Abstract. Five distinct patterns of DNA replication have been identified during S-phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus . In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin . As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase . At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of D URING S-phase of the mammalian cell cycle, the cell must copy the millions ofnucleotides which form its genome with precise fidelity within a complex three dimensional network to maintain genetic integrity (Jackson, 1990). DNA replication initiates at origins ofreplication that occur in clusters or replication units (Cairns, 1966;Huberman and Riggs, 1968 ;Hand, 1978) which are activated at different times throughout S-phase (Taylor, 1960;Hand, 1978) . The timing of replication of some multicopy genes (Stambrook, 1974;D'Andrea et al ., 1983) (reviewed by Balazs et al ., 1974) and some single copy genes (Epner et al ., 1981; Furst etal ., 1981;Braunstein et al ., 1982;Calza et al ., 1984;Goldman et al., 1984;Brown et al., 1987; Hatton et al., 1988a,b, Dhar et al., 1989Taljanidisz et al., 1989) during S-phase has been demonstrated at the biochemical level. Several factors have been shown to influence the timing of replication ofdifferent genes. The transcriptional activity of a gene appears to be important in the timing ofits replication (Goldman et al., 1984; Hatton et al., 1988a,b ;Dhar et al ., 1989;Taljanidisz et al., 1989) . In general, actively transcribed genes seem to replicate early in S-phase . electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin . Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the MU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific a-satellite DNA sequences indicates that the ot-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.have been shown to replicate at other times during S-phase . The location ofa geneona chromosome has also been shown to affect its timing of r...

Background The UK 100,000 Genomes Project is in the process of investigating the role of genome sequencing of patients with undiagnosed rare disease following usual care, and the alignment of research with healthcare implementation in the UK’s national health service. (Other parts of this Project focus on patients with cancer and infection.) Methods We enrolled participants, collected clinical features with human phenotype ontology terms, undertook genome sequencing and applied automated variant prioritization based on virtual gene panels (PanelApp) and phenotypes (Exomiser), alongside identification of novel pathogenic variants through research analysis. We report results on a pilot study of 4660 participants from 2183 families with 161 disorders covering a broad spectrum of rare disease. Results Diagnostic yields varied by family structure and were highest in trios and larger pedigrees. Likely monogenic disorders had much higher diagnostic yields (35%) with intellectual disability, hearing and vision disorders, achieving yields between 40 and 55%. Those with more complex etiologies had an overall 25% yield. Combining research and automated approaches was critical to 14% of diagnoses in which we found etiologic non-coding, structural and mitochondrial genome variants and coding variants poorly covered by exome sequencing. Cohort-wide burden testing across 57,000 genomes enabled discovery of 3 new disease genes and 19 novel associations. Of the genetic diagnoses that we made, 24% had immediate ramifications for the clinical decision-making for the patient or their relatives. Conclusion Our pilot study of genome sequencing in a national health care system demonstrates diagnostic uplift across a range of rare diseases. (Funded by National Institute for Health Research and others)

Abstract. We have examined the functional significance of the organization of pre-mRNA splicing factors in a speckled distribution in the mammalian cell nucleus. Upon microinjection into living cells of oligonucleotides or antibodies that inhibit pre-mRNA splicing in vitro, we observed major changes in the organization of splicing factors in vivo. Interchromatin granule clusters became uniform in shape, decreased in number, and increased in both size and content of splicing factors, as measured by immunofluorescence.These changes were transient and the organization of splicing factors returned to their normal distribution by 24 h following microinjection. Microinjection of these oligonucleotides or antibodies also resulted in a reduction of transcription in vivo, but the oligonucleotides did not inhibit transcription in vitro. Control oligonucleotides did not disrupt splicing or transcription in vivo. We propose that the reorganization of splicing factors we observed is the result of the inhibition of splicing in vivo.T RANSCRIPTION in mammalian cells by RNA polymerase II (Pol II) ~ results mostly in pre-messenger RNAs (pre-mRNAs) that contain intron sequences. These introns must be efficiently removed before the pre-mRNA is transported from the nucleus to the cytoplasm, where it is translated. The removal of introns and the ligation of the remaining exons is catalyzed by small nuclear ribonucleoprotein particles (snRNPs) and a number of non-snRNP protein splicing factors (reviewed in Green, 1991;Guthrie, 1991).The pre-mRNAs produced by Pol II transcription contain conserved sequences at the 5' and 3' splice sites and a conserved region near the 3' splice site in the intron called the branchpoint. These conserved sequences are recognized by snRNPs and non-snRNP splicing factors as they assemble onto the pre-mRNA to form a spliceosome (reviewed in Green, 1991;Guthrie, 1991). The assembly of snRNPs onto pre-mRNA to form a spliceosome occurs in an ordered pathway that culminates in the removal of intron sequences and ligation of exon sequences. A large number of experiments in yeast and mammalian systems have led to a model for the assembly of snRNPs onto pre-mRNA (reviewed in McKeown, 1993). First, the U1 snRNP recognizes sequences at the 5' splice site in the pre-mRNA. Next, the U2 snRNP binds to Address all correspondence to Dr. D. L. Spector, Cold Spring Harbor Laboratory, P.O. Box 100, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Abbreviations used in this paper:AdML, adenovirus major late; m3G, 2,2,7-trimethylguanosine cap; Pol II, RNA polymerase II; snRNP, small nuclear ribonucleoprotein particle. the branchpoint region. Following the binding of the U1 and U2 snRNPs to the pre-mRNA, a pre-assembled U4/U6/U5 particle associates with the bound snRNPs and the premRNA, with U5 interacting with 5' and 3' exon sequences. The U1 association is then destabilized, U5 binds to the 5' end of the intron, and the U4/U6 helix becomes partially unpaired resulting in the association of U6 with U2 forming a U2/U4/U6 complex...

We have developed an in vitro reconstitution system to investigate the role of U5 snRNA in the two catalytic steps of pre-mRNA splicing. The invariant U5 loop 1 is known to interact with exon sequences at the 5' splice site before the first catalytic step. Remarkably, analysis of U5 mutations in vitro reveals that the first transesterification occurs accurately in the absence of the U5 loop. Therefore this sequence is not an essential component of the spliceosomal active site for the first catalytic step. The second catalytic step, although strongly dependent on the presence of a U5 loop to tether the exon 1 splicing intermediate, is surprisingly tolerant of mutations in the invariant sequence.

Although ribosomes are ubiquitously expressed and essential for life, recent data indicate that monogenic causes of ribosomal dysfunction can confer a remarkable degree of specificity in terms of human disease phenotype. Box C/D small nucleolar RNAs (snoRNAs) are evolutionarily conserved non-protein encoding RNAs involved in ribosome biogenesis. Here we show that biallelic mutations in the gene SNORD118, encoding the box C/D snoRNA U8, cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts (LCC), presenting at any age from early childhood to late adulthood. These mutations affect U8 expression, processing and protein binding and thus implicate U8 as essential in cerebral vascular homeostasis.