A method that avoids culture was devised to determine serovars of Chlamydia trachomatis. Polymerase chain reaction was used first to amplify a part of the chlamydial genome that included the leader sequence and all four variable domains of the major outer membrane protein (MOMP) of the 15 serovars of C. trachomatis. The amplified DNA was then digested simultaneously with restriction endonucleases AluI and MspI and the resulting fragments separated on 10% polyacrylamide gels. After silver staining, a total of 13 characteristic patterns were observed for the 15 serovars, leaving two ambiguities that were resolved using alternate enzymes. Analysis of 40 clinical isolates revealed patterns indistinguishable from those of the prototype serovars including, unexpectedly, 5 Ba serovars. The same PCR procedure also allowed amplification of the MOMP gene of two avian Chlamydia psittaci and one Chlamydia pneumoniae isolates.
The chromatin remodeling process that takes place during spermiogenesis in mammals is characterized by a transient increase in DNA single‐strand breaks (SSB). The mammalian transition proteins (TPs) are expressed at a high level at mid‐spermiogenesis steps coincident with chromatin remodeling and could be involved in the repair of these lesions since SSB are no longer detected in terminally differentiated spermatids. We report that TP1 can stimulate the repair of SSB in vitro and demonstrate that in vivo repair of UV‐induced DNA lesions is enhanced in mammalian cells stably expressing TP1. These results suggest that, aside from its role in DNA compaction, this major transition protein may contribute to the yet unidentified enzymatic activity responsible for the repair of SSB at mid‐spermiogenesis steps. These results also suggest that the TP1 proteins have the potential to participate in the repair process following genotoxic insults and therefore may play an active role in the maintenance of the integrity of the male haploid genome during spermiogenesis. Mol. Reprod. Dev. 58:437–443, 2001. © 2001 Wiley‐Liss, Inc.
Mammalian spermiogenesis is characterized by a striking restructuring of the spermatid chromatin caused by the replacement of nucleohistones with transition proteins and their subsequent replacement with nucleoprotamines. The onset of nuclear elongation and chromatin condensation in spermatids is accompanied by a general decrease in the transcriptional activity of the DNA. A recently identified testis-specific high-mobilitygroup (tsHMG) protein, similar to the human mitochondrial transcription factor I and to the linker-associated protein ␦ of Tetrahymena thermophila micronuclei, is thought to play a structural role in this process. We confirm by immunoblot analysis of fractionated germ cells that the presence of tsHMG is restricted to transcriptionally quiescent elongating and condensing spermatids. Purified recombinant tsHMG protein displays preferential binding to supercoiled plasmid DNA, which reversibly protects the DNA against the DNA-relaxing activity of eukaryotic topoisomerase I and also impairs the transcriptional activity of this template when assayed in vitro. The tsHMG protein can also introduce negative supercoils into a relaxed plasmid substrate in a topoisomerase I-dependent manner. We also show that the tsHMG protein is the substrate of a Ca 2؉ -phospholipid-dependent protein kinase (protein kinase C) present in testis extracts of adult mice and demonstrate that phosphorylation by protein kinase C is required for both the DNA-binding and the topoisomerase I-dependent supercoiling activities of tsHMG. Our results support the hypothesis that the spermatid tsHMG protein is a topological factor (transition protein) that can modulate the activity of topoisomerase I. This activity could contribute to the important transition in chromatin structure which leads to the decrease in DNA metabolism observed at the early stages of spermatid elongation.The highly condensed state of mature mammalian sperm DNA results from the replacement of the somatic-like histones in the nucleosomes by more basic transition proteins initially and the subsequent replacement of these proteins with highly basic, arginine-and cysteine-rich protamines. This protein replacement permits a much higher level of DNA packaging than is observed in the somatic nucleus (56). This complex transition process produces remarkable changes in chromatin structure that greatly improve the mechanical and chemical stability of the DNA (3). The chromatin restructuring process begins when the spermatid nucleus adopts its species-specific elongated shape. It is known that this event coincides with a general shutoff of both DNA transcription and repair activity (17,20). The transition in nuclear proteins requires several steps that include posttranslational modifications of histones (37) as well as changes in the activities of topology-modifying enzymes such as topoisomerase I (30). Major changes in the pattern of the DNase I-hypersensitive sites have also been demonstrated and likely reflect this alteration in nucleosome organization (31).Although the...
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.
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