Purification and characterization of actin from maize pollen

Pollen is an excellent source of actin for biochemical and physiological studies of the actomyosin system in higher plants. We have developed an efficient method to prepare relatively high levels of actin from the pollen of maize (Zea mays L.). The procedures of purification include acetone powder preparation, saturated ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, a cycle of polymerization-depolymerization, and Sephacryl S-200 gel filtration. The average yield of actin is 19 milligrams per 100 grams of pollen grains extracted. This is comparable with those of Acanthamoeba castellanii and human platelets. The purified pollen actin is electrophoretically homogeneous and its molecular mass is 42 kilodaltons. The amino acid composition and circular dichroism spectrum of pollen actin are identical to those of muscle actin. The actin purified from pollen is able to polymerize to F-actin. The pollen F-actin activated the activity of the muscle myosin ATPase sevenfold.     

Release of different fractions of epidermal growth factor from human platelets in vitro: preferential release of 140 kDa fraction.

Platelet-rich plasma in acidic-citrate-dextrose anticoagulant was kept for 5 days in an oxygen-permeable bag at 22 degrees C in an incubator/rotator. Platelet count remained stable throughout the experiment. On days 0, 3 and 5, aliquots were removed; platelets were isolated by centrifugation at 22 degrees C, 1500 g for 20 min, reconstituted to the original volume with PBS buffer, and the contents of alpha-granules were released by repeated freezing and thawing. Epidermal growth factor (EGF) and beta-thromboglobulin (beta-TG) in the platelet-poor plasma and platelet lysates were determined by radioimmunoassays. Results indicated that in platelet-free plasma, both total EGF and beta-TG increased 3-5-fold after 5 days; this amount represented 10-20% of the factors stored in the platelets. Correspondingly, the EGF and beta-TG contents of the platelet lysates exhibited accompanying decreases. HPLC fractionation showed that the main EGF fraction which progressively decreased in the lysates and increased in plasma had a molecular mass of 140 kDa. The contents of the 67 kDa and 6 kDa fractions did not change substantially. We conclude that under these conditions, the 140 kDa fraction was released preferentially. In view of these and previous experiments, it seems likely that different organs contribute to plasma EGF fractions.     

Degradation of intracellular protein in Salmonella typhimurium peptidase mutants

Multiply peptidase-deficient mutant strains of Salmonella typhimurium fail to carry out normal protein degradation during starvation for a carbon source. In these mutants, the extent of protein breakdown during starvation is about fourfold less than in the wild type. The products of protein breakdown in the mutant are mainly small, trichloroacetic acid-soluble peptides, not free amino acids as in the wild type. The carbon-starved mutant strain produces only about one thirtieth as much free amino acid from protein as the wild type. As a result, protein synthesis during starvation is reduced in the mutant compared to the wild type and the mutant strain shows a greatly prolonged lag phase after a nutritional shift-down.     

Peptide accumulation during growth of peptidase deficient mutants

Mutant strains of Salmonella typhimurium simultaneously lacking peptidases N, A, B and D accumulate a heterogeneous mixture of small, trichloroacetic acid-soluble peptides during growth in minimal medium. Approximately 20% of the labelled leucine supplied to a growing culture of the mutant strain is converted to peptides. These peptides accumulate inside the cells before being released into the growth medium. Although the origin of these peptides has not been established, there are several processes that might contribute peptides to this pool. These include (1) turnover of signal sequences, (2) turnover of attenuator peptides, and (3) degradation of prematurely terminated proteins. These results indicate that the same family of peptidases that catabolizes exogenously supplied peptides and functions in carbon-starvation-induced protein turnover also hydrolyzes peptides generated during normal exponential growth.     

Kinetics of bacteriophage lambda repressor synthesis directed by the PRE promoter: influence of temperature, multiplicity of infection, and mutation of PRM or the cro gene

Summary Expression of the P _RE (establishment) pathway for repressor synthesis is regulated both by phage-specific genetic elements and by physiological conditions. Here we describe the effects of temperature, multiplicity of infection, mutations in the cro gene, and a mutation in P _RM on P _RE-directed repressor synthesis. As Reichardt (1975a) has shown, repressor synthesis begins 5–15 min after infection by wildtype phage, and is shut off at 20–30 min after infection, depending on the temperature. At 43°, synthesis starts sooner, shuts off earlier, and leads to lower repressor levels than are attained at lower temperatures. Experiments with the temperature sensitive mutant crots20 demonstrate that, as had been shown previously in experiments at 30° and 37° C, cro protein is responsible for the shut-off of repressor synthesis at 43°. In addition to the effects of temperature, the kinetics of repressor synthesis are strongly affected by multiplicity of infection (moi). At mois greater than 10, repressor synthesis after infection by wildtype at 30° is dramatically inhibited. Unexpectedly, the P _RM mutation prm116, under certain conditions, can alleviate both cro-mediated shutoff and the inhibition of P _RE-directed repressor synthesis at high moi. These effects of prm116 are observed only at low temperature (30°–32° C) and at mois of about 6–10 or greater; they also appear to be cis-specific. Possible mechanisms for the effects of the prm116 mutation are discussed. Finally, these studies demonstrate that crots20, which was isolated as a temperature-sensitive lethal mutation in the cro gene (Herskowitz, unpublished), is temperature-sensitive with respect to the ability to shutoff P _RE-directed repressor synthesis; however, even at low temperature (30° C), the crots20 gene product is only partially active.     

Plasma and platelet associated factors act in G1 to maintain proliferation and to stabilize arrested cells in a viable quiescent state : A temporal map of control points in the G1 phase

In medium supplemented with defibrinogenated, platelet-poor human plasma and a low molecular weight growth factor derived from human platelets (PDGF), Swiss 3T3 cells proliferate exponentially with the same cell cycle kinetics as cells cultured in medium supplemented with commercial calf serum. Removal of PDGF from the culture medium arrests proliferating cells in a stable, reversible G0/G1 quiescent state. This arrested state is similar to the known quiescent state induced by deprivation of calf serum in cell exit kinetics and cytoplasmic proteins synthesized. Cells are sensitive to PDGF deprivation only at the beginning of G1. Reduction of the plasma concentration in the culture medium also arrests cells in G1. The resulting arrested population is unstable and exhibits progressive cell death. Reduced levels of plasma block cellular transit through the cell cycle at a median time of approx. 2.1 h following mitosis, approx. 3.3 h prior to S phase initiation. In addition to being required by cycling cells, plasma associated factors are required to maintain G1 cells blocked by PDGF deprivation in a stable quiescent state. Establishment of a stable, viable G0/G1 growth-arrested state, therefore, apparently involves two distinct processes: arrest of cellular proliferation in G1 and stabilization of the arrested cells in a viable quiescent state. Together with previously reported findings on serum and isoleucine starvation, these results provide a temporal map of growth control points in the G1 phase.     

Release and uptake of gene transfer agent by Rhodopseudomonas capsulata.

Many strains of Rhodopseudomonas capsulata are capable of exchanging genetic information via a recently discovered gene transfer process involving the release and subsequent uptake from the medium of particles containing genetic information (gene transfer agents, GTAs). No viral activities are observed to be associated with this system. An assay has been developed to quantitate gene transfer in R. capsulata. Conditions are described for which the number of cells acquiring a new genetic trait is direcly proportional to the number of GTAs and independent of the number of receipient cells. These conditions were used for the assay of the uptake and release of GTAs by cells. The maximum fraction of recipients that acquire a given genetic marker is approximately 4 X 10(-4). Free GTA appears in a growing culture in one or two abrupt waves near the time of transition from exponential to stationary phase. During these waves, the titer of GTA for a given marker may reach 2 X 103/ml. A comparison of the frequency of single- and double-marker transfers suggests that most of the cells in early-stationary-phase cultures are active recipients. The ultraviolet inactivation spectrum of GTA resembles that of the small ribonucleic acid phages. The inactivation cross section section beta, for GTA was calculated to be 1.7 X 10(-16) cm2/photon at 265 nm.     

Mutants of Salmonella typhimurium deficient in an endoprotease.

Three bands of hydrolytic activity toward the chromogenic protease substrate N-acetyl-DL-phenylalanine beta-naphthyl ester (NAPNE) can be observed after gel electrophoresis of crude extracts of Salmonella typhimurium or Escherichia coli. Mutants deficient in one of these three activities have been isolated using a staining procedure that identifies colonies that show reduced ability to hydrolyze NAPNE. These mutants lack the strongest of the three bands of activity. The Salmonella mutations (designated apeA) are all co-transducible with purE, and the order (pro)-apeA-Hfr K17 origin-purE has been established. Strains carrying apeA mutations have wild-type doubling times. None of the apeA mutants isolated gains an auxotrophic requirement as a result of loss of the apeA gene product. The rates and extents of protein degradation during starvation for a carbon source or during growth after exposure to the amino acid analogue canavanine do not seem to be affected by apeA mutations. Revertants of apeA mutations (selected by screening for clones that have regained the ability to hydrolyze NAPNE) frequently contain a new enzymatic activity not found in wild-type cells.