Reprogramming and differentiation in mammals: motifs and mechanisms.

The natural reprogramming of the mammalian egg and sperm genomes is an efficient process that takes place in less than 24 hours and gives rise to a totipotent zygote. Transfer of somatic nuclei to mammalian oocytes also leads to their reprogramming and formation of totipotent embryos, albeit very inefficiently and requiring an activation step. Reprogramming of differentiated cells to induced pluripotent stem (iPS) cells takes place during a period of time substantially longer than reprogramming of the egg and sperm nuclei and is significantly less efficient. The stochastic expression of endogenous proteins during this process would imply that controlled expression of specific proteins is crucial for reprogramming to take place. The fact that OCT4, NANOG, and SOX2 form the core components of the pluripotency circuitry would imply that control at the transcriptional level is important for reprogramming to iPS cells. In contradistinction, the much more efficient reprogramming of the mammalian egg and sperm genomes implies that other levels of control are necessary, such as chromatin remodeling, translational regulation, and efficient degradation of no longer needed proteins and RNAs.

Pooling and PCR as a method to combat low frequency gene targeting in mouse embryonic stem cells.

The introduction of germ line modifications by gene targeting in mouse embryonic stem (ES) cells has proven a fundamental technology to relate genes to mammalian biology. Critical aspects required for successful gene targeting have traditionally been experimental enhancements that increase the frequency or detection of homologous recombination within ES cells; however, the utilization of such methods may still result in the failed isolation of a positively targeted ES cell clone. In this study, we discuss the current enhancement methods and describe an ES cell pooling strategy that maximizes the ability to detect properly targeted ES cells regardless of an inherent low targeting efficiency. The sensitivity required to detect correctly targeted events out of a pool of ES cell clones is provided by polymerase chain reaction (PCR), and only those pools containing positives need to be expanded and screened to find individually targeted clones. This method made it possible to identify targeted clones from a screen of approximately 2,300 ES cell colonies by performing only 123 PCR reactions. This technically streamlined approach bypasses the need to troubleshoot and re-engineer an existing targeting construct that is functionally suitable despite its low targeting frequency.

In silico detection of control signals: mRNA 3'-end-processing sequences in diverse species.

We have investigated mRNA 3'-end-processing signals in each of six eukaryotic species (yeast, rice, arabidopsis, fruitfly, mouse, and human) through the analysis of more than 20,000 3'-expressed sequence tags. The use and conservation of the canonical AAUAAA element vary widely among the six species and are especially weak in plants and yeast. Even in the animal species, the AAUAAA signal does not appear to be as universal as indicated by previous studies. The abundance of single-base variants of AAUAAA correlates with their measured processing efficiencies. As found previously, the plant polyadenylation signals are more similar to those of yeast than to those of animals, with both common content and arrangement of the signal elements. In all species examined, the complete polyadenylation signal appears to consist of an aggregate of multiple elements. In light of these and previous results, we present a broadened concept of 3'-end-processing signals in which no single exact sequence element is universally required for processing. Rather, the total efficiency is a function of all elements and, importantly, an inefficient word in one element can be compensated for by strong words in other elements. These complex patterns indicate that effective tools to identify 3'-end-processing signals will require more than consensus sequence identification.

PCR based targeted genomic and cDNA differential display.

We previously described a targeted genomic differential display method (TGDD: Broude NE, Chandra A, Smith CL. Differential display of genomic subsets containing specific interspersed repeats. Proc. Natl. Acad. Sci. USA 1997;94:4548-53). In that method, presently characterized as method I, targeting was accomplished by capturing DNA fragments containing specific a sequence by hybridization with complementary single-stranded DNA. The captured fragments were amplified by PCR. Here, we describe method II where targeting is accomplished by PCR using primers specific to the target sequence. Method II takes advantage of PCR suppression to eliminate fragments not containing the target sequence (Siebert PDA, Chenchik A, Kellogg DE, Lukyanov KA and Lukyanov SA. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 1995;23:1087-1088). Targeting focuses analysis on and around interesting areas and additionally serves to reduce the complexity of the amplified subset. These approaches are useful to amplify genome subsets containing a variety of targets including various conserved sequences coding for cis-acting elements or protein motifs.

Differential sequencing with mass spectrometry.

Differential or genetic sequencing requires searching sample DNA for variations with respect to a reference sequence. Conventional detection techniques are too labor and cost expensive for use in diagnostic applications, therefore new technologies will be required. Measurement techniques based on mass spectrometry (MS) possess the potential for high-throughput, high fidelity measurement of sequence variation. Unambiguous detection of polymorphic sequences has been demonstrated, even in heterozygous samples. Automated reproducible measurements of microscopic arrays of samples will enable the high-throughput detection required for large-scale applications. Computational simulation and analysis of experimental parameters prior to experimentation will provide the optimization necessary for development of robust, reproducible measurements.

Genomic detection of new yeast pre-mRNA 3'-end-processing signals.

To investigate Saccharomyces cerevisiae 3'-end-processing signals, a set of 1352 unique pre-mRNA 3'-end-processing sites, corresponding to 861 different genes, was identified by alignment of expressed sequence tag sequences with the complete yeast genome. Nucleotide word frequencies in the vicinity of the cleavage sites were analyzed to reveal the signal element features. In addition to previously recognized processing signals, two previously uncharacterized components of the 3'-end-processing signal sequence were discovered, specifically a predominance of U-rich sequences located on either side of the cleavage site. One of these, the downstream U-rich signal, provides a further link between the 3'-end-processing mechanisms of yeast and higher eukaryotes. Analysis of the complete set of 3'-end-processing sites by means of a discrimination function supports a 'contextual' model in which the sum total effectiveness of the signals in all four elements determines whether or not processing occurs.

Advances in DNA diagnostics.

The key advances in DNA diagnostics during the past year are techniques which will lead to advanced throughput without sacrificing sensitivity: miniaturization of samples to reduce material cost and preparation time, and parallelization through use of measurement arrays. The most promising gains have come in the areas of DNA arrays and mass spectrometry, where differential sequencing measurements are now possible.