Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex

Summary Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells ofZea mays — a species in which endomitosis occurs — andTulipa kaufmanniana — in which this process does not occur. InTulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. InZea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with³H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone.³H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments inZea mays and decreases slightly inTulipa kaufmanniana.It is argued that the differences between the incorporation of³H uridine and that or³H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

Underrepresentation of nuclear DNA sequences in differentiating root cells ofVicia faba

Summary Data from cytological and biochemical analyses are presented on the behaviour of nuclear DNA during the differentiation ofVicia faba root cells. From the terminal 10.5 mm of the root, three segments were dissected by cutting transversely the root at 0.5 (segments I, meristematic cells), 4.5 (segment II, both meristematic and differentiating cells) and 10.5 mm (segment III, differentiating and/or differentiated cells) from the tip. Cytophotometric determinations of Feulgen absorptions in cell nuclei of the three root segments, carried out in preparations subjected to hydrolysis curve, revealed a lesser amount of nuclear DNA in differentiating cells when compared to the meristematic ones. Analyses of the reassociation kinetics of the DNAs extracted separately from the three root segments showed differences in the frequency of highly repeated sequences, which amount to 11.0, 8.6, and 7.5% of the total DNA in segments I, II, and III, respectively. Density gradient centrifugations in CsCl revealed a lighter satellite in the DNAs from segments I and II (ca. 5.4 and 3.8% of the total DNA, respectively) and no satellite in the DNA from segment III. It is suggested that underrepresentation of repeated DNA sequences occurs in differentiating cells and is a determining factor of the discharge of a cell from the mitotic activity.     

Expression in different populations of cells of the root meristem is controlled by different domains of the rolB promoter

Selective gene expression in different populations of cells of the root apex of transgenic tobacco could be evidenced by means of GUS constructs with deletions of the rolB promoter and fusions with the CaMV 35S minimal promoter. Five regulatory regions have been broadly identified in the rolB 5 non-coding region. The presence of all five domains (A to E) directs gene expression in the root cap, in the protoderm and in the different tissues within the root meristematic region: the dermatocalyptrogen, the cortex and the vascular cylinder. Deletion of domain A (–623 to –471) selectively suppresses expression in non-meristematic cells, i.e. the root cap and the protoderm. Deletion of either domain B (–341 to –306) or E (80 bp around the TATA box) causes loss of expression in all cells of the root apex: constructs C+D+E, B+C+D, B+C are inactive. Domain D (70 bp around the CAAT box) is necessary for gene expression in the dermatogen and in meristematic cells of the cortex but not in the innermost meristematic layer: construct B+C+E is active only in vascular meristematic cells. Domain C (–216 to –158) seems to have a double regulatory role as construct B+E is no longer expressed in meristematic cells of the vascular cylinder but is very active in the protoderm. Constructs allowing gene expression in meristematic cells are also inducible by auxin in leaf protoplasts, while activation of the regulatory elements necessary for gene expression in the non-meristematic cells of the root apex do not seem to depend upon the hormone. The connection between auxin induction and meristematic expression is discussed.     

Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex.

Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells of Zea mays - a species in which endomitosis occurs - and Tulipa kaufmanniana - in which this process does not occur. In Tulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. In Zea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with 3H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone. 3H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments in Zea mays and decreases slightly in Tulipa kaufmanniana. It is argued that the differences between the incorporation of 3H uridine and that or 3H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

Auxin transport in roots

Summary The reasons underlying the initial increase and subsequent decrease in the amount of radioactivity in the receiver block at the apical end of a Zea root segment supplied with a basal donor block containing labelled IAA have been investigated.The phenomenon was observed in segments supplied with IAA-1-¹⁴C, IAA-2-¹⁴C and IAA-5-³H. An acropetal polarity in the movement of radioactivity into the receiver blocks was observed using donor blocks containing IAA-5-³H at concentrations as low as 10^-10M.The decrease in the amount of radioactivity in the receiver block begins after 6–8 h of transport at 25° C, and is unaffected by renewal of the donor block every 2 h, or the presence of 2% sucrose in the donor and receiver blocks.The net export of radioactivity into the receiver block at the apical end of the segment virtually ceases after 6–8 h of transport at 25° C, and is not prolonged by the presence of 2% sucrose in the donor and receiver blocks. At 10° C, net export of radioactivity continues for at least the first 50 h of transport, and the amount of radioactivity in a continuously applied receiver block continues to increase over this period.Receiver blocks removed from the apical end of segments after 8 h of transport and placed on planchettes show little or no decrease in the amount of radioactivity they contain as a function of time, in marked contrast to those left in contact with the segment.There is a marked, and metabolically dependent, resorption of radioactivity from the receiver block at the apical end of the segment after about 8 h of transport at 25° C; most of the resorbed radioactivity remains in the apical 2–4 mm of the segment.There is a loss of radioactive CO₂ from segments supplied with a basal donor block containing 10^-6M IAA-1-¹⁴C at 25° C, the emission beginning after 6–8 h of transport. Segments similarly supplied with 10^-6M IAA-2-¹⁴C did not begin to lose radioactive CO₂ until after about 10–12 h of transport.The ability of the segments to transport radioactivity in a polar manner declines with time after they are excised from the root, regardless of whether their cut ends are kept in the intervening period in contact with plain agar blocks, or ones containing unlabelled IAA at 10^-6M. By the 6th h after excision at 25° C no transport of radioactivity through the segments and into the receiver blocks could be detected in either the aropetal or basipetal direction.The decrease in radioactivity in the receiver block after transport periods of 6–8 h at 25° C is therefore due to (1) a cessation of net export of radioactivity into the block, and (2) the onset of a metabolically-dependent, net resorption of radioactivity. At this time substantial amounts of radioactive CO₂ begin to be evolved from segments supplied with IAA-1-¹⁴C, whereas with IAA-2-¹⁴C radioactive CO₂ is not evolved for a further 4–6 h.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Distribution and Toxic Effects of Cadmium and Lead on Maize Roots

Two-day-old seedlings of maize (Zea mays L.) were incubated on Cd and Pb nitrate solutions at the concentrations that inhibited root growth approximately by 50% after two-day-long incubation (LC₅₀; 10^–4 and 10^–3 M, respectively) or completely terminated growth of the primary root after one-day-long incubation (LC; 5 × 10^–4 and 10^–2 M, respectively). Cd and Pb contents were measured using an anodic inversion voltammetric technique in a flow injection system and a histochemical method. At LC₅₀, Cd and Pb were discerned, by histochemical techniques, in all root apical tissues, whereas in the root hair zone, the heavy metals were primarily accumulated in the apoplast of the rhizodermis and cortex and to a lesser extent, in the vascular tissues and parenchyma cells surrounding the metaxylem vessels. Insignificant accumulation of Cd and Pb in the pericycle probably explains why root branching was tolerant to these agents. At LC, Cd and Pb were found in the apoplast of all root tissues, in accordance with the practically complete inhibition of root growth and branching. Irrespectively of Cd and Pb concentrations in the external solution, the metal contents in the root apex exceeded those in the basal region. Procion dyes were used to assess cell death inflicted by Cd and Pb. At LC, the root cap and meristematic cells perished, together with the rhizodermal cells and the outer cortical cells of the root apex, whereas only the rhizodermal cells in the root apical region died at LC₅₀. The evidence that Cd and Pb cross the endodermal barrier at LC presumes that, at lower metal concentrations, the Casparian strip and plasmalemma of the endodermis regulate the transport of these metals into the central cylinder. The authors conclude that the identical barriers control Cd and Pb transport in root tissues.     

Replication of DNA in mammalian chromosomes

DNA replication has been studied in cells (CHO) synchronized by mitotic selection from roller cultures. A study of the incorporation of ³H supplied as uridine indicates that cells cannot be blocked precisely at the beginning of the S phase, but DNA synthesis can be stopped in early S by treating with F-dU in G₁. After blockage potential initiation sites continue to increase at a linear rate for atleast 13 hours after division. Incorporation of ³H-thymidine begins at most of these sites within seconds after thymidine is supplied in the medium and incorporation continues at a linear rate for 20–24 minutes. There appears to be a pause after this interval before synthesis is resumed at about two times the initial rate. ³H-bromodeoxyuridine can be substituted for thymidine without affecting the kinetic pattern over a similar period. The increased rate is probably an increase in sites of chain growth rather than a change in rate of chain growth. A study of the labeled DNA segments by band sedimentation in a preformed NaClO₄ isokinetic gradient shows that two distinctly different sized segments can be released from the chromosomes by lysis at submelting conditions. One is the previously reported single chain segments averaging about one-half micron in length, but the other is a much larger segment (26S) which is native DNA with perhaps small regions of single chains presumably at the ends. Primarily single chain DNA is released after 1–2 minute pulse labeling, but after 2 minutes the larger segments (26S) contain most of the newly formed DNA except that attached to the chains of the major part of the template DNA which exhibits a discontinuous distribution, sedimenting far faster than either newly replicated segment. A consideration of the kinetics of formation of the 26S component indicates that is may contain the replicating fork. If this proves to be the correct interpretation the template chains would both have non-adjacent nicks preceeding the fork and also in a post-fork site at a mean distance of about 2 microns in both directions. The isolation of the growing points of DNA replication in chromosomes is now possible and the study of properties of the newly replicated regions should be greatly facilitated.     

Relationship between intracellular pH and N metabolism in maize (Zea mays L.) roots

In vivo ³¹P nuclear magnetic resonance (NMR) was used to characterize the effect of the N form (NO₃ vs. NH₄) and the external pH (4, 6, and 8), on the intracellular pH of root tips (0–5 mm) and root segments (5–30 mm). Ammonium-grown root tips were the most sensitive to changes in the external pH. In vivo ¹⁵N NMR was used to characterize the pathway of primary ammonium assimilation in the ammonium-grown roots and to compare the activity of the apical and more-basal root parts. The kinetics of ¹⁵NH₄ ⁺ incorporation showed that primary assimilation in both root tips and root segments followed the glutamine synthetase (GS) pathway. In agreement with the reported gradient of GS along the seminal root of maize, incorporation of label into glutamine amide was more rapid in tips than in segments. It is suggested that this higher GS activity increases the endogenous proton production and thus contributes to the greater dependence of the cytoplasmic pH on the external pH in the ammonium-treated root tips.     

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

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.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

Patterns of auxin and abscisic acid movement in the tips of gravistimulated primary roots of maize

Because both abscisic acid (ABA) and auxin (IAA) have been suggested as possible chemical mediators of differential growth during root gravitropism, we compared with redistribution of label from applied ³H-IAA and ³H-ABA during maize root gravitropism and examined the relative basipetal movement of ³H-IAA and ³H-ABA applied to the caps of vertical roots. Lateral movement of ³H-ABA across the tips of vertical roots was non-polar and about 2-fold greater than lateral movement of ³H-IAA (also non-polar). The greater movement of ABA was not due to enhanced uptake since the uptake of ³H-IAA was greater than that of ³H-ABA. Basipetal movement of label from ³H-IAA or ³H-ABA applied to the root cap was determined by measuring radioactivity in successive 1 mm sections behind the tip 90 minutes after application. ABA remained largely in the first mm (point of application) whereas IAA was concentrated in the region 2–4 mm from the tip with substantial levels found 7–8 mm from the tip. Pretreatment with inhibitors of polar auxin transport decreased both gravicurvature and the basipetal movement of IAA. When roots were placed horizontally, the movement of ³H-IAA from top to bottom across the cap was enhanced relative to movement from bottom to top whereas the pattern of movement of label from ³H-ABA was unaffected. These results are consistent with the hypothesis that IAA plays a role in root gravitropism but contrary to the idea that gravi-induced asymmetric distribution of ABA contributes to the response.     

PHYLOGENETIC COMPARISON OF LARGE NUCLEAR DNA CONTENTS OF DIFFERENTIATED CELLS IN THE ROOTS OF EQUISETUM,TRADESCANTIA, AND HORDEUM

Nuclear DNA of meristematic, epidermal and root cap cells from the roots of three vascular plants—the cryptogam, Equisetum hyemale L, and the phanerogams, Tradescantia Clone 02 and Hordeum vulgare L.—was measured with quantitative Feulgen microspectrophotometry. Epidermal cells of all three species and root cap cells in both phanerogams contained up to 8fold the amount of nuclear DNA found in their respective meristematic telophase nuclei. In general, the large amounts of nuclear DNA parallel development and differentiation in the epidermis regardless of phylogeny, habitat, or degree of domestication. However, comparisons of the increase in nuclear DNA contents in the various epidermal cell types among these three species suggest that the mechanisms giving rise to these increases may differ phylogenetically and may represent another character in which cryptogams and phanerogams diverged in their evolution.     

Differentiation of metaxylem cell line in the root ofAllium cepa

Summary DNA was extracted from three root segments ofAllium cepa: i) an apical portion 500 m long from the tip (meristem); ii) a second portion 4 mm long (I root segment containing metaxylem cells in the initial stages of differentiation); iii) a third portion 6 mm long (II root segment containing metaxylem cells in further stages of differentiation). A mixture of homologous 18 S and 25 S³H-rRNA was used for invitro DNA-rRNA hybridization. The following percent saturation values were detected in the three samples: 0.08 in meristem DNA (samplea), 0.129 in I root segment DNA (sampleb), and 0.105 in II root segment DNA (samplec).Thermal denaturation of DNA and the derivative curves of the melting profiles evidenced five DNA families which were differently represented in the three DNA samples. DNA elution by thermal chromatography on hydroxyapatite followed by hybridization with³H-rRNA, revealed that ribosomal cistrons melt between 90 and 91 °C, corresponding to a G-C content of 50.7%. Moreover, the amount of the DNA family containing ribosomal cistrons was greater in sampleb andc, in sampleb to a greater extent, as compared with samplea. On the other hand, one DNA family melting at a higher temperature (92–93 °C) was drastically increased in samplec.Buoyant density profiles of unsonicated DNA showed no peaks in the three DNA samples. Upon somcation, a heavy shoulder was observed in the profile of sampleb. As the density of ribosomal cistrons and that of shoulder were very similar, it seems possible that the two fractions contain many DNA sequences in common.The present studies demonstrate that the proportion of ribosomal cistrons and other DNA families does not keep constant during the development of the metaxylem cell line.     

Fibrous carrot root responses to irrigation and compaction of sandy and organic soils

Field experiments were performed in Southern Finland on fine sand and organic soil in 1990 and 1991 to study carrot roots. Fall ploughed land was loosened by rotary harrowing to a depth of 20 cm or compacted under moist conditions to a depth of 25–30 cm by three passes of adjacent wheel tracks with a tractor weighing 3 Mg, in April were contiguously applied across the plot before seed bed preparation. Sprinkler irrigation (30 mm) was applied to fine sand when moisture in the 0–15 cm range of soil depth was 50% of plant-available water capacity. For root sampling, polyvinyl chloride (PVC) cylinders (30 × 60 cm) were installed in the rows of experimental plots after sowing, and removed at harvest. Six carrot plants were grown in each of in these soil colums in situ in the field.Fine root length and width were quantified by image analysis. Root length density (RLD) per plant was 0.2–1.0 cm cm^-3 in the 0–30 cm range. The fibrous root system of one carrot had total root lengths of 130–150 m in loose fine sand and 180–200 m in compacted fine sand. More roots were observed in irrigated than non-irrigated soils. In the 0–50 cm range of organic soil, 230–250 m of root length were removed from loosened organic soils and 240–300 m from compacted soils. Specific root surface area (surface area divided by dry root weight) of a carrot fibrous root system averaged 1500–2000 cm² g^-1. Root length to weight ratios of 250–350 m g^-1 effectively compare with the ratios of other species.Fibrous root growth was stimulated by soil compaction or irrigation to a depth of 30 cm, in both the fine sand and organic soils, suggesting better soil water supply in compacted than in loosened soils. Soil compaction increased root diameters more in fine sand than it did in organic soil. Most of the root length in loosened soils (fine sand 90%, organic soil 80%) and compacted soils (fine sand 80%, organic soil 75%) was composed of roots with diameters of approximately 0.15 mm. With respect to dry weight, length, surface area and volume of the fibrous root system, all the measurements gave significant resposes to irrigation and soil compaction. Total root volumes in the 0–50 cm of soil were 4.3 cm³ and 9.8 cm³ in loosened fine sand and organic soils, respectively, and 6.7 cm³ and 13.4 cm³ in compacted sand and organic soils, respectively. In fine sand, irrigation increased the volume from 4.8 to 6.3 cm³.     

The Root Cap: Cell Dynamics, Cell Differentiation and Cap Function

The root cap is a universal feature of angiosperm, gymnosperm, and pteridophyte roots. Besides providing protection against abrasive damage to the root tip, the root cap is also involved in the simultaneous perception of a number of signals – pressure, moisture, gravity, and perhaps others – that modulate growth in the main body of the root. These signals, which originate in the external environment, are transduced by the cap and are then transported from the cap to the root. Root gravitropism is one much studied response to an external signal. In the present paper, consideration is given to the structure of the root cap and, in particular, to how the meristematic initial cells of both the central cap columella and the lateral portion of the cap which surrounds the columella are organized in relation to the production of new cells. The subsequent differentiation and development of these cells is associated with their displacement through the cap and their eventual release, as border cells, from the cap periphery. Mutations, particularly in Arabidopsis, are increasingly playing a part in defining not only the pattern of genetic activity within different cells of the cap but also in revealing how the corresponding wild-type proteins relate to the range of functions of the cap. Notable in this respect have been analyses of the early events of root gravitropism. The ability to image auxin and auxin permeases within the cap and elsewhere in the root has also extended our understanding of this growth response. Images of auxin distribution may, in addition, help extend ideas concerning the positional controls of cell division and cell differentiation within the cap. However, firm information relating to these controls is scarce, though there are intriguing suggestions of some kind of physiological link between the border cells surrounding the cap and mitotic activity in the cap meristem. Open questions concern the structure and functional interrelationships between the root and the cap which surmounts it, and also the means by which the cap transduces the environmental signals that are of critical importance for the growth of the individual roots, and collectively for the shaping of the root system. Current address (Peter W. Barlow): School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK     

Effect of Water Stress on Cell Division and Cdc2-Like Cell Cycle Kinase Activity in Wheat Leaves

In wheat (Triticum aestivum) seedlings subjected to a mild water stress (water potential of −0.3 MPa), the leaf-elongation rate was reduced by one-half and the mitotic activity of mesophyll cells was reduced to 42% of well-watered controls within 1 d. There was also a reduction in the length of the zone of mesophyll cell division to within 4 mm from the base compared with 8 mm in control leaves. However, the period of division continued longer in the stressed than in the control leaves, and the final cell number in the stressed leaves reached 85% of controls. Cyclin-dependent protein kinase enzymes that are required in vivo for DNA replication and mitosis were recovered from the meristematic zone of leaves by affinity for p13^suc1. Water stress caused a reduction in H1 histone kinase activity to one-half of the control level, although amounts of the enzyme were unaffected. Reduced activity was correlated with an increased proportion of the 34-kD Cdc2-like kinase (an enzyme sharing with the Cdc2 protein of other eukaryotes the same size, antigenic sites, affinity for p13^suc1, and H1 histone kinase catalytic activity) deactivated by tyrosine phosphorylation. Deactivation to 50% occurred within 3 h of stress imposition in cells at the base of the meristematic zone and was therefore too fast to be explained by a reduction in the rate at which cells reached mitosis because of slowing of growth; rather, stress must have acted more immediately on the enzyme. The operation of controls slowing the exit from the G1 and G2 phases is discussed. We suggest that a water-stress signal acts on Cdc2 kinase by increasing phosphorylation of tyrosine, causing a shift to the inhibited form and slowing cell production.     

Metabolically stable non-histone chromosomal proteins in growing maize roots

DNA and non-histone chromosomal proteins (NHCP) of meristematic cells of maize primary roots were double labelled in vivo with [³H]- or [¹⁴C] thymidine and [¹⁴C]- or [³H]-tryptophan respectively. The ratio of labelled tryptophan to labelled DNA was followed during the transition of the meristematic cells of the root tip into the distal zones of finally differentiated cells. It was found that only 20% of the newly synthesized NHCP in the proliferating cells were turned over, while the rest were preserved and found as metabolically stable proteins in the zone of final differentiation. This result is consistent with the hypothesis that some NHCP remain permanently associated with chromatin of non-dividing differentiated cells in order to maintain the genomic characteristics of a given cellular type.     

Postreplication Repair of Ultraviolet Damage to DNA, DNA-Chain Elongation, and Effects of Metabolic Inhibitors in Mouse L Cells

Alkaline sucrose sedimentation studies of DNA from mouse L cells have demonstrated the following effects of several inhibitors of nucleic acid and protein synthesis on postreplication repair of ultraviolet (UV) damage to their DNA. The DNA newly synthesized by a 2 h [³H]thymidine (dThd) label following 254 nm UV irradiation of 20 J/m² is made in smaller segments of the number average mol wt (Mn) of ~10 × 10⁶ than the control of ~40 × 10⁶. The presence of caffeine at a concentration of 2 mM during the labeling of the irradiated cells reduces the Mn value to 5.8 × 10⁶, which is nearly comparable to, but somewhat larger than the expected distance between dimers in parental DNA. Afterwards, such an interrupted DNA made in the irradiated cells is completely repaired to the present maximum Mn value of 40 × 10⁶ in the consecutive 4 h chase in unlabeled dThd. The presence of the nucleic acid inhibitor, either 2 mM hydroxyurea, 50 μM arabinofuranosyl cytosine, 2 mM excess dThd or 5 μg/ml of actinomycin D (AMD) during 2- to 24-h chase periods after a 2 h postirradiation label prevents the repair to various extents, while 2 mM caffeine completely inhibits it. In the unirradiated cells, these agents except excess dThd and caffeine also interfere severely with normal elongation of nascent DNA made by a 3 min pulse label, but do not appreciably induce single chain breaks of either newly synthesized or parental DNA. The inhibition of the repair by AMD suggests that de novo elongation of DNA to close the gaps in new DNA made in the irradiated cells requires at least a template-dependent DNA polymerase. In contrast, 100 μg/ml of cycloheximide allows to complete the gap-filling repair, while it simply reduces the rates of chain growth for the repair and normal replication. Therefore, the similar sensitivity of gap-filling repair and normal replication towards the above inhibitors indicates that a preexisting DNA polymerizing system appears to be responsible and to play a common role without new protein synthesis, as far as the repair at early time after UV is concerned.     

Epifluorescent microscopic studies on the mechanism of preferential destruction of chloroplast nucleoids of male origin in young zygotes ofChlamydomonas reinhardtii

Summary The studies on the kinetics of nucleoid destruction reported here showed that destruction of chloroplast nucleoids (ct nucleoids) of male origin began to occur at about 30 minutes after mixing of male (mt^–) and female (mt⁺) gametes. The timing of initiation of the destruction differed among zygotes but usually occurred during 50–120 minutes after mixing. About 10 minutes was required for complete digestion of the ct nucleoids. UV irradiation on young zygotes or addition of an RNA-synthesis inhibitor, actinomycin D, to the incubation medium during the first 0–30 minutes after mixing almost completely inhibited the incorporation of³H uridine into the cell nuclei and the preferential destruction without inhibiting cell nuclear fusion. These results suggest that soon after mating,de novo RNA synthesis is concerned for the preferential destruction of ct nucleoids. To determine in which of the two cell nuclei in the zygotes the RNA is synthesized, each gamete (mt^–, mt⁺) was irradiated with UV and mated with unirradiated gametes of opposite mating type. This treatment of the male gametes had no effect on the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids but UV irradiation of female gametes almost completely inhibited the incorporation of³H uridine into cell nuclei and the preferential destruction of ct nucleoids. Similar phenomena occurred in other crosses. The UV effect was photoreactivated in about 50% by white light, suggesting that the UV target is DNA. Thus, RNA synthesized in the cell nucleus of female origin soon after mating may be responsible for the preferential destruction of ct nucleoids of male origin