Deletion mapping of kil and kor functions in the trfA and trfB regions of broad host range plasmid RK2

Figurski et al. (1982) have reported that certain loci on the broad host range plasmid RK2 (kil functions) can be cloned only in the presence of other trans-acting segments of the plasmid genome (kor functions). They have suggested that the presence of these functions may in part account for the structure of mini RK2 replicons which were constructed in order to define the regions of the plasmid which encode replication/maintenance functions (Thomas et al. 1980). We have therefore investigated the relationship between these two sets of kil and kor loci and the loci implicated in the replication/maintenance of RK2. We find that, whilst the three kil loci reported by Figurski et al. (1982) are absent from these derivatives, a fourth such locus (kilD) is closely linked to trfA, a gene essential for RK2 replication. The kilD locus was probably responsible for the inclusion in mini replicons of a segment of RK2 DNA which carries both korD and korA in addition to trfB, a gene defined by a temperature-sensitive maintenance defect, but which can be deleted leaving a functional RK2 replicon (Thomas 1981 b). The kilB locus is situated on the opposite side of kilD from trfA, all three loci lying within a 3.6 kb segment of RK2 DNA. The korA, korD and trfB functions all map within a 900 bp segment of DNA, while korB requires sequence information at least 1.5 kb from this segment.     

Cloning of the RHO1 gene from Candida albicans and its regulation of beta-1,3-glucan synthesis.

The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazur and W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota et al., Science 272:279-281, 1996). From the opportunistic pathogenic fungus Candida albicans, we cloned the RHO1 gene by the PCR and cross-hybridization methods. Sequence analysis revealed that the Candida RHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDa peptide. The deduced amino acid sequence predicts that Candida albicans Rho1p is 82.9% identical to Saccharomyces Rho1p and contains all the domains conserved among Rho-type GTPases from other organisms. The Candida albicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1 deletion. Furthermore, recombinant Candida albicans Rho1p could reactivate the beta-1,3-glucan synthesis activities of both C. albicans and S. cerevisiae membranes in which endogenous Rho1p had been depleted by Tergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified with the beta-1,3-glucan synthase putative catalytic subunit, Candida albicans Gsc1p, by product entrapment. Candida albicans Rho1p was shown to interact directly with Candida albicans Gsc1p in a ligand overlay assay and a cross-linking study. These results indicate that Candida albicans Rho1p acts in the same manner as Saccharomyces cerevisiae Rho1p to regulate beta-1,3-glucan synthesis.     

DNA replication origin of polyoma virus: early proximal boundary.

We constructed a series of deleted polyoma genomes by Bal 31 nuclease digestion from the unique Bg/I site at nucleotide 86 on the "early" side of the origin of DNA replication. The ability of the cloned deleted genomes to replicate was tested after transfection into mouse 3T6 fibroblasts or into the polyomatransformed C127 (COP5) mouse cell line (Tyndall et al., Nucleic Acids Res. 9:6231-6251, 1981). Deletions up to nucleotide 64-had no effect on the amount of replicated DNA accumulated, but larger deletions, extending up to nucleotide 42, decreased this amount 7- to 10-fold. By nucleotide 38, the quantity of detected DNA was down 100-fold, and by nucleotide 20, no replication could be detected. The minimum origin segment does not contain any known high-affinity, large tumor antigen binding site.     

Evolution along the parasitism-mutualism continuum determines the genetic repertoire of prophages

Integrated into their bacterial hosts’ genomes, prophage sequences exhibit a wide diversity of length and gene content, from highly degraded cryptic sequences to intact, functional prophages that retain a full complement of lytic-function genes. We apply three approaches—bioinformatics, analytical modelling and computational simulation—to understand the diverse gene content of prophages. In the bioinformatics work, we examine the distributions of over 50,000 annotated prophage genes identified in 1384 prophage sequences, comparing the gene repertoires of intact and incomplete prophages. These data indicate that genes involved in the replication, packaging, and release of phage particles have been preferentially lost in incomplete prophages, while tail fiber, transposase and integrase genes are significantly enriched. Consistent with these results, our mathematical and computational approaches predict that genes involved in phage lytic function are preferentially lost, resulting in shorter prophages that often retain genes that benefit the host. Informed by these models, we offer novel hypotheses for the enrichment of integrase and transposase genes in cryptic prophages. Overall, we demonstrate that functional and cryptic prophages represent a diversity of genetic sequences that evolve along a parasitism-mutualism continuum.     

Horizontal gene transfer in microbial genome evolution

Horizontal gene transfer is the collective name for processes that permit the exchange of DNA among organisms of different species. Only recently has it been recognized as a significant contribution to inter-organismal gene exchange. Traditionally, it was thought that microorganisms evolved clonally, passing genes from mother to daughter cells with little or no exchange of DNA among diverse species. Studies of microbial genomes, however, have shown that genomes contain genes that are closely related to a number of different prokaryotes, sometimes to phylogenetically very distantly related ones. (Doolittle et al., 1990, J. Mol. Evol. 31, 383-388; Karlin et al., 1997, J. Bacteriol. 179, 3899-3913; Karlin et al., 1998, Annu. Rev. Genet. 32, 185-225; Lawrence and Ochman, 1998, Proc. Natl. Acad. Sci. USA 95, 9413-9417; Rivera et al., 1998, Proc. Natl. Acad. Sci. USA 95, 6239-6244; Campbell, 2000, Theor. Popul. Biol. 57 71-77; Doolittle, 2000, Sci. Am. 282, 90-95; Ochman and Jones, 2000, Embo. J. 19, 6637-6643; Boucher et al. 2001, Curr. Opin., Microbiol. 4, 285-289; Wang et al., 2001, Mol. Biol. Evol. 18, 792-800). Whereas prokaryotic and eukaryotic evolution was once reconstructed from a single 16S ribosomal RNA (rRNA) gene, the analysis of complete genomes is beginning to yield a different picture of microbial evolution, one that is wrought with the lateral movement of genes across vast phylogenetic distances. (Lane et al., 1988, Methods Enzymol. 167, 138-144; Lake and Rivera, 1996, Proc. Natl. Acad. Sci. USA 91, 2880-2881; Lake et al., 1999, Science 283, 2027-2028).     

Mini-Mu-duction as a test for genetic complementation in Escherichia coli.

In mini-Mu-duction, segments of host DNA bracketed between two copies of an internally deleted Mu phage (a mini-Mu) can be packaged within Mu phage particles. Upon infection of a second host strain, the DNA injected by these particles can insert into the chromosomal DNA in a reaction catalyzed by the phage A gene product (transposase), which is independent of homologous recombination. This results in a partially diploid host strain in which the duplicated host DNA is bracketed by two copies of the mini-Mu phage (Faelen et al., Mol. Gen. Genet. 176:191-197, 1979). The frequency of mini-Mu-duction reported previously was low (10(-8) to 10(-9) per recipient cell) thus limiting its use to rather stable mutational lesions. I have increased the frequency of mini-Mu-duction 10- to 100-fold by use of a helper phage lacking the kil gene and by UV irradiation of the phage stocks. I have also shown that mini-Mu-duction is a reliable complementation assay in rec+ as well as recA recipient strains. This genetic complementation test does not require prior gene localization and (due to the extended host range of phage Mu) should be applicable to many enterobacterial species.     

Lack of an Additive Effect between the Deletions of Klf5 and Nkx3-1 in Mouse Prostatic Tumorigenesis

Prostate cancer is one of the most common malignancies.The development and progression of prostate cancer are driven by a series of genetic and epigenetic events including gene amplification that activates oncogenes and chromosomal deletion that inactivates tumor suppressor genes.Whereas gene amplification occurs in human prostate cancer,gene deletion is more common,and a large number of chromosomal regions have been identified to have frequent deletion in prostate cancer,suggesting that tumor suppressor inactivation is more common than oncogene activation in prostatic carcinogenesis (Knuutila et al.,1998,1999;Dong,2001).Among the most frequently deleted chromosomal regions in prostate cancer,target genes such as NKX3-1 from 8p21,PTENfrom 10q23 andATBF1 from 16q22 have been identified by different approaches (He et al.,1997;Li et al.,1997;Sun et al.,2005),and deletion of these genes in mouse prostates has been demonstrated to induce and/or promote prostatic carcinogenesis.For example,knockout of Nkx3-1 in mice induces hyperplasia and dysplasia (Bhatia-Gaur et al.,1999;Abdulkadir et al.,2002) and promotes prostatic tumorigenesis (Abate-Shen et al.,2003),while knockout of Pten alone causes prostatic neoplasia (Wang et al.,2003).Therefore,gene deletion plays a causal role in prostatic carcinogenesis (Dong,2001).     

Partial deletion of the Rhizobium phaseoli CFN23 symbiotic plasmid implies a concomitant amplification of plasmid DNA sequences

Rhizobium leguminosarum biovar phaseoli CFN23 loses its ability to nodulate beans at a high frequency because of a deletion of part of its symbiotic (pSym) plasmid (Soberón-Chávez et al., 1986). We report here that at least 80 kb of pSym are deleted upon loss of the symbiotic phenotype; the deletion removes the nitrogenase structural nifHDK and the common nodABC genes. The size of the deleted pSym is not reduced, since it is accompanied by an amplification of other pSym plasmid sequences. This genetic rearrangement is similar to the deletion and amplification of yeast mitochondrial DNA leading to 'petite' mutations.     

Epstein-Barr virus DNA XII. A variable region of the Epstein-Barr virus genome is included in the P3HR-1 deletion.

The P3HR-1 subclone of Jijoye differs from Jijoye and from other Epstein-Barr virus (EBV)-infected cell lines in that the virus produced by P3HR-1 cultures lacks the ability to growth-transform normal B lymphocytes (Heston et al., Nature (London) 295:160-163, 1982; Miller et al., J. Virol. 18:1071-1080, 1976; Miller et al., Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974; Ragona et al., Virology 101:553-557, 1980). The P3HR-1 virus was known to be deleted for a region which encodes RNA in latently infected, growth-transformed cells (Bornkamm et al., J. Virol. 35:603-618, 1980; Heller et al., J. Virol. 38:632-648, 1981; King et al., J. Virol. 36:506-518, 1980; Raab-Traub et al., J. Virol. 27:388-398, 1978; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934, 1980). This deletion is now more precisely defined. The P3HR-1 genome contains less than 170 base pairs (and possibly none) of the 3,300-base pair U2 region of EBV DNA and is also lacking IR2 (a 123-base pair repeat which is the right boundary of U2). A surprising finding is that EBV isolates vary in part of the U2 region. Two transforming EB viruses, AG876 and Jijoye, are deleted for part of the U2 region including most or all of a fragment, HinfI-c, which encodes part of one of the three more abundant cytoplasmic polyadenylated RNAs of growth-transformed cells (King et al., J. Virol. 36:506-518, 1980; King et al., J. Virol. 38:649-660, 1981; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934).     

Two poles and a compass

Rho GTPases control fundamental aspects of neutrophil chemotaxis: establishment of front and back and orientation toward the chemoattractant. Two reports in this issue show that activated Cdc42 at the leading edge helps orient the cell's axis in a signaling complex with G beta gamma, PAK1, and PIX alpha; while Rho, activated via G alpha 13, mediates formation of the uropod, which then interacts by mutual negative feedback with the front to reinforce polarization (Li et al., 2003 [this issue of Cell]; Xu et al., [this issue of Cell]).     

Retention of cryptic genes in microbial populations

Cryptic genes are silenced genes that can still be reactivated by mutation. Since they can make no positive contribution to the fitness of their carriers, it is not clear why many cryptic genes in microbial populations have not degenerated into useless DNA sequences. Hall et al. (1983) have suggested that cryptic genes have persisted because of occasional strong environmental selection for reactivated genes. The present mathematical study supports their suggestion. It shows that a cryptic gene can be retained without having any selective advantage over a useless DNA sequence, if selection for the reactivated gene occasionally occurs for a substantially long time.      

Altered responses of regenerating hepatocytes to norepinephrine and transforming growth factor type beta

Norepinephrine (NE), acting through the alpha 1-adrenergic receptor, modules the response of rat hepatocytes in primary culture to transforming growth factor type beta 1 (TGF beta) by increasing the amount of TGF beta required for a given degree of inhibition of epidermal growth factor (EGF)-induced DNA synthesis (Houck et al., J. Cell. Physiol. 135:551-555, 1988). This effect was also found in hepatocytes isolated from regenerating livers but was greatly magnified in cells isolated between 12 and 18 hr after two-thirds partial hepatectomy (PHX). During this period of enhanced sensitivity, NE was equally potent in terms of dose but more efficacious in the regenerating hepatocytes. As it did in control hepatocytes (Cruise et al., Science 227:749-751, 1985), the alpha 1-adrenergic receptor mediated the activity of NE in regenerating hepatocytes. Vasopressin (VP) and angiotensin-II (AG) also antagonized the effect of TGF beta and showed increased activity in regenerating hepatocytes but at only 50% or less of the maximal effect reached by NE. Regenerating hepatocytes isolated 24-72 hr after PHX exhibited decreased sensitivity to inhibition by TGF beta, with a nadir in 48-hr-regenerating cells. These findings suggest that NE may be involved in triggering the early phase of DNA synthesis during liver regeneration, with the subsequent acquisition of innate resistance to TGF beta responsible for continued proliferation at a time when TGF beta mRNA is known to be increasing in the liver (Braun et al., Proc. Natl. Acad. Sci. USA 85:1539-1543, 1988). EGF induced increased DNA and protein synthesis in cultures of control hepatocytes; TGF beta inhibited the EGF-induced DNA synthesis but had no effect on protein synthesis. This may be relevant to the latter stages of liver regeneration, when high levels of TGF beta mRNA are detected in liver and cellular hypertrophy predominates over hyperplasia.     

Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed.