyll cycle, exhibited the same type of fluorescence quenching as that observed in leaves. Two groups of blue-green algal lichens were used for a comparison with these green algal lichens. A group of zeaxanthin-free blue-green algal lichens did not exhibit the type of chlorophyll fluorescence quenching indicative of energy dissipation in the pigment bed. In contrast, a group of blue-green algal lichens which had formed zeaxanthin slowly through reactions other than the xanthophyll cycle, did show a very similar response to that of leaves and green algal lichens. Fluorescence quenching indicative of radiationless energy dissipation in the antenna chlorophyll was the predominant component of high-energy-state quenching in spinach leaves under conditions allowing for high rates of steady-state photosynthesis. A second, but distinctly different type of high-energy-state quenching of chlorophyll fluorescence, which was not inhibited by DTT (i.e., it was zeaxanthin independent) and which is possibly associated with the photosystem II reaction center, occurred in addition to that associated with zeaxanthin in leaves under a range of conditions which were less favorable for linear photosynthetic electron flow. In intact chloroplasts isolated from (zeaxanthin-free) spinach leaves a combination of these two types of rapidly reversible fluorescence quenching occurred under all conditions examined.Abbreviations DTT dithiothreitol - F₀ (or F₀) yield of instantaneous fluorescence at open PS II reaction centers in the dark (or during actinic illumination) - F_M (or F_M) yield of maximum fluorescence induced by a saturation pulse of light in the dark (or during actinic illumination) - F_V (or F_V) yield of variable fluorescence induced by a saturating pulse of light in the dark (or during actinic illumination) - k _D rate constant for radiationless energy dissipation in the antenna chlorophyll - SV Stern-Volmer equation - PFD photon flux density - PS I photosystem I - PS II photosystem II - Q_A acceptor of photosystem II - q_N coefficient of nonphotochemical chlorophyll fluorescence quenching - q_P coefficient of photochemical chlorophyll fluorescence quenching     

Identification of consensus patterns in unaligned DNA sequences known to be functionally related

We have developed a method for identifying consensus patternsin a set of unaligned DNA sequences known to bind a common proteinor to have some other common biochemical function. The methodis based on a tnatrix representation of binding site patterns.Each row of the matrix represents one of the four possible bases,each column represents one of the positions of the binding siteand each element is determined by the frequency the indicatedbase occurs at the indicated position. The goal of the methodis to find the most significant matrix-i.e. the one with thelowest probability of occurring by chance-out of all the matricesthat can be formed from the set of related sequences. The reliabilityof the method improves with the number of sequences, while thetime required increases only linearly with the number of sequences.To test this method, we analysed 11 DNA sequences containingpromoters regulated by the Escherichia coli LexA protein. Thematrices we' found were consistent with the known consensussequence, and could distinguish the generally accepted LexAbinding sites from other DNA sequences. Received on November 6, 1989; accepted on December 20, 1989     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

Sulfonates: novel electron acceptors in anaerobic respiration

The enrichment and isolation in pure culture of a bacterium, identified as a strain of Desulfovibrio, able to release and reduce the sulfur of isethionate (2-hydroxyethanesulfonate) and other sulfonates to support anaerobic respiratory growth, is described. The sulfonate moiety was the source of sulfur that served as the terminal electron acceptor, while the carbon skeleton of isethionate functioned as an accessory electron donor for the reduction of sulfite. Cysteate (alanine-3-sulfonate) and sulfoacetaldehyde (acetaldehyde-2-sulfonate) could also be used for anaerobic respiration, but many other sulfonates could not. A survey of known sulfate-reducing bacteria revealed that some, but not all, strains tested could utilize the sulfur of some sulfonates as terminal electron acceptor. Isethionate-grown cells of Desulfovibrio strain IC1 reduced sulfonate-sulfur in preference to that of sulfate; however, sulfate-grown cells reduced sulfate-sulfur in preference to that of sulfonate. Received: 2 May 1996 / Accepted: 8 June 1996     

Effects of light,moisture, temperature,and litter on the regeneration of five tree species in the tropical montane wet forest of Puerto Rico

Field experiments were conducted to investigate the effects of light, moisture, temperature, and litter on the regeneration of two early-, one mid-, and two late-successional tropical tree species. High light and litter seem to be universally good cues for regeneration, increasing seed/seedling survival for all species except for Cecropia (an early-successional species) whose small seeds may not be able to penetrate the litter layer. In addition, the high temperature environment in both artificially shaded and nonshaded areas of a natural gap exhibits less seed loss, an increase in the percent and rate of germination, and an increase in seedling survival for Dacryodes (a late-successional species), than the lower temperature environment under an intact canopy. Low soil water is also a good cue for Dacryodes germination as it is for Prestoea and Cecropia. Finally, the lower temperature environment found under the forest canopy (compared to the natural gap) leads to less seed loss and more germination for Guarea (a mid-successional species). Our results suggest that a good patch for regeneration of many species in this forest, early- as well as late-successional species, would have high light and a litter layer that moderates temperature and moisture extremes. The substantial variation in suitability among regeneration filters and species could: (1) contribute to low establishment success, i.e., most dispersed propagules do not become trees, (2) make it difficult to group species into germination strategies, and (3) make it hard to generalize about a net effect of any specific environmental variable on establishment. We suggest that tropical disturbances should be viewed in terms of their impact on a variety of environmental cues, which may signal germination and impact subsequent growth and survival.