Total Uptake and Incorporation into DNA of 3H-Thymidine, 3H-Deoxyuridine and 3H-Thymine in the Primary Root of Vicia faba L.: I. INTACT ROOTS

Total uptake and incorporation of ³H-thymidine, ³H-thymine,and ³H-deoxyuridine into DNA have been investigated in the apical3 cm of the primary root of Vicia faba. Evidence has been obtainedthat endogenous TdR in these roots may be transported eitherapically or basally; apical movement being greater than movementfrom the apex towards the base of the root. The results havebeen discussed with respect to the possible distribution ofendogenous pools of thymidine, thymine, and deoxyuridine inthe primary root of V. faba.     

Organelle DNA Synthesis in the Quiescent centre of Arabidopsis thaliana (Col.)

The behaviour of cell nuclei and organelle nucleoids (organellenuclei) was studied in the root apical meristem of 3-d-old seedlingsof Arabidopsis thaliana (Col.). Samples were embedded in Technovit7100 resin, cut into thin sections and stained with 4'-6-diamidino-2-phenylindole(DAPI) for observation of DNA. DNA synthesis in cell nucleiand organelle nucleoids was investigated using the incorporationof [³H] thymidine or 5-bromo-2'-deoxyuridine (BrdU). Incorporated[³H] thymidine and BrdU were detected by microautoradiographyor immunofiuorescence microscopy, respectively. Central cellsand cells just above the central cells of the quiescent centre(QC) showed an extremely low activity of DNA synthesis. However,DNA synthesis occurred in at least one organelle nucleoid ofall cells in the QC within 24 h. This suggests the cells inthe QC are quiescent with regard to nuclear DNA synthesis, butnot with regard to the organelle nucleoids. Key words: Arabidopsis thaliana, quiescent centre, root apical meristem, mitochondrial nucleoid (nuclei), plastid nucleoid (nuclei)     

The Expression Pattern of the Gene for NPK1 Protein Kinase Related to Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK) in a Tobacco Plant: Correlation with Cell Proliferation

Mitogen-activated protein kinase (MAPK) cascades consist ofmembers of three families of protein kinases: the MAPK family,the MAPK kinase family, and the MAPK kinase kinase (MAPKKK)family. Some of these cascades have been shown to play centralroles in the transmission of signals that control various cellularprocesses including cell proliferation. Protein kinase NPK1is a structural and functional tobacco homologue of MAPKKK,but its physiological function is yet unknown. In the presentstudy, we have investigated sites of expression of the NPK1gene in a tobacco plant and developmental and physiologicalcontrols of this expression. After germination, expression ofNPK1 was first detected in tips of a radicle and cotyledons,then in shoot and root apical meristems, surrounding tissuesof the apical meristems, primordia of lateral roots, and youngdeveloping organs. No expression was, however, observed in matureorgans. Incubation of discs from mature leaves of tobacco withboth auxin and cytokinin induced NPK1 expression before thedivision of cells. It was also induced at early stages of thedevelopment of primordia of lateral roots and adventitious roots.Thus, NPK1 expression appears to be tightly correlated withcell division or division competence. Even when an inhibitorof DNA synthesis was added during the germination or the inductionof lateral roots by auxin, NPK1 expression was detected. Theseresults showed that the NPK1 expression precedes DNA replication.We propose that NPK1 participates in a process involving thedivision of plant cells. (Received January 26, 1998; Accepted April 9, 1998)     

Auxin Transport in Roots: VI. MOVEMENT OF IAA THROUGH DIFFERENT ZONES OF ZEA ROOTS

The movement of IAA through 6-mm segments excised 1 mm, 7 mm,and 13 mm behind the apex of the primary root of Zea mays seedlingshas been investigated at temperatures between 10 and 25°C. In all segments, and at all temperatures, the movement of IAAwas polarized acropetally, more IAA being found in apical receiverblocks than in basal ones after transport periods of up to 24h. The amounts of IAA which moved acropetally through a segmentdecreased as the segment was taken at an increasing distancebehind the root apex. Similarly, at least after transport periodsof 8 h, more IAA moved basipetally through the apical segmentthan through the basal ones. At 10°C the velocity of acropetal movement was similar inall three segments, but the acropetbut the acropetal flux wasgreatest in the apical segment and smallest in the most basalone. The same situation appears to exist at the other temperatures. The flux and velocity of the acropetal movement of IAA througha 6-mm segment taken 7 mm behind the apex of the root were similarto those previously reported for the acropetal movement througha 12-mm segment excised 1 mm behind the apex. The smaller amountsof IAA which move acropetally through longer root segments aretherefore attributable to a limitation of the flux in the mostbasal regions of the segment.     

cAMP-sensitive endocytic trafficking in A6 epithelia

Blocker-induced noise analysis and laser scanning confocalmicroscopy were used to test the idea that cAMP-mediated vesicle exocytosis/endocytosis may be a mechanism for regulation of functional epithelial Na⁺ channels (ENaCs) at apical membranes of A6epithelia. After forskolin stimulation of Na⁺ transport andlabeling apical membranes with the fluorescent dyeN-(3-triethylammoniumpropyl)4-(6-4 diethylaminophenyl)hexatrienyl pyridinium dibromide (FM 4-64), ENaC densities(N_T) decreased exponentially (time constant~20 min) from mean values of 320 to 98 channels/cell within 55 minduring washout of forskolin. Two populations of apical membrane-labeledvesicles appeared in the cytosol within 55 min, reaching mean valuesnear 18 vesicles/cell, compared with five vesicles per cell in control,unstimulated tissues. The majority of cAMP-dependent endocytosedvesicles remained within a few micrometers of the apical membranes forthe duration of the experiments. A minority of vesicles migrated to >5µm below the apical membrane. Because steady states require identicalrates of endocytosis and exocytosis, and because forskolin increased endocytic rates by fivefold or more, cAMP/protein kinase A acts kinetically not only to increase rates of cycling of vesicles at theapical membranes, but also principally to increase exocytic rates.These observations are consistent with and support, but do not prove,that vesicle trafficking is a mechanism for cAMP-mediated regulation ofapical membrane channel densities in A6 epithelia.

     

Thymidine kinase activity in mouse embryo fibroblast cells infected with murine cytomegalovirus

Thymidine kinase activity was found in whole cell extracts of growing and stationary mouse embryo fibroblast cells after infection with murine cytomegalovirus. Determination of the kinetic constants and heat stability characteristics indicated that the enzyme activity from infected cells was different to that found in uninfected cells in the growth phase. The expression of thymidine kinase activity during virus replication was reflected by the incorporation of (6-³H) thymidine into acid precipitable fractions of infected cell cultures. Preliminary data from kinetic studies showed a reduction in the phosphorylation of thymidine by this enzyme activity in the presence of Acyclovir, a potent inhibitor of herpes virus replication.     

Structural Aspects of Cluster Root Development and their Possible Significance for Nutrient Acquisition in Grevillea robusta (Proteaceae)

Light microscopy (LM), scanning (SEM) and transmission electronmicroscopy (TEM) were used to study structure and function ofcluster roots inGrevillea robusta . These roots were developedduring growth ofG. robustaseedlings in modified Hoagland's solutionlacking phosphate. Cluster rootlets formed root hairs, basipetally,only after completing their determinate development. The rootlethairs branched in two ways and some had apical swelling. Rootletswith hairs produced two different forms of exudate, one fibrousand the other globular in nature. The fibrous material appearedto be synthesised in the cortical cells. It is released by exocytosisfrom the epidermis. Rootlet hairs produced only fibrous exudate.They attached firmly to pieces of vermiculite. The significanceof cluster roots is discussed within the context of patchy soilresources. Grevillea robusta ; Silky oak; Proteaceae; cluster roots; morphology; mucilage; nutrient acquisition; root exudates     

Effects of Cytokinins on the Growth of Primary Roots of Zea mays

Intact primary roots of Zea mays seedlings, apical 6-mm segmentsisolated from the intact primary roots and 5-mm detipped segments,prepared from the 6-mm apical segments by removal of the apical1-mm meristematic region, were incubated in potassium-phosphatebuffer that contained various concentrations of kinetin, 6-benzylaminopurine(BAP) or zeatin. These cytokinins inhibited the growth of intactprimary roots but they promoted the growth of both tipped anddetipped apical segments. In other words, they promoted thegrowth of root segments irrespective of the presence or absenceof apical meristematic regions. Detipped segments were stoodvertically, with their apical or basal cutends in contact withan agar plate that contained the abovementioned buffer and variousconcentrations of kinetin, BAP or zeatin so that cytokininswere supplied either from the apical or basal cut-ends. Cytokininssupplied from the top promoted the growth of the segments, whilethose supplied from the base did not. These results indicate that the response of roots to the exogenouslyapplied cytokinins is not influenced by the presence of theroot meristem but is significantly affected by the way in whichcytokinins are supplied. (Received October 17, 1995; Accepted August 28, 1995)     

Expression of herpes virus thymidine kinase in Neurospora crassa.

The expression of thymidine kinase in fungi, which normally lack this enzyme, will greatly aid the study of DNA metabolism and provide useful drug-sensitive phenotypes. The herpes simplex virus type-1 thymidine kinase gene ( tk ) was expressed in Neurospora crassa. tk was expressed as a fusion to N.crassa arg-2 regulatory sequences and as a hygromycin phosphotransferase-thymidine kinase fusion gene under the control of cytomegalovirus and SV40 sequences. Only strains containing tk showed thymidine kinase enzyme activity. In strains containing the arg-2 - tk gene, both the level of enzyme activity and the level of mRNA were reduced by growth in arginine medium, consistent with control through arg-2 regulatory sequences. Expression of thymidine kinase in N.crassa facilitated radioactive labeling of replicating DNA following addition of [3H]thymidine or [14C]thymidine to the growth medium. Thymidine labeling of DNA enabled demonstration that hydroxyurea can be used to block replication and synchronize the N.crassa mitotic cycle. Strains expressing thymidine kinase were also more sensitive than strains lacking thymidine kinase to anticancer and antiviral nucleoside drugs that are activated by thymidine kinase, including 5-fluoro-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine and trifluorothymidine. Finally, expression of thymidine kinase in N. crassa enabled incorporation of bromodeoxyuridine into DNA at levels sufficient to separate newly replicated DNA from old DNA using equilibrium centrifugation.     

Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus.

Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity.     

The Involvement of Glucose-6-Phosphatase in Mucilage Secretion by Root Cap Cells of Zea mays

In order to determine the involvement of glucose-6-phosphatasein mucilage secretion by root cap cells, we have cytochemicallylocalized the enzyme in columella and peripheral cells of rootcaps of Zea mays. Glucose-6-phosphatase is associated with theplasmalemma and cell wall of columella cells. As columella cellsdifferentiate into peripheral cells and begin to produce andsecrete mucilage, glucose-6-phosphatase staining intensifiesand becomes associated with the mucilage and, to a lesser extent,the cell wall. Cells being sloughed from the cap are characterizedby glucose-6-phosphatase staining being associated with thevacuole and plasmalemma. These changes in enzyme localizationduring cellular differentiation in root caps suggest that glucose-6-phosphataseis involved in the production and/or secretion of mucilage byperipheral cells of Z. mays. Zea mays, corn, glucose-6-phosphatase, columella cell, peripheral cell, mucilage, secretion, cytochemistry     

Thymidine phosphorylation in synchronous cultures of Tetrahymena pyriformis GL

Recently it has been established that thymidine can be phosphorylated in two ways in Tetrahymena pyriformis

1. 1. by action of thymidine kinase

2. 2. by action of nucleoside phosphotransferase.

The present report confirms that thymidine kinase is a peak enzyme during S phase. It is suggested that a different thymidine concentration in the thymidine kinase assay might explain why previous workers have been unable to find thymidine kinase in Tetrahymena.     

Long-term stability of root cultures of tomato transformed with Agrobacterium rhizogenes R1601

Tomato explants were transformed with Agrobacter rhizogenesR1601 and root clones used to initiate longterm cultures inliquid and on solid media. The liquid cultures were maintainedfor 50 passages over 25 months in the presence and absence ofkanamycin. During this time the clones retained their high growthrates and antibiotic resistance phenotypes. The nptll gene wasdetected by PCR and dot blot hybridization in DNA from clonesat the 21st passage, and NPT-II enzyme activity was detectedin cell extracts prepared at the 45th passage. The solid cultureswere main tained for 12 passages over 12 months, either withcontinual selection, or with alternation during the last sixpassages between selective and non-selective media. The nptllgene was detected by PCR in DNA from cultures at the 5th passageand NPT-II enzyme activity was detected in cell extracts ofvigorously growing clones from the 12th passage. Passages inthe absence of selection had no discernible effect on the stabilityof the transformed state. The results indicate that the Ri-transformedstate can be maintained in tomato root clones during long periodsof culture. Key words: Agrobacterium rhizogenes, long-term cell culture, root clones, tomato, transformation     

Independent characterization of thymidine transport and subsequent metabolism in Hymenolepis diminuta--II. Purification and preliminary analysis of thymidine kinase

1. An affinity column for the purification of thymidine kinase (TK) from the cestode Hymenolepis diminuta is described. Using an epoxy-activated Sepharose 6B affinity column containing thymidine as a ligand, a 698-fold purification of thymidine kinase was obtained. 2. Thymidine kinase eluted from this affinity column was partially characterized as having an apparent Km value of 3.94 microM thymidine. This value is very similar to those observed in mammalian systems. 3. Thymidine kinase appears to be an extremely active and ubiquitous enzyme, whose primary function is to rapidly phosphorylate incoming thymidine and thus "trap" it for the cell's use, reducing efflux to a minimum. 4. The apparent Km for TK is two orders of magnitude lower than the Kt for thymidine transport. Thus, theories postulating that long-term (2 min) uptake kinetics for thymidine actually represent subsequent metabolism must look further along the thymidine phosphorylating pathway, beyond TK and its very active role.     

Duration of the Mitotic Cycle in a Meristem

The length of the mitotic cycle and the time spent in mitosisare calculated for six regions of the apical meristem of rootsof Zea. The two methods used are the measurement of the ratesof labelling nuclei with radioactive thymidine and the ratesof accumulation of metaphases in roots grown in colchicine.The mitotic cycle lasts from 12 hours in the cap initials toabout 200 hours in the quiescent centre of the same roots. Somepoints about the organization of apices are discussed in thelight of these estimates.     

Purification of thymidine kinase by affinity chromatography with an enzyme inhibitor as the ligand

An affinity column for the purification of thymidine kinase is described. The ligand in this column is a glycoprotein isolated from rat kidney. This glycoprotein inhibits phosphorylation of thymidine in cultured cells and in a cell-free assay system. With an affinity column containing the glycoprotein as a ligand, a 24-fold purification of thymidine kinase from an ammonium sulfate fraction of a crude tissue extract can be obtained. Thymidine kinase eluted from the affinity column migrates as one major band on polyacrylamide and as one diffuse major band on sodium dodecyl sulfate-polyacrylamide. The affinity column, with thymidine kinase bound to the inhibitor, can also be used as an assay system. When the glycoprotein is covalently attached to Sepharose, it retains its binding capacity for thymidine kinase but has apparently lost its ability to inhibit the enzyme. Thymidine kinase eluted from the affinity column is again sensitive to the glycoprotein. It seems to be a carbohydrate moiety of the glycoprotein that is responsible for the inhibition.     

Origin of Exogenous Adventitious Roots in Rhizomes of the Fern Microsorium scolopendria (Polypodiaceae)

Adventitious roots of rhizomes of the fern Microsorium scolopendriaarise exogenously from apical initials which have their originin the cell layer directly beneath the epidermis. Divisionsof the apical initial and its derivatives produce the adventitiousroot apical meristem. Cells between the apical cell and themeristele of the rhizome develop into the root trace. Anatomy, light microscopy, Microsorium scolopendria Burm.) Copel