The chromosome of the murine pathogen Mycoplasma pulmonis undergoes rearrangements at a high frequency. We show that some of these rearrangements regulate the phase-variable expression of a cluster of genes (the vsa locus) that encode the variable V-1 surface antigens. Only one vsa gene is associated with an expression site; the other vsa genes are transcriptionally silent. The silent genes lack the 5' end region (promoter and ribosome-binding site) that is present in the expressed gene, and DNA rearrangements regulate gene expression by reassorting the 5' end region from an expressed gene with the 3' end region from a previously silent gene. All vsa rearrangements identified so far are site-specific DNA inversions that occur between copies of a specific 34 bp sequence that is conserved in each vsa gene. Interestingly, DNA inversions within the vsa locus apparently occur in concert with inversion of the hsd1 element, which regulates restriction and modification activity in M. pulmonis.

We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tightbinding substrate of cyclin E͞cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1͞E2 complex, and A-and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin͞Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replicationinitiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates.Cell cycle transitions are coordinated in large part through the action of a family of cyclin-dependent kinases (Cdks), enzymes composed of a catalytic Cdk subunit, and a regulatory cyclin subunit (1). Much of what we know concerning Cdk action in the G 1 ͞S transition comes from analysis of the Rb͞E2F pathway, which has led to the identification of Rb, p107, p130, E2F-1, and DP-1 as Cdk substrates (1, 2). Phosphorylation of these proteins typically is linked with formation of tight complexes between the kinase and the substrate, most frequently through a motif in the substrate (the RXL motif) that interacts with the cyclin box. The RXL motif forms the basis for interaction of the p21 family of Cdk inhibitors with cyclins (3-6) and is important for Cdk-mediated phosphorylation of p107 (6, 7), E2F-1 (6, 8, 9), and Rb (10). Cyclin E͞Cdk2 is a key regulator of S-phase initiation. Removal of cyclin E͞Cdk2 activity from Xenopus egg replication systems (reviewed in ref. 11) or mammalian cells (12) blocks S-phase entry, and inXenopus, this blockade can be overcome by addition of cyclin E͞Cdk2. Conversely, ectopic expression of cyclin E can initiate replication independent of Rb inactivation in mammalian cells and in Drosophila (ref. 13, and reviewed in ref. 2). However, targets of this kinase in the preinitiation complex are unknown. We and others (14) have taken advantage of the tight association of cyclins with their substrates to identify cyclin E͞Cdk-binding proteins and substrates via expression cloning techniques. Here we report the identification of human papillomavirus (HPV) replication-initiation protein E1 as a tightbinding substrate of cyclin͞Cdk complexes.Papill...

The vsa genes of Mycoplasma pulmonis encode the V-1 lipoproteins. Most V-1 proteins contain repetitive domains and are thought to be involved in mycoplasma-host cell interactions. Previously, we have reported the isolation and characterization of six vsa genes comprising a 10-kb region of the genome of M. pulmonis strain KD735-15. In the current study, vsa-specific probes were used to clone several fragments from a genomic library of KD735-15 DNA and assemble a single 20-kb contig containing 11 vsa genes. The middle region of the vsa locus contains a large open reading frame (ORF) that is not a vsa gene and has undergone an internal deletion in some strains. The ORF is predicted to encode a membrane protein that may have a role in disease pathogenesis. To examine vsa genes in a strain of M. pulmonis that is unrelated to KD735-15, strain CT was studied. Through Southern hybridization and genomic cloning analyses, CT was found to possess homologs of the KD735-15 vsaA, -C, -E, and -F genes and two unique genes (vsaG and vsaH) that were not found in KD735-15. High-frequency, site-specific DNA inversions serve to regulate the phase-variable production of individual V-1 proteins. As a result of the sequence analysis of vsa recombination products, a model in which DNA inversion arises from strand exchange involving at least six nucleotides of the vrs box is proposed.Mycoplasmas cause slowly progressive, chronic diseases in human and animals. The mechanisms of mycoplasmal disease pathogenesis are poorly understood, and there are no effective control measures. Mycoplasma pulmonis is the etiologic agent of murine respiratory mycoplasmosis and can also cause genital disease and arthritis in rats and mice (31). Thus, M. pulmonis can colonize a variety of epithelial surfaces. Rat isolates of M. pulmonis such as strains UAB 5782 and UAB 6510 are generally more virulent in rats than in mice (10,11,24). In the mouse, UAB 5782 and UAB 6510 colonize the respiratory tract without usually causing lesions (10). In contrast, the mouse isolate strain CT causes severe respiratory disease in the mouse (6,7,10,12). Mycoplasma factors that contribute to the host specificity of disease are unknown. A comparison of the proteins produced by 18 strains of M. pulmonis revealed mostly conserved proteins that were invariant among strains (38). An exception was the V-1 family of surface proteins that are encoded by the vsa (variable surface antigen) genes (4,21,33,35,39). Variation in the V-1 proteins may contribute to the host specificity of the mycoplasma and to the chronicity and severity of disease.The chronic nature of mycoplasmal diseases indicates that mycoplasmas can adapt to the rapidly changing conditions in the host. Previous studies had shown that phenotypic variation and genetic recombination occur at high frequencies in M. pulmonis (3). The vsa genes comprise one of the highly recombinogenic loci in this species. Recombination between vsa genes involves site-specific DNA inversions occurring at a 34-bp sequence that defines the vsa ...

Human papillomaviruses (HPVs) establish long-term infections in patients. The mechanism for extrachromosomal HPV DNA persistence in cycling cells is unknown. We show that HPV origincontaining plasmids partition as minichromosomes, attributable to an association of the viral origin recognition protein E2 with mitotic spindles. ␣-, ␤-, and ␥-tubulins were pulled down with a tagged E2. The N-terminal transacting and C-terminal protein dimerization͞DNA binding domains independently associated with the spindles. We suggest that this E2 property enables these viruses to establish persistence. Its implication for HPV oncogenesis is presented.F or any extrachromosomal DNA virus to establish a persistent infection in cycling host cells, the viral genome must replicate and partition into both daughter cells during division. The E2 origin (ori)-binding protein of bovine papillomavirus type 1 (BPV-1) associates with mitotic chromosomes (1-3), thus providing a mechanism for viral DNA segregation. Comparable mechanisms have been demonstrated for the Epstein-Barr virus through the Epstein-Barr virus-encoded nuclear antigen 1 protein and the Kaposi's Sarcoma virus (human herpesvirus 8) through the latency-associated nuclear antigen 1 protein (4, 5). In contrast, the mechanism by which human papillomavirus (HPV) DNA partitions during cell division has not been elucidated. In this report, we demonstrate that HPV ori-containing DNA segregates as minichromosomes by association with mitotic spindles and this association is mediated by the HPV origin recognition protein E2.HPVs are medically important pathogens that establish persistent infections in long-living basal keratinocytes. Infections typically cause benign hyperproliferation of squamous epithelia in the form of cutaneous warts, laryngeal papillomas, and anogenital condylomata. Over time, infections can become subclinical, but may reactivate during episodes of immune suppression. The HPV genome is a double-stranded, circular DNA of Ϸ7,900 bp and replicates extrachromosomally in the nucleus of infected keratinocytes. Low copy numbers of the mucosotrophic HPV DNA plasmids are maintained in the basal and parabasal cells that divide, whereas the productive phase takes place only in postmitotic, differentiated cell strata and progeny virus shed within the sloughing superficial cells (6). Thus, it is paramount that, in either latent or active infections, HPV DNA must partition into the two daughters of dividing cells for viral persistence. To support viral DNA amplification in postmitotic cells, the viral E6 and E7 proteins inactivate the host tumor suppressor proteins p53 and pRB (retinoblastoma protein), respectively, reestablishing an S-phase environment. Elevated transcription of these oncogenes is normally limited to the differentiated compartment. However, if inappropriately expressed in the basal cells, such as during repeated wounding and healing, the high-risk HPV oncoproteins can promote excessive cell cycling and host chromosome instability. Indeed, a small fraction of ...