Noncoding RNAs are recognized increasingly as important regulators of fundamental biological processes, such as gene expression and development, in eukaryotes. We report here the identification and functional characterization of the small noncoding human Y RNAs (hY RNAs) as novel factors for chromosomal DNA replication in a human cell-free system. In addition to protein fractions, hY RNAs are essential for the establishment of active chromosomal DNA replication forks in template nuclei isolated from late-G 1 -phase human cells. Specific degradation of hY RNAs leads to the inhibition of semiconservative DNA replication in late-G 1 -phase template nuclei. This inhibition is negated by resupplementation of hY RNAs. All four hY RNAs (hY1, hY3, hY4, and hY5) can functionally substitute for each other in this system. Mutagenesis of hY1 RNA showed that the binding site for Ro60 protein, which is required for Ro RNP assembly, is not essential for DNA replication. Degradation of hY1 RNA in asynchronously proliferating HeLa cells by RNA interference reduced the percentages of cells incorporating bromodeoxyuridine in vivo. These experiments implicate a functional role for hY RNAs in human chromosomal DNA replication.In recent years, it has become apparent that noncoding RNAs are regulating many biological processes, from gene expression and chromatin dynamics to complex developmental programs (reviewed in references 2, 26, and 35). A fundamental process for which an involvement of noncoding RNAs has not been reported to date is the replication of chromosomal DNA in eukaryotes.Chromosomal DNA replication is initiated at the G 1 -to-S phase transition of the cell division cycle. Regulators for this transition have been identified genetically and biochemically as proteins that interact with chromosomal DNA replication origins during G 1 phase, directing the stepwise formation of preinitiation complexes (reviewed in references 1, 13, 25, 33, and 39). These protein factors are functionally conserved through evolution. The six-protein subunit origin recognition complex is assembled on origin DNA, from which Cdc6 and Cdt1 proteins recruit six minichromosome maintenance proteins (MCM2 to MCM7) to form a prereplicative complex, or replication license, in G 1 phase. Conversion of this complex into active replication forks marks the entry into S phase, which is under the temporal and spatial control of S-phase cyclin-dependent kinase Cdk2 and Dbf4-dependent kinase Cdc7. Additional initiation proteins, including MCM10, Cdc45, GINS complex, Mus101 (Dbp11 and Cut5 in yeasts), and replication protein A (RPA) are recruited in this process to unwind origin DNA (1, 25, 39). Active DNA replication forks are established from there by the stepwise recruitment of DNA polymerase ␣/primase and the replicative DNA polymerases ␦ and ε, together with replication factor C and proliferating nuclear antigen (PCNA). This elaborate pathway has been worked out predominantly in the model systems of amphibian egg extracts and unicellular yeasts; later stage...

Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.

SummaryNon-coding Y RNAs are required for the initiation of chromosomal DNA replication in mammalian cells. It is unknown how they perform this function or if they associate with a nuclear structure during DNA replication. Here, we investigate the association of Y RNAs with chromatin and their interaction with replication proteins during DNA replication in a human cell-free system. Our results show that fluorescently labelled Y RNAs associate with unreplicated euchromatin in late G1 phase cell nuclei before the initiation of DNA replication. Following initiation, Y RNAs are displaced locally from nascent and replicated DNA present in replication foci. In intact human cells, a substantial fraction of endogenous Y RNAs are associated with G1 phase nuclei, but not with G2 phase nuclei. Y RNAs interact and colocalise with the origin recognition complex (ORC), the pre-replication complex (pre-RC) protein Cdt1, and other proteins implicated in the initiation of DNA replication. These data support a molecular 'catch and release' mechanism for Y RNA function during the initiation of chromosomal DNA replication, which is consistent with Y RNAs acting as replication licensing factors.

The effects of cyclic shear strain-range, shear strain-rate and temperature on the accumulation of damage during constant and variable shear strain range fatigue tests on a 1 per cent Cr-MeV steel are reported.The hypothesis of linear accumulation of damage and the summation of cyclic fractions to unity are shown to be incorrect for interactions of time, cyclic and environmental processes which are considered in detail.Stage I cracks may be accelerated, retarded or stopped and new cracks nucleated. Nevertheless only 8 per cent of the cumulative damage tests in the high strain fatigue regime gave unsafe linear summation factors less than 0.8. None fell below 0.7.

A labile virus has been identified in white clover in New Zealand. The virus was mechanically transmitted to nine species of herbaceous test plants. No viruslike particles were identified by electron microscopy in ultrathin sections or in negatively stained sap extracts, although in infected Chenopodiurn quinoa there were prominent membraneous inclusion bodies in the cell cytoplasm and membrane-bound structures c. SO nm in diameter associated with the tonoplast in cell vacuoles. Double-stranded RNA species of approximately 6800, 3.500 and 3300 bp were isolated from infected tissues. DsRNA denatured-by boiling was infectious to C. quinoa, but undenatured dsRNA was not infectious. Total nucleic acid preparations from infected leaves were highly infective without boiling, indicating that most of the infectivity was single-stranded RNA. Infectivity was recovered in the poly (A)-faction following oligo (dT)-cellulose chromatography, indicating that the RNA probably lacks a 3' tract of poly (A). The labile white clover virus is tentatively named white clover virus L (WCIVL). IntroductionMost pasture farmlands in New Zealand are mixed swards containing ryegrass and white clover. White clover (Trifolium repens) is grown for its nutritive value and for its ability to fix atmospheric nitrogen. To date, seven viruses have been identified in white clover in New Zealand: alfalfa mosaic virus (AMV; Fry, 19-52), clover yellow vein potyvirus (CYVV; Forster & Musgrave, 1985), lucerne Australian latent nepovirus (LALV; Forster & Morris-Krsinich, 198.5), red clover necrotic mosaic dianthovirus (RCNMV; Morris-Krsinich, Forster . 1990. Infectious transcripts and nuclcotide sequence of cloned cDNA of the potexvirus white clover mosaic virus. Virology 177: 152-15%. Bergh S T, Koziel M G , Huang S-C, Thomas R A, Gilley D P, Siege1 A. 1985. The nucleotide sequence of tobacco rattle virus RNA-2 (CAM strain). Nucleic Acids Research 13:8507-85 18.