Preparation of Chromosomes of Tetrahymena Pyriformis for Photomicrography

Conjugating animals of the protozoan, Tetrahymena pryiformis, were affixed to cover slips by means of Nissenbaum's fluid, followed immediately by 1:3 acetic-alcohol for 18–24 hr. After fixation, the material was transferred through a descending alcohol series to water, then hydrolyzed in 1 N HCl, washed in water, followed by immersion in 45% acetic acid and subsequent mounting in aceto-carmine. Photomicrographs were made using a phase-contrast microscope and Microfile film. The schedule resulted in preparations with abundant material, adequate spacing of chromosomes in a single plane, and excellent differentiation of the chromosomes from the cytoplasm.     

Quinacrine Fluorescence for Identifying Metaphase Chromosomes, with Special Reference to Photomicrography

Metaphase chromosomes of humans and other species can be identified by their quinacrine fluorescence patterns. For the study of human chromosomes, standard phytohemagglutinin-stimulated leukocytes are used. A 45-60 min interval of colchicine treatment is used to obtain a higher proportion of extended chromosomes with contiguous chromatids which have generally proved to be most informative. Slides are stained with 0.5% quinacrine for 6 min, rinsed in running tap water for 3 min, and rinsed and mounted in a Tris-maleate buffer at pH 5.6. For photomicrography, a microscope with an HBO 200 w high-pressure mercury vapor lamp, 3 mm BG 12 excitor filter, darkfield condenser, fluorite 95x objective, K510 barrier filter and a nonautomatic exposure microphotographic attachment with an 8x eyepiece is used. With this system, images of adequate brightness reach the film plane, thus making possible the use of fine-grain, high definition 35 mm films, such as Kodak Panatomic-X and H & W Control VTE panchromatic. Photographic prints of moderately high contrast are used for preparing karyotypes in which every human chromosome can usually be readily identified.     

Preparation of Insect Chromosomes for Immunolabeling

We describe a method for isolating chromosomes from testes of the desert locust, Schistocerca gregaria, and their subsequent incubation with antibodies directed against chromosomal proteins. The procedure involves hypotonic pretreatment of the germ cells, centrifugation onto coverslips in a cytocentrifuge and immunolabeling, while still unfixed, using a chromatin-stabilizing buffer. In the present case, an antibody specific for the acetylated isoforms of his tone H4 was tested. After the antibody treatment, the preparations are fixed using formaldehyde, stained with a DNA-specific fluorescent dye and mounted. Analysis of the preparations revealed good preservation of chromosome structure in prophase spermatogonia and late prophase I spermatocytes. Fully condensed chromosomes were not observed and are probably lost during preparation. The bright fluorescence of the autosomes indicates that the reaction between the antibody against acetylated histone H4 and its chromosomal antigen is not impeded. In contrast, the X univalent remained unstained with the exception of a small terminal band. Thus, cytospin preparations of locust germ cells allow high resolution immunolabeling with antibodies against chromosome-associated proteins.     

Interactions of Pseudomonas Aeruginosa Hemagglutinins With Euglena Gracilis, Chlamydomonas Reinhardi, and Tetrahymena Pyriformis

SYNOPSIS The galactosephilic and mannosephilic hemagglutinins of Pseudomonas aeruginosa adsorbed onto Euglena gracilis, Chlamydomonas reinhardi , and Tetrahymena pyriformis . Furthermore, peroxidase binding to the 3 protozoan species was shown to be mediated by these lectins. Binding of Pseudomonas lectins to E. gracilis and C. reinhardi caused their specific agglutination, whereas no agglutination was observed with T. pyriformis , even after treatment by papain or by NaF. Added to the culture medium, the Pseudomonas hemagglutinins stimulated growth of E. gracilis and T. pyriformis due to their binding to these protozoa: this effect was partly inhibited by the specific sugar.     

Ultradian Photosynchronization in Tetrahymena Pyriformis Glc is Related to Modal Cell Generation Time: Further Evidence for a Common Timer Model

This study contains the first report of the photosynchronization of Tetrahymena in the ultradian mode of cell division. Ultradian mode cultures of T. pyriformis GLC were grown at low cell titers in a nephelostat under five different ultradian photocycles and also under constant conditions of illumination.

Entrainment was achieved only when the period of the synchronizer did not exceed the nearest modal generation time observed in free-running single cells. Thus, the discrete ranges for photentrainment of ultradian rhythms in Tetrahymena were restricted to modal windows for the generation times in free-run. Cell division was found to be a function of the phase of the ultradian zeitgeber cycle. The cells did not behave as if they had been forced into synchrony by physiological shock; the synchronous populations obtained by this technique behaved like the populations commonly used in circadian studies, which had been phased by a cyclic variation within the tolerance range of the organism.     

Studies On Cyclic Nucleotide Metabolism In Tetrahymena Pyriformis: Partial Characterization of Cyclic Amp- and Cyclic Gmp-Dependent Phosphodiesterases*

SYNOPSIS. Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in Tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction: the activities were optimal at pH 8.0–9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg²⁺ or Mn^2-. A kinetic analysis of the properties of the enzymes yielded 2 apparent K_III values ranging in concentration from 0.5 to 50 μM and from 0.1 to 62 μ M for cyclic AMP and GMP. respectively. A Ca²⁺-dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, Tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca²⁺, although this activator did not stimulate the phosphodiesterase. the results suggested that Tetrahymena might contain 2 types of Ca²⁺-dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Nullisomic Tetrahymena. II. a Set of Nullisomics Define the Germinal Chromosomes

Crosses of a diploid Tetrahymena thermophila to a strain with a haploid germinal nucleus result in chromosome loss during meiosis in the haploid. The resulting monosomics can be made nullisomic by a special cross that induces homozygosis of a meiotic product of the germinal nucleus, but retention of the parental somatic nucleus. The creation and testing of single nullisomics for three of the five chromosome pairs and a triple nullisomic missing another pair is presented. Taken together, these strains make possible a series of crosses in which all but one of the chromosomes is missing in one parent. This set of nullisomics can, therefore, be used to map any mutation in Tetrahymena to a specific chromosome.     

The Preparation of Congenic Strains of Tetrahymena1

SYNOPSIS. Congenic strains of syngen 1, Tetrahymena pyriformis, were produced by backcrossing the F¹ hybrid between inbred strains C2 and D to strain D in 12 consecutive backcrosses, with selection for certain C2 genes, and then using genomic exclusion to induce homozygosity. Six congenic strains of high breeding performance are available. Five differ from strain D in single genes at 5 different loci. The 6th strain differs at all 5 loci. Assuming the size of the Drosophila gene (4 × 10⁴ nucleotide pairs), we can calculate that strains differing from D by single genes have a heterozygous segment 3 genes long while The strain which differs from D by 5 genes has 5 heterozygous segments and 15 genes contributed from strain C2. A 40-fold increase in heterozygosity would be found with a gene size of 10³ nucleotide pairs. This means that in using these strains for biochemical work we must be aware that some genetic noise still remains.     

利用新生子叶制备牡丹染色体的研究

首次报道了利用牡丹(P．suffruticosa)种子的新生子叶制备染色体,同时比较了根尖染色体的核型,结果表明:牡丹子叶和根尖染色体核型数据一致,2种方法获得的染色体数均为2n=10,核型公式为2n=2x=10=6m 2sm 2st,全套染色体未见随体;2种方法获得的染色体形态结构基本相同。相对根尖而言,牡丹种子的新生子叶面积大,容易获得更多的试材,说明利用新生子叶制备染色体更适合遗传学实验研究。     

The Histidine Decarboxylase (HDC) Gene of Tetrahymena Pyriformis is Similar to the Mammalian One. A Study of HDC Expression

RNA was isolated from Tetrahymena pyriformis GL and using human histidine decarboxylase (HDC) gene primers, the RT-PCR product was sequenced. A fraction containing 207 base pairs was compared to the published sequences of prokaryotic and mammalian (rat, mouse and human) HDC cDNA (exons). The HDC-cDNA fraction of Tetrahymena was similar to the mammalian cDNA-s and it was completely different from the prokaryotic HDC-gene. The results indicate the presence of a mammalian-like HDC-gene already in a unicellular eukaryote organism and demonstrates also that the divergence of the prokaryotic–eukaryotic common gene took place already at this low evolutionary level.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).     

Interval Scanning Photomicrography of Microbial Cell Populations

A procedure is presented for photographically recording growth and other changes occurring within microbial populations over a period of time.