The structure of Tetrahymena pyriformis mitochondrial DNA. II. The complex structure of strain GL mitochondrial DNA.

1. Isolated mtDNA from Tetrahymena pyriformis strain GL is a linear duplex molecule with an average molecular weight of 32.6 - 10(6) and without internal gaps or breaks. Denaturation of this DNA results in single strands with a duplex hairpin at one end. The length of this hairpin varies between 0 and 5 micrometer within one preparation. 2. Uder renaturation conditions the single strands with hairpins are able to circularize in two ways, depending on the length of the hairpin. Circularization is also observed after partial digestion with exonuclease III of native strain GL mtDNA. 3. All these data fit a model (see Fig.2) in which the DNA is heterogeneous in length at both ends. At the left end a 10-micrometer duplication-inversion is present; part of this duplication-inversion is complementary to a region at the right end of the molecule. 4. The analogy between the structural peculiarities of strain GL mtDNA and of some linear viral DNAs is stressed.

The organization of ribosomal RNA genes in the mitochondrial DNA of Tetrahymena pyriformis strain ST.

1. We have constructed a physical map of the mtDNA of Tetrahymena pyriformis strain ST using the restriction endonucleases EcoRI, PstI, SacI, HindIII and HhaI. 2. Hybridization of mitochondrial 21 S and 14 S ribosomal RNA to restriction fragments of strain ST mtDNA shows that this DNA contains two 21-S and only one 14-S ribosomal RNA genes. By S1 nuclease treatment of briefly renatured single-stranded DNA the terminal duplication-inversion previously detected in this DNA (Arnberg et al. (1975) Biochim. Biophys. Acta 383, 359--369) has been isolated and shown to contain both 21-S ribosomal RNA genes. 14 S ribosomal RNA hybridizes to a region in the central part of the DNA, about 8000 nucleotides or 20% of the total DNA length apart from the nearest 21 S ribosomal RNA gene. 3. We have confirmed this position of the three ribosomal RNA genes by electron microscopical analysis of DNA . RNA hybrid molecules and R-loop molecules. 4. Hybridization of 21 S ribosomal RNA with duplex mtDNA digested either with phage lambda-induced exonuclease or exonuclease III of Escherichia coli, shows that the 21-S ribosomal RNA genes are located on the 5'-ends of each DNA strand. Electron microscopy of denaturated mtDNA hybridized with a mixture of 14-S and 21-S ribosomal RNAs show that the 14 S ribosomal RNA gene has the same polarity as the nearest 21 S ribosomal RNA gene. 5. Tetrahymena mtDNA is (after Saccharomyces mtDNA) the second mtDNA in which the two ribosomal RNA cistrons are far apart and the first mtDNA in which one of the ribosomal RNA cistrons is duplicated.

Conservation of the sequence and position of the ribosomal RNA genes in Tetrahymena pyriformis mitochondrial DNA.

1. We have done cross-hybridizations between the mitochondrial ribosomal RNAs and DNAs from strains ST and PP of Tetrahymena pyriformis. DNA . ribosomal RNA hybrid formation can be completely prevented by an excess of the heterologous ribosomal RNA and the heterologous hybrids melt 6 degrees C below the homologous hybrids. This shows that the ribosomal RNA cistrons can account for the 5% cross-hybridization previously observed between the mtDNAs of strains PP and ST (Goldbach et al. (1977) Biochim. Biophys. Acta 477, 37--50). 2. By electron microscopy of DNA . ribosomal RNA hybrids we have determined the position of the ribosomal RNA cistrons on the mtDNA of strain GL, a mtDNA which we have shown to contain a sub-terminal 1 micron duplication-inversion and a terminal palindrome at one end which varies in length from 0 to 5 micron and which includes the 1 micron duplication-inversion (Arnberg et al. (1977) Biochim. Biophys. Acta 477, 51--69). The 21 S ribosomal RNA cistron overlaps the 1 micron duplication-inversion and as a result two or three cistrons are present, depending on the size of the terminal palindrome. Only one 14 S ribosomal RNA cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron is found, located about 10 000 base pairs away from the nearest 21 S cistron and with the same polarity as this cistron. 3. We conclude from these results and those in the preceding paper that the sequence of the ribosomal RNAs and the position of the ribosomal RNA genes in the mtDNA is strongly conserved in Tetrahymena. Possible reasons for the duplication of 21-S ribosomal RNA genes and the terminal heterogeneity of Tetrahymena mtDNA are discussed.

The structure of Tetrahymena pyriformis mitochondrial DNA. I. Strain differences and occurrence of inverted repetitions.

We have analysed the structure of the mtDNAs of six amicronucleate Tetrahymena pyriformis strains, belonging to at least four phenosets, as defined by Borden et al. (Borden, D., Whitt, G.S. and Nanney, D.L. (1973) J. Protozool. 20, 693--700). 2. The mtDNAs of all strains are linear, but they differ in size, in their fragmentation by endonuclease EcoRI and in overall sequence; less than 20% sequence homology was found by DNA-DNA hybridization in all combinations tested, except for the mtDNAs from strains T and ST which are indistinguishable. 3. In spite of these marked sequence differences the mtDNAs of all strains share two structural peculiarities: ragged (gnawed) duplex ends and a duplication-inversion, which varies in length between 0.3 and 1.2 micrometer, depending on the strain. In four strains the duplication-inversion is terminal, allowing formation of single-stranded DNA circles with a duplex tail; in two strains it is subterminal. 4. The ragged ends and sub-terminal position of the duplication-inversion in some of the Tetrahymena mtDNAs do not fit any of the current models for the replication of linear mtDNAs.

Replication of the linear mitochondrial DNA of Tetrahymena pyriformis.

1. Electron micrographs of the linear mtDNA from Tetrahymena pyriformis strain GL show linear molecules with a duplex 'eye' of variable size in the middle. This indicates that replication of this DNA starts near the middle of the molecule and proceeds bidirectionally to the ends, as previously shown for the mtDNA of strain ST (Arnberg, A.C., Van Bruggen, E.F.J., Clegg, R.A., Upholt, W.B. and Borst, P. (1974) Biochim. Biophys. Acta 361, 266-276). The mtDNAs of these two strains have little base sequence homology beyond the ribosomal RNA cistron (Goldbach, R.W., Bollen-De Boer, J.E., Van Bruggen, E.F.J. and Borst, P. (1978) Biochim. Biophys. Acta 521, 187-197). 2. Electron micrographs of mtDNA from strain ST, spread under non-denaturing conditions, contain only molecules with fully duplex ends. mtDNA spread under conditions of early denaturation contains duplex loops on one end (40% of all molecules) or both ends (37%). The loops are stable to partial denaturation and vary in size from 0.15 to approximately 1.0 micron, most loops measuring 0.25--0.40 micron. No loops are formed with single-stranded DNA under analogous conditions and we conclude from this result that loop formation is based on the presence of straight, rather than inverted, duplications near the ends. 3. When full-length 3H-labelled mtDNA from strain ST, 32P-labelled at the 5'-termini with T4 polynucleotide kinase, was sedimented in alkaline sucrose gradients, greater than 70% of the 3H and less than 30% of the 32P cosedimented with full-length molecules; the remaining 32P sedimented heterogeneously and predominantly with the DNA less than 10% the size of intact single strands. Brief incubations of full-length mtDNA with DNA polymerase I from Escherichia coli and labelled dNTPs at 15 degrees C did not lead to preferential labelling of terminal EcoRI fragments of the DNA. From these results we infer that the DNA contains nicks or gaps near the termini and that these are not bordered by free 3'-OH groups. 4. A model is presented in which straight sequence repetitions at the termini of Tetrahymena pyriformis mtDNA are involved in the later stages of replication. This model can also account for the pronounced terminal heterogeneity previously observed in this DNA.

Mitochondrial DNA of Tetrahymena pyriformis strain ST contains a long terminal duplication-inversion.

1. We have studied denatured Tetrahymena mtDNA by electron microscopy using the formamide technique. 2. After denaturation all DNA is single stranded, but within a few minutes single-stranded circles with a duplex tail are formed. 3. The duplex tail is 1.3 mum long, i.e. 8 percent of the length of native mtDNA, and it often contains a small single-stranded eye. 4. Digestion of the duplex DNA with exonuclease III of Escherichia coli abolishes its ability to form circles and duplex tails after denaturation. 5. Renaturation of denatured mtDNA leads to the formation of duplex circles with single-stranded section and/or duplex tails. In addition, a minority of duplex circles without apparent tails is formed, but these circles contain a small ambiguous section. 6. We conclude that this mtDNA contains a long terminal duplication-inversion, that could be involved in the replication of this linear mtDNA.

A highly conserved genomic region in baculoviruses: sequence analysis of an 11.3 kbp DNA fragment (46.5-55.1 m.u.) of the Spodoptera exigua multicapsid nucleopolyhedrovirus.

A DNA fragment of 11.3 kilobase pairs (kbp) in size of the baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) genome (46.5 to 55.1 m.u.) was completely sequenced. Analysis of the sequence revealed eleven potential open reading frames (ORF). Ten of these ORFs showed significant amino acid identity to Autographa californica MNPV (AcMNPV) and Orgyia pseudotsugata MNPV (OpMNPV) genes p6.9, lef5, 38K, p19, p143, p25, p18, vp33, lef4, and vp39. One ORF (XC12) has no homolog in other baculoviruses and may be unique to SeMNPV. All but three of these putative genes are preceded by the consensus baculovirus late promoter element (5'-ATAAG-3'). The genetic organization and the putative map of transcripts of this fragment suggested that this region is highly similar to a region in AcMNPV fragment EcoRI-D. Comparison of the genetic organization of this 11.3 kbp fragment with the genomes of AcMNPV, OpMNPV, Bombyx mori NPV (BmNPV) and SeMNPV revealed that this region is highly conserved among baculovirus genomes. This is in contrast to the genetic organization of the polyhedrin-p10 region, which is much more diverged, but has been taken as point of reference to orient baculovirus physical maps. Through its diversity the latter region, however, would be an excellent candidate to determine baculovirus relatedness and phylogeny. The presence of conserved and diverged regions in baculovirus genomes with respect to gene order is reminiscent to the situation in other large DNA viruses, such as herpes- and poxviruses, where conserved central and diverged terminal parts are common characteristics. The role of this feature in the genomic organization of large DNA viruses is discussed with particular emphasis on virus replication and evolution.

Genetic organization of the HindIII-I region of the single-nucleocapsid nucleopolyhedrovirus of Buzura suppressaria.

In order to investigate the genomic organization of the single-nucleocapsid nucleopolyhedrovirus (SNPV) of Buzura suppressaria (BusuNPV), the HindIII-I fragment located at map units (mu) 26.6-29.4 of the viral genome was sequenced. The fragment contained two partial and three complete open reading frames (ORFs) representing the 3' end of a polyhedron envelope protein gene (pep), a homologue of the AcMNPV ORF117, a conotoxin-like protein gene (ctl), an inhibitor of apoptosis gene (iap) and a superoxide dismutase gene (sod), respectively. These five genes were identified for the first time in a SNPV. Sequence analysis further revealed that these ORFs have the same conserved motifs and gene structure as those observed in their homologues from other baculoviruses. Between ctl and iap, an intergenic region of about 700 basepairs with structure similar to non-hr origins of DNA replication was observed. The genomic arrangement of the ORFs in the BusuNPV HindIII-I fragment is very different from the arrangement of their homologues in the genome of Autographa californica multiple nucleocapsid (M) NPV and other baculoviruses to date. Our data suggest that on the basis of gene arrangement, BusuNPV belongs to a distinct taxon within the Baculoviridae family, corroborating our previous conclusions derived from phylogeny analysis of several BusuNPV genes.

Immuno-electron microscopical detection of tomato spotted wilt virus and its nucleocapsids in crude plant extracts.

Tomato spotted wilt virus (TSWV) particles were identified in thin sections and in crude extracts of leaves from plants infected with different TSWV isolates, using gold labelled protein A and antibodies prepared against purified virus particles or against nucleocapsid preparations. In addition, both in thin sections and in dip preparations aggregates were detected using either gold-labelled antiserum. These aggregates were not detected using antisera against envelope proteins or against the non-structural protein NSs. They were the only detectably labelled material in preparations made from plants infected by morphologically defective isolates of TSWV, i.e. isolates that did not produce enveloped particles. The aggregates were discerned in dip preparations as cloudy amorphous structures having a cottonwool boll-like appearance. These 'cotton bolls' were interpreted as being the in vitro structures of the nucleocapsid aggregates occurring in plant cells infected by isolates maintained by mechanical inoculation and are the only structures in cells infected by morphologically defective isolates (Ie, 1982; Kitajima et al., 1992). The detection of the 'cotton boll-like' structures in crude extracts provides an adequate and rapid method to establish the presence of nucleocapsid aggregates in cells of infected plants, irrespective of whether they are infected by morphologically defective isolates or not.

Thrips Resistance in Pepper and Its Consequences for the Acquisition and Inoculation of Tomato spotted wilt virus by the Western Flower Thrips.

ABSTRACT Different levels of thrips resistance were found in seven Capsicum accessions. Based on the level of feeding damage, host preference, and host suitability for reproduction, a thrips susceptible and a resistant accession were selected to study their performance as Tomato spotted wilt virus (TSWV) sources and targets during thrips-mediated virus transmission. Vector resistance did not affect the virus acquisition efficiency in a broad range of acquisition access periods. Inoculation efficiency was also not affected in short inoculation periods, but was significantly lower on plants of the thrips resistant accession during longer inoculation access periods. Under the experimental conditions used, the results obtained show that transmission of TSWV is little affected by vector resistance. However, due to a lower reproduction rate on resistant plants and a lower preference of thrips for these plants, beneficial effects of vector resistance might be expected under field conditions.

Restricted Spread of Tomato spotted wilt virus in Thrips-Resistant Pepper.

ABSTRACT Spread of Tomato spotted wilt virus (TSWV) and population development of its vector Frankliniella occidentalis were studied on the pepper accessions CPRO-1 and Pikante Reuzen, which are resistant and susceptible to thrips, respectively. Viruliferous thrips were released on plants of each accession (nonchoice tests) or on plants in a 1:1 mixture of both accessions (choice tests) in small cages containing 8 or 16 plants. Significantly fewer CPRO-1 plants became infected in the primary infection phase in both tests. In the nonchoice test, virus infection of the resistant plants did not increase after the initial infection, but all plants eventually became infected when mixtures of both cultivars were challenged in the secondary infection phase. Secondary spread of TSWV from an infected resistant or susceptible source plant was significantly slower to resistant plants than to susceptible plants, independent of source plant phenotype. The restricted introduction and spread of TSWV in the thrips-resistant cultivar was confirmed in a large-scale greenhouse experiment. The restricted and delayed TSWV spread to plants of the resistant accession in both the cage and the greenhouse experiment was explained by impeded thrips population development. The results obtained indicate that thrips resistance may provide a significant protection to TSWV infection, even when the crop is fully susceptible to the virus.

Tomato spotted wilt virus Infection Improves Host Suitability for Its Vector Frankliniella occidentalis.

ABSTRACT The effect of Tomato spotted wilt virus (TSWV) infection on plant attractiveness for the western flower thrips (Frankliniella occidentalis) was studied. Significantly more thrips were recovered on infected than were recovered on noninfected pepper (Capsicum annuum) plants in different preference tests. In addition, more offspring were produced on the virus-infected pepper plants, and this effect also was found for TSWV-infected Datura stramonium. Thrips behavior was minimally influenced by TSWV-infection of host plants with only a slight preference for feeding on infected plants. Offspring development was positively affected since larvae hatched earlier from eggs and subsequently pupated faster on TSWV-infected plants. These results show a mutualistic relationship between F. occidentalis and TSWV.

Application of Phage Display in Selecting Tomato spotted wilt virus-Specific Single-Chain Antibodies (scFvs) for Sensitive Diagnosis in ELISA.

ABSTRACT A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S, which allowed the scFvs to be expressed as alkaline phosphatase fusion proteins, 17 different scFv antibodies were obtained. Of these, 12 scFvs were directed against the nucleoprotein (N) and 5, putatively, against the glycoproteins (G1 and G2). Five of the N-specific antibodies cross-reacted with two other tospoviruses (Tomato chlorotic spot virus and Groundnut ringspot virus), but none recognized the more distantly related tospoviruses Impatiens necrotic spot virus, Watermelon silverleaf mottle virus, Iris yellow spot virus, or Physalis severe mottle virus. The successful use of one of the antibodies as coating and detection reagent in a double-antibody sandwich enzyme-linked immunosorbent assay showed the potential of the phage display system in obtaining antibodies for routine TSWV diagnosis.

Hexachlorobutadiene residues in aquatic fauna from surface water fed by the river rhine.

Hexachlorobutadiene (HCBD) was identified as a component of the residue mixture of chlorinated hydrocarbons in fish from inland waters fed by the Rhine, particularly in the Ketelmeer. The residue level in fish is related to the fat content; no bioaccumulation in the food chain to higher trophic levels was observed. HCBD concentrations in the water of the Ketelmeer appeared to be three orders of magnitude lower than those found in aquatic animals.

Tomato yellow leaf curl virus Can Be Acquired and Transmitted by Bemisia tabaci (Gennadius) from Tomato Fruit.

The whitefly Bemisia tabaci is an insect pest causing worldwide economic losses, especially as a vector of geminiviruses such as Tomato yellow leaf curl virus (TYLCV). Currently, imported and exported tomato fruit are not monitored for TYLCV infection because they are not considered to represent a potential risk as a virus source for whiteflies. A survey of tomato fruit imported into Réunion Island indicated that more than 50% of the fruit contained TYLCV as determined by DNA blot analysis. Moreover, we showed that TYLCV was present at a high titer in tomato fruit, and demonstrated that it can be acquired by whiteflies and subsequently transmitted to healthy tomato plants. Potential risk of the spread of TYLCV by tomato fruit in natural conditions needs to be further assessed.

A hybrid baculovirus-bacteriophage T7 transient expression system.

A hybrid recombinant baculovirus-bacteriophage T7 expression system was developed for transient expression in insect cells of plasmids with foreign genes provided with a T7 promoter. The coding sequence for T7 RNA polymerase, with or without a nuclear localization signal, was inserted into the genome of Autographa californica nuclear polyhedrosis virus. Recombinant viruses stably expressed T7 RNA polymerase in insect cells. Upon transfection of infected insect cells with plasmids containing the genes for chloramphenicol acetyltransferase (CAT), the hepatitis B virus precore-, core- or e- antigens under control of the T7 promoter, transient expression of these genes was detected by ELISA. The results obtained indicate that this baculovirus/T7 system provides a simple and widely applicable tool for transient gene expression studies.

Identification of potyvirus isolates from faba bean (Vicia faba L.), and the relationships between bean yellow mosaic virus and clover yellow vein virus.

Clover yellow vein virus (CYVV) isolate H and the related potyvirus isolates E178, E197, and E242 could be distinguished from bean yellow mosaic (BYMV) isolates by their wider host range among non-legume test plant species, the peculiar enlargement of the nucleolus in infected plants, and the larger size of their coat protein as evidenced by slower migration in SDS-PAGE. Serologically, they are qualitatively indistinguishable in electro-blot immunoassay (EBIA) also with antibodies specific to the N-terminal part of BYMV-B25 coat protein, implying therefore that CYVV and BYMV coat proteins contain identical amino acid sequences in the N-terminal region. The faba-bean virus isolates from Sudan, Syria, and The Netherlands could be identified as BYMV isolates especially adapted to faba bean. All of them were weakly pathogenic to Phaseolus bean with the exception of SV205, assuming an intermediate position between Phaseolus-bean isolates, with low pathogenicity to faba bean, and faba-bean isolates, usually having low pathogenicity to Phaseolus bean. Strains of BYMV are thus hard to delimit.

A putative non-hr origin of DNA replication in the HindIII-K fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus.

In addition to the seven known homologous regions (hrs) of Autographa californica multiple nucleocapsid polyhedrosis virus (AcMNPV) the HindIII-K fragment was also found to carry a putative ori, although this fragment does not contain an hr. Deletion analysis showed that this ori contains several segments essential for its activity and other 'auxiliary' sequences that enhance the ori activity. Sequence analysis identified several structures often found in other viral replication oris, such as palindromes and other repeated motifs. Although most of the auxiliary sequences of this ori were found to be deleted in the Bombyx mori nucleocapsid polyhedrosis virus genome, the essential part of this ori, containing the palindromes and the A/T-rich region, was retained. This and the fact that after prolonged serial passage of AcMNPV large replicating DNA molecules are found in which repeated sequences derived from the HindIII-K fragment accumulate are consistent with this region being a putative origin of AcMNPV DNA replication.

Virus-ribosome complexes from cell-free translation systems supplemented with cowpea chlorotic mottle virus particles.

When particles of cowpea chlorotic mottle virus (CCMV) were added to cell-free extracts from wheat germ, the encapsidated viral genome was translated into polypeptides similar to the translation products specified by unencapsidated viral RNA (as shown before by M.J. Brisco, R. Hull, and T.M.A. Wilson, 1986, Virology 148, 210-217). The rate of protein synthesis observed upon addition of virus particles was much slower than that of extracted RNA and the quantity of protein formed was only 10% of that of extracted RNA. Using sucrose and cesium-chloride gradient analysis, virus-ribosome complexes, containing up to four ribosomes per virus particle, were isolated from translation mixtures supplemented with CCMV particles. These complexes, with densities intermediate of those of virus (1.36 g cm-3) and ribosomes (1.58 g cm-3), were analyzed and quantified in the electron microscope. Less than 5% of the particles was found in association with ribosomes. To verify whether these complexes were involved in the process of cotranslational disassembly, tobacco mosaic virus was analyzed with the same techniques and methods. The results found for TMV were similar to those found for CCMV except that virus-ribosome complexes with up to 20 ribosomes per virus particle were observed. The implications of the process of virion-directed translation for the structure of the particle as well as the role of this process in vivo are discussed.