ALBERT

Notes: Flow cytometry, which requires individualized or single cell preparations, is particularly adapted to studies of protoplast biology. It has been developed at three levels: descriptions of populations, measurement of cell function, and positive or negative selection of rare protoplast types. It enables measurement of several parameters in single protoplasts at a high rate of analysis. Without protoplast purification, excellent resolution of subpopulations is still possible. The quantitative data obtained on each protoplast normally have relative units but absolute calibration of intensity scale is available. Specific probes allow one to correlate multiparameter data on each cell. DNA, RNA, proteins, metabolites, organelles, virus and plasmid content of protoplasts have been described.The essential conceptual advantage of flow cytometry is the processing of heterogeneous material, typical of biological samples, but inadequately handled through overall mean values. Biological properties of protoplast subpopulations are followed using fluorescence intensity as a measure of DNA versus RNA or protein synthesis, cytoplasmic and nuclear antigens, membrane potentials, membrane viscosity, cell wall regeneration and pH.

Notes: Abstract Acridine orange simultaneously stains DNA and RNA. Using flow cytometry, synthesis of these nucleic acids can be related throughout a culture time-course. This technique has been used with nuclei isolated from Petunia hybrida protoplasts during 48 h of culture. Nuclear RNA content has been evaluated with respect to DNA levels, namely the cell-cycle phase. Nuclear RNA synthesis was not dependent upon exogeneous hormones during the first 18 h of culture, but either auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) or cytokinin (N6-benzyladenine) were necessary for entry into the S phase. Cytokinin alone could stimulate maximal RNA synthesis within each cell-cycle phase up to 24 h. In complete medium, DNA synthesis only began from a phase “G1B” having substantial RNA, although a subnormal amount of RNA (in protoplasts cultivated only with 2,4-D) did not prevent protoplast entry into the S phase. However, both hormones were necessary for highest RNA levels and G2 frequencies after 48 h. As in mammalian cells, the mean RNA level in plant 4C nuclei is double that of 2C nuclei. G2 nuclei are larger than G1 nuclei, but upon activation G1 nuclei in fact diminsh in size. This study aimed to identify restriction points in the cell cycle as affected by growth regulators and the specific synthesis of nucleic acids. For example, the RNA levels induced by N6-benzyladenine, although similar to those in complete medium, were not sufficient to induce mitosis. Conversely, 2,4-D action was probably limited by low nucleotide synthesis in the absence of cytokinin.

Notes: [Auszug] Chromosome imprinting is the process by which one of two genetically homologous chromosomes is predetermined to function differently from the other at a subsequent stage in development. In the coccid insects, imprinting occurs in the egg, at the time of fertilisation; it probably occurs at the same ...

Notes: [Auszug] Except for a few tissues, the paternal set maintains its heterochromaticity throughout the life of the male; at spermatogenesis it is segregated from the maternal, or euchromatic, set, and is not included in the sperm. In certain related forms, however, the heterochromatic state is reversed during ...

Notes: [Auszug] In the Coccoidea, the haploid chromosome number ranges from 2 to 20 (ref. 1). Since the basic number for the superfamily is in the range of n - 5 to 7, both increase and decrease have occurred in the evolution of the karyotype. The chromosomes of the coccids lack localized centromeres and fragments ...

Notes: [Auszug] Fig. 1. Fibroblast-like testis cells of Potorous tridactylis in tissue culture Fig. 2. Epithelial-like kidney cells of the same animal in tissue culture Fig. 3. Somatic chromosome complement of a tissue culture cell of the male Potorous tridactylis. A, the X, B, the Yl and C, the F2 chromosomes ...

Notes: Summary 1. Chromosome behavior at spermatogenesis has been described for one species each of three families of coccids not previously studied in this regard: Cerococcus quercus Comstock of the Asterolecaniidae, Conchaspis lepagei Hempel of the Conchaspidae, and Tachardiella sp. of the Lacciferidae. All three families belong to Balachowsky's “lecanoid” section of the coccid superfamily, and the first two have been suggested, by various systematists, as close allies of the armored scales (Diaspididae). 2. All three species showed lecanoid chromosome behavior, as originally described by the Sohraders, in that one haploid set of chromosomes is heterochromatic in the males and eliminated at spermatogenesis. The exceedingly clear cytology of C. quercus permitted a detailed study of the critical second division stages when the euchromatic and heterochromatic sets are separated. Although considerably more difficult cytologically, both the other species showed a typical lecanoid pattern of behavior; in addition, both showed an accessory structure, of unknown origin and function, which was present during the meiotic sequence. 3. The cytological and taxonomic implications of the results are considered, largely on a comparative basis, in the discussion. Special attention is given to the problem of the origin of the armored scales and also to Buchner's recent suggestion of the possible influence of symbionts on sex determination.

Notes: Abstract A survey of chromosome systems in the Eriococcidae shows that many of them are intermediates between two systems occurring elsewhere in the coccids. These two systems differ in the number of paternal chromosomes which remain heterochromatic during spermatogenesis and are then discarded. In the lecanoid system, all are discarded; in the Comstockiella system only one, the DH chromosome is discarded. Evolutionary steps from the lecanoid to the Comstockiella systems had previously been postulated with the intermediates having from n-1 down to 2 chromosomes remaining heterochromatic during spermatogenesis. The transition was believed to be reversible and the systems therefore inherently unstable. Most of the armored scales with Comstockiella systems have not shown intermediacy but one case has been recently reported. The eriococcids as a family are characteristically intermediate with frequently extreme variation from cyst to cyst in number of chromosomes remaining heterochromatic. Species with an unmodified Comstockiella system may occur but are in a minority. One unanticipated system is reported for two species, the “complete” Comstockiella system in which all the heterochromatic chromosomes are reversed at spermatogenesis; this system is also theoretically unstable. Indirect evidence for the great antiquity of the eriococcids is presented, and the question raised as to the maintenance of instability over several epochs. Comparative cytology may provide part of the answer to this question. The most common, and probably the basic diploid chromosome number of the eriococcids is 18, with a range from 12 to 28 and about 48; decreases in number are more frequent than increases.

Notes: Abstract Conventional types of cytogenetic studies with the mealybug, Planococcus citri (Risso), are possible with the use of genetic markers and meiotic analysis in the female. The loci of an eye-color mutant, salmon, and a wing-shape mutant, banjo, are linked with about 22 per cent recombination. These markers have been used in the identification and maintenance of lethals and rearrangements. All the cytologically identifiable rearrangements have proved to be reciprocal translocations, some symmetric, others, grossly asymmetric or otherwise complicated. No simple breakage products have been recovered. On the basis of their effects on crossing over, some of the lethals are believed to be associated with small rearrangements. The bivalents normally have one chiasma; only 1.2 per cent have two. Interference is decidedly decreased in chiasma formation in translocation heterozygotes, and in genetic recombination with suspected small rearrangements associated with lethals; it is also decreased, but less markedly, in genetic recombination with lethals in translocations. These various results are discussed in relationship to the holokinetic nature of the coccid chromosome, and natural increases in coccid chromosome number, as well as in regard to the effect of rearrangements on interference.

Notes: Summary The Comstockiella chromosome system occurs in the armored scale insects and the closely allied palm scales. During development of the males, the paternal chromosome set becomes heterochromatic and remains so until spermatogenesis. With the exception of one chromosome, the heterochromatic complement loses its differential aspect during early spermatogenesis and its members pair with their euchromatic homologues There is but one division during which the two components of each bivalent separate to opposite poles. Both division products form sperm. One pair of chromosomes, the D pair, always shows differential behavior. The D pair usually does not form a bivalent. The heterochromatic homologue, DH, divides equationally and is eliminated by anaphase lagging or telophase ejection; its daughter halves remain as pycnotic residues during the early phases of spermiogenesis. The euchromatic homologue, DE, also divides equationally to contribute to both of the telophase nuclei. Compensation for the division of the DE univalent may occur during either the early or late phases of spermatogenesis. In some species the D pair is a fixed entity, analogous to the sex chromosomes in this regard. In other species, more than one pair may be elected to the D role, but only one at a time, and always the same one within each cyst. Taxonomic evidence indicates the Comstockiella system was derived from the lecanoid system, previously known from the work of the Schraders and others. In the lecanoid system, the paternally derived heterochromatic set divides equationally, along with the euchromatic set, during the first spermatogenic division. During the second spermatogenic division, the two sets are segregated from each other. The two euchromatic derivatives form sperm while the heterochromatic derivatives persist for a while as pycnotic residues. Both the lecanoid and Comstockiella systems occur in some species often in the same testis, but only one of the two systems within any one cyst. The discussion is devoted to an analysis of the mode of inheritance expected in the Comstockiella system and its evolutionary derivation. The Comstockiella system may have been derived in a step-by-step fashion from the lecanoid. The two systems differ by four processes which occur at spermatogenesis in the Comstockiella but not the lecanoid system; these are (1) deheterochromatization, (2) chromosome pairing, (3) compensation for the extra division of the DE chromosome, and (4) lagging or ejection to eliminate the DH chromosome. In addition, the residual genetic effects of the heterochromatic set may have undergone considerable change before the lecanoid system could evolve into a Comstockiella. Once the evolutionary step were otherwise possible, mechanistic features would aid and abet the emergence of the new system even though it lacked immediate selective advantage. The variable-D aspect of some examples of the Comstockiella system cannot be readily understood in terms of known examples of chromosome behavior; an admittedly highly speculative hypothesis is offered in an attempt to explain the situation. The diaspidid system, in which the paternal chromosomes are eliminated at late cleavage, is believed on taxonomic grounds to have stemmed from the Comstockiella, and forms the final stage of the four-step evolutionary sequence. Necessary changes for the derivation of the diaspidid system from the Comstockiella are discussed.