Presence of an SAR-like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency

A 12.5-kb DNA fragment with junction regions between the transgene and genomic DNA was cloned from a transgenic tobacco cell line obtained by microprojectile bombardment of plasmid pCaMVNEO. Nucleotide sequence analysis of the fragment (DDBJ accession no. D84238) showed that it carried a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and two identical 1.3-kb junction sequences that flanked both the 5' and 3' ends of the core sequence and had inverted orientations. These sequences had topoisomerase II (Topo II) cleavage sites and adenine and thimine-rich sequences known to be specific to nuclear scaffold-attachment regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated TJ1) from the 1.3-kb sequence had the ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirmation that the 1.3-kb sequence is an SAR. Insertion of TJ1 at the 5' and 3' sides of the expression cassette for the npt II gene increased transformant yields 5- to 10-fold and the NPT II enzyme activity per copy of the gene 5-fold. TJ1 enhances the integration or expression of the transgene, or both. Clearly, TJ1 is very useful for producing transgenic plants. This is the first report on an SAR-like sequence that is located in the transgene locus and enhances transformation efficiency in eukaryotic cells. The possible role of TJ1-SAR in the molecular evolution of plant genome is discussed.     

Serial Alu sequence transposition interrupting a human B creatine kinase pseudogene.

We have isolated, sequenced, and characterized a single-copy B creatine kinase pseudogene. The chromosomal assignment of this gene is 16p13 and a unique sequence probe from this locus detects EcoRI restriction fragment length polymorphisms of 7.8 and 5.4 kb. In 26 unrelated individuals, the frequencies for the 7.8- and 5.4-kb B creatine kinase pseudogene alleles were calculated to be 17.3 and 82.7%, respectively. The B creatine kinase pseudogene is interrupted by a 904-bp DNA insertion composed of three Alu repeat sequences in tandem flanked by an 18-bp direct repeat, derived from the pseudogene sequence. Nucleotide sequence analysis of the Alu elements suggests that the Alu sequences were incorporated into this locus in three separate integration events. Several complex clustered Alu repeat sequences without defined integration borders have been previously identified at different genomic loci. This is the first evidence that complex tandem Alu elements can integrate in an apparently serial manner in the human genome and supports the contention that Alu repeats integrate nonrandomly into the human genome.     

Cutting edge: distal regulatory elements are required to achieve selective expression of IFN-gamma in Th1/Tc1 effector cells

Using a transgenic approach, we analyzed the contribution of introns located within the IFN-gamma gene and distal regulatory regions to IFN-gamma gene expression. Intron 1 and 3 from the IFN-gamma gene displayed strong enhancer activity. This activity appeared to be dependent upon integration into the genome but resulted in a loss of Th1 selectivity. We also found that distal regulatory elements are not required for high level expression of the human IFN-gamma gene, but rather for cell lineage-specific expression. An 8.6-kb human IFN-gamma transgene was sufficient to yield high level expression but a 191-kb IFN-gamma transgene with approximately 90 kb of flanking 5' and 3' sequence was necessary to achieve both high level and Th1 selective expression of human IFN-gamma.     

Development of an all-fish gene cassette for gene transfer in aquaculture.

To develop an all-fish gene cassette suitable for gene transfer in aquaculture, the antifreeze protein (AFP) gene promoter from the ocean pout (Macrozoarces americanus) was analyzed for its ability to direct exogenous gene expression both in vitro and in vivo. The ocean pout AFP (opAFP) gene promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) was functionally analyzed in two fish cell lines and in Japanese medaka embryos. The opAFP gene promoter was active in these systems, as demonstrated by the transient expression of CAT activity. These results suggest that the opAFP gene promoter is useful for many other gene transfer experiments. To facilitate use of the opAFP gene promoter as a common and versatile vehicle for fish gene transfers, an expression vector, opAFP-V, was constructed by linking the 2.1-kb opAFP gene promoter, the 63-bp opAFP gene 5' untranslated sequence, and the 1.2-kb opAFP gene 3' sequence by two unique restriction sites, Bg/II and HpaI, respectively. Thus, genes of interest can be inserted into either the Bg/II site or the HpaI site depending on the length of their 5' untranslated sequence. The complete DNA sequence of opAFP-V was determined to facilitate future detailed analysis of integration and expression of the transgene.     

A single human keratin 18 gene is expressed in diverse epithelial cells of transgenic mice

The expression of keratin 18 (K18) is restricted in humans primarily to a variety of single layered or simple epithelia. However, direct introduction of a cloned K18 gene into cultured, somatic cells by DNA transfection has been shown to result in the promiscuous expression of K18 even while the endogenous mouse form of K18 (Endo B) remains silent. To determine if the cloned K18 genomic DNA fragment contains sufficient information to be regulated appropriately when subjected to a normal developmental environment, and to determine if the cloned gene is expressed in diverse epithelia, the K18 gene, including 2.5 kb of 5' flanking sequence and 3.5 kb of 3' flanking sequence, has been introduced into the germ line of mice. Mice from all three resulting K18 transgenic lines express the gene in an appropriate tissue-specific pattern that includes hepatocytes, simple epithelia of the intestinal tract, ductal cells of several glands and epithelial cells of the thymus. No expression of K18 was found in muscle, heart, or in most of the brain even in mice carrying 18 copies of the K18 gene. In most tissues, the level of K18 RNA was directly proportional to copy number and was as efficiently expressed as the endogenous Endo B gene. The K18 protein was identified by both protein blotting methods and indirect immunofluorescence staining. No pathological consequences of overexpression of the K18 gene were observed. The cloned K18 gene appears to contain all cis-acting DNA sequences necessary for appropriate expression. In addition, diverse epithelial cell types are able to express this single human gene.     

Regulation of BN115, a low-temperature-responsive gene from winter Brassica napus.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the beta-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2 degrees C. No expression was observed after incubation at 22 degrees C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.