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The difference between primary and secondary metabolites is explained. Common characteristics of the secondary metabolites that are secreted by various species of bacteria are summarized. Detailed descriptions are provided for secondary metabolites expressed by aquatic bacteria, bacteria that grow in the intestines of animals and a bacterium that infects crop plants. The roles of these metabolites in a wide variety of biological phenomena are discussed. Intercellular communication by means of quorum sensing, induction of bioluminescence, and regulation of the transcription of an operon are described. Acquisition of iron by an organic ligand, degradation of chloroplasts, and infection of plants are also discussed.

Precautions for the safe handling of microorganisms in a laboratory are presented. The two biosafety levels that are most pertinent to the exercises in this book are summarized. The personal protective equipment required to work safely with chemicals in a laboratory is described. The use of safety data sheets to understand the hazards of chemicals is explained. Summaries of the hazards presented by the chemicals that are used in the exercises in this book are given. The proper way to use laboratory devices to avoid accidents is discussed. The materials necessary for containing and cleaning spills of chemicals are mentioned. Instructions are given for the proper response to emergencies in the laboratory such as contact of skin or eyes with chemicals, injury, nausea, fainting, fire, and earthquake. A guide to creating a written record of the plan for each experiment and summarizing the results in a laboratory notebook is provided.

Techniques for growing crystals of organic metabolites that are suitable for examination with X-ray diffraction are given. The theory of X-ray diffraction as it pertains to these crystals is explained. The characteristics of the X-rays, including the wavelength, are provided. The relationship between reflection planes, angle of incidence, wavelength, and order of diffraction, which is the basis of Bragg’s law, is discussed. Identification of the lattice system, the Bravais lattice, and the space group of the unit cell in a crystal is explained. The reciprocal lattice that specifies the position and orientation of each of the parallel reflection planes that include all of the atoms within each unit cell are discussed. Sources are provided for the various types of computational software that are necessary for solving the structure of an organic metabolite.

The theoretical basis of absorption of ultraviolet (UV), visible, and infrared (IR) radiation by organic molecules and metallic ions is discussed. The terms for the devices used to quantify absorption of radiation, and the types of data that are collected, are defined. The mathematical equation for the relationship between data quantified as absorbance and data quantified as transmittance is given. Insight into the structure of an organic molecule from the spectrum of the absorption of UV radiation is discussed. The mathematical equation for the calculation of the concentration of a pure substance in solution from a spectrophotometric measurement is given. Preparation of analytes for assays is explained. Spectra of the absorption of visible radiation that provides evidence for the chelation of a metal by an organic molecule are included. Spectra of the absorption of IR radiation that provides insight into the structure of organic molecules are also provided.

In Photobacterium, the flavin reductase encoded by luxG regenerates the reduced form of flavin mononucleotide (FMN). Reduced FMN is one of the substrates of the luciferase enzyme that catalyzes a light-emitting reaction. A set of experiments, that employs a luxG-expression plasmid construct (pGhis) and is suitable for an undergraduate laboratory course, is presented. Hexahistidine-tagged protein is expressed in E. coli from pGhis, with the T7 RNA polymerase/lac repressor induction system. Bacteria are lysed by sonication and the tag allows for purification by immobilized metal ion affinity chromatography. A gel filtration column is used to remove ions and the other small molecules. The Bradford assay, with multiwell plates and an automated plate reader, is used to identify protein concentration peaks from both columns. The concentration of purified enzyme is then calculated from its A(280) using the predicted extinction coefficient. Yield and purity are further assayed with SDS-PAGE. Activity of purified enzyme is measured with riboflavin or FMN as substrate. Reaction rate is quantified by monitoring decrease in A(340) as the redox partner, NADH, is oxidized.

The bromodomain, an evolutionarily conserved motif that binds acetyl-lysine on histones, is found in many chromatin-associated proteins, transcription factors, and in nearly all known histone acetyltransferases. The BET subclass of bromodomain-containing proteins contains two bromodomains and one ET domain and consists of at least four members in mouse and human, Brd2, Brd3, Brd4, and Brdt. We isolated mouse cDNAs for these genes and studied their expression patterns with particular focus on the testis. Northern hybridization revealed that Brd3 is most abundant in testis, ovary, placenta, uterus, and brain; that Brd4 is rather ubiquitously expressed but is most abundant in mid-gestation embryo, testis, ovary, and brain; and that Brdt is specifically expressed in testis. In situ hybridization and immunostaining on histological sections of mouse testes revealed a strikingly specific and dynamic change of cellular specificity in the germ line during the progression of spermatogenesis. Brd4 is expressed in spermatogonia, Brdt is only expressed in mid- to late-spermatocytes, Brd2 is expressed in diplotene spermatocytes and round spermatids and at low levels in spermatogonia, and Brd3 is expressed in round spermatids. This unique expression pattern suggests that genes in this subclass are not simply redundant. Rather, their expression is tightly regulated in the male germ cell lineage, suggesting that they likely have specific roles in different developmental stages and/or cell types.

Fsrg1 (female sterile homeotic-related gene 1) is the mouse homolog of the human RING3 protein, which has been shown to associate with the E2 promoter binding factor (E2F) transcription factor and to have a possible role in cell cycle-linked transcriptional regulation. The Fsrg1 protein is 60% identical in sequence to the RNA polymerase II mediator subunit Fsrg4, another member of this subfamily of double bromodomain-containing proteins that are homologs of Drosophila female sterile homeotic. Antibodies against murine Fsrg1 were generated and used in immunoblot and immunoprecipitation experiments to identify proteins interacting with Fsrg1 and RING3. In the presence of acetylated but not nonacetylated histone H3 and H4 peptides, RING3 was shown to interact with E2F, mediator components cyclin-dependent kinase 8 and thyroid receptor-associated protein 220, and the RNA polymerase II large subunit. Fsrg1 mRNA had been previously shown to be expressed at high levels in the epithelium of the adult mouse mammary gland. To determine the physiological relevance of these potential associations, we examined the patterns of expression of Fsrg1 mRNA and protein in the adult mammary epithelia during the reproductive cycle as the tissue is responding to estrogen, progesterone, and prolactin. Changes in the nuclear vs. cytoplasmic localization of Fsrg1 were observed and correlated with physiological changes in mammary gland function. The observations suggested that Fsrg1 may be involved in the transcriptional activities of genes involved in proliferation of the mammary epithelia during pregnancy and in orchestrating postlactation involution and apoptosis. Localization of Fsrg1 on euchromatin, the transcribed portion of the chromosomes, is consistent with its hypothesized function as a transcription regulator.

The student is guided in the design of his/her own experiments. Each exercise addresses an issue related to one of the three enzymes examined in the author-directed experimental sections earlier in the book. These enzymes are: ferredoxin-NADP+ reductase, flavin mononucleotide reductase (NADH) and l-lactate dehydrogenase (FNR, LuxG and l-LDH respectively). The issue of whether or not FNR associates with the thylakoid, the membranous structure in the chloroplast, is addressed. Hints regarding appropriate methods are provided. In the exercise that pertains to LuxG, the question addressed is whether a related enzyme, riboflavin reductase [NAD(P)H], is expressed in the luminescent bacterium Photobacterium leiognathi. This related enzyme is usually referred to as Fre and is known to be expressed in other bacterial species. The challenge for the student is to decide which enzymatic characteristic will allow for distinction of Fre activity from LuxG activity. The exercise that pertains to l-LDH addresses tissue-specific expression in mammals of the enzyme that acts on d-lactate, the stereoisomer of l-lactate. Evidence has been published that this enzyme, d-LDH, is expressed in mitochondria in rat liver but expression in other mammalian tissues has not been documented. The student must devise an experiment to assay for the activity of d-LDH in mitochondria from a mammalian tissue other than liver.

This section includes eleven exercises that are focused on the catabolic enzyme that catalyzes the interconversion of pyruvate, the three-carbon acid generated by glycolysis, and l -lactate. This enzyme is l -lactate dehydrogenase ( l -LDH). The importance of this oxidation–reduction reaction, and the variation in the direction of the reaction, in aerobic and anaerobic catabolism, is discussed. A comparison of the primary and secondary structures of related enzymes from a bacterium and a plant is provided. The expression of various isoenzymes, due to tetramers being assembled from two types of subunits, is also discussed. Procedures for preparing extracts from various bovine tissues such as kidney, liver, skeletal muscle and heart are provided. The student is guided in the electrophoretic separation of the isoenzymes of l -LDH in unfractionated extracts. A series of exercises that direct the student in purification of l -LDH from bovine heart are also included. Precipitation with sulfate anions and purification by affinity adsorption are employed. A kinetic experiment to measure the Michaelis constant, K mNAD , of this enzyme for the nicotinic adenine dinucleotide (NAD) substrate is then suggested. The student is then guided in obtaining evidence for the quaternary structure (tetramer configuration) of bovine l -LDH by means of molecular exclusion chromatography. A procedure for an immunoblot to distinguish isoenzymes of l -LDH is also provided.

The six exercises provided are focused on LuxG, a flavin mononucleotide reductase (NADH) involved in luminescence in marine bacteria. The role of this enzyme in providing reduced flavin mononucleotide for the light-generating reaction is discussed. The coordinated expression of the lux operon that encodes LuxG and the other enzymes that generate luminescence is described. The exercises guide the student in the use of a recombinant expression system for purification of an enzyme to which a nickel-binding sequence has been fused (LuxG-hexahistidine in this case). A procedure for growth of a culture of Escherichia coli harboring plasmid DNA that includes luxG under the control of an inducible promoter is provided. The student is then directed to use adsorption to a solid phase to which nickel ions have been bound to purify LuxG-hexahistidine. The student is also guided in performance of various analytical procedures to characterize the purified enzyme. A kinetic experiment to measure the Michaelis constant, K mNAD , of LuxG-hexahistidine for the nicotinic adenine dinucleotide (NAD) coenzyme is suggested. Procedures for other analytical techniques, such as electrophoresis of denatured LuxG-hexahistidine to measure the length of the polypeptide, quantification of protein concentration by means of the Bradford staining method and quantification of protein concentration by measuring absorbance of ultraviolet light, are also included.

Seven exercises focused on ferredoxin-NADP + reductase (FNR) are provided. FNR is an oxidation-reduction enzyme that functions in photosynthetic electron transport. Background is provided regarding the significance of the function of this enzyme in the final step of linear electron transport. The role of the prosthetic, flavin adenine dinucleotide, in electron transfer is discussed. A comparison of the primary and secondary structures of related bacterial and human enzymes is presented to emphasize the value of studying enzymes that function in fundamental metabolic processes. The exercises guide the student in purification of the enzyme from the chloroplasts in leaves of spinach and in analytical experiments to elucidate the enzyme’s properties. Methods used for purification include anion exchange-adsorption and affinity-adsorption. The issue of whether FNR functions as a monomer or dimer in the chloroplast is addressed with a chromatography experiment. The Michaelis constant, K mNADP , of FNR for the substrate nicotinic adenine dinucleotide phosphate (NADP) is measured in a kinetic experiment.

The Drosophila exuperantia gene (exu) functions in both oogenesis and spermatogenesis. Alternative RNA processing and promoter usage generates sex-specific transcripts which differ in their 5' and 3' untranslated regions, but encode the same predicted protein. We have sequenced the breakpoints of an exu allele which is defective in spermatogenesis but functions normally in oogenesis. This allele deletes most of the sequence specific to the male 3'-UTR, together with some flanking DNA, and causes a reduction in steady-state level of exu mRNA in the testis. In addition, we find that a smaller deletion which removes only sequence within the male 3'-UTR reduces the steady-state level of the mRNA and prevents an exu transgene from rescuing male sterility. Males carrying multiple copies of this transgene are fertile, suggesting that the male-specific 3'-UTR functions to maintain a proper level of exu product in the germline.

The genes responsible for luminescence in various species of the marine microorganism Photobacterium, have been used for many years as a tool by researchers and instructors. In particular, the lux operon of Photobacterium fischeri has been used by many instructors to teach recombinant DNA techniques. Two methods using an automated plate reader and multiwell plates were applied to a set of previously-published exercises. In these exercises that involve transfer of lux genes to Escherichia coli to create a luminescent phenotype, this technology was used to screen for Lux(+) colonies. It was found to be more convenient and more sensitive than the previously used method; that is, assaying bacterial plates by direct observation. Eight students synthesized four genomic libraries and isolated six Lux(+) clones. The fluorescent-detection feature of the plate reader was used to verify amplification of target sequence in polymerase chain reaction (PCR) reactions. Lux(+) E. coli colony lysates were examined. An exonuclease-activated, fluorescent DNA probe generated a signal on hybridization to an amplified portion of the luxA gene in each lysate tested. This method is suggested as a means of demonstrating the concept of real-time PCR without the expense of the specialized device typically used for this technique.

Purpose Disruption of the mouse gene encoding the gap junction subunit α3 connexin 46 (α3Cx46) results in the formation of lens cataracts that have a severity affected by the genetic background of the mouse strain. To identify the genes that influence the severity of the nuclear opacity, global gene expression was analyzed in lenses from the 129SvJae strain and compared to the C57BL/6J strain. Methods Lens transcripts were subjected to cDNA microarray analysis. Results on selected genes were confirmed by real-time PCR. Results Genes that were determined to be altered in expression levels as a result of strain differences could be clustered into three groups: energy metabolism, stress response, and cell growth. Conclusions There were no observed changes in gene expression as a result of the lack of α3Cx46 in the different mouse strains, suggesting that the pathways mediated by this connexin do not influence gene transcription in the lens. Analysis of the transcript changes due to strain differences provides new insights into potential genetic modifiers of cataractogenesis. More detailed experimentation will be needed to determine if these observed changes do indeed affect cataractogenesis.

The human Brd2 (Bromodomain-containing 2) gene codes for a double-bromodomain protein that associates with the cell cycle-driving transcription factors E2F-1 and E2F-2. Expression of mouse Brd2 has been shown previously to be expressed in specific patterns in proliferating cells in the developing alveoli in the mammary gland. In the present study, in situ hybridization and immunohistochemical analyses were used to examine expression of Brd2 in developing neural tissues. Brd2 mRNA was detected in brain vesicles, neural tube, spinal cord and dorsal root ganglia (DRG). Immunostaining proved that the message is translated in these tissues and further revealed that Brd2 protein localizes to the nucleus in proliferating cells, but is cytoplasmic in differentiated neurons that are no longer cycling. Brd2 protein in the nuclei of the proliferating neuronal precursors is excluded from the heterochromatin. These observations are consistent with our previous finding that nuclear localization of Brd2 protein correlates with an active cell cycle in mouse mammary alveoli during the reproductive cycle, and similar results from others in cultured fibroblasts. Our findings are also consistent with the cell cycle progression/transcription coactivator function suggested by the association of Brd2 with E2F-1 and E2F-2.

The TATA-binding protein TBP is necessary for the transcription of eukaryotic genes. Multi-protein complexes formed by TBP and different TBP-associated factors are involved in the initiation of transcription by polymerases I and II, and probably III as well. During the formation of an active initiation complex, TBP makes specific contacts with other proteins, for example TFIIB and RNA polymerase II (refs 2-4). Here we describe the cloning and characterization of a Drosophila gene product with considerable sequence similarity to TBP and a highly restricted expression pattern in the embryo. This TBP-related factor is a DNA-binding protein but is not likely to be a basal transcription factor. Our results suggest that TBP-related factor is a sequence-specific transcription factor that shares the DNA-binding properties of TBP.

The polytene chromosome puff at 68C on the Drosophila melanogaster third chromosome is thought from genetic experiments to contain the structural gene for one of the secreted salivary gland glue polypeptides, sgs-3. Previous work has demonstrated that the DNA included in this puff contains sequences that are transcribed to give three different polyadenylated RNAs that are abundant in third-larval-instar salivary glands. These have been called the group II, group III, and group IV RNAs. In the experiments reported here, we used the nucleotide sequence of the DNA coding for these RNAs to predict some of the physical and chemical properties expected of their protein products, including molecular weight, amino acid composition, and amino acid sequence. Salivary gland polypeptides with molecular weights similar to those expected for the 68C RNA translation products, and with the expected degree of incorporation of different radioactive amino acids, were purified. These proteins were shown by amino acid sequencing to correspond to the protein products of the 68C RNAs. It was further shown that each of these proteins is a part of the secreted salivary gland glue: the group IV RNA codes for the previously described sgs-3, whereas the group II and III RNAs code for the newly identified glue polypeptides sgs-8 and sgs-7.

The 68C region of the Drosophila melanogaster salivary gland polytene chromosomes harbors the structural genes for the three salivary gland glue proteins sgs-3, sgs-7, and sgs-8. This region is puffed during the third larval instar, the stage when glue proteins are being produced in the salivary glands. The puff regresses near the end of the third instar as a result of an increased titer of the steroid hormone ecdysterone in the larval hemolymph. The experiments reported here were designed to determine whether the ecdysterone effect on puffing at 68C is correlated with hormone effects on expression of the three puff RNAs. In the first series of experiments, it is shown that there is a more rapid disappearance of 68C RNA transcripts from salivary glands cultured in the presence of ecdysterone than from glands cultured in its absence. The second set of experiments, in which 68C transcripts were pulse-labeled in salivary glands cultured in the presence or absence of hormone, demonstrates that one effect of ecdysterone is to cause a sharp reduction in the rate at which newly synthesized 68C transcripts accumulate. The final experiments follow the time required for ecdysterone to produce this effect, and show that it occurs in salivary glands exposed to the hormone for as little as 15 min. In all of the experiments, the RNA products of the Sgs-3, Sgs-7, and Sgs-8 genes acted coordinately.

The mutation l(1)npr-1 is located at cytological location 2B5 on the X chromosome in Drosophila melanogaster. We have found that this mutation causes absence of the normal product of the 2B5 locus and that it has the following phenotypes: the 68C glue puff on the third chromosome does not regress when mutant salivary glands are cultured in the presence of ecdysterone; the three 68C glue protein mRNAs are not synthesized; and a transformed Drosophila strain carrying both a normal resident 68C Sgs-3 gene and an introduced functional Sgs-3 gene with only a few kb of flanking sequences expresses neither Sgs-3 RNA if the l(1)npr-1 mutation is crossed into the stock. Thus the normal product of the l(1)npr-1 gene is required for regression of the 68C puff, and the l(1)npr-1 gene product allows expression of the Sgs-3 gene by interacting, either directly or indirectly, with DNA sequences near this glue protein gene.

Using an antisense construct of the discoidin gene transfected into Dictyostelium, we have repressed the expression of the three endogenous discoidin genes. Transformants exhibit a greater than 90% reduction in accumulated discoidin mRNA and protein. Nuclear run-on assays show that both the endogenous and the antisense genes are transcribed. Since only minor amounts of endogenous gene transcripts and none from the antisense gene can be detected on blots, we suggest that hybrids are formed within the nucleus and are rapidly degraded. Discoidin is believed to play a role in cell-substratum interaction and exhibits homologies to fibronectin. Discoidin-minus mutants exhibit the developmental phenotype of not streaming on a plastic surface. Antisense transformants show a similar phenotype and are thus phenocopies of these mutants.

Publisher Summary This chapter discusses that the Dictyostelium transformation system provides a tool to study gene regulation in Dictyostelium at the molecular level. The major factors influencing molecular biological techniques in Dictyostelium are its genome organization and DNA composition. Gene fusions carrying the promoter regions of developmentally regulated Dictyostelium genes are expressed and regulated in a manner similar to their genomic counterparts. Up to now, this has been shown for an actin gene, two discoidin genes, the Dictyostelium ras gene, and pst-cath , a prestalk-specific gene. The first major application of the transformation system is the mapping of promoters and sequences essential for regulated gene expression. Trans-acting factors are investigated using cotransformation. It is possible that extrachromosomal vectors containing the promoter of a differentially regulated gene can be isolated at time points during development while maintaining their chromatin structure. The investigation of the protein organization in the promoter region on such a DNA gives insight into the DNA protein interactions during gene regulation. The antisense transformation provides means to study the phenotype of cells deficient in a known gene product, thus mimicking a loss of function mutant.