Temporal order of replication of mouse ribosomal RNA genes during the cell cycle

The timing of replication of mouse ribosomal RNA (rRNA) genes was determined in cultured cells by using 5-bromodeoxyuridine labeling of DNA coupled with synchronization. Two subclasses of rRNA genes were characterized that differ in their temporal order of replication during S-phase. Approximately half of the rDNA repeat units replicated primarily during the first half of S-phase and the other 50% preferentially in the second half. This difference in replication timing was consistently observed for the approximately 400 rDNA repeat units of NIH3T3 fibroblasts, but not for plasmid DNA containing fragments of rRNA genes that had been stably transfected into the genome of these cells. The rDNA fragments inserted into these transfection vectors contained the recently mapped origin of bidirectional replication with or without amplification-promoting sequences, or none of the above. Since the plasmid DNA that was integrated into the host cell genome replicated randomly during S-phase we conclude that the integrated plasmid DNA is either replicated from a chromosomal origin in the neighborhood of its integration site or that inserts are replicated from their own origins and the timing of replication is determined by flanking sequences. Received: 7 July 1997; in revised form: 1 October 1997; Accepted: 1 October 1997     

DNA repair replication by soluble extracts from human lymphoid cell lines

A system is described in which extracts from human cells can perform repair replication on DNA damaged by ultraviolet light or chemical carcinogens. Whole cell extracts from lymphoid cell lines are incubated with damaged plasmid DNA circles at 30 degrees C in the presence of ATP and the four deoxynucleoside triphosphates. Repair synthesis is monitored by the incorporation of alpha-32P-dATP into closed circular plasmid molecules. Analysis of the time course of the reaction suggests that the slowest step in repair is incision, rather than polymerization or ligation. The size of repair patches inserted into ultraviolet-irradiated DNA during a reaction was estimated by substitution of thymidine triphosphate with 5-bromodeoxyuridine triphosphate and sedimentation in alkaline cesium chloride gradients. Patches with heterogeneous sizes of less than 120 bases were observed.     

Profiling conserved microRNA expression in recombinant CHO cell lines using Illumina sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate multiple aspects of cell physiology. The differential expression of conserved miRNAs in two Chinese hamster ovary (CHO) cell lines producing recombinant proteins was examined relative to the CHO-K1 cell line. A total of 190 conserved CHO miRNAs were identified through homology with known human and rodent miRNAs. More than 80% of these miRNAs showed differential expression in recombinant CHO cell lines. The small RNA sequencing data were analyzed in context of the CHO-K1 genome to examine miRNA organization and develop sequence-specific miRNA resources for CHO cells. The identification and characterization of CHO miRNAs will facilitate the use of miRNA tools in cell line engineering efforts to improve product yield and quality.     

Fine mapping of the ribosomal RNA genes of HeLa cell mitochondrial DNA

A fine mapping study of the ribosomal RNA region of HeLa cell mitochondrial DNA has been carried out by using as an approach the protection by hybridized 12 S and 16 S rRNA of the complementary sequences in DNA against digestion with the single strand-specific Aspergillus nuclease S₁ or Escherichia coli exonuclease VII. No inserts have been detected in the main body of the 12 S and 16 S rRNA cistrons, in contrast to the situation described in the large mitochondrial ribosomal RNA gene of some strains of yeast and of Neurospora crassa. Furthermore, it has been possible to assign more precisely than previously the positions of the 5′ and 3′-ends of the 12 S rRNA and 16 S rRNA genes in the HpaII restriction map of HeLa cell mitochondrial DNA.     

DNA replication in synchronized cultured mammalian cells : VI. The temporal replication of ribosomal cistrons in synchronized cell lines

The time of synthesis of ribosomal genes was studied in a haploid (Rana pipiens), and a pseudodiploid (Chinese hamster) cell line. R. pipiens cells were synchronized by amethopterin block. Chinese hamster cells were synchronized by isoleucine starvation followed by hydroxyurea treatment. DNA replicated during three or four selected intervals of the S period was separated from the remainder of the DNA by bromodeoxyuridine density labeling. Purified bromodeoxyuridine substituted DNA was annealed with radioactive-labeled 28S ribosomal RNA (rRNA) to determine when, during different intervals of S, the nuclear DNA homologous to rRNA was replicated. In the R. pipiens and Chinese hamster cell lines, the percent of nuclear DNA homologous to 28S rRNA is highest in the DNA replicated during the first half of the S period.     

The ribosomal RNA genes of Drosophila mitochondrial DNA.

The nucleotide sequence of a segment of the mtDNA molecule of Drosophila yakuba which contains the A+T-rich region and the small and large rRNA genes separated by the tRNAval gene has been determined. The 5' end of the small rRNA gene was located by S1 protection analysis. In contrast to mammalian mtDNA, a tRNA gene was not found at the 5' end of the D. yakuba small rRNA gene. The small and large rRNA genes are 20.7% and 16.7% G+C and contain only 789 and 1326 nucleotides. The 5' regions of the small rRNA gene (371 nucleotides) and of the large rRNA gene (643 nucleotides) are extremely low in G+C (14.6% and 9.5%, respectively) and convincing sequence homologies between these regions and the corresponding regions of mouse mt-rRNA genes were found only for a few short segments. Nevertheless, the entire lengths of both of the D. yakuba mt-rRNA genes can be folded into secondary structures which are remarkably similar to secondary structures proposed for the rRNAs of mouse mtDNA. The replication origin-containing, A+T-rich region (1077 nucleotides; 92.8% A+T), which lies between the tRNAile gene and the small rRNA gene, lacks open reading frames greater than 123 nucleotides.     

DNA double-strand break repair in eukaryotic cell lines having radically different radiosensitivities

TN-368 lepidopteran insect cells are on the order of 100 times more resistant to the lethal effects of ionizing radiation than cultured mammalian cells. DNA double-strand breaks (DSB) are believed by many to be the critical molecular lesion leading to cell death. We have therefore compared the rejoining of DSB in TN-368 and V79 Chinese hamster cells. Cells were irradiated on ice with 137Cs gamma rays at a dose rate of 2.5 Gy/min, incubated for various periods of time, and assayed for DNA DSB using the method of neutral elution. The kinetics of DSB rejoining following a dose of 90.2 Gy is similar for both cell lines with 50% of the rejoining completed in about 12 min. Approximately 83 and 87% of the DSB are rejoined in the TN-368 and V79 cells, respectively, by 1 h postirradiation. However, no further rejoining occurs in the TN-368 cells through at least 6 h postirradiation, whereas approximately 92% of the DSB are rejoined in the V79 cells by 2 h postirradiation. Other studies (from 22.6 to 226 Gy) demonstrate that the amount of rejoining of DSB varies inversely with dose for both cell lines, but this relationship is not as pronounced for the TN-368 cells. In general, these findings do not support the hypothesis that unrejoined DNA DSB represent the critical molecular lesion responsible for cell death.     

Differential repair and recombination of psoralen damaged plasmid DNA in Saccharomyces cerevisiae.

Summary Psoralen photoreaction with DNA produces interstrand crosslinks, which require the activity of excision and recombinational pathways for repair. Yeast replicating plasmids, carrying the HIS3, TRP1, and URA3 genes, were photoreacted with psoralen in vitro and transfected into Saccharomyces cerevisiae cells. Repair was assayed as the relative transformation efficiency. A recombination-deficient rad52 strain was the least efficient in the repair of psoralen-damaged plasmids; excision repair-deficient rad1 and rad3 strains had repair efficiencies intermediate between those of rad52 and RAD cells. The level of repair also depended on the conditions of transformant selection; repair was more efficient in medium lacking tryptophan than in medium from which either histidine or uracil was omitted. The plasmid repair differential between these selective media was greatest in rad1 cells, and depended on RAD52. Plasmid-chromosome recombination was stimulated by psoralen damage, and required RAD52 function. Chromosome to plasmid gene conversion was seen most frequently at the HIS3 locus. In RAD and rad3 cells, the majority of the conversions were associated with plasmid integration, while in rad1 cells most were non-crossover events. Plasmid to chromosome gene conversion was observed most frequently at the TRP1 locus, and was accompanied by plasmid loss.     

Combining high-throughput screening of caspase activity with anti-apoptosis genes for development of robust CHO production cell lines

A set of anti-apoptotic genes were over-expressed, either singly or in combination, in an effort to develop robust Chinese Hamster Ovary host cell lines suitable for manufacturing biotherapeutics. High-throughput screening of caspase 3/7 activity enabled a rapid selection of transfectants with reduced caspase activity relative to the host cell line. Transfectants with reduced caspase 3/7 activity were then tested for improved integrated viable cell count (IVCC), a function of peak viable cell density and longevity. The maximal level of improvement in IVCC could be achieved by over-expression of either single anti-apoptotic genes, e.g., Bcl-2Δ (a mutated variant of Bcl-2) or Bcl-XL, or a combination of two or three anti-apoptotic genes, e.g., E1B-19K, Aven, and XIAPΔ. These cell lines yielded higher transient antibody production and a greater number of stable clones with high antibody yields. In a 5 L fed-batch bioreactor system, BΔ31-1, a stable clone expressing Bcl-2Δ, had a product titer that was 180% as compared to an optimal clone (Con-1) from the control cell line. Although lactate accumulated to more than 5 g/L in the control culture, its concentration was reduced in the anti-apoptotic BΔ31-1 cultures to below 1 g/L, confirming our earlier findings that cells over-expressing anti-apoptotic genes consume the lactate that would otherwise accumulate as a by-product in the culture medium. To the best of our knowledge, this is the first study to use the high throughput caspase screening method to identify CHO host cell lines with superior anti-apoptotic characteristics.     

Recombinational repair and restart of damaged replication forks

Genome duplication necessarily involves the replication of imperfect DNA templates and, if left to their own devices, replication complexes regularly run into problems. The details of how cells overcome these replicative 'hiccups' are beginning to emerge, revealing a complex interplay between DNA replication, recombination and repair that ensures faithful passage of the genetic material from one generation to the next.