Detection of T-cell surface determinants in three Marek's disease lymphoblastoid cell lines.

The T-cell surface antigens on three lymphoblastoid cell lines, MOB1, MOB-2 and MSB-1, derived from Marek's disease lymphomas were examined by the cytotoxicity test and the indirect membrane immunofluorescent test. These cell lines reacted specifically with antisera prepared against chicken thymus cells, although their reactivities were less than that of typical thymus cells. These three cell lines were of thymus origin.     

Propagation and infectivity titration of the Gifu-1 strain of chicken anemia agent in a cell line (MDCC-MSB1) derived from Marek's disease lymphoma

Chicken anemia agent (CAA) propagated in an established cell line derived from Marek's disease (MD) lymphoma (MDCC-MSB1). When passaged 19 times in MDCC-MSB1 cell cultures, it produced anemia of the same severity in chicks as it did before passage. Titration of the infectivity of CAA was performed successfully with subcultures of MDCC-MSB1 cell cultures which had been inoculated with serial tenfold dilutions of infected material. In it, no infected cultures could be subcultured. The propagation of CAA was also proved in the MD cell line, MDCC-JP2, and the avian lymphoid leukosis (LL) cell line, LSCC-1104B1, but not in the two MD cell lines, MDCC-RP1 and MDCC-BP1, or in the two LL cell lines, LSCC-1104X5 and LSCC-TLT. No CAA propagated in cell cultures prepared from skin and muscle, liver, or brain of chick embryos, or kidney, thymus, bursa of Fabricius, bone marrow, or white blood cells of chickens.     

Low glucosinolate Brassica juncea breeding line revealed to be nullisomic.

The low glucosinolate Brassica juncea breeding line 1058 was derived from a BC1F3 plant of an interspecific cross between high glucosinolate Indian B. juncea (genome AABB, 2n = 36) line 60143 and B. rapa (genome AA, 2n = 20) canola strain CZY. Line 60143 had 2n = 36 chromosomes (18 bivalents at metaphase I) and strain CZY had 2n = 20 chromosomes (10 bivalents). Line 1058 was nullisomic, with 2n - 2 = 34 chromosomes, with 17 bivalents formed at metaphase I and an even chromosomal segregation of 17:17 at anaphase I. In F1 hybrid plants of the cross 1058 x CZY, 98.3% of the pollen mother cells had 10 bivalents and seven univalents. This is evidence that plants of line 1058 are nullisomic, missing one pair of B-genome chromosomes.     

Characterization of six wheat x Thinopyrum intermedium derivatives by GISH, RFLP, and multicolor GISH.

Restriction fragment length polymorphism (RFLP) analysis and multicolor genomic in situ hybridization (GISH) are useful tools to precisely characterize genetic stocks derived from crosses of wheat (Triticum aestivum) with Thinopyrum intermedium and Thinopyrum elongatum. The wheat x Th. intermedium derived stocks designated Z1, Z2, Z3, Z4, Z5, and Z6 were initially screened by multicolor GISH using Aegilops speltoides genomic DNA for blocking and various combinations of genomic DNA from Th. intermedium, Triticum urartu, and Aegilops tauschii for probes. The probing (GISH) results indicated that lines Z1 and Z3 were alien disomic addition lines with chromosome numbers of 2n = 44. Z2 was a substitution line in which chromosome 2D was substituted by a pair of Th. intermedium chromosomes; this was confirmed by RFLP and muticolour GISH. Z4 (2n = 44) contained two pairs of wheat--Th. intermedium translocated chromosomes; one pair involved A-genome chromosomes, the other involved D- and A- genome chromosomes. Z5 (2n = 44) contained one pair of wheat--Th. intermedium translocated chromosomes involving the D- and A-genome chromosomes of wheat. Z6 (2n = 44) contained one pair of chromosomes derived from Th. intermedium plus another pair of translocated chromosomes involving B-genome chromosomes of wheat Line Z2 was of special interest because it has some resistance to infection by Fusarium graminearum.     

Pathogenicity for chicks of line cells from lymphoma of Marek's disease.

Pathogenicity for chicks of the MSB-1 line, a cell line derived from the tumorous tissue of a chick with Marek's disease (MD) and established by Akiyama & Kato, was studied. Five groups, including a control one, of 20 chicks each were inoculated with 1 X 10(3), 1 X 10(4), 1 X 10(5), 1 X 10(6) and no cells of a 180-day culture of the cell line at one day of age. They were housed all together in an isolation unit. An attempt was first made successfully to isolate MD virus (MDV) directly in culture of kidney cells 3 weeks after inoculation. Horizontal infection was first detected 4 weeks after inoculation. From 3 weeks after inoculation on, the disease with almost the same clinical and pathological pictures as the infection with a virulent strain of MDV showed a high incidence. Morbidity was closely related to the number of MSB-1 line cells inoculated. Parenchymal destruction was conspicuous in the central lymphoid organs of four chicks given the largest number of MSB-1 line cells and sacrificed in extremis about 4 weeks after inoculation. Establishment of MD in chicks inoculated with MSB-1 line cells carrying MDV genome seemed to be initiated under the circumstances where the line cells which had come into contact with susceptible cells in the peritoneal cavity released virulent MDV per se. Then host chicks might be infected with MDV and suffer from MD at a high rate. There was no great difference in oncogenic potential between MSB-1 line cells cultivated in vitro for 180 days and virulent MDV serially passaged through one-day-old chicks.     

Karyology and tumorigenicity of a simian virus 40-transformed Chinese hamster cell clone.

A pseudodiploid clone of chinese hamster cells tranformed in vitro with Simian virus 40 (SV40) was isolated from soft agar and injected into nude mice through three successive passages with a short in vitro cultivation between each animal inoculation. the original clone and the three subsequent tumor populations were characterized in regard to SV40 T antigen staining, modal chromosome number, and Giemsa-banded karyotype. All tumor cell lines maintained the pseudodiploid mode, as well as the positive sv40 t antigen staining. Nonrandom chromosomal changes included loss of one of the X chromosomes, additions of abnormal variants of chromosomes No. 1 and No. 2, the appearance of unidentified marker chromosomes, and the loss of autosomes No. 5, No. 6, and No. 11. The deletion of one of the X chromosomes occurred with about the same frequency in all cell lines. Additions of abnormal chromosomes No. 1 and No. 2 tended to recur more often in the tumor cell lines than in the original clone. the appearance of marker chromosomes, as well as the loss of autosomes No. 5, No. 6, and No. 11 demonstrated a correlation with tumorigenicity. Yet, the three successive passages of the cells through the animal did not select for a tumor population with a single, homogeneous karyotype.     

Sex determination in the European eel (Anguilla anguilla, L.). A hypothesis based on cytogenetic results, correlated with the findings of skewed sex ratios in eel culture ponds

Metaphase chromosomes from cultured blood cells of female, male, and hermaphroditic European eels were analyzed. In addition, both gonads from each of the specimens were examined microscopically to ensure correct sexing. The karyological investigation revealed that in some of the specimens a heteromorphic chromosome pair was present. This heteromorphism appeared in both sexes and in the hermaphrodite. C-banding and silver nitrate staining demonstrated that the heteromorphism was due to quantitative differences in constitutive heterochromatin and nucleolar organizing regions in the short arm of chromosome 8. In G-banded preparations it was demonstrated that, except for the heteromorphism mentioned, the karyotypes from both sexes and the hermaphrodite were identical. With the G-band technique it was also easily demonstrated that both the largest metacentric (No. 1) and the smallest metacentric (No. 11) had homologs. Therefore, in contrast to some earlier reports which claimed that these two chromosomes were a heteromorphic pair of sex chromosomes, it is concluded that Anguilla anguilla has no heteromorphic sex chromosomes. The implication of these findings are discussed in relation to the many reports of strongly skewed sex ratios found in commercial eel farms. It is tentatively hypothesized that sex determination in A. anguilla may be metagamic and that sex inversion may occur in this species.     

Genomic constitution and variation in five partial amphiploids of wheat–<Emphasis Type="Italic">Thinopyrum intermedium</Emphasis> as revealed by GISH,multicolor GISH and seed storage protein analysis

Genomic in situ hybridization (GISH) and multicolor GISH (mcGISH) methodology were used to establish the cytogenetic constitution of five partial amphiploid lines obtained from wheat × Thinopyrum intermedium hybridizations. Line Zhong 1, 2n=52, contained 14 chromosomes from each of the wheat genomes plus ten Th. intermedium chromosomes, with one pair of A-genome chromosomes having a Th. intermedium chromosomal segment translocated to the short arm. Line Zhong 2, 2n=54, had intact ABD wheat genome chromosomes plus 12 Th. intermedium chromosomes. The multicolor GISH results, using different fluorochrome labeled Th. intermedium and the various diploid wheat genomic DNAs as probes, indicated that both Zhong 1 and Zhong 2 contained one pair of Th. intermedium chromosomes with a significant homology to the wheat D genome. High-molecular-weight (HMW) glutenin and gliadin analysis revealed that Zhong 1 and Zhong 2 had identical banding patterns that contained all of the wheat bands and a specific HMW band from Th. intermedium. Zhong 1 and Zhong 2 had good HMW subunits for wheat breeding. Zhong 3 and Zhong 5, both 2n=56, possessed no gross chromosomal aberrations or translocations that were detectable at the GISH level. Zhong 4 also had a chromosome number of 2n=56 and contained the complete wheat ABD-genome chromosomes plus 14 Th. intermedium chromosomes, with one pair of Th. intermedium chromosomes being markedly smaller. Multicolor GISH results indicated that Zhong 4 also contained two pairs of reciprocally translocated chromosomes involving the A and D genomes. Zhong 3, Zhong 4 and Zhong 5 contained a specific gliadin band from Th. intermedium. Based on the above data, it was concluded that inter-genomic transfer of chromosomal segments and/or sequence introgression had occurred in these newly synthesized partial amphiploids despite their diploid-like meiotic behavior and disomic inheritance.     

Chromosomal instability of SV40-transformed human prostatic epithelial cell lines

Three SV40-transformed derivatives (G1, T2, and T5) of human prostatic epithelial cells were analyzed karyotypically. For comparison, an SV40 derivative (TA) obtained from isogenic fibroblasts, was also studied. The chromosome complements of these cells, as well as a sub-clone of T2 isolated in soft agar (T2-A5), were analyzed using banding techniques. Numerical as well as structural changes were observed in all transformed cultures. Karyotypic changes in all cells at a given passage level appeared to be random. On the other hand, characteristic differences in modal chromosome number, and type and number of abnormal chromosomes were observed among the different lines. Most cells of two of the three epithelial lines (T1 and T2) were either hypo- or pseudodiploid, whereas T5 consisted of a mixed hypodiploid and hypotetraploid population. The TA subline was also predominantly hypo- and pseudodiploid. Dicentrics, telomeric associations, translocations, and loss of chromosomes were the most prominent abnormalities. The loss of chromosome 18 was characteristic for all epithelial lines. All T1 and T5 cells had lost either one or both copies of the 18. While individual cells of the original T2 line had random karyotypes, most of T2-A5 cells had a relatively uniform karyotypic pattern. They also had a similar pattern of abnormal chromosomes. These observations suggest that culture in soft agar may have selected a particular chromosomal variant. We conclude that transformation of prostatic epithelial cells by SV40 may bring about site-specific as well as random chromosomal changes. These changes could reflect either intermediate or sequential stages in progression to neoplasia.     

Condensation of chromatin into chromosomes preserves an open configuration but alters the DNase I hypersensitive cleavage sites of the transcribed gene

DNase I was used to probe the molecular organization of the chicken ovalbumin (OV) gene and glyceraldehyde 3-phosphate dehydrogenase (GPD) gene in interphase nuclei and in metaphase chromosomes of cultured chicken lymphoblastoid cells (MSB-1 line). The OV gene was not transcribed in this cell line, whereas the GPD gene was constitutively expressed. The GPD gene was more sensitive to DNase I digestion than the OV gene in both interphase nuclei and metaphase chromosomes, as determined by Southern blotting and liquid hybridization techniques. In addition, we observed DNase I hypersensitive sites around the 5' region of the GPD gene. These hypersensitive sites were not always at the same locations between the interphase nuclei and metaphase chromosomes. Our results suggest that chromatin condensation and decondensation during cell cycle alters nuclease hypersensitive cleavage sites.     

Establishment of the AU-bek cell line and comparison with two other bovine cell lines

Summary Establishment of a new bovine cell line, AU-BEK, is reported. The cell line developed in a culture initiated from bovine embryonic kidneys by spontaneous cultural alteration to epithelioid cells that are indefinitely propagable. Epithelioid cells gradually increased to become the predominant cell. Whereas normal bovine cells have a diploid number of 60 chromosomes, of which only the two sex chromosomes are biarmed, AU-BEK cells at the 80th passage had a modal chromosome number of 84 and an average of 30 biarmed chromosomes per cell. AU-BEK cells are now in their 220th passage. Of the AU-BEK, MDBK, and CKT-1 bovine cell lines, the CKT-1 cell line had a karyotype closest to that of normal bovine cells. Their modal chromosome number was 57, and only three biarmed chromosomes were usually present. The bovine character of AU-BEK and CKT-1 cells was established by cytotoxic and viral susceptibility tests. Supported by the Alabama Agricultural Experiment Station. Publication No. 1115, School of Veterinary Medicine, Auburn University.     

Isolation of cell lines from embryos of the cockroach,Blattella germanica

Summary Cell lines were isolated from three stages of embryos ofBlattella germanica dissociated with trypsin. The lines have been subcultured 50 to 134 times in 3 years. Line UM-BGE-1 was isolated from germ band embryos at stages of segmentation and limb-bud formation (5 days old). Line UM-BGE-2 was derived from embryos at dorsal closure (7 days old). Line UM-BGE-4 arose from embryos in the germ band and dorsal closure stages (5 and 7 days old); these cells colonize as hollow spheres or vesicles. Line UM-BGE-5, isolated during organogenesis (10 days old), developed into two distinct sublines. Subline α is composed of round cells that do not attach to the flask. Subline β grows as an attached monolayer; the cells can be removed with a saline solution containing 20mM disodium dihydrogen Versenate^?. Most of the cells of these lines have the diploid chromosome number (23 or 24) excepting line UM-BGE-1 in which the tetraploid number predominates. Presented in part at the 26th Annual Meeting of the Tissue Culture Association, at Montreal, Canada, 2–5 June 1975. This investigation was supported by U.S. Public Health Service Research Grant No. AI 09914 from the National Institute of Allergy and Infectious Diseases. This is Paper No. 9416, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Inhibition of melanoma cell proliferation by resveratrol is correlated with upregulation of quinone reductase 2 and p53

Resveratrol (trans-3,4',5-trihydroxystilbene) is a grape-derived polyphenol under intensive study for its potential in cancer prevention. In the case of cultured human melanoma cells, no one to our knowledge has investigated whether resveratrol exerts similar anti-proliferative activities in cells with different metastatic potential. Therefore, we examined the effects of this polyphenol on the growth of weakly metastatic Line IV clone 3 and on autologous, highly metastatic Line IV clone 1 cultured melanoma cells. Comparable inhibition of growth and colony formation resulted from treatment by resveratrol in both cell lines. Flow cytometric analysis revealed that resveratrol-treated clone 1 cells had a dose-dependent increase in S phase and a concomitant reduction in the G(1) phase. No detectable change in cell cycle phase distribution was found in similarly treated clone 3 cells. Western blots demonstrated a significant increase in the expression of the tumor suppressor gene p53, without a commensurate change in p21 and several other cell cycle regulatory proteins in both cell types. Chromatography of Line IV clone 3 and clone 1 cell extracts on resveratrol affinity columns revealed that the basal expression of dihydronicotinamide riboside quinone reductase 2 (NQO2) was higher in Line IV clone 1 than clone 3 cells. Levels of NQO2 but not its structural analog NQO1 were dose-dependently increased by resveratrol in both cell lines. We propose that induction of NQO2 may relate to the observed increased expression of p53 that, in turn, contributes to the observed suppression of cell growth in both melanoma cell lines.     

中国两种掌突蟾(锄足蟾科Pelobatidae,无尾目Anura)的细胞遗传学研究

本文对分布于云南境内的两种Leptolalax——L.ventripunctatus和L.alpinus的常规Giemsa核型、C-带和Ag-NORs作了研究,结果表明L.ventripunctatus的2n=22,20M 2T,NF=42,1对Ag-NORs位于5(?),并呈现异形现象,该区域亦显C-带正染;L.alpinus 2n=24,14M 4SM 6T,NF=42,1对Ag-NORs位于No.8短臂端部,并有随体联合现象。两种的着丝点区域均呈现C-带正染。     

Brain cell karyotype of the phlebotomine sand fly Lutzomyia shannoni (Dyar) (Diptera: Psychodidae)

The brain cell karyotype of New World sand fly Lutzomyia shannoni was described. This species has four pairs of chromosomes, 2N=8, with one pair of heteromorphic chromosomes.     

Variation of wheatgrass chromosomes in wheat-wheatgrass alien addition line "TAI-27"revealed by fluorescence in situ hybridization (FISH)

The Karyotyp of the primary wheat-whastgrass alien addition line TAI-27 was 2n = 44 in which all d the chromosomes were metacentric and subrmetacentric. However, in the progeny of TAI-27 a pair of chromosomes had become small chromosomea in the two morphologically different plants. Fluorescence in situ hybridizstionm (FISH) technique was used to analyze the two different plants. The observations indicate that a pair of small chromosomes in one varietion line are from wheatgrass. In another variation line, a pair of small chromosomes are also from whest-grass, while another pair of wheatgrass chromosomes have substituted the wheat chromosomes. TAI-27 and its variant lines showed a high level of resistance to barley yellow dwarf virus (BYDV). The pessible explanation for such a variation and the potential use of the variant lines were discussed briefly.     

Variation of wheatgrass chromosomes in wheat-wheatgrass alien addition line “TAI-27” revealed by fluorescencein situ hybridization (FISH)

The Karyotyp of the primary wheat-whastgrass alien addition line TAI-27 was 2n = 44 in which all d the chromosomes were metacentric and subrmetacentric. However, in the progeny of TAI-27 a pair of chromosomes had become small chromosomea in the two morphologically different plants. Fluorescence in situ hybridizstionm (FISH) technique was used to analyze the two different plants. The observations indicate that a pair of small chromosomes in one varietion line are from wheatgrass. In another variation line, a pair of small chromosomes are also from whest-grass, while another pair of wheatgrass chromosomes have substituted the wheat chromosomes. TAI-27 and its variant lines showed a high level of resistance to barley yellow dwarf virus (BYDV). The pessible explanation for such a variation and the potential use of the variant lines were discussed briefly.