and NEGRUTIU, I. 1990. Karyotyping Melandrium album, a dioecious plant with heteromorphic sex chromosomes. Genome, 33: 556-562. Mitotic metaphase chromosomes of Melandrium album obtained from root protoplasts were studied. Morphologically, the chromosomes were either metacentrics or submetacentrics. They were classified into three distinct groups: group A comprising six pairs of autosomal metacentrics, group B comprising five pairs of autosomal submetacentrics, and the sex chromosomes: X and Y. The X chromosome is a metacentric (r = 1.44), which accounts for more than 14% of the genome. The Y chromosome is a metacentric with, virtually, equal arms (r = 1.09) and accounts for 21 % of the genome, being the largest of the complement. The Y:X ratio was 1.4. Ethidium bromide, caffeine, and vinblastine were used to obtain a better resolution and higher frequency of satellited chromosomes 7q and 9p. The proposed karyotype of M. album is 2n = 24, XX, s(7q;9p) for female and 2n = 24, XY, s(7q;9p) for male plants. Nucleolus organizer regions (NORs) were present at the telomeric sites of three chromosome pairs: 7q, 9p, and lop. The NORs were polymorphic, particularly between the nonhomologous chromosomes. The in situ hybridization technique localized the rRNA genes on four chromosome pairs: 5p, 7q, 9p, and lop. The discrepancy between the NORs and the hybridization signals was probably due to the fact that NORs were restricted only to transcriptionally active rRNA genes. It was concluded that for a complete description and characterization of rRNA genes, both NOR detection and in situ hybridization techniques, as cpplementary methods, shouldfie e m p l y d . J I / J Key words: Melandrium album, karyotype, satellites, idiogram, nucleolus organizer regions, in situ hybridization. CIUPERCESCU, D. D., VEUSKENS, J., MOURAS, A., YE, D., BRIQUET, M., et NEGRUTIU, 1. 1990. Karyotyping Melandrium album, a dioecious plant with heteromorphic sex chromosomes. Genome, 33 : 556-562. Les chromosomes en metaphase mitotique de Melandrium album ont ete etudies dans des protoplastes de racines. Morphologiquement, ces chromosomes sont soit metacentriques, soit submetacentriques. 11s ont ete classes en trois groupes distincts : un groupe A comprenant six paires d'autosomes metacentriques, un groupe B constitue de cinq paires d'autosomes submetacentriques, et les chromosomes du sexe X et Y. Le chromosome X est un metacentrique (r = 1,44) representant plus de 14% du genome. Le chromosome Y est metacentrique avec bras virtuellement egaux (r = 1,09); il represente 21010 du genome, etant le plus gros du complement. Le ratio de X:Y est de 1,4. Du bromure d'ethidium, de la cafeine et du vinblastine ont ete utilises pour obtenir une meilleure resolution et une frequence plus elevee des chromosomes 7q et 9p, porteurs de satellites. Le caryotype propose pour le M. album femelle est 2n = 24, XX, s(7q;9p) et, pour le miile, 2n = 24, XY, s(7q;9p). Les regions organisatrices de nucleoles (RONs) ont occupe des sites telomeriques chez trois paires de chromoso...

SummaryThe average cellular positions of the ftsQAZ region (2 min) and the minB region (26.5 min) during the cell cycle was determined by fluorescent in situ hybridization using the position of oriC as a reference point. At the steady-state growth conditions used, newborn cells had replicated about 50% of the chromosome. By measuring the distances of the labelled oriCs with respect to mid-cell, we found two well-separated average oriC positions in cells of newborn length. These average oriC positions moved further apart along with cell elongation. The cellular position of the ftsQAZ gene region resembled the position of oriC, although its average position was closer to mid-cell. In contrast, a single minB focus was observed at cell birth. Separated minB foci appeared towards the end of DNA replication. The average positions of oriC, ftsQAZ and minB relative to each other fitted a model in which DNA replication takes place in the cell centre and subsequent gene regions pass sequentially through this centre. We have interpreted the polarized orientation of the studied gene regions as a consequence of the mode of DNA segregation.

Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, platinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyrin-labeled streptavidin conjugates were evaluated for DNA in situ hybridization on metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not only in mammals but also in plants. A modular- based time-resolved microscope was constructed and used for the evaluation of porphyrin-stained samples. The time-resolved module was found suitable for detection of antigens and DNA targets in an autofluorescent environment. Higher image contrasts were generally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase).

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The preparation of stable chromosome suspensions and flow cytometric sorting of both the Y sex chromosome of the white campion, Melandrium album, and the deleted Y chromosome of an asexual mutant, 5K63, is described. The principle has been to maintain transformed roots in vitro, synchronise and block mitosis, reduce cells to protoplasts, and lyse these to release chromosomes. Such in vitro material, unlike many cell suspensions, showed a stable karyotype. Factors critical to producing high-quality chromosome suspensions from protoplasts include osmolality of isolation solutions and choice of spindle toxin and of lysis buffer. Agrobacterium rhizogenes transformed young growing root cultures were synchronised at Gl/S with 50 pM aphidicolin for 24 h and released to a mitotic block with 30 pM oryzalin for 11 h. Protoplast preparations from such tissue routinely had metaphase indices reaching 15%. Suspensions of intact metaphase chromosomes, with few chromatids, were obtained by lysing swollen mitotic protoplasts in a citric acidldisodium phosphate buffer. Except for the presence of clumps of autosoma1 chromosomes near the X and Y chromosome zones, monoparametric histograms of fluorescence intensities of suspensions stained with 4',6-diamidino-2-phenylindole showed profiles similar to theoretical flow karyotypes. Two types of Y chromosomes, one full-length and one partially deleted (from the asexual mutant), could be sorted at 90% purity (21-fold enrichment of Y). These results are discussed in the context of sex determination and differentiation in higher plants. The availability of purified individual chromosomes facilitates the study of molecular properties of eukaryotic genomes. This is based mainly on the enrichment of potentially interesting DNA as compared with the total genomic content. The construction of chromosome-specific DNA libraries greatly facilitates the isolation and ordering of DNA sequences located on these chromosomes. Flow cytometry offers the possibility to isolate specific chromosomes. Individual chromosomes differing sufficiently in DNA content (generally proportional to relative length) and/or base pair composition are represented as separate peaks in the flow karyotype (18).

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Anther-derived plants of Melandrium album were analysed by flow-cytometric, phenotypic, cytological, and genetic means: more than 90% were classified as females, while no male plants were observed. Instead, one hermaphrodite and several atypical female parental plants were identified. One highly androgenic plant (M5045) turned out to possess two extra autosomes (2n = 26,XY) instead of the ordinary male formula, 2n = 24 (24,XY). The results are discussed in light of possible developmental interactions among the three classes of chromosomes in this dioecious species: male (Y), female (X), and autosomes.Key words: Melandrium album, dioecious, haploids, sex chromosomes, embryogenic development, hormones.