The somatic replication of DNA methylation

We have tested the hypothesis that DNA methylation patterns are replicated in the somatic cells of vertebrates. Using M-Hpa II, the modification enzyme from Haemophilus parainfluenzae which methylates the internal cytosine residues in the sequence 5'CCGG 3' GGCC, we methylated bacteriophage phi X174 RF DNA and the cloned chicken thymidine kinase (tk) gene in vitro and then introduced these DNAs and unmethylated controls into tk- cultured mouse cells by DNA-mediated transformation. Twenty-five cell generations later, the state of methylation of transferred DNA was examined by restriction endonuclease analysis and blot hybridization. We conclude that methylation at Hpa II sites is replicated by these cultured cells but not with 100% fidelity. We have also noted that methylation of the cloned chicken tk gene decreases its apparent transformation efficiency relative to unmethylated molecules.     

Mitochondrial DNA heteroplasmy in calves cloned by using adult somatic cell

Adult somatic cell cloned calves were produced by somatic cell nuclear transfer prepared by fusion of cultured ear fibroblast from a Holstein cow into enucleated oocytes of Luxi Yellow cow. In order to determinate the source of mitochondrial DNA of cloned calves, we designed the breed-specific PCR primers by aligning the known D-loop sequences of Bos taurus and analyzed the displacement loop sequences of five live cloned calves by breed-specific primers PCR. The results demonstrated that mtDNA originated from Holstein breed and that from Luxi breed co-exist in all five live calves.     

Retention of mitochondrial DNA species in somatic cell hybrids using antibiotic selection

Interspecific cell hybrids were constructed by fusion of an antimycin-resistant, thymidine kinase- (TK-) Chinese hamster cell line with a chloramphenicol-resistant, hypoxanthine-guanine phosphoribosyl transferase- (HPRT-) mouse cell line. Hybrids were selected in HAT medium alone, or HAT supplemented with chloramphenicol, antimycin, or both antibiotics. Analysis of the mitochondrial DNA (mtDNA) of these hybrids indicates that antibiotic selection directed at the mitochondrial populations results in retention of the resistant parental genome and loss of the sensitive parental genome.     

The transformation of legionellae by plasmid DNA

For the first time the possibility of the genetic transformation of L. pneumophila and L. bozemanii strains with the use of purified DNA of plasmids pUC19, pUC4K, pSC101 and RSF1010-pBR322 was shown. The frequency of transformation varied from 5.2 x 10(-6) to 5.8 x 10(-7), depending on the strain used in the experiment and plasmid DNA. In some of the transformants obtained in this investigation plasmid DNA whose molecular weight was similar to that of the plasmid DNA used for transformation was detected. The relatively stable preservation of plasmids pSC101 and RSF1010 in Legionella strains and the loss of plasmids pUC19, pUC4K and pBR322 in 80% of transformants during storage were shown.     

Regional localization of DNA sequences on chromosome 21 using somatic cell hybrids.

We have used a panel of Chinese hamster X human somatic cell hybrids, each containing various portions of chromosome 21 as the only detectable human chromosome component, for regional mapping of cloned, chromosome 21-derived DNA sequences. Thirty unique and very low-repeat sequences were mapped to the short arm and three sections of the long arm. Three unique sequences map to the proximal part of the terminal band 21q22.3, and five to the distal part of this band. Some of these may represent parts of gene sequences that may be relevant to the pathogenesis of Down syndrome, as 21q22 is the area required to be present in triplicate for the full clinical picture.     

Efficient transformation of filamentous fungus Pleurotus ostreatus using single-strand carrier DNA

The effects of carrier DNAs on the transformation of the basidiomycete Pleurotus ostreatus were analyzed. When lambda phage DNA was added to a transformation mixture containing protoplasts and CbxR vector plasmid, an increased number of drug-resistant transformants was observed on a screening plate containing 2 microg carboxin/ml. The highest efficiency (about 200 transformants/microg vector plasmid) was obtained by the addition of heat-denatured lambda DNA, which gave yields approximately 50-fold higher than the control experiment without a carrier DNA. To our knowledge, this is the first report on enhancement in transformation efficiency of fungal protoplasts by single strand carrier DNA.     

Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajearska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l^–1 sucrose, 1 g l^–1 yeast extract and 0.05 mg l^–1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l^–1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or -glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - Km kanamycin - NPTII neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid - BM basal medium     

Genetic transformation of somatic cells. XIII. Limited replication of DNA containing a fragment of human satellite DNA III in mouse cells

The plasmids containing the variant sequence of human satellite III "rescued" after replication in Chinese hamster cells were transfected into Chinese hamster, mouse and human cells by DEAE-dextran method. Several days after transfection extrachromosomal fractions were isolated, treated with DpnI, transformed into E. coli. In mouse cells, transformed with oncogene v-myc, after transient transfection of HS3-containing plasmids the appearance of rearranged and non-rearranged DpnI-resistant plasmids has been found. At the same time MboI-sensitive plasmids were not found in this material. The data suggest a limited replication (1 round) of HS3-containing plasmids in mouse cells transformed with oncogene v-myc.     

Random sequencing of Paramecium somatic DNA

We report a random survey of 1 to 2% of the somatic genome of the free-living ciliate Paramecium tetraurelia by single-run sequencing of the ends of plasmid inserts. As in all ciliates, the germ line genome of Paramecium (100 to 200 Mb) is reproducibly rearranged at each sexual cycle to produce a somatic genome of expressed or potentially expressed genes, stripped of repeated sequences, transposons, and AT-rich unique sequence elements limited to the germ line. We found the somatic genome to be compact (>68% coding, estimated from the sequence of several complete library inserts) and to feature uniformly small introns (18 to 35 nucleotides). This facilitated gene discovery: 722 open reading frames (ORFs) were identified by similarity with known proteins, and 119 novel ORFs were tentatively identified by internal comparison of the data set. We determined the phylogenetic position of Paramecium with respect to eukaryotes whose genomes have been sequenced by the distance matrix neighbor-joining method by using random combined protein data from the project. The unrooted tree obtained is very robust and in excellent agreement with accepted topology, providing strong support for the quality and consistency of the data set. Our study demonstrates that a random survey of the somatic genome of Paramecium is a good strategy for gene discovery in this organism.     

The extra DNA of brain cortex neurons is qualitatively indistinguishable from other somatic DNA

Brain cortex neurons from adult mammals contain amounts of DNA elevated to between the diploid and tetraploid level. In the rat, the rise in DNA starts rapidly a few hours before birth, then slows down to taper off at 3 weeks after birth. In view of the possibility that the extra amount of DNA may reflect the specific amplification of a restricted set of functionally important genes, we have searched for qualitative differences distinguishing the extra DNA of rat cortex neurons from other somatic DNA, the latter being represented by prenatally synthesized neuronal DNA and liver DNA. However, all DNAs examined were found to be indistinguishable by multiple criteria, such as base composition, secondary structure content, appearance of native and heat-denatured DNA in the electron microscope, and proportion of sequences with varying degrees of repetition. Further, no evidence for selective gene amplification was obtained. Thus, at the present level of analysis the postnatally synthesized extra DNA of cortex neurons seems not to differ qualitatively from other somatic DNA.     

植物体细胞原生质体遗传转化研究

重点介绍了植物体细胞原生质体遗传转化的方法和当前已经取得的成果,同时提出了目前原生质体遗传转化中存在的问题,展望了今后的工作重点。植物原生质体遗传转化的方法主要有：PEG介导转化法、电击穿孔转化法、脂质体介导转化法、农杆菌共培养转化法等。     

The somatic embryogenesis and establishment of transformation experiment system in Larix principis-Rupprechtii]

Larix principis-Rupprechtii is one of the superior afforestation forest trees growing in north China. Embryogenic cultures were initiated from immature zygotic embryos of Larix principis-Rupprechtii on S culture medium containing 2, 4-D 0-2.2 mg/L, KT and BA each at 0-0. 8 mg/L. Embryogenic calli were subcultured and multiplicated on S + B culture medium containing dropping off each hormone concentration. We set up 33 steady-going embryogenic cell lines; We studied on the growth stage and genotype differences of every embryogenic cell lines; and Finded more than 10 high-frequency somatic embryogenesis cell lines such as 2K, 2T, 2I, 2J, 3C etc.. The number of 2T somatic embryos reaches 314/per gram of embryogenic tissue and the number of 3C somatic embryos is 185/per gram of embryogenic tissue. The re-induction method of Larix principis-Rupprechtii from somatic embryos was used to produce renewable embryogenic cultures and steady-going embryogenic cell lines effectively. Mature somatic embryos can germinate and develop further into plantlets when they are isolated and cultured on a hormone-free WPM culture medium. The regeneration plantlets were obtained. Furthermore, the transformation with a truncated gene of Bacillus thuringensis (B. t) were carried out, the PCR showed positive results, because of this, embryogenic cell line of Larix principis-Rupprechtii can be used for transformation experiments to support further breeding in forestry.     

Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos

Summary Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 g·ml^–1 kanamycin. Four putative transgenic plant lines were obtained from the cp gene^– vector and two from the cp gene⁺ vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene⁺ plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.Journal Series no. 3757 of the Hawaii Institute of Tropical Agriculture and Human Resources     

农杆菌介导的苜蓿次级体细胞胚的遗传转化

采用农杆菌菌株GV3101感染子叶期苜蓿体细胞胚来研究苜蓿次级体细胞胚的遗传转化方法。农杆菌菌株GV3101双相载体pCAMBIA2301,此双相载体具有gus报告基因和nptⅡ抗卡那霉素筛选基因。感染的子叶期苜蓿体细胞在75 mg/L卡那霉素筛选压下,经过一系列诱导培养,最终获得转基因植株。然后,通过GUS组织化学定位分析来检测转基因植株不同器官中的GUS表达,并进一步通过PCR和Southern杂交确定转基因的稳定整合和转化率。结果表明转基因植株不同器官均有GUS表达,整合的nptⅡ基因的拷贝数是1~4,获得的转基因植株的转化率是65.82%。     

DNA delivery into rice cells and transformation using silicon carbide whiskers

A plasmid (p Act1-F), containing b-glucuronidase (GUS) as a reporter gene, was delivered into embryogenic rice cells by using silicon carbide whiskers (SCW). The cells were thoroughly mixed with the plasmid and SCW, incubated for 1 day and transient GUS activity was revealed histochemically. Under optimal conditions, 533 transformants per 1 g wet cells were observed.     

The relationship between transformation and somatic mutation in human and Chinese hamster cells

The frequencies of transformations of primary human and Chinese hamster fibroblasts have been compared with the spontaneous and induced frequencies of mutation for resistance to thioguanine and ouabain, and for ability to use fructose, using the carcinogens benzo (alpha) pyrene and urethane. Whereas the rates and frequencies of mutation were similar in the two cell systems, transformations to morphologically altered cells was observed only in hamster cells. The frequency of this latter transformation event in hamster cells was abour 10(3) greater than the frequencies of mutation in these cells. The morphologically altered cells formed in the above transformation process cannot grow in agar (aga-) and do not produce tumors when injected into animals. The frequency of transition of these latter cells to aga+ cells which produce tumors in animals is similar to the mutation-like events.