Library-independent cloning of genomic fragments adjacent to vector integration sites: isolation of the EB4-PSV gene from a Dictyostelium gene disruption transformant.

We have designed an efficient procedure to clone genomic DNA adjacent to the integration site of transformation vectors. Using this method on a Dictyostelium gene disruption transformant, we have cloned a 5kb fragment which has previously escaped isolation by conventional library screening. Our protocol eliminates recloning of the original vectors which are often integrated in multiple tandem copies (1) but specifically recovers vectors containing genomic fragments adjacent to the integration site. The protocol is useful to isolate flanking fragments in gene disruption transformants to characterize flanking regions which may influence the expression of transformed genes by position effects and to identify sites of insertional mutagenesis.     

Structure, expression, and inactivation by gene disruption of the Dictyostelium discoideum prespore gene EB4

We present the nucleotide sequence of the cell type specific prespore gene EB4 which encodes a protein containing a hydrophobic leader sequence and two distinct domains of amino acid repeats. By RNase protection experiments we have determined the genomic organization of the gene which differs substantially from the previously published data. An antibody directed against one of the repeat structures (hexamer repeat) specifically reacts with a developmentally regulated antigen of 58 kd. Gene disruption transformants were obtained by transformation with a genomic DNA fragment. The transformants express a 3' truncated mRNA and do not react with the anti-hexamer antibody. So far, we could not detect any phenotypic aberrations in the transformed cell line.     

Odorant receptor gene regulation: implications from genomic organization.

Odorant receptor genes comprise the largest known family of G-protein-coupled receptors in vertebrates. These receptor genes are tightly clustered in the genomes of every vertebrate organism investigated, including zebrafish, mice and humans, and they appear to have expanded and duplicated throughout evolution. In a mechanism that has yet to be elucidated, each olfactory neuron expresses a single receptor gene. This highly restricted expression pattern underlies the ability to distinguish between a wide variety of odorants. Here, we address the evolutionary expansion of odorant receptor genes and the role genomic organization of these genes might have in their tightly regulated expression.