Steven Dooley

Isolation and characterization of the human mismatch repair gene hMSH2 promoter region

Hereditary nonpolyposis colorectal cancer (HN-PCC) is one of mans commonest hereditary diseases. Several studies have identified four responsible genes that are involved in a process known as DNA mismatch repair; hMSH2 is the most important of these four genes. In addition to mutational analysis of these genes, investigations of transcriptional regulatory mechanisms are important. Therefore, our purpose has been to isolate the hMSH2 promoter region. Using direct sequencing of P1 recombinant DNA we have characterized 1100 bp of the hMSH2 promoter.

Isolation and characterization of the human genomic locus coding for the putative metastasis control gene nm23-H1.

Nm23-H1 gene expression is inversely correlated with tumor metastatic potential in certain tumors, including melanomas, breast carcinomas, and hepatocellular carcinomas. Using nm23-H1 c-DNA primer and genomic polymerase chain reaction (PCR) amplification, we purified three PCR fragments (one of 4kb and two of 2 kb) covering the whole human genomic locus of the gene (8.460bp). We recombined the PCR products into pUC18 and produced a restriction map to perform subcloning. Complete sequencing of genomic PCR fragments, including the whole coding region of nm23-H1, revealed that the gene consists of five exons and four introns spanning 8.5kb. A sequence homology analysis between human nm23-H1 and the homolog gene of the rat (NDP-Kβ) shows that exon-intron boundaries are well conserved between these two species.

A second trefoil protein, ITF/hP1.B, is transcribed in human breast cancer

SummaryTrefoil proteins form a specific group of stable secreted polypeptides. They are expressed in a lot of human cancers and during inflammatory processes of the gastrointestinal tract. Recently a new human trefoil protein, ITF/hP1.B, was isolated. Until now no studies of the activity of this gene in human solid tumors exist. In our examination we show for the first time that this gene is transcribed in human breast cancer. In contrast to another trefoil protein, pS2, the expression of ITF/hP1.B is not under control of estrogen in the human breast cancer cell line MCF-7. We suggest that the gene activity of ITF/hP1.B in addition to pS2 expression may be an improved prognostic marker in human breast cancer.

A non-alphoid repetitive DNA sequence from human chromosome 21

SummaryA non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40–50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specifity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.

c-myb Intron I protein binding and association with transcriptional activity in leukemic cells

Specific binding of nuclear proteins to the region of transcriptional attenuation has been shown to modulate the expression of c-myb, a nuclear proto-oncogene preferentially expressed in lympho-hematopoietic cells. Here, it plays an important role in processes of differentiation and proliferation. The mechanism that regulates c-myb expression is not yet fully understood. The block of transcriptional elongation which has been mapped to a 1 kb region within murine intron 1 may represent one regulatory pathway. The DNA sequences containing the transcriptional pause site are well conserved between murine and human species, thus Implying similar transcription-control strategies. We compared the binding potential of nuclear extracts (from human fibroblasts and MOLT4 as well as murine NIH3T3- and 70Z/3B- cell lines) to oligonucleotide sequences previously shown to be target binding sites in the murine system. One complex containing a 70 D protein was found to be associated specifically with transcriptionally active leukemia cells. We performed transient expression studies with a CAT reporter construct containing this putative enhancer sequence and yielded significant CAT activity. We identified further a putative 20 kD repressor protein in transcriptionally silent cells and demonstrated that c-Jun is part of an ubiquitously present complex. Our results confirm the participation of intron 1 in transcriptional regulation of the c-myb gene (in mouse and human) and implicate multiple and complex regulatory mechanisms of activation during myelomonocytic differentiation and leukemic cell growth control.

Cross-hybridization between the avian myeloblastosis oncogene and eukaryotic 28S ribosomal RNA

In Northern blots, avian myeloblastosis (myb) oncogene probes (genomic or cDNA) cross-hybridize to the 28S rRNA band mimicking a myb-specific transcript. A misinterpretation of the hybridization data can be avoided by using an oligodeoxyribonucleotide probe.

Expression of hMSH-2 Mismatch-Repair Gene in Epithelial and Melanocytic Skin Tumors: Regulation by P53 Protein and UV-B Irradiation

Microsatellite instability (MSI) secondary to replication errors can be detected in various malignant human epithelial and melanocytic skin tumors.1 Microsatellite instability is a recently recognized genetic mechanism important in the development of various human cancers that is characterized by length changes at repetitive loci scattered throughout the genome.2,3 In most patients with hereditary nonpolyposis colon cancer (HNPCC), where almost every tumour reveals a high incidence of mutations in microsatellite repeat sequences, it was shown that cancer predisposition is attributable to defects in any one of four genes, all of which encode homologs of the microbial mismatch repair proteins mutS and mutL.3,4 The hMSH-2 gene specifies a mutS homolog, whereas hMLH1, hPMS1, and hPMS2 encode homologs of mutL.5 Analysis of all the 16 exons of hMSH2 in 34 unrelated HNPCC kindreds has revealed a heterogeneous spectrum of mutations.3,6 Tumour cells that display microsatellite instability are typically defective in mismatch correction, thus providing a direct link between DNA-mismatch repair enzymes and genetic stability afforded by this DNA repair system.7 Recently, we have cloned the promoter region of hMSH-2 and detected a site with homology to the p53 consensus binding sequence.8 Using gel mobility shift assays we were able to show that purified p53 has at least in vitro the potential to bind specifically the hMSH-2 motif.9