C. A. Bjerknes

The size and number of replicon families of chromosomal DNA of Arabidopsis thaliana

Analysis of the replicon properties and the cell cycle ofArabidopsis thaliana (col.) at 22° C were performed via autoradiography of isolated chromosomal DNA fibers and single cells of seedlings. The cell cycle was 8.5 h and G1, S, and G2+1/2 M were 1.7, 2.8, and 4 h respectively. The average single fork rate was 5.8 μm/h and the average replicon size was 24 μm. The data best support the hypothesis that A. thaliana has two replicon families, one with approximately 687 and another with 1888 members per genome and that the families initiate replication in sequence separated by a 36 min interval. Replication of an average single replicon required a little more than 2 h or 74% of S and the 36 min interval between the initiation of replication by the two families constituted 21% of S.

Similar replicon properties of higher plant cells with different S periods and genome sizes

Abstract Root meristematic cells of nine unrelated diploid higher plants with genome sizes that differ 82-fold and with S periods that differ 4-fold have similar replicon sizes and single replication fork rates that average 22 μm and 8 μm/h respectively. The average replicon size of 22 μm is near the 18 μm obtained by extrapolation of measurements, taken from DNA fiber autoradiograms, to zero pulse time with [3H]thymidine. The data suggest that the duration of S is determined by the minimal number of replicon families that function sequentially during DNA replication.

18 μm replication units of chromosomal DNA fibers of differentiated cells of Pea (Pisum sativum)

Explants of stelar and cortical mature pea root tissues were cultured on solid agar synthetic medium for up to 6 d. On the 3rd and 6th day the tissue was pulsed with 3H-thymidine from 15 up to 180 min to label the DNA. Subsequently the nuclei were isolated, lysed, and the DNA spread on microscope slides. The autoradiograms of labeled DNA fibers showed that the average replication fork traveled at 9 μm/h, and that an average of 18 μm of DNA separated activated origins. Nearly identical parameters were obtained from DNA of all callus independent of tissue source and independent of the presence or absence of kinetin in the medium.

Replication termini in the rDNA of synchronized pea root cells (Pisum sativum).

In synchronized root cells of Pisum sativum (cv. Alaska) the joining of nascent replicons is delayed until cells reach the S–G2 boundary or early G2 phase. To determine if the delayed ligation of nascent chains occurs at specific termination sites, we mapped the location of arrested forks in the ribosomal DNA (rDNA) repeats from cells in late S and G2 phases. Two‐dimensional (neutral‐alkaline) agarose electrophoresis and Southern blot hybridization with specific rDNA sequences show that only cells located at the S–G2 boundary and early G2 phase produce alkali‐released rDNA fragments of discrete size. The released fragments are from a particular restriction fragment, demonstrating that the replication forks stop non‐randomly within the rDNA repeats. Indirect end‐labeling with probes homologous to one or the other end of the fork‐containing restriction fragment shows that there are two termination regions, T1 and T2, where forks stop. T1 is located in the non‐transcribed spacer and T2 is at the junction between the non‐transcribed spacer and the 18S gene. The two termini are separated by ˜1.3 kb. Replication forks stop at identical sites in both the 8.6‐ and 9.0‐kb rDNA repeat size classes indicating that these sites are sequence determined.

Nascent replicons are synchronously joined at the end of S phase or during G2 phase in peas.

In synchronized meristematic cells of Pisum sativum replicon-size DNA-fragments ((14--27) . 10(6) daltons) are not joined until the cells achieve a 4C DNA content. The combined use of a pulse-chase labeling protocol, single-cell autoradiography, cytophotometry, and velocity sedimentation in alkaline sucrose gradients showed that, unlike low molecular weight DNA (i.e., Okazaki fragments) which is ligated soon after replication starts, the joining of nascent replicon-size DNA is delayed until G2. In addition, this observation argues for the existence of replicon termini, although the data are insufficient to determine the true nature of these termini, i.e., whether they are coded in the DNA itself or simply arise as a consequence of the convergence of two replicating forks.

Proximity of an ARS consensus sequence to a replication origin of pea (Pisum sativum).

The replication origin (ori-r9) of the 9.0 kb rDNA repeats of pea (Pisum sativum, cv. Alaska) was cloned and found to reside in a 1.5 kb fragment of the non-transcribed spacer region located between the 25 S and 18 S genes. Labeled rDNA rich in replication forks, from cells positioned at the G1/S phase boundary, was used to map ori-r9 by hybridization procedures. Ori-r9 is in a 210-base fragment that is 1.6 kb from the 5′ end of the 18 S gene and about 1.5 kb from the 3′ end of the 25 S gene. The same procedures, using labeled synthetic ARS consensus sequence as a probe, showed than an ARS consensus sequence is located 3′ to ori-r9 in a 710-base fragment. An ARS consensus sequence is, therefore, adjacent to ori-r9 but not coincidental with it.

Cells of pea (Pisum sativum) that differentiate from G2 phase have extrachromosomal DNA.

Velocity sedimentation in an alkaline sucrose gradient of newly replicated chromosomal DNA revealed the presence of extrachromosomal DNA that was not replicated by differentiating cells in the elongation zone. The extrachromosomal DNA had a number average molecular weight of 12 X 10(6) to 15 X 10(6) and a weight average molecular weight of 25 X 10(6), corresponding to about 26 X 10(6) and 50 X 10(6) daltons, respectively, of double-stranded DNA. The molecules were stable, lasting at least 72 h after being formed. Concurrent measurements by velocity sedimentation, autoradiography, and cytophotometry of isolated nuclei indicated that the extrachromosomal molecules were associated with root-tip cells that stopped dividing and differentiated from G2 phase but not with those that stopped dividing and differentiated from G1 phase.

Location of the replication origin in the 9-kb repeat size class of rDNA in pea (Pisum sativum)

The replication origin of the 9-kb rDNA repeat size class of pea (Pisum sativum cv. Alaska) was identified by benzoylated naphthoylated DEAE-cellulose column chromatography and Southern blotting procedures. The origin is located at or near a 0.19-kb EcoR I fragment in the non-transcribed spacer region between the 25S and 18S rRNA genes. Identification of the origin was based on three criteria: (i) an enrichment of the 0.19-kb fragment in replicating rDNA from asynchronously dividing root meristematic cells, (ii) the scarcity of the 0.19-kb fragment in rDNA from non-dividing carbohydrate starved cells, and (iii) a 60-min periodic enrichment of the 0.19-kb fragment in replicating rDNA that temporally coincides with the sequential initiation of replication of replicon families in synchronized pea root cells.

Replicon properties of chromosomal DNA fibers and the duration of DNA synthesis of sunflower root-tip meristem cells at different temperatures

Chromosomal DNA fiber autoradiography was used to examine the replicon properties of root-tip meristem cells of Helianthus annuus intact seedlings grown at temperatures from 10 to 38° C and those of root-tip cells grown in vitro at 23°. The average replicon size was approximately 22 μm and it did not change with temperature nor when the roots were grown in culture. The average fork rate was 6 μm/h at 10° and it rose gradually to 12 μm/h at 38°. The responses of replication fork movement and of the duration of S to temperature were of three types: those in which change in fork rate was primarily (more than 90%) responsible for change in the duration of S, those in which the fork rate remained constant while S increased nearly twofold, and those in which the duration of S increased even though the replication forks were moving faster. The first type of response listed was observed at temperatures from 20 to 35°, the second type listed was observed at 10 to 15°, and the third, was produced at 38°.

Extrachromosomal DNA of pea-root (Pisum sativum) has repeated sequences and ribosomal genes.

SummaryRestriction endonuclease digestion and Southern blotting procedure were used to determine differences between extrachromosomal, nuclear, plastid, and mitochondrial DNAs from meristematic cells of cultured pea roots.Extrachromosomal and nuclear DNA are highly methylated and neither DNA is homologous to plastid or mitochondrial DNA. Hybridization of extrachromosomal DNA to nuclear DNA indicated that extrachromosomal DNA differed quantitatively from total nuclear DNA in repetitive sequences. Cloned rDNA showed that extrachromosomal DNA contains rRNA genes but the hybridization signal indicated that the copy number was less than that expected if the molecules were amplified. These and cytological findings suggest that extrachromosomal DNA is involved in or a product of genomic changes associated with the onset of differentiation by precursor cells of vascular parenchyma and the root cap.