Disruption of the Rad52 Gene Alters the Spectrum of Spontaneous Sup4-O Mutations in Saccharomyces Cerevisiae

Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of stream order and season on mineralization of [14C]-phenol in streams

Mineralization of trace levels of [¹⁴C]-phenol by heterotrophic microorganisms was quantified at 4 sites along a river continuum in southwestern Virginia. Significant phenol mineralization rates were detected in surface sediment and seston samples at all sites from August 1985 through May 1986. Phenol degradation was strongly affected by season (ANOVA; P < 0.0001). From a baseline rate in August (range: 1.19 × 10^-5 to 897 × 10^-4 mg phenol mineralized mg AFDW^-1 h^-1) phenol mineralization rose to a yearly maximum in October (range: 1.21 × 10^-4 to 1.16 × 10^-3 mg phenol mineralized mg AFDW^-1 h^-1) despite decreasing stream temperatures. This autumnal peak in phenol degradation was attributed to the pulsed input of allochthonous detritus, especially leaf litter, which contains substantial quantities of phenols and related compounds. Although phenol mineralization was significant in these streams, phenols were metabolized at much slower rates than more labile compounds present in the dissolved organic matter (DOM) pool. Estimates of turnover rates for three major components of DOM revealed that glucose and glutamate turnover rates (0.064–0.140 h^-1 mg sediment AFDW^-1 and 0.140–0.610 h^-1 mg sediment AFDW^-1, respectively) were, respectively, 2.2–4.7 × and 9.6–16.9 × greater than phenol turnover rates (0.015–0.064 h^-1 mg sediment AFDW^-1). Although the relatively low rates of utilization of refractory phenolic materials suggest that these compounds may accumulate and become more prevalent components of the DOM pool, phenol concentrations at the 4 study sites remained below detectable levels (i.e., < 1 g 1^-1) throughout the study. Consequently, it seems that although phenolic materials are metabolized more slowly than labile DOM, phenols are degraded at rates which preclude accumulation in the water column.     

Disruption of pattern formation in palatal rugae in fetal mice heterozygous for First arch (Far)

The purpose of this study was to document the extent of disruption in the pattern of palatal rugae caused by the presence of one copy of the First arch mutation. The palatal ruga pattern was found to be disrupted in 86% of 15- to 17-day mouse fetuses that were heterozygous for the First arch mutation in the ICR/Bc strain, compared with 9% in ICR/Bc fetuses of normal (+/+) genotype. This new observation in First arch heterozygotes, together with the previously reported dominant effects of the First arch mutation, particularly the bifurcation of the maxillary nerve (100% in both BALB/cGaBc and ICR/Bc strains), the disruption of maxillary vibrissa pattern (80% in ICR/Bc), and the hemifacial deficiency (38% in ICR/Bc), has led us to redefine the First arch mutation as a semidominant, Far. Like the other defects caused by Far, the rugal defects are in tissue derived from the embryonic maxillary prominence. The rugal defects observed in +/Far palates were always asymmetrical and most often involved fragmentation and misalignment of two or more of rugae 4-7. The relatively large degree of variation in ruga pattern observed in fetuses of normal genotype suggests that it is a less well canalized trait than the normal pattern of maxillary vibrissae which varies only in a few very specific and minor ways. The First arch mutation, which in heterozygotes disrupts pattern formation in both palatal rugae and maxillary vibrissae, can be used to study genetic control of pattern formation in mammalian embryos.     

Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse.

The proto-oncogene int-2 has been implicated in the formation of mouse mammary-tumour-virus-induced mammary tumours. Analysis of the predicted coding sequence indicates that int-2 is a member of the fibroblast growth factor family. Previous studies using Northern blot analysis suggested that normal expression of int-2 may be confined to extra-embryonic endoderm lineages of embryonic stages of mouse development. We have used in situ hybridization and Northern blot analysis to examine directly int-2 expression in embryo stem cells and in the developing embryo from early gastrulation to midsomite stages. Complex patterns of accumulation of int-2 RNA were observed in embryonic and extra-embryonic tissues. The data suggest multiple roles for int-2 in development which may include migration of early mesoderm cells and induction of the otocyst.     

Restriction pattern and polypeptide homology among plasmid-borne mercury resistance determinants

The structural and functional properties of mercury resistance determinants cloned from a series of independently isolated conjugative plasmids were compared with those of the prototype HgR determinants from Tn501 and plasmid R100 (containing Tn21). Restriction endonuclease mapping classified the HgR determinants into at least three different but related structural groups which are distantly related to those from Tn501 and R100. These relationships were confirmed by the functional analysis of sub-clones and gamma delta insertion mutations and from the polypeptides specified by the cloned HgR determinants. Each mercury resistance clone synthesized polypeptides equivalent in size to the merA, merT, and merP gene products. However, those for merA and merT showed considerable size variation. No polypeptide equivalent to merD or merC of R100 was detected.     

Inverse relationship between proliferation and differentiation in a human TNP-specific B cell line. Cell cycle dependence of antibody secretion

Studies of an EBV-transformed and TNP-specific human B cell line revealed that, unlike myeloma or hybridoma cell lines that consist mainly of fully differentiated cells, most of the cloned EBV-transformed cells were not fully differentiated, as judged by inability to bind TNP-SRBC and to secrete anti-TNP antibody. The minority of more differentiated cells were selected by TNP-SRBC rosetting. They were found to proliferate to a lesser extent than nonrosetting cells and to contain increased numbers of antibody-secreting cells. This inverse relationship between proliferation and differentiation was also shown to be cell cycle related in that the TNP-SRBC rosetting cells resided, to a greater extent than the nonrosetting cells, in the G1 phase of the cell cycle. The finding that the G1 phase of the cell cycle was associated with differentiation into anti-TNP secreting cells was confirmed by demonstrating that treatment with hydroxyurea, which arrests the cells in G1, resulted in decreased proliferation and an increased proportion of antibody-secreting cells. Similarly, addition of phorbol ester resulted in increased antibody secretion and decreased proliferation, suggesting a role for protein kinase C in this differentiation pathway. The strategy of increasing the number of antibody-producing cells in this human EBV line, by promoting differentiation of the cells in G1, may be relevant to the large scale production of specific human mAb for the treatment and diagnosis of human diseases.     

Biochemical analysis of the activation of adherent neutrophils in vitro

The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo.     

Studies on the System Regulating Proton Movement across the Chloroplast Envelope : Effects of ATPase Inhibitors, Mg, and an Amine Anesthetic on Stromal pH and Photosynthesis

Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H⁺ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg²⁺ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg²⁺ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg²⁺ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H⁺ fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg²⁺ modulated) H⁺ flux linked to monovalent cation antiport, and ATPase-dependent H⁺ efflux.     

Ultrastructure of the approximately 26S complex containing the approximately 20S cylinder particle (multicatalytic proteinase/proteasome).

We have isolated a large protein complex of approximately 26S from Xenopus laevis oocytes and eggs which is composed of the approximately 20S cylinder particle (multicatalytic proteinase/proteasome) and additional proteinaceous components. In its polypeptide composition and sedimentation coefficient this approximately 26S complex closely resembles the 26S ubiquitin-dependent protease, a high molecular weight multienzyme complex recently described in the literature. Specific antibodies directed against a single subunit of the approximately 20S cylinder particle retain, on affinity columns, the large approximately 26S complex, and on sucrose gradients up to approximately 50% of the approximately 20S cylinder particles present in oocyte extracts sedimented with approximately 26S, suggesting that a large proportion of the approximately 20S particles exists in the cell as a component of the approximately 26S complex. Electron microscopy reveals the approximately 26S complex to be a symmetrical elongated macromolecular assembly of at least three protein particles. The central core of the complex is formed by the approximately 20S cylinder particle to which two other large components are attached at the ends, yielding a dumbbell-shaped complex of approximately 40 nm in length. Dissociation of the approximately 26S complexes releases in addition to approximately 20S cylinder particles a novel type of a disc-shaped particle of approximately 15 nm diameter which may represent the attached components or subcomplexes of them. Based on its structural and biochemical properties we postulate that the approximately 26S complex identified here is identical to the ubiquitin-dependent protease.