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The synthesis of the glycosidic prodrugs (1S)‐30a, (1S,10R)‐30band (1S,10R)‐32labelled with different fluorescence dyes at different positions at the aromatic A‐ring in 2is described; the compounds are structurally based on the cytotoxic antibiotic duocarmycin SA. For binding, the amino compounds (1S)‐3aand (1S,10R)‐3bwere treated with the commercially available succinimides of the dyes 5‐SFX (29) and D10162 (31), respectively.

ortho‐Haloarylcarbamates like 1–4show a high rotational barrier about the Naryl bond of up to 91.6 kJ mol−1as found for 1, which was determined by 2D exchange NMR spectroscopy (EXSY). It was further demonstrated that the height of the barrier not only depends on the substituents at the axis of chirality, but is also influenced by electronic effects.

Cytotoxicities almost a million times lowerthan the prevailing active compounds, which can have IC50values of about 100 fM, are displayed by new glycosidic prodrugs for selective tumor therapy (see example; gray C, white H, green Cl, blue N, red O). The cytotoxicity of these new active compounds is presumably not attributable to DNA intra‐ or DNA inter‐strand cross‐linking, but might be based on an as yet unknown mechanism.

Fast eine Million Mal weniger zytotoxischsind neue glycosidische Prodrugs zur selektiven Tumortherapie (siehe Beispiel; grau C, weiß H, grün Cl, blau N, rot O) als die jeweiligen Wirkstoffe, die IC50‐Werte um 100 fMaufweisen können. Die Zytotoxizität der neuen Wirkstoffe ist vermutlich nicht auf DNA‐Intra‐ oder DNA‐Interstrangvernetzungen zurückzuführen, sondern beruht möglicherweise auf einem noch unbekannten Mechanismus.

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.

In synchronized root cells of Pisum sativum(cv. Alaska) the joining of nascent replicons is delayed until cells reach the S–G2boundary or early G2phase. 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 G2phases. Two‐dimensional (neutral‐alkaline) agarose electrophoresis and Southern blot hybridization with specific rDNA sequences show that only cells located at the S–G2boundary and early G2phase 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, T1and T2, where forks stop. T1is located in the non‐transcribed spacer and T2is 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.

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.

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.

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 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.

Restriction 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.

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.

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°.

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.

In cultured pea roots there is extrachromosomal DNA associated with cells that differentiate from the G2phase of the cell cycle that is absent from those that differentiate from the G1phase. We examined this extrachromosomal DNA by electron microscopy and found that it consisted of three types: (i) double-stranded linear molecules with single-stranded branches (74%), (ii) double-stranded molecules without branches (26%), and (iii) free single-stranded molecules. The double-stranded molecules with or without branches were similar in length, having a modal length of 10-15 μm. The free single-stranded molecules were shorter and had a mean length of 3.8 μm. The length of the branches attached to the duplex molecules was only slightly less than that of the free form. The duplex molecules with branches were interpreted as configurations reflecting an ongoing strand-displacement process that results in free single-stranded molecules. Finally, measurements on duplex molecules with multiple branches suggested that the extrachromosomal DNA may exist in the form of tandemly repeated sequences.

Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80% of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 X 10(6) to 140 X 10(6) daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 X 10(6) daltons and another of 7 X 10(6) daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 X 10(6) and 7 X 10(6) daltons.

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.

A tetraploid cell population was produced in the primary root meristem of Pisum sativum by one-half hour treatments with various concentrations of colchicine. The tetraploid population so produced was found to be reasonably synchronous in its passage through successive mitotic cycles with the degree of synchrony being more or less proportional to the concentration of colchicine used. The average time between mitoses appears to be of the order of 12 hours which agrees well with previous estimates. Treatments of roots containing tetraploid populations with 2.52 × 10-5 M. actidione for 15 minutes were used to demonstrate the possibility of using the system for studies on the differential susceptibility of cells at different stages of the mitotic cycle.

1.Treatment of a tetraploid population of proliferating cells with DNP or DCP in early interphase caused a delay in appearance in the subsequent mitosis and an improvement in the synchronization.2.Treatment of the population with DNP or DCP when it was in late interphase resulted in a decrease in synchrony and a slight delay in its entrance into division.3.When the population was treated with DNP, DCP, or cyanide at the fifth hour after polyploidizing, appearance in the second division was early as compared to the controls, thus indicating a decrease in the mitotic cycle time.4.Neither cyanide nor fluoride in low concentrations affected the degree of synchrony of the population.5.The acceleration of the second cycle following treatment was prevented to a greater or lesser degree by combining fluoride with either DNP or cyanide.6.The decrease in cycle time of a treated population appears to be the result of creating cellular conditions favorable to an increase in the rate of anaerobic glycolysis at the fifth hour of interphase.

Two cell populations in sunflower root meristems are described. Most cells stop in G1 after being cultured in sucrose‐deficient medium, but “leaky” cells continue through DNA synthesis and stop in G2. A comparison of “leaky” and arrested cells is reported on the basis of radiosensitivity, and cytological and biochemical responses to metabolic inhibitors. “Leaky” cells are randomly distributed throughout primary meristematic tissues. They are not inhibited from initiating DNA synthesis by exposure to doses of γ‐irradiation ranging from 300–7200 R; arrested cells, depending upon the dose, are inhibited partially or completely. “Leaky” cells do, however, show a dose‐dependent mitotic delay in G2, which is the same as arrested cells. Treatment with puromycin and actidione does not inhibit “leaky” cells from initiating DNA synthesis but does inhibit them from mitosis. Arrested cells are inhibited from advancing to S and M by both inhibitors. Also, puromycin and actidione cause a decrease in protein and RNA synthesis, demonstrating a possible protein dependent RNA synthesis necessary for cell cycle progression. Actinomycin D (10 μg/ml) inhibits neither “leaky” nor arrested cells from entering S and M. At 30 μg/ml, however, arrested cells are partially inhibited. “Leaky” cell metabolism is unique in preparation for and initiation of DNA synthesis but similar to that of the remaining cells of the meristem in terms of requirements for progression through the rest of the mitotic cycle.

Experiments were performed with cultured excised primary root tips of Vicia faba‘Longpod’ to determine: (1) the proportion of meristematic cells arrested in Gl and in G2 during carbohydrate starvation, and to determine if the proportion is fixed or can be varied experimentally; (2) the effect of increased starvation on the ability of arrested cells in Gl and G2 to initiate DNA synthesis and mitosis, respectively, when exogenous sucrose was supplied; and (3) whether puromycin, cycloheximide, or actinomycin D prevented the initiation of DNA synthesis and the onset of mitosis. Microspectrophotometry of nuclear DNA and autoradiographic measurements of incorporated 3H‐thymidine showed that 72 hr of starvation immediately after excision produced tissue with more than 70 % of the cells arrested in G2 and less than 30 % in Gl. If cultured for three days and then starved for 72 hr, the tissue had nearly equal numbers of cells arrested in Gl and G2. As the duration of starvation increased, the time required to initiate DNA synthesis and to divide when carbohydrate was replenished also increased. Inhibition of protein synthesis by puromycin and cycloheximide prevented the initiation of DNA synthesis and mitosis, but actinomycin D, an inhibitor of RNA synthesis, did not prevent division of cells from G2 nor DNA synthesis by cells from Gl. The experiments demonstrated that the mitotic cycle of Viciahas two major controls, one in Gl and another in G2, and that other factors determine how many cells are affected by either of these cycle controls.

In a family, two children exhibited chromosome aberrations: a male infant had trisomy G1 and his sister Bp deficiency (cri du chat-syndrome). Screening for proteinpolymorphisms within three generations revealed heterozygosity for two enzyme variants, LDH BB' and 6-PGD AB, in a number of family members, some of whom were double heterozygotes. Linkage can not be tested since these variants are derived from different sibships. An aunt of the affected children exhibited a LDH isoenzyme pattern as it is to be expected in a protein chimaera. In addition, a high number of aneuploid and polyploid cells, including endomitoses, were present in the blood of this proband, and similarly in her mother. The origin of the chimaera is discussed.

The enzyme 6-PGD exhibits a genetically determined polymorphism which results in a variability of the isoenzyme pattern. In mammals so far examined the findings are to be interpreted according to the model of multiple alleles at one single gene locus. In order to test whether the mammalian genome is uniformly endowed with one single locus for this enzyme or whether a duplication of this locus is present in certain species, we examined various mammals of different orders. Our results in this first communication on 6 different Microtinae species are all in agreement with the assumption of a single 6-PGD locus. In Microtus oeconomus 2 alleles could be demonstrated and in Microtus ochrogaster 3 different alleles. Family studies performed with both species revealed the occurrence of all the expected combinations of these alleles.

Electrophoretic analysis of the sorbitol dehydrogenase isozymes (SDH) in various mammalian species including man revealed the existence of only 1 gene locus for this enzyme. As a rule, the same is true for Teleostean fishes. Some species of the fish order Isospondyli, however, represent an exception. Within this order, a diploid-tetraploid relationship exists. Species exhibiting diploid characteristics as the herring (Clupea harengus), are endowed with a single SDH gene locus at which 3 different alleles were observed. In some Salmonoid fish having passed through tetraploid evolution, a duplication of the SDH gene can be demonstrated. While in Salmo trutta the duplicated genes evolved divergently and became diploidized, in Salmo irideus and Coregonus lavaretus tetrasomic phenotypes occur. In Coregonus, the predominance of heterozygotes is to be interpreted as the consequence of preferential pairing of meiotic chromosomes endowed with identical alleles. These findings give some insight in the diploidization mechanism which may have played an important role during evolution of higher vertebrates.

In swine, the enzyme sorbitol dehydrogenase exhibits a genetically determined polymorphism as identified by multiple electrophoretic bands suggesting a tetrameric structure (Op't Hof, 1969). In order to obtain further information on the genetics of this polymorphism, a number of other vertebrate species were examined. Multiple alleles at the SDH gene locus were found in the Cyprinid fishes Leuciscus and Rutilus, and in the Salmonid fish Salmo trutta. The electrophoretic patterns found in these species are compatible with the model of a tetrameric structure of the enzyme. This model was further verified by dissociation-reassociation studies with the homomeric enzymes of swine and Leuciscus. Since in the heterozygote an intensity gradient of the isozymes was seen, the three phenotypes observed in swine were further characterized by means of activity measurements and application of different substrates. The results point to a differential activity of the two different homomeres.

Following provision of sucrose to starved, stationary phase pea root meristems, G1and G2cells enter DNA synthesis and mitosis, respectively. Puromycin (450 μg/ml) and cycloheximide (5 μg/ml) completely prevent this initiation of progression through the cell cycle. Actinomycin D (10 μg/ml) has no effect on the initial entry of G1and G2cells into S and mitosis, although later entry is prevented. The resistance of the cells to actinomycin D is lost slowly with time in medium without sucrose, suggesting that an RNA required for the resumption of proliferative activity is being gradually lost. The effects of the inhibitors on transitional and proliferative phase meristem cells indicate that such dividing cells do indeed have sufficient of the requisite RNA for 8‐12 hr progression through the cycle, but that protein synthesis is required continuously. It is suggested that this RNA is the one lost slowly during starvation, allowing starved cells to reinitiate progression through the cycle in the presence of actinomycin D.

Two callus tissues, one composed of diploid and the other of a mixture of diploid and polyploid cells, were derived by culturing 1‐mm pea root segments; the mixed callus tissues were obtained by incorporation of kinetin in the culture medium. The callus tissues were used to determine (a) if cell proliferation was altered with the change in cell constituents of a callus; (b) the rate at which polyploid cells increased after kinetin stimulation; (c) the nature of the mitotic cycle in the diploid and mixed polyploid callus tissues; and (d) if the mitotic cycle changed as the tissue aged. Histological, cytological, radioisotope, and radioautographic analyses were made on callus tissue ranging from 1 to 4 days old. The results indicated that gross morphological changes were associated with the anatomical location of the proliferative cells. They showed that the percentage of polyploid division figures after stimulation by kinetin increased rapidly during the first 6 to 7 days in culture and then continued to increase at a much reduced rate. Cell counts revealed that cell proliferation in the mixed callus tissue was initially delayed when compared with the diploid tissue, but that after the delay was overcome cell number increased in each in similar manner. Analysis of the number of DNA‐synthesizing cells showed that their percentage was highest during the first 2 days of culture and then leveled off at a value of about 10%. Mitotic cycle analysis indicated that it could be accurately measured only in the younger diploid callus tissues and that it increased in variability with increased age.

During the placement of a mould for a hearing-aid by a hearing-aid dispenser, moulding material entered the middle ear through pre-existent perforations in the tympanic membrane in both ears. Besides hearing loss, there were no other symptoms. Surgical removal of the moulding material by tympanotomy was necessary, and was complicated by encirclement of the ossicles by the material. All the material could be removed and the hearing was saved. Recommendations for an improved procedure of mould-making are made including more detailed information of the otoscopic findings at the prescriptions for hearing-aid moulds.

The 6-PGD isoenzyme system in the four Ungulata species horse, cattle, swine and sheep is controlled by one autosomal gene locus. At this locus two alleles have been found in the horse and swine.

Within 2455 individuals two have a faster moving variant 6-PGD AF. The zymogram is similar to the Friendship variant but not altered after addition of NADP to the starch gel. The allele is segregating in the two families. The gene frequency of 6-PGDf is 0.0004.

Zusammenfassung 1. Die MDH-Aktivität von Herz-, Carotis-, Lebervenen- und Milzvenenblut bei 30 Rindern wurde mit Hilfe der optischen Methode nachKubowitz undOtt getestet. Dabei zeigten sich signifikante Unterschiede zwischen Milzvenenblut einerseits und Herz-, Carotis- und Lebervenenblut andererseits.

Zusammenfassung Es wird über die verschieden starke Schutzwirkung pathologischer Blutseren auf die Bienengifthämolyse berichtet. Von 80 Seren durchweg schwerer Fälle zeigten 20 Seren eine deutliche Abschwächung der Hämolysehemmung, während ein Serum eine deutliche (8fache) Verstärkung der Hämolysehemmung aufwies. Die anderen Seren verhielten sich normal.

A method is described for detecting the enzyme sorbit dehydrogenase (SDH) after starchgel electrophoresis. Activity of this enzyme occurs predominantly in liver, kidney, thyroid and testis. It was found that this enzyme exists in multiple molecular forms. Three electrophoretic phenotypes were observed apparently representing two different homozygotes and the corresponding heterozygote. These findings can be interpreted in terms of two alleles at one SDH locus.

The 6-PGD phenotypes of 404 unrelated individuals from South-Western Germany were determined. The frequency of the 6-PGBb1allele is 0.031.

In addition to the loci excluded from the distal part of the short arm of chromosome 5 in the literature the genes coding for PGM1, LDH B, and 6-PGD B do not lie on this segment.