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Miledi R, Palma E, Eusebi F Methods Mol Biol. 2006;322:347–55 The Xenopus oocyte is largely used as a cell expression system for studying both structure and function of transmitter receptors and ion channels. Messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functionalion channels. A new method was developed further to transplant neurotransmitter receptors from human brain or cultured cell lines to the membrane of Xenopus oocytes. This method represents a modification of the method used many years ago of injecting into oocytes membrane vesicles from Torpedo electroplaques, yielding the expression of functional Torpedo acetylcholine receptors. We describe this approach by extracting membrane vesicles from human hippocampus or temporal neocortex and from mammalian cell lines stably expressing glutamate or neuronal nicotinic receptors. Because the human neurotransmitter receptors are "microtransplanted" with their native cell membranes, this method extends the usefulness ofXenopus oocytes as an expression system for addressing issues in many fields, including channelopathies.

Stahl FW, Housworth EA Methods Mol Biol. 2009;557:35–53 Interest in crossover interference in yeast has been spurred by the discovery and characterization of mutants that alter it as well as by the development and testing of models to explain it. This chapter describes methods for detecting and for measuring interference, with emphasis on those that exploit the ability to examine all four products of individual acts of meiosis.

Costa WS, Liewald J, Gottschalk A Methods Mol Biol. New York, NY: Springer New York; 2014;1148(Chapter 11):161–75 In recent years, optogenetic methods became invaluable tools, particularly in neurobiological research. Most prominently, optogenetic methods utilize microbial rhodopsins to elicit neuronal de- or hyperpolarization. However, other optogenetic tools have emerged that allow influencing neuronalfunction by different approaches. In this chapter we describe the use of photoactivated adenylyl cyclases (PACs) as modulators of neuronal activity. Using Caenorhabditis elegans as a model organism, this chapter shows how to measure the effect of PAC photoactivation by behavioral and electrophysiological assays, as well as their significance to neurobiology.

Huberts DHEW, González J, Lee SS, Litsios A, Hubmann G, Wit EC, Heinemann M Proc Natl Acad Sci USA. National Acad Sciences; 2014 Aug 12;111(32):11727–31 Calorie restriction (CR) is often described as the most robust manner to extend lifespan in a large variety of organisms. Hence, considerable research effort is directed toward understanding the mechanisms underlying CR, especially in the yeast Saccharomyces cerevisiae. However, the effect of CR on lifespan has never been systematically reviewed in this organism. Here, we performed a meta-analysis of replicative lifespan (RLS) data published in more than 40 different papers. Our analysis revealed that there is significant variation in the reported RLS data, which appears to be mainly due to the low number of cells analyzed per experiment. Furthermore, we found that the RLS measured at 2% (wt/vol) glucose in CR experiments is partly biased toward shorter lifespans compared with identical lifespan measurements from other studies. Excluding the 2% (wt/vol) glucose experiments from CR experiments, we determined that the average RLS of the yeast strains BY4741 and BY4742 is 25.9 buds at 2% (wt/vol) glucose and 30.2 buds under CR conditions. RLS measurements with a microfluidic dissection platform produced identical RLS data at 2% (wt/vol) glucose. However, CR conditions did not induce lifespan extension. As we excluded obvious methodological differences, such as temperature and medium, as causes, we conclude that subtle method-specific factors are crucial to induce lifespan extension under CR conditions in S. cerevisiae.

Charron G, Leducq J-B, Landry CR Mol Ecol. 2014 Sep;23(17):4362–72 Reproductive isolation is a critical step in the process of speciation. Among the most important factors driving reproductive isolation are genetic incompatibilities. Whether these incompatibilities are already present before extrinsic factors prevent gene flow between incipient species remains largely unresolved in natural systems. This question is particularly challenging because it requires that we catch speciating populations in the act before they reach the full-fledged species status. We measured the extent of intrinsic postzygotic isolation within and between phenotypically and genetically divergent lineages of the wild yeast Saccharomyces paradoxus that have partially overlapping geographical distributions. We find that hybrid viability between lineages progressively decreases with genetic divergence. A large proportion of postzygotic inviability within lineages is associated with chromosomal rearrangements, suggesting that chromosomal differences substantially contribute to the early steps of reproductiveisolation within lineages before reaching fixation. Our observations show that polymorphic intrinsic factors may segregate within incipient speciesbefore they contribute to their full reproductive isolation and highlight the role of chromosomal rearrangements in speciation. We propose different hypotheses based on adaptation, biogeographical events and life history evolution that could explain these observations.

Schricker R, Magdolen V, Bandlow W Molecular and General Genetics MGG. Springer; 1992;233(3):363–71 Making use of the polymerase chain reaction primed by oligonucleotides corresponding to regions conserved between members of the nucleoside monophosphate kinase family, we have isolated the yeast gene PAK3. Pak3p belongs to the subgroup of long-form adenylate kinase isozymes (deduced molecular mass 25.3 kDa) and exhibits highest sequence similarity to bovine AK3 rather than to the yeast isozyme, Aky2p. The gene is shown to be non-essential because haploid disruption mutants are viable, both in the presence and absence of a functional AKY2 allele. It maps on chromosome V upstream of RAD3. Its expression level is low when cells are grown on glucose or other fermentable carbon sources and about threefold higher on glycerol, but can be significantly induced by ethanol. A PAK3/mouse dihydrofolate reductase fusion construct expressed in yeastis targeted to mitochondria. Transformation with PAK3 on a multicopy plasmid complements neither adenylate kinase deficiency in an aky2-disruptedyeast strain nor in Escherichia coli cells conditionally defective in adenylate kinase.

Konrad M J Biol Chem. 1992 Dec 25;267(36):25652–5 Guanylate kinase catalyzes the reversible transfer of the terminal phosphoryl group of ATP to the acceptor molecule GMP. Detailed analysis of the in vivo function of this enzyme has been limited by the lack of any genetic data. Using oligonucleotides based on amino acid sequence information of the yeast enzyme, the Saccharomyces cerevisiae gene, GUK1, was isolated and characterized. The gene is present in single copy and maps to chromosome IV. Insertional mutagenesis of the GUK1 locus caused recessive lethality, indicating that this enzyme is necessary for vegetative cell growth. Using inducible expression systems, guanylate kinase was produced in large amounts both in S. cerevisiae and in Escherichia coli.

Konrad M J Biol Chem. 1993 May 25;268(15):11326–34 The enzyme-catalyzed transfer of the terminal phosphoryl group from ATP to an acceptor molecule is an important reaction in a wide variety of biological processes. I demonstrate here the essential function of an ATP:AMP phosphotransferase (adenylate kinase) in the fission yeastSchizosaccharomyces pombe. A cDNA clone encoding immunoreactive adenylate kinase from S. pombe was isolated from a lambda gt11 expression library by cross-reaction with antibodies raised against the recently characterized ADK1 enzyme from the budding yeast Saccharomyces cerevisiae. Subsequent cloning and nucleotide sequence analysis of the S. pombe adenylate kinase gene, adk1, revealed a coding region of 660 nucleotides. The alignment of the two amino acid sequences from S. cerevisiae and S. pombe shows 67% identity. By gene disruption and tetradanalysis it is demonstrated that adk1 is absolutely essential for cell viability. This is in contrast to the ADK1 gene of S. cerevisiae, the deletion of which was shown to lead to a slower cell growth rate rather than to a lethal phenotype. Expression of adk1 in the S. cerevisiae ADK1 deletion strain restored normal cell growth, demonstrating that ADK1 and adk1 are functionally interchangeable. However, despite lack of absolute substrate specificity of adenylate kinases, adk1 could not complement the loss of function of the guanylate kinase encoding gene in a S. cerevisiae null mutant strain, thus highlighting the functional uniqueness of each nucleoside monophosphate kinase. Using suitable expression vectors, large amounts of active adk1 enzyme were produced in either yeast species and in E. coli. The purified enzyme exhibits a high preference for adenine nucleotides, with ATP being a 10 times more efficient phosphoryl donor than GTP.

Zahn-Zabal M, Lehmann E, Kohli J Genetics. 1995 Jun;140(2):469–78 The M26 mutation in the ade6 gene of Schizosaccharomyces pombe creates a hot spot of meiotic recombination. A single base substitution, theM26 mutation is situated within the open reading frame, near the 5' end. It has previously been shown that the heptanucleotide sequence 5' ATGACGT 3', which includes the M26 mutation, is required for hot spot activity. The 510-bp ade6-delXB deletion encompasses the promoter and the first 23 bp of the open reading frame, ending 112 bp upstream of M26. Deletion of the promoter in cis to M26 abolishes hot spot activity, whiledeletion in trans to M26 has no effect. Homozygous deletion of the promoter also eliminates M26 hot spot activity, indicating that the heterology created through deletion of the promoter per se is not responsible for the loss of hot spot activity. Thus, DNA sequences other than the heptanucleotide 5' ATGACGT 3', which must be located at the 5' end of the ade6 gene, appear to be required for hot spot activity. While the M26hotspot stimulates crossovers associated with M26 conversion, it does not affect the crossover frequency in the intervals adjacent to ade6. The flanking marker ura4-aim, a heterology created by insertion of the ura4+ gene upstream of ade6, turned out to be a hot spot itself. It shows disparity of conversion with preferential loss of the insertion. The frequency of conversion at ura4-aim is reduced when the M26 hot spot is active 15 kb away, indicating competition for recombination factors by hot spots in close proximity.

Gos P, Eicher B, Kohli J, Heyer WD Bioelectromagnetics. 1997;18(2):142-55 Exponentially growing cells of the yeast Saccharomyces cerevisiae were exposed to electromagnetic fields in the frequency range from 41.682 GHz to 41.710 GHz in 2 MHz increments at low power densities (0.5 microW/cm2 and 50 microW/cm2) to observe possible nonthermal effects on the division of this microorganism. The electronic setup was carefully designed and tested to allow precise determination and stability of theelectromagnetic field parameters as well as to minimize possible effects of external sources. Two identical test chambers were constructed in one exposure system to perform concurrent control and test experiments at every frequency step under well-controlled exposure conditions. Division of cells was assessed via time-lapse photography. Control experiments showed that the cells were dividing at submaximal rates, ensuring the possibility of observing either an increase or a decrease of the division rate. The data from several independent series of exposure experiments and from control experiments show no consistently significant differences exposed and unexposed cells. This is in contrast to previous studies claiming nonthermal effects of electromagnetic fields in this frequency range on the division of S. cerevisiae cells. Possible reasons for this difference are discussed.