The nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae: a potential adenine nucleotide binding amino acid sequence and a nonessential acidic carboxyl terminal region.

The RAD3 gene of Saccharomyces cerevisiae is required for excision of pyrimidine dimers and is essential for viability. We present the nucleotide sequence of the RAD3 protein coding region and its flanking regions, and the deduced primary structure of the RAD3 protein. In addition, we have mapped the 5' end of RAD3 mRNA. The predicted RAD3 protein contains 778 amino acids with a calculated molecular weight of 89,779. A segment of the RAD3 protein shares homology with several adenine nucleotide binding proteins, suggesting that RAD3 protein may react with ATP. The twenty carboxyl terminal amino acids of RAD3 protein are predominantly acidic; however, deletion of this acidic region has no obvious effect on viability or DNA repair.     

Effects of particle concentration and season on the filtration rates of the freshwater clam,Sphaerium striatinum Lamarck (Bivalvia: Pisidiidae)

The effects of particle concentration and season on the filtration rates of the freshwater clamSphaerium striatinum Lamarck were assessed by measuring clearance rates of small (2.02 μm) latex beads from dilute suspensions. Filtration rates decreased as particle concentration increased over a range of 2–64 mg 1⁻¹, with rates decreasing in similar proportion for clams of all sizes. For a 1-mg clam, rates decreased from approximately 8.4 to 0.57 ml clam⁻¹ h⁻¹. Seasonal filtration rates for adult clams peaked during periods of greatest reproduction. The patterns for smaller clams are similar, though proportional changes in filtration rates differ for various sizes of clams. It is estimated that clams occupying 1 m² of stream substrate removed about 3.67 gCa⁻¹. This is equivalent to 0.0004% of the carbon that flows past them annually. Filter-feeding provided only 24% of the calculated energy needs of the population, suggesting that another mode of feeding (e.g. deposit-feeding) may provide an important energy source for these forms. Funded in part by a grant-in-aid to D. J. Hornbach from Sigma-Xi, The Research Society. Funded in part by a grant-in-aid to D. J. Hornbach from Sigma-Xi, The Research Society.     

Populational dynamics of three annual species of alpine plants in the Rocky Mountains

Summary Survivorship, fecundity, and seed bank size were measured over two years in alpine populations of the annuals, Koenigia islandica, Polygonum confertiflorum, and P. douglasii. The major loss of individuals from all populations occurred between seed dispersal and germination. Survival of vegetative plants to maturity was high in all species, usually above 80%, and the average number of seeds produced per plant was less than 10 in all species in both years. Seed banks existed for all three species but were small since more than 80% of the viable seeds in the soil germinated in the spring. Both survival and fecundity were negatively correlated with density in P. confertiflorum. These patterns of population dynamics are similar to those found in many annuals of temperate environments.     

Papovaviral sialoadenitis in athymic nude rats

A wasting disease was found in 32 athymic nude rats. The rats had parotid sialoadenitis with intranuclear inclusion bodies in ductal and acinar epithelial cells. Other common lesions included bronchitis, bronchiolitis and secondary bacterial pneumonia. Less commonly, rhinitis and Harderian adenitis were seen. Intranuclear inclusions were also seen in bronchial epithelium of 1 rat, Harderian gland acini of 1 rat and laryngeal glands of 2 rats. Viral particles, averaging 45 nm in diameter, sometimes in crystalline arrays, were found in the nucleus of parotid epithelial cells. By the use of the avidin-biotin-peroxidase complex (ABC) immunoperoxidase technique, antibodies to disrupted SV40 virus (the group specific antigen of the polyomavirus (miopapovavirus) genus of the papovavirus family) reacted with intranuclear inclusions and cytoplasm of parotid epithelium and inclusions in lung and Harderian gland. The viral antigen did not cross react with antibodies to mouse polyoma, mouse K or disrupted bovine papilloma viruses.     

Calcium transients during early development in single starfish (Asterias forbesi) oocytes

Maturation and fertilization of the starfish oocyte are putative calcium-dependent events. We have investigated the spatial distribution and temporal dynamics of this calcium dependence in single oocytes of Asterias forbesi. We used the calcium photoprotein, aequorin, in conjunction with a microscope-photomultiplier and microscope-image intensifier. Surprisingly, in contrast to earlier work with Marasthenias glacialis, there is no detectable increase in intracellular-free calcium in the oocyte of A. forbesi in response to the maturation hormone 1-methyl adenine. During fertilization of the same, matured, A. forbesi oocyte there is a large increase in intracellular-free calcium. The calcium concentration increases to approximately 1 microM at the point of insemination and the region of elevated free calcium expands across the oocyte in approximately 20 s (17-19 degrees C). After the entire oocyte reaches an elevated concentration of free calcium, the concentration decreases uniformly throughout the oocyte over the next several minutes.     

Relationship of thyrotropin-releasing hormone-induced spike and plateau phases in cytosolic free Ca2+ concentrations to hormone secretion. Selective blockade using ionomycin and nifedipine

In clonal rat pituitary cells (GH cells), thyrotropin-releasing hormone (TRH) induced a pattern of changes in cytosolic free calcium concentrations [( Ca2+]i) composed of two phases: an acute spike phase to micromolar levels which decayed (t1/2 = 8 s) to a near-basal concentration and then rose to a prolonged plateau phase of elevated [Ca2+]i (as measured using Quin 2). Closely following these changes in [Ca2+]i, TRH stimulated a rapid "spike phase" of pronounced, but brief, enhancement of the rate of prolactin and growth-hormone secretion and then a "plateau phase" of prolonged enhancement. These two phases were dissociated using two classes of pharmacologic agents: the ionophore ionomycin, and a calcium channel antagonist nifedipine. Ionomycin (100 nM) specifically blocked (less than 90%) the spike phase of TRH action by rapidly emptying the TRH-regulated reservoir of cellular Ca2+ to generate a TRH-like spike in [Ca2+]i; nifedipine inhibited (less than 50%) the plateau phase of TRH-induced changes in [Ca2+]i and hormone secretion by preventing Ca2+ influx through voltage-dependent Ca2+ channels. These agents demonstrated that the TRH-induced spike in [Ca2+]i in GH cells is caused by release of an ionomycin-sensitive pool of cellular Ca2+ with a small component (10%) due to influx of extracellular Ca2+. The TRH-induced plateau in [Ca2+]i is due to influx of extracellular Ca2+, about half of which enters through voltage-dependent calcium channels and half of which enters via nifedipine/verapamil-insensitive influx. The TRH-induced spike in [Ca2+]i led to a burst in hormone secretion, and the plateau in [Ca2+]i produced a prolonged enhancement of secretion; the spike and plateau phases were generated independently by TRH. A spike in [Ca2+]i is necessary, but not sufficient, to induce burst release of hormone, while the prolonged rate of hormone secretion is intimately related to the steady-state [Ca2+]i.     

Natural killer activity in the rat. III. Characterization of transplantable large granular lymphocyte (LGL) leukemias in the F344 rat

Six transplantable large granular lymphocyte (LGL) tumor lines in F344 rats were examined for natural killer (NK) and antibody-dependent cell-mediated cytotoxicity. Tumor cells from all six lines were highly cytotoxic, even at low effector to target ratios, when tested against NK-susceptible targets, but were unreactive against an NK-resistant target (C58NT)D) and a macrophage-susceptible target (P815). Three lines showed significant levels of lysis against antibody-coated tumor cells. After in vivo transplantation, the levels of cytotoxicity steadily increased in three lines and decreased in one. The cytotoxic activity of one line (RNK-16) remained high through 12 transplant generations. Tumor cells injected i.p. spread via the lymphatics to regional lymph nodes, mediastinal nodes, blood, and eventually the bone marrow. Leukemia occurred concurrently with organ enlargement and increased levels of NK. Studies in (F344 X W/Fu)F1 rats clearly demonstrated that the cytotoxic cells from leukemic animals were the transplanted tumor cells themselves and not merely the activation of normal host LGL. These results demonstrate that naturally occurring, transplantable LGL leukemias are an easily obtainable and excellent source of materials for those studies requiring a large number of functionally active LGL.     

Purification and properties of cytoplasmic granules from cytotoxic rat LGL tumors

To evaluate the role of NK cell granules in the lytic activity of NK cells, cytoplasmic granules of rat NK tumors were purified by centrifugation of the cell homogenates in a Percoll gradient. Analysis of such gradients showed a band of light-scattering material near the bottom of the tube; assay of gradient fractions for lytic activity against SRBC showed a potent lytic activity giving a sharp peak in this region. Complete lysis of SRBC was achieved with less than 1 microgram/ml protein of the most active fractions. Examination in the electron microscope showed that a pool of fractions containing lytic activity consisted of pure cytoplasmic granules showing similar morphology to those found in the LGL tumors. The lytic band was associated with a peak in the activity of four different lysosomal enzymes. Analysis of Percoll gradient fractions showed that marker enzymes for mitochondria, plasma membrane, and cytosol were well separated from this activity peak. Analysis of the Percoll gradient fractions by SDS gel electrophoresis showed that this granule fraction was free of contamination of proteins from other parts of the gradient. The granules contained major protein bands of 62, 58, 30, 29, and 28 kilodaltons. In addition to protein, the purified granule fractions contain hexose and uronic acid, but no nucleic acids or phospholipids were detected in chemical assays. Major amounts of chymotryptic, tryptic, and elastase activities were not present, nor were peroxidase or lysozyme activities detectable in substantial amounts. These data show that NK tumor cell cytoplasmic granules contain a potent lytic activity and have biochemical properties that distinguish them from granules present in granulocytes and mast cells.     

Continuity of arterial and venous extra-alveolar interstitium in excised rabbit lungs

We studied the interdependence of arterial and venous extra-alveolar vessel (EAV) leakage on the rate of pulmonary vascular fluid filtration (measured as the change in lung weight over time). Edema was produced in continually weighed, excised rabbit lungs kept in zone 1 (alveolar pressure = 25 cmH2O) by increasing pulmonary arterial (Ppa) and/or venous (Ppv) pressure from 5 to 20 cmH2O (relative to the lung base) and continuing this hydrostatic stress for 3-5 h. Raising Ppa and Ppv simultaneously produced a lower filtration rate than the sum of the filtration rates obtained when Ppa and Ppv were raised separately, while the lung gained from 20 to 95% of its initial weight. When vascular pressure was elevated in either EAV segment, fluid filtration always decreased rapidly as the lung gained up to 30-45% of its initial weight. Filtration then decreased more slowly. The lungs became isogravimetric at 60 and 85% weight gain when the Ppa or Ppv was elevated, respectively; when Ppa and Ppv were raised simultaneously substantial fluid filtration continued even after 140% weight gain. We conclude that the arterial and venous EAV's share a common interstitium in the zone 1 condition, this interstitium cannot be represented as a single compartment with a fixed resistance and compliance, and arterial and venous EAV leakage influences leakage from the other segment.