Optimizing the expression of chimeric genes in plant cells

Summary The Serratia marcescens chiA gene encodes a secreted chitinase activity which contributes to the fungal growth inhibition exhibited by this bacterium. The coding region from the chiA gene was fused to the promoter and 3 polyadenylation region of the Agrobacterium nopaline synthase gene. Site-directed mutagenesis of specific nucleotides surrounding the initiating AUG of the coding sequence of this chimeric gene resulted in up to an eight-fold increase in the amount of chitinase protein detected in transformed plant tissue. Analysis of the chiA mRNA indicated that these nucleotides also affected mRNA levels. At least 50% of the chitinase protein produced in transformed tobacco cells was the same molecular weight as the S. marcescen secreted protein.     

Evolution,pollination, and systematics of the tribeNeottieae (Orchidaceae)

The various classifications of the orchid tribeNeottieae are reviewed and a new classification is proposed that divides the tribe into three subtribes,Neottiinae, Limodorinae, andCephalantherinae, based primarily on characters of the column (gynostemium). A cladistic analysis illustrates that these three subtribes are more closely related to one another than either is to any other group in subfam.Neottioideae, although there are very few apomorphic characters for the tribe. Pollination biology is also discussed showing links between breeding systems and distribution. There is also a possible role between column and labellum morphology and the emergence of a deceptive pollination syndrome from one of reward.     

Rapid detection of alpha-1-antitrypsin deficiency by analysis of a PCR-induced TaqI restriction site

Summary A single base substitution is responsible for the PI-Z mutation in alpha-1-antitrypsin (AAT) deficiency. The Z mutation, which is in exon V of the AAT gene, was analysed directly using a primer designed with a single base substitution in the DNA sequence. During the polymerase chain reaction with this primer, a restriction enzyme site was created in the exon-V-amplified DNA sequence; this site was present in the normal allele (M form) but absent in the Z form. Here, the design of the primer and the application of the designer primer for prenatal diagnosis of chorion villus samples (CVS) for AAT deficiency is described. The method provides a simple rapid means of prenatal diagnosis of AAT deficiency within a day of the collection of the CVS. The detection of the nucleotide base change in AAT deficiency at the Z mutation site provides the opportunity for accurate prenatal diagnosis where no tissue is available from an AAT-affected individual.     

Fetal lung surfactant lipid synthesis from glycogen during organ culture

The role of fetal lung glycogen as a precursor for lipids during late gestational development was explored by a combination of in vivo labeling with [U-14C]glucose, administered directly to rat fetuses at 18.5 days, and in vitro assessment using an organ explant culture system. Our major objectives were to demonstrate that radioactivity was transferred specifically and preferentially to surfactant lipids, as glycogenolysis occurred, and to determine the molecular distribution of 14C labeling in newly synthesized phosphatidylcholine (PC). Surfactant and residual (non-surfactant) lipids were separated by sucrose density gradient centrifugation, and other subcellular fractions such as microsomes were isolated by subsequent centrifugations. After 72 h of culture, there was a 5.7-fold increase in the concentration of PC in the surfactant fraction, which contributed 8.8% of total PC at the beginning and 29.6% (P less than 0.001) at the end of the 72 h period. The labeling of PC in the surfactant fraction increased markedly during culture, but there was no significant change in the residual fraction or microsomal PC. Hydrolysis of surfactant PC indicated that the radioactivity was predominantly located in the fatty acyl portion of the molecule, both before and after culture; however, PC glycerol labeling also increased significantly during culture. The distribution of PC radioactivity was similar in the residual fraction and microsomes, with the majority of 14C in the fatty acids. Neutral lipid radioactivity also increased significantly in both the surfactant (240%) and residual (136%) fractions. Quantitation of the changes in radioactivity among subcellular components during lung explant culture indicated that the greatest decrease occurred in glycogen, whereas only lipids, particularly those of the surfactant fraction, were found to show significant increases. These results support the hypothesis that glycogen, which accumulates in fetal lung prior to augmented surfactant production, can supply precursors for synthesis of functionally essential pulmonary phospholipids.     

Proteolytic enzymes and arachidonic acid metabolites produced by MRC-5 cells on various microcarrier substrates

Summary Human diploid fibroblasts were cultured on microcarriers made from DEAE-dextran, denatured collagen, DEAE-dextran linked to denatured collagen, and glass. Cells grown on these four substrates were examined for the production of proteolytic enzymes and arachidonic acid metabolites. Culture fluids from cells grown on the DEAE-dextran microcarriers contained the highest amounts of proteolytic enzyme activity. Both plasminogen-independent and plasminogen-dependent fibrinolytic activities were present and the plasminogen-dependent activity seemed to result from the presence of both urokinase and tissue plasminogen activator. Culture fluid from the cells grown on the glass microcarriers contained the least amount of protease activity, and nearly all of the plasminogen-activator activity seemed to be of the urokinase type. Protease activity in the culture fluids of cells grown on the other two substrates were intermediate. With regard to arachidonic acid metabolites, cells grown on the DEAE-dextran microcarriers produced the highest amounts of cyclooxygenase products but very low levels of lipoxygenase metabolites. Cells grown on the other three substrates produced comparable amounts of various cyclooxygenase products (lower than that produced by cells on the DEAE-dextrans substrate). Cells grown on the glass microcarriers also produced detectable amounts of two lipoxygenase metabolites—leukotriene B₄ and leukotriene C₄. Inasmuch as both proteolytic enzymes and arachidonic acid metabolites regulate basic cell properties, the differential amount of these metabolites observed in the culture fluids on the various substrates may contribute to the biological differences that exist on these substrates. This study was supported in part by grants R44 CA 36656 and IK08HL01332-01 from the Public Health Service, U. S. Department of Health and Human Services and by grant BC-512 from the American Cancer Society. JDH is a research fellow of the American Lung Association.     

Splenic natural killer-cell activity in mice infected with Leishmania donovani

Several strains of inbred mice were infected with the protozoan parasite Leishmania donovani, and, at several points during the infection, spleens of groups of these mice were tested for natural killer (NK)-cell activity vs lymphoma target cells in vitro and were evaluated for parasite burdens. Generally, elevated followed by normal (compared to uninfected control mice) or subnormal NK responses occurred as the result of infection. Elevated NK responses were not accompanied by high circulating levels of interferon, yet infected mice responded to an injection of an interferon inducer with interferon production as great as control mice. No consistent correlations among susceptibility phenotype to L. donovani infection, spontaneous NK activity phenotype, and infection-induced NK activation/depression patterns were detected among the various strains of mice.     

Tubulin-colchicine complexes differentially poison opposite microtubule ends

The kinetics of radiolabeled guanosine 5'-triphosphate-tubulin dimer addition to preformed microtubule copolymers, containing large numbers of tubulin-colchicine complexes (TCs), were examined at apparent equilibrium. The results indicated that radiolabeled dimer addition to copolymers occurs predominantly by a "treadmilling" reaction, analogous to that described for unpoisoned microtubules, and that some labeled dimer uptake also occurs by equilibrium exchange. The data further showed that TCs decrease the steady-state treadmilling reaction in a concentration-dependent manner. Since microtubule copolymers exhibited a treadmilling reaction, it was possible to differentially radiolabel opposite copolymer ends with [3H]- and [14C]guanine nucleotides and thus to measure the effects of TCs on dimer loss from opposite copolymer ends upon copolymer dilution. Dimer loss from both copolymer ends was inhibited in a concentration-dependent manner, but dimer loss from copolymer net assembly (A) ends (defined under steady-state conditions) was inhibited to a far greater extent than that from the opposite, net disassembly (D) copolymer ends. TCs therefore exhibited a graded, polar poisoning action, with copolymer A-end association and dissociation rate constants being far more susceptible to TC inhibition than those at the opposite copolymer D ends. The potential significance of this TC effect for regulating microtubule spatial orientation in vivo is discussed.