Biomedical subjects

J A McClary

Publications and source records attributed to J A McClary.

3 recordsLinked to original sources

PubMed2001

Cloning and genetic characterization of an evolutionarily conserved human olfactory receptor that is differentially expressed across species.

We have cloned the full-length cDNA and genomic region of a human prostate specific G-protein coupled receptor with properties characteristic of an olfactory receptor. A partial cDNA sequence of this gene, called PSGR, was recently cloned. The gene contains two exons and one intron of 14.9 kb in its 5'untranslated region, and was mapped to human chromosome 11p15.2. A cluster of transcription initiation sites for the 2.8 kb PSGR mRNA was identified. Cloning of the homologous gene from the mouse revealed 93% amino acid homology between the human and mouse or rat (previously cloned as RA1c) proteins, and 99% identity between the rat and mouse homologs. Although northern analysis indicated expression of the human PSGR homolog was prostate specific, its mRNA could also be detected in the olfactory zone and the medulla oblongata of the human brain. In the mouse, the PSGR gene is predominantly expressed in the brain and colon. In the rat, the PSGR homolog is expressed in the liver in addition to the brain. These data add to the growing body of evidence suggesting that olfactory receptors may have functional roles in tissues other than the olfactory organ, and further, suggest that these functions may vary across species.

5' Flanking Region↗

PubMed1991

Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template.

A simple, rapid method is presented for the enzymatic sequence analysis of nanogram amounts of single-stranded or double-stranded DNA. This approach employs the thermostable DNA polymerase from Bacillus sterothermophilus and exploits its ability to efficiently extend all of the template-primer complex, even at low substrate concentrations. The procedure requires few pipetting steps, no preannealing step and very short reaction time. This method can significantly reduce the cost associated with DNA polymerase and the amount of template and time required to perform the enzymatic sequencing reactions. As little as a 10-ng aliquot of such sequencing reactions can be analyzed on a fluorescence-based capillary gel electrophoresis instrument recently developed in our laboratory. This highly sensitive detection, in conjunction with the ability to efficiently sequence nanogram amounts of template, strongly suggests the feasibility of direct DNA sequencing of single bacteriophage M13 plaques without prior amplification.

Bacteriophages↗

PubMed1989

Efficient site-directed in vitro mutagenesis using phagemid vectors.

Several methods have been developed that enhance the efficiency of in vitro, site-directed mutagenesis. Kunkel (8,9) has developed a method which uses a strong selection for the mutated strand and, hence, is highly efficient, but yet simple and rapid. This method originally used M13 phage as the vector. In this paper, we describe a refinement of this method using phagemid vectors, which combine the advantages of plasmids (such as high copy number and stability of cloned DNA) with the single-stranded DNA generating capability of M13 phage. We demonstrate that high efficiency of mutant production can be obtained with these vectors. We also analyzed by sequencing 11 mutated clones and found no second-site mutations, suggesting that alterations other than the site-directed mutation rarely occur in our system.

Escherichia coli↗