The timing of synthesis of proteins required for mitosis in the cell cycle of the sea urchin embryo

The protein synthesis inhibitor emetine was used to establish the times of synthesis of mitotic proteins, whose presence in the cell are essential in the mitotic processes of chromosome condensation, nuclear membrane breakdown, and possibly, chromosome alignment at metaphase. In embryos of the purple sea urchin, Strongylocentrotus purpuratus, protein synthesis required for chromosome condensation and nuclear membrane breakdown occurs between 20 and 35 min after fertilization. In Lytechinus variegatus embryos the time of synthesis of the mitotic proteins is more variable, occurring between 4 and 15 min after fertilization. Furthermore, in both species the mitosis of each cell cycle requires new synthesis of these proteins with the synthesis occurring at the beginning of each cycle. This observation indicates that the mitotic proteins, which are active at prophase and metaphase, lose their activity at late ana- and telophase.     

Rapid cloning and characterization of new chromosome 10 DNA markers by Alu element-mediated PCR

Alu element-mediated polymerase chain reaction is a strategy for rapidly cloning and mapping human DNA markers from mixed DNA sources. A novel primer homologous to the 3′ end of the human Alu repeat element provides the basis for preferential synthesis of human DNA fragments from human/rodent somatic cell hybrid DNA template. This approach has been used to isolate a series of new markers from chromosome 10. The Alu element-mediated PCR probes were regionally assigned on chromosome 10 by hybridization to Southern blots of Alu PCR-synthesized DNA derived from somatic cell hybrid template DNA. Alu element-mediated PCR is generally applicable and makes possible the analysis of complex genomes with a speed and sensitivity that has not been previously possible.     

Imparired DNA synthesis and envelope defect in a mutant of Salmonella typhimurium

Summary S. typhimurium mutants with temperature-sensitive synthesis of DNA have been isolated. One of these mutants,dna-26, has been studied in detail. DNA synthesis is stopped indna-26 without any residual replication after shift to 42° though increase in cell mass is not inhibited. Mutantdna-26 shows increased sensitivity to deoxycholate, to nalidixic acid and rifampicin. This suggests a cell envelope defect. Inhibition of DNA synthesis at 42° can be phenotypically cured indna-26 by 0.25 M NaCl and KCl and 0.44 M sucrose but not by 0.44 M glycerol. This DNA synthesis induced by hypertonic medium proceeds at a slower rate than increase in cell mass but is predominantly due to normal sequential chromosome replication. The position of mutationdna-26 has been approximately mapped in thepurD region of the chromosome.     

Mechanism of conjugation

Summary A new conditional thermosensitive Hfr mutant of Escherichia coli K-12 was isolated. The ts mutation is cotransducible with purE and tsx loci on the E. coli chromosome. Upon temperature shift to 42° C the DNA synthesis and transfer of chromosome is stopped immediately and RNA, protein synthesis in about ten minutes.     

Cloning and Characterization of the Murine GPI Anchor Synthesis GenePigf,a Homologue of the HumanPIGFGene

Many eukaryotic proteins are bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Its core backbone, which is conserved in different organisms, is synthesized in the endoplasmic reticulum by the sequential addition of glycan components to phosphatidylinositol. One of the human GPI synthesis genes,PIGF(phosphatidylinositol glycan complementation class F), which is involved late in the synthesis pathway, has been cloned. In this study, we isolated complementary and genomic clones ofPigf,a murine counterpart ofPIGF. Pigfencodes a 219 amino acid protein that complements a class F mutation. ThePigfgene consists of six exons spanning 30 kb and was mapped to chromosome 17 at 17E4–E5. These features are very similar toPIGF,thus demonstrating the interspecies conservation of structure, function, gene organization, and genetic locus between these GPI synthesis genes. The results also extend a region in murine distal chromosome 17 that is syntenic to human chromosome 2p16–p22.     

Multiple nucleoli and enhanced nucleolar activity in the nurse cells of the insect ovary

Origin and function of the nucleolar apparatus in nurse cell nuclei of Calliphora erythrocephala have been investigated by cytological and autoradiographic methods in some inbred lines of laboratory blowflies with well paired polytene chromosomes in the nurse cell nuclei. Besides the nucleolus at chromosome VI large numbers of multiple free nucleoli develop in the highly polyploidized nurse cells during oocyte growth. The nucleoli incorporate H³-uridine in a considerable amount producing a homogeneous and RNase-sensitive label even after short time incubation. Their capacity of RNA synthesis is independent of their spatial relationships to other nuclear components. DNA particles in the nucleoli could be identified by the Feulgen reaction and by fluorescence staining with N,N'-diethylpseudoisocya-ninchloride, which also demonstrates the existence of own templates for autonomous RNA synthesis. There are indications that the nucleolus' own DNA is produced by gene amplification beyond the level of endomitotic polyploidization in the nurse cell nuclei. A quantitative estimation of grain density in the autoradiograms shows a rigorous shift of rRNA synthesis: at least 72% of all newly synthesized macromolecular RNA in nurse cell nuclei as contrasted to 13 % of nucleolar RNA synthesis in bristle forming cells with a similar degree of polyploidy. It seems that the nurse cell nuclei of Calliphora in addition to polyploidization increase their template capacity for synthesizing rRNA in a similar way as has repeatedly been demonstrated for Amphibia. Cytological and physiological peculiarities of the nurse cells have been discussed from the viewpoint of their functional similarity to the oocyte nucleus.     

Establishment of a cell-free translation system from rice callus extracts

ABSTRACT

Eukaryotic in vitro translation systems require large numbers of protein and RNA components and thereby rely on the use of cell extracts. Here we established a new in vitro translation system based on rice callus extract (RCE). We confirmed that RCE maintains its initial activity even after five freeze-thaw cycles and that the optimum temperature for translation is around 20°C. We demonstrated that the RCE system allows the synthesis of hERG, a large membrane protein, in the presence of liposomes. We also showed that the introduction of a bicistronic mRNA based on 2A peptide to RCE allowed the production of two distinct proteins from a single mRNA. Our new method thus facilitates laboratory-scale production of cell extracts, making it a useful tool for the in vitro synthesis of proteins for biochemical studies.     

Timing of mitochondrial DNA synthesis during meiosis in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) synthesis was examined during meiosis in Saccharomyces cerevisiae using an aneuploid strain disomic (n + 1) for chromosome III. The aneuploid has the advantage over true diploid strains in that it completes early meiotic events, including premeiotic chromosome replication, but does not form mature ascospores. Thus, differential extraction problems, resulting from the simultaneous presence of both unsporulated cells and spores in the population, are eliminated. The kinetic of mtDNA synthesis was monitored by determining the actual mtDNA content of cells following analytical CsCl centrifugation of cell extracts. MtDNA synthesis started soon after the cells were placed in sporulation medium and continued at an approximately constant rate until 24 h, resulting in slightly more than a doubling of the mitochondrial DNA content per cell. [¹⁴C]uracil was incorporated into mtDNA during the entire developmental period. Extensive preferential labeling of mtDNA occurred between 24 and 50 h, when no net DNA synthesis was observed.     

Chronology and pattern of replication in the bone marrow chromosomes of Gallus domesticus

The duration of the cell generation, the chronology, and the pattern of chromosome duplication was studied in the bone marrow of Gallus domesticus. The duration of the phases of the cell cycle is: cell generation 17.5 hours, S period 9 hours. G₂ period plus prophase stage 2.5 hours, G₁ period 6 hours. Chromosome replication begins at many sites. During middle S it extends to the whole complement and finally finishes in small, late replicating regions of the macrochromosomes. Interchromosomal asynchrony of duplication at the initiation or at the end of the S period was not observed. Z-chromosomes begin and finish DNA synthesis synchronously with the other macrochromosomes. The W-chromosome in females is the last microchromosome to finish replication. However it ends DNA synthesis at about the same time as the macrochromosomes. Similarities and differences between chromosome replication in Aves and Mammalia are considered.     

Control of membrane protein synthesis in bacillus subtilis

In synchronous cultures of Bacillus subtilis 168/S grown on succinate as a sole carbon source (mean generation time 115 min), chromosome initiation occurs at the beginning of the cell cycle but the rate of membrane protein synthesis doubles in mid-cycle more or less coincident with nuclear segregation. In glucose-grown cultures, the doubling in rate of membrane protein synthesis occurs at about the same time as nuclear segregation and DNA initiation at the beginning of the cycle. Control of the rate of membrane synthesis by the chromosome has been demonstrated by inhibiting DNA synthesis using thymine starvation and showing that membrane protein synthesis continues at a constant rate, whereas the rate of cytoplasmic protein synthesis almost doubles.I suggest that the replication of a region at or close to the chromosome terminus is required to allow the doubling in rate of membrane synthesis.     

Septation and chromosome segregation during sporulation in Bacillus subtilis

The early stages of sporulation in Bacillus subtilis incorporate a modified, highly asymmetric cell division. It is now clear that most, if not all, of the components of the vegetative division machinery are used also for asymmetric division. However, the machinery for chromosome segregation may differ significantly between vegetative growth and sporulation. Several interesting checkpoint mechanisms couple cell cycle events to gene expression early in sporulation. This review summarises important advances in the understanding of chromosome segregation and cell division at the onset of sporulation in B.subtilis in the past three years.     

Macromolecular synthesis in chromosome initiation mutants of Bacillus subtilis.

Summary Inactivation of the dna B or dna D gene product in Bacillus subtilis stimulates RNA and protein synthesis. Strains containing ts dna B and D mutations have been constructed by introducing the mutations by transformation into a thymine requiring strain which does not lyse during thymine starvation. The consequences of inactivation of these gene products have been assessed by comparing RNA and protein synthesis during thymine starvation at the restrictive temperature with the recipient strain. In the ts ⁺ strain, there is a doubling in rate of RNA synthesis during thymine starvation. In the ts dna B and D mutations at the restrictive temperature the rate of RNA synthesis increases four fold. By preincubating the mutants in the absence of thymine for one generation at the permissive temperature the two fold increase in rate of RNA synthesis associated with inactivation of the initiation complex can be demonstrated under conditions where the ts ⁺ strain shows a decrease in rate of RNA synthesis. The rate of protein synthesis observed largely reflects the rate of RNA synthesis in all strains. Completion of the chromosome at the restictive temperature has no significant effect on the rate of RNA synthesis. It is suggested that inactivation of the initiation complex after chromosome initiation could play an important role in control of RNA synthesis in relation to the cell cycle.     

The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis

Summary The oligopeptide permease is encoded by at least four genes which are transcribed as a single operon. We cloned and characterized this operon from Salmonella typhimurium, as well as the flanking genes, tonB, ana and a new gene, cwd, which affects cell wall synthesis. We correlated the physical map of opp DNA with a detailed genetic map of the opp operon and the individual opp genes were accurately located with respect to various restriction sites by Southern blotting. The region of the chromosome near opp was found to be highly unstable with deletions arising at a high frequency. The operon also contains hot-spots for IS1 and IS5 insertions.     

Staphylococcus aureus requires at least one FtsK/SpoIIIE protein for correct chromosome segregation

Faithful coordination between bacterial cell division and chromosome segregation in rod‐shaped bacteria, such as Escherichia coli and Bacillus subtilis, is dependent on the DNA translocase activity of FtsK/SpoIIIE proteins, which move DNA away from the division site before cytokinesis is completed. However, the role of these proteins in chromosome partitioning has not been well studied in spherical bacteria. Here, it was shown that the two Staphylococcus aureus FtsK/SpoIIIE homologues, SpoIIIE and FtsK, operate in independent pathways to ensure correct chromosome management during cell division. SpoIIIE forms foci at the centre of the closing septum in at least 50% of the cells that are close to complete septum synthesis. FtsK is a multifunctional septal protein with a C‐terminal DNA translocase domain that is not required for correct chromosome management in the presence of SpoIIIE. However, lack of both SpoIIIE and FtsK causes severe nucleoid segregation and morphological defects, showing that the two proteins have partially redundant roles in S. aureus.     

The diurnal pattern of protein and photopigment synthesis in the retina of the crayfish,Procambarus clarkii

Summary The interrelationship between the diurnal cycle of membrane loss and synthesis of new rhabdom components remains a key element in forming a complete picture of the turnover of photopigment-containing membrane in the crayfish photoreceptor cell. In order to examine this aspect of the turnover process, the diurnal pattern of photopigment synthesis was examined using an in vitro incubation system for incorporation of³H-leucine into photoreceptor protein. The incorporation of³H-leucine into total protein and photopigment specifically was measured in photoreceptors isolated from incubated retinas. The results indicate that for both total protein and photopigment there is no significant variation in the rate of synthesis during the 12-12 light-dark cycle. These data combined with earlier data on diurnal membrane loss from the rhabdom suggest that light-stimulated rhabdom membrane loss is superimposed on a diurnally constant level of synthesis and assembly of new rhabdom constituents.Abbreviations dpm disintegration per minute - LRB lysosome related body - TCA trichloroacetic acid     

Differences in the degree of inhibition of NDP reductase by chemical inactivation and by the thermosensitive mutation <Emphasis Type="Italic">nrdA101</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> suggest an effect on chromosome segregation

NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdA _ts mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the non-permissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS. In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation machinery through the maintenance of the proposed replication hyperstructure.     

Late replication patterns in adult and embryonic mice carrying Searle's X-autosome translocation

Bromodeoxyuridine-dye technique analysis of X chromosome DNA synthesis in female adult and fetal mice carrying the balanced form of the T(X; 16) 16H translocation demonstrated that the structurally normal X chromosome was late replicating (and hence presumably inactive) in 93% of the adult cells and 99% of the 9-day embryo cells, with the X¹⁶ chromosome late replicating in the remaining cells. We conclude from these results that in T16H/+ females either there is preferential inactivation of the normal X chromosome or that, if inactivation is random, cell selection takes place before 9 days of development. Two 9-day female embryos with an unbalanced karyotype were also studied; both had two late-replicating chromosomes in most of their cells, one being the chromosome 16^X, the other a normal X chromosome. These results, together with the presence of a late-replicating X¹⁶ chromosome in T16H/+ adult and fetal mice, support the concept that more than one inactivation center is present on the X chromosome of the mouse because the X¹⁶ and the 16^x chromosomes can be late replicating.     

Discovery of a bifunctional cardiolipin/phosphatidylethanolamine synthase in bacteria

Phosphatidylethanolamine (PE) and cardiolipin (CL) are major components of bacterial and eukaryotic membranes. In bacteria, synthesis of PE usually occurs via decarboxylation of phosphatidylserine (PS) by PS decarboxylases (Psd). CL is produced by various CL synthases (Cls). Membranes of the plant pathogen Xanthomonas campestris predominantly contain PE, phosphatidylglycerol (PG) and CL. The X. campestris genome encodes one Psd and six putative CLs. Deletion of psd resulted in loss of PE and accumulation of PS. The mutant was severely affected in growth and cell size. PE synthesis, growth and cell division were partially restored when cells were supplied with ethanolamine (EA) suggesting a previously unknown PE synthase activity. Via mutagenesis, we identified a Cls enzyme (Xc_0186) responsible for EA‐dependent PE biosynthesis. Xanthomonas lacking xc_0186 not only lost its ability to utilize EA for PE synthesis but also produced less CL suggesting a bifunctional enzyme. Recombinant Xc_0186 in E. coli and in cell‐free extracts uses cytidine diphosphate diacylglycerol (CDP‐DAG) and PG for CL synthesis. It is also able to use CDP‐DAG and EA for PE synthesis. Owing to its dual function in CL and PE production, we consider Xc_0186 the founding member of a new class of enzymes called CL/PE synthase (CL/PEs).