The 3' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.

Isolation of the LEMMI9 gene and promoter analysis during a compatible plant-nematode interaction.

Plant-endoparasitic root-knot nematodes feed on specialized giant cells that they induce in the vascular cylinder of susceptible plants. Although it has been established that a number of plant genes change their expression pattern during giant cell differentiation, virtually no data are available about the mechanisms involved in that change. One possibility is differential promoter recognition by the transcription factor(s) responsible for the expression of specific genes. We have isolated and characterized a genomic clone from tomato containing the promoter region of LEMMI9, one of the few plant genes that have been reported to be highly expressed in galls (predominantly in giant cells). The analysis of transgenic potato plants carrying a LEMMI9 promoter-beta glucuronidase (GUS) fusion has demonstrated that the tomato promoter was activated in Meloidogyne incognita-induced galls in a heterologous system. We have located putative regulatory sequences in the promoter and have found that nuclear proteins from the galls formed specific DNA-protein complexes with the proximal region of the LEMMI9 promoter. The nuclear protein-binding sequence mapped to a region of 111 bp immediately upstream from the TATA box. This region contains a 12-bp repeat possibly involved in the formation of DNA-protein complexes, which might be related to the LEMMI9 transcriptional activation in the giant cells.

Cloning and nucleotide sequence of different iso-IS231 elements and their structural association with the Tn4430 transposon in Bacillus thuringiensis.

A family of five repetitive sequences (RS) has been isolated from a plasmid DNA library of Bacillus thuringiensis strain berliner 1715. In a previous paper [Mahillon et al., EMBO J. 4(1985)3895-3899] one of these was shown to harbor all the features of an IS element (IS231). Further nucleotide sequence analysis revealed that two other RS, flanking the delta-endotoxin gene, are actually variants of IS231. Comparison of the nucleotide sequences surrounding the iso-IS231 elements showed a unique structural association between some of these elements and the transposon Tn4430. Although these IS231 elements have transposed into Tn4430, both these IS231 s and the transposon Tn4430 remain structurally intact.

Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana.

The plant, Arabidopsis thaliana, contains two S-adenosylmethionine synthetase-encoding genes (sam). Here, we analyze the structure and expression of the sam-2 gene and compare it with the previously described sam-1 gene. Northern-blot analysis using gene-specific probes shows that both sam-1 and sam-2 are highly expressed in stem, root, and callus tissue. This similar expression pattern might be mediated by the presence of three highly conserved sequences in the 5' region of both sam genes. Using a chimeric beta-glucuronidase (GUS)-encoding gene, we show that in transgenic tobacco plants, 748 bp of 5' sam-1 sequences generate high GUS activity in the same type of tissues as previously observed in transgenic A. thaliana plants. A deletion analysis of these 5' sam-1 sequences indicates that 224 bp of 5' sam-1 sequences can still induce higher expression of the gene in stem and root relative to leaf. However, the level of expression is reduced when compared to the expression level obtained with the full-length promoter.

Characterization and heterologous expression of the tetL gene and identification of iso-ISS1 elements from Enterococcus faecalis plasmid pJH1.

The tetracycline-resistance (TcR) determinant of the Enterococcus faecalis plasmid pJH1 has been identified and located on a 2.2-kb RsaI-EcoRI fragment. The fragment was cloned in Escherichia coli, and specified TcR in this host. The nucleotide (nt) sequence of the cloned fragment showed the presence of an open reading frame (ORF) of 1374 bp, designated tetL. The nt sequence of tetL from pJH1 was identical to that of the tetL present on pLS1 from Streptococcus agalactiae. Upstream of the pJH1 tetL, part of another ORF was found that, except for two single-nt substitutions, was identical to an iso-ISS1 element from Lactococcus lactis. Hybridization studies indicated the presence of several ISS1-like elements in plasmid pJH1, but not on the En. faecalis chromosome. To study its usefulness as a marker in Gram+ organisms, the pJH1 tetL was cloned on the broad-host-range plasmid pNZ124, resulting in pNZ280, that was found to give resistance to 40 micrograms Tc/ml in Lc. lactis and Bacillus subtilis.

Nucleotide sequence of gene cryIIID encoding a novel coleopteran-active crystal protein from strain BTI109P of Bacillus thuringiensis subsp. kurstaki.

The nucleotide sequence of a novel insecticidal crystal protein(Cry)-encoding gene from a Bacillus thuringiensis serotype kurstaki isolate is described. The gene is related to the known coleopteran-active cryIII genes and encodes a CryIIID that is much more active against Colorado potato beetle than other CryIII.

Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons.

The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43 degrees C).

Developmental expression of tobacco pistil-specific genes encoding novel extensin-like proteins.

We have sought to identify pistil-specific genes that can be used as molecular markers to study pistil development. For this purpose, a cDNA library was constructed from poly(A)+ RNA extracted from tobacco stigmas and styles at different developmental stages. Differential screening of this library led to the isolation of cDNA clones that correspond to genes preferentially or specifically expressed in the pistil. Seven of these cDNA clones encode proteins containing repetitions of the pentapeptide Ser-Pro4, which is a typical motif found in extensins. Unlike extensin genes, the extensin-like genes described here are not induced under stress conditions. RNA gel blot hybridizations demonstrated the organ-specific expression of the extensin-like genes and their temporal regulation during pistil development. After pollination, the transcript levels of the pistil-specific extensin-like genes change relative to levels in unpollinated pistils. In situ hybridization experiments showed that at least one of these pistil-specific genes is specifically expressed in cells of the transmitting tissue. The possible roles of the extensin-like proteins in pistils are discussed.

A new restriction endonuclease from Acetobacter pasteurianus.

A restriction endonuclease, ApaI, has been partially purified from Acetobacter pasteurianus. This enzyme cleaves bacteriophage lambda DNA and Simian virus 40 DNA at one site, adenovirus-2 DNA at more than nine sites, but it does not cleave phi X174 DNA nor plasmid pBR322 DNA. This enzyme recognizes the sequence (formula; see text) and cuts at the sites indicated by the arrows.

Intron-mediated enhancement of transgene expression in maize is a nuclear, gene-dependent process.

In monocots, transgene expression can be stimulated by over two magnitudes by including an intron in the 5' untranslated region (UTR). The underlying mechanism is presently unknown. Inclusion of the salT intron into the 5' UTR of cat and bar genes stimulated expression of the first gene only, indicating that intron-mediated enhancement of expression (IME) is gene-dependent. Stimulation was associated with increased cat RNA levels, which did not result from a reduced cytoplasmic turnover and were not associated with increased translation. This implies that IME acts in the nucleus. Importantly, the cytoplasmic accumulation of spliced cat transcripts, even with IME, is less than that encoded by the intronless bar gene. As the cat and bar genes were flanked by identical regulatory signals, and the transcripts had a similar cytoplasmic stability it may mean that IME rescues rather than stimulates gene expression.

Genome structure of tobacco necrosis virus strain A.

An almost complete sequence of the RNA genome of tobacco necrosis virus (TNV) strain A has been determined. The genome organization is very similar to that of carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 5'-proximal open reading frame (ORF) encodes a 23-kDa protein and read-through of its amber codon into the second ORF is presumably used for the translation of a 82-kDa protein. The third large ORF encodes the 30-kDa coat protein. Two small ORFs are located upstream and one immediately downstream of this coat protein cistron. Extensive sequence similarity was found between the TNV 82-kDa protein and the putative polymerases of TCV, CarMV, cucumber necrosis virus (CNV), maize chlorotic mottle virus (MCMV), red clover necrotic mosaic virus (RCNMV), and barley yellow dwarf virus (BYDV). The TNV coat protein is very similar to southern bean mosaic virus (SBMV) capsid protein. Of the predicted small proteins only a 7.9-kDa protein shows some sequence similarity with a corresponding protein of MCMV, CarMV, and TCV. The others are unique to TNV. Except for the first four nucleotides at the 5' end no homology was found with the RNA of STNV (satellite of TNV).

Structural similarities between the RNAs of two satellites of tobacco necrosis virus.

The complete nucleotide sequence of satellite tobacco necrosis virus 2 (STNV-2) RNA has been determined. It has the same organization as the previously studied STNV-1 RNA. The 5' untranslated regions (about 30 nt) are nearly identical, while the coat protein coding regions (about 600 nt) have 55% nucleotide sequence similarity. The 620-nt-long trailer sequences, with 64% nucleotide sequence conservation, can fold into a phylogenetically conserved secondary structure consisting of three pseudoknots followed by a long-range interaction-born hairpin structure. The significance of these elements is discussed in view of the particular properties (stability, translational competitiveness, and replication) that characterize these RNAs.

Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera.

The complete nucleotide sequence of a cloned gene encoding a 130-kDa crystal protein of Bacillus thuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito-specific toxin with a LC50 value of 43 ng/ml to third-instar larvae of Aedes aegypti. Bt8 is processed by proteases or midgut extracts of mosquito larvae into toxic fragments of 68-78 kDa. Deletion mapping indicated that the active fragment of Bt8 is localized in the N-terminal half of the protoxin molecule. The deduced amino acid sequence of Bt8 has been compared with that of Bt2, a Lepidoptera-specific toxin, previously cloned from Bacillus thuringiensis berliner. Highly homologous amino acid stretches are present in the C-terminal half of the proteins. The N-terminal parts show much less sequence homology but they display a strikingly similar distribution of hydrophilic and hydrophobic amino acids. In addition, Bt8 and Bt2 show a significant immunological cross-reaction. The data indicate that although these B.t. delta endotoxins exhibit a different insect-host specificity, they are structurally related and might use a similar mechanism to interact with insect cell membranes.

Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene.

We have determined the complete nucleotide sequence of the gene for the crown gall enzyme, octopine synthase. The sequence was derived from cloned fragments of the Agrobacterium tumefaciens Ti plasmid Ach5. It displayed a continuous open reading frame encoding a polypeptide chain of 358 amino acids. The nucleotide positions corresponding to the 5' end and poly(A) addition site of the mature octopine synthase mRNA from a tobacco tumor cell line were determined by S1 nuclease mapping. Two sequences closely resembling transcriptional control regions found in eukaryotic genes transcribed by RNA polymerase II were identified in the flanking genomic DNA: a sequence 5'-TATTTAAA-3' was located 32 base pairs upstream from the initiation site of transcription, and a hexanucleotide 5'-AATAAT-3' occurred 17 base pairs in front of the poly(A) addition site. No Shine-Dalgarno sequence was present in the untranslated 5' leader sequence. The observations indicate that this DNA sequence, although naturally carried by a bacterial plasmid, is programmed as a functional plant gene.

A tobacco flower-specific gene encodes a polyphenol oxidase.

Identification of pistil-expressed genes is an important step in understanding pistil development and function in plant reproduction. A tobacco stigma/style cDNA library was differentially screened and several cDNA clones were isolated. One of these tobacco genes, designated tobP1, is characterized here. TobP1 encodes a protein highly homologous to plant polyphenol oxidases. Northern blot analysis of total RNA extracted from different organs and probed with tobP1 cDNA identified a single transcript that is exclusively present in flower organs (petals, stamens, and predominantly in pistils). The tobP1 gene is co-ordinately regulated during development in pistils and stamens, and is not induced in mature leaves even under stress conditions. TobP1 belongs to a multigene family, as reported for PPO in other plant species.

The tobacco luminal binding protein is encoded by a multigene family.

We have cloned cDNAs of the tobacco homolog of the luminal binding protein (BiP) that has been described in other higher eukaryotes. In contrast to the mammalian and yeast protein, tobacco BiP is encoded by a multigene family. The gene products of all the cloned members of this family contain a carboxy-terminal His-Asp-Glu-Leu peptide that may form the signal for retention in the endoplasmic reticulum. Analysis of expression patterns revealed that BiP transcripts are predominantly present in tissues with high rates of cell divisions, in secretory tissues, and in cells treated with tunicamycin. We also show that a chimeric gene containing the coding region of one of the tobacco BiP genes is able to complement a mutation in the Saccharomyces cerevisiae BiP gene.

Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana.

The multigene family encoding the small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase/oxygenase in the crucifer Arabidopsis thaliana has been isolated and the organization and structure of the individual members determined. The family consists of four genes which have been divided into two subfamilies on the basis of linkage and DNA and amino acid sequence similarities. Three of the genes, designated ats1B, ats2B, and ats3B, reside in tandem on an 8 kb stretch of the chromosome. These genes share greater than 95% similarity in DNA sequence and encode polypeptides identical in length and 96.7% similar in amino acid sequence. The fourth gene, ats1A, is at least 10 kb removed from, or completely unlinked to the B subfamily. The B subfamily genes are more similar to each other than to ats1A in nucleotide and amino acid sequence. All four genes are interupted by two introns whose placement within the coding region of the genes is conserved. The introns of the B subfamily genes are similar in length and nucleotide sequence, but show no similarity to the introns of ats1A. Comparison of the DNA sequences within the immediate 5' and 3' flanking sequences among the genes revealed only limited regions of homology. S1 analysis shows that all four genes are expressed.

Differential expression of five Arabidopsis genes encoding glycine-rich proteins.

Five cDNA clones coding for glycine-rich proteins in Arabidopsis thaliana were isolated. The corresponding genes are present in the genome as single copies. The derived protein sequences contain highly repetitive glycine-rich motifs. There is, however, little homology among them, nor with previously described glycine-rich proteins from other species. All five genes are expressed in leaves and stems of 6-week-old plants but show different patterns of expression in other organ systems. Analysis of the effect of different external stimuli on the expression pattern showed that, in most cases, the transcript levels were moderately but selectively affected. With flooding stress, the accumulated level of the transcript from one of the genes was remarkably increased.

Nucleotide sequence and mutational analysis of an immunity repressor gene from Bacillus subtilis temperate phage phi 105.

We have identified and sequenced a bacteriophage phi 105 gene encoding an immunity repressor, the first to be characterized from a temperate phage infecting a Gram-positive host. Using superinfection immunity as an assay for repressor function, the phi 105 repressor gene was located within a 740-bp PvuII-HindIII subfragment near the left end of the phi 105 EcoRI-F fragment. We show that the repressor is specified by the 5'-proximal coding sequence of a translationally overlapping gene pair, transcribed from right to left on the conventional phi 105 map. Comparison of its amino acid sequence (146 residues) with that of a large number of Gram-negative bacterial and phage repressors revealed a putative DNA-binding region between positions 20 and 39. The coding region is preceded by a strong Shine-Dalgarno sequence 5' AAAGGAG 3'. Deletion analysis of the 5'-flanking DNA allowed to identify transcriptional control elements. Their structure, 5' TTGTAT 3' at -35 and 5' TATAAT 3' at -10, strongly suggests that the phi 105 repressor gene is transcribed by the major vegetative form of B. subtilis RNA polymerase, as would be expected for an early phage gene.

Nucleotide sequence and structural organization of an insertion sequence element (IS231) from Bacillus thuringiensis strain berliner 1715.

This paper describes the structural organization of a repetitive DNA sequence isolated from plasmids of Bacillus thuringiensis strain berliner 1715. DNA sequence analysis of this repetitive sequence (RS) revealed all the characteristic features of an insertion sequence (IS). This 1656-bp element is delineated by two 20-bp inverted repeats which are flanked by two 11-bp direct repeats. A long open reading frame spans almost the entire sequence and is preceded by potential transcriptional and translational signals. A structural homology was observed between this RS and the Escherichia coli IS4 element in the length of their direct repeats, the sequence of their inverted repeats and the sequence of their putative transposases. These data strongly suggest that this sequence is an authentic insertion sequence. Therefore the name IS231 is proposed for this first Bacillus IS element. Further relationships with other known IS sequences were also found and are discussed.