Autoradiographic study of the effect of 5-aminouracil on the S phase and mitosis of barley root meristems

The effect of 5-aminouracil on the S phase and mitosis in root meristems of barley embryos cultivated in the liquid nutrient solution was followed. Embryos were cultivated in different concentrations of 5-aminouracil (200 ppm, 400 ppm and 750 ppm) for 48 h. The drug postponed the onset of mitosis. In the lowest concentration used, synchronization was observed even in the presence of 5-aminouracil. In higher concentrations, mitosis was suppressed irregularly with increasing concentration. 5-aminouracil slowed down the rate of DNA synthesis during S phase and prolonged the S phase, as measured by the utilization of [³H] thymidine. The drug does not influence considerably the entry of cells into the S phase. The transition from G₂ to mitosis is delayed in the presence of 5-aminouracil, especially in higher concentrations. After prolonged treatment with 5-aminouracil, all the effects of the drug on the mitotic cycle decrease continuously.     

Autoradiographic studies of human chromosomes

Chromosomal sites of DNA synthesis during the final 30 minutes or less of the S-phase of the cell division cycle of fibroblasts were delinated autoradiographically. Very light labeling was found, indicating that a recognizable but very minor portion of the cell's DNA is synthesized during a few minutes at the extreme end of S. This interval immediately follows those periods near the end of S when prominent synthetic asynchrony exists in different chromosomal regions. A non-random distribution of label, but one different from the more familiar end-of-S pattern, was detected during this final interval. The late-replicating X was less heavily labeled than some autosomes during the final minutes of S, while sites in chromosome No. 3 were somewhat more heavily labeled than those in other chromosomes. The biological significance of these minute, last-to-replicate chromosomal regions is unknown.     

Autoradiographic studies of human chromosomes

Certain differences in the location of chromosomal material completing DNA synthesis late in the S-period of the cell cycle were demonstrated when a comparison was made between human blood lymphocytes and epithelial cells derived from term amnion grown in vitro for short periods of time. The differences in the patterns of synthesis between these two differentiated diploid cells, each from the same species but of different embryonic origins (mesodermal vs. ectodermal), functions in vivo, and appearances and growth characteristics in vitro, may be reflections of distinctive patterns of condensed interphase chromatin, i.e. a characteristic distribution of heterochromatin, and possibly also of different cellular functions in the organisms.Supported by research grants from the U.S. Public Health Service (HD 04134) and the National Science Foundation (GB 6282). These data were presented at the Fourth Basel Colloquium on Mammalian Sex Chromosomes in Differentiation and Development, March, 1967.     

Autoradiographic studies of human chromosomes

The DNA content of the elongated long arm of an inherited variant No. 16 chromosome was compared with that of the non-elongated long arm of the other No. 16 in the same chromosomal complement. An indirect method of estimation of DNA content was employed, based on the number of autoradiographic grains produced by the segments after they had been labeled with H³-thymidine throughout an S-period. The method proved adequately sensitive to detect a difference in number of grains—and presumably in DNA content—between the short arm and the long arm of a normal chromosome No. 16. The failure to detect an increased number of grains over the elongated long arm of the variant No. 16, in comparison with the other No. 16's long arm in the same cells, favors explanations other than an increase in content of DNA to account for this well-known morphological variation of the human No. 16 chromosome.This research was supported by National Institutes of Health Grant HD 04134-01 and American Cancer Society Grant E-461.     

Effects of 5-Fluorodeoxyuridine and Related Halogenated Pyrimidines on the Sand-Dollar Embryo

The embryo of the sand-dollar (Echinarachnius parma) was exposed to various concentrations of fluorinated pyrimidines immediately after fertilization. FUDR (5-fluorodeoxyuridine) was most active, and a concentration of 2 to 4 mγ/10 cc. (0.8 to 1.6 x 10^-6 m.eq./liter) blocked development at the early blastula stage. Larger doses interrupted development at the same stage. This effect was prevented by thymidine (TDR) and thymine (T); and these pyrimidines protected against many times the minimal lethal concentration of FUDR. TDR was active as a protective agent if added just before early blastula formation. The other fluorinated pyrimidines, 5-fluorouracil (FU), 5-fluorouridine (FUR), 5-fluorocytidine (FCR), 5-fluorodeoxycytidine (FCDR), and 5-fluoroorotic acid (FO), were also studied. These drugs produced effects on embryonic development similar to those seen with FUDR. The effective concentrations, however, varied greatly. T and TDR provided protection against these drugs, but in most cases they were not so effective as against FUDR. 5-Bromodeoxyurdine (BrUDR), beginning at the early blastula stage, caused a random pattern of embryonic death up to the pluteus stage. This drug has been shown to be incorporated into bacterial DNA. BrUDR protected embryos against the early lethal effects of FUDR presumably acting as a thymidine substitute, but the embryos died subsequently in a pattern similar to that seen with BrUDR alone. FUDR and BrUDR appear to inhibit the formation and alter the structure of DNA, respectively, distinctive effects whch may provide a means for studying the role of DNA in embryonic development.     

Effects of 5-Fluorouracil and 5-Fluorodeoxyuridine on Growth and Tumor-Inducing Ability of Agrobacterium tumefaciens

Beardsley, Robert E. (Manhattan College, Bronx, N.Y.), and Jacques Lipetz Effects of 5-fluorouracil and 5-fluorodeoxyuridine on growth and tumor-inducing ability of Agrobacterium tumefaciens. J. Bacteriol. 92:346-348. 1966.-Agrobacterium tumefaciens B6, grown in the presence of 5-fluorouracil or 5-fluodeoxyuridine, exhibited a prolonged lag phase. The tumor-inducing ability of bacteria grown in the presence of these compounds was decreased even after exposures as short as 40 min. A positive correlation was found between the growth-inhibitory effects of these compounds and their effects on the tumor-inducing ability of the bacteria.     

Telomere-mediated truncation of barley chromosomes

Engineered minichromosomes offer an enormous opportunity to plant biotechnology as they have the potential to simultaneously transfer and stably express multiple genes. Following a top-down approach, we truncated endogenous chromosomes in barley (Hordeum vulgare) by Agrobacterium-mediated transfer of T-DNA constructs containing telomere sequences. Blocks of Arabidopsis-like telomeric repeats were inserted into a binary vector suitable for stable transformation. After transfer of these constructs into immature embryos of diploid and tetraploid barley, chromosome truncation by T-DNA-induced de novo formation of telomeres could be confirmed by fluorescent in situ hybridisation, primer extension telomere repeat amplification and DNA gel blot analysis in regenerated plants. Telomere seeding connected to chromosome truncation was found in tetraploid plants only, indicating that genetic redundancy facilitates recovery of shortened chromosomes. Truncated chromosomes were transmissible in sexual reproduction, but were inherited at rates lower than expected according to Mendelian rules.     

Effect of different concentrations of 5-fluordeoxyuridine on mitosis and chromosomes of barley root meristems

Excised barley embryos were cultivated in a liquid complete medium for 48 h and then 5-fluordeoxyuridine was added. The concentrations of 5-fluordeoxyuridine differed in the range of 6 orders of magnitude (10^-8 to 10^-3 M). All concentrations, except 10^-8 M led to a total inhibition of mitosis during 24 h. 5-fluordeoxyuridine induced chromosomal aberrations of the non-exchange type in all concentrations used. The frequencies of induced breaks increased during the interval of 12 h in whích they were followed and, in samples fixed at later intervals after the beginning of the treatment, there was a tendency to clustering of the induced fragments in some mitotic figures. The most striking feature of the effects oliowed is the relatively small dependence on the concentration used.     

Further evidence for a high position-specific effect in the action of chemical mutagens on the chromosomes of barley

Summary B1 and B2 are small, circular, mitochondrial plasmid-like DNAs found in male-sterile cytoplasm (cms-Bo) of rice. In this study, nuclear sequences homologous to these DNAs were investigated among a number of rice cultivars. Several copies of nuclear B1-and B2-homologous sequences were detected in all examined cultivars, regardless of the presence or absence of the B1 and B2 DNAs in mitochondria, indicating that the existence of the B1- and B2-homologous sequences in the rice nuclear genome was widespread. A restriction fragment length polymorphism (RFLP) was detected for both sequences, and we propose that these DNAs could be useful RFLP markers for the rice nuclear genome. To analyze these nuclear homologues genetically, segregation analysis of the RFLP was carried out in the F₂ progenies of an Indica-Japonica rice hybrid. Of the B1 homologues, there were two nonallelic fragments, one specific to the Indica parent and the other to the Japonica. These results indicate that the B1 and B2 homologues were dispersed in the nuclear genome. The integration of B1-homologous DNA into the nuclear DNA may have occurred independently after sexual isolation of the Indica and Japonica rice varietal groups, or a intranuclear transposition of these sequences took place during the process of rice differentiation into the varietal groups.     

5-Fluorodeoxyuridine inhibition of photoperiodically induced flowering inChenopodium rubrum L.

Flowering in the short day plantChenopodium rubrum was inhibited by 5-fluoro-deoxyuridine (FDU) at a concentration of 4×10^?6 M and higher when applied during photoperiodic induction or immediately afterwards. This inhibition is always accompanied by a general reduction of growth (e.g. a decrease in the first leaf length). The mitotic activity within the shoot apex is completely blocked by FDU application during the photoperiodic treatment. The floral induction (evocationsensu Evans) was not cancelled in this situation as was revealed when reversing the FDU effect by thymidine application. One day after the end of the photoperiodic treatment (the plants were transferred to continuous light again) the FDU inhibition becomes irreparable. The results indicate that DNA synthesis and hence the mitotic activity are not obligatory prerequisites for photoperiodic floral induction inChenopodium. Low concentrations of FDU may promote flowering under suboptimal floral induction.     

Orcein staining and the study of the effect of chemicals on chromosomes

Summary Plant materials of both dicotyledonous and monocotyledonous groups were subjected to treatment with alloxan followed by coumarin, phenols, oxyquinoline etc. for 3 to 4 hours.Root tips were then heated in an orcein-acid mixture for 10 to 40 seconds and studied after mounting in 1% orcein. Clear metaphase plates were obtained after 10 seconds of heating, whereas after 20 and 30 seconds considerable erosion and fragmentation respectively were recorded. Complete loss of stainability resulted after heating for 40 seconds.Fragmentation is caused only by orcein under heat conditions. This is demonstrated by control series checking all variables.Pretreatment with the chemicals stated is of absolute necessity for fragmentation. It is suggested that this pretreatment causes depolymerisation of DNA and lability of nucleoprotein linkage at certain segments from which the DNA becomes detached during subsequent heating.Of all the chemicals tested for pretreatment effects, marked positive results were obtained with alloxan (1 hour) followed by coumarin (2 hours at 16–20 ° C.). Phenols also gave positive results. The other chemicals tried need slightly longer time of treatment for fragment induction.     

Autoradiographic study of the DNA synthesis process along metaphasic chromosomes of Pleurodeles waltlii Michah (urodele amphibian)]

Chromosomes from embryos (stage 34) of the amphibian urodele Pleurodeles waltlii were labeled by 3H-thymidine at different times of the DNA synthesis cycle and studied at metaphase. Terminal segments of the chromosomes, induced secondary constrictions, and satellites were found to be late-labeling. Therefore, they may be considered as formed at least partially by heterochromatin. However, after other treatments or stainings, they do not present other features considered characteristic of heterochromatin. The question of the heterogeneity of their structures is discussed and the ambiguity of the term heterochromatin is emphasized. The proximal part of the long arm of chromosome VII shows characteristics similar to satellites, which may indicate the presence of a previously unrecognized satellite.     

Autoradiographic study on the synthesis of nuclear proteins

Experiments were carried out on cultures of Chinese hamster fibroblasts. Cells were short-labelled with H³-Iysine or H³-tryptophane. The kinetics of silver grain number over interphase nuclei, metaphases, and individual chromosomes of the first pair were studied. — The grain count distribution over chromosomes of different groups and over sister-chromatids was analyzed. The data presented allow us to conclude that: 1. There is equal distribution of nuclear and chromosomal proteins between daughter nuclei and sister-chromatids, resp., during each mitotic cycle. 2. The synthesis of chromosome proteins, in general, and nonhistone proteins, in particular, takes place at all stages of the cycle. 3. At each stage of the cycle, proteins are incorporated into chromosomes of different groups simultaneously and at equal rate. The rate of this process in S and G2 is two times that in G1.