Abstract. Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were used in competition assays. Modification of the free amino groups of peptide F-9 by acetylation abolished its ability to inhibit the binding of [3H]hepafin to laminin on polystyrene surfaces. Peptide F-9 promoted the adhesion of various cell lines (melanoma, fibrosarcoma, glioma, pheochromocytoma) and of aortic endothelial cells. Furthermore, when peptide F-9 was present in solution, it inhibited the adhesion of melanoma cells to laminin-coated substrates. These findings suggest that peptide F-9 defines a novel heparin-binding and cell adhesion-promoting site on laminin.

Agrobacterium tumefaciens VirD2 polypeptide, in the presence of VirD1, catalyzes a site- and strand-specific nicking reaction at the T-DNA border sequences. VirD2 is found tightly attached to the 5' end of the nicked DNA. The protein-DNA complex is presumably formed via a tyrosine residue of VirD2 (F. Durrenberger, A. Crameri, B. Hohn, and Z. Koukolikova-Nicola, Proc. Natl. Acad. Sci. USA 86:9154-9158, 1989). A mutational approach was used to study whether a tyrosine residue(s) of VirD2 is required for its activity. By site-specific mutagenesis, a tyrosine (Y) residue at position 29, 68, 99, 119, 121, 160, or 195 of the octopine Ti plasmid pTiA6 VirD2 was altered to phenylalanine (F). The Y-29-F or Y-121-F mutation completely abolished nicking activity of VirD2 in vivo in Escherichia coli. Two other substitutions, Y-68-F and Y-160-F, drastically reduced VirD2 activity. A substitution at position 99, 119, or 195 had no effect on VirD2 activity. Additional mutagenesis experiments showed that at position 29, no other amino acid could substitute for tyrosine without destroying VirD2 activity. At position 121, only a tryptophan (W) residue could be substituted. This, however, yielded a mutant protein with significantly reduced VirD2 activity. The nicked DNA from strains bearing a Y-68-F, Y-99-F, Y-119-F, Y-160-F, Y-195-F, or Y-121-W mutation in VirD2 was always found to contain a tightly linked protein.

Abstract. We have previously identified three distinctive amino acid sequences from type IV collagen which specifically bound to heparin and also inhibited

Genetic studies indicate that three of the four polypeptides encoded within the virD operon of the Agrobacterium tumefaciens Ti plasmid are essential for virulence. In order to determine whether the fourth polypeptide, VirD3, has any role in virulence, complementation analysis was used. An A. tumefaciens strain, A348AD, which lacked the entire virD operon in the Ti plasmid pTiA6, was constructed. Plasmids containing defined regions of the virD operon were introduced into this strain, and virulence was tested by the strains' abilities to form tumors on Kalanchoe leaves, tomato stems, and potato tubers. As expected, deletion of the virD operon led to an avirulent phenotype. The virulence of this strain could be restored by providing virDI, virD2, and virD4 in trans. No requirement for virD3 in tumor formation was observed in these assays.Agrobactenum tumefaciens is a phytopathogenic bacterium responsible for causing crown gall tumors on plants.Tumor formation involves the transfer of a segment of a tumor-inducing (Ti) plasmid from the bacterium to the plant cell followed by the stable integration of the transferred DNA (T-DNA) into the plant nuclear genome (reviewed in references 16 and 28). The T-DNA is flanked by a 24-bp imperfect direct-repeat sequence, the border sequence, at its termini. The DNA transfer process is catalyzed by the Ti plasmid virulence (vir) genes that are arranged in at least eight operons, virA through virH (3,10,13,18). Of these, virA and virG are the regulatory loci (20) and are members of the bacterial two-component regulatory systems. These genes are constitutively expressed in the bacterium and coordinately induce expression of all of the virulence genes when a bacterium encounters a susceptible host. Under these conditions, the Ti plasmid DNA is processed to form a single-stranded T-strand DNA that is believed to be an intermediate in the DNA transfer process (1,19).The initial reaction for the T-strand DNA synthesis is catalyzed by the enzymes encoded within the virD locus (26). DNA sequence analysis studies indicated that virD can potentially encode five polypeptides, VirDl to VirD5 (9,15,26 site-and strand-specific nicking reaction at the T-DNA borders (9, 26) and becomes covalently attached to the 5' end of the nicked DNA (6,8,22,24,27). This reaction is the first step for T-strand DNA synthesis. VirDl has been shown to possess a DNA-relaxing activity (4). The functions of the other two VirD proteins, VirD3 and VirD4, remain unknown. However, mutational studies demonstrate that VirD4 is required for agrobacterial pathogenicity (18). Stud-* Corresponding author. ies to date have established the requirement of virDl and virD2 in T-DNA processing and that of virD4 in the pathogenic function of Agrobactenium species. The role of virD3 in pathogenicity is not known. In the present study, we demonstrate that virD3 is not essential for tumorigenicity on several plant species.Construction of a Ti plasmid lacking virD. A deletion of the pTiA6 virD locus was introduced by replacing the virD...

Mutagenesis experiments were used to identify functionally important regions of Agrobacterium tumefaciens pTiA6 VirD1. Random mutations were introduced by using Taq polymerase in a mutagenic reaction buffer containing manganese and altered nucleotide ratios to increase errors during the polymerase chain reaction (PCR). The mutants were assayed for VirD1-, VirD2-dependent border-nicking activity in Escherichia coli harbouring a border-containing substrate plasmid. Analysis of the mutants led to the identification of a region from amino acids 45-60 that is important for VirD1 activity. This region corresponds to a previously postulated potential DNA-binding domain. Deletion mutagenesis indicated that amino acids 2-16 could be deleted without affecting VirD1 function, whereas a larger deletion, amino acids 5-27, completely inactivated VirD1.