Linker Ligation Research Articles

Cloning of the shark Po promoter using a genomic walking technique based on the polymerase chain reaction

We have cloned the putative shark promoter of protein zero (Po) using a novel application of the ligation mediated single-sided polymerase chain reaction (PCR) method. This method uses linker ligation and subsequent amplifications with a linker primer and multiple specific primers to generate specificity. The method allowed us to amplify approximately 305 base pairs of shark genomic DNA sequence immediately upstream from the 5' end of our full-length Po cDNA. The Po promoter was shown to be directly linked to its first exon, contain a transcription initiation start site and sequences commonly found in eukaryotic promoters. This genomic walking technique will be useful for cloning promoters, insertion sites, and other sequences of interest without the need for constructing and screening genomic libraries.

Structure in the precore region of hepatitis B core gene affecting its expression in E. coli.

Restriction fragments of HBV-DNA, cleaved by endonuclease HhaI, containing HBcAg gene were trimmed by BAL-31 exonuclease to remove different lengths of the precore sequence. They were inserted into plasmid pUR222 at EcoRI site through synthetic linker ligation. Transformants in E. coli BMH7118 showing different levels of HBcAg gene expression were screened and analyzed for their nucleotide sequences in the junction region both by Maxam and Gilbert's chemical degradation method and by M13 chain termination method. Results of sequence analysis of different transformants revealed a partial palindromic (loop and stem) structure, at -7 to -35 nucleotide with regard to ATG of the HBcAg gene as position +1, which has dramatic effect on the level of expression of the inserted gene using the same promoter, SD sequence and identical N-terminus. The amount of HBcAg synthesized differed from 9% in the high expressing plasmid to less than 0.01% of the total cell proteins in the low expressing transformants. The findings were compared to results obtained by other workers in studies of HBcAg expression in procaryotes and their significance in the expression of eucaryotic genes in procaryotic cells were discussed.

A new method for linker ligation

A new linker ligation procedure that does not require recleavage with a restriction enzyme to generate cohesive ends is described. The unique aspect of this method is that unphosphorylated linkers are utilized in the ligation reaction. As a result, only one strand of the linker covalently joins to each end of a DNA fragment. Removal of the unligated complementary single-stranded linker by heating and quick cooling results in the generation of short tails at the 5′ ends of the DNA fragment. The linker used in this method does not have to be palindromic, and any unphosphorylated double-stranded DNA longer than a pentamer may be used for tailing purposes.

A general method for the transfer of cloned genes to plant cells

This paper describes a method for the transfer to plant cells of any cloned gene, regardless of its termini or internal restriction enzyme cleavage sites. A broad host-range intermediate vector, pGV1117, was constructed containing HindIII-23, a right-end T-region fragment of the nopaline plasmid pTiC58. Using in vivo protection by EcoRI methylase and EcoRI linker ligation, a fragment of rabbit chromosomal DNA, carrying the β-globin gene, was inserted into plasmid pGV1117. Following transmission to Agrobacterium tumefaciens, insertion of the gene into the T-region of pTiC58 occurred via in vivo recombination. Infection of axenic tobacco seedlings resulted in the transfer to the plant genome of an intact β-globin gene, as part of the T-DNA. Although the gene was stably maintained during tissue culture, β-globin-specific transcripts were not detected in the transformed plant cells.

Asymmetric linker molecules for recombinant DNA constructions

Asymmetric EcoRI DNA linkers consisting of an AATTC(A) 7 dodecamer and a complementary G(T) 7 octamer were synthesized. Ligation of such linkers to DNA fragments obviates the need for EcoRI digestion prior to cloning in EcoRI-cleaved vectors.