Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen.

BACKGROUND: The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B- and T-cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an anti-TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex. RESULTS: The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two-hybrid screening as the gene products which directly interacted with TdT. The interaction between PCNA and TdT was confirmed by co-immunoprecipitation, both in vitro and in vivo. TdT binds directly to a PCNA trimer, as shown by gel filtration. TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA-1 C-terminal (BRCT) domain. TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation. CONCLUSION: TdT interacts directly with PCNA through its DPD. A functional consequence of this interaction is the negative regulation of TdT activity. These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination.

Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane.

The virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls. Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium. We have examined the localization and orientation of this protein in the cell membrane. When the nucleotides encoding the first 30 to 41 amino acids from the N-terminus of the VirD4 protein were fused to the gene for alkaline phosphatase from which the signal sequence had been removed, alkaline phosphatase activity was detectable under appropriate conditions. Immunoblotting with VirD4-specific antiserum indicated that the VirD4 protein could be recovered exclusively from the membrane fraction of Agrobacterium cells. Moreover, when the membrane fraction was separated into inner and outer membrane fractions by sucrose density-gradient centrifugation, VirD4 protein was detected in the inner-membrane fraction and in fractions that sedimented between the inner and outer membrane fractions. By contrast, the VirD4'/alkaline phosphatase fusion protein with the N-terminal sequence from VirD4 was detected only in the inner membrane fraction. Treatment of spheroplasts of Agrobacterium cells with proteinase K resulted in digestion of the VirD4 protein. These results indicate that the VirD4 protein is transported to the bacterial membrane and anchored on the inner membrane by its N-terminal region. In addition, the C-terminal portion of the VirD4 protein probably protrudes into the periplasmic space, perhaps in association with some unidentified cellular factor(s).

Isolation of an auxin-regulated gene cDNA expressed during the transition from G0 to S phase in tobacco mesophyll protoplasts.

A cDNA clone for an auxin-regulated gene was isolated from a tobacco mesophyll protoplast cDNA library by differential screening. Nucleotide, sequence analysis showed that the deduced product of the gene, which we have designated par, is hydrophilic and is composed of 220 amino acids. No significant homology to other known proteins was detected. The mRNA of the par gene is approximately 900 bases long and its accumulation was detected in cultured mesophyll protoplasts as early as 30 min after the addition of 2,4-dichlorophenoxyacetic acid to the culture medium. The par mRNA was not detected in leaves or freshly prepared protoplasts or in protoplasts in the absence of 2,4-dichlorophenoxyacetic acid. Expression of the par gene was detected at a low level in actively dividing BY-2 tobacco suspension culture cells. The conspicuous accumulation of par mRNA before the initiation of DNA synthesis in tobacco mesophyll protoplasts suggests that the par gene product could play a role in the initiation of meristematic activity in differentiated mesophyll cells.

Subcellular localization of tobacco mosaic virus minus strand RNA in infected protoplasts.

Radioactive RNA probes were prepared which specifically hybridize with sequences complementary to 5' and 3' regions of tobacco mosaic virus (TMV) RNA. These probes were used in Northern hybridization to locate TMV-RNA minus strands in the subcellular fractions of infected tobacco protoplasts. When the protoplasts were lysed with Triton X-100, full-length minus strands were present in the cytoplasmic but not in the nuclear fraction. With mechanically broken protoplasts, the crude nuclear fraction (250 g pellet) contained small amount of minus strands which appeared to derive from unbroken protoplasts, but most of minus strands were recovered in a fraction sedimented between 250 and 2500 g, little if any being found in lighter fractions. The results indicate that TMV-RNA replicates in association with an extranuclear structure.

Right-hand border regions of octopine T-DNA are recognized by RNA polymerase of Agrobacterium as well as by VirD1 and VirD2 proteins.

The T-DNA of octopine Ti plasmid of Agrobacterium tumefaciens contains TL- and TR-DNA regions each bounded by 25 base-pair-repeats (designated A, B, C and D from left to right). Short DNA segments containing the borders B, C and D were found to function as promoter when placed in the rightward orientation upstream of promoter-less lacZ. Promoter consensus sequence of Agrobacterium were found within these border repeats and in their adjacent regions. The expression of lacZ was low when the segments contained the overdrive, a sequence known to enhance T-DNA transfer. Simultaneous overproduction of VirD1 and D2 proteins, endonuclease acting on the border repeats, interfered with the promoter functions of the border segments. In spite of their activity under these conditions, the border regions do not seem to be involved in the gene expression, because they are not followed by appropriate open reading frames. We propose that RNA polymerase of Agrobacterium competes with VirD products for T-DNA borders and thereby affects the transfer of T-DNA.

Escherichia coli lacZ gene as a biochemical and histochemical marker in plant cells.

Several lacZ chimeric genes were constructed by fusing the truncated lacZ sequence of Escherichia coli to N-terminal sequences of few other genes. Promoters used to direct expression of the chimeric genes were the promoter for 35S RNA of cauliflower mosaic virus (P35S) as well as those of the small subunit gene of ribulose bisphosphate carboxylase and the octopine synthase gene. These constructs were introduced into tobacco cells using a Ti plasmid of Agrobacterium tumefaciens, and beta-galactosidase activity in uncloned and cloned calli derived from the crown galls were examined. The results showed that the P35S-linked lacZ chimeric gene is expressed very efficiently. When slices of the crown gall carrying this chimeric gene were placed on plates containing indicator XGal, localized areas of the outgrowth turned deep blue, whereas no such areas were found in the crown gall having promoter-less lacZ. Calli from galls containing this construct expressed beta-galactosidase activity at an eight-fold higher level (approx. 7000 units/mg protein) than the endogenous activity (approx. 900 units/mg protein). Some of the calli displayed over 20-fold higher activity. Actively growing mini calli expressing activity higher than 4000 units/mg protein dyed deep blue on XGal agar medium such that they were distinguishable from calli having no lacZ. Half of the uncloned P35S-lacZ transformant calli showed activity higher than this level. These results indicate that the lacZ gene linked to a strong promoter such as P35S is useful as a biochemical and histochemical marker gene in plant cells.

Factor inducing Agrobacterium tumefaciens vir gene expression is present in monocotyledonous plants.

Agrobacterium tumefaciens harboring the tumor-inducing Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, Ti plasmid DNA sequence is stably transferred (T-DNA) by unknown mechanisms to plant cells to be integrated into nuclear DNA. The T-DNA processing and transfer require the expression of vir (virulence) genes on the Ti plasmid, which are known to be induced by certain phenolic compounds released from cells at the wounded inoculation site. The results of the present study demonstrate that wheat and oats contain a substance(s) that induces vir gene expression, yet the inducing substance of wheat differs from the phenolic inducers in that it is hydrophilic and has a molecular weight of several thousand. The novel inducer was not detectable in the exudates of seedlings of these plants but was found in an extract from the transition region between shoot and root of the seedlings and also in extracts from the seeds, bran, and germ. This finding suggests that T-DNA processing and possibly its transfer should take place when Agrobacterium invades suitable tissues of monocotyledonous plants.

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses.

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.

Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium.

T-DNA circularization is one of the molecular events specifically induced in agrobacterial cells upon their infection of dicotyledonous plant cells. We developed a seedling co-cultivation procedure to determine whether or not monocotyledonous plants have the ability to induce T-DNA circularization and vir gene expression. Co-cultivation of Agrobacterium tumefaciens with seedlings of dicotyledonous plants showed that the circularization event takes place efficiently. The exudates and extracts of the seedlings also effectively induced T-DNA circularization and vir gene expression, indicating that dicotyledonous seedlings contain diffusible factors capable of inducing these molecular events. In contrast, neither T-DNA circularization nor vir gene expression was detectable when Agrobacterium was incubated with seedlings of monocotyledonous plants. Supplementing with acetosyringone, a known inducer of vir gene expression and T-DNA circularization, resulted in the induction of circularization during co-cultivation with moncotyledonous seedlings. These results indicate that the seedlings of monocotyledonous plants have no detectable amounts of diffusible inducers, unlike dicotyledonous seedlings. Therefore, it is unlikely that the vir genes are expressed in Agrobacterium inoculated in monocotyledonous plants. This may be one of the blocks in tumorigenesis of monocotyledonous plant by Agrobacterium.

The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA.

The formation of crown gall tumours involves the transfer of the T-DNA region of the Ti plasmid from Agrobacterium to plant cells and its subsequent integration into plant chromosomes. When agrobacteria are incubated with plant protoplasts or exudates of plants, the T-DNA region is circularized by recombination or cleavage and rejoining between the 25 bp terminal repeats; the formation of circular T-DNAs is thought to be one step in T-DNA transfer (Koukolikova-Nicola et al. 1985; Machida et al. 1986). We previously showed that the virulence region of the Ti plasmid is required for T-DNA circularization. In the present paper, we examined the circularization event in agrobacteria harbouring octopine Ti plasmids with mutations in various loci of the virulence region. The results clearly demonstrate that the gene(s) encoded in the virD locus are necessary for T-DNA circularization. In particular, the gene(s) present in the region proximal to the virD promoter are essential. We propose that product(s) of this gene have recombinase or endonuclease activity which specifically recognizes the 25 bp terminal repeats of T-DNA.

An ultrastructural study of the interaction of liposomes with plant protoplasts.

A one-step procedure using a mixture of glutaraldehyde and osmium tetroxide was devised to fix in situ large unilamellar liposomes of phosphatidylserine for transmission electron microscopy (TEM), since the conventional fixation method was found to be inadequate in this respect. The new fixation procedure enabled us to visualize the sequence of events in the interaction of liposomes with protoplasts from Vinca rosea suspension cultures in the presence of polyethylene glycol. Liposomes were thus found adhering to the surface of protoplasts, in association with invaginating plasmalemma, and within intracellular vesicles. These observations showed that liposomes enter plant protoplasts via endocytosis. Ultrastructural profiles indicating fusion of liposomes with protoplasts were not observed.

Mitotic protoplasts and their infection with tobacco mosaic virus RNA encapsulated in liposomes.

M-phase and S-phase protoplasts were prepared from tobacco cells in suspension culture after a high degree of synchronization using aphidicolin, a specific inhibitor for eukaryotic DNA polymerase. When TMV-RNA was introduced into these protoplasts mediated by REV liposomes, 37% of M-phase and 26% of S-phase protoplasts were infected as determined by the fluorescent antibody technique. After the 24 hr interval between the introduction of TMV-RNA into protoplasts and the determination of infection, half of the infected mitotic protoplasts formed dumbell-shaped daughter cells. The significance of synchronized protoplasts in genetic engineering of plant cells is discussed in reference to the delivery of DNA into the nucleus.

Liposome-mediated infection of plant protoplasts with tobacco mosaic virus RNA.

Tobacco mosaic virus (TMV) RNA was encapsulated in large unilamellar vesicles (LUV) of phosphatidylserine and was introduced into protoplasts from Vinca rosea suspension cultures. Infection could be effected by brief incubation of protoplasts with the LUV containing TMV-RNA in the presence of either polyethylene glycol or polyvinyl alcohol. The presence of these polymers was essential for infection, and postinoculation washing with the high pH-high Ca2+ buffer enhanced infection significantly. Up to 80% of protoplasts could be infected under the optimal conditions as demonstrated by immunofluorescence technique. Calculations showed that around 2 x 10(6) TMV-RNA molecules were added per infected protoplast, indicating that the efficiency of infection by this method compares favorably with those by the existing methods for inoculating protoplasts from mesophyll cells with TMV-RNA. The significance of using protoplasts from cultured cells for infection with plant viruses and their nucleic acids is discussed.

Correlation between particle multiplicity and location on virion RNA of the assembly initiation site for viruses of the tobacco mosaic virus group.

The initiation site for reconstitution on genome RNA was determined by electron microscopic serology for a watermelon strain of cucumber green mottle mosaic virus (CGMMV-W), which is chemically and serologically related to tobacco mosaic virus (TMV). The initiation site was located at the same position as that of the cowpea strain, a virus that produces short rods of encapsidated subgenomic messenger RNA for the coat protein (a two-component TMV), being about 320 nucleotides away from the 3' terminus, and hence within the coat protein cistron. Although CGMMV-W was until now believed to be a single-component TMV, the location of the initiation site indicated the presence of short rods containing coat protein messenger RNA in CGMMV-W-infected tissue, as in the case for the cowpea strain. We found such short rods in CGMMV-W-infected tissue. The results confirmed our previous hypothesis that the site of the initiation region for reconstitution determines the rod multiplicity of TMV. The finding of the second two-component TMV, CGMMV, indicates that the cowpea strain of TMV is not unique in being a two-component virus and that the location of the assembly initiation site on the genome RNA can be a criterion for grouping of viruses.

The site of initiation of rod assembly on the RNA of a tomato and a cowpea strain of tobacco mosaic virus.

Genome RNA of a tomato (T) and a cowpea (Cc) strain of tobacco mosaic virus (TMV), as well as RNA of the short particles of cowpea strain, were tritium labeled at the termini. Chromatographic analyis of the alkali-hydrolysate of these RNAs showed that they all have A(OH) at the 3'-terminus and are capped with m7G ppp at the 5'-terminus. The site of initiation of rod assembly for these strains was determined by sequential reconstitution of virus rods with proteins of two different strains followed by examination of distribution of the proteins on the reconstituted rods by electron microscopic serology. The initiation site on T-RNA was located at the same position as that of a common strain previously studied, being about 800 nucleotides away from the 3'-terminus. In contrast, the initiation site on Cc-RNA was found to be much closer to the 3'-terminus, only about 320 nucleotides away from the terminus, and hence within the coat protein cistron. The results showed that the internal initiation and the bidirectional elongation are a universal mechanism of assembly among TMV strains, but different strains may use different initiation sites. The location of the initiation site of the cowpea strain explained why the coat protein messenger RNA of this strain, but not that of the common and the tomato strains, is encapsidated to form short particles.

Infectivity of partially encapsidated tobacco mosaic virus RNA.

Tobacco mosaic virus was reconstituted in vitro, and the infectivity of intermediate products at various stages of reconstitution was investigated in relation to their rod length. The infectivity of intermediates was exponentially related to their rod length throughout the reconstitution process, indicating that no particular region of TMV-RNA is preferentially susceptible to inactivation or more important for infection.