Nigel Burns

We have developed a large-scale screen to identify genes expressed at different times during the life cycle of Saccharomyces cerevisiae and to determine the subcellular locations of many of the encoded gene products. Diploid yeast strains containing random lacZ insertions throughout the genome have been constructed by transformation with a mutagenized genomic library. Twenty-eight hundred transformants containing fusion genes expressed during vegetative growth and 55 transformants containing meiotically induced fusion genes have been identified. Based on the frequency of transformed strains producing beta-galactosidase, we estimate that 80-86% of the yeast genome (excluding the rDNA) contains open reading frames expressed in vegetative cells and that there are 93-135 meiotically induced genes. Indirect immunofluorescence analysis of 2373 strains carrying fusion genes expressed in vegetative cells has identified 245 fusion proteins that localize to discrete locations in the cell, including the nucleus, mitochondria, endoplasmic reticulum, cytoplasmic dots, spindle pole body, and microtubules. The DNA sequence adjacent to the lacZ gene has been determined for 91 vegetative fusion genes whose products have been localized and for 43 meiotically induced fusions. Although most fusions represent genes unidentified previously, many correspond to known genes, including some whose expression has not been studied previously and whose products have not been localized. For example, Sec21-beta-gal fusion proteins yield a Golgi-like staining pattern, Ty1-beta-gal fusion proteins localize to cytoplasmic dots, and the meiosis-specific Mek1/Mre4-beta-gal and Spo11-beta-gal fusion proteins reside in the nucleus. The phenotypes in haploid cells have been analyzed for 59 strains containing chromosomal fusion genes expressed during vegetative growth; 9 strains fail to form colonies indicating that the disrupted genes are essential. Fifteen additional strains display slow growth or are impaired for growth on specific media or in the presence of inhibitors. Of 39 meiotically induced fusion genes examined, 14 disruptions confer defects in spore formation or spore viability in homozygous diploids. Our results will allow researchers who identify a yeast gene to determine immediately whether that gene is expressed at a specific time during the life cycle and whether its gene product localizes to a specific subcellular location.

The polymerase chain reaction is an immensely powerful technique for identification and detection purposes. Increasingly, competitive PCR is being used as the basis for quantification. However, sequence length, melting temperature and primary sequence have all been shown to influence the efficiency of amplification in PCR systems and may therefore compromise the required equivalent co-amplification of target and mimic in competitive PCR. The work discussed here not only illustrates the need to balance length and melting temperature when designing a competitive PCR assay, but also emphasises the importance of careful examination of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of amplification by serving as pause or termination sites. We present data confirming that under particular circumstances such localised sequence, high melting temperature regions can act as permanent termination sites, and offer an explanation for the severity of this effect which results in prevention of amplification of a DNA mimic in competitive PCR. It is also demonstrated that when Taq DNA polymerase is used in the presence of betaine or a proof reading enzyme, the effect may be reduced or eliminated.

Amplification techniques such as the Polymerase Chain Reaction (PCR) are held to be largely qualitative procedures and are widely used as such. Since the efficiency of amplification is less than perfect, small changes in efficiency can yield dramatic differences in the final amount of product generated. Despite this unpredictability the exquisite sensitivity of PCR makes the demanding goal of absolute quantification highly desirable. Consequently, the use of this technique for the quantification of nucleic acids has increased at an exponential rate. However, the ability of PCR to accurately quantify absolute levels of DNA is still not universally accepted. <br><br>The overall aim of this investigation was to determine the critical factors affecting the quantification of DNA using PCR and to use these findings to develop an assay for the absolute quantification of DNA in a model system. The novel work presented here illustrates the need for careful examination of sequences for GC-rich domains which could give rise to stable secondary structures and reduce the efficiency of amplification by serving as termination sites. <br><br>To determine the accuracy of competitive PCR, CE and IP-RP-HPLC were employed to quantify PCR- products. These two techniques provided valuable information on the identification and elimination of sources of error which led to improvements in speed, accuracy and precision, as well as ease of quantification by PCR. They also yielded information on the process of heteroduplex formation whilst simultaneously revealing assay limitations. <br><br>Consequently, the on-line fluorescence monitoring of PCR was used as an alternative method for the quantification of <i>Legionella pneumophila</i>. This technique was highly reproducible however, mispriming and the subsequent amplification of non-specific PCR products limited the level of detection. The Y-end labelling of degraded DNA with DIG prevented short DNA fragments from mispriming (and consequently extending) allowing the amplification of DNA targets. Therefore, to reduce mispriming and hence improve assay sensitivity, this approach was adapted for the first time to produce 5'-degenerate, 3'- DIG-terminated competitive primer analogues. These analogues, coupled with the use of the Lightcycler^TM, allowed the detection and absolute quantification of a single cell of <i>Legionella pneumophila</i>. This is the first time that this level of sensitivity has been achieved using this type of assay. <br><br>This technique should provide a very rapid and sensitive alternative for quantification compared to the other, more expensive technologies available at present.