Exfoliative cytologic evaluation of primary cultured lung carcinomas

This paper describes a tissue culture and exfoliative cell culture system that enables one to (1) evaluate the adequacy of primary lung carcinoma cultures for cytogenetic analysis, and (2) predict the likelihood of viable cells and type of differentiation present in the primary lung tumor cultures used for cytogenetics. Primary lung carcinomas were established from explant outgrowths and maintained in serum supplemented or serum free media on plastic or basement membrane associated protein coated dishes in order to obtain cells for karyotypic analysis (Miura et al., 1990). The media from these cultures that would ordinarily have been discarded was aspirated at each media change and used to prepare cytocentrifuge cytology preparations. Papanicolaou stained cells from the preparations were evaluated by cytotechnologists in order to assess (1) the cellularity and presence of cancer cells in the sample, (2) differentiation of the malignant cells, and (3) adequacy for chromosomal studies. We determined that cytology preparations of cell and explant outgrowth cultures from primary lung tumors are a reliable method for screening and evaluating the suitability of primary lung carcinoma cultures for cytogenetic analysis.Supported in part by an NCI grant CA-45745 (JRT). JRT is a Scholar of the Leukemia Society of America.     

Artificial hybridization in the Hawaiian endemic genus Labordia (Loganiaceae)

Cross-pollinations were performed within and among eight species of Labordia from Oahu, Molokai, and Hawaii, and one species from Guam of the closely related genus Geniostoma. Detailed floral examination confirmed that the species are functionally dioecious, i.e., a given individual lacks either ovules or pollen grains. Female inflorescences bagged to prevent pollination never produced seed, but intraspecific crosses between male and female individuals nearly always yielded fruits with viable seed (>80%). Interspecific crosses between species from different islands and separate taxonomic sections of the genus also yielded good fruit set (>55%). Interspecific F1 hybrids were vigorous and appeared to be morphologically intermediate to their parents. Intergeneric crosses between Geniostoma and Labordia failed. Chromosome counts from Labordia species were found to be 2n = 80 or 2n =~80, twice the chromosome complement of two collections examined from its postulated ancestor, Geniostoma rupestre. Labordia species are distinct morphologically, ecologically, and geographically but apparently lack genetic barriers to interbreeding. This suggests that geographical and ecological isolation, recentness of colonization, and/or rapid speciation have been important factors in the origin of species of Labordia.     

Conventional karyotype, nucleolar organizer regions and genome size in five Mediterranean species of Syngnathidae (Pisces, Syngnathiformes)

Conventional karyotypes, NOR-bearing chromosomes by means of silver impregnation and genome size were investigated in five Mediterranean species in three genera of the Syngnathidae. A karyotype of 48 subtelocentric-acrocentric chromosomes was found in the seahorse Hippocampus hippocampus (FN=48) while a diploid value of 44 occurred in H. guttulatus (2 sm-m+42 a; FN=46) and the pipefish Syngnathus abaster (44 a; FN=44) and S. typhle (44 a; FN=44). The pipefish Nerophis ophidion , possessing a diploid chromosomal set of 58 made up of 50 meta-submetacentric and eight subteloacrocentric elements (FN=108) and a genome size three to four times larger than those known to date, differs cytogenetically from all other Syngnathids studied so far. A single pair of active NOR-bearing chromosomes was found in both species of the genus Hippocampus while in Syngnathus and Nerophis species more than two silver positive chromosomes were found to be involved in nucleolus organization giving rise to NOR-bearing chromosome polymorphism. The possible evolutionary routes of quantitative and qualitative changes in chromosome and DNA are discussed. The resulting phylogenetic scheme is shown to coincide with that constructed from morphological characters.     

小麦K、V型胞质雄性不育育性恢复的细胞学研究

以Ｋ、Ｖ型１Ｂ／１Ｒ不育系分别和四个非１Ｂ／１Ｒ恢复系杂交,观察了异质杂种小麦的花粉母细胞减数分裂。以Ｔ型细胞质和普通小麦胞质作比较,研究了Ｋ、Ｖ型异源细胞质对１Ｂ·１Ｂ／１Ｒ杂合核型染色体配对的影响。实验结果表明,Ｋ、Ｖ胞质背景下,育性恢复度与１Ｂ·１Ｂ／１Ｒ杂合核型染色体配对行为不直接相关。减数分裂中期Ⅰ、Ｋ、Ｖ、Ｔ型异源细胞质对杂合核型染色体配对的影响随父本遗传背景不同而异。后期Ⅰ,三类异质小麦杂种Ｆ１代均出现了不正常分离,后期Ⅰ和后期Ⅱ出现了落后染色体,四分体时期出现了微核。三类异源细胞质对落后染色体率和微核率的影响因不同遗传背景而异。     

Metaphase karyotypes and G-banding in sandflies of the Lutzomyia longipalpis complex

Mitotic metaphase chromosomes (2n = 8) from brain cells of fourth instar sandfly larvae of four geographical strains of the Lutzomyia longipaplis complex were examined microscopically, with bright-field illumination, after staining by a new G-banding technique involving exposure of air-dried chromosome preparations to quinacrine and ultraviolet light. Differences of G-banding and/or position of the centromere on chromosome 4 (the smallest chromosome pair) distinguished four putative sibling species from Costa Rica, Colombia and Brazil (distinctive populations from Jacobina and Lapinha Caves). The karyotype of the population from Jacobina, Brazil, showed an apparently plesiomorphic pattern of G-banding. On the basis of their recognizably different mitotic karyotypes, cytogenetic identification of separate taxa in the L. longipalpis complex should be useful for specific female vector competence and ecology studies.     

The evolution of hypervariable sex and supernumerary (B) chromosomes in the relict New Zealand frog,Leiopelma hochstetteri

Chromosomes exhibiting elevated levels of differentiation are termed hypervariable but no proposed mechanisms are sufficient to account for such enhanced evolutionary divergence. Both hypervariable sex and supernumerary (B) chromosomes were investigated in the endemic New Zealand frog, Leiopelma hochstetteri, which is chromosomally polymorphic both within and between populations and has sufficiently elevated variation that different populations can be identified solely by their C-banded karyotypes. This frog is further distinguished by the univalent, female-specific W-chromosome (0W/00 sex determination) uniquely possessed by North Island populations. This sex chromosome exhibited variation in morphology, size, and heterochromatin distribution, sufficient to resolve 11 different types, including isochromosomes. Five of the 12 populations examined also had supernumerary chromosomes that varied in number (up to 15 per individual) and morphology. Specific variations seen among the hypervariable chromosomes could have resulted from heterochromatinisation, chromosome fusions, loss-of-function mutations, deletions, and/or duplications. Frogs of the same species from Great Barrier Island, however, had neither supernumeraries nor the female-specific chromosome. The 0W/00 sex chromosome system must have been derived after the isolation of Great Barrier Island from North Island populations by raised sea levels between 14 000 and 8000 years ago. Furthermore, biochemical divergence between populations is minor and therefore the chromosomal variation seen is comparatively recent in origin. The one characteristic common to all known hypervariable chromosomes is curtailment or lack of recombination. Their accelerated evolution therefore is possible via the mechanism of Muller's ratchet, either alone or in concert with other factors.     

Effect of Fixation on Interphase Cytogenetic Analysis by Direct Fluorescence in Situ Hybridization on Cell Imprints

The direct fluorescent in situ hybridization (FISH) technique using a centromere-specific DNA probe for chromosome 11 was applied on fresh tissue imprints from melanomas and pituitary adenomas. The cell imprints were fixed in acetone and formalin, and the influence of fixation was evaluated. Acetone fixed imprints gave a sharp and intense fluorescent signal in a large number of cells from both tumors without any pretreatment. In contrast, formalin fixed imprints disclosed a weak hybridization signal in a few cells even after careful enzymatic digestion. Direct FISH on acetone fixed cell imprints represents a simple and time saving technique applicable to any laboratory for the study of numerical chromosomal abnormalities.     

An Improved Method for Chromosome Counting in Maize

An improved method for counting chromosomes in maize (Zea mays L.) is presented. Application of cold treatment (5C, 24 hr), heat treatment (42 C, 5 min) and a second cold treatment (5C, 24 hr) to root tips before fixation increased the number of condensed and dispersed countable metaphase chromosome figures. Fixed root tips were prepared by the enzymatic maceration-air drying method and preparations were stained with acetic orcein. Under favorable conditions, one preparation with 50-100 countable chromosome figures could be obtained in diploid maize using this method. Conditions affecting the dispersion of the chromosomes are described. This technique is especially useful for determining the somatic chromosome number in triploid and tetraploid maize lines.     

Banding Human Chromosomes Using a Combined C-Banding-Fluorochrome Staining Technique

Slides pretreated for C-banding and stained with DAPI or CMA3 show different banding patterns in human metaphase chromosomes compared to those obtained with either standard Giemsa C-banding or fluorochrome staining alone. Human chromosomes show C-plus DA-DAPI banding after C-banding plus DAPI and enhanced R-banding after C-banding plus Chromomycin A3 staining. If C-banding preferentially removes certain classes of DNA and proteins from different chromosome domains, C-banding pre-treatment may cause a differential DNA extraction from G- and R-bands in human chromosomes, resulting in a preferential extraction of DNA included in G-bands. This hypothesis is partially supported by the selective cleavage and removal of DNA from R-bands of restriction endonuclease HaeIII with C-banding combined with DAPI or Chromomycin A3 staining. Structural factors relating to regional differences in DNA and/or proteins could also explain these results.     

When is a species not a species? Uncoupled phenotypic, karyotypic and genotypic divergence in two species of South African laminate-toothed rats (Murinae: Otomyini)

Chromosomal polytypy, morphological conservatism and absence of data have frustrated the taxonomic revision of two species of southern African-endemic laminate-toothed rats ( Otomys irroratus and Otomys saundersiae s.l.). New cytogenetic (G-banding and fluorescence in situ hybridization), DNA sequence [cytochrome b (cyt b ) gene] and geometric morphometric data demonstrate the synonymy of O. saundersiae from Grahamstown (Eastern Cape, South Africa) under O. irroratus , and the validity of Otomys karoensis from the Fynbos Biome of the Western Cape. Phenotypic dimorphism in pelage colour and cranial morphology in O. irroratus from the climatically unpredictable Albany Thicket (=Savanna) Biome of the Eastern Cape results from the retention of allometric paedomorphic traits in some adults ( saundersiae morph) but not others. The same paedomorphic traits are associated with speciation and karyotypic and genetic differentiation in O. karoensis . Within O. irroratus , two phenotypically and genotypically (cyt b divergence=6.4%) divergent lineages correspond with the Fynbos/Albany Thicket and Grassland biomes. Incipient speciation in O. irroratus seems to be associated with ecology rather than karyotype.