James J. Murtagh

Human 15-Lipoxygenase gene promoter: analysis and identification of DNA binding sites for IL-13-induced regulatory factors in monocytes

In order to study the transcriptional control of 15-LO expression, we have cloned and sequenced the human 15-LO promoter region. The 15-LO promoter is associated with a CpG island at the 5′-end of the gene, and sequence analysis reveals putative Sp1 and Ap2 binding site/s and absence of TATA or CAAT motifs. Transcription is initiated at one major site. Using deletion constructs, we have defined an active promoter region of 1056 bp. Gel-shift assays revealed that transcriptional factor(s) induced only in response to IL-13 treatment of human peripheral blood monocytes bind to the 15-LO promoter DNA. Two regions, DP1 (−140 to −92 bp) and DP2 (−353 to −304 bp) of the promoter were essential for transcription in HeLa cells and human peripheral monocytes. Hela nuclear extracts contained a specific nuclear factor(s) binding to 15-LO promoter DNA which are distinct from those derived from IL-13-treated human peripheral monocyte nuclear extracts. In addition, fluorescent in situ hybridization (FISH) results refined the previous localization of 15-LO to human chromosome 17p13.3.

Exonuclease enhances hybridization efficiency: Improved direct cycle sequencing and point mutation detection

Solution hybridization is an essential step in sequencing and some point mutation detection methods. In practice, this hybridization is hampered resulting in the need of additional purification of the amplification products. The use of T7 gene 6 exonuclease may lead to efficient production of single-stranded DNA. In this study, the effect of pretreatment with exonuclease on direct cycle sequencing and point mutation detection was analyzed. Exonuclease-treated products were directly cycle sequenced without further purification. This resulted in highly efficient quality improvement for sequencing allowing detection of heterozygotes. Point mutation detection by Point-EXACCT (exonuclease-amplification coupled capture technique) demonstrated detection of one cell containing a mutation in an excess of 75000 wild type cells. Exonuclease-enhanced detection methods offer simple, rapid detection strategies that are easily adaptable for widespread clinical laboratory use. With the use of exonuclease, the detection of heterozygosity using fluorescent cycle sequencing is becoming more reliable. The high sensitivity of Point-EXACCT due to the use of exonuclease makes it a highly promising method for large-scale screening of (pre)malignant changes in patients with a high risk for developing cancer.

Identification and mapping of a putative bombesin receptor gene on human chromosome 17q21.3

A mouse bombesin receptor cDNA was used as a probe to screen a human P1 genomic library. Clone HBR1 was isolated and used to localize a putative human bombesin receptor gene (HBRKS) on human chromosome 17q21.3 by fluorescent in situ hybridization (FISH). HBRKS was identified and mapped by polymerase chain reaction (PCR) amplification from a Yeast artificial chromosome (YAC) contig spanning 17q21-q23. In addition, a few candidate genes were found by exon-trapping from HBR1.

The multigene families of guanine nucleotide-binding proteins: Evolutionary and computational analysis☆

Abstract The multigene families of guanine nucleotide-binding proteins (G proteins) regulate numerous cell functions including transmembrane signal transduction and oncogenesis. These gene families have been intensively studied in a wide variety of eukaryotic systems, resulting in rich substrate for computational analysis in an evolutionary and developmental context. Signal transducing GTP-binding proteins are often considered in three general categories: (1) the heterotrimeric G proteins—e.g. G s and G i , the stimulatory and inhibitory regulators of adenylyl cyclase that transmit signals from transmembrane receptors to intracellular effectors and contain distinctive 40 kDa α subunits; (2) the family of smaller (∼20 kDa) monomeric proteins related to the oncogene product ras , including rab, rac, ypt , and sec 4; and (3) the ADP-ribosylation factors (ARFs). Analysis of these multigene G protein families present instructive examples of the increasing sophistication in computational sequence analysis that will be necessary to keep pace with the expanding number of gene families that will be characterized in the course of the Human Genome Project. Convenient computational analysis is critical to multiple phases of the current work: (1) to design PCR primers for effective cross-species amplification; (2) to analyze the gene structures of sequence obtained; (3) to identify functional domains in protein coding exons and in untranslated regions (UTRs); and (4) to discern evolutionary relationships between cloned sequences.