Variety of Hybrid Characters among Recombinants Obtained by Interspecific Protoplast Fusion in Streptomycetes

To discover whether the protoplast fusion method is useful or not for interspecific breeding, some methods were devised, and the appearance of various hybrids with different characters and the change of antibiotic activities in the recombinants obtained by the protoplast fusion were investigated. The purification of protoplasts, the choice of parental natural characters as selection markers, and the adoption of a replica method for selecting all types of recombinants were devised and used for these experiments. Protoplast fusion was done between S. griseus KCC S-0644 and each strain of 5 species that were clearly different species from S. griseus, in addition to being streptomycin sensitive (SM^s) and capable of L-arabinose utilization for growth (Ara⁺). Recombinants (SM^r, Ara⁺) obtained by protoplast fusion displayed a great variety of hybrids in their taxonomic characters, e.g., 21 recombinant strains obtained by the cross between S. griseus and S. griseoruber consisted of 14 types of hybrids. Antibiotic productivity was examined in all recombinants obtained. Although both parental species produced their respective antibiotics, 60% of the recombinants did not produce any antibiotic and 24% produced different antibiotics from those of their parents. Among those recombinants, it was also found that the distribution of the productivity of each antibiotic among the recombinants was entirely different from that of the allelo-character in each taxonomic feature.     

Induction of premature chromosome condensation in Drosophila melanogaster — rat heterokaryons

Summary After fusion mediated by polyethylene glycol (PEG) plus dimethyl sulfoxide (DMSO) between rat and Drosophila melanogaster cells cultured in vitro, the phenomenon of premature chromosome condensation (PCC) induced by mitotic rat chromosomes was detected in Drosophila nuclei. Exceptionally PCC was induced in rat nuclei but only in the presence of a very high ploidy level of Drosophila mitotic chromosomes. This provides further evidence of the lack of species specificity and of the effect of dosage of the PCC inducing factors, even among very distant species.     

Evidence for the priming role of the central retinula cell in ommatidium differentiation of Ephestia kuehniella

Summary In the developing compound eye of Ephestia kuehniella, within the advancing front of differentiation, regular cell clusters arise which consist of a central cell and two flanking cells. The central cell is destined to become the basal retinula cell later in development. Its crucial role in ommatidium formation is confirmed by ³H-thymidine labelling. Eye anlagen labelled early in the pupal stage incorporate thymidine within two distinct zones along the front of differentiation. After the ommatidia are completely differentiated, both zones contain labelled nuclei of all cell types which participate in ommatidia formation. Within the posterior zone, however, the basal retinula cells are always unlabelled, whereas in the anterior they show labelled nuclei. From this observation it must be concluded that the basal retinula cell first terminates proliferation (either alone or together with a few other cells) to become differentiated as the central retinula cell. These results agree with those found in Drosophila and indicate that the ordered stepwise addition of cells to a central founder cell is a widespread principle of ommatidia formation in insects.     

Absence of nucleolar dominance in mouse-human heterokaryons

Autoradiographic analysis of [³H]uridine incorporation 48 h after polyethylene glycol-mediated cell fusion indicates that nucleolar RNA synthesis persists in both human and mouse nuclei in interspecific heterokaryons. The absence of nucleolar dominance in heterokaryons has been confirmed by zinc-dithizone nucleolus-specific staining, and is true even when there are considerably more nuclei of one species than of the other in the heterokaryon. Studies of actinomycin D-induced nucleolar segregation indicate that the zinc-binding proteins responsible for zinc-dithizone staining are located in a different nucleolar component than the protein responsible for silver staining.     

Cell cycle-related transfer of proteins between the nucleus and cytoplasm of Physarum polycephalum

The proteins of wild-type and polyploid plasmodia of P. polycephalum were prelabelled with [³H]leucine and [¹⁴C]leucine. The two types of plasmodia were then fused for 2 h. Following fusion the nuclei were isolated and the smaller wild-type cell nuclei separated from the larger polyploid cell nuclei. The proteins were isolated from the recipient cell nuclei and the recipient nuclear proteins extracted. Ratios of ³H/¹⁴C in the various nuclear protein fractions show that during fusion differential transfer of labelled preformed proteins from the donor cell into the recipient cell nucleus occurs. The quantity of proteins transferred varies among the different fractions and with the phase of the cell cycle. Isotopic dilution experiments indicate that these differences in protein transfer are, in part, due to a high rate of synthesis and turnover of the nuclear proteins.     

Mitochondrial Fragmentation Due to Inhibition of Fusion Increases Cyclin B through Mitochondrial Superoxide Radicals

During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G₁-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G₂- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the ‘mitotic mitochondrial fragmentation’ checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R⁺ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. We provide evidence for the importance of mitochondrial superoxide in this process. Our results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.     

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with ³H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.     

Nucleolar number variation in Hordeum species,their haploids and interspecific hybrids

Nucleolar number variation has been investigated in root tip cells by cytologically determining the number of nucleoli per cell in autoploids and alloploids of Hordeum species, their haploids and interspecific hybrids. The nucleolus organisers in autoploid types of H. vulgare or H. bulbosum did not show any alterations irrespective of the ploidy level. The nucleolar number variation in these species results from a definite pattern of fusion and the maximum number of nucleoli per nucleus corresponds to the number of secondary constrictions. Nucleolus formation in alloploids and interspecific hybrids is impaired on some of the NORs, suggesting differential amphiplasty or nucleolar dominance. A comparison of nucleolar formation in the alloploid species (brachyantherum, arizonicum, procerum and parodii), their haploids, and the interspecific hybrids revealed different degrees of variation from the expected mean and maximum numbers of nucleoli. While the deviations in hybrids between alloploids (H. arizonicum and H. brachyantherum or H. procerum and H. brachyantherum) are marginal, nucleolar dominance is more pronounced in hybrids involving H. vulgare or H. bulbosum as one of the parents and is invariably associated with the disappearance of the secondary constriction(s) from the NOR(s) contributed by one of the parents, and the number of nucleoli is appropriately reduced.     

Morphogenic effects of halogenated thymidine analogues on Drosophila. I. Quantitative analysis of lesions induced by 5-bromodeoxyuridine and 5-fluorouracil

When 5-fluorouracil (FU) is offered simultaneously with 5-bromodeoxyuridine (BUdR) to Drosophila larvae, a variety of bristle modifications and hyperplastic growths are found on the wings of the adult flies. Administration of FU alone will not stimulate growth in Drosophila, while high concentrations of BUdR offered alone will induce a lower frequency of growth modifications than induced by BUdR plus FU. Comparison of the morphological response induced by sequential treatment with the two analogues and that by simultaneous treatment with the analogues at the same concentrations indicates that maximum response is achieved by the presence of both analogues at the same time. These observations suggest that BUdR may be the primary agent in upsetting morphogenesis in Drosophila, while FU plays a subsidiary role leading to intensification of the morphogenic effects when it is present during the treatment period. The incorporation of BUdR-H³ and FU-H³ in Drosophila tissues was demonstrated by autoradiography. BUdR-H³ was incorporated in nuclei of both larval and imaginal disc cells, and the isotopic label was removable by deoxyribonuclease. Following dietary administration of FU-H³, tritium was found in RNA, primarily in cytoplasmic regions. Since BUdR is a known mutagen, consideration was given to the hypothesis that the altered growth patterns in Drosophila wings are the result of somatic cell mutational events induced by BUdR. Validity of the argument that recessive mutations on the X chromosomes can be readily expressed in the somatic cells of the male with one X chromosome as opposed to the female with two X chromosomes was tested by comparing the frequency of the induced somatic cell lesions in male and female zygotes. The males showed a higher frequency of induced supernumeraries, while the icidence of bristle effects and total wings affected was the same in both sexes.This research was supported by grants to T. M. R. from the National Science Foundation (GB-11745) and by an Institutional Research Grant (No. IN-40H) to the University of Michigan from the American Cancer Society.     

The potentialities of transplanted early gastrula nuclei ofDrosophila melanogaster. Production of their imago descendants by germ-line transplantation

Summary Wild-type nuclei, taken out of cells from five regions of early gastrula embryos, were implanted singly into unfertilizedy w sn ³lz^50e eggs ofDrosophila melanogaster. The different types of nuclei initiated development with nearly equal frequencies of about 60%. 2.9% of the 1073 nuclear transfers developed as far as one of the three larval instars, and one reached the pupal stage.All individuals showed stage-specific patterns of defect. Most of these abnormalities were probably due to some inevitable damage caused by the implantation procedure such as disarrangement of the internal egg morphology and loss of peripheral egg substance. The proportions of individuals arrested at different embryonic and larval stages were similar for the five nuclear groups.Fertile imagos, descendants of all five types of donor nuclei, were produced via germ-line mosaics in two ways: (1) Pole cells of nuclear-transplant blastoderm stages were implanted into the pole cell region of host blastoderm eggs. (2) Gonads were taken from nuclear-transplant larvae and implanted into host larvae. In both cases gametes developed from the transplants as could be recognized from the genotypes of their progeny. By means of suitable crosses it was possible to get clones of flies whose large chromosomes were descended from the chromosomes of only one transplanted nucleus, that is, each clone was the descendant of one somatic nucleus. The data presented show that the nuclei remain omnipotent until the early gastrula stage.     

Isozyme variability in species of the genusDrosophila. V. ejaculatory bulb esterases inDrosophila phylogeny

The presence or absence of certain esterase types in ejaculatory bulbs of 93 Drosophilaspecies was determined by appropriate staining after starch gel electrophoresis. One entire subgenus (Sophophora)and several primitive species were found lacking in one type of esterase. This esterase was present in numerous species of the large Drosophilasubgenus, but presence and absence did not follow closely the lines of taxonomic subdivisions in the subgenus. Correlation of presence-absence of the bulb esterase and previous results in interspecific hybridization tests indicated a greater chance for a successful interspecific cross when males possessed the bulb esterase.     

Evidence for asynchronous mitotic cell divisions in secondary spermatogonia of Drosophila

Examination of germ cell numbers within premeiotic as well as postmeiotic cysts of various Drosophila species gave evidence against any strict synchrony of mitotic cell division in secondary spermatogonia. The evidence was based on numbers of germ cells in primary spermatocyte cysts and spermatid bundles. Each species examined had its own distribution of primary spermatocyte cyst types in pupal testes, and the most common cyst type did not necessarily contain 2, 4, 8, 16 or (2) ⁿ germ cells which implies asynchrony of the previous spermatogonial divisions. Similar but not exactly the same distributions of germ cells were found in adult spermatid bundles, if allowances were made for a 4-fold increase in germ cell number during meiosis. This observation gives support to the operation of an age-dependent factor which controls germ cell numbers within cysts [1], The data thus suggest that the commonly accepted concept of a (2) ⁿ increase of spermatogonia via synchronous mitotic divisions is not true for the species of Drosophila studied.     

The Hippo pathway controls polar cell fate through Notch signaling during Drosophila oogenesis

During Drosophila oogenesis, the somatic follicle cells form an epithelial layer surrounding the germline cells to form egg chambers. In this process, follicle cell precursors are specified into polar cells, stalk cells, and main-body follicle cells. Proper specification of these three cell types ensures correct egg chamber formation and polarization of the anterior–posterior axis of the germline cells. Multiple signaling cascades coordinate to control the follicle cell fate determination, including Notch, JAK/STAT, and Hedgehog signaling pathways. Here, we show that the Hippo pathway also participates in polar cell specification. Over-activation of yorkie (yki) leads to egg chamber fusion, possibly through attenuation of polar cell specification. Loss-of-function experiments using RNAi knockdown or generation of mutant clones by mitotic recombination demonstrates that reduction of yki expression promotes polar cell formation in a cell-autonomous manner. Consistently, polar cells mutant for hippo (hpo) or warts (wts) are not properly specified, leading to egg chamber fusion. Furthermore, Notch activity is increased in yki mutant cells and reduction of Notch activity suppresses polar cell formation in yki mutant clones. These results demonstrate that yki represses polar cell fate through Notch signaling. Collectively, our data reveal that the Hippo pathway controls polar cell specification. Through repressing Notch activity, Yki serves as a key repressor in specifying polar cells during Drosophila oogenesis.     

Increase in DNA synthesis in Werner''s syndrome cells by hybridization with normal human diploid and HeLa cells

The dominance or recessiveness of the senescent phenotype in cells from patients with Werner's syndrome (WS cells) was investigated using cell fusion. The [³H]thymidine labeling index of normal human diploid fibroblast cell X WS cell heterodikaryons was considerably lower than that of normal homodikaryons, but was significantly higher than that of WS homodikaryons. The labeling index of WS cell X HeLa cell heterodikaryons was the same as that of HeLa homodikaryons. The labeling indices of heterodikaryons obtained by fusion between various strains of premature aging cells were as low as those of parental homodikaryons. These results indicate: (1) the senescent phenotype of WS cells appears to be partially recessive to the phenotype of normal cells and completely recessive to that of HeLa cells; (2) the marked inhibition of DNA synthesis in normal nuclei in heterodikaryons with WS cells could be due to ‘senescent factor(s)’ in WS cells; and (3) no complementation phenomenon was observed among genetically different premature aging cells, probably due to ‘senescent factor(s)’.     

Studies on graft unions. I. Plasmodesmata between cells of plants belonging to different unrelated taxa

Summary In order to answer the question whether plasmodesmata exist between the cells of stock and scion in a graft union of higher plants, a heteroplastic graft has been selected with species-specific cell markers.Vicia faba was used as scion andHelianthus annuus as stock. In the mixed callus of the graft union, individual cells of the two partners differ in the structure of the nuclei, plastids and microbodies. In the fused cell walls betweenVicia andHelianthus cells, plasmodesmata interconnect the protoplasts of the unrelated cells. Two types of plasmodesma occur: single strands and branched connections. The fine structure of the interspecific cell bridges, which have been secondarily formed in non-division walls, is similar to that of normal plasmodesmata in division walls. Some questions concerning compatibility/incompatibility in the heterograft are discussed.     

DNA polymerase α and the regulation of entry into S phase in heterokaryons

We have previously reported that the DNA polymerase α activity/unit cellular protein is decreased in latepassage (senescent) human diploid fibroblast-like (HDFL) cultures due to the cellular enlargement associated with in vitro aging. In the studies described here, we have used cell fusion technology to investigate the formal kinetic relationship between the concentration of DNA polymerase α and the rate of reinitiation of DNA synthesis in nuclei from senescent cells. Heterokaryons were derived from the fusion of senescent cells to a series of actively dividing cell types with inherently different DNA polymerase α activities per cell. A kinetic analysis revealed a first-order relationship between the entry into S phase of senescent nuclei and the concentration of DNA polymerase a activity calculated to be in heterokaryons. This result suggests that increases in cell volume may be related to the decline in proliferative activity of late-passage HDFL cells, via “dilution” of factors essential for cellular replication.     

Nuclear heterogeneity and proliferation activity of human adipose derived MSC-like cells

Adipose tissue (AT) is an easily available source of mesenchymal-like stem cells (MSCs) that are appropriate for applications in regenerative medicine. There is conflicting evidence on the morphology of AT-derived stem cells (ADSCs). Here, we described the morphology and proliferation activity of human ADSCs. The cells were plated at a density of 10 cells/sm² and cultivated for 1 month. Twenty-one colonies were grown. In nine out of 17 analyzed colonies, few atypical cells (large nuclei and cytoplasm) were found. ANOVA demonstrated that colonies also differed (p = 0.0025) in the size and diameter of cell nuclei. The size of nuclei and logarithm of cell density were correlated in the reverse proportion (−0.7; p = 0.002). Thus, a culture obtained from the stromal vascular fraction (SVF) is heterogeneous and composed of two types of cells, i.e., highly proliferative and large, low proliferative cells. These cells are typical of the MSC and ADSC cultures described in literature. The cell heterogeneity observed in some colonies probably resulted from variations in the cell-cycle phases.