Regulation of the F-factor pif operon: pifO, a site required in cis for autoregulation, titrates the pifC product in trans.

F factor pifC, pifA, and pifB gene expression is subject to negative regulation by the product of the pifC locus (J.F. Miller and M. H. Malamy, J. Bacteriol. 156:338-347, 1983). In this paper, we describe the properties of a new regulatory site in the pif region, pifO, which is required in cis for autoregulation of pif gene expression. Spontaneous pifO mutations were isolated that allow expression of a pifC-lacZ protein fusion in the presence of pifC product in trans. Recombination of these pifO mutations onto F'lac results in increased pifA and pifB activity. Thus, a single regulatory element, pifO, regulates pifC, pifA, and pifB expression in cis. The presence of multiple copies of a fragment from the pif region carrying wild-type pifO sequences (F coordinates 42.9 to 42.43 kilobases) in trans to F'lac results in an increase in pifA and pifB activity as measured by inhibition of T7 plating. When the pifO mutations are recombined onto a plasmid carrying pifO, the resulting recombinants are greatly decreased in the ability to increase F'lac pif expression. These results suggest that increased F'lac pifA and pifB expression caused by pifO sequences in trans is a consequence of titration of pifC product and derepression of the pif operon.     

Indirect SOS induction is promoted by ultraviolet light-damaged miniF and requires the miniF lynA locus

Indirect prophage induction is produced by transfer to recipients of u.v.-damaged F plasmid (95 kb). We tested whether the SOS signal can be produced by miniF, a 9.3 kb restriction fragment, coding for the replication and segregation functions of plasmid F. We used λminiF, a hybrid phage-plasmid. u.v.-irradiated λminiF induced prophages φ80 or λ and sfiA, a chromosomal SOS gene, in more than 50% of the infected cells. The maximal inducing dose produced about 0.5 pyrimidine dimers per kb and left 1% of λminiF survivors. Thus, the SOS signal produced by u.v.-damaged λminiF was almost as potent as that resulting from direct u.v.-irradiation of the lysogens. The u.v.-damaged vector λ, devoid of miniF, failed to promote SOS induction. In contrast, efficient induction was observed when u.v.-damaged λminiF infected a λ immune host, in which replication and expression of the phage genome were repressed. When replication and expression of the miniF genome was repressed by Hfr incompatibility, SOS induction was largely prevented. All these facts indicate that, in the hybrid λ-miniF, it is the u.v.-damaged miniF that generates an SOS signal.To locate on the miniF genome the loci that are involved in the production of the SOS signal, we isolated deletions spanning all the miniF restriction fragments. We characterized six mutant phenotypes (Par⁺, Rep^?, Fid^?, Par-2, Par-1 and SOS^?) related to four functions; partition, copy number, replication and SOS induction. A locus, we call lynA, 800bp long, located by deletion mapping between the two origins of replication oriP and oriS is required for the production of an inducing signal.We postulate that indirect SOS induction by u.v.-damaged miniF results from the disturbance of the lynA function that may be involved in the co-segregation of F plasmid with the host chromosome.     

A replication region of the IncHI plasmid, R27, is highly homologous with the RepFIA replicon of F

A region of the IncHI plasmid R27 has been found to share very close nucleotide sequence homology with the RepFIA replicon of F. This region has been located on a 1.6 kb segment of R27 plasmid DNA, and corresponds to ori-2 and the E gene of F. The incC repeat sequence region shows reduced homology, with the F repeats being an imperfect subset of a larger repeated sequence found in R27. The E gene homologue of R27 is able to initiate replication from the F ori-2 sequence and to repress the E gene promoter of F. The results are consistent with the observed incompatibility behaviour of R27, and have a bearing on the specificity of interaction of E protein with its DNA-binding sites.     

Identification of a multimer resolution system involved in stabilization of the Salmonella dublin virulence plasmid pSDL2.

The Salmonella dublin virulence plasmid pSDL2 is a low-copy-number plasmid that is highly conserved in its host. Deletion of the 8-kb EcoRI C fragment downstream of the virulence region leads to plasmid instability and formation of multimers. We identified a multimer resolution system in the EcoRI C fragment composed of a trans-acting resolvase gene and a cis-acting resolution site. The resolvase gene, rsd, maps within a 2-kb EcoRV fragment and appears to be part of a multicistronic unit together with at least two other genes of unknown function. The derived protein, 28.7-kDa in size, is almost identical to the D protein of miniF. The C-terminal region was shown to have substantial similarity to the conserved C-terminal domains of the site-specific recombinases of the integrase family. The cis-acting resolution site, crs, is located upstream of rsd within a 628-bp SmaI-HpaI fragment. It contains eight direct incomplete 17-bp repeats followed by a segment rich in indirect repeats, the latter being homologous to the oriV1 sequence of miniF. crs contains the crossover site for specific recombination and mediates bidirectional promoter activity. A replicative function in analogy to that of oriV1 of F could not be demonstrated. The multimer resolution system was shown to stabilize pACYC184 and is dependent on the recA-mediated formation of multimeric plasmids. Screening different Salmonella serovars with a pSDL2-specific recombination assay revealed that only strains harboring a virulence plasmid encode for resolvase activity. Our results suggest that site-specific recombination contributes to the stable inheritance of pSDL2 and other Salmonella virulence plasmids.     

L-arabinose binding protein from Escherichia coli B-r

A protein which is capable of binding l-arabinose-1-(14)C has been isolated from l-arabinose-induced cultures of Escherichia coli B/r. Analysis for this l-arabinose-binding protein (ABP) in a number of l-arabinose-negative mutants suggests that the ABP is not coded for by any of the known genetic units of the l-arabinose complex yet is under the control of the regulator gene araC. The ABP has been purified and found to bind l-arabinose, d-fucose, d-xylose, and l-ribulose with decreasing affinities. The K(m) for l-arabinose is 5.7 x 10(-6)m. The molecular weight, as determined by equilibrium centrifugation, was found to be 32,000. The protein was observed to have many features that liken it to other recently isolated binding proteins that have been implicated in the active transport of small molecules.     

A plant gene encoding a Myb-like protein that binds telomeric GGTTTAG repeats in vitro

A gene (AtTRP1) encoding a telomeric repeat-binding protein has been isolated from Arabidopsis thaliana. AtTRP1 is a single copy gene located on chromosome 5 of A. thaliana. The protein AtTRP1 encoded by this gene is not only homologous to the Myb DNA-binding motifs of other telomere-binding proteins but also is similar to several initiator-binding proteins in plants. Gel retardation assay revealed that the 115 residues on the C terminus of this protein, including the Myb motif, are sufficient for binding to the double-stranded plant telomeric sequence. The isolated DNA-binding domain of AtTRP1 recognizes each telomeric repeat centered on the sequence GGTTTAG. The almost full-length protein of AtTRP1 does not form any complex at all with the DNA fragments carrying four or fewer GGTTTAG repeats. However, it forms a complex with the sequence (GGTTTAG)(8) more efficiently than with the sequence (GGTTTAG)(5). These data suggest that the minimum length of a telomeric DNA for AtTRP1 binding consists of five GGTTTAG repeats and that the optimal AtTRP1 binding may require eight or more GGTTTAG repeats. It also implies that this protein AtTRP1 may bind in vivo primarily to the ends of plant chromosomes, which consist of long stretches of telomeric repeats.     

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium‐dependent membrane‐binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5‐Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca²⁺‐binding region in C2A domain is longer than classical C2 domains and a novel Ca²⁺ binding site in the vWA domain. The structure of BON1 bound to Mn²⁺ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid‐binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca²⁺. These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.     

Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene

A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells. Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA. From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein. These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd. The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established. When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related. The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence. When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins. The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved. Secondary structure predictions indicate that the two proteins contain similar structural domains. It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.     

Analysis of the F plasmid centromere

Summary The nucleotide sequence of the cis-acting partition site (centromere) of the miniF plasmid has been determined. Its most notable feature is a reiterated 43 base pair unit. A series of plasmids deleted for portions of the repeat region was constructed and tested for incompatibility with R386 and for stability of inheritance. The extent of incompatibility with R386 was correlated with the number of repeat units. In contrast, the great majority of the repeats were not needed for miniF stability. An adjacent region of unique sequence was also found to be involved in centromere function.     

Plasmid pColBM-Cl139 does not encode a colicin lysis protein but contains sequences highly homologous to the D protein (resolvase) and the oriV region of the miniF plasmid

Colicins are usually released from producing cells by so-called lysis proteins. No sequence homologous to the structurally very similar colicin lysis genes was found in the gene cluster cmi cma cbi cba, which determines the activity and immunity proteins of colicin B and M on pColBM-Cl139. Instead, the region upstream of cmi contained sequences that showed 91% homology to the structural gene of protein D (resolvase) and 75.5% homology to the rfsF sequence of the Escherichia coli miniF plasmid. It is concluded that colicins B and M are not released via the activity of lysis proteins and that the highly homologous regions encode a resolvase and its target respectively.     

Characterisation of chicken erythroid nuclear proteins which bind to the nuclease hypersensitive regions upstream of the beta A- and beta H-globin genes.

Chicken erythrocyte sequence-specific nuclear DNA-binding proteins, which bind to the 5'-flanking DNAseI hypersensitive sites of the erythrocyte chromosomal beta A- and beta H-globin genes, have been fractionated by HPLC gel filtration. Three beta A-globin gene DNA binding activities (to sites A, B and B' (10-12)) were separated. The erythroid precursor cell line HD3 has beta A-globin gene sites B and B' binding activities, but binding to site A is detected only after the HD3 cells are induced to differentiate. The fractionated protein binds to a redefined site B', which contains at its center the globin CACCC consensus sequence. The chromosomal beta H-globin gene has two 5'-flanking DNAseI hypersensitive sites which bracket two sequences (H and H') bound by erythrocyte and HD3 nuclear protein in vitro. The beta H- and beta A-globin gene binding sites (H and B) contain variants of the sequences bound by Nuclear Factor 1 and the TGGCA-binding protein, and their protein binding activity(ies) co-purify after HPLC gel filtration.     

Mechanical measurement of single-molecule binding rates: kinetics of DNA helix-destabilization by T4 gene 32 protein

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein, and is essential for DNA replication, recombination and repair. While gp32 binds preferentially and cooperatively to ssDNA, it has not been observed to lower the thermal melting temperature of natural double-stranded DNA (dsDNA). However, in single-molecule stretching experiments, gp32 significantly destabilizes lambda DNA. In this study, we develop a theory of the effect of the protein on single dsDNA stretching curves, and apply it to the measured dependence of the DNA overstretching force on pulling rate in the presence of the full-length and two truncated forms of the protein. This allows us to calculate the rate of cooperative growth of single clusters of protein along ssDNA that are formed as the dsDNA molecule is stretched, as well as determine the site size of the protein binding to ssDNA. The rate of cooperative binding (ka) of both gp32 and of its proteolytic fragment *I (which lacks 48 residues from the C terminus) varies non-linearly with protein concentration, and appears to exceed the diffusion limit. We develop a model of protein association with the ends of growing clusters of cooperatively bound protein enhanced by 1-D diffusion along dsDNA, under the condition of protein excess. Upon globally fitting ka versus protein concentration, we determine the binding site size and the non-cooperative binding constants to dsDNA for gp32 and I. Our experiment mimics the growth of clusters of gp32 that likely exist at the DNA replication fork in vivo, and explains the origin of the "kinetic block" to dsDNA melting by gene 32 protein observed in thermal melting experiments.     

Characterization of C14orf4, a novel intronless human gene containing a polyglutamine repeat, mapped to the ARVD1 critical region

Within the ARVD1 (arrhythmogenic right ventricular dysplasia/cardiomyopathy, type 1) critical region, mapped to 14q24.3, we detected an intronless gene of 4859 bp, predominantly expressed in the heart tissue. This gene encodes a 796-amino-acid, proline-rich protein showing polyglutamine and polyalanine tracks with variable length at the N-terminus and a C3HC4 RING finger domain at the C-terminus. CREB and AP-2 binding sites are present in the promoter region. The 5' flanking region contains neither a TATA box nor a CAAT box, but it is high in GC content and includes several Sp1 binding sites. Protein similarity searches revealed a significant match between the C-terminus and a human hypothetical protein, whose gene is located on the chromosome 19 long arm. The predicted protein shows PEST sequences, suggesting its rapid degradation. The novel intronless gene, provisionally named C14orf4 and probably encoding a nuclear protein, was excluded from being the ARVD1 gene.