Round Of Synthesis Research Articles

Simultaneous rapid chemical synthesis of over one hundred oligonucleotides on a microscale

An inexpensive, extremely rapid manual method for simultaneous synthesis of large numbers of oligodeoxyribonucleotides on 50 or 150 nanomole scale is described. The oligonucleotides are assembled in parallel by the phosphotriester method on small cellulose paper disks in a simple gas pressure-controlled continuous-flow system. For each addition of a nucleotide the disks are sorted into four sets which are placed in four columns for addition of A, C, G and T, respectively. During one 2-week period, three rounds of synthesis by this method yielded 254 oligomers (8- to 22-mers), many of which were also purified during this time. Using 50 nanomole scale reactions the yields for 17-mers, for example, were in the range of 0.5 O.D.(260) units ( 5 nmol, i.e., 10% yield), an amount sufficient for most purposes. The equipment required is relatively inexpensive and for the most part usually already available in molecular biology laboratories. All chemicals are commercially available and the current reagent cost per oligonucleotide (25 mug, average length 17-mer) is 3 US dollars.

Control of wall band splitting in Streptococcus faecalis.

Computer reconstructions of 659 and 1325 whole mounted, shadowed cells, randomly chosen from cultures of Streptococcus faecalis undergoing balanced growth and doubling in mass every 83 min and 30 min, respectively, were used to analyse the cell cycle. The size limits and duration of phases of the cell cycle were estimated by applying a method previously described by the authors, details of which are given here to allow others to use the method. Deeply constricted cells whose primary septal radius, Rs, was less than or equal to 0.18 micron were considered as belonging to an E-phase ending the cell cycle. The statistical parameters of these E-phase cells were used to calculate the mean and coefficient of variation of dividing cells. These latter values, in turn, predicted the moments of the total population well enough so that the method's assumptions were judged adequately satisfied. Therefore, the method was considered applicable to other phases and sub-phases of the cell cycle of these two cultures. The E-phase cells were further classified as having either 0, 1 or 2 secondary growth zones, allowing us to calculate the percentage of newborn cells without growth zones. In the slow-growing cells, 69% of the cells arose with no growth zone. On the other hand, in more rapidly growing cells 16% of the cells or less arose with no growth zone. Our calculations showed that they could exist without a growth zone for only 2 and 0.1 min, respectively. We also classified cells as possessing a 'birth site' if the volume between the two daughter bands was greater than 0, but less than 0.06 micron3. From the statistical properties of such cells with new growth zones, the mean pole time, W, was estimated. We also estimated W from the size of cells in E-phase. The major conclusion is that the pole time is only slightly greater than the mass doubling time at both growth rates. Since DNA synthesis in S. faecalis takes longer (C = 50 to 52 min) than the mass doubling time in rich medium (30 min), a new round of chromosome replication must be initiated before the old round of synthesis is completed (dichotomous replication). Consequently, wall band splitting and initiation of chromosome replication do not occur simultaneously. It was also concluded that the cell initiates wall band splitting, resulting in pole formation and cell division, when the growth zones cannot function rapidly enough to allow the increase of surface area required to accommodate continuing production of cytoplasm.

Non-enzymatic template-directed synthesis of RNA copolymers.

Cytosine-rich RNA copolymers facilitate the template-directed synthesis of complementary oligomers from mononucleotide 5'-phospho-2-methylimidazolides (Fig. 1). The efficiency of this reaction falls sharply as the ratio of cytosine to non-cytosine in the template is decreased. This is a severe limitation for self-replication because it means that any cytosine-rich polynucleotide that can serve as a good template will produce a cytosine-poor complementary strand that is unable to direct further rounds of synthesis. Studies with low-ratio random copolymer templates have shown that the efficiency can be increased by adjusting initial monomer concentrations and by providing additional activated monomers during later stages of the reaction. The oligomeric reaction products can be studied in detail using high performance liquid chromatography. It is possible to separate oligonucleotides on the basis of chain length and base composition. Thus a wealth of information is available to characterize the distribution of products over the course of the reaction and under a variety of reaction conditions.

Study of Pole Assembly in Bacillus subtilis by Computer Reconstruction of Septal Growth Zones Seen in Central, Longitudinal Thin Sections of Cells

The septal growth of Bacillus subtilis 168/s has been studied by making a number of observations from thin sections of cells from exponentially growing cultures. The process was initiated by the formation of a new cross wall under a preexisting layer of cylindrical wall. An annular notch appeared to cut through the overlying wall and presumably allowed the cross wall to split into two layers of peripheral wall. During this initial notching process, two raised bands of wall material were produced which resembled those previously observed in morphological studies of Streptococcus faecalis. Through an improved fixation technique, it was possible to preserve the bands seen in B. subtilis to the extent that they were used as markers to study the subsequent stages of septal growth. These stages included (i) the continued displacement of the two bands from the cross wall (as the two nascent polar surfaces enlarged and as the diameter of the cross wall decreased), (ii) the closure of the cross wall, and (iii) the final severance of the common cross wall connection between two completed poles. To study this process in a more quantitative manner, three-dimensional reconstructions of the envelope observed between pairs of the raised bands were made from axial thin sections of cells. The process of reconstruction was based on a technique by which x, y coordinates were taken from thin sections and were rotated around the cell's central axis. These reconstructions were used to estimate the surface area or volume of the reconstructed zones or their parts. A round of septal growth was then simulated by arranging 118 reconstructions in order of increasing surface area or volume. The topology of the process was studied by noting how various measurements of septal thickness, length, surface area, and volume varied as a function of increasing septal zone size. This analysis was based on several assumptions, of which three of the most important are: (i) the bands produced by the initial notching process are markers which separate septal from cylindrical wall growth; (ii) a septal zone observed between pairs of bands is made up of two nascent poles and a single cross wall; and (iii) as septal zones develop in terms of relative age they increase in size (volume or surface area) or amount of wall. The data suggested that the S. faecalis model of surface growth (in which polar growth occurs through a regulated constrictive separation and expansion of a cross wall) also seems applicable to the pattern of septal growth observed here for B. subtilis. This was indicated from measurements which showed that increases in the size of nascent polar surfaces were correlated with decreases in cross wall diameter. An explanation of these observations may be that decreases in cross wall diameter were due to a progressive splitting of the cross wall that removed surface from the outer circumference of the cross wall and converted it into new polar surface. Calculations further suggested that if the poles of B. subtilis were made by this model a sizeable and variable increase in surface area of the cross wall would also be required to convert these separating cross wall layers into two curved polar structures. Measurements of wall thickness taken from various locations within septal zones indicated that while the thickness of the polar wall of B. subtilis was constant over its surface, the width of the cross wall varied considerably during a round of synthesis. Again, one of the simplest explanations compatible with these observations and those previously made in S. faecalis is that the B. subtilis cross wall is brought to a constant thickness (possibly by remodeling or precursor addition) before or during separation. Although most observations made from the reconstruction of the septal zones of B. subtilis may fit the S. faecalis model of surface growth, differences in the pattern of septal growth were seen when the two organisms were compared. These have been discussed in terms of differences in the regulation of their respective septal growth sites and basic mechanisms of wall assembly and modification.

Production of DNA bifilarly substituted with bromodeoxyuridine in the first round of synthesis: branch migration during isolation of cellular DNA.

Incubation of human lymphoid cells with bromodeoxyuridine (BrdUrd) for short periods produces three classes of DNA containing analog: DNAHL (hybrid DNA, density approximately equal to 1.75 g/cm3), DNAint (intermediate density DNA, density approximately equal to 1.71 g/cm3), and DNAHH (DNA with both strands containing analog, density approximately equal to 1.80 g/cm3). Preparations of DNAint yield DNAHH after extensive shearing and/or treatment with single strand specific endonuclease. Cross-linking of pulse-labeled (BrdUrd + 3HdT) DNA in cells by treatment with trioxsalen and near UV light before lysis prevents the appearance of DNAHH.Cross-linking after lysis has little effect. A large fraction of DNAHH is obtained after incubation of cells with caffeine. Extraction of DNA at high salt concentration or cross-linking with trioxsalen and near UV light drastically reduced the amount of DNAHH obtained from caffeine-treated cells. We conclude that most DNAHH arises from in vitro branch migration in isolated DNA growing points.

Chromosomal isolabelling caused by three rounds of synthesis in late replicating regions.

Isolabelling only occurs in CHO cells that have been allowed to replicate for more than 2 but less than 3 cell divisions in the presence of BrdU. The isolabelling is confined to late replicating regions of the chromosomes. The staining patterns obtained indicate that BrdU was incorporated three times in these regions and that the isolabelling did not come from the segregation of label in polynemic chromosomes.

Characterization of Simian Virus 40 tsA 58 Transcriptional Intermediates at Restrictive Temperatures: Relationship Between DNA Replication and Transcription

When nuclei from simian virus 40 (SV40)-infected cells are lysed with Sarkosyl and the chromatin is pelleted, the supernatant fluid contains a nucleoprotein complex capable of synthesizing viral RNA (Laub and Aloni, Virology 75:346-354, 1976; Gariglio and Mousset, FEBS Lett. 56:149-155, 1975). The level of activity of the RNA polymerase in the complex increased during infection in parallel with the amount of viral DNA that had been synthesized. If cells infected at 33 degrees C with the SV40 mutant tsA 58 were shifted to the nonpermissive temperature of 40 degrees C at any time between 18 and 48 h postinfection, no viral DNA replication was detected after 45 min and no new rounds of synthesis were initiated after 20 to 30 min. However, after this shift, polymerase activity associated with the nucleo-protein complex did continue to increase for 5 h, at which time it reached a plateau. There was an increase of RNA synthesized from both the early (E) and late (L) SV40 DNA strands, and there was a threefold increase in the ratio of early-to-late RNA species after the shift. In comparable experiments with cells infected with wild-type virions, no increase in polymerase activity occurred because of the temperature change alone. At 33 degrees C, the relative amount of RNA transcribed from the wild-type E-strand was less than tsA 58 at 33 degrees C and did not increase after a shift to 40 degrees C. The tsA 58 transcriptional complexes extracted from cells grown at 33 degrees C sedimented heterogeneously in sucrose gradients, with a peak near 26S. There were no detectable alterations in the sedimentation properties of the complexes when tsA 58-infected cells were shifted to 40 degrees C for 2 h. We conclude that continued synthesis of viral DNA is not an obligatory prerequisite for maintenance of late viral transcription nor is the sedimentation of the transcriptional complex at 26S related to actively replicating DNA molecules serving as templates for transcription. Further, an increase in late transcription can occur under conditions where reinitiation of viral DNA synthesis is prevented. The increase in the synthesis of early and late RNA at the restrictive temperature without concurrent DNA synthesis is discussed in relationship to the function of the A gene product.

Study of cycle of cell wall assembly in Streptococcus faecalis by three-dimensional reconstructions of thin sections of cells.

A new ultrastructural method was used to study rounds of envelope synthesis that occur in Streptococcus faecalis in "growth zones" found between pairs of naturally occurring surface markers. The technique consists of producing three-dimensional reconstructions of these growth zones from the mathematical rotation, about a central axis, of measurements taken from central, longitudinal thin sections of cells. A cycle of exponential-phase envelope growth was then simulated by arranging a series of these reconstructions in increasing order of the amount of peripheral wall surface area or the amount of cell volume that each was calculated to contain. Using this simulated cycle of growth, the geometry of a single growth zone during a round of synthesis was studied. Based on this analysis, a model was developed for the assembly of the cell wall of S. faecalis. The model states that new cell wall surface is synthesized by the regulated flow of essentially two channels of cell wall precursors into a single growth zone. One channel of precursors would be involved in the assembly of a bilayered cross wall that would proceed at a fairly constant rate until the cross wall closes. The second channel of precursors would be involved in the separation of the bilayered cross wall into two segments of peripheral wall. These precursors would intercalate into and thicken the separating layers of the cross wall. The flow of precursors through this channel would be progressively reduced through a cycle. These decreases, when coupled with internal hydrostatic pressure, apparently would result in the enlarging peripheral wall becoming increasingly more curved and would also promote cell division by reducing the total amount of cell wall that must be assembled in order for septation to occur.

Accumulation of the capacity of initiation of deoxyribonucleic acid replication in Escherichia coli.

Several temperature-sensitive initiation mutants of Escherichia coli were examined for the ability to initiate more than one round of replication after being held at nonpermissive temperature for approximately 1.5 generation equivalents. The capacity for initiation was measured by residual synthesis experiments and rate experiments under conditions where protein synthesis and ribonucleic acid synthesis were inhibited. Results of the rate and density transfer experiments suggest that the cells may initiate more than one round of replication in the absence of protein or ribonucleic acid synthesis. This contrasts with the results of the residual synthesis experiments which suggest that, under these conditions, only one round of synthesis is achieved. These findings suggest that the total amount of residual synthesis achieved in the presence of an inhibitor may be both a function of the number of initiation events which occur and the effect of the inhibitor of protein or ribonucleic acid synthesis on chain elongation.

Regulation of four functionally related enzymes during shifts in the developmental program of Dictyostelium discoideum

Partially developed cell aggregates of Dictyostelium discoideum engaged in fruiting body construction can be disaggregated to single cells and redeposited on solid substratum. The cells almost immediately reaggregate, recapitulate within two to three hours a previous course of morphogenesis that might have taken as much as 18 to 20 hours the first time, and then complete the construction of fruiting bodies with approximately normal timing. During particular stages of fruit construction, four functionally related enzymes accumulate, reach characteristic levels of specific activity, and then disappear partly or completely. They are: UDP-glucose pyrophosphorylase, trehalose-6-phosphate synthetase, UDP-galactose-4-epimerase and UDP galactose: polysaccharide galactosyl transferase. The disaggregated cells discontinued the initial rounds of enzyme synthesis, but when permitted to reaggregate and develop further, they accomplished complete second rounds of enzyme accumulation. If disaggregated and redeposited a second time, they accomplished complete third rounds of enzyme synthesis as well. All such additional rounds required new periods of RNA synthesis. A characteristic level of each enzyme activity accumulated during each round of synthesis regardless of the amount previously accumulated. In these additional rounds the temporal relations between transcription and enzyme synthesis were drastically altered.

Control of initiation of DNA synthesis in Escherichia coli B-r.

The pattern of new growing forks in the Escherichia coli genome after a period of inhibition of DNA synthesis has been examined. Synchronous cultures of E. coli B/r were treated with nalidixic acid to stop DNA synthesis for various lengths of time. After removal of the nalidixic acid the location of active growing forks was determined by measuring the reversion rate of a histidine requirement induced by the "growing-point mutagen," nitrosoguanidine. It was found that the ability to initiate a new round of DNA synthesis upon release from the nalidixic acid block was attained at the cell age at which a new round of replication would have been initiated had DNA synthesis not been blocked. However, if the nalidixic acid was removed before the normal initiation age, the initiation of the next round of synthesis was delayed past the normal time, which indicates that initiation of new rounds is not completely independent of DNA synthesis.

Studies on the Synthesis of Deoxyribonucleic Acid: II. Studies with Biologically Active Templates and the Stimulation of Synthesis by Oligonucleotides

Abstract When purified T5-induced deoxyribonucleic acid polymerase is incubated with transforming deoxyribonucleic acid from Bacillus subtilis, there is no detectable loss of transforming ability. By using 5-bromodeoxyuridine triphosphate triphosphate as a density marker and 32P-labeled transforming deoxyribonucleic acid as template, it was shown that after two rounds of synthesis, fractions isolated from the regions of a density gradient equivalent to deoxyribonucleic acid fully substituted with bromuracil and containing no detectable 32P had low but significant transforming activity. When deoxyribonuclease digests are added to incubation mixtures containing single stranded deoxyribonucleic acid and purified T5-induced polymerase, there is a marked stimulation of both the rate and yield. Digests prepared from the same type of deoxyribonucleic acid used as template are more effective than digests of heterologous nucleic acid. From analysis of sedimentation patterns in alkaline sucrose, and from experiments involving gel filtration, it has been found that oligonucleotides present in the digest are incorporated into the product in covalent linkage and probably act as initiators for deoxyribonucleic acid synthesis in this system.