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In classical genetics, inferences are based on the phenotypic differences between a wild-type and a mutant organism. This old-fashioned systems biology continues to provide deep insights into the genetic basis of fundamental biological processes mainly at the phenomenological level. This limitation was partly overcome by the revolutionary developments in recombinant DNA technology (Chap. 5). With this, it was possible to isolate, manipulate in vitro, and reintroduce a gene into the organism to determine and evaluate its effects on the performance at the phenotypic level. The impact of these combined approaches in deciphering the mechanistic details of molecular interactions has been impressive. However, biological problems are too challenging to be understood, even with these powerful approaches. Nevertheless, the classical and molecular genetic approaches culminated in a large body of data including the whole genome sequence of different organisms resulting in a paradigm shift in experimental biology beyond expectations. In no other experimental organism genomic approach has been so successfully used than in S. cerevisiae. This is because yeast has been the vanguard of biochemical, genetic, and cell biological studies. An existing wealth of knowledge of yeast biology was put to great advantage in the context of sequence information. One of the immediate outcomes of the genome sequence is "expression profiling" using micro-array analysis. This technique has become quite routine and has yielded valuable information not only in yeast but also in almost all organisms whose genome sequence is known. [ABSTRACT FROM AUTHOR]

In eucaryotes, three distinct species of RNA polymerases (RNA Pol) have been identified while prokaryotes and archaea have only one RNA polymerase. In eukaryotes, RNA PolI, transcribes the large rRNA precursor that is processed leading to the production of 28 S, 18 S and 5.8 S rRNAs. RNA PolII, transcribes heterogeneous nuclear RNAs (hnRNAs) and small nuclear RNAs (sn RNAS). hnRNAs are processed to mature mRNA, which requires snRNAs. RNA PolIII transcribes 5 S rRNA and tRNAs. The identity of these three RNA polymerases has been confirmed by molecular genetic and biochemical analysis. Although these enzymes share a common property of transcribing DNA, they lack the ability to identify the transcription initiation sites for which they depend on additional proteins. As expected, the promoters of genes transcribed by these RNA polymerases also have unique features. Of the three polymerases, PolII has attracted considerable attention owing to the fact that its activity is highly regulated. The fundamental question is: How does the transcriptional machinery direct the RNA PolII to accurately transcribe the diverse set of genes depending upon the physiological need? In general, promoters served by the RNA PolII have a common architecture consisting of core elements required for the promoter function and for the assembly and orientation of the pre-initiation complex. The most significant structural elements are the TATA sequences located 25 nucleotides upstream of the transcription initiation site. The transcription initiation site is generally pyrimidine-rich. In addition, the promoters contain regulatory sequences that govern the expression status by interacting with specific transcriptional regulators. GAL promoter is an archetypical PolII promoter that is turned OFF by glucose and ON by galactose. We shall focus on how Gal4p, the DNA-binding transcriptional activator, activates PolII to transcribe GAL genes in response to the inducer. [ABSTRACT FROM AUTHOR]

The signal from galactose is transmitted to Gal80p-Gal4p transcriptional switch through Gal3p/Gal1p signaling system. Based on the genetic experiments discussed previously, it appeared unlikely that signaling is due to a derivative of galactose catalyzed by Gal3p. Attempts to dissociate Gal4p-Gal80p complex in presence of galactose derivatives were unsuccessful, but it was clear that at least galactokinase activity is not required for signaling. Therefore, an idea that necessitates the presence of galactose for induction but does not warrant that the signal transducer to be an enzyme was considered. [ABSTRACT FROM AUTHOR]

In general, a replica of a living or a non-living entity is called a clone. In biology, a clone refers to a progeny genetically identical to its parents. Clones are produced during vegetative or mitotic or asexual reproduction. Occasionally, during asexual reproduction, genetic variants arise at a frequency of 10−6 due to errors in copying the genetic information. Such variants are not the clone of the parent cell. In singlecelled organisms like yeast, such variants can be isolated using genetic screens or selection, and this process is called "cloning of an organism". We have learned how to clone yeast strains that exhibit different phenotypes. Cloning multicellular organisms entails different approaches (Box 5.1.1). By definition, descendents of sexual reproduction are not clones because they are not genetically identical to the parents. This definition breaks down when one considers the haploid spores of a homo-diploid yeast cell. Here, the haploid spores produced through sexual reproduction are genetically identical to one another. [ABSTRACT FROM AUTHOR]

We learned from the previous chapters that in strains bearing recessive mutations in GAL4, Leloir enzymes are not induced in response to galactose. On the other hand, recessive mutation in GAL3 leads to a delayed kinetics of induction of the Leloir enzyme in response to galactose. In a broad sense, GAL4 and GAL3 are regulators of the expression of a family of genes required for galactose utilization and such genes are called regulatory genes. The fundamental question that needs to be understood is how do Gal3p and Gal4p interact to perceive the presence of galactose in the medium? What prevents Gal4p from activating the GAL genes in the absence of galactose? Is there another protein that represses Gal4p from activating transcription in the absence of galactose? If so, can we identify such a gene and study how galactose inactivates this gene function? In essence, we wish to understand the strategy employed by yeast to translate the three-dimensional information of galactose into a specific biological signal. If a negative regulator prevents GAL4 function in the absence of galactose, then it is possible to isolate a mutant strain defective in such a repressor. Such a mutant strain would express GAL genes constitutively. In this case, one is looking for a strain that expresses Leloir enzymes even if galactose is not present. How do we detect the presence of such rare variants in the population? For example, if the frequency of occurrence of such mutants in a mutagenized population of cells is 10−3, then, one has to screen at least 1,000 independent mutant strains for enzyme expression with the hope of finding one such mutant, which is obviously a difficult task. Developing a selection or screening strategies to identify mutant strains with novel phenotypes is at the heart of genetic analysis. These rare variants provide initial clues that help us reveal mechanisms of regulation which are otherwise difficult to discover. The following example illustrates this point. [ABSTRACT FROM AUTHOR]

Of the many carbon sources, yeast prefers glucose. In the presence of glucose, enzymes of the galactose metabolic pathway are not expressed. Even a disaccharide containing glucose moiety such as sucrose, is not utilized until all the available free glucose is completely consumed. Yeast maintains a strict hierarchy in terms of sugar utilization and glucose is at the top. Does it offer any advantage to yeast despite the free energy content between say galactose and glucose is the same? Growth is a resultant of all the biological activities of a cell. Quantitative analysis of growth provides insights into the metabolic strategies adapted by different organisms or same organisms under different experimental or physiological conditions. In this section, I shall briefly discuss the basic aspects of cell growth with a focus on what a cell or a living organism considers important for its evolutionary success. [ABSTRACT FROM AUTHOR]

Robert Hooke presented an account of the cells of cork to the members of the Royal Society in 1660. Although Jan Swammerdam had observed blood cells around this same time, it was only documented almost 50 years later after his death. As early as 1700, using a tiny sphere of polished glass as a microscope, which had a magnification power of 275, Antony van Leeuwenhoek observed live yeast cells as globular bodies in a drop of fermenting beer and called them "animalcules". Both Robert Hook and Antony van Leeuwenhoek had doubted the validity of the spontaneous theory, but a new approach was necessary in order to abandon the spontaneous generation theory. In 1838, while outlining the importance of the cell nucleus over dinner, Matthias Jakob Schleiden (a botanist) prompted Theodor Schwann (a zoologist) to recall observing similar structures in the notochordal cells of the tadpole. The perceived commonality of the plant and animal world, in having nucleated cells, led to the "grand unification theory" of biology, the cell theory. Identified as the common denominator of "life", the cell became its fundamental building block. However, in Schwann and Schleiden's "cell theory", cells arise spontaneously, contrary to the subsequent recognition that a new cell arises from pre-existing cells. Since the time of "grand unification" much progress has been made in dissecting cells down to the atomic constituents. Paradoxically, the fundamental essence of "life" is not fully understood. The second half of the 18th century was a turning point. The cause and significance of heat production during fermentation was discovered by Louis Pasteur, who argued that fermentation is a process involving chemical transformation of glucose to ethanol and is intricately linked to life. Later, using the famous swan-neck experiment, Pasteur demonstrated that a cell arises only from a pre-existing cell, putting to rest the long-held belief of "spontaneous" origin. Justus von Leibig did not accept that fermentation of glucose was in any way fundamental to "life". Later, Hans and Eduard Buchner demonstrated that even yeast extract, although lacking a "living" cell, ferments glucose. Both Leibig and Pasteur were right in their own ways; "Biochemistry" was thus born, being given this name by Carl Alexander Neuberg in 1903. [ABSTRACT FROM AUTHOR]

How does yeast translate the three-dimensional information of galactose into a specific biological signal that activates transcription of GAL genes? Genetic analysis is the method of choice for exploring the molecular basis of such phenomena that are otherwise not amenable for routine biochemical analysis. The success of genetic analysis depends on the availability of a large number of genetic variants which have a discernible phenotype. In our example, yeast strains unable to utilize galactose as the sole carbon source, serve as a starting point for genetic analysis. This chapter describes the genetic analysis that led to the identification of genes responsible for galactose utilization. [ABSTRACT FROM AUTHOR]