Screening for trbB- and traG-like sequences by PCR for the detection of conjugative plasmids in bacterial soil isolates.

The transfer regions of different conjugative plasmids show significant similarities in the genetic organization and in the amino acid sequence of some gene products, especially of proteins from the traG or trbB family. These similarities are also evident on the level of the nucleotide sequences. On the basis of conserved DNA regions we designed degenerate PCR primer pairs to detect specifically tra regions within a collection of bacterial clones isolated from an agricultural soil. Most of the potential transfer-proficient indigenous bacterial isolates were able to mobilize a derivative of the nonconjugative IncQ plasmid RSF1010 into recipient strains. With the help of the primers it should be possible to evaluate the genetic potential for horizontal gene transfer carried out by conjugative plasmids.

The cytoplasmic DNA-binding protein TraM binds to the inner membrane protein TraD in vitro.

The cytoplasmic protein TraM is one of four essential gene products of the F factor which are involved in DNA transfer after mating pair formation. TraM binds to three specific sites within the oriT region. Besides regulation of its own synthesis, the precise function of TraM during conjugation is not yet known. In the present work, the affinity of TraM to TraD was studied in vitro by an overlay assay and by affinity chromatography. Whether the interaction between TraM and TraD causes a transient or permanent anchoring of the F factor to the site of transfer is discussed. A 35-kDa host membrane protein of yet unknown function also shows affinity to TraM and may be involved in this anchoring process as well.

The human gene ZFP161 on 18p11.21-pter encodes a putative c-myc repressor and is homologous to murine Zfp161 (Chr 17) and Zfp161-rs1 (X Chr)

A clone from a lambda gt11 cDNA expression library of HeLa cells was isolated, sequenced, and shown to encode a new human zinc finger protein. The cDNA of the gene termed ZFP161 has an open reading frame of 1347 bp. The predicted protein comprises 449 amino acid residues and contains five zinc finger motifs of the Krüppel type near the C-terminus and a BTB/POZ domain in the N-terminal region. The protein is 98% homologous to a murine zinc finger protein, ZF5 (M. Numoto et al., 1993, Nucleic Acids Res. 21: 3767-3775), which is a putative transcriptional repressor of c-myc and exhibits growth-suppressive activity in mouse cell lines. Through the use of a panel of somatic cell hybrids for chromosomal assignment and DNAs of somatic cell hybrids containing a deleted chromosome 18 for fine mapping, the human gene ZFP161 was localized to 18p11.21-pter. Therefore, ZFP161 is a candidate gene by position for the holoprosencephaly type 4 gene, HPE4, which is involved in congenital malformations. With DNAs from an interspecific backcross, two homologous mouse genes, Zfp161 and Zfp161-rs1, were mapped to chromosome 17 and the X chromosome, respectively. Mapping of Zfp161 confirms and extends a region of homology between distal mouse chromosome 17 and human 18p.

Mutations resulting in a defective mini-F replicon in the presence of both origins, oriI and oriII.

Within the region of the origin of replication (oriII) of plasmid mini-F, we introduced a set of deletions around the XhoI site (45.080F) and an insertion of 4 nucleotides at the Bg/II site (45.213F). These alterations had the effect that replication from oriII as well as from the alternative origin, oriI, was abolished. The analysis suggests that an AT-rich region which may function as entry site for DnaB-DnaC protein is an essential structural requirement for replication of plasmid mini-F.

Purification and DNA binding of the D protein, a putative resolvase of the F-factor of Escherichia coli.

The D protein encoded by plasmid mini-F promotes resolution of plasmid cointegrates or dimers of the F-factor or mini-F. In addition, two rfsF sequences are essential for this site-specific, recA-independent recombination event. The D gene was cloned into an expression vector and the gene product was overproduced in Escherichia coli and purified to homogeneity. The sequence of the N-terminus of the D protein was determined, thus permitting identification of the correct translational start codon in the nucleotide sequence that results in a 29.6 kDa protein. The binding site for the purified D protein is located within the mini-F NcoI-HpaI DNA fragment (192 bp). Binding seems to be affected by DNA methylation, since the protein did not bind to DNA isolated from a dam mutant of E. coli. The binding site, which is a region of approximately 28 bp and is located 160 bp downstream of the rfsF site, was identified by DNase I footprinting using fluorescence labelled DNA.

The repeated sequences (incB) preceding the protein E gene of plasmid mini-F are essential for replication.

At the XhoI site (45.08F) of plasmid mini-F a deletion of 649 bp was generated employing exonuclease Bal31. By this deletion nucleotide sequences functioning as origin II and the four 19 bp direct repeats constituting the incB region in front of the E protein gene were removed from the plasmid. Analysis of proteins radioactively labelled in Escherichia coli mini-cells indicated that all mini-F encoded proteins are expressed. However, the plasmid carrying the deletion was not capable of replicating from the primary origin (origin I, 42.6F). Recently a smaller deletion at the XhoI site (45.08F) of about 300 bp, removing only the region functioning as origin II and replicating from origin I, was described by Tanimoto and Iino (1984, 1985). The data presented suggest that the incB repeats are essential for the initiation of replication from origin I, and possibly also from origin II, and seem not to be engaged in the autoregulation of E protein expression.