ChvD, a chromosomally encoded ATP-binding cassette transporter-homologous protein involved in regulation of virulence gene expression in Agrobacterium tumefaciens.

A yeast two-hybrid screen searching for chromosomally encoded proteins that interact with the Agrobacterium tumefaciens VirB8 protein was carried out. This screen identified an interaction candidate homologous to the partial sequence of a gene that had previously been identified in a transposon screen as a potential regulator of virG expression, chvD. In this report, the cloning of the entire chvD gene is described and the gene is sequenced and characterized. Insertion of a promoterless lacZ gene into the chvD locus greatly attenuated virulence and vir gene expression. Compared to that of the wild-type strain, growth of the chvD mutant was reduced in rich, but not minimal, medium. Expression of chvD, as monitored by expression of beta-galactosidase activity from the chvD-lacZ fusion, occurred in both rich and minimal media as well as under conditions that induce virulence gene expression. The ChvD protein is highly homologous to a family of ATP-binding cassette transporters involved in antibiotic export from bacteria and has two complete Walker box motifs. Molecular genetic analysis demonstrated that disruption of either Walker A box, singly, does not inactivate this protein's effect on virulence but that mutations in both Walker A boxes renders it incapable of complementing a chvD mutant strain. Constitutive expression of virG in the chvD mutant strain restored virulence, supporting the hypothesis that ChvD controls virulence through effects on virG expression.

Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas).

Conserved gene sequences, including 16S rRNA and ribosomal protein gene sequences, were used to evaluate genetic variations in phytoplasma strains belonging to 16S rRNA groups I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). We used PCR to amplify the sequences of the 16S ribosomal DNA and a segment of the ribosomal protein gene operon (encoding the 3' region of rps19, all of rp122, and rps3) from diverse phytoplasma group I and III strains. Additional chromosomal gene sequences of group I strains were also amplified. The PCR products amplified from members of each group of phytoplasmas were compared by performing restriction fragment length polymorphism (RFLP) analyses. On the basis of the RFLP patterns observed and similarity coefficients derived from combined RFLP analyses, the phytoplasma strains belonging to groups I and III were placed in distinct 16S rRNA, ribosomal protein, and 16S rRNA-ribosomal protein subgroups. Analyses of two or more conserved gene sequences revealed that members of the two groups were more diverse than previously thought. Subgroup differentiation on the basis of our combined analyses of 16S rRNA and ribosomal protein gene sequences seemed to adequately reflect the levels of chromosomal homology determined by DNA-DNA hybridization assays. On the basis of unique RFLP profiles, we identified new, previously unclassified group I phytoplasma strains, including the organisms that are associated with Ipomoea obscura witches'-broom [subgroup 16SrI-F(rr-rp)], maize bushy stunt [subgroup 16SrI-I(rr-rp)], and Mexican periwinkle virescence [subgroup 16SrI-J(rr-rp)], and new, previously unclassified group III phytoplasma strains, including the organism that is associated with pecan bunch [subgroup 16SrIII-H(rr-rp)]. On the basis of the results of our analyses of 16S rRNA and ribosomal protein conserved gene sequences, we recognized 9 group I subgroups and eight group III subgroups. We propose that phytoplasma strains belonging to each group I and III subgroup should be distinguished taxonomically at a level equivalent to the subspecies level.

The adenine phosphoribosyltransferase-encoding gene of Arabidopsis thaliana.

The apt gene, coding for adenine phosphoribosyltransferase (APRT), has been isolated from the plant Arabidopsis thaliana. Data from both Southern analysis and characterization of apt clones isolated from a genomic library is consistent with the occurrence of one apt within the A. thaliana genome. Comparison of the nucleotide sequence of the apt gene with its corresponding cDNA indicates that the gene contains five introns, whereas all other apt isolated to date have fewer introns (four in mammals, two in Drosophila). The locations of the introns within the plant apt coding region are not consistent with the placement of introns in the previously isolated apt of murines, human and Drosophila species. In agreement with its expression pattern in vivo, the upstream region of this plant apt is able to express the beta-glucuronidase-encoding gene (gus) in an apparently constitutive manner in transgenic A. thaliana plants. The apt promoter region is notable for its lack of conventional promoter elements such as TATA, CCAAT or G+C-rich sequence elements.

Mouse transgenes in human cells detect specific base substitutions.

We describe a system of transgenic human cell lines that detects and identifies specific point mutations at defined positions within a gene. The target transgenome is a mouse adenine phosphoribosyltransferase (APRT) gene rendered nonfunctional by introduction of a substitution at either of two bases that comprise a splice acceptor site. Reversion at a mutated site results in the expression of wild-type mouse APRT and consequent growth of APRT+ transgenic cell colonies. Site-specific reversion to wild-type sequence is confirmed by regeneration of a previously destroyed diagnostic Pst I site. Two independent cell clones, each with mutant transgenomes bearing an A----G transition, exhibited an up to 7500-fold, dose-dependent induction of reversion following treatment with ethyl methanesulfonate. Treatment of these clones with 2-aminopurine resulted in no induction of revertants. In contrast, another transgenic cell clone, bearing a G----A transition, reverted as a consequence of 2-aminopurine, but not ethyl methanesulfonate, treatment. These data confirm for human cells the proposed mechanisms of action of these mutagens and provide evidence for the utility of our site-specific reversion method for mutagenesis studies.

Denaturing gradient gel analysis of single-base substitutions at a mouse adenine phosphoribosyltransferase splice acceptor site.

Denaturing gradient gel electrophoresis (DGGE) can detect single-base changes in DNA. We used site-directed mutagenesis to produce all six possible single-base substitutions at a splice acceptor consensus AG dinucleotide within the mouse adenine phosphoribosyltransferase (aprt) gene. Studies of mouse and Chinese hamster cell aprt indicate a high level of both spontaneous and induced mutations in this region. We systematically evaluated each of the six mutations by DGGE. Five of the six mutant sequences could be distinguished from wildtype by DGGE analysis of a 560-bp fragment containing the mutation. However, one mutant could not be distinguished from wild-type, and some of the mutant constructs could not be distinguished from each other. Analysis of DNA heteroduplexes consisting of wild-type and mutant strands or two different mutant strands enabled all mutant constructs to be distinguished from wild-type and from each other. The pairwise mixtures resulted in 24 different heteroduplexes, all of which were less stable than the parental homoduplexes. End labeling of DNA prior to heteroduplex formation and subsequent DGGE analysis enabled us to determine the relative destabilization caused by different types of single-base mismatches.

Identification of DNA sequences required for mouse APRT gene expression.

The mouse aprt promoter contains four GC boxes, which bind transcription factor Spl in vitro, and lacks both TATA and CCAAT boxes. Removal of the two most distal GC boxes of this promoter had little effect on APRT enzyme levels produced in a transient expression assay. Deletion of the distal three GC boxes resulted in a 50% reduction, and deletion of all GC boxes resulted in essentially complete loss of APRT activity. There are two predominant transcription start sites which are located within the region containing the GC boxes. The promoter behaved as a relatively strong promoter when compared to the RSV LTR promoter in a transient CAT assay, and operated in one orientation only. No upstream anti-sense transcripts were detected in either mouse CAK or liver cells, confirming that the mouse aprt promoter, unlike some other GC-rich promoters appears not to support bidirectional transcription.

Comparative anatomy of the human APRT gene and enzyme: nucleotide sequence divergence and conservation of a nonrandom CpG dinucleotide arrangement.

The functional human adenine phosphoribosyltransferase (APRT) gene is less than 2.6 kilobases in length and contains five exons. The amino acid sequences of APRTs have been highly conserved throughout evolution. The human enzyme is 82%, 90%, and 40% identical to the mouse, hamster, and Escherichia coli enzymes, respectively. The promoter region of the human APRT gene, like that of several other "housekeeping" genes, lacks "TATA" and "CCAAT" boxes but contains five GC boxes that are potential binding sites for the Sp1 transcription factor. The distal three, however, are dispensable for gene expression. Comparison between human and mouse APRT gene nucleotide sequences reveals a high degree of homology within protein coding regions but an absence of significant homology in 5' flanking, 3' untranslated, and intron sequences, except for similarly positioned GC boxes in the promoter region and a 26-base-pair region in intron 3. This 26-base-pair sequence is 92% identical with a similarly positioned sequence in the mouse gene and is also found in intron 3 of the hamster gene, suggesting that its retention may be a consequence of stringent selection. The positions of all introns have been precisely retained in the human and both rodent genes, as has an unusual AG/GC donor splice site in intron 2. Particularly striking is the distribution of CpG dinucleotides within human and rodent APRT genes. Although the nucleotide sequences of intron 1 and the 5' flanking regions of human and mouse APRT genes have no substantial homology, they have a frequency of CpG dinucleotides that is much higher than expected and nonrandom considering the G + C content of the gene. Retention of an elevated CpG dinucleotide content, despite loss of sequence homology, suggests that there may be selection for CpG dinucleotides in these regions and that their maintenance may be important for APRT gene function.

Computational procedure for a weighted diallel analysis.

A diallel analysis is described for the case in which the values of the characteristic used in an inheritance study have unequal variances. Such a characteristic can be a mean, a slope of a regression line, or the estimates of some parameter of a linear or nonlinear model. Computational formulae are presented which incorporate the necessary weighting along with the statistics of the Hayman-Jinks method for diallel analysis. The method described can also be used to perform a weighted diallel analysis based on means when there are unequal numbers of replications per cross. A simple example demonstrates the computations necessary to complete a weighted diallel analysis.

Hybridization and fertility of hybrid derivatives of Solatium melongena L. and Solanum macrocarpon L.

Eleven genotypes of Solanum melongena L. and one genotype tentatively identified as Solanum macrocarpon were reciprocally intercrossed. Three patterns of the crossability were determined: a) reciprocally crossable, b) reciprocally non-crossable, and c) unidirectionally crossable. In toto 524 F1 interspecific hybrids were grown during one season under open pollination conditions in the field. A large proportion of the F1 hybrids produced seed set. The highest degree of seed set was recorded in the reciprocal F1 hybrid of S. melongena (cv. 'Burpee Hybrid') and S. macrocarpon (Acc. 21-73). In addition, a limited number of back-cross progeny have been produced. The germinating seeds produced an F2 generation of which some recombinants showed a considerably higher degree of fertility than the F1. This finding suggests the possibility of the transfer of genes for resistance to two-spotted spider mite from S. macrocarpon to S. melongena.