S. S. Lamm

Measurements of wet metaphase chromosomes in the scanning transmission X-ray microscope

Radiation damage to Vicia faba chromosome structure, as measured by the mass loss, was determined in the scanning transmission X‐ray microscope for unstained specimens in both the wet and dry states. Dried specimens remain undamaged after either single or multiple images at doses up to 2400 Mrad at wavelengths of 3·15 or 3·64nm. In contrast, wet specimens are damaged irrespective of the imaging protocol. The damage induced by multiple exposures is greater than that seen in a single exposure of the same total dose. Thus, the rate of data collection is greater than or equal to the rate of damage. The damage during multiple exposures of wet chromosomes is influenced by several factors. First, the fixative used influences the extent of radiation damage. Wet chromosomes fixed with glutaraldehyde are more resistant than those fixed with formaldehyde or osmium tetroxide. A second factor is ionic strength. Damage to wet chromosomes increases if the ionic strength decreases below that at which chromatin undergoes a conformational transition. The mass of wet and dry chromosomes is the same, and consequently quantitative measurements can be made on wet specimens. Such measurements give a DNA mass fraction of 39 ± 8% for V. faba chromosomes.

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.

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.

Site of initiation of replication of the ribosomal genes of pea (Pisum sativum) detected by two-dimensional gel electrophoresis

The time course of replication of the 9 kb ribosomal DNA repeats of synchronized root cells of pea was followed by two-dimensional gel electrophoresis. The temporal order of appearance of single-stranded replication intermediates shows that replication begins within the subrepeats located in the intergenic spacer region about 1.5 kb downstream from the 3′ end of the 25S gene. Hybridization to specific probes indicated that this location is identical to that established earlier by a different method.

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.

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.

Detection of replication initiation by a replicon family in DNA of synchronized pea (Pisum sativum) root cells using benzoylated naphthoylated DEAE-cellulose chromatography

Fractionated replicating DNA from pea was obtained from both synchronized cells just starting replication and from carbohydrate-starved cells ending replication. Benzoylated naphthoylated DEAE-cellulose chromatography of pulse-labeled DNA digested with EcoR I gave evidence that a family of replicons initiated replication 45 to 60 min after synchronized cells were released from the G1/S phase boundary. DNA from cells labeled in late S phase, on the other hand, showed no signs of additional replication initiations before entering G2 phase. Results with DNA from both early and late S phase cells comply with a model based on the premise that with short pulses of [3H]-thymidine the isotope is localized at replication forks and that longer pulses label both replication forks and recently replicated segments of double-stranded DNA. The model applies only to DNA subjected to fragmentation before chromatography.The results also suggest that benzoylated naphthoylated DEAE-cellulose chromatography is a useful means to isolate origins and replication forks from synchronized plant cells.

Single-stranded replication intermediates of ribosomal DNA replicons of pea.

Replication of ribosomal DNA replicons in cells of Pisum sativum (cv. Alaska) occurs bidirectionally by displacement loops. Replication is initiated on opposite parental strands and nascent chains are elongated moving 5′‐‐‐‐3′ along each parental template. Replicative intermediates were analyzed by 2‐dimensional agarose gel electrophoresis under neutral‐‐neutral and neutral‐‐alkaline conditions. Southern blots of ribosomal DNA fragments separated in the second dimension under neutral conditions show slowly migrating replicative fragments that hybridize with specific probes in a manner consistent with bidirectional replication. The replicative fragments are present in root meristems with cells in S phase; they are absent or few in number in meristems with cells in G2 phase. The following observations indicate that the replicative fragments are single stranded. The apparent length of the replicative fragments is not the same when separated under neutral and alkaline conditions. They contain rDNA without breaks and they do not exhibit the smaller nascent chains expected from replication bubbles and forks. They are not cleaved by restriction enzymes that require duplex DNA as substrate and they are digestible by S1 nuclease.

Replication of the rRNA and legumin genes in synchronized root cells of pea (Pisum sativum): evidence for transient ECOR I sites in replicating rRNA genes

SummaryThe temporal pattern of replication of the rRNA and legumin genes differs in synchronized pea root cells. The relative number of rRNA genes replicated hourly during the first five hours of S phase ranges between 5 and 10 percent. In late S phase, during hours six through nine, the number of rRNA genes replicated increases reaching a maximum of about 25 percent at the ninth hour. Unlike the rRNA genes, the legumin genes have a wave-like pattern of replication peaking in early S phase at the third hour and again in late S phase at the eighth hour.Replicating rDNA, isolated by benzoylated naphthoylated DEAE-column chromatography, has EcoR I restriction sites that are absent in non-replicating rDNA sequences. The cleavage of these sites is independent of the time of rDNA replication. The transient nature of the EcoR I sites suggests that they exist in a hemimethylated state in parental DNA.The two Hind III repeat-size classes of rDNA of var. Alaska peas are replicated simultaneously as cells progress through S phase. Thus, even if the 9.0 kb and 8.6 kb repeat classes are located on different chromosomes, their temporal order of replication is the same.

Meristematic precursors of vascular parenchyma differentiate from G2 phase after replicating DNA discontinuously

A small population of cells representing 1% or less of those in the root-tip meristem was identified as the precursor of vascular parenchyma and certain root-cap cells in carbohydrate starved cultured pea roots. Autoradiography and cytophotometric measurements of nuclei labeled with [3 H]-thymidine showed that in the absence of carbohydrate the precursor cells replicate their DNA discontinuously accumulating temporarily in late S phase prior to differentiating from the G2 phase. Besides discontinuity of DNA synthesis, the nuclei of precursor cells undergo a change in morphology. The nuclei are shaped round when replicating DNA but later on, while differentiating, they become oblong. This transformation occurs within 72 hr after the starved roots are fed sucrose. Autoradiograms of serial cross-sections of pulse-labeled roots indicate that the cells in late S phase differentiate forming a ring around the stelar cylinder and a ring around the periphery of the root. These observations suggest that during the last half of the final S phase the precursor cells modify their chromosomal DNA and that this modification is associated with the initial steps of differentiation.