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Mutations of the ADAR1gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi–Goutières syndrome and bilateral striatal necrosis1–3. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA4,5. Hemizygous ADAR1mutations in the Zα domain cause type I IFN-mediated pathologies in humans2,3and mice6–8; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains9, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1mZα/–mice). Adar1mZα/–mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology in Adar1mZα/–mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused by ADAR1mutations.

Crown gall, a neoplasmic transformation of plant cells, is caused by transfer and integration into nuclear plant DNA of T-DNA, a segment of the Ti plasmid of Agrobacterium tumefaciens1–8. In plant cells, this T-DNA is expressed9–12and determines the morphogenetic growth properties of crown gall cells12–16and the synthesis of opines17–21. The T-DNA in octopine crown galls is composed of either one or two fragments derived from a continuous Ti sequence. Thus far, all octopine tumour lines contain the TL fragment (14 kilobases (kb)), whereas TR-DNA (∼6 kb) can be present as an additional sequence22–24. Foreign DNA, inserted in the T region of a Ti plasmid, is co-transferred to the plant genome17,18,21. However, an important requirement for the use of T-DNA as vectors for plant genetic engineering is the regeneration of normal plants which stably maintain, express and sexually transmit the transferred DNA. We report here the isolation, from tumour tissues induced by mutant Ti plasmids, of morphologically normal plants that produce octopine synthase in all tissues. This enzymatic activity is inherited as a dominant mendelian marker25. These regenerated plants retained and expressed only the octopine synthase gene of the T-DNA.

LARGE plasmids, called Ti-plasmids1, are responsible for the transformation of dicotyledonous plants by Agrobacterium tumefaciens2–5. A comparison of Ti-plasmids derived from different Agrobacterium strains6–10revealed two major groups of homology. These correlate with the Ti-plasmid-determined properties of octopine or nopaline utilisation by Ti-plasmid-harbouring agrobacteria2,5,11–13, and octopine or nopaline synthesis by Ti-plasmid-transformed plant cells11,13. It was originally suggested that octopine or nopaline synthesis in crown-gall tissues might be mediated by bacterial genes, transferred to the transformed plant cells14; this can now be restated as being mediated by Ti-plasmid genes, and is supported both by genetic data15and by hybridisation studies16. The capacity to determine both the catabolism and the synthesis of one or the other of these or similar compounds seems to be the dominant evolutionary significance of the Ti-plasmids and one can therefore speak of ‘octopine’- and ‘nopaline’- Ti-plasmids. In view of the fact that the different types of Ti-plasmids only have a limited degree of DNA sequence homology6, but produce transformed plant cells with similar phenotypes, it was conceivable that at least part of the common DNA sequences would be directly and/or indirectly involved in oncogenicity. We report here the finding of a specific DNA segment which is highly conserved among several different Agrobacterium tumefaciens Ti-plasmids. We also give genetic evidence that this region is essential for oncogenicity and overlaps with the Ti-plasmid DNA found in transformed plant cells.

Foreign genes introduced into plant cells with Ti-plasmid vectors are not expressed. We have constructed an expression vector derived from the promoter sequence of nopaline synthase, and have inserted the coding sequences of the octopine synthase gene and a chloramphenicol acetyltransferase gene into this vector. These chimaeric genes are functionally expressed in plant cells after their transfer via a Ti-plasmid of Agrobacterium tumefaciens.

Light regulates many varied physiological and developmental phenomena during plant growth and differentiation, including the formation of a photosynthetically competent chloroplast from a proplastid1. The expression of ribulose 1,5-bisphosphate carboxylase small subunit (rbcS) genes is regulated by light in a development- and tissue-specific manner2,3. In some plant species, phytochrome has been demonstrated to mediate this response4–7, and photoregulation of rbcS expression occurs at least in part at the level of transcription8,9. We have shown previously that a 5′-noncoding fragment (4–973 base pairs (bp) upstream of the messenger RNA cap site) of the pea rbcS ss3.6 gene contains all of the nucleotide sequence information necessary to direct the photoregulated expression of a bacterial chloramphenicol acetyltransferase (cat) gene in tobacco10. Consistent with these findings, Morelli et al.11have shown by deletion analysis of a second rbcS gene promoter, that the sequences required for photo-regulated expression of rbcS E9 reside within the 5′-noncoding region. They identified an upstream region of ∼700 bp needed for maximum transcription but not light–dark regulation, and a region from −35 to −2 bp which included the TATA box and contained the necessary information for light responsiveness. We now demonstrate that regulatory sequences 5′ distal to the rbcS ss3.6 TATA box and transcriptional start site not only contain the information necessary for maximum expression, but also confer photoregulation. These upstream regulatory sequences function independently of orientation when fused to their homologous promoter or a heterologous promoter.

Studies in viral and mammalian cell systems have identified regulatory elements, collectively termed enhancers, which are cis-acting elements effective in either orientation and at both 5′ and 3′ ends of a gene, and which increase transcription when linked to either their own or a heterologous promoter gene system1–5. Certain upstream elements in yeast have also been shown to exert a negative effect on the rate of transcription of cell-cycle-specific genes and these have been called silencers6. Two of the best characterized plant gene families are those of the small subunit of the ribulose 1,5-bisphosphate carboxylase (rbcS) genes7and the lightharvesting chlorophyll a/b-bind ing protein (lhcp) genes8. The first enhancer element in plants has recently been described showing that an upstream element of an rbcSgene could, in both orientations, confer light-inducible expression on a heterologous promoter/gene system9. We report here that a 247-base pair (bp) element from an lhcpgene acts not only as a light-inducible enhancer but also as a tissue-specific ‘silencer’.

When a chimaeric soybean leghaemoglobin gene was introduced into the genome of another legume species, Lotus corniculatus, nodule-specific expression of the chimaeric gene was found in root nodules formed on fully regenerated plants inoculated with the Lotus microsymbiont, Rhizobium loti. Expression under control of the 5′ upstream region of the soybean gene was regulated at the level of RNA and followed the correct developmental timing. This indicates a conserved induction mechanism for leghaemoglobin genes in legumes.

A chimaeric gene, consisting of the 5′-flanking region of a member of the Pisum sativum gene family encoding ribulose 1,5-bisphosphate carboxylase linked to the coding region of a bacterial chloramphenicol acetyltransferase gene, has been introduced into the genome of the plant Nicotiana tabacum using a Ti plasmid of Agrobacterium tumefaciens. The expression of the chimaeric gene is light-inducible in chloroplast-containing transformed tissue.

The Gram-negative soil bacterium Agrobacterium tumefaciens induces crown gall tumours on most dicotyledonous plants1,2, its Ti plasmids causing the neoplastic transformation of plant cells3–5. Molecular hybridization has shown that transformed cells contain a segment of the Ti plasmid (T-DN A) of molecular weight 8–15 × 106(refs 6–9), of which at least part is transcribed10,11. Some copies of the T-DNA are integrated into the plant DNA (unpublished work). In view of the prokaryotic origin of the T-DNA, the possibility of its localization within either mitochondria or chloroplasts must be considered. We present here evidence for the absence of T-DNA from mitochondria and chloroplasts. Our results clearly show its presence within the nucleus.

The molecular basis of the neoplasmic transformation of plant cells by the crown gall bacterium Agrobacterium tumefaciens is the transfer to and stable maintenance of T DNA—a well defined segment of the bacterial Ti plasmid—in plant cells1–4. Transformed cells express new biosynthetic capacities, such as the synthesis of opines5, of which octopine6and nopaline7are well known examples. Ti plasmids harbour genes enabling the bacteria to degrade opines and use them as carbon and nitrogen sources8. pTi-linked genes also determine the specificity of synthesis of opines in the plant tumour cells8. On this basis the Ti plasmid can be considered an unusual type of catabolic plasmid, which induces the synthesis of its substrate in transformed plant cells. This relationship, called genetic colonization2, has opened prospects for the use of Ti plasmids as vectors for genetic manipulation of plants. To evaluate this possibility we inserted a well defined DNA segment, the bacterial transposon Tn7 (ref. 9), into the Ti-plasmid DNA sequence that determines nopaline synthesis in A. tumefaciens strain T37 Nocc1 (ref. 10). As we report here, the inserted Tn7 DNA segment became part of the T DNA because the 9.6 × 106molecular weight (MW) Tn7 DNA sequence was transferred to, and maintained in, the DNA of tumour tissue cultures induced by this mutant strain.

MEASUREMENTS of the radioactivity in water and biological samples from Bikini and Eniwetok lagoons indicate that although the samples were collected from the most plutonium-polluted waters of the world, the values of the naturally produced radionuclide, polonium-210 were usually greater than the values of plutonium-239 and 240, which are produced by nuclear detonations by factors as great as 100.

CONJUGATIVE plasmids of bacteria are extrachromosomal genetic elements able to bring about DNA transfer by conjugation. The best-studied system is that of F-like plasmids in Escherichia coli, where at least 12 genes are involved1. Expression of the transfer system in this case is subject to negative control2,3. We report here that the newly discovered conjugative activity of Ti plasmids is inducible by specific amino acid derivatives that are also the substrates for plasmid-coded catabolic enzymes. This finding further extends the analogy between the regulation of the lactose operon and of transfer functions.

THE relationship of the plant-oncogenic properties of Agrobacterium tumefaciens to the presence in these strains of large plasmids (the Ti plasmids) has now been convincingly demonstrated in several ways: oncogenic strains cured of the Ti plasmid lose their oncogenicity irreversibly1–3; transfer of Ti plasmids to non-oncogenic plasmidless strains confers oncogenicity to these strains3–7. The first method used to transfer plasmids was based on observations made by Kerr8,9, who had developed a method for the transfer of oncogenicity before the association of oncogenicity with large plasmids was known. It was later demonstrated that this transfer of oncogenicity was due to the transfer of the Ti plasmid3,4. These observations suggested that in certain circumstances the Ti plasmids could behave as conjugative plasmids in promoting bacterial conjugation and plasmid transfer. This paper and its companion10describe experiments confirming that suggestion, making use of the fact that the capacity of Agrobacterium strains to utilise the unusual amino acids octopine (N2-(D-1-carboxyethyl)-L-arginine) or nopaline N2-(1, 3-dicarboxypropyl)-L-arginine) is coded for by Ti plasmid linked genes3,5–7,11, and indicate that octopine and nopaline may act in inducing plasmid transfer.

The most successful method currently used to produce transgenic plants is based on the ability of Argobacterium tumefaciens to infect a number of higher plant species and transfer a defined DNA fragment (T-DNA) to the genome of the infected cells1. This procedure cannot be used with the agriculturally important cereals, although preliminary data have suggested2infection of maize seedlings by A. tumefaciens strains. Purified exogenous DNA can be taken up, integrated and expressed in cells of a variety of plant species including some cereals following direct gene transfer into isolated protoplasts3–7. However, although it is possible to grow isolated cereal protoplast into unorganized tissue (calli)5,7,8, only rice protoplasts have so far been shown to regenerate mature plants9–11. Here we report an alternative approach to transformation of cereal plants which does not involve tissue culture techniques.

THE gram-negative bacterium Agrobacterium tumefaciens induces crown gall tumours in many, mostly dicotyledonous, plants. Zaenen et al.1demonstrated the presence of one or more large plasmids in a number of crown gall-inducing Agrobacterium strains belonging to seven different Agrobacterium groups. They were not able to find such plasmids in eight non-pathogenic Agrobacterium strains belonging to four of the same groups2,3. They therefore formulated the hypothesis that the genetic information for the tumour-inducing principle4in crown gall-inducing Agrobacterium strains is carried by one or several large plasmids.

IT has been suggested that the tumour-inducing capacity of Agrobacterium is determined by plasmid genes1,2. The acquisition of virulence by non-pathogenic agrobacteria from pathogenic agrobacteria has been demonstrated by Kerr3,4. We investigated whether this phenomenon could be the result of transfer of a plasmid. Besides providing further evidence for the hypothesis that plasmid genes carry the genetic information for the tumour-inducing ability of Agrobacterium, the system offers the possibility of studying the genetics and functions of the plasmid.

RIBOSOMES obtained from Escherichia coli contain ribonuclease (RNase) which is latent if the ribosome is intact1. It has been reported that this enzyme originates in the cell wall and adds to the particle only during preparation2. This finding is in contradiction to earlier work3,4and the work of Anraku5which suggest that the enzyme is an intrinsic part of the ribosome. The experiments of Neu et al.2showed addition of RNase to ribosomes but no inactivation had been mentioned. If ribosomes are treated with 0.5 M ammonium chloride solution the RNase is released6,7and subsequently separated from the ribosome by ‘Sephadex’ filtration8.

THE production of rubella complement fixing antigen in BHK-21 cell culture has been reported in a separate publication1. The ease of production of antigen in the BHK-21 host cell system2and the greatly increased yield made this antigen available for experimental manipulation. This communication describes the properties of rubella complement fixing antigen under various conditions of maintenance and storage.

SIMIAN virus 5 (S V5) (ref. 1), a member of the parainfluenza-mumps-Newcastle disease subgroup of myxoviruses2, is a common contaminant of primary cultures of monkey kidney cells. S V5virus multiplies to high titre in monkey kidney cells, but causes only minimal cytopathic effects3. Haemadsorption is necessary to show the presence of the virus4. In contrast, in BHK-21 cells, it produces extensive fusion and disintegration, yielding relatively little virus5.

MAYER et al.1describe the preparation of complement-fixing antigen of vacuolating agent SV-40 in monkey kidney cell cultures. Strain No. 776 of SV -40 used for the work described here was obtained from Dr. H. M. Meyer. In our attempts to prepare a satisfactory complement-fixing antigen, two types of cell cultures were used: primary African green monkey kidney (AGMK) and serially propagated BS–C–l (ref. 1). These cultures were obtained from Microbiological Associates, Inc., and maintained at 34° C on Eagle's basal medium containing 2 per cent agamma ealf serum, 100 mg of streptomycin per ml., 100 units of penicillin per ml. and 2 mM of glutamine per ml.