Mutational Analysis of Branching in Pea. Evidence That Rms1 and Rms5 Regulate the Same Novel Signal

The fifth increased branching ramosus (rms) mutant, rms5, from pea (Pisum sativum), is described here for phenotype and grafting responses with four other rms mutants. Xylem sap zeatin riboside concentration and shoot auxin levels in rms5 plants have also been compared with rms1 and wild type (WT). Rms1 and Rms5 appear to act closely at the biochemical or cellular level to control branching, because branching was inhibited in reciprocal epicotyl grafts between rms5 or rms1 and WT plants, but not inhibited in reciprocal grafts between rms5 and rms1 seedlings. The weakly transgressive or slightly additive phenotype of the rms1 rms5 double mutant provides further evidence for this interaction. Like rms1, rms5 rootstocks have reduced xylem sap cytokinin concentrations, and rms5 shoots do not appear deficient in indole-3-acetic acid or 4-chloroindole-3-acetic acid. Rms1 and Rms5 are similar in their interaction with other Rms genes. Reciprocal grafting studies with rms1, rms2, and rms5, together with the fact that root xylem sap cytokinin concentrations are reduced in rms1 and rms5 and elevated in rms2 plants, indicates that Rms1 and Rms5 may control a different pathway than that controlled by Rms2. Our studies indicate that Rms1 and Rms5 may regulate a novel graft-transmissible signal involved in the control of branching.     

Deletion mutants from R plasmids with increased copy number.

Rms 201-12 and Rms 201-46 are R mutants with increased copy number, and are derived from a conjugative plasmid Rms 201 that encodes resistance to five drugs, ampicillin (Apc), tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), and sulfonamides (Sa). The mutants expressed increased levels of resistance to Apc, Sm, and Cm, and a decreased level of resistance to Tc than those of the parent Rms 201 plasmid. When the Rms 201-12+ or Rms 201-46+ cells were inoculated onto plates containing a high concentration of Tc, colonies developed on the plate at a frequency of 10-3 to 10-4 after overnight incubation. The cells grown on the Tc plate carried a tet (Tc gene)-deleted R mutant besides tet-possessing Rms 201-12 or Rms 201-46, and we isolated the tet-deleted R mutant by purifying the R+ cells on drug-free plates. On the other hand, various deletion mutants possessing tet were isolated by prolonged culture of the cells. We have presented a circular gene order of Rms 201 by comparing the genetic markers of all deletion mutants derived from Rms 201, Rms 201-46, and Rms 201-12. The gene(s) regulating the copy number was closely linked to the rep gene. The gene(s) specifying entry exclusion was jointly lost with the tra region.     

Regular segregation of composite plasmid Rms201.

Copy number mutants Rms201ts15 and Rms201ts16 were isolated at 30 degrees C from a temperature-sensitive replication mutant (Rms201ts14) of the conjugative plasmid Rms201. The numbers of plasmids per chromosome of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) grown at 30 degrees C were 2.2, 7.4, and 20, respectively. The synthesis of covalently closed circular plasmid deoxyribonucleic acid stopped in Rms201ts14, Rms201ts15, and Rms201ts16 immediately after a "shift-up" in temperature (42 degrees C). At 42 degrees C, antibiotic-sensitive derivatives appeared after a certain lag time: the lag times of ML1410(Rms201ts14), ML1410(Rms201ts15), and ML1410(Rms201ts16) were 2.5, 5, and 6.8 generations, respectively. After these times, plasmid-positive cells in the populations decreased at a rate of about 50% per generation in all of the mutants. From these results we conclude that plasmid segregation (partition) of Rms201 occurs by regular segregation (partition).     

A mutant defective in partitioning of composite plasmid Rms201.

Escherichia coli harboring mutant plasmids defective in maintenance stability (from the conjugative plasmid Rms201) showed a wide distribution of ampicillin resistance levels, as well as increased frequency of plasmid loss from the cell. The amounts of covalently closed circular deoxyribonucleic acid of mutant plasmid Rms268 and parental plasmid Rms201 per chromosome were 5.3 and 6.1%, respectively. The beta-lactamase activities of strains W3630(Rms268) and W3630(Rms201) were 0.56 and 0.44 U/mg of protein, respectively. Frequency of plasmid loss from W3630(Rms268) was about 0.8 to 1.2% per cell generation, 100 times more than that of the wild-type strain. Ampicillin resistance levels of the colonies harboring the mutant plasmid showed a wide distribution, from low (100 micrograms/ml) to high (1,600 micrograms/ml). A miniplasmid (pMS268) with a mass of 7 X 10(6) daltons and encoding ampicillin resistance was isolated from Rms268. Frequency of pMS268 loss from W3630(pMS268) was about 0.8 to 1.9% per cell generation. W3630(pMS268) also showed a wide range of distribution in the levels of ampicillin resistance. These results indicated that the copies of Rms268 in E. coli did not segregate evenly between daughter cells at cell division and that the gene involved was located on the miniplasmid.     

Isolation and Characterization of a Composite Plasmid Rms201 Mutant Temperature Sensitive for Replication

A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37°C. When Escherichia coli ML1410(Rms201ts14)⁺ was grown at temperatures between 40 and 42°C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)⁺ in L-broth was shifted to 42 from 30°C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42°C. At 30°C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42°C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42°C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42°C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 × 10⁶ and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 × 10⁶. These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.     

Effect of phospholipase C treatment on the activity of the particulate cyclic nucleotide phosphodiesterase of rat brain

1. The activity of cAMP phosphodiesterase (PDE) was studied in a 10,000 g particulate fraction prepared from rat brain. 2. Phospholipase C such as sphingomyelin choline phosphodiesterase (SMase), phosphatidylinositol phosphodiesterase (PIase) and phosphatidylcholine phosphohydrolase (PCase) were used to deplete phospholipid(s) from the particulate fraction and their effects on PDE activity were investigated. 3. Treatment with SMase or PIase did not affect PDE activity whereas treatment with PCase resulted in inhibition. 4. It was also found that the PCase used not only hydrolyzed phosphatidylcholine but also other phospholipids such as phosphatidylethanolamine, phosphatidylserine and sphingomyelin.     

Reduction of cyclic AMP phosphodiesterase activity of several subcellular fractions of rat brain after pretreatment with phospholipase C

Cyclic AMP phosphodiesterase (PDE) activity was assayed in the plasma membrane, mitochondrial and microsomal fractions of rat brain. The specific activity of the enzyme was highest in the plasma membrane fraction followed by mitochondrial and then the microsomal fraction. Phosphodiesterase activity of all three fractions was reduced after pretreatment with lecithinase C (PCase) from Clostridium perfringens but less markedly affected by the pretreatment with sphingomyelinase (SMase) from human placenta. The PDE activity of the plasma membrane fraction was more sensitive to PCase treatment compared with the other two particulate fractions, which showed only a slight loss of activity. Temperature seemed to affect PDE activity of the plasma membrane. The enzyme was quite stable at 30 degrees C but its activity dropped by approximately 46% at 37 degrees C after 90 min of incubation. Pretreatment of the plasma membrane at 30 degrees C with PCase at a concentration of more than 5 U caused a marked loss of PDE activity and the decrease in activity reached a plateau at concentrations above 10 U.     

Stimulation of the lysogenization of Pseudomonas aeruginosa cells by phage transposon B39 induced by plasmid Rms163

The influence of Rms163 plasmid on lysogenization of Pseudomonas aeruginosa cells by B39 phage was studied. Plasmid Rms163 was shown to increase the frequency of lysogenization of PAO1 cells 7-8 times. C-mutants of B39 phage were isolated. According to complementation test, c-mutants were distributed into two groups--cI and cII/III. The product of cI is essential for establishment and maintenance of lysogenic state, cII/cIII product being only necessary for establishment of lysogenization. The mutants with special characteristics were isolated: B39cx1 phage carries a mutation which seems to be located on a regulatory site essential for establishment of lysogenic state. The region of the B39 genome responsible for interaction with Rms163 plasmid was mapped. Possible mechanisms of Rms163 plasmid interference with transposable B39 phage are discussed.     

Auxin inhibition of decapitation-induced branching is dependent on graft-transmissible signals regulated by genes Rms1 and Rms2

Decapitation-induced axillary bud outgrowth is a vital mechanism whereby shoots are able to continue normal growth and development. In many plants, including wild-type garden pea (Pisum sativum L.), this process can be inhibited by exogenous auxin. Using the ramosus (rms) increased branching mutants of pea, we present evidence that this response to auxin is dependent on graft-transmissible substance(s) regulated by the genes Rms1 and Rms2. The response to exogenous auxin is massively diminished in decapitated rms1 and rms2 mutant plants. However, basipetal auxin transport is not reduced in intact or decapitated mutants. Grafting rms1 or rms2 shoots onto wild-type rootstocks restored the auxin response, indicating that Rms1 and Rms2 gene action in the rootstock is sufficient to enable an auxin response in mutant shoots. We conclude that Rms1 and Rms2 act in the rootstock and shoot to control levels of mobile substance(s) that interact with exogenous auxin in the inhibition of bud outgrowth after decapitation. At least for rms1, the reduced auxin response is unlikely to be due to an inability of auxin to decrease xylem sap cytokinin content, as this is already low in intact rms1 plants. Consequently, we have genetic evidence that auxin action in decapitated plants depends on at least one novel long-distance signal.     

Pea rms6 mutants exhibit increased basal branching

Our studies on two branching mutants of pea ( Pisum sativum L.) have identified a further Ramosus locus , Rms6, with two recessive or partially recessive mutant alleles: rms6-1 (type line S2-271) and rms6-2 (type line K586). Mutants rms6-1 and rms6-2 were derived from dwarf and tall cultivars, Solara and Torsdag, respectively. The rms6 mutants are characterized by increased branching from basal nodes. In contrast, mutants rms1 through rms5 have increased branching from both basal and aerial (upper stem) nodes. Buds at the cotyledonary node of wild-type (WT) plants remain dormant but in rms6 plants these buds were usually released from dormancy. Their growth was either subsequently inhibited, sometimes even prior to emergence above ground, or they grew into secondary stems. The mutant phenotype was strongest for rms6-1 on the dwarf background. Although rms6-2 had a weak single-mutant phenotype, the rms3-1 rms6-2 double mutant showed clear transgression and an additive branching phenotype, with a total lateral length almost 2-fold greater than rms3-1 and nearly 5-fold greater than rms6-2 . Grafting studies between WT and rms6-1 plants demonstrated the primary action of Rms6 may be confined to the shoot. Young WT and rms6-1 shoots had similar auxin levels, and decapitated plants had a similar magnitude of response to applied auxin. Abscisic acid levels were elevated 2-fold at node 2 of young rms6-1 plants. The Rms6 locus mapped to the R to Gp segment of linkage group V (chromosome 3). The rms6 mutants will be useful for basic research and also have possible agronomical value.     

Purification and some properties of a chloramphenicol acetyltransferase mediated by plasmids from Vibrio anguillarum

Vibrio anguillarum strains were isolated from chloramphenicol-resistant bacteria in diseased fish. Plasmid Rms418, which confers chloramphenicol resistance, was transferred from V. anguillarum GN11379 to Escherichia coli K12 by conjugation. The Rms418-encoded chloramphenicol acetyltransferase (CAT) [EC 2.3.1.99] was isolated and purified to homogeneity using affinity chromatography on immobilized p-amino-chloramphenicol or ATP. The general CAT could be adsorbed by a matrix with a chloramphenicol base ligand (Zaidenzaig, Y. & Shaw, W.V. (1976) FEBS Lett. 62,266-271), but the Rms418-encoded CAT was not bound under these conditions. The specific activity of the enzyme, when measured by the spectrophotometric assay, was 71.4 units/mg protein at 37 degrees C. The molecular weight of the enzyme treated with SDS and 2-mercaptoethanol was shown to be approximately 22,000. The molecular weight of the native enzyme, as determined by gel filtration, was approximately 69,000, and the optimal pH was 7.8. The Km values for chloramphenicol and CoASAc were 34.5 and 150 microM, respectively. Enzyme activity was inhibited by HgCl2, p-chloromercuribenzoate (p-CMB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and ethylendiaminotetraacetic acid (EDTA). The half life at 53 degrees C was approximately 100 min.     

Long-distance signalling and a mutational analysis of branching in pea

Four ramosus mutants with increased branching at basal andaerial nodes have been used to investigate the genetic regulation of budoutgrowth in Pisum sativum L. (garden pea). Studies oflong-distance signalling, xylem sap cytokinin concentrations, shootauxin level, auxin transport and auxin response are discussed. A modelof branching control is presented that encompasses twograft-transmissible signals in addition to auxin and cytokinin. Mutantsrms1 through rms4 are not deficient in indole-3-aceticacid (IAA) or in the basipetal transport of this hormone. Three of thefour mutants, rms1, rms3 and rms4, have veryreduced cytokinin concentrations in xylem sap from roots. This reductionin xylem sap cytokinin concentration appears to be caused by a propertyof the shoot and may be part of a feedback mechanism induced by anaspect of bud outgrowth. The shoot-to-root feedback signal is unlikelyto be auxin itself, as auxin levels and transport are not correlatedwith xylem sap cytokinin concentrations in various intact and graftedmutant and wild-type plants. Rms1 and Rms2 act inshoot and rootstock to regulate the level or transport ofgraft-transmissible signals. Various grafting studies and double mutantanalyses have associated Rms2 with the regulation of theshoot-to-root feedback signal. Rms1 is associated with a secondunknown graft-transmissible signal that is postulated to move in thedirection of root-to-shoot. Exogenous auxin appears to interact withboth of the signals regulated by Rms1 and Rms2 in theinhibition of branching after decapitation. The action of Rms3and Rms4 is less apparent at this stage, although both appearto act largely in the shoot.     

Gentic properties of an R factor carrying resistance to aminolgycoside antibiotics.

R factor Rms 151 is an fi+ R factor and belongs to a incompatibility group FII. It carries the genes governing resistance to various aminoglycoside antibiotics, i.e., kanamycin (KM), lividomycin (LV), gentamicin C complex (GM), and 3',4'-dideoxykanamycin B (DKB), in addition to those governing to tetracycline (TC), chloramphenicol (CM), sulfanilamide (SA), and ampicillin (APC). Electron microscopy observation disclosed that the Rms151 deoxyribonucleic acid was a circular form with length of 31.2 mum. A probable circular genetic map of Rms151 was proposed by genetic and biochemical studies, the genes being in the order of -tet-tra-amp-aad-sul-aph-cml-, in which aad and aph confer resistance to KM.GM.DKB by adenylytransferase or resistance to KM.LV by phosphotransferase, respectively.     

Branching in Pea (Action of Genes Rms3 and Rms4)

The nonallelic ramosus mutations rms3-2 and rms4 of pea (Pisum sativum L.) cause extensive release of vegetative axillary buds and lateral growth in comparison with wild-type (cv Torsdag) plants, in which axillary buds are not normally released under the conditions utilized. Grafting studies showed that the expression of the rms4 mutation in the shoot is independent of the genotype of the root-stock. In contrast, the length of the branches at certain nodes of rms3-2 plants was reduced by grafting to wild-type stocks, indicating that the wild-type Rms3 gene may control the level of a mobile substance produced in the root. This substance also appears to be produced in the shoot because Rms3 shoots did not branch when grafted to mutant rms3-2 rootstocks. However, the end product of the Rms3 gene appears to differ from that of the Rms2 gene (C.A. Beveridge, J.J. Ross, and I.C. Murfet [1994] Plant Physiol 104: 953-959) because reciprocal grafts between rms3-2 and rms2 seedlings produced mature shoots with apical dominance similar to that of rms3-2 and rms2 shoots grafted to wild-type stocks. Indole-3-acetic acid levels were not reduced in apical or nodal portions of rms4 plants and were actually elevated (up to 2-fold) in rms3-2 plants. It is suggested that further studies with these branching mutants may enable significant progress in understanding the normal control of apical dominance and the related communication between the root and shoot.     

Long-distance signaling and the control of branching in the rms1 mutant of pea

The ramosus (rms) mutation (rms1) of pea (Pisum sativum) causes increased branching through modification of graft-transmissible signal(s) produced in rootstock and shoot. Additional grafting techniques have led us to propose that the novel signal regulated by Rms1 moves acropetally in shoots and acts as a branching inhibitor. Epicotyl interstock grafts showed that wild-type (WT) epicotyls grafted between rms1 scions and rootstocks can revert mutant scions to a WT non-branching phenotype. Mutant scions grafted together with mutant and WT rootstocks did not branch despite a contiguous mutant root-shoot system. The primary action of Rms1 is, therefore, unlikely to be to block transport of a branching stimulus from root to shoot. Rather, Rms1 may influence a long-distance signal that functions, directly or indirectly, as a branching inhibitor. It can be deduced that this signal moves acropetally in shoots because WT rootstocks inhibit branching in rms1 shoots, and although WT scions do not branch when grafted to mutant rootstocks, they do not inhibit branching in rms1 cotyledonary shoots growing from the same rootstocks. The acropetal direction of transport of the Rms1 signal supports previous evidence that the rms1 lesion is not in an auxin biosynthesis or transport pathway. The different branching phenotypes of WT and rms1 shoots growing from the same rms1 rootstock provides further evidence that the shoot has a major role in the regulation of branching and, moreover, that root-exported cytokinin is not the only graft-transmissible signal regulating branching in intact pea plants.     

Salmonella typhimurium LT2 mutation affecting the deletion of resistance determinants on R plasmids.

Plasmid Rms312, specifying resistance to tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), sulfonamide (Su), and mercury chloride (Mer), deletes both Tc and Cm Sm Su Mer determinants at a high frequency in Salmonella typhimurium LT2. S. typhimurium mutants that were stable carriers of Rms312 were isolated by alternate culture of R-bearing cells in a medium containing either tetracycline or chloramphenicol. In one of these mutants the deletion frequency of drug resistance determinants was decreased by about 100-fold not only Rms312, but also in R100, R1, and R6-5. This mutation caused a slight reduction of ultraviolet resistance but did not affect generalized genetic recombination, indicating that the mutation is different from recA. The mutation, designated dor (deletion of r-determinants), was mapped to a position near 57 units in the new linkage map of S. typhimurijm LT2 (K. E. Sanderson and P. E. Hartman, Microbiol. Rev. 42:471-519, 1978). The dor mutation had no effect on IS1-mediated illegitimate deletion, indicating that the dor mutation is different from the del mutation described by Nevers and Saedler (P. Nevers and H. Saedler, Mol. Gen. Genet. 160:209-214, 1978).     

鲇鱼成鱼和幼鱼中含色素的巨噬细胞集结的诱导发生

通过腹腔注射不同剂量的牛血清白蛋白（ＢＳＡ）或墨汁以刺激巨噬细胞活动,研究了鲇鱼成鱼和幼鱼头肾、肾、脾和肝中含色素的巨噬细胞集结（ＰＭＡ）的发生。鲇鱼淋巴样组织中存在巨噬细胞的不均一性,可分网状／纤维状巨噬细胞,梭状巨噬细胞,圆形红色巨噬细胞,圆形黄红色巨噬细胞和含色素的巨噬细胞。注射ＢＳＡ或墨汁后,头肾、肾,脾和肝中ＰＭＡ和圆形巨噬细胞的数量最终均有不同程度的增加。通过诱导发生实验,表明含色素的     

Transposition of the carbenicillin-hydrolyzing beta-lactamase gene

We isolated a new transposon Tn2101, from plasmid Rms433 in Enterobacter cloacae. Tn2101 encoded the formation of type IV (carbenicillin-hydrolyzing) beta-lactamase and multiple resistance to streptomycin, sulfanilamide, spectinomycin, and mercury in addition to ampicillin. Tn2101 was transposable between conjugative (or nonconjugative) plasmids and the host chromosome. Transposition occurred independently of the general recombination ability of the host cell. Tn2101 had a molecular size of 9.5 x 10(6) and contained short inverted repeat terminal sequences.     

Two loci in the genome of the transposable phage B39 of Pseudomonas aeruginosa affecting the process of integration. I. Study of the properties of phages with the Pde- phenotype and mapping of the rms site

Mutants and recombinants of transposable Pseudomonas aeruginosa bacteriophage B39 with a specific phenotype Pde- (pleiotropic developmental effect) were studied. Pde- phages produce clear minute plaques on lawns of P. aeruginosa PAO1 and fail to grow in cells of PAO1 harbouring Rms 163 (Inc P5) plasmid. Pde+ character is under control of the two loci in phage genome which were designated pdeX and pdeY. In hybrid phages the pdeX and pdeY loci originating from different transposable phages (pdeX from B39 and pdeY from PH132) do not accomplish their function and, as a result, the hybrid phages have the Pde- phenotype. The frequency of integration (f.o.i.) of Pde- phages into bacterial chromosome is lower than f.o.i. for Pde+ phages, as well as the frequency of stable lysogenization of infected bacteria; lytic development of the Pde- phages is also limited. The great difference among the transposable phages in their reaction to the presence of Rms163 plasmid is caused by some differences in the specific rms site in the phage genome. The site is located inside the interval 1.1-3.9 kb of the physical genome map, being closely linked to cI gene of phage B39. The growth of Pde- phages in cells with Rms163 can be restored, due to additional mutations in phage genes affecting lysogenization.