Kinetic heterogeneity of the replication of genetically inactive X chromosome in human cells

Kinetics of DNA replication in genetically non-active X chromosome was studied in peripheral lymphocytes and skin fibroblasts from four phenotypically normal women and one fetus using BrdU 33258 Hoechst-Giemsa techniques. The localization of the earliest replicated chromosomal segment was shown to be unstable, varying from cell to cell in both lymphocytes and fibroblasts of all persons examined. Several variants of replication sequence in the X chromosome were found in both types of cells. The variants revealed were classified, according to Willard. The statistically significant differences in replication sequence were found between blood lymphocytes and skin fibroblasts in two individuals. The problem of tissue specificity in replication kinetics of the genetically non-active X chromosome is discussed.     

The coupling of epigenome replication with DNA replication

In multicellular organisms, each cell contains the same DNA sequence, but with different epigenetic information that determines the cell specificity. Semi-conservative DNA replication faithfully copies the parental nucleotide sequence into two DNA daughter strands during each cell cycle. At the same time, epigenetic marks such as DNA methylation and histone modifications are either precisely transmitted to the daughter cells or dynamically changed during S-phase. Recent studies indicate that in each cell cycle, many DNA replication related proteins are involved in not only genomic but also epigenomic replication. Histone modification proteins, chromatin remodeling proteins, histone variants, and RNAs participate in the epigenomic replication during S-phase. As a consequence, epigenome replication is closely linked with DNA replication during S-phase.     

Replication variants of the human inactive X chromosome

Replication variants of the inactive X chromosome were investigated in lymphocytes from six donors by means of terminal BrdU or thymidine incorporation. There were interindividual differences in the incidence of particular variants. In endoreduplicated and tetraploid cells both allocyclic X chromosomes showed the same replication sequence. The Xp22 band of the allocyclic X chromosome seemed to replicate later than the homologous material in some cells. Initiation time of DNA synthesis within the inactive X chromosome was found to be stable; termination time, however, varied greatly relative to the other chromosomes. Early completion of replication within the heterochromatic X chromosome could be demonstrated preferentially for the Xq25–27 terminal sequence, but other variants expressed the phenomenon also. A variable replication rate of the inactive X chromosome is believed to be responsible for its asynchronous, independent replication. The biological significance of the phenomenon is discussed with respect to cell differentiation.     

Naturally arising recombinants that are missing portions of the simian virus 40 regulatory region.

When simian virus 40 (SV40) is serially passaged at high multiplicity, a heterogeneous collection of naturally arising variants is generated. Those which are the most abundant presumably have a selective replicative advantage over other defective and wild-type helper SV40s. Two such naturally arising host-substituted variants of SV40 have been characterized in terms of complete nucleotide sequence determination. Evolutionary variant ev-1101 (previously isolated by Lee et al., Virology 66:53-69, 1975) is from undiluted serial passage 13, whereas ev-2101 is newly isolated from undiluted serial passage 6 of an independently-derived evolutionary series. Both variants contain a five-times tandemly repeated segment of DNA consisting of viral Hin C and Hin A sequences that have recombined with a segment of host DNA that is not highly reiterated in the monkey genome. The monkey segment differs in the two variants as does the size of the viral segment retained. In two additional host-substituted variants, ev-1102 (previously isolated from serial passage 20 by Brockman et al., Virology 54:384-397, 1973) and ev-1108 (newly isolated from serial passage 40), the SV40 sequences derived from the replication origin are present as inverted repetitions. The inverted repeat regions of these two variants have been analyzed at the nucleotide sequence level and are compared with SV40 variant ev-1104 from passage 45 (previously characterized by Gutai and Nathans, J. Mol. Biol. 126:259-274, 1978). The viral segment containing the regulatory signals for replication and viral gene expression is considerably shortened in later serial passages as demonstrated by these five variants. It is of interest that the variants presumably arose due to their enhanced replication efficiency, yet are missing some of the sequence elements implicated in the regulation of replication. Furthermore, a comparison of the structure of the replication origin regions indicates that additional changes occur in the SV40 regulatory region with continued undiluted serial passage.     

Pausing of simian virus 40 DNA replication fork movement in vivo by (dG-dA)n·(dT-dC)n tracts

We have earlier demonstrated that a sequence bordering an amplified DNA segment and containing the unusual sequence (dG-dA)_n·(dT-dC)_n could slow replication fork movement [Rao et al., Nucleic Acids Res. 16 (1988) 8077–8094]. This was done by cloning the unusual sequence in simian virus 40 (SV40) and following the rate of incorporation of radioactively labeled nucleotides into various regions of the SV40 genome. In the present study, we have analyzed the in vivo replicative intermediates of the SV40 variants containing the unusual sequences by a two-dimensional gel electrophoretic technique. We found that the technique can be used to detect minor pauses in DNA replication and demonstrated that the cloned (dG-dA)_n·(dT-dC)_n tracts, that can potentially adopt triplex structures, could slow DNA replication fork movement. A sequence from the plasmid pUC18 did not slow fork movement when cloned in the same locus of SV40. The pause caused by the alternating guanosine-adenosine repeats might play a role in the regulation of DNA replication and gene amplification in vivo.     

Enhancer dependence of polyomavirus persistence in mouse kidneys.

We previously showed that alterations in the enhancer sequence of polyomavirus DNA can alter both the level and the organ specificity of viral DNA replication during the acute phase of infection of newborn mice (R. Rochford, B. A. Campbell, and L. P. Villarreal, J. Virol. 64:476-485, 1990). In this study, we examined whether these enhancer sequence alterations can also affect polyomavirus replication during the persistent phase of infection in vivo. After infection of newborn mice with a mixture of three enhancer variants, the individual organs could select for enhancer-specific viral DNA replication during both the acute and the persistent phases of infection. Contrary to expectations, the ability of some variants to establish a high-level acute infection in some organs (e.g., the pancreas) did not necessarily lead to a persistent infection in those organs. Thus, enhancers can affect acute and persistent infections differently. In addition, some enhancer variants tended to establish a high-level persistent infection in the kidneys immediately following an acute infection; however, in all cases considerable histopathology was associated with these elevated long-term infections, and these mice were always runty. A persistent infection in the kidneys thus appears able to exist in two distinguishable states, a high-level pathological state and a low-level nonpathological state, which can be affected by the viral enhancer sequence.     

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.     

Evolutionarily selected replication origins: functional aspects and structural organization.

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.     

Replication of human chromosomes in human-mouse hybrids: evidence that the timing of DNA synthesis is determined independently in each human chromosome.

The terminal phase of DNA replication was studied by autoradiography in hybrids between human lymphocytes and mouse fibroblasts. The hybrids contained on the average only 11 human chromosomes. It was found that the sequence of terminal DNA replication for the human chromosomes in the hybrids was the same as the sequence of terminal replication for the corresponding chromosomes in the human lymphocytes. Furthermore, it was shown that the maintenance of the normal terminal replication sequence of the human chromosomes in the hybrids was not dependent on the presence of any specific human chromosome. The results suggest that the timing of terminal DNA replication is determined independently in each human chromosome.     

Recombination in simian virus 40-infected cells. Structure of naturally arising variants ev-2114, ev-2102, and ev-1110

The complete nucleotide sequence has been determined for three newly cloned evolutionary variants from two different independently generated evolutionary series (1100 and 2100 series) of simian virus 40 (SV40). These naturally arising variants, designated ev-1110, ev-2102, and ev-2114, were isolated after five high multiplicity serial passages. The structure of the variants consists of a monomeric unit tandemly repeated four times (ev-2102 and ev-2114) or six times (ev-1110) in the variant genome; the variants have four or six copies, respectively, of the viral origin signal for DNA replication. The DNA content in the three variants is vastly different in that the genome of variant ev-2114 contains only rearranged viral sequences, while variant ev-2102 contains a substitution with monkey DNA sequences consisting of a nearly complete dimeric unit of Alu family sequences as well as less repetitive sequences and variant ev-1110 contains monkey DNA sequences derived solely from repetitive alpha-component DNA. Recombination events, cellular sequences, and structural features of these and other naturally arising SV40 variants are compared.     

Changes in the H-1 histone complement during myogenesis. II. Regulation by differential coupling of H-1 variant mRNA accumulation to DNA replication

We have shown that changes in proportions of the four chicken H-1's during in vitro myogenesis are primarily the result of differential coupling of their synthesis to DNA replication (see the previous paper). We show here that the four major chicken H-1's are encoded by distinct mRNAs which specify primary amino acid sequence variants. Accumulation of the H-1-variant mRNAs is coupled to DNA replication to different extents. The level of mRNA encoding H-1c (the H-1 variant that increases relative to the other H-1's in nondividing muscle cells) is completely uncoupled. In contrast, the level of mRNAs encoding H-1's a, b, and d (which have levels that decrease in nondividing muscle cells) are more tightly coupled. Polyadenylation is not involved in uncoupling H-1c mRNA accumulation from DNA replication.     

Interference between M13 and oriM13 plasmids is mediated by a replication enhancer sequence near the viral strand origin

The origin of replication for the viral strand of bacteriophage M13 DNA is contained within a 507 base-pair intergenic region of the phage chromosome. The viral strand origin is defined as the specific site at which the M13 gene II protein nicks the duplex replicative form of M13 DNA to initiate rolling-circle synthesis of progeny viral DNA. Using in vitro techniques we have constructed deletion mutations in M13 DNA at the unique AvaI site which is located 45 nucleotides away on the 3' side of the gene II protein nicking site. This deletion analysis has identified a sequence near the viral strand origin that is required for efficient replication of the M13 genome. We refer to this part of the intergenic region as a "replication enhancer" sequence. We have also studied the function of this sequence in chimeric pBR322-M13 plasmids and found that plasmids carrying both the viral strand origin and the replication enhancer sequence interfere with M13 phage replication. Based upon these findings we propose a model for the mechanism of action of the replication enhancer sequence involving binding of the M13 gene II protein.     

Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.     

Cellular transformation by adenovirus type 5 is influenced by the viral DNA polymerase.

Early region 2B (E2B) of the group C adenoviruses encodes a number of proteins, including the 140-kilodalton DNA polymerase, which plays a role in the initiation of viral DNA replication. Temperature-sensitive (ts) mutants with mutations mapping to E2B are conditionally defective for both DNA replication in human cells and transformation of rat cells. Nucleotide sequence analysis shows that the E2B mutant ts36 possesses a single point mutation specific to the viral DNA polymerase; this transition of a C to a T at position 7623 changes leucine residue 249 in the polymerase to a phenylalanine. A wild-type (ts+) revertant possesses a codon specifying the original leucine at position 249. Phenotypic analysis of revertant and wild-type viruses derived by marker rescue from ts36 shows that these variants are wild type for both viral DNA replication and transformation. Thus, the single point mutation in the polymerase gene of ts36 is responsible for both defects.     

The replication behavior of Saccharomyces cerevisiae DNA in human cells

We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells. Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells. Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA. This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells. To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells. We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells. This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.by J.A. Huberman