Evidence for chromosomal replicons as units of sister chromatid exchanges

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 m with a peak value around 0.3 to 0.6 m regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 m was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 m in size. In addition, we have found that this chromosomal unit of replication or chromosomal replicon does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 m in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.     

Replication in Drosophila chromosomes

DNA fibre autoradiography of highly polytenized nuclei in salivary glands of Drosophila nasuta larvae reveals two distinct types of active replicons. Type I replicons are longer (mean size=64 m), have a very high rate of fork migration (average rate=0.95 m/min) and generally occur in large arrays often extending over several thousand m. In contrast, the type II replicons are smaller (mean size= 20 m), slow replicating (average rate=0.07 m/min) and occur in short arrays containing only a few closely spaced active replicons. Evidence is presented that type I replicons are active in the early S and type II in the late S. Observations on autoradiographic labelling of partially lysed polytene chromosomes provide evidence for a lack of temporal and spatial agreement in the activation of origin points in homologous regions of the lateral polytene strands; these observations also suggest local variations in levels of polyteny within a chromosome. On the basis of this and other available information on replication in polytene chromosomes the possible roles of the two replicon types in the generation of the different ³H-thymidine labelling patterns of polytene chromosomes are discussed.We take pleasure in dedicating this paper to our inspiring teacher Prof. S.P. Ray Chaudhuri on his completing 75 years of fruitful life     

Replication forks are not found in a Drosophila minichromosome demonstrating a gradient of polytenization

Differential DNA replication is widely held to influence polytene chromosome structure by causing the dramatic reductions in heterochromatic DNA content that are characteristic of most endopolyploid cells. The underreplication model of heterochromatic sequence underrepresentation predicts that replication intermediates should populate regions of DNA between fully polytenized euchromatic sequences and underpolytenized heterochromatic sequences. We directly tested this prediction using Dp1187, a 1300 kb Drosophila minichromosome containing well-defined heterochromatic regions. DNA from a euchromatic/heterochromatic junction region of Dp1187, demonstrating a significant gradient of underrepresentation in larval salivary glands, lacked the stalled replication forks predicted by the underreplication model. We consider an alternative mechanism leading to heterochromatic sequence underrepresentation involving a process of DNA elimination.by W. Hennig     

Replicon size of yeast ribosomal DNA

Summary The ribosomal RNAs of the yeast Saccharomyces cerevisiae are transcribed from a 9Kbp stretch of DNA which is reiterated about 120-fold in a continuous array, about 360 m long, on chromosome XII. Although ARS activity has been detected in the repeat unit, the size and disposition of replicons along this array of identical genes has not hitherto been determined. We have used immobilised rRNA as a probe to examine the size of radioactively labelled rDNA replicons resolved on alkaline sucrose gradients. The replicons were found to be uniformly sized, about 5 repeat units in length, and groups of 4 adjacent replicons may be activated simultaneously. These observations suggest that replicon initiation events are not determined solely by the recognition of specific DNA sequences that function as origins of replication.     

Electron microscopic band-interband pattern of the X chromosome in Drosophila hydei

The band-interband pattern of the salivary gland X chromosome in Drosophila hydei was studied by electron microscopy (EM) using the technique of surface-spread polytene (SSP) chromosome preparation. We observed 526 chromosome bands, i.e. 135 additional bands as compared with the original light microscopic chromosome map (Berendes 1963). Individual interband lengths and band thicknesses were measured for the entire X chromosome in electron micrographs of ten SSP chromosome preparations. Average values were used to plot an EM chromosome map. The average interband had an axial length of 0.38 m. Depending upon the extension of the DNA packing ratio in interbands, this indicates 1.1 kb of totally extended DNA or 3.8 kb, if a DNA packing ratio of 0.10 m/kb is assumed for SSP chromosomes (Kress et al. 1985).     

6-Methylpurine-induced inhibition of sclerotia morphogenesis in Sclerotium rolfsii and its reversal by adenosine

In liquid synthetic medium inoculated with Sclerotium rolfsii (SR), addition of 6-methylpurine (MP, 50g/ml) immediately after inoculation led to approximately 100% reduction in sclerotia production. Adenosine, and to a lesser extent guanosine, each at final concentration of 100g/ml significantly reduced inhibition of sclerotia formation by SR in presence of 50g/ml MP. Uridine and cytidine each at 100g/ml had no such effect. The inhibition of sclerotia morphogenesis could be prevented by addition of 800g/ml of adenosine together with 50g/ml MP. Reversal by adenosine of MP-induced inhibition of sclerotia development was concentration dependent.     

Cyclin-dependent kinase inhibitor,roscovitine, in combination with exogenous cytokinin,N6-benzyladenine,causes increase of cis-cytokinins in immobilized tobacco cells

Dynamics of the response of tobacco cells (line BY-2) to exogenous cytokinin, N ⁶-benzyladenine, and cyclin-dependent kinase inhibitor, roscovitine, was followed using alginate-immobilized cells packed into a column. N ⁶-Benzyladenine (1.25 M) increased the synthesis of the physiologically-active endogenous cytokinin, isopentenyladenosine, in the effluent up to 0.1 nM. Simultaneously, conversion of the excess of endogenous cytokinins to biologically inactive derivatives of cis-zeatin occurred, up to 0.8 nM. Roscovitine (50 M) further increased cis-cytokinins, up to 2.2 nM.     

The units of DNA replication in the mammalian chromosomes: Evidence for a large size of replication units

The replication of chromosomal DNA in human and Chinese hamster cell populations has been studied by means of the DNA fiber autoradiography. It was found that the rate of DNA replication for one fork in human cells varies from 0.2 to 0.9 m/min, the average being 0.6 m/min. In the Chinese hamster cells the rate of DNA replication is greater, varying from 0.3 to 1.2 m/min, the average being 0.8 m/min. There are no clusters containing a great number of replication units in human and Chinese hamster cells. Sequences consisting of two or three replicons which belong to single DNA molecule have been observed, but their frequency was relatively low. The distances between the initiation points in such sequences of replicons vary from 40 to 280 m, the average value being 130 m. This value represents the minimum size of the replication units which have completed the DNA synthesis within 3 h of the S-period. The DNA synthesis in most replication units fails to be accomplished within the three hours of labelling. The process can be completed only in the fragments of DNA molecules of 40 to 200 m (the average value being 100 m) in human cells, whereas in the Chinese hamster cells the fragments of 40 to 250 m (the average being about 140 m) are completely replicated. Provided that the replication is bidirectional the complete replicons are supposed to contain two such fragments. Consequently, the greater part of replication units in mammalian cells covers the pieces of a few hundred microns in DNA molecules. The relation between replication process at the DNA molecules level and that at the metaphase chromosome level is discussed.     

Canavanine synthesis in thein vitro propagated tissues ofCanavalia lineata

Maximum shoot induction from stem explants ofCanavalia lineata was obtained with an agar-solidified PC medium containing 10 M benzylaminopurine and 1 M naphthaleneacetic acid. Rooting of thesein vitro produced shoots was achieved with hormone-free PC medium. Canavanine was produced almost exclusively in the leaves and was not detected in the roots ofin vitro propagatedC. lineata. To exclude the possibility of imminent translocation of canavanine from the root to leaf, adventitious roots were induced from leaf explants in PC medium supplemented with 1 M kinetin and 20 M indole-3-acetic acid and subcultured in medium lacking growth regulators, and the roots excised from germinated seedlings were cultured in hormone-free PC medium. All the roots were incapable of accumulation of canavanine. These results suggest that leaves ofC. lineata are the possible site of canavanine synthesis.     

Mean rate of DNA replication and replicon size in the shoot apex ofSilene coelirosa L. During the initial 120 minutes of the first day of floral induction

Summary 28-day-old plants ofSilene coeli-rosa were exposed, at 1,700 hours, to long day (LD) conditions comprising light of low fluence rate provided by tungsten bulbs, or maintained in darkness as short day (SD) controls. All plants were exposed at 1,700 hours to tritiated-(methyl-³H)-thymidine for 30, 45, 60, 90, or 120 minutes. Apical domes were isolated and prepared as fiber autoradiographs from which replicon size and rates of DNA replication, per single replication fork were recorded. In SD, replicon size was between 15–20 m and exposure to LD conditions altered neither replicon size nor the pattern of deployment of replicons during S-phase relative to the SD controls. However, the mean rate of replication in LD was 8.7 m h^–1 compared with 5.2 m h^–1 in SD. Thus, exposure to LD resulted in a 1.7-fold increase in the rate of DNA replication relative to the SD controls. This rapid increase in replication rate, detectable within 30 minutes of the start of the LD is discussed in relation to changes known to occur to the cell cycle inSilene during the first day of floral induction.     

Fine structure of the spermatophore and intradermic penetration of sperm cells inMyzostoma cirriferum (Annelida,Myzostomida)

Summary The spermatophore ofMyzostoma cirriferum is a white V-shaped structure up to ca. 500 m long. It is formed by a translucent matrix which includes numerous cysts of two types that are very close together and tend to form interlacing twists. According to their contents, three spermatophoral regions can be distinguished: the body with the horns, the foot and the basal disc. The body-horns region forms the upper part of the spermatophore and extends over ca. 400 m. This region includes mature spermiocysts which are formed by one cyst cell each including one to three groups of rolled up spermatozoons. Features of these cyst cells are their great length (up to 25 m), their euchromatic nuclei each provided with a large nucleolus, their numerous mitochondria and osmiophilic vesicles included in the cytoplasm as well as cytoplasmic remnants of the residual bodies of the spermatids. Spermatozoons appear to be well adapted to the intradermic penetration occurring in this species in that all of them possess nuclei provided with dense nuclear grains, a hairpin-bent flagellum and a microtubular palissade. The spermatophore foot is located just below the body and extends over ca. 90 m. It contains exclusively spermiocysts which include one to three abortive germinal cells. They differ also from the previous cysts by their smaller length (ca. 6–10 m) and their more heterochromatic nuclei. The basal disc is the lower part of the spermatophore. It extends over ca. 10 m and contains electron-dense vesicles in its upper part and vesicles with fibrillar material in its lower part. When mature myzostomids contact each other, a spermatophore is expulsed from one seminal vesicle of the donor myzostomid to the integument of the receiver myzostomid. The vesicles with fibrillar content are the first in contact with the cuticle of the receiver myzostomid. The material they include is supposed to have a histolytic action and to be responsible for the lysis of the cuticle and epidermal cells thus providing a passage for the spermatophore contents. Afterwards, cysts move as a result of the spermatozoons' beating and pass through the receiver's integument. At the time of penetration, cytoplasmic membranes of the cyst cells merge together forming an enormous syncytium extending into the whole receiver's body. This syncytium surrounds the spermatozoons and the abortive germinal cells. The whole process of intradermic penetration (i.e. from the fixation of the spermatophore to its reduction to an empty matrix) lasts from 1–5 h.     

Ultrastructural analysis ofSpinacia oleracea sperm cells isolated from mature pollen grains

Summary In isolated condition, the sperm cells ofSpinacia oleracea are no longer arranged in pairs as in the pollen grain. The vegetative membrane, which surrounds a sperm cell pair in a mature pollen grain, is lost during the isolation procedure. The sperm cells become spherical in shape.The isolated sperm cell is surrounded by an intact plasma membrane. The heterochromatic or euchromatic sperm cell nucleus is located in the cell center. Mitochondria are round to oval and have distinct cristae. Often they are clustered in groups of 5 to 10 mitochondria. Dictyosomes are present in the cytoplasm and consist of 4 to 5 cisterns. Endoplasmatic reticulum is mostly situated at the sperm cell periphery, as single cisterns very near the plasma membrane.From diameters of sectioned sperm cells in electron micrographs, it is possible to calculate the average diameter of the whole sperm cell. This average diameter is 3.66 m with a variation of 3.0 m to 4.2 m, resulting in an average volume of 25.6 m³. The nuclear volume is 12.8 m³ (50.0% of the whole cell) and the mitochondrial volume is 0.7 m³ (2.5% of the whole cell). The frequency distribution of the isolated sperm cells diameters shows only one peak with a normal distribution, indicating that there is no dimorphism in volume.     

Replicon size and mean rate of DNA synthesis in rye (Secale cereale L. cv. Petkus Spring)

Chromosomal DNA replication was investigated in root meristem cells of Secale cereale L. cv. Petkus Spring using DNA fibre auto-radiography. At 23 ° the mean rate of replication, per single replicon fork, was 12.1 m/h. Replicon size was between 20–25 m. These results are compared with corresponding measurements for other angiosperm species.     

Replicating DNA molecules from eggs ofDrosophila melanogaster

Eggs ofDrosophila melanogaster were lysed with sodium dodecyl sulphate within 110 minutes after laying and the lysate prepared for electron microscopy by the protein monolayer technique. Long, non-circular DNA molecules were found with a form suggesting they contained either a single replicated region, or two, three or four replicated regions arranged in tandem. Each replicated region was delimited by two forks. The two segments of DNA spanning the region between the forks were approximately equal in length and appeared to be totally or almost totally double-stranded. The appearance of replicating molecules was not altered by digestion with pronase or treatment with phenol or chloroform. The lengths of replicated regions varied from 0.2 to 22.1 with a mean value of 2.97 . The distances between midpoints of adjacent tandemly arranged replicated regions ranged from 1.2 to 9.7 with a mean value of 3.87 . Circular molecules found in these preparations, and presumed to be of mitochondrial origin, were estimated from comparative length measurements with circular double-stranded DNA molecules from the bacteriophages lambda, X174 and fd to have a molecular weight of 12.36 X 10⁶ daltons.     

Eubacteria have 3 growth modes keyed to nutrient flow

Aerobic growth of Escherichia coli and Paracoccus denitrificans has been studied in chemostat, fed batch, and recycling fermentor modes under carbon and energy limitation. Two abrupt drops or discontinuities in molar growth yield, Y, have been found that occur over relatively short ranges in the value of specific growth rate.Before the first discontinuity, Y is constant and maximal. After the first discontinuity, at a doubling time of 33 h, Y becomes constant again and independent of until the second discontinuity appears at a doubling time of about 50 h, corresponding to a of about 0.014. At this point, Y drops to a lower value that is constant at doubling times longer than 100 h, corresponding to a of about 0.007.The second discontinuity is associated in Paracoccus with elevated levels of guanosine tetraphosphate (ppGpp) that impose stringent regulation as has been found previously with Bacillus and Escherichia species. It is thus likely that the stringent response generally occurs in bacteria in vivo at a doubling time of about 50 h. The cause of the first discontinuity is unknown. All experiments indicate that Pirt-type calculations relating , Y, and maintenance energy demand are no longer valid. In chemostat experiments, the intercept of the relationship between specific substrate utilization and specific growth rate is defined as maintenance. However, this intercept most probably is caused by stringent regulation at low dilution rates. Three regions of bacterial growth rates are defined by this study, corresponding to doubling times of 0.5 to 15 h, 33 to 50 h, and >100 h. Some growth behavior in each region is unique to that region.Abbreviations ppGpp guanosine 5 diP 3 diP - pppGpp guanosine 5 triP 3 diP - SPR substrate provision rate (mol/l h)     

Effect of chloramphenicol and the recB gene product on DNA metabolism in Escherichia coli K12 strains defective in DNA ligase

Summary We have examined DNA strand breakage, DNA degradation, and the rate of DNA synthesis in lig and lig-recB strains of Escherichia coli K12 incubated in the presence and absence of 3 g/ml chloramphenicol. Substantial DNA strand breakage and DNA degradation is observed in the lig strain upon growth at 40°C; however, such strand breakage and DNA degradation is not observed in the lig-recB strain. Incubation of the lig strain at 40°C in the presence of 3 g/ml chloramphenicol reduces the amount of DNA strand breakage and DNA degradation to the level observed in the lig-recB strain. Together, these results demonstrate that exonuclease V (the recBC gene product) is responsible for the increased DNA degradation associated with DNA ligase deficiency.     

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 m/h and the average replicon size was 24 m. 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.     

Effects of nucleotides on assembly of the 26S proteasome and degradation of ubiquitin conjugates

We have investigated three aspects of nucleotide usage by the 26S proteasome and its regulatory complex (RC). Both particles hydrolyze the four major ribonucleotides, but ATP and CTP have substantially lower K _s for hydrolysis than do GTP and UTP. The K _ for ATP hydrolysis is 15 m for the 26S proteasome and 30 m for the regulatory complex. Formation of the 26S proteasome from the RC and the 20S proteasome requires about 5 m ATP. Although measurable degradation of Ubiquitin(Ub)-lysozyme conjugates occurs in the presence of CTP, GTP, and UTP, the best nucleotide for Ub-conjugate degradation by the 26S proteasome is ATP, with an estimated K _ of 12 m. In summary, our studies show that micromolar concentrations of ATP are sufficient for several 26S proteasome activities.     

Resistance ofBradyrhizobium japonicum strains to selected antibiotics

The majority of the 50Bradyrhizobium japonicum strains tested were resistant to ampicillin, kanamycin, streptomycin and tetracycline in concentrations below 100 g/ml but resistant to chloramphenicol in concentrations equal to or above 100 g/ml. Two strains had high levels of resistance to ampicillin and to streptomycin and six strains were very sensitive to several antibiotics.

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Résumé La majorité de la cinquantaine de souches deBradyrhizobium japonicum s'est révélée résistante à l'ampicilline, à la kanamycine, à la streptomycine et à la tétracycline aux concentrations intérieures à 100g/ml mais au chloramphenicol aux concentrations égales ou supérieures à 100 g/ml. Deux souches présentalent un haut degré de résistance à l'ampicilline et à la streptomycine. Six souches étaient très sensibles à plusleurs antibiotiques.

     

Optimization of an electroporation procedure for Kluyveromyces lactis transformation

Summary An electroporation method using a Bio-Rad Gene Pulser has been optimized for introducing heterologous DNA into Kluyveromyces lactis yeasts. The plasmid pCR1, derived from a native Kluyveromyces plasmid, was used to transform K. lactis. This plasmid produces a wheat -amylase and contains both the biosynthetic marker URAA and G418 resistance genes. Transformation was optimal at 4500 V/cm, 25 F, and with 0.2 g plasmid DNA. Transformation efficiencies in the range 10⁴–10⁵ transformants/10⁷ cells/g DNA were obtained.