Yunzheng Le | PubMed Health

PubMed2003

Far-upstream elements are dispensable for tissue-specific proenkephalin expression using a Cre-mediated knock-in strategy.

Several cis-regulatory DNA elements are present in the 5' upstream regulatory region of the enkephalin gene (ENK) promoter. To determine their role in conferring organ-specificity of ENK expression in mice and to circumvent the position effects from random gene insertion that are known to often frustrate such analysis in transgenic mice, we used a Cre-mediated gene knock-in strategy to target reporter constructs to a "safe haven" loxP-tagged locus in the hypoxanthine phosphoribosyltransferase (HPRT) gene. Here we report reliable and reproducible reporter gene expression under the control of the 5' upstream regulatory region of the mouse ENK gene in gene-modified mice using this Cre-mediated knock-in strategy. Comparison of two 5'ENK regulatory regions (one with and the other without known cis-regulatory DNA elements) in the resulting adult mice showed that conserved far-upstream cis-regulatory DNA elements are dispensable for correct organ-specific gene expression. Thus the proximal 1.4 kb of the murine ENK promoter region is sufficient for organ-specificity of ENK gene expression when targeted to a safe-haven genomic locus. These results suggest that conservation of the far-upstream DNA elements serves more subtle roles, such as the developmental or cell-specific expression of the ENK gene.

5' Flanking Region↗

PubMed1996

Streptomyces akiyoshiensis differs from other gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids.

A partial Sau3Al digest of genomic DNA from Streptomyces akiyoshiensis was cloned in a Streptomyces-Escherichia coli shuttle vector, and the recombinant plasmids were used to transform E. coli CGSC 6212, which carries a mutation in the gene for aspartate semialdehyde dehydrogenase (Asd). One of 39,000 transformants tested grew on LB medium lacking diaminopimelate. A 17 kb plasmid (pJV21) isolated from this strain conferred prototrophy when used to transform E. coli CGSC 6212. The gene responsible was located on a 2.2 kb DNA fragment by subcloning. Nucleotide sequencing and codon preference analysis of the subcloned insert and of the 3.3 kb insert in the Asd(-)-complementing plasmid pJV36 located three complete and two incomplete open reading frames (ORFs). One of these (ORF3), encoding a polypeptide of 338 amino acids (Mr 35484), was identified as the gene for Asd by comparing its sequence with database sequences of asd from other bacteria. The inability of pJV30, in which a segment of ORF3 had been deleted, to transform E. coli CGSC 6212 to prototrophy supported this assignment. Southern hybridization indicated that the sequenced region of the cloned DNA fragment represented a continuous segment of the S. akiyoshiensis chromosome. The deduced amino acid sequences of the ORFs adjacent to asd showed no similarity to sequences for aspartate kinase (Ask); also, transformation with plasmids containing asd and adjacent regions from the S. akiyoshiensis chromosome did not complement the ask mutant E. coli CGSC 5074. It is concluded that asd and ask in S. akiyoshiensis are not present in an operon, and thus are organized differently from these genes in the Gram-positive bacteria previously examined.

Amino Acid Sequence↗