Methylation is co-ordinated on the putative replication origins of Physarum ribosomal DNA

In Physarum polycephalum, the ribosomal DNA is found as 60,000 base-pair palindromes. Each rDNA has four symmetrically arranged replication origins flanked by ribosomal RNA genes. A particular sequence, the putative replication origin, is repeated at the approximate position of each origin and nowhere else in the molecule. On a typical rDNA molecule, only one origin is active per replication cycle. We show that both the level and co-ordination of methylation result in asymmetrically methylated rDNA molecules that are particularly hypomethylated at one of their four putative replication origins. This pattern of methylation on a typical rDNA molecule is consistent with a model where hypomethylation is a determinant of origin activity.     

Analysis of spreading of inactivation in eight X autosome translocations utilizing the high resolution RBG technique

Summary Eight X autosome translocations were studied with replication banding to localize spreading of late replication into the autosomal segments. Partial spreading into the autosomal segment was seen in four translocations and no spreading of late replication was seen in four translocations. In those translocations with partial spreading of late replication into the autosomal segment, late replication did not always spread continuously from the X chromosome breakpoint throughout the autosome. Instead, it appeared to skip some bands and affect others. The data on the pattern of replication, taken to indicate also a spread of inactivation into these autosomal segments, correlated well with the clinical data in most cases and suggest that spreading of late replication is often incomplete and may be discontinuous.     

What causes the abnormal phenotype in a 49,XXXXY male?

Summary The chromosome replication pattern of a man with 49,XXXXY was analyzed using ³H-thymidine and autoradiography as well as BrdU and acridine orange. The former technique showed a highly irregular replication pattern; the latter revealed one early replicating X chromosome, and the other three more or less asynchronously replicating. Two hypotheses seem to explain best the abnormal phenotype of males with an XXXXY sex chromosome constitution: (1) The number of the always active regions (tip of Xp) and of the possibly always active regions (the Q-dark regions on both sides of the centromere) is increased from one to four. (2) The replication pattern of the late-replicating X chromosomes is highly asynchronous, which might affect the phenotype. The possibility that more than one X chromosome might remain active in some cells, an even more abnormal and obviously deleterious situation, is still open.     

Autoradiographic studies of the human Y chromosome

An autoradiographic analysis (using continuous labeling with tritiated thymidine) was made on 317 cells from four normal males. The labeling pattern of the Y chromosome was compared to the first and the last chromosomes to complete replication as well as to G21–22. The Y chromosome was never found to be the last chromosome in the cell to complete replication. Instead, it completed DNA synthesis relatively early (usually among the first 10 chromosomes) but had a distinctively heavy label during the earliest stages of late-S. In 51% of those cells with one labeled G+Y chromosome, a G21–22 was labeled and the Y was not.—It was concluded, therefore, that the human Y chromosome is not a late-replicating chromosome but terminates replication earlier than most of the autosomes. In addition, the Y chromosome cannot be distinguished from the G chromosomes on the basis of a consistent and differential labeling pattern.Supported by USPHS Grant GM 15361.     

Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo.

Initiation of simian virus 40 (SV40) DNA replication is facilitated by two auxiliary sequences that flank the minimally required origin (ori) core sequence. In monkey cells, the replication rate of each of the four ori configurations changed with time after transfection in a characteristic pattern. This pattern was reproduced in an extract from SV40-infected monkey cells by varying the ratio of DNA substrate to cell extract; DNA replication in vitro depended on ori auxiliary sequences to the same extent as they did in vivo. Facilitation by ori auxiliary sequences was lost at high ratios of DNA to cell extract, revealing that the activity of these sequences required either multiple initiation factors or a molar excess of one initiation factor bound to ori. This parameter, together with ionic strength and the method used to measure DNA replication, determined the level of facilitation by ori auxiliary sequences in vitro. The activity of ori auxiliary sequences was not diminished in vivo or in vitro by increasing amounts of large tumor antigen. Therefore, ori auxiliary sequences promoted initiation of replication at some step after tumor antigen binding to ori. Furthermore, although cellular factors could modulate the activity of ori auxiliary sequences in vitro, these factors did not appear to involve nucleosome assembly because no correlation was observed between the number of nucleosomes assembled per DNA molecule and facilitation by ori auxiliary sequences. These results demonstrate that SV40 ori auxiliary sequences can function in vitro as they do in vivo and begin to elucidate their role in initiating DNA replication.     

Genetics of 51d-52a, a Region Containing Several Maternal-Effect Genes and Two Maternal-Specific Transcripts in Drosophila

Two genomic clones exhibiting a maternal-specific pattern of expression map to cytological region 52A. To elucidate the function of these clones we have undertaken a mutagenesis of the cytological region 51D-52A. This paper presents the results of this screen and the preliminary analysis of female-sterile and lethal mutations isolated. A total of twelve complementation groups have been identified, four of which are defined exclusively by female-sterile alleles. Only one visible mutation was isolated, a recessive temperature-sensitive allele of Thickened-arista (Tarts). Several of the seven lethal loci display an embryonic lethal phase. Three of the four female-sterile loci affect chorion structure with one resulting in underamplification of the chorion genes, and two (possibly three) of the four female-steriles affect nuclear division/DNA replication. Thus it appears that this is a "developmentally important" region, possibly representing a clustering of genes involved in either DNA replication or nuclear division.     

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbillus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially reinactivated, suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.Deceased on 2 Jan. 1987     

Replication and subnuclear location dynamics of the immunoglobulin heavy-chain locus in B-lineage cells

The murine immunoglobulin heavy-chain (Igh) locus provides an important model for understanding the replication of tissue-specific gene loci in mammalian cells. We have observed two DNA replication programs with dramatically different temporal replication patterns for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines and in ex vivo-expanded pro-B cells, the entire locus is replicated early in S phase. In three cell lines that exhibit the early-replication pattern, we found that replication forks progress in both directions through the constant-region genes, which is consistent with the activation of multiple initiation sites. In contrast, in plasma cell lines, replication of the Igh locus occurs through a triphasic pattern similar to that previously detected in MEL cells. Sequences downstream of the Igh-C alpha gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An approximately 500-kb transition region connecting sequences that replicate early and late is replicated progressively later in S. The formation of the transition region in different cell lines is independent of the sequences encompassed. In B-cell lines that exhibit a triphasic-replication pattern, replication forks progress in one direction through the examined constant-region genes. Timing data and the direction of replication fork movement indicate that replication of the transition region occurs by a single replication fork, as previously described for MEL cells. Associated with the contrasting replication programs are differences in the subnuclear locations of Igh loci. When the entire locus is replicated early in S, the Igh locus is located away from the nuclear periphery, but when Vh gene sequences replicate late and there is a temporal-transition region, the entire Igh locus is located near the nuclear periphery.     

Decrease of DNA synthesis in amniotic fluid cells during the middle part of S-phase revealed by differential chromosome staining after incorporation of BrdU

The time sequence of DNA replication in partially synchronized human amniotic fluid cells has been analysed, employing BrdU incorporation techniques. —Regardless of the interval between removal of the methotrexate/uridine block and addition of BrdU during S-phase, the treatment results in an R-type replication pattern. Conversely, replacement of BrdU containing medium by another one with thymidine yields G-type replication patterns. A thymidine pulse during the first 4 h of S-phase results in R-type replication patterns; from 7–10 h after block removal it produces G-type pattern. In between, only faint red staining dots can be found indicating a marked decrease of replicational activity during the middle part of the S-phase.     

Isochores and replication time zones: a perfect match

The mammalian genome is not a random sequence but shows a specific, evolutionarily conserved structure that becomes manifest in its isochore pattern. Isochores, i.e. stretches of DNA with a distinct sequence composition and thus a specific GC content, cause the chromosomal banding pattern. This fundamental level of genome organization is related to several functional features like the replication timing of a DNA sequence. GC richness of genomic regions generally corresponds to an early replication time during S phase. Recently, we demonstrated this interdependency on a molecular level for an abrupt transition from a GC-poor isochore to a GC-rich one in the NF1 gene region; this isochore boundary also separates late from early replicating chromatin. Now, we analyzed another genomic region containing four isochores separated by three sharp isochore transitions. Again, the GC-rich isochores were found to be replicating early, the GC-poor isochores late in S phase; one of the replication time zones was discovered to consist of one single replicon. At the boundaries between isochores, that all show no special sequence elements, the replication machinery stopped for several hours. Thus, our results emphasize the importance of isochores as functional genomic units, and of isochore transitions as genomic landmarks with a key function for chromosome organization and basic biological properties.     

Multiple sites of replication initiation in the human beta-globin gene locus

The cell cycle-dependent, ordered assembly of protein prereplicative complexes suggests that eukaryotic replication origins determine when genomic replication initiates. By comparison, the factors that determine where replication initiates relative to the sites of prereplicative complex formation are not known. In the human globin gene locus previous work showed that replication initiates at a single site 5′ to the β-globin gene when protein synthesis is inhibited by emetine. The present study has examined the pattern of initiation around the genetically defined β-globin replicator in logarithmically growing HeLa cells, using two PCR-based nascent strand assays. In contrast to the pattern of initiation detected in emetine-treated cells, analysis of the short nascent strands at five positions spanning a 40 kb globin gene region shows that replication initiates at more than one site in non-drug-treated cells. Quantitation of nascent DNA chains confirmed that replication begins at several locations in this domain, including one near the initiation region (IR) identified in emetine-treated cells. However, the abundance of short nascent strands at another initiation site ~20 kb upstream is ~4-fold as great as that at the IR. The latter site abuts an early S phase replicating fragment previously defined at low resolution in logarithmically dividing cells.     

Induction of endoreduplication in double minutes of the human neuroblastoma cells and the replication pattern.

Induction of endoreduplication (ERD) using Hoechst 33258 as well as colcemid was carried out in cultured neuroblastoma (NB) line cells. In these endoreduplicated cells, the majority of double minutes (DMs) appeared to take a diplochromosome like configuration to form a cluster consisting of four minute elements, assuming a complex DM. Sister chromatid differential staining (SCD) using 5-bromo-2'-deoxyuridine (BrdUrd) revealed the non-random distribution of the stained chromatids among four chromatids composing each diplochromosome, suggesting the occurrence of so-called "outside replication" of DNA strands during the process of ERD. The same pattern of differential staining was also found in the quadruple minutes of each endoreduplicated DM. Since DMs are acentric, the present results suggest that centromeres do not play any essential role in the formation of diplochromosomes observed in the conventional cytologic preparations and that centromeres are probably not responsible for the phenomenon of the "outside replication" of DNA strands.     

Effect of growth rate and incC mutation on symmetric plasmid distribution by the IncP-1 partitioning apparatus

The incC and korB genes of IncP-1 plasmid RK2 encode homologues of ubiquitous ParA and ParB partitioning proteins of bacterial plasmids and chromosomes. Using immunofluorescence microscopy, we found that KorB, which binds to 12 widely distributed sites on the genome, is located in symmetrically placed foci in cells containing IncP-1 plasmids. When maintained by the low-copy-number P7 replicon, an RK2 segment including incC, korB and the kla, kle and korC regions encodes an efficient partitioning system that gives a pattern of foci similar to RK2 itself. Symmetrical distribution of KorB foci correlates with segregational stability conferred by either the IncP-1 or P7 partitioning systems; KorB distribution follows plasmid distribution. In the absence of a second partitioning system, incC inactivation resulted in paired or clumped foci that were not symmetrically distributed. At a slow growth rate, position analysis of foci showed a cycle from one central focus to two foci (at one- and three-quarter positions) and back, and at a high growth rate it showed a cycle from two foci to four and back. This pattern fits with the plasmid being coupled to the replication zones in the cell and being moved to successively younger zones by active partitioning, indicating a tight association between replication and partitioning.     

Isolation and properties of temperature-sensitive mutants of the trfA gene of the broad host range plasmid RK2

Two small plasmid RK2 derivatives, pSV6 and pSV16, were constructed and used for the isolation and characterization of trfA mutants temperature-sensitive (ts) for replication in Escherichia coli. Four of the mutants were examined for their ability to initiate replication from the RK2 replication origin in E. coli when present in cis with respect to the origin and in trans when present on a multicopy pBR322 replicon. Each of the mutant trfA genes exhibited temperature-sensitivity in supporting replication from the RK2 origin when present in cis, and the lowest nonpermissive temperature varied depending on the mutant. When the mutant trfA genes were present on the multicopy replicon (in trans), three of the four mutant genes could support replication of the RK2-oriV plasmid pSV16 at all temperatures tested. However, with the exception of one of the mutants, the activity was reduced when compared to wild-type. The increased activity in trans possibly is the result of the increased cellular level of the TrfA protein when compared with the in cis situation where the mutant trfA gene is at a much lower copy-number. Two of the mutants also were tested in cis for temperature sensitivity in Pseudomonas aeruginosa. One of the mutants did not exhibit temperature sensitivity under the conditions employed. The second mutant showed some temperature sensitivity but the nonpermissive temperature pattern was different than that found in E. coli.     

Characterization of the putative replisome organizer of the lactococcal bacteriophage r1t

Analysis of the nucleotide sequence of the genome of the lactococcal bacteriophage r1t showed that it may encode at least two proteins involved in DNA replication. On the basis of its similarity with the G38P protein encoded by the Bacillus subtilis phage SPP1, the product of orf11 (Pro11) is thought to be involved in the initiation of phage DNA replication. This protein was overexpressed in Lactococcus lactis and partially purified. Gel retardation analysis using various r1t DNA fragments indicates that Pro11 specifically binds to a sequence located within its cognate gene. DNase I footprinting showed that Pro11 protects a stretch of DNA of 47 bp. This region spans four 6-bp short direct repeats, which suggests that the region contains four binding sites for Pro11. 1,10-Phenanthroline-copper footprinting confirmed the protection of the hexamers. An asymmetric protection pattern of each strand was observed, suggesting that Pro11 contacts each DNA strand separately at contiguous hexamers. We propose a model for the binding of Pro11 to its target sites that may account for the torsion strain required for strand opening at the origin of replication.     

Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin.

Our laboratory has previously shown that replication of a small plasmid, p174, containing the genetically defined Epstein-Barr virus (EBV) latent origin of replication, oriP, initiates within oriP at or near a dyad symmetry (DS) element and terminates specifically at a family of repeated sequences (FR), also located within oriP. We describe here an analysis of the replication of intact approximately 170-kb EBV genomes in four latently infected cell lines that uses two-dimensional gel replicon mapping. Initiation was detected at oriP in all EBV genomes examined; however, some replication forks appear to originate from alternative initiation sites. In addition, pausing of replication forks was observed at the two clusters of EBV nuclear antigen 1 binding sites within oriP and at or near two highly expressed viral genes 0.5 to 1 kb upstream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV genome, the relative abundance of these stalled forks and the direction in which they are stalled indicate that most replication forks originate upstream of oriP. We thus searched for additional initiation sites in the Raji EBV and found that the majority of initiation events were distributed over a broad region to the left of oriP. This delocalized pattern of initiation resembles initiation of replication in several well-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with which to investigate factors influencing the selection of replication initiation and termination sites in mammalian cells.     

Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins

Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures ("bobbins"). These bobbins constitute a major portion of the mass of the full replication complex.     

Regional and temporal changes in the pattern of X-chromosome replication during the early post-implantation development of the female mouse

We have made a detailed study of the X-chromosome replication pattern during the period when X-inactivation is occurring in the mouse embryo. Our observations show unequivocal regionalization of the embryo with respect to the temporal appearance, parental origin and DNA replication pattern of the allocyclic X-chromosome. The switch from isocyclic to allocyclic replication occurs in the embryonic ectoderm at approximately 6 days of development and is random with respect to parental origin of the X-chromosome. In the extra-embryonic tissues, however, the switch to allocyclic replication has apparently occurred prior to 5.3 days of development and almost exclusively involves the paternally-derived X-chromosome. Since these findings are consistent with results obtained in biochemical studies of X-chromosome activity in female embryos, we conclude that there is a close temporal relationship between the cytogenetic and biochemical manifestations of the X-inactivation process. In addition, we have observed a pattern of early paternal X-chromosome replication, transitory in some cases, that is unique to extra-embryonic tissues. These results suggest that there may be some differences in the mechanism by which X-inactivation occurs in the extra-embryonic tissues as compared with the embryonic ectoderm.     

Radiation sensitive mutants of phage T4

Summary A comparative series of phage survival, multiplicity reactivation and radiation stability experiments have been performed with single and double combinations of the four UV-sensitive mutations v, x, y and 1206. These mutations appear to fall into two groups with v (which is associated with excision repair) in one, and x, y and 1206 (associated with what can be non-committally termed replication repair) in the other. The pattern of the results suggest that the extent of the initial shoulder exhibited by multiplication reactivation curves is determined by replication repair, while the slope of the subsequent linear portion depends on excision repair. The development of the characteristic radiation stability observed in Luria-Latarjet experiments is shown to depend to a major extent on replication repair and only in a minor way on excision repair.