The distribution of x-ray induced chromosomal abnormalities in rat kangaroo (Potorous tridactylis) cells in vitro

Synchronized cells of Potorous tridactylis (female origin) were irradiated with X-rays in vitro at different times during the cell cycle. Chromosome aberrations were scored in the first metaphase after irradiation.It is shown that the distribution of breaks and gaps in the chromosome arms is different at different cell-cycle times, and is non-random.     

The development of thermoregulation in the rat kangaroo,Potorous tridactylus

• 1.1. The young were first seen out of the pouch on day 103.2 ±2.0 post-partum, and were permanently out of the pouch on day 119.7 ± 1.6, several days prior to the birth of the next young on day 122.8 ± 2.2.
• 2.2. The average mother's pouch temperature and the young's rectal temperature while in the pouch showed similar ranges—however, further analysis demonstrated that the pouch temperature decreased towards the end of lactation.
• 3.3. The difference in rectal temperature of the young in the pouch from 90–107 days to 108–123 days was 0.01 ± 0.34°C, whereas the mother's pouch temperature decreased 0.68 ± 0.37°C.
• 4.4. The results of this study suggests that curiosity and the need for solid food are the primary reasons for the young leaving the pouch and temperature does not play a primary role in the evacuation of the pouch.

     

Characteristics of quantitative karyotypic variability in cell line of kidney from rat kangaroo (Potorous tridactylis)

The goal of this work was to investigate the numeral karyotypic variability in different sublines (MT, M2). These sublines are formed spontaneously from the main cell line (M) and have modal number of chromosomes 9 and 10, MSVK (main structural variant karyotype)--3 + 3 + 1 + 2 and 3 + 4 + 2 + 1. There are general regulations which were originally got for the line M. In particular: 1) nonrandom character of cell distribution according to the number of chromosomal deviations from MSVK; 2) specific character of deviations of each chromosome from MSVK; 3) presence of significant connections between separate chromosomes by simultaneous, mainly single directed numeral deviations. These three lines (M, MT, M2) were compared and some differences were found: 1) different frequencies of deviations from MSVK; 2) the same chromosomes have tendency to different numeral deviation; 3) the specificity of some significant connections between separate chromosomes by simultaneous numeral deviations. These results lead us to a conclusion that the balance of numerical karyotypic structure in cell populations depends on the regulations connected with the character of deviations according to the number of chromosomes from MSVK which has the largest selected advantage. Each line has its own specific limits of karyotypic variability.     

Distribution of fluorescent α-amanitin (FAMA) during mitosis in cultured rat kangaroo (PtK1) cells

The mushroom toxin α-amanitin is known to possess a high affinity to eukaryotic RNA polymerase II (or B) [1–3]. To pursue the question where these enzymes are located during mitosis of cells, a fluorescent derivative of α-amanitin (FAMA) was prepared. The affinity of FAMA to RNA polymerase II is 18 times lower than that of α-amanitin which is, however, sufficient for bright staining of nuclei of interphase rat kangaroo (PtK1) cells. During mitosis a large part of the fluorescent stain was distributed over the cytoplasm, while the chromosomes were never found to be stained. An accumulation of the fluorescent toxin during metaphase was observed in the spindle, particularly in the centrioles. Fluorescence of the centrioles persists also during anaphase. It is concluded that during mitosis of PtK1 cells the RNA polymerase II is distributed in the cytoplasm rather than bound to chromosomes. The accumulation of fluorescent toxin in the spindle and centrioles may speculatively be explained by the presence of another protein with high affinity to amatoxins, which has recently been isolated from calf thymus by Brodner & Wieland [4].     

Chromosomes and DNA-replication of rat kangaroo cells (PtK2)

The karyotype, chromosomal measurements, and the time course of DNA replication during the S-phase were determined in metaphase chromosomes of non-synchronized monolayer cultures of PtK₂ cells (CCL 56) derived from Potorous tridactylis. The karotype was the same as originally determined for this cell line. Chromosomal measurements differed from data for primary bone marrow cells of this species published by Shaw and Krooth. PtK₂ cells and chromosomes showed maximal incorporation of tritiated thymidine (³H-TdR) halfway through the S-phase. Chromosome Y₁ showed a second peak of ³H-TdR-incorporation at the end of the S-phase in addition to the peak halfway through S. Comparison of grain densities for chromosomal arms showed late replication of the short arms of chromosomes 1, 3, and X. The time course of incorporation of ³H-TdR was changed when cells were treated for 1 h with fluorodeoxyuridine (FUdR) prior to the ³H-TdR-pulse. FUdR-treated cells showed maximum incorporation of ³H-TdR immediately after the beginning of the S-phase, which was followed by a second peak halfway through the S-phase. This indicated that ³H-TdR-incorporation was partially synchronized by treatment of cells with FUdR. Total radioactivity of FUdR-treated cells had increased by 77% in comparison to cells not treated with FUdR, which indicates that approximately 44% of the TdR-precursors of the latter cells may have originated from cellular precursor pools.     

Centriole behavior in early mitosis of rat kangaroo cells (PTK2)

Behavior of the centriolar duplexes during early mitosis was investigated. In general, both duplexes are capable of migrating about the cell as a unit with little change in their center to center spacing prior to separation to form the spindle poles. This duplex separation may occur at any point within the mid-prophase-prometaphase period. If it is delayed to prometaphase, transitory monopolar spindles were observed.     

Cyclin O (Ccno) functions during deuterosome-mediated centriole amplification of multiciliated cells

Mucociliary clearance and fluid transport along epithelial surfaces are carried out by multiciliated cells (MCCs). Recently, human mutations in Cyclin O (CCNO) were linked to severe airway disease. Here, we show that Ccno expression is restricted to MCCs and the genetic deletion of Ccno in mouse leads to reduced numbers of multiple motile cilia and characteristic phenotypes of MCC dysfunction including severe hydrocephalus and mucociliary clearance deficits. Reduced cilia numbers are caused by compromised generation of centrioles at deuterosomes, which serve as major amplification platform for centrioles in MCCs. Ccno-deficient MCCs fail to sufficiently generate deuterosomes, and only reduced numbers of fully functional centrioles that undergo maturation to ciliary basal bodies are formed. Collectively, this study implicates CCNO as first known regulator of deuterosome formation and function for the amplification of centrioles in MCCs.     

Nuclear asynchrony in multinucleate rat kangaroo cells

Multinucleate (MN) cells were induced in PtK1 cells by colcemid treatment. A large percentage of cells developed nuclear asynchrony both in relation to DNA synthesis and mitosis within one cell cycle. Asynchrony could be traced even in metaphase and anaphase cells in which interphase nuclei, PCC of S-phase nuclei and less condensed prophase-like chromosomes could be observed along with normally condensed chromosomes. The occurrence of such abnormalities in these large MN cells may be explained on the basis of an uneven distribution of inducer molecules of DNA synthesis and mitosis due to cytoplasmic compartmentation. The less condensed form of all the chromosomes except chromosome 4 could be traced in asynchronous metaphase. The failure of the less condensed chromosomes to undergo complete condensation does not always appear to result from late entry of nuclei containing these chromosomes into G2 phase. It is likely that chromosome 4 carries gene(s) for chromosome condensation, as this chromosome itself never appears in a less condensed form. The inducers for chromosome condensation may not always be available at equal concentrations to all chromosomes located in separate nuclei, thus they may sometimes fail to undergo complete condensation before other nuclei reach the end of prophase, when the nuclear envelopes of all nuclei present in the cell break down simultaneously.     

Nucleoli and ploidy in Potorous cells (PTK2) in vitro

Most cells of the male PTK₂ cell line contain one nucleolus, and they are diploid. However, a proportion of cells have more than one nucleolus. Cells with two and three nucleoli were isolated and cloned into viable populations. Greater than 90% of the cells in these clonal populations maintained the abnormal nucleolar number of the originally isolated cell. Karyotype analysis of cells with two and three nucleoli demonstrated that the cells were respectively tetraploid and hexaploid. It was concluded that in PTK₂ cells, nucleolarity is a good index of ploidy even if the ploidy level is abnormal. Furthermore, long term monitoring of the tetraploid cells demonstrated virtually no tendency towards reversion to the diploid condition as suggested by other studies in Potorous.     

Damage and repair in mammalian cells after exposure to non-ionizing radiations. II. Photoreactivation and killing of rat kangaroo cells (Potorous tridactylus) and Herpes simplex virus-1 by exposure to fluorescent "white" light or sunlight

Photoreactivation (PR) of ultraviolet (254 nm)-inactivated cornea cells of the potoroo (or rat kangaroo; Potorous tridacylus) has been studied at wavelengths greater than 375 nm from either fluorescent "white" light or sunlight. In both cases the PR kinetics curves pass through maxima, which most likely result from the superposition of concomitant inactivation by the photoreactivating light. The inactivating effect of light was directly demonstrated for non-UV-irradiated cells, permitting correction of the PR curves. Wavelengths greater than 475 nm, and even greater than 560 nm, which do not noticeably damage cells, still photoreactivate, though less effectively than shorter wavelengths. Light treatment of UV-inactivated Herpes simplex Virus-1 (HSV-1) after infection leads to PR effects resembling those observed for cells, while light treatment of unirradiated virus after infection likewise causes inactivation. The "fluence-reduction factor" of PR, which is greater than 3 for the virus, exceeds that for the cells, where it decreases with increasing UV fluence. In vitro tests have indicated that sunlight greater than 375 nm causes photorepairable DNA lesions which are virtually fully repaired by the same light. Thus cell inactivation resulting from these solar wavelengths must be due to non-photorepairable damage.     

Vinblastine-induced paracrystals and unusually large microtubules (macrotubules) in rat renal cells

Summary Vinblastine sulfate was administered to adult rats by intravenous injections. Kidney cortex was fixed after 1, 2, or 5 hours of treatment and studied by routine transmission electron microscopy.In control animals, cells of distal convoluted tubules possessed numerous microtubules with an average diameter of 280 Å. In treated animals, the microtubules of these cells were reduced in number, and paracrystalline inclusions characteristic of vinblastine treatment were common. Macrotubules (570 Å average diameter) were also present and often were seen close to, or in apparent continuity with, paracrystals. Since the work of others indicates that vinblastine-induced paracrystals contain microtubular protein (tubulin), observation of continuities between paracrystals and macrotubules is interpreted as evidence that macrotubules are also composed of tubulin and that macrotubules may become incorporated into paracrystals.Unlike the ordinary microtubules of cells of the distal tubules, vinblastine-induced macrotubules exhibited cross-striations in longitudinal view and subunit structure in cross section.Macrotubules and paracrystals were also observed in cells of the proximal convoluted tubule, mesangium, glomerular endothelium, parietal epithelium of Bowman's capsule, and visceral epithelium of Bowman's capsule. Continuities between macrotubules and paracrystals, although relatively common in occurrence in distal tubule cells, were only rarely seen in the other kinds of cells examined. Acknowledgements. The authors gratefully acknowledge the technical help of Mrs. Dawn Bockus, Miss Judy Groombridge, Mrs. Jeri Hunter, Mrs. Jolan Pinter, Miss Franque Remington, Miss Mary Stewart, Miss Louise Young, Mr. Reginald Pickering, and Mr. W. J. Masten. This research was supported by N.I.H. grants AM 16 236, GM 00 100, and HE 03 174, by Institutional Cancer Grant IN-26L from the American Cancer Society, and by the Graduate School Research Fund of the University of Washington.     

Photoreactivation and repair replication in rat kangaroo cells

Potorous tridactylis cells can perform photoreactivation, i.e., the visible light- catalyzed reversal of UV-induced pyrimidine dimers in DNA. UV-induced inhibitions of total RNA and DNA synthesis can also be partially reversed by exposure to visible light. P. tridactylis cells can also perform repair replication, but the extent of the latter is reduced if the cells are exposed to visible light (VL). None of these effects are observed in mouse L cells, which cannot perform photoreactivation. The results are consistent with the concept of pyrimidine dimers are one of the main substrates for repair replication.     

Distribution and orientation of microtubules in milk secreting epithelial cells of rat mammary gland

Summary Quantitative ultrastructural analysis of mid-lactation rat mammary gland demonstrated that cytoplasmic microtubules were present in nearly all secretory epithelial cells examined. Most microtubules were oriented perpendicular to the apical membrane and were found in the apical and medial portions of the cell cytoplasm. There was no statistical difference between the number of microtubules associated with vesicles and the number that were not. Most vesicles which were in contact with microtubules were small (50 to 150 nm), appeared electron lucent and were located in a supra-Golgi complex position. Many of these vesicles were seen to be aligned along the axis of longitudinally sectioned microtubules oriented perpendicular to the apical plasma membrane. As measured by a colchicine binding assay, the total tubulin content of mammary tissue from mid-lactation rats was about 107 g/100 mg wet weight. Approximately 19% of the total tubulin was in polymerized form. This study provides evidence that microtubules may be involved in guiding transport of small secretory vesicles to the apical regions of cells for exocytosis.     

Light and electron microscopy of rat kangaroo cells in mitosis

Kinetochores in rat kangaroo (PtK₂) cells in prophase of mitosis are finely fibrillar, globular bodies, 5000–8000 Å in diameter. Sister kinetochores are attached to opposite lateral faces in the primary constriction of chromosomes. No microtubules (MTs) occur in prophase nuclei. During prometaphase the ball-shaped kinetochores differentiate into trilaminar plaques. An outer kinetochore layer, less electron dense than chromatin, appears first in the fibrillar matrix. The inner layer, continuous with, but more electron dense than the chromosome, is formed later. Kinetochore-spindle MT interaction is evident at the very beginning of prometaphase. As a result, kinetochore shape is very variable, but three types of kinetochores can be distinguished by fine structure analysis. A comparison of kinetochore structure and chromosome position in the mitotic spindle yielded clues regarding initial orientation and congression. At the time the nuclear envelope (NE) breaks down chromosomes near asters orient first. Chromosomes approximately equidistant from the two spindle poles amphi-orient immediately. Chromosomes closer to one pole probably achieve mono-orientation first, then amphi-orient and congress. In normal metaphase all the chromosomes lie at or near the spindle equator and kinetochores are structurally uniform. Paraxial and para-equatorial sections revealed that they are trilaminar, roughly circular plaques of 4000–6000 Å diameter. Inner and outer layers are 400 Å, and the electron translucent middle layer which separates them is 270 Å thick. From 16 to 40 MTs are anchored in the outer layer. In cold-treated cells the kinetochores are trilaminar, but in colcemid-treated cells the inner layer is lacking. Both kinetochores and their MTs are disorganized beginning in late anaphase. In telophase the inner layer persists for some time as an electron dense patch apposed to the NE, while the outer layer disintegrates.     

Distribution of microtubules in prolactin cells of lactating rats

The intracellular distribution of microtubules was studied using serial sections of prolactin cells in anterior pituitary glands from lactating rats. Numerous microtubules were present in these cells following fixation with glutaraldehyde and osmium tetroxide. The greatest number of microtubules were present in the Golgi complex, situated around the perimeter and in association with the cisternae, vesicles and developing secretory granules. Microtubules were found in channels between groups of parallel cisternae of rough surfaced endoplasmic reticulum and in close proximity to small vesicles. They were also located adjacent to mitochondria, the plasmalemma, the nuclear envelope, and among mature secretory granules. Due to their orientation within the cell, it is suggested that the microtubules may act to direct the movement of organelles from one region of the cell to another and to give internal support to the cell.