David M. Stalker

Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2.

SummaryA DNA sequence cosisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc2 incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc2 incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G+C content is present. Deletion evidence indicates that the A-T rich and possibly the G+C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.

Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers

In animals, the small GTP-binding proteins, Rac and Rho, of theras superfamily participate in the signal rransduction pathway that regulates the organization of the actin cytoskeleton. We report here on the characterization of two distinct cDNA clones isolated from a cotton fiber cDNA library that code for homologs of animal Rac proteins. Using gene-specific probes, we have determined that amphidiploid cotton contains two genes that code for each of the two Rac proteins, designated Rac13 and Rac9, respectively. The gene for Rac13 shows highly enhanced expression in developing cotton fibers, with maximal expression occurring at the time of transition between primary and secondary wall synthesis. This is also the time at which reorganization of the cytoskeleton occurs, and thus the pattern of expression of Rac13 is consistent with its possible role, analogous to animal Rac, in the signal transduction pathway that controls cytoskeletal organization.

Plasmid R6K DNA replication: I. Complete nucleotide sequence of an autonomously replicating segment☆

Abstract A 1565-base segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia coli. This segment contains a functional origin of replication and the structural gene pir for a protein, designated π, that is required for the initiation of R6K DNA replication. The nucleotide sequence of this 1565 base-pair replicon was determined. From the sequence and the analysis of proteins produced by minicells of E. coli strains carrying the wild-type pir gene and a deletion of the pir gene, it can be concluded that the π structural gene encodes for a protein of a molecular weight of approximately 35,000. On the basis of the nucleotide sequence, the π protein is the only protein containing more than 50 amino acid residues that is encoded by this 1583 base-pair replicon. The nucleotide sequence also contains eight 22 base-pair direct repeats. Seven of the direct repeats are in tandem and located in the origin region, while the eighth repeat is near or part of the promoter for the π structural gene. This eighth repeat may play a role in the autoregulated expression of the π structural gene.

Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2

SummaryReplication and incompatibility properties in Escherichia coli of DNA segments from the replication origin region of plasmid RK2 have been investigated. A 393 bp HpaII fragment, derived from the region of the RK2 origin of replication, carries an active origin when essential RK2 encoded functions are provided in trans and will form a mini RK2 replicon when linked to a non-replicating selective fragment. This small oriRK2 plasmid cannot stably coexist with other functional RK2 replicons and is thus ‘incompatible” with RK2 replicons. However, the 393 bp oriRK2 segment when cloned into a high copy number plasmid, where plasmid maintenance is no longer dependent on oriRK2, does not interfere with maintenance of a resident RK2 replicon. This is in contrast to larger segments from the origin region that, when cloned at high copy number, cause the loss of a resident RK2 replicon. The apparent ability of the small HpaII oriRK2 plasmid to displace resident RK2 replicons may indicate the turning on of one incompatibility mechanism only when replication from oriRK2is required or may simply reflect the strong selective pressure for establishment of the incoming oriRK2 plasmid and poor ability of the HpaII oriRK2 plasmid to replicate in the presence of another RK2 replicon. The incompatibility expressed by the functional HpaII oriRK2 may be designated inc1. The activity of a segment of RK2, cloned at high copy number, to eliminate a resident RK2 plasmid has been localized to a region of RK2 DNA, designated the inc2 region, to distinguish it from inc1, above, that overlaps but does not coincide with the 393 bp HpaII oriRK2. This inc2 region also appears to be involved in segregation of RK2 derivatives since removal of a portion of this region results in both higher copy number and increased instability of the RK2 derivative. In addition to defining the region of the RK2 origin of replication, these results indicate that the ability to eliminate a resident RK2 replicon can be expressed by fragments, cloned at high copy number, that do not contain the complete oriRK2. Also, only part of the inc2 region that appears to be responsible for efficient elimination of RK2 replicons by fragments cloned at high copy number is required for oriRK2.

Characterization of fruit specific cDNAs from tomato

SummaryDifferential hybridization was used to screen a cDNA library made from ripe tomato fruit poly(A+)RNA. Clones were identified representing genes expressed predominantly at the unripe and/or ripe stage of the fruit development. Northern analysis was used for further characterization of the clones and in this report we describe four cDNA clones expressed at varying stages of fruit development. Three of these cDNAs were found to represent low-copy number genes and one was found to represent a gene family. Dot blot analysis revealed that the expression of these four genes was reduced between 2-fold and 100-fold in three ripening mutants of tomato.

Plasmid R6K DNA replication: III. Regulatory properties of the π initiation protein☆

Abstract The pir locus of R6K encodes a protein (π) that is essential for the initiation of R6K DNA replication. The promoter and coding sequences of this locus were separated and cloned into different plasmids. These various plasmid constructs were used to study the role of π both in regulating the initiation of R6K DNA replication and in regulating its own expression. The π protein was found to reduce levels of β-galactosidase expression from the lacZ gene fused to the pir promoter. In addition, a relatively small change in the cellular concentration of π is observed in response to a 20-fold increase in pir gene dosage. These findings suggest that the expression of the π protein is regulated autogeneously. By fusing the π coding sequences to the tryptophan and pBR322 promoters, the π concentration in vivo could be varied over a wide range. The copy number of an R6K origin plasmid remained essentially the same over a broad range of π concentration, indicating that, although the π protein is required for the initiation of R6K DNA replication, it does not regulate the frequency of this event.

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.

STRUCTURE OF THE REPLICATION ORIGINS OF PLASMIDS RK2 AND R6K AND THE REGULATION OF PLASMID DNA REPLICATION

ABSTRACT Regions of the plasmid genome essential for plasmid replication and maintenance in Escherichia coli have been identified for two antibiotic resistance Plasmids, RK2 and R6K. In both cases a small segment of the plasmid, containing an origin of replication, has been maintained as an extrachromosomal element when another essential region(s) of the plasmid that specifies a trans-acting product is present in the host cell. Nucleotide sequence analysis of the origin regions of plasmids RK2 and R6K revealed seven 22 base pair (bp) direct repeats in tandem for the R6K origin region and eight 17 bp direct repeats in clusters of three and five for RK2. A model is presented involving the interacting of the π protein, the trans-acting product of the R6K genome that is required for the initiation of R6K replication, with the 22 bp direct repeats as a necessary step in the initiation of plasmid R6K replication.