Identification of potential diagnostic markers of prostate cancer and prostatic intraepithelial neoplasia using cDNA microarray.

The identification of novel genes or groups of genes expressed in prostate cancer may allow earlier diagnosis or more accurate staging of the disease. We describe the assembly and use of a 1877-member microarray representing cDNA clones from a range of prostate cancer stages and grades, precursor lesions and normal tissue. Using labelled cDNA from tumour samples obtained from TURP or radical prostatectomy, analysis of expression patterns identified many up-regulated transcripts. Cell lines were found to over-express fewer genes than diseased tissue samples. 17 known genes were found to over-express more than 4-fold in 4 or more cancers out of 15 cancers. Only 2 genes were over-expressed in 6 out of 15 cancers or more, whilst no genes were consistently found to be over-expressed in all cancer samples. Novel prostate cancer associations for several well characterized genes or full length cDNAs were identified, including PLRP1, JM27, human UbcM2, dynein light intermediate chain 2 and human homologue of rat sec61. Novel associations with high-grade PIN include: breast carcinoma fatty acid synthase and cDNA DKFZp434B0335. We shortlist and discuss the most significant over-expressed genes in prostate cancer and PIN, and highlight expression differences between malignant and benign samples.

Adapting in situ polymerase chain reaction for genotyping of cells in suspension.

An approach is described for in situ polymerase chain reaction (ISPCR) based on cycling primed in situ synthesis (PRINS) conditions defined for alpha-satellite DNA. Using blood cell preparations subjected to limited fixation with paraformaldehyde, ISPCR cycling resulted in a gradual buildup of amplicon at the site of synthesis, as judged by the characteristic presence of paired nuclear spots corresponding to specific centromeres. Using longer cycling regimens, primers for single copy genes also generated paired nuclear spots in a primer-pair--specific manner. In this context, the amplification refractory mutation system (ARMS) was evaluated for in situ applications. In ARMS, allele-specific primers are used in such a manner that PCR proceeds only when an exact 3' match between annealed primer and template is recognized by DNA polymerase. Using normal and mutant primers for the delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene as a model system, it was not possible to reliably differentiate between ARMS reactions by accumulation of direct labeled reaction product in cells, because of ARMS-independent nonspecific labeling. However, by DNA extraction and reamplification with ARMS primers, it was shown that amplicon accumulates in cells in the expected primer/template-dependent manner crucial to mutation detection by ARMS. It was also shown that nonspecific signal is due to primer dimer formation, especially in the absence of true template DNA. The impact of primer dimer formation in generating a false-positive signal is discussed. The method described here enables a cell population to be analyzed for a given point mutation.

Beta-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes.

A cosmid clone containing closely linked beta-lactam antibiotic biosynthetic genes was isolated from a gene library of Flavobacterium sp. SC 12,154. The location within the cluster of the DNA thought to contain the gene for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), the first step in the beta-lactam antibiotic biosynthetic pathway, was identified by a novel method. This DNA facilitated the isolation, by cross-hybridization, of the corresponding DNA from Streptomyces clavuligerus ATCC 27064, Penicillium chrysogenum Oli13 and Aspergillus nidulans R153. Evidence was obtained which confirmed that the cross-hybridizing sequences contained the ACVS gene. In each case the ACVS gene was found to be closely linked to other beta-lactam biosynthetic genes and constituted part of a gene cluster.

Amplification of the isopenicillin N synthetase gene in a strain of Penicillium chrysogenum producing high levels of penicillin.

The isopenicillin N synthetase (IPNS) gene has been isolated from wild-type Penicillium chrysogenum and used as a probe to detect the equivalent gene on Southern blots of genomic DNA from a mutant producing high levels of penicillin. The IPNS gene in this strain is contained within a region of DNA of wild-type restriction pattern that extends for at least 39 kb and is present at between 8 and 16 copies. The steady state level of IPNS mRNA in the mutant producing high levels of penicillin is between 32- and 64-fold of that of the wild type, suggesting that the rate of transcription of some or all of the copies has been increased. In addition we have also shown that both the IPNS mRNA and enzyme is present throughout the growth phase in both strains under the culture conditions used. IPNS enzyme activity is greatly increased in the strain with the high penicillin titre.

Transformation of Penicillium chrysogenum with a dominant selectable marker.

We have cloned a mutant oligomycin resistance allele of the mitochondrial ATP synthase subunit 9 gene from the filamentous fungus Penicillium chrysogenum. The gene was isolated using the equivalent gene from Aspergillus nidulans as a hybridisation probe. Using the cloned gene it is possible to select for oligomycin resistance in P. chrysogenum transformation experiments. This transformation system was used to introduce further copies of the P. chrysogenum isopenicillin N synthetase gene, which were stably maintained without selection. An assessment of the frequency with which homologous integration occurs was also made. With this system, it should prove possible to transform any strain of P. chrysogenum.

Heavily methylated amplified DNA in transformants of Neurospora crassa.

Substantial DNA methylation occurs in higher eukaryotes and in some cases affects gene expression. However, the genomes of some fungi, Drosophila and other lower eukaryotes have an extremely low 5-methylcytosine content, suggesting that DNA methylation might not have a general role in gene control. We have now found heavy methylation of transforming DNA which has become stably amplified in complex tandem arrays in the fungus Neurospora crassa. Rearranged amplified arrays of this type have not previously been found in fungi, but resemble those of animal system. Our results demonstrate that this lower eukaryote normally maintains only very low levels of 5-methylcytosine in its genome, but possesses a mechanism for substantial methylation of DNA de novo. This heavy methylation, which lacks the preference for CG sequences found in higher eukaryotes, does not apparently affect gene expression and might be involved in a recombination or repair process for which the amplified DNA is a target.