Biomedical subjects

Michael C MacLeod

Publications and source records attributed to Michael C MacLeod.

6 recordsLinked to original sources

PubMed2007

High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes.

Site-specific recognition of duplex DNA by triplex-forming oligonucleotides (TFOs) provides a promising approach to manipulate mammalian genomes. A prerequisite for successful gene targeting using this approach is that the targeted gene must contain specific, high-affinity TFO target sequences (TTS). To date, TTS have been identified and characterized in only approximately 37 human or rodent genes, limiting the application of triplex-directed gene targeting. We searched the complete human and mouse genomes using an algorithm designed to identify high-affinity TTS. The resulting data set contains 1.9 million potential TTS for each species. We found that 97.8% of known human and 95.2% of known mouse genes have at least one potential high-affinity TTS in the promoter and/or transcribed gene regions. Importantly, 86.5% of known human and 83% of the known mouse genes have at least one TTS that is unique to that gene. Thus, it is possible to target the majority of human and mouse genes with specific TFOs. We found substantially more potential TTS in the promoter sequences than in the transcribed gene sequences or intergenic sequences in both genomes. We selected 12 mouse genes and 2 human genes critical for cell signaling, proliferation, and/or carcinogenesis, identified potential TTS in each, and determined TFO binding affinities to these sites in vitro. We identified at least one high-affinity, specific TFO binding site within each of these genes. Using this information, many genes involved in mammalian cell proliferation and carcinogenesis can now be targeted.

Animals↗

PubMed2006

A web-based search engine for triplex-forming oligonucleotide target sequences.

Triplex technology offers a useful approach for site-specific modification of gene structure and function both in vitro and in vivo. Triplex-forming oligonucleotides (TFOs) bind to their target sites in duplex DNA, thereby forming triple-helical DNA structures via Hoogsteen hydrogen bonding. TFO binding has been demonstrated to site-specifically inhibit gene expression, enhance homologous recombination, induce mutation, inhibit protein binding, and direct DNA damage, thus providing a tool for gene-specific manipulation of DNA. We have developed a flexible web-based search engine to find and annotate TFO target sequences within the human and mouse genomes. Descriptive information about each site, including sequence context and gene region (intron, exon, or promoter), is provided. The engine assists the user in finding highly specific TFO target sequences by eliminating or flagging known repeat sequences and flagging overlapping genes. A convenient way to check for the uniqueness of a potential TFO binding site is provided via NCBI BLAST. The search engine may be accessed at spi.mdanderson.org/tfo.

Animals↗

PubMed2005

Quantitative high-throughput measurement of gene expression with sub-zeptomole sensitivity by capillary electrophoresis.

Microarray technologies have provided the ability to monitor the expression of whole genomes rapidly. However, concerns persist with regard to quantitation and reproducibility, and the detection limits for individual genes in particular arrays are generally unknown. This article describes a semiautomated PCR-based technology, Q-RAGE, which rapidly provides measurements of mRNA abundance with extremely high sensitivity using fluorescent detection of specific products separated by capillary electrophoresis. A linear relationship between template concentration and fluorescent signal can be demonstrated down to template concentrations in the low aM region, corresponding to approximately 0.04 zmol (24 molecules) per reaction. The technique is shown to be quantitative over five orders of magnitude of template concentration, and average mRNA abundances of approximately 0.01 molecule per cell can be detected. A single predefined set of 320 primers provides 90-95% coverage of all eukaryotic genomes. Analysis of a set of 19 p53-regulated genes in untreated cultures of normal human epithelial cells, derived from three different tissues, revealed a 600-fold range of apparent constitutive expression levels. For most of the genes assayed, good correlations were observed among the expression levels in normal mammary, bronchial, and epidermal epithelial cells.

Bronchi↗

PubMed2005

Expression of common chromosomal fragile site genes, WWOX/FRA16D and FHIT/FRA3B is downregulated by exposure to environmental carcinogens, UV, and BPDE but not by IR.

Common chromosomal fragile sites are unstable genomic loci susceptible to breakage, rearrangement, and are highly recombinogenic. Frequent alterations at these loci in tumor cells led to the hypothesis that they may contribute to cancer development. The two most common chromosomal fragile sites FRA16D and FRA3B which harbor WWOX and FHIT genes, respectively, are frequently altered in human cancers. Here we report that environmental carcinogens, ultraviolet (UV) light, and Benzo[a]pyrene diol epoxide (BPDE), significantly downregulate expression of both genes. On the other hand, we observe that ionizing radiation (IR) does not affect expression of these genes, suggesting that the effect of repression exerted by UV and BPDE is not just a consequence of DNA damage but may be a result of different signaling pathways triggered by specific DNA lesions. Such downregulation correlates with an induction of an S-phase delay in the cell cycle. Treatment of UV-irradiated cells with caffeine abrogates the S-phase delay while concomitantly overcoming the repression phenomenon. This suggests the involvement of unique cell cycle checkpoint mechanisms in the observed repression. Therefore, it is hypothesized that protracted downregulation of the putative tumor suppressor genes WWOX and FHIT by environmental carcinogens may constitute an additional mechanism of relevance in the initiation of tumorigenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2005

Late cornified envelope family in differentiating epithelia--response to calcium and ultraviolet irradiation.

The late cornified envelope (LCE) gene cluster within the epidermal differentiation complex on human chromosome one (mouse chromosome three) contains multiple conserved genes encoding stratum-corneum proteins. Within the LCE cluster, genes form "groups" based on chromosomal position and protein homology. We link a recently accepted nomenclature for the LCE cluster (formerly XP5, small proline-rich-like, late-envelope protein genes) to gene structure, groupings, and chromosomal organization, and carry out a pan-cluster quantitative expression analysis in a variety of tissues and environmental conditions. This analysis shows that (i) the cluster organizes into two "skin" expressing groups and a third group with low-level, tissue-specific expression patterns in all barrier-forming epithelia tested, including internal epithelia; (ii) LCE genes respond "group-wise" to environmental stimuli such as calcium levels and ultraviolet (UV) light, highlighting the functional significance of groups; (iii) in response to UV stimulation there is massive upregulation of a single, normally quiescent, non-skin LCE gene; and (iv) heterogeneity occurs between individuals with one individual lacking expression of an LCE skin gene without overt skin disease, suggesting LCE genes affect subtle attributes of skin function. This quantitative and pan-cluster expression analysis suggests that LCE groups have distinct functions and that within groups regulatory diversification permits specific responsiveness to environmental challenge.

Calcium↗

PubMed2003

Response of human mammary epithelial cells to DNA damage induced by BPDE: involvement of novel regulatory pathways.

The responses of a line of normal human mammary epithelial cells, HME87, to treatment with the ultimate carcinogen benzo[a]pyrene diol epoxide (BPDE) were analyzed using a directed gene expression analysis technique, RAGE. Under conditions where cell number was decreased by 50% 24 or 48 h post-treatment, flow cytometry demonstrated no establishment of a G(1)/S arrest nor induction of apoptosis; cells continued to enter S phase from G(1) for at least 24 h but were blocked at G(2)/M. Using the RAGE technique, changes in gene expression were assayed for over 1000 genes, and multiple time-point data were collected for approximately 90 genes. In accord with the cell cycle studies, expression of the p21-WAF1 gene, the major mediator of p53-dependent G(1)/S arrest, did not increase until 24 h post-treatment. The expression of other target genes for transactivation by p53 was increased at early time points, including GADD45, an effector of the G(2)/M checkpoint, and WIP1. Analyses of proteins in treated cells indicated that p53 was phosphorylated at Ser15 but not at Ser20 within 30 min of treatment, and this correlated with an increase in the total amount of p53 protein. Significant expression changes were noted in a number of transcription factor genes, including ATF3 and E2A, genes that have not been previously connected to a response to DNA damage involving bulky chemical adducts. In addition, expression of the XPC gene was induced by BPDE treatment; the XPC product is thought to be involved in recognition of DNA damage by the nucleotide excision repair system.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗