A restriction endonuclease cleavage map of mitochondrial DNA from transformed hamster cells.

Mitochondrial DNA from cultured C13/B4 hamster cells was cleaved by the restriction endonucleases Hpa II, Hind III, Eco RI and Bam HI into 7, 5, 3 and 2 unique fragments, respectively. The summed molecular weights of fragments obtained from electrophoretic mobilities in agarose-ethidium bromide gels (with Hpa I-cleaved T7 DNA as standard) and electron microscopic analysis of fragment classes isolated from gels (with SV40 DNA as standard) were in good agreement with the size of 10.37 +/- 0.22 x 10(6) daltons (15,700 +/- 330 nucleotide pairs) determined for the intact circular mitochondrial genome. Cyclization of all Hind III, Eco RI and Bam HI fragments was observed. A cleavage map containing the 17 restriction sites (+/- 1% s.d.) was constructed by electrophoretic analysis of 32P-labeled single- and double-enzyme digestion products and reciprocal redigestion of isolated fragments. The 7 Hpa II sites were located in one half of the genome. The total distribution of the 17 cleavages around the genome was relatively uniform. The position of the D-loop was determined from its location and expansion on 3 overlapping restriction fragments.     

Production and characteristics of seven rat embryo cell lines transformed by adenovirus type 5 and its DNA

Seven cell lines transformed by adenovirus type 5 and its DNA were obtained. It was shown that different cell lines contain the fragments of viral DNA which differ in length and number of copies per DNA of diploid cells. They contain from the left end 6% of the viral DNA to complete or almost complete viral genome. All studied cell lines were sensitive to reinfection with adenovirus type 5. They produced no virus being cocultivated with cell sensitive to the virus. No cell line was able to induce tumors even in immunosuppressed newborn rats. All cell lines formed colonies in soft agar. The level of virus-specific antigens was higher in cells that contained a large part of the viral genome. The methods used did not allow to correlate the biological properties of the transformed cells with the length and the number of copies of the integrated part of the viral genome.     

In vitro synthesis of adenovirus type 5 T antigens. II. Translation of virus-specific RNA from cells transformed by fragments of adenovirus type 5 DNA.

Virus-specific cytoplasmic RNA was isolated from rat cell lines transformed by fragments of adenovirus type 5 DNA, and the RNAs were translated in cell-free systems derived from wheat germ or rabbit reticulocytes. RNA was isolated from cell lines transformed by the following fragments: XhoI-C (leftmost 15.5%), HindIII-G (leftmost 8%), and HpaI-E (leftmost 4.5%). In addition, the adenovirus type 5-transformed human embryonic kidney line 293.C31 was investigated. The products were immunoprecipitated with serum from tumor-bearing hamsters and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The results show that all transformed cells investigated contain early region 1a-specific RNAs which can be translated into proteins with molecular weights of 34,000 (34K), 36K, 40K, and 42K. Transformed cells that also contain an intact early region 1b synthesized RNA which can be translated into proteins with molecular weights of 19K and 65K. Minor proteins of 15K, 16K, 17.5K, 18K, 25K, and 29K were also observed, but these proteins could not be mapped unambiguously. Cells transformed by the 8% HindIII-G apparently lack RNA encoding the 65K protein, but they do contain RNA coding for the 19K protein.     

Characterization of yeast ribosomal DNA fragments generated by EcoR1 restriction endonuclease

Summary The action of Escherichia coli restriction endonuclease R1 (EcoR1) on DNA isolated from Saccharomyces cerevisiae (strain MAR-33) generates three predominent homogenously sized DNA fragments (species of 1.8, 2.2 and 2.5 kilo nucleotide base pairs (KB). Many DNA species of molecular weight greater than 2 million daltons can be recognized upon incomplete EcoR1 digestion of yeast DNA. Four additional DNA species ranging from 0.3–0.9 KB can be identified as the second major class of EcoR1-yeast DNA products.Hybridization with radioactive ribosomal RNA (rRNA) and competition with nonradioactive rRNA show that of the three predominent EcoR1-yeast DNA species, the 2.5 KB species hybridizes only with the 25S rRNA while the lighter 1.8 KB species hybridizes with the 18S rRNA. The intermediate DNA species of 2.2 KB hybridizes to a small extent with the 25S rRNA and could be a result of the presence of the 2.5 KB DNA species. The mass proportions and hybridization values of these 3 DNA species account for about 60% of the total ribosomal DNA (rDNA).The 5EcoR1-yeast DNA species of less than 0.9 KB (4 major and 1 minor species) hybridize to varying degrees with the 2 rRNA and can be grouped in two classes. In one class there are 3 DNA species that hybridize exclusively with the 18S rRNA. In the second class there are 2 DNA species that besides hybridizing predominently with the 25S rRNA also hybridize with the 18S rRNA. The 7 EcoR1-yeast DNA species (excluding the 2.2 KB DNA species) that hybridize with the two rRNA account for nearly a 5 million dalton DNA segment, which is very close to the anticipated gene size of rRNA precursor molecule. If the 2.2 KB DNA species is a part of the rDNA that is not transcribed or 5 sRNA then the cistron encoding the rRNA in S. cerevisiae has at least 8 EcoR1 recognition sites resulting in 8 DNA fragments upon digestion with the EcoR1. Consideration is given to the relationship of the rRNA species generated by EcoR1 digestion and the chromosomes containing ribosomal cistrons.     

Fish cell lines: Establishment and characterization of nine cell lines from salmonids

Summary Nine permanent cell lines have been established from five species of salmonids native to America's Pacific Northwest. With the exception of a hepatoma from an adult trout, the lines were derived from normal tissues of embryonic or juvenile fish. Cells were routinely grown in Eagle's minimum essential medium with 10% fetal bovine serum. Optimum growth temperatures for these lines ranged from 21 to 24°C. All survived storage for at least 1 yr at −65°C and at least 5 yr in liquid nitrogen. Six of the lines were demonstrably free of any microbial contamination but mycoplasmas were found in three. Eight of the lines were heteroploid. The morphology of only one was fibroblastic. All the lines effectively replicated one or more of the common salmonid viruses. Isozyme patterns were consistent with those of the species of origin. These cell lines have significant application in fish virology. This work is a result of research sponsored in part by the Oregon State University Sea Grant College Program supported by NOAA Office of Sea Grant, U.S. Department of Commerce, under Grant NA79AA-D-0016 and by the Oregon Department of Fish and Wildlife under PL-89304 Anadromous Fish Act and is Oregon Agricultural Experiment Station Technical Paper 6857.     

Viral gene products in adenovirus type-2 transformed hamster cells.

I have analyzed viral gene products expressed in five adenovirus type 2 (Ad2)- cytoplasmic, viral RNA which was selected by hybridization to cloned restriction endonuclease fragments of Ad2 DNA. Proteins synthesized in vitro were analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and compared with those directed by RNAs prepared from productively infected cells. The early regions E1 and E4 of adenovirus type 2 (Ad2) were found to be expressed in all of five Ad2-transformed hamster embryo cells lines. RNA transcribed from early region E2, which codes for the 72,000-molecular-weight (72K) DNA-binding protein was detected in cell line HE1 only, and early region E3 was expressed exclusively in cell line HE4. RNA transcribed from the region between approximately 12 and 35 map units, coding for immediate early (13.5K, 52/53K) and immediate early proteins (13.6K, 16K, 17K, 87K), as well as RNA from late genes, was not found in any of the cell lines HE1 to HE5 had electrophoretic mobilities similar to those programmed by RNA from productively infected cells.     

Identification of a new strain of adenovirus type 2 by restriction endonuclease analysis

Restriction endonuclease analysis has been used to identify a new strain Ad2a of human adenovirus type 2 (Ad2). The sites for six restriction endonucleases have been mapped on the Ad2a genome and compared with those for the Ad2 prototype.     

Establishment and characterization of bovine mammary epithelial cell lines

Summary One bovine mammary epithelial cell clone, designated PS-BME-C1, and two bovine mammary epithelial cell lines, designated PS-BME-L6 and PS-BME-L7, were derived from mammary tissue of a pregnant (270 day) Holstein cow. The cells exhibit the distinctive morphologic characteristics of mammary epithelial cells and express the milk fat globule membrane protein, PAS-III. They form domes when cultured on plastic substrata and acinilike aggregates when cultured on a collagen matrix. These cells are capable of synthesizing and secretingα-lactalbumin andα-s₁-casein when cultured on a collagen matrix in the presence of insulin, cortisol, and prolactin. The cells have a near-normal diploid number and do not grow in suspension culture. When transplanted to the cleared mammary fat pads of female athymic nude mice, the cells readily proliferate forming noninvasive palpable spherical cellular masses within 8 wk after inoculation. The cells may become a useful tool to study the regulation of ruminant mammary epithelial cell growth and differentation. This work was supported by the Pennsylvania State University Experiment Station. The PS-BME cells are the property of The Pennsylvania Research Corporation. Scientists interested in obtaining the PS-BME clone or cell lines for their research may request them from the corresponding author.     

人胚胎干细胞建系和鉴定

人胚胎干细胞是一种取自人囊胚内细胞团且具有形成所有三个胚层细胞能力的全能细胞。建立一个理想的人胚胎干细胞培养系统是研究和利用这种具有巨大潜力细胞的首要条件。本文讨论了目前建立的人胚胎干细胞培养系统,阐述了其有利的和不利的一面,并着重讨论其体外培养方法和鉴定策略。     

Purification and characterization of the FokI restriction endonuclease

The restriction endonuclease FokI from Flavobacterium okeanokoites was purified to homogeneity. Based on gel filtration, sedimentation and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the following properties of the enzyme were determined: FokI exists in one active monomeric form, and has an Mr of 64-65.4 x 10(3).FokI is a strongly basic protein with an isoelectric point of 9.4. The enzyme exhibits restriction activity in the pH range 5.0 to 10.5 (maximum level at pH 7.0-8.5) and its divalent cation requirement is satisfied not only by Mg2+, but also by Co2+, Mn2+, Ni2+, Cd2+, Zn2+ and Fe2+.     

Establishment and characterization of five human myeloma cell lines

Since 1980 five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12-PE and KMS-12-BM, have been established in Kawasaki Medical School. Histologically, all the cell lines resembled plasma cells and were EBNA negative. KMM-1, KMS-11, KMS-12-PE and KMS-12-BM reacted with PCA-1, while KMM-1, KMS-12-PE and KMS-12-BM with CD38. KMM-1 and KMS-11 secreted immunoglobulins into culture medium. Karyologically, all the cell lines were abnormal. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.     

Functional macrophage cell lines transformed by Abelson leukemia virus.

Three cloned cell lines have been established from murine tumors induced with Abelson leukemia virus which express properties of macrophages. Two of the three original tumors in addition yielded lymphocyte cell lines, one typical of the Abelson virus disease and the other a thymic lymphoma. Two of the macrophage lines are tumorigenic when placed in syngeneic mice. All of the macrophage lines pinocytose neutral red, phagocytose zymosan and latex beads, mediate antibody-dependent killing and phagocytosis of sheep erythrocyte targets, and secrete high levels of lysozyme. None of these properties was exhibited by the lymphocyte lines. Of the two macrophage cell lines tested, neither was capable of replacing the adherent cell population required for the induction of in vitro immune responses. An agent that activates normal macrophages, bacterial lipopolysaccharide, specifically inhibits the growth of the transformed macrophages in culture. Secretion of infectious Abelson leukemia virus by two of the macrophage lines, RAW 309Cr and WR 19M, provides conclusive evidence that the Abelson virus is capable of productively infecting the macrophage cell type. The other macrophage line, RAW 264, fails to secrete detectable virus particles and is negative in the XC plaque formation assay, as well as the fibroblast transformation assay for Abelson virus, but becomes positive for Abelson virus production after rescue by Moloney leukemia virus.     

Isolation and characterization of vitamin A-sensitive Chinese hamster lung cell lines

Retinyl acetate (RA)-sensitive variants (RAs-2 and RAs-3) of V79 cell line were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The variants were stable and showed a 3- to 4-fold increase in sensitivity to RA compared to parental V79 cells. The RAs-2 clone was also sensitive to retinol and retinol palmitate. The RA-sensitivity behaves as a recessive trait in all hybrids of RAs-2 and V79. A number of physiological parameters were indistinguishable in V79 and RAs-2 cells, including the extent of uptake of [3H]retinol, the release of K+ from the cells induced by RA, and the levels of retinol and retinoic acid binding proteins. However, one possible correlation with the RA-sensitive phenotype was observed: Gomori acid-phosphatase staining of RA-treated RAs-2 and V79 cells indicated that lysosomal membrane of RAs-2 cells was more labile than those of the parental V79 cells.