The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells.

Here we show that the VirD2 protein of A. tumefaciens functions as a nuclear localizing protein in plant cells. The nuclear localization signal of VirD2 consists of two regions containing 4-5 basic amino acids (KRPR and RKRER), located within the C-terminal 34 amino acids. These regions conform to the KR/KXR/K motif required for numerous nuclear localized nonplant eukaryotic proteins. Each region independently directs a beta-glucuronidase reporter protein to the nucleus; however, both regions are necessary for maximum efficiency. VirD2 has been shown to be tightly bound to the 5' end of the single-stranded DNA transfer intermediate, T-strand, transferred from Agrobacterium to the plant cell genome. The present results imply that T-strand transport to the plant nucleus is mediated by the tightly attached VirD2 protein via an import pathway common to higher eukaryotes.

Activation of the T-DNA transfer process in Agrobacterium results in the generation of a T-strand-protein complex: Tight association of VirD2 with the 5' ends of T-strands.

The T-DNA transfer process of Agrobacterium is activated following induction of expression of the Ti plasmid virulence (vir) genes. The virD1 and virD2 gene products are required for the production of nicks at the T-DNA borders and for the generation of free linear single-stranded copies of the T-DNA region, T-strands. T-strands are complexed with proteins in vir-induced bacteria, since T-strands partition to the aqueous/phenol interface in non-Pronasetreated total cell extracts. To determine whether the proteins are tightly associated with T-strands, DNA-protein complexes were purified away from bulk proteins by adsorption to glass beads. A 58-kDa protein was specifically released from vir-induced DNA-protein complexes after treatment with S1 nuclease to digest single-stranded DNA. The 58-kDa protein was identified as VirD2 by using VirD2-specific antibodies. The tight association of VirD2 with T-strands was shown directly by using VirD2-specific antibody to isolate T-strands. The 5' side of the border nick sites on the Ti plasmid was also shown to be tightly associated with protein. The data suggest that after VirD1/VirD2-dependent nicking at the T-DNA borders, the VirD2 protein remains bound to the 5' end of the nick, and the VirD2 protein continues to bind tightly to this 5' end during unwinding (or displacement) of the T-strand from the Ti plasmid T-DNA region. The tight binding of VirD2 to T-strands suggests that this protein has additional functions in T-strand generation and potentially in the later steps of T-DNA transfer.

DNA sequences required for anaerobic expression of the maize alcohol dehydrogenase 1 gene.

Expression of the maize alcohol dehydrogenase 1 (Adh1) gene is transcriptionally regulated under conditions of anaerobic stress. DNA sequences required for the expression of Adh1 have been identified by a functional analysis of in vitro constructed hybrid genes consisting of the Adh1 promoter fused to the chloramphenicol acetyltransferase coding region. A series of 5' deletions, 3' deletions, hybrid promoters, and linker scanning mutants of the Adh-CAT hybrid gene were introduced into maize protoplasts by electroporation and assayed for chloramphenicol acetyltransferase activity after incubation of the protoplasts under different oxygen tensions. The results indicate that a 40-base-pair DNA sequence within the Adh1 promoter is required for anaerobically regulated expression of the hybrid gene. Clustered point mutations in this sequence show that it is composed of two essential regions, each approximately 15 base pairs, separated by a 10-base-pair DNA sequence that does not appear to be important for anaerobic expression. Attachment of this 40-base-pair element to an unrelated promoter shows that this DNA sequence is both necessary and sufficient for induction of gene expression by low oxygen stress.

Regulated expression of an alcohol dehydrogenase 1 chimeric gene introduced into maize protoplasts.

Transient-expression analysis has shown anaerobic regulation of the alcohol dehydrogenase (Adh1) promoter in a chimeric construct. Chimeric plasmids containing the promoter for the Adh1 gene of maize (Zea mays L.) linked to the coding sequence of the chloramphenicol acetyl transferase (cat) gene were introduced into maize protoplasts using electroporation. Both the introduced Adh1 promoter and the endogenous Adh1 gene promoter are regulated at the RNA level, the O2-tension optimum for induction is the same for both, and both promoters initiate transcription from the same site. The demonstration of regulation of a plant gene promoter in a transient expression system will allow the identification of cis acting sequences responsible for regulation.

Reproducible and variable genomic rearrangements occur in the developing somatic nucleus of the ciliate Tetrahymena thermophila.

We analyzed the extent, reproducibility, and developmental control of genomic rearrangements in the somatic macronucleus of the ciliate Tetrahymena thermophila. To exclude differences caused by genetic polymorphisms, we constructed whole-genome homozygotes, and we compared the homozygous progeny derived from single macronuclear differentiation events. This strategy enabled us to identify a novel form of variable rearrangement and to confirm previous findings that rearranged sequences occur at a high frequency in the Tetrahymena genome. Rearrangements studied here were deletions of both unique and interchromosomally dispersed repetitive DNA sequences involving DNA rejoining of internal, nontelomeric regions of macronuclear DNAs. We showed that although rearrangements of some sequence classes are reproducible among independently developed macronuclei, other specific sequence classes are variably rearranged in macronuclear development. The variable somatic genomes so produced may be the source of phenotypically variant cell lines.