A system for tissue-specific copper-controllable gene expression in transgenic plants: nodule-specific antisense of aspartate aminotransferase-P2.

A vector system, based on copper controllable gene expression, has been developed to give control over place as well as time of expression of an introduced gene. This system consists of two elements: (1) the yeast ace1 gene encoding a metallo-regulatory transcription factor, ACE1, under control of either an organ-specific or a constitutive promoter; and (2) a gene of interest under control of a chimaeric promoter consisting of the 46 bp TATA fragment of the CaMV 35S RNA promoter linked to four repeats of the ACE1 binding site. The functioning of the system in an organ-specific manner was tested in nodulated Lotus corniculatus plants which consisted of non-transformed shoots plus transformed hairy root tissue 'wild-type tops/transgenic roots'. After addition of copper ions to the plant nutrient solution, beta-glucuronidase (GUS) expression was visualized either specifically in nodules or in both roots and nodules when the ace1 gene was placed under control of the nod45 promoter or the CaMV 35S RNA promoter, respectively. The nodule-specific system was used to express antisense constructs of aspartate aminotransferase-P2 in transgenic Lotus corniculatus plants. When expression was induced by the addition of copper ions to the plant nutrient solution aspartate aminotransferase-P2 activity declined dramatically, and a decrease of up to 90% was observed in nodule asparagine concentration.

Copper-controllable gene expression system for whole plants.

We describe a system for gene expression in plants based on the regulation mechanism of the yeast metallothionein (MT) gene. The system consists of two elements: (i) the yeast ace1 (activating copper-MT expression) gene encoding a transcription factor under control of the cauliflower mosaic virus (CaMV) 35S RNA promoter, and (ii) a gene of interest under control of a chimeric promoter consisting of the 90-base-pair domain A of the CaMV 35S RNA promoter linked to the ACE1 transcription factor-binding site. At elevated copper ion concentrations, the ACE1 protein changes conformation, binds to, and activates transcription from the chimeric promoter. To test the functioning of the system in plants, a construct containing the beta-glucuronidase (GUS) reporter gene under control of the chimeric promoter was prepared, and transgenic tobacco plants were produced. It was shown that GUS activity in the leaves of transgenic plants increased up to 50-fold, either after addition of 50 microM CuSO4 to the nutrient solution or after application of 0.5 microM CuSO4 to the plants in a foliar spray. This GUS expression was repressed after the removal of copper ions. The results show that the activity of the described chimeric promoter directly depends on copper ion concentration and that this system can be used in experiments that demand precise timing of expression.

The use of bacterial genes encoding herbicide tolerance in constructing transgenic plants.

The modes of action of some of the best-studied and widespread herbicides are briefly reviewed. Particular attention is given to those herbicide-inhibited processes that bacteria and plants have in common. We describe bacterial mutant genes of herbicide resistance, peculiarities of their introduction into plants, and success in the construction of transgenic resistant plants.

[Regions of human genome-containing analogs of oncogenes and retroviral genes. II. A new class of retrovirus-like elements]. / Uchastki genoma cheloveka, soderzhashchie analogi onkogenov i genov retrovirusov. II. Novyi klass retrovirusopodobnykh élementov.

Earlier we have found that recombinant phage lambda gp5 contains the sequences homologous to v-mos oncogene and retroviruses. After the nucleotide sequence determination we have found the region with homology to U5 part of retroviral LTR. Adjacent to this region are sequences complementary to 3'-end of tRNAMet. Numerous transcripts reacting with subcloned U5 probe from gp5 are present in polyadenylated RNA fraction from human cells. The humane genome contains several copies of these region, with two of them residing in gp5 locus. On the basis of these data we deduced the presence in the human genome of a new class of retroviral-like elements, existing probably as part of new human endogeneous retrovirus (HuEV).

Human genome: proto-oncogenes and proretroviruses.

A brief review of the studies undertaken at the Laboratory for Molecular Bases of Oncogenesis (Institute of Molecular Biology, Moscow) till middle of 1984 is presented. The human genome contains multiple dispersed nucleotide sequences related to the proto-oncogene mos and to proretroviral sequences in tight juxtaposition to each other. From sequencing appropriate cloned fragments of human DNA in phage and plasmid vectors it follows that one of these regions, NV-1, is a pseudogene of proto-mos with partial duplications and two Alu elements intervening its coding sequence, and the other, CL-1, seems to be also a mos-related gene with a deletion of the internal part of the structural gene. CL-1 is flanked by a proretroviral-like sequence including tRNAiMet binding site and U5 (part of the long terminal repeat). The proretroviral-like sequences are transcribed in 21-35S poly(A)+RNA abundant in normal and malignant human cells. Two hypotheses are proposed: endogenous retroviruses take part in amplification of at least some proto-oncogenes; proto-oncogenes are inactivated via insertion of movable genetic elements and conversion into pseudogenes. Potential oncogenicity of a normal human genome undergoes two controversial influences: it increases due to proto-oncogene amplification and decreases due to inactivation of some of them.

Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs.

A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions. From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences. This mos-specific probe has been used for screening a human gene library cloned in bacteriophage lambda Charon 4A. Of these, 19 clones have been isolated containing mos-related sequences. By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus. Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene. The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed. By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen. The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.