The gene encoding the major viral structural protein stimulates recombination in polyomavirus DNA.

RmI is a chimeric DNA molecule consisting of a polyoma genome in which a partly duplicated VP1-coding region brackets an insert of murine DNA (Ins); when transfected into mouse cells, RmI recombines intramolecularly to yield infectious, unit-length, polyoma DNA. We report here that RmI encodes a polypeptide of 337 amino acids (designated VmP1) which includes the N-terminal 328 amino acids of VP1 and 9 amino acids specified by Ins. Mutating the VmP1-coding sequence strongly reduces the ability of RmI to yield polyoma DNA. In contrast, mutating the portion of the VP1-coding sequence which is not part of the VmP1-coding sequence has little or no impact on the ability of RmI to yield polyoma DNA, even though it renders such DNA noninfectious. Thus, release of polyoma DNA from RmI involves a function of VP1 distinct from that ensuring virus assembly and propagation; since VP1 can arise only after recombination has occurred, VmP1, but not VP1, could carry such a function. We suggest that VmP1 acts in concert with VP2, which we have already reported to stimulate recombination in RmI.

Involvement of minor structural proteins in recombination of polyoma virus DNA.

We have previously observed that a polyoma-mouse chimeric DNA molecule (RmI) in which the murine DNA insert is flanked by directly repeated viral sequences is effectively converted into unit-length polyoma DNA upon transfection of permissive mouse cells. This intramolecular recombination event appears to be dependent on VmP1, a protein encoded by RmI which includes the 328 N-terminal amino acids of polyoma VP1, and nine amino acids of murine origin carrying the C-terminus of the protein. We report here that introducing mutations into the VP2/VP3 coding sequence reduces the ability of RmI to generate polyoma DNA, even though the same mutations seem to exert little or no effect on the ability of polyoma DNA to either replicate or accumulate inside transfected cells. A mutation affecting VP2 alone being as effective as one that affects both VP2 and VP3, VP2 appears to be playing a critical role in recombination.

Rescue of polyomavirus DNA after co-transfection of recombinant plasmids with viral DNA fragments.

Plasmid DNA bearing a single copy of the mouse polyomavirus (Py) genome (template A) was transfected into murine cells together with another DNA (template B) carrying intact the viral sequence interrupted in template A. Rescue of unit-length Py DNA including markers from both templates was observed as long as the viral DNA in B overlapped that split in A by one kbp or more. Such rescue was not detectably enhanced by linearizing either or both template(s), and occurred in the absence of template replication. These findings are suggestive of an intermolecular recombination process taking place soon after transfection and starting with homologous pairing between A and B. Such pairing would facilitate removal of vector DNA from one template (A), followed by closure of the resulting break or gap through recombination with the other template (B). Since B may consist of a PCR-synthesized DNA fragment, these observations could conceivably serve as the basis for a method of generating mutant viral genomes.

p53-mediated DNA renaturation can mimic strand exchange.

The process of strand exchange is considered to be the hallmark of DNA recombination. Proteins known to carry out such exchange are believed to operate via one or the other of two mechanisms. RecA-like proteins promote the formation of a three-stranded or triplex synaptic intermediate in which strand exchange occurs, whereas other proteins would allow the coordinated exonucleolytic degradation of one strand in the duplex DNA and its replacement by an invading strand of similar sequence and polarity. In view of properties ascribed to it, we have attempted to determine whether p53 belongs to one or the other of these groups of proteins. The in vitro assay used relies on a double-stranded (ds) oligonucleotide (oligo 1+2) and a single-stranded (ss) oligonucleotide (oligo 3), part of which is complementary to oligo 1. The data collected suggest that, under the conditions of the assay, oligo 1+2 undergoes partial denaturation; p53 then catalyzes renaturation of oligo 1 with oligo 3, rather than true strand exchange. Since p53 is not known for being able to 'melt' DNA, it would seem unlikely that this protein would effect strand exchange in vivo without assistance from another, denaturing, protein.

An enhancer of recombination in polyomavirus DNA.

Previous work from this laboratory has indicated that intramolecular homologous recombination of polyomavirus (Py) DNA is dependent upon promoter structure or function. In this report, we demonstrate that Py DNA contains not two but three binding sites for transcription factor YY1, all located on the late side of viral origin of replication (ori) and the third well within the VP1 coding sequence. This third site (Y3), which may or may not play a role in transcription regulation, is immediately adjacent to a previously described recombination hot spot (S1/S2). We found that Py replicons carrying an altered Y3 site recombined in a manner suggesting partial inactivation of the S1/S hot spot. Point mutations precluding the binding of YY1 to Y3 in vitro depressed hot spot activity in vivo; however, of the two reciprocal products reflecting recombination at this spot, only that carrying the mutated Y3 site arose at a reduced rate. These results are interpreted in light of a model assuming that recombination occurs within a transcriptionally active viral chromatin tethered to the nuclear matrix by YY1.

Quantitation of Chlamydia trachomatis by culture, direct immunofluorescence and competitive polymerase chain reaction.

OBJECTIVES: Methods to quantitate Chlamydia trachomatis have never been compared although it would be relevant to periodically evaluate the sensitivity of a detection system. We compared the sensitivity and reproducibility of culture, direct immunofluorescence and the polymerase chain reaction (PCR) to quantitate C trachomatis. METHODS: A competitive semiquantitative PCR procedure was developed. The number of inclusions in culture, particles by direct immunofluorescence and DNA copies by PCR were measured for 12 patient specimens. Variation was determined by measuring a sample 10 times for each method. RESULTS: Patient C trachomatis major outer membrane protein gene DNA was measured semiquantitatively by amplifying together with reference DNA. DNA molecules, particles and infectious units were quantitated in clinical samples with, on average, 595 DNA molecules and 87 immunofluorescent particles observed per inclusion-forming-unit. Similar coefficients of variation (47-52%) were observed for the 3 procedures. CONCLUSION: Competitive PCR and counting immunofluorescent particles provide reproducible and sensitive methods of quantitating C trachomatis.

Topoisomerase activity associated with polyoma virus large tumor antigen.

Polyomavirus (Py) large tumor antigen (LT) was produced in mammalian or insect cells infected with a suitable viral expression vector, and purified by a procedure combining immunoprecipitation with ion-exchange chromatography. Fractions containing the bulk of LT displayed a DNA-relaxing activity (LT-topo) which could be attributed neither to topoisomerase II (topo II) nor to topoisomerase I (topo I) encoded by the cell or the viral vector. On the one hand, LT-topo relaxed pBR322 DNA in a reaction which, unlike that characteristic of topo II, was ATP-independent and inhibited by camptothecin. On the other hand, serum from scleroderma patients which strongly inhibited calf thymus topo I had no effect on LT-topo, which absolutely required Mg2+ ions to relax DNA. Thus, LT-topo is either inherent to LT or belongs to a LT-bound enzyme similar to, but distinct from, topo I.

A hotspot for promoter-dependent recombination in polyomavirus DNA.

We have engineered polyomavirus (Py) DNA molecules carrying two large direct repeats within the late coding region, as well as a deletion encompassing the TATA box in the early promoter. Such constructs recombine less readily than a construct containing the same duplication of late sequences, but an intact early promoter. Furthermore, residual recombination in the molecules with a deletion occurs between homologous sites which differ from those used in the molecule without deletion. These findings are consistent with recombination being stimulated by transcription originating from the early promoter, rather than facilitated by the "openness" of viral chromatin undergoing transcription.

Variation outside variable segments of the major outer membrane protein distinguishes trachoma from urogenital isolates of the same serovar of Chlamydia trachomatis.

OBJECTIVES--Whereas serovars A, B, Ba and C of Chlamydia trachomatis are usually associated with trachoma, two of these serovars (Ba and C) are occasionally observed in urogenital infections. Variation in the gene encoding the major outer membrane protein (MOMP) was explored to distinguish urogenital from trachoma specimens of the same serovar. METHODS--A large portion of the MOMP gene was amplified by nested PCR directly from clinical samples from trachoma or urogenital infection and the serovar of the infecting C trachomatis was determined by restriction fragment length polymorphism (RFLP). Amplified DNA from trachoma serovars B, Ba and C and from urogenital serovars Ba, C, D and E was sequenced by the dideoxy chain termination method. RESULTS--While almost identical in variable segment (VS)I, three urogenital Ba samples differed from all trachoma B and Ba samples at eight nucleotides including two sites which changed amino acids in the constant region upstream of VSI. An identical sequence in this region was observed for the reference urogenital D serovar. Variation in this same region upstream of VSI also distinguished 40% of serovar D samples from prototype D including three that were sequenced. Two urogenital C differed from trachoma C samples at four sites that changed the MOMP amino acid sequence including two changes in the constant region between VSII and III and single changes in VSII and III. On the basis of these sequence determinations, RFLP was predicted which allowed extension of these observations to 20 other urogenital Ba, 12 trachoma B or Ba, seven variant D, 12 D, four urogenital C and three trachoma C samples without further sequencing. CONCLUSION--Urogenital Ba and C samples have VSI or II and III sequences identical or very similar to trachoma strains of the same serovar, but resemble more closely other serovars in the constant regions. Urogenital serovar D samples can also be divided into two genotypes on the basis of sequence differences in the constant region preceding VSI.

Intramolecular recombination in polyomavirus DNA is a nonconservative process directed from the viral intergenic region.

Previously, we have studied intramolecular homologous recombination in polyomavirus replicons under conditions allowing only one amplifiable recombination product to be generated from a single precursor molecule. In order to detect putative reciprocal product(s), we have now constructed precursor polyomavirus replicons which contain two copies, instead of one copy, of the viral intergenic region, including the origin of replication as well as both promoters. Upon transfection of mouse cells, constructs containing directly repeated intergenic regions yielded distinct amplifiable products, in number depending upon the functional integrity of both intergenic regions. Our data indicate that of two possible reciprocal products, a given precursor molecule would yield either one or the other but never both at the same time. Most striking, however, is the observation that promoter function is required for recombination, while the origin of replication function may be needed only for amplification of the recombination product once it has been formed. The data reported here confirm and extend previous data suggesting that (i) transcription is instrumental in recombination between direct repeats and (ii) nonconservative recombination involving direct repeats relies upon two promoters of opposing polarities.

Intramolecular recombination in polyomavirus DNA is controlled by promoter elements.

We show here that intramolecular homologous recombination in polyomavirus (Py) DNA depends upon discrete sequence elements of the viral regulatory region which are believed to regulate transcription initiation and exert little or no cis-control over replication. Either deleting the viral early promoter (EP) or inverting the viral late promoter (LP) strongly impairs viral DNA recombination under conditions allowing viral DNA replication to proceed undisturbed. These findings suggest that bi-directional transcription proceeding from the intergenic region favors intramolecular recombination.

Topoisomerase activity associated with SV40 large tumor antigen.

Purified preparations of simian virus 40 (SV40) large tumor antigen (LT) from three different sources, including LT expressed from a recombinant baculovirus, were found to relax negatively supercoiled cyclic DNA molecules, whether or not they contained SV40 sequences. Relaxation was stimulated by MgCl2 but not by ATP, and inhibited by camptothecin, suggesting the involvement of an enzymatic activity similar to that of topoisomerase I (topo I). However, the pH requirements for relaxation by respectively LT and topo I are different. Also, antibodies reacting with LT inhibited relaxation by preparations of LT but not topo I, whereas antibodies inhibiting relaxation by topo I had no effect on relaxation by LT. Reconstruction experiments suggested that both procedures used to purify LT, immunoaffinity chromatography and DEAE-Sepharose chromatography, separate topo I from LT. Finally, relaxing activity was found in over 40 preparations of LT, and in the few instances where activity could not be found, it probably had been lost during storage, rather than absent from the start. Whereas these results seem to exclude that the activity being detected is that of a contaminant of LT, they would be consistent with this activity being that of a stable topo-LT complex, or else intrinsic to LT itself.

Crossover site selection during recombination of polyomavirus replicons.

Two hybrid replicons containing polyomavirus (Py) genomes with large duplications of the viral late coding sequences were transfected into various permissive mouse cell lines. In all cell lines, either replicon yielded the sole amplifiable product expected from intramolecular homologous recombination, unit-length Py DNA (P155). In normal and in Py-transformed cells, such recombination was highly effective and involved sequences previously found to act as recombination hot spots (S repeats). In cells transformed by simian virus 40, however, these hot spots were inoperative in the generation of P155, which occurred with a reduced efficiency. These data confirm and extend earlier data indicating that the nature of products arising from recombination in Py replicons is tightly controlled by both cis- and trans-acting factors.

DNA nicking favors PCR recombination.

We attempted to use the polymerase chain reaction (PCR) to monitor in vitro recombination in a plasmid containing directly repeated sequences. Some of the plasmid preparations which had not been exposed to recombination conditions were however found to behave in the PCR test as if they had undergone homologous recombination. We show here that such false positives are attributable to a small degree of nicking and/or breaking of the DNA template. Presumably, such damage allows the formation of hybrid parental duplexes containing at least one truncated strand, the 3' end of which maps within the homology; extension of this 3' end by the polymerase then results in a linkage of sequences identical to that arising from homologous recombination.

The same mammalian replicon yields distinct recombination products in different cell lines.

We have observed previously that some chimeric replicons inclusive of a partly duplicated polyomavirus (Py) genome yield unit-length Py DNA (P155) at high frequency when transfected into normal or Py-transformed mouse cells. We demonstrate here that one such replicon generates either P155 or illegitimate recombination products in other mouse cells, transformed by simian virus 40. Use of the polymerase chain reaction indicates that each of the illegitimate products carried a different deletion, but that all deletions mapped within a rather well defined portion of the precursor replicon. Thus, these products were organized as if two hotspots for recombination existed in the Py late-coding region, one being located within or near one of the duplicated sequences characteristic of the chimeric replicon. Since this particular hotspot has already been shown to be involved in the generation of P155, the data reported here could indicate that a single recombination mechanism can yield either homologous (P155) or illegitimate products. How the DNA interacts with certain proteins, such as papovavirus large tumor antigen, could explain why one or the other type of product is formed.

Alternative homologous and nonhomologous products arising from intramolecular recombination.

RmI, a chimeric DNA molecule containing polyomavirus (Py) and mouse sequences, generates unit-length Py DNA via intramolecular recombination between two directly repeated viral sequences of 182 base pairs (S repeats). To analyze the contribution of the S repeats in this process, we produced mutants of RmI carrying deletions in either one or both S repeats and tested them for their ability to recombine in mouse 3T6 cells. Mutant DNAs were found to yield unit-length Py DNA as long as they carried a minimal internal homology of 40 to 50 base pairs. Unlike RmI itself, however, the mutants also gave rise to nonhomologous recombination products. These results suggest that when the generation of homologous products is hampered by a limiting homology, nonhomologous products may arise instead of homologous ones. Therefore, the initial step(s) in the mechanisms yielding the two kinds of products could be identical.

Preferred crossover sites on polyomavirus DNA.

RmI is a hybrid replicon consisting of polyomavirus (Py) and mouse sequences that yields unit-length polyomavirus DNA via recombination between two directly repeated viral sequences of 182 base pairs (S repeats). To define the contribution of the S repeats in this intramolecular recombination, we derived from RmI a series of replicons containing the original S repeats as well as additional direct viral repeats which were 1 to 2 kilobases in length (L repeats). After mouse 3T6 cells were transfected with these constructs, recombination products that displayed the physical properties of homologous recombinants were detected. The structures of these recombinants indicated that whereas repeat length influences the likelihood of recombination, crossover occurs preferentially near the S repeats, provided that one of them is proximal to the viral origin of replication. This finding suggests that recombination near the S repeats depends on a process initiated near the viral origin of replication.

A substitution in a nonconserved region of polyomavirus large T antigen which causes a thermosensitive mutation.

The temperature-sensitive defect of the tsP155 mutant of polyomavirus (Py) maps in the large T antigen (LT) coding sequence of a viral DNA diverging markedly from that of extensively characterized wild-types (WTs) such as A2 and CSP. We have sequenced about 600 base pairs (bp) "early" DNA encompassing the mutated site in tsP155, as well as the corresponding DNA segment from a revertant virus (RtsP155). As expected, tsP155 was found to be more closely related to CSP than to A2. Out of 3 single bp differences between tsP155 and CSP, 2 were common to tsP155 and RtsP155. The only substitution exclusive to tsP155 was a G----C transversion at bp 2658 which canceled the HaeIII site at bp 2657. Heteroduplexes inclusive of tsP155 DNA and of a 312-bp-long fragment of RtsP155 DNA yielded recombinant viruses growing under restrictive conditions whose DNAs had all regained the HaeIII site at bp 2657. These findings clearly identify the ts mutation with the tranversion at bp 2658, which is expected to change Ala 701 for a Pro in LT. We discuss this substitution in relation to the phenotype of tsP155.

Major rearrangement of cellular DNA in the vicinity of integrated polyomavirus DNA.

The Cyp cell line was produced by transforming mouse embryo cells at the restrictive temperature with an early thermosensitive mutant of polyomavirus (Py). Transfer of Cyp cells to the nonrestrictive temperature causes excision to occur at a single chromosomal site carrying viral DNA, and leads to the production of infectious virus. We have attempted to elucidate the recombination event that occurred during the integration of Py DNA in this inducible line. Physical characterization of two recombinant DNAs-one selected from a genomic library of normal mouse DNA and the other constructed from the unoccupied allele of the Cyp integration site-indicates that generation of the Cyp line has involved the joining of not only viral DNA to a cellular alpha site, but also the cellular alpha site to a cellular alpha site to cellular beta site. Hence, previously described hybrid excision products from the Cyp line were made of mouse DNA segments representing two distinct cellular sites. The alpha-beta joining may play a role in the expression of integrated Py DNA.