Cucurbitacins are insect steroid hormone antagonists acting at the ecdysteroid receptor.

Two triterpenoids, cucurbitacins B and D, have been isolated from seeds of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)-induced morphological changes in the Drosophila melanogaster BII permanent cell line. With a 20E concentration of 50 nM, cucurbitacins B and D give 50% responses at 1.5 and 10 microM respectively. Both cucurbitacins are able to displace specifically bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone A) from a cell-free preparation of the BII cells containing ecdysteroid receptors. The Kd values for cucurbitacins B and D (5 and 50 microM respectively) are similar to the concentrations required to antagonize 20E activity with whole cells. Cucurbitacin B (cucB) prevents stimulation by 20E of an ecdysteroid-responsive reporter gene in a transfection assay. CucB also prevents the formation of the Drosophila ecdysteroid receptor/Ultraspiracle/20E complex with the hsp27 ecdysteroid response element as demonstrated by gel-shift assay. This is therefore the first definitive evidence for the existence of antagonists acting at the ecdysteroid receptor. Preliminary structure/activity studies indicate the importance of the Delta23-22-oxo functional grouping in the side chain for antagonistic activity. Hexanorcucurbitacin D, which lacks carbon atoms C-22 to C-27, is found to be a weak agonist rather than an antagonist. Moreover, the side chain analogue 5-methylhex-3-en-2-one possesses weak antagonistic activity.

The mosquito ultraspiracle homologue, a partner of ecdysteroid receptor heterodimer: cloning and characterization of isoforms expressed during vitellogenesis.

We report the cloning and characterization of two isoforms of the Ultraspiracle homologue (AaUSP) from the mosquito, Aedes aegypti. The 2.33-kb AaUSPa cDNA has an open reading frame (ORF) of 484 amino acids encoding a polypeptide of 54 kDa, whereas the 2.14-kb AaUSPb ORF of 459 amino acids encodes a 51.3 kDa polypeptide. The AaUSPa and AaUSPb proteins differ only in the N-terminal portion of the variable A/B domain. The AaUSP DNA-binding domain shares 92% and 97% identities with the respective domains of the Drosophila (DmUSP) and Bombyx (BmUSP) Ultraspiracles. However, the AaUSP ligand-binding domain is only 57% and 52% identical to those of DmUSP and BmUSP, respectively. In spite of the relatively low level of sequence conservation, electrophoretic mobility shift assay (EMSA) and hormone-binding assay clearly demonstrated that the products of the AaUSPa and AaUSPb cDNAs are functional heterodimeric partners of the mosquito ecdysteroid receptor. In vitellogenic tissues, each of the two AaUSP isoforms is expressed differently: the AaUSPa is predominant in the fat body and the AaUSPb in the ovary. The kinetics of ovarian AaUSP mRNA coincide with those of the ecdysteroid receptor, being elevated during the previtellogenic period and shortly after the onset of vitellogenesis. In contrast, the level of the AaUSP in the fat body remains relatively constant throughout most of the vitellogenic cycle.

Site-specific mutagenesis of potato spindle tuber viroid cDNA: : Alterations within premelting region 2 that abolish infectivity.

The infectivity of cloned viroid cDNAs permits investigation of structure/function relationships in these unusual pathogenic RNAs by systematic site-specific mutagenesis of the cDNAs and subsequent bioassay. We have used three different strategies to create nucleotide substitutions within premelting region 2, a region of potato spindle tuber viroid (PSTV) believed to be important in viroid replication: sodium bisulfitecatalyzed deamination of deoxycytosine residues, oligonucleotide-directed mutagenesis, and construction of chimeric viroid cDNAs from fragments of infectious PSTV and tomato apical stunt viroid cDNAs. Although their effects upon the rod-like native structure of PSTV should be minimal, C → U transitions at positions 92 or 284 appeared to be lethal. When inoculation with PSTV cDNA containing a single nucleotide substitution was mediated by the Ti plasmid of Agrobacterium tumefaciens, PSTV progeny with an unaltered 'wild type' sequence was obtained. Two factors, the high error frequency characteristic of RNA synthesis and the use of a systemic bioassay for PSTV replication, may explain such sequence reversion and emphasize the importance of an appropriate bioassay system for screening mutant viroid cDNAs.

Genotypic variability of soybean response to agrobacterium strains harboring the ti or ri plasmids.

Twenty four diverse cultivars of soybean (Glycine max [L.] Merrill) and three lines of its annual wild progenitor Glycine soja Sieb and Zucc. were tested for their response to Agrobacterium strains harboring either the Ti (tumor-inducing) plasmid (pTi) from Agrobacterium tumefaciens or the Ri (root-inducing) plasmid (pRi) from Agrobacterium rhizogenes following uniform wounding and inoculation. Based upon gall weight at 8 weeks postinfection, three G. max cultivars (Biloxi, Jupiter, and Peking) and one G. soja line, Plant Introduction (PI) 398.693B, were judged highly susceptible to A. tumefaciens strain A348 (pTiA6), ten genotypes moderately susceptible, 11 weakly susceptible, and two nonsusceptible. Of 26 genotypes inoculated with strain R1000 (pRiA4b), only seven responded in a clearly susceptible fashion by forming small, fleshy roots at internodal infection sites. Cotyledons excised from 1- or 3-day old seedlings of Peking and Biloxi cultivars also formed galls when infected in vitro with agrobacteria carrying either the Ti or Ri plasmid. Tumor lines established from cotyledon and stem galls induced by A. tumefaciens A348 (pTiA6) exhibited the T-DNA borne traits of phytohormone-independent growth and octopine synthesis. Additionally, DNA isolated from cultured tumors hybridized with labeled T-DNA probe.

Construction of infectious potato spindle tuber viroid cDNA clones.

Contiguous restriction fragments from two cloned partial-length potato spindle tuber viroid (PSTV) cDNAs were used to construct recombinant DNAs containing full-length monomeric and dimeric PSTV cDNA. When five different PSTV cDNA plasmids and RNA isolated from E. coli cells harboring these plasmids were tested for infectivity on tomato, plasmid DNAs containing PSTV cDNA dimers were infectious. RNA transcripts containing the sequence of PSTV from these plasmids were also infectious. The sequences of the viroid progeny and the cloned DNA were identical. In vitro mutagenesis of infectious PSTV cDNAs will allow systematic investigation of the role of specific sequences in viroid replication and pathogenesis.

Osmotic stress inhibits thymidine incorporation into soybean protoplast DNA.

DNA synthesis in protoplasts isolated from soybean cell suspension cultures has been investigated by [(3)H] thymidine uptake and incorporation kinetics. Initial rates of incorporation in exponential and 5-fluorodeoxyuridine synchronized protoplasts are inhibited by increased osmolarities of the medium. The inhibition was not readily reversible during 3 h culture in low osmotic medium. Velocity sedimentation analyses of replicating DNA from such protoplasts shows a complex pattern of inhibition. The inhibition probably effects replicon initiation as well as strand elongation and ligation of replication intermediates.

Nuclear DNA from uninfected or potato spindle tuber viroid-infected tomato plants contains no detectable sequences complementary to cloned double-stranded viroid cDNA.

High molecular weight tomato nuclear DNA was isolated from uninfected and potato spindle tuber viroid (PSTV)-infected tomato leaves. Restriction digests were fractionated on agarose gels, denatured and transferred to diazobenzyloxymethylpaper, and hybridized to 32P-labeled cloned double-stranded PSTV cDNA. No hybridization to DNA from either uninfected or infected tissue could be detected under conditions that permitted detection of cloned double-stranded PSTV cDNA at a concentration equivalent to one-fifth copy of PSTV-related DNA per haploid tomato genome. Hybridization of tomato DNA to 32P-labeled cloned soybean 18S and 28S ribosomal DNA sequences showed that the restricted nuclear DNA was suitable for hybridization to probes containing homologous sequences. Our results indicate that neither PSTV nor its complementary strand is transcribed from nuclear DNA but do not rule out the possibility of sequence homology between host DNA and a small portion of PSTV or its complement.

Liposome-mediated delivery of DNA to carrot protoplasts.

The encapsulation of DNA within liposomes and subsequent fusion of the liposomes with carrot (Daucus carota L.) protoplasts were examined to determine optimum conditions for effective liposome-mediated delivery of DNA to protoplasts. Escherichia coli [(3)H]DNA could be encapsulated with 50% efficiency using encapsulation volumes as low as 0.5 ml. Incorporation of liposome-encapsulated [(3)H]DNA by carrot protoplasts increased linearly for 2.5 h, and increasing the ratio of protoplasts to liposomes increased the total amount of radioactive label incorporated within the protoplasts. Liposome-mediated incorporation of [(3)H]DNA by protoplasts increased over a range of polyethylene glycol concentrations up to 20%, but Ca(2+) did not increase liposome-mediated incorporation when present in the liposome-protoplast incubation mixture. Optimum incorporation was observed when the pH of the liposome-protoplast incubation medium was decreased to 4.8. Encapsulation experiments using DNA of the plasmid pBR322 indicated that an average of 200-1,000 intact copies of pBR322 were sequestered within each nucleus after liposome delivery.

Molecular cloning and characterization of potato spindle tuber viroid cDNA sequences.

Double-stranded cDNA has been synthesized from a polyadenylylated potato spindle tuber viroid (PSTV) template and inserted in the Pst I endonuclease site of plasmid pBR322 by using the oligo(dC).oligo(dG)-tailing procedure. Tetracycline-resistant ampicillin-sensitive transformants contained sequences complementary to PSTV [(32)P]cDNA, and one recombinant clone (pDC-29) contains a 460-base-pair insert. This cloned double-stranded PSTV cDNA contains the cleavage sites for six restriction endonucleases predicted by the published primary sequence of PSTV as well as one additional site each for Ava I, Hae III, Hpa II, and Sma I. The additional Ava I, Hpa II, and Sma I sites are explained by the presence of a second C-C-C-G-G-G sequence in the cloned double-stranded cDNA. The largest fragment released by Hae III digestion contains approximately 360 base pairs. These results suggest that we have cloned almost the entire sequence of PSTV, but the sequence cloned differs slightly from that published. Hybridization probes derived from pDC-29 insert have allowed detection and preliminary characterization of RNA molecules having the same size as PSTV but the opposite polarity. This RNA is present during PSTV replication in infected tomato cells.

DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures.

Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 µm. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4-6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4-6S) and large (20-30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm(3). Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 µm.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.

Nonintegrated plasmid-chromosome complexes in Escherichia coli.

A number of plasmid systems have been examined for the ability of their covalently closed circular deoxyribonucleic acid (CCC DNA) forms to cosediment in neutral sucrose gradients with the folded chromosomes of their respective hosts. Given that cosedimentation of CCC plasmid and chromosomal DNA represents a bound or complexed state between these replicons, our results can be expressed as follows. (i) All plasmid systems complex, on the average, at least one plasmid per chromosomal equivalent. (ii) Stringently controlled plasmids exist predominantly in the bound state, whereas the opposite is true for plasmids that exist in multiple copies or are under relaxed control of replication. (iii) The degree to which a plasmid population binds to host chromosomes appears to be a function of plasmid genotype and not of plasmid size. (iv) For the colicin E1 plasmid the absolute number of plasmids bound per folded chromosome equivalent does increase as the intracellular plasmid/chromosome ratio increases in cells starved for required amino acids or in cells treated with chloramphenicol; however, the ratio of bound to free plasmids remains constant during plasmid copy number amplification.

Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number.

We have isolated chromosomal mutants of an Escherchia coli K-12 strain that maintain higher levels of an F' plasmid. The mutants are designated as plasmid copy number (pcn) mutants. They were detected by selecting for increased lactose fermentation in bacteria deleted for the lac operon but harboring an F'lacI,P pro+ plasmid. When examined for the amount of F' plasmid deoxyribonucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show two to seven times as much covalently closed, circular (CCC) DNA as does the parental strain. The increased plasmid level in one mutant strain (pcn-24) was confirmed by DNA-DNA hybridization; however, this latter technique indicated about a twofold lower increase when compared with the increase measured for pcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F' DNA reflects a proportional increase in monomeric-size plasmid molecules because oligomeric forms are not found. Also, in mutant pcn-24 the extra CCC plasmid copies do not seem to be randomly distributed throughout the cell's cytoplasm but appear complexed in situ with their host's folded chromosome. In all pcn mutants examined to date, the classical sex factor F is maintained at normal levels, whereas the viral plasmid Pl CM is maintained at two to three times the normal level. In all 17 pcn mutants isolated, the pcn mutation maps on the chromosome and not on the plasmid. Finally, the absolute amount of CCC F' DNA detectable in lysates of the six different pcn mutants examined decreased 50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNA occurs when lysates of the parental F' strain are incubated at 37 C.