Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5'' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.     

Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize

An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25–30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences.     

Transgene expression produced by biolistic-mediated, site-specific gene integration is consistently inherited by the subsequent generations

The efficient production of stable transgenic plants is important for both crop improvement and functional genomics. Site-specific integration of foreign genes into a designated genomic position is an attractive tool for minimizing expression variability between transgenic lines. Here, we studied the utility of a Cre-mediated, site-specific integration approach, facilitated by particle bombardment, for streamlining the production of stable transgenic plants, using rice as a model species. Using this method, we generated 18 different transgenic lines containing a precise integration of a single copy of beta-glucuronidase gene (gusA) into a designated genomic location. Eleven of these lines contained no illegitimate integration in the background (single-copy lines), and seven contained illegitimate integrations in addition to the site-specific integration (multicopy lines). We monitored gusA expression in these lines up to three to four successive generations. Each of the single-copy lines expressed the gusA gene at consistent levels and nearly doubled the expression level in the homozygous state. In contrast, multicopy lines displayed expression variation and gene silencing. In about half of the multicopy lines, however, expression of the site-specific integration locus could be reactivated and stabilized on segregation of the illegitimate integrations, whereas, in the remaining half, expression could not be restored, as they contained genetically linked illegitimate integrations. This study demonstrates that biolistic-mediated, site-specific gene integration is an efficient and reliable tool for streamlining the production of stable transgenic plants.     

异源DNA导入水稻诱发活跃反转子Tos17发生可遗传DNA甲基化变异

用水稻(Oryza sativa L.)内源反转座子Tos17为探针,经Southern杂交在5种含有野生稻(Zizania latifolia Griseb)(菰)DNA片段的水稻渐渗杂交系中检测到了可遗传DNA甲基化变异.在分析的4种甲基化敏感限制性内切酶中,每种酶切都发生了亲本杂交片段的消失和新片段的出现.发生甲基化变异的位点包括对称和不对称的胞嘧啶碱基,也包括腺嘌呤碱基.序列分析表明,与水稻亲本比较,所研究的5种渐渗杂交系在Tos17的2个重要区域(5'-LTR和RT)均未发生序列变异.但甲基化敏感-序列特异性PCR分析证实,每种渐渗杂交系在这2个区域内均发生了广泛的DNA甲基化变异.而且,在2种渐渗杂交系中发现5'-LTR和RT区域的甲基化变异存在协同性.甲基化变异可稳定遗传给后代.因为已有的研究表明,在这5种渐渗杂交系中异源DNA导入均导致了Tos17的激活和转座,因此可以推测DNA甲基化在调控Tos17活性中可能具有一定作用.但反转座子激活和甲基化变异之间的确切关系尚有待进一步研究.     

Transgenic mice harboring SV40 t-antigen genes develop characteristic brain tumors

A high percentage of transgenic mice developing from eggs microinjected with plasmids containing the SV40 early region genes and a metallothionein fusion gene develop tumors within the choroid plexus. A line of mice has been established in which nearly every affected animal succumbs to this brain tumor. Thymic hypertrophy and kidney pathology are also observed in some mice. SV40 T-antigen mRNA and protein are readily detected in affected tissues; however, SV40 T-antigen gene expression is barely detectable in unaffected tissues or in susceptible tissues prior to overt pathology, suggesting that tumorigenesis depends upon activation of the SV40 genes. Comparison of DNA from tumor tissue (or cell lines derived from tumors) with DNA from unaffected tissues reveals structural rearrangements as well as changes in DNA methylation of the foreign DNA. The SV40 genes are frequently amplified in tumor tissue, which further indicates that their expression is intimately involved in tumorigenesis in transgenic mice.     

Gene stability in transgenic aspen (Populus). II. Molecular characterization of variable expression of transgene in wild and hybrid aspen

In many annual plant species, transgene inactivation occurs most often when multiple incomplete/complete copies of the transgene are present in a genome. The expression of single-copy transgene loci may also be negatively influenced by the flanking plant DNA and/or chromosomal location (position effect). To understand transgene silencing in a long-lived tree system, we analyzed several wild (Populus tremula L.) and hybrid (P. tremula L. x P. tremuloides Michx.) aspen lines transgenic to the rolC phenotypical marker system and grown under in vitro, greenhouse and field conditions. The morphological features of the 35S-rolC gene construct were used to screen lines with altered transgene expression, which was later confirmed by Northern experiments. Molecular analyses of hybrid aspen revealed that transgene inactivation was always a consequence of transgene repeats. In wild non-hybrid aspen, however, multiple-insertion-based altered or loss of rolC expression was observed only in three out of six lines showing transgene inactivation. Sequencing analysis revealed AT-rich patches at the transgene flanking genomic regions of some of the wild aspen transgenic lines. One wild aspen line showing variable rolC expression revealed characteristic integration of the transgene into genomic regions containing a high AT content (85% or more). In the remaining two wild aspen transgenic lines unstable for rolC expression, single-copy integration and non-AT-rich or repeat-free transgene flanking regions were found. A partial suppression of rolC was observed in some plants of one of the field-grown wild aspen transgenic lines. In the other wild aspen transgenic line an additional mutant phenotype along with transgene inactivation was found. This indicates that the host genome has some control over expression of a transgene, and the possible role of AT-rich regions in defense against foreign DNA.     

A 5' differentially methylated sequence and the 3'-flanking region are necessary for H19 transgene imprinting.

The mouse H19 gene is expressed exclusively from the maternal allele. The imprinted expression of the endogenous gene can be recapitulated in mice by using a 14-kb transgene encompassing 4 kb of 5'-flanking sequence, 8 kb of 3'-flanking sequence, which includes the two endoderm-specific enhancers, and an internally deleted structural gene. We have generated multiple transgenic lines with this 14-kb transgene and found that high-copy-number transgenes most closely follow the imprinted expression of the endogenous gene. To determine which sequences are important for imprinted expression, deletions were introduced into the transgene. Deletion of the 5' region, where a differentially methylated sequence proposed to be important in determining parental-specific expression is located, resulted in transgenes that were expressed and hypomethylated, regardless of parental origin. A 6-kb transgene, which contains most of the differentially methylated sequence but lacks the 8-kb 3' region, was not expressed and also not methylated. These results indicate that expression of either the H19 transgene or a 3' DNA sequence is key to establishing the differential methylation pattern observed at the endogenous locus. Finally, methylation analysis of transgenic sperm DNA from the lines that are not imprinted reveals that the transgenes are not capable of establishing and maintaining the paternal methylation pattern observed for imprinted transgenes and the endogenous paternal allele. Thus, the imprinting of the H19 gene requires a complex set of elements including the region of differential methylation and the 3'-flanking sequence.     

Generation of marker‐free transgenic hexaploid wheat via an Agrobacterium‐mediated co‐transformation strategy in commercial Chinese wheat varieties

Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker‐free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium‐mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker‐free transgenic wheat plants from various commercial Chinese varieties and their F₁ hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T‐DNA regions. The average co‐integration frequency of the gus and the bar genes located on the two independent T‐DNA regions was 49.0% in T₀ plants. We further found that the efficiency of generating marker‐free plants was related to the number of bar gene copies integrated in the genome. Marker‐free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T₁ positive plants, but the gus gene was never found to be silenced in T₁ plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants.     

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.     

转基因水稻中外源基因的荧光原位杂交（FISH）分析

Using fluorescence in situ hybridization (FISH) with metaphase preparations, we localized a transferred barnase-psl DNA sequence onto chromosomes in 8 rice transgenic plants. All the tested rice transgenic lines showed hybridization signals on the middle and terminal regions of chromosome arms except for those close to centromeres. In two lines, two different integration sites were identified, and the other lines showed only one integration site. With the aid of Southern analysis and expression detection, we found that the barnase tended to show a higher level expression in the lines whose integration sites near the distal regions of chromosomes, while the expression level became lower in the lines whose integration sites near the centromeres. This result suggested a possible relationship between chromosomal location of transgenes and the expression level. However it showed no obvious relationship between copy numbers and expression levels. In most cases, the results of multi-color FISH showed that barnase-ps1 always integrated at the same position on the chrmosome as the reporter genes(pHctinG).     

Divergent DNA methylation contributes to duplicated gene evolution and chilling response in tea plants

The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H = A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.